key: cord- -uit nm authors: arnold, theresa; gulati, mitu; panizza, ugo title: how to restructure euro area sovereign debt in the era of covid- date: - - journal: nan doi: . /cmlj/kmaa sha: doc_id: cord_uid: uit nm nan facing holdout issues on the local-law debt. this was also the case with barbados' debt restructuring in , which followed the greek template. however, both greece and barbados faced significant holdout problems in their foreign-law-governed debt; many of the foreign-law creditors refused to take the same offers that were made to the local-law creditors and ended up receiving higher recoveries. in the analysis that follows, we focus on three strategies a euro area sovereign might utilize in a possible future crisis to solve the primary source of disruption to modern day sovereign debt restructurings: the holdout creditor problem. therefore, in the analysis that follows, we assume that the sovereign and its official sector backers have been able to negotiate a restructuring deal with the majority of creditors, but those creditors do not want to enter into an agreement where either their recoveries are at risk of disruption from litigation by holdout creditors or they are in danger of being embarrassed by having suffered much bigger haircuts than the holdouts. we also put to the side the possibility of legal changes at the broader european level that might ameliorate the holdout problem more directly, and abstract from the possibility of reforms that will create a clear seniority structure within euro area bonds. although the analysis that follows might be applied to other euro area sovereigns, we look specifically at italy for the purposes of our analysis. by using italy as an example, we do not mean to suggest that it needs to restructure its debt pre-emptively. however, italy did face a confidence crisis in late and the coronavirus has hit it particularly hard in . italy's gargantuan debt stock of around e . trillion and a debt to gdp ratio of per cent make italy vulnerable to another confidence crisis. the economic recession and ). an alternative proposal which is fiscally neutral but could reduce the likelihood of rollover risk is the padre plan. see pierre pâris and charles wyplosz, padre: politically acceptable debt restructuring in the eurozone, geneva special report on the world economy , icmb and cepr ( ) . for a discussion of the risks associated with a pre-emptive italian debt restructuring, see giampaolo galli, why restructuring of the italian public debt should be avoided ( ) (osservatorio cpi <https://osservatoriocpi.unicatt.it/cpi-why_a_restructuring_ should_be_avoidced_gg_ocpi.pdf>); ugo panizza, 'come risolvere il problema del debito pubblico italiano: un'analisi critica delle soluzioni facili' (forthcoming ) rivista di politica economica. for a prior discussion by one of us, see ugo paniza, public debt in italy: myths, facts and policies ( draft) <http://repec. graduateinstitute.ch/pdfs/working_papers/heidwp - .pdf> ferdinando giugliano, the euro's $ . trillion dollar problem, bloomberg, april ( ) <https://www.bloomberg.com/ news/articles/ - - /the-euro-s- - -trillion-italy-problem> accessed july ; peter s goodman, 'italy's biggest economic problem? it's still italy', ny times, august ( ), <https://www.nytimes.com/ / / /business/italy-economydebt.html> theresa arnold, mitu gulati and ugo panizza • euro area sovereign debt in the era of ballooning expenditure needs associated with covid- are expected to bring the debt-to-gdp ratio well above per cent. quite possibly, such a crisis could be dealt with through some combination of fiscal adjustment, economic reform and official (esm/ omt) financial support. this said, it is also conceivable that such a crisis might lead to a situation in which italy might have to restructure its debts. we thus ask two related questions: in such a situation, what restructuring options might italy have? and could italy do something now to put itself in a better position to deal with such a situation in the future? two considerations for any government in undertaking a debt restructuring are: (i) how to minimize the impact of that restructuring on its costs of future borrowing in situations where the sovereign needs to be able to tap those markets regularly; and (ii) how to minimize the domestic economic and political costs in situations where a large portion of the debt is held domestically. the balance of the foregoing considerations, along with the interests of official sector sponsors, will determine how the choice among restructuring options is made. we do not attempt the exercise of prescribing a specific restructuring plan for italy. instead, we put what we see as the most plausible options on the table. the options to prepare for a possible future debt restructuring fall into three categories: do nothing; do a little; or do a lot. one can think of these options in terms of the choice of what type of crisis insurance to buy: none at all; cheap, partial-coverage insurance; or expensive, full-coverage insurance. the article proceeds as follows. section describes the basic data on italian sovereign debt. section works through the three options noted above. section concludes. according to italian treasury and bank of italy data, as of mid- , italian public debt amounts to approximately e . trillion (table ) . the main holders of italian public debt are domestic banks and other domestic financial institutions (e . trillion, corresponding to per cent of the total), non-resident entities (e billion, corresponding to per cent of the total) and the bank of italy (e billion, corresponding to per cent of the total). italian households, which in the early s held about per cent of the italian public debt, now hold less than per cent of the total. about per cent of the total stock of italian debt (e trillion) consists of tradable instruments (government bonds and bills), which we refer to as "bonded debt". the data show that per cent of bonded debt is held by domestic banks and other domestic financial institutions, per cent by non-residents, per cent by the bank of italy and the for an early discussion of the economic impact of the covid- crisis on italy, see ugo panizza, 'europe's ground zero', in richard baldwin and beatrice weder di mauro, mitigating the covid- economic crisis: act fast and do whatever it takes (cepr press ). the most recent bank of italy data are available at <https://www.bancaditalia.it/pubblicazioni/finanza-pubblica/index.html>. treasury data on the composition of bonded debt are available at: <http://www.dt.tesoro.it/en/debito_pubblico/dati_statistici/sca denze_titoli_suddivise_anno/ . bank of italy data> used in this paper refer to the end of july , while italian treasury data refer to the end of september . there is a small discrepancy between bank of italy data, which report a total for bonded debt of e , billion, and treasury data which report a total for bonded debt of e , billion. remaining per cent by households. the remaining debt is held by italian banks (e billion, corresponding to per cent of the non-bonded debt), resident households (e billion, corresponding to per cent of the non-bonded debt) and non-residents (e billion, corresponding to per cent of the non-bonded debt). more than per cent of italian bonded debt is denominated in euro (issues in us dollars amount to e . billion, issues in british pounds amount to e . billion and issues in japanese yen amount to e million; table ). most bonded debt consists of treasury bonds (btp, per cent of the total), treasury certificates (cct, per cent of the total) and treasury bills (bot, per cent of the total), all of which are governed by local italian law. a small share of bonds are listed on the luxembourg exchange, under a medium term note program (emtn). most of these bonds are denominated in euro, but a small amount are in british pounds and japanese yen. all of these luxembourg-listed bonds, as best we can tell, are governed by local italian law, although as we detail later, these bonds do have different (and better) legal protections for creditors than the aforementioned treasury bonds, treasury certificates and treasury bills. finally there is a small amount of debt in the form of global bonds, issued under a us programme and governed by new york law (roughly e billion, denominated in us dollars). in sum, about per cent of italian debt is governed by local law and, therefore, benefits from the local-law advantage ( . per cent if we include emtn and . per cent excluding emtn; figure ). about one-third (e billion) of outstanding btps were issued before and, hence, do not include collective action clauses (cacs) that are now required by the eu in order to provide an orderly path to restructuring. other bonds that do not include cacs include foreign-law bonds issued before , ispa bonds (these were bonds issued before to finance infrastructure projects), and bot. in total, bonds and bills without government bonds of the euro zone with maturities in excess of one year, the treasury will albeit continue to issue, with its usual regularity, bonds without the above clauses, these being outstanding bonds at / / and off-the-run bonds, so as to guarantee the necessary liquidity.' hence, the share of bonds without cacs is likely to be slightly higher than what is reported here. cacs amount to approximately e billion (ie, per cent of total outstanding bonded debt; per cent of the total if we exclude bot, which amount to approximately e billion). figure shows the maturity profile of outstanding italian bonds. during - , the italian treasury will need to roll over about e billion per quarter, with large amounts of btp without cacs that need to be rolled over in q and q . figure shows the residual stock of bonds without cacs. while the curve is steep, starting with e billion in q and dropping to e billion in q , this is mostly due to maturing bot. after the bot effect ends, the curve flattens and by , italy will still have more than e billion of bonds without cacs. the top panel of figure plots the expected composition of italian bonded debt over the period - . it shows that in , more than per cent of bonded debt will still consist of long-term bonds without cac, if we add bots and hard-to-restructure foreign law and emtn bonds, we find that in , almost one-quarter of italian bonds are hard to restructure. if to this debt we add loans (ie non-bond debt), we find that only two-thirds of italian public debt consists of bonded debt with cacs (bottom panel of figure ). the do nothing approach is for the italian authorities to literally do nothing. that is, to take no steps to utilize the local-law advantage in advance of a potential crisis. this is perhaps the most likely outcome, since governments are loath to admit that a debt crisis might be in the offing. the main reason for a country to take the do nothing approach is the concern that taking steps to prepare for a crisis will send a negative signal to the market and either lead to an increase in sovereign yields or, worse, cause the crisis to happen. in most cases, this head-in-the-sand approach is far from ideal. but, as explained below, it may not be a terrible approach in the italian case, given the advantages the legal terms governing the current italian debt stock provide. as a starting point, italy will have first-generation euro cacs in over per cent of its bonded debt stock by mid- ( per cent of its total debt stock, see figure ). these clauses were put in place starting in january , mandatorily, for all euro area sovereign debt issuances of over a year in maturity. simplifying somewhat, the presence of these clauses means that all of the bonds containing them can be modified in a single transaction, so long as the approval of . per cent of the holders of each bond and per cent of the holders in all the bonds put together (in principal amount) is obtained. however, there are issues. the requirement that the approval of . per cent of the creditors in each bond be obtained to effect a consensual debt restructuring gives rise to the risk of holdout creditors targeting a subset of the smaller bonds and accumulating blocking positions in them. in the greek restructuring of , where the majority of foreign-law bonds were governed by english law and had cacs that could only be activated with super-majority votes, there were holdouts in separate sovereign and sovereign guaranteed bonds (amounting to about e . billion in face amount of debt). the holdouts, after blocking the votes in their bonds, got paid in full. and this was in large part because greece did not want to risk the type of litigation that argentina was suffering at that same time. for the italian local-law bonds with cacs, the vote requirement is lower than what was required in the greek foreign-law bonds ( per cent, with a quorum requirement in the context of a bondholder meeting). but this is unlikely to be a significant barrier for a wellcapitalized hedge fund seeking to utilize the holdout strategy. in addition, if a creditor can figure out what the holdings of the european central bank (ecb) are in particular bonds (and they are substantial in many bonds), he/she will be in an even stronger position to hold out. the reason is that the ecb has asserted a , , , , source: own elaboration based on imf data and italian treasury data. in order to build the debt composition for - , we started with imf forecasts for the total stock of debt and then assumed that the shares of loans and bot remain constant. data for external debt, and domestic bonds are instead taken from the flow of maturing bonds data used for figure . the graph is built under the conservative assumption that no external bonds will be issued in the next three years; while italy issued dollar bonds in october , this was a small amount ($ billion) and these issuances-and possible future external issuances-are unlikely to substantially change the figure given their relatively small amount. commitment to not voting in favour of any debt restructuring plan. in a cac that calls for an affirmative vote of the creditors of a certain percentage of principal amount, abstention is scored as a no vote. the ecb's commitment, therefore, effectively lowers the percentage the holdout would be required to purchase in order to block a modification. the foregoing has already caused concern in policy circles-and has resulted in proposals for the euro area to reform its cacs starting in . that said, an argument could be made that the potential holdout problem is less serious than it has been made out to be. one way to see this is by detailing the steps a holdout creditor seeking to litigate against a distressed sovereign generally needs to take. assuming that the bond in question has not yet come due but the sovereign is in default on other indebtedness, the holdout will typically utilize her bond's cross default provision to accelerate the payments owed on her bond, demanding immediate payment of principal and accrued interest. the ability to declare a default and accelerate is key in the litigation context because suing the sovereign on small unpaid coupon payments is unlikely to be worthwhile when measured against legal costs. italian local-law bonds though, except for the sliver listed in luxembourg under the emtn program, have neither cross default nor acceleration provisions. in other words, the only bonds worth holding out on will be the ones where a substantial principal amount is due. worse, from the holdout creditor's perspective, those bonds (except again for the luxembourg-listed ones) lack explicit waivers of sovereign immunity from either suit or execution, which means that the italian government lawyers can potentially use that as a defence. put that together with the dysfunction of the italian legal system, and it is hard to imagine one of the specialist hedge funds that won large recoveries against argentina in the new york courts wanting to invest in holding out on the non-luxembourg listed italian local-law bonds. in contrast to italian creditors, the foreign-law holdout creditors in the greek restructuring in had the rights to declare cross defaults, accelerate the debt and sue for recovery in english, swiss or japanese courts under the explicit consents to jurisdiction and waivers of immunities from suit and execution in the contracts. absent those rights to a key question will be whether there is an implicit waiver of sovereign immunity under italian law. ordinarily, we would presume that an italian court would find such an implicit waiver as a matter of customary international law since sovereign bond issuances are considered a commercial activity. however, italian courts have shown a willingness to reinstate immunity for actions of the state that are necessary to tackle an extreme crisis (and covid- appears to have created just such a situation). - . in the most recent doing business ranking from the world bank, italy ranks nd out of countries in terms of ease of contract enforcement. average enforcement time is , days and the cost of litigation, as a fraction of the claim is . %. those do not strike us as numbers that would attract litigation oriented holdout creditors. for details, see <https://www.doingbusiness. org/content/dam/doingbusiness/country/i/italy/ita.pdf> theresa arnold, mitu gulati and ugo panizza • euro area sovereign debt in the era of declare defaults, accelerate, bring suit and execute against the state's assets in a jurisdiction where the legal system moves quickly and efficiently, a sovereign such as italy might hope that it would simply be unattractive for any investors in its bonds to hold out. if so, doing nothing might work. that said, the costs of default for a country that needs to regularly tap the borrowing markets can be substantial. and potential holdout creditors, especially if they watched what happened in greece a few years prior, might recognize their potential to impose costs on italy even in the absence of substantial litigation rights. this is because during the restructuring period the country is likely to be given a selective default rating by the credit rating agencies, which will make new borrowing hard. plus, its bonds are unlikely to be eligible for delivery at the central bank's discount window, making them an unattractive investment. the end result will probably be an increase in sovereign borrowing costs beyond the usual default penalty. all in all, a country like italy may not want to have a number of defaulted bonds outstanding after its restructuring. let us assume that the prospect of being in a long-term payment default vis-à-vis a substantial number of creditors, even if their litigation options are not attractive, is a prospect that the italian government does not relish. the question then is whether there are some non-obvious options available under existing italian law. we see two: (a) an extension of maturities of those bonds that were issued at low rates in prior years and that are coming due; and (b) the imposition of a withholding tax equivalent to the haircut taken voluntarily by non-holdouts. as we discuss further below, these options pose some litigation risk, and italy might choose to 'do a little' by taking steps to mitigate that risk. as a threshold question to examining these options, we must ask whether euro area countries contemplating a restructuring are constrained to utilizing only the current euro cacs and no other technique. the answer here, we think, is no for a number of reasons, including the explicit language of the cacs and the policy intent behind them. to clarify the question, let us say that the italian government decides that it wants to reduce its debt burden by imposing a withholding tax (more on this later). do the euro cacs in its existing bonds bar the use of this strategy? no. there is nothing in the mandate requiring the inclusion of euro cacs that constrains any individual euro area country from using its tax authority. indeed, there could not be, as italy has retained the authority to levy taxes, even as it has delegated other aspects of sovereignty to the euro area. italy is constrained in terms of the contract provisions it has agreed to with bondholders, but the vast majority of its local-law bonds (except those pesky luxembourg-listed ones) do not contain the protections against the sovereign imposing withholding taxes that are standard in foreign-law bond issuances (including in those issued by italy). a similar analysis applies to other routes to enable an easier restructuring, such as making it harder to sue in italian courts; increasing the costs of litigating a contracts case, for example. that would not be particularly good for the italian legal system and economy generally, but it would deter holdout creditors with debt instruments whose holders were required to litigate in italian courts under italian law. and there is nothing in the body of european law respecting cacs that constrains italy from taking such actions. the language of the cacs themselves signals the possibility of other restructuring techniques being used. in relevant part, they say: in the case of a cross-series modification, the terms and conditions of the bonds and debt securities of any other series . . . may be modified in relation to a reserved matter with the consent of the issuer and [the requisite proportion of bondholders]. the word 'may' is crucial; it tells us that the cacs are a debt restructuring option that the sovereign may use. but just one option. if these cacs were meant to say that sovereigns were constrained from using other restructuring options, they would have said 'must' instead of 'may'. one might ask whether there is anything in the esm treaty, whose article mandated that all euro area nations utilize cacs, suggesting that future debt restructurings of the bonds with cacs could only proceed by utilizing the cacs. there is not. nor is there anything in the history of why these cacs were put in place by the european authorities that suggests so. the history tells us that, starting with the infamous deauville beach walk on october , the utilization of cacs was meant to set up an orderly mechanism for future debt restructurings in europe and to signal to investors that the purchasers of euro area sovereign bonds that those bonds could be restructured in the future. mark weidemaier, in his paper, sums this up: [a euro area] sovereign that has issued local-law debt [with cacs] remains free to alter its law to facilitate restructuring, although it will encounter various legal constraints in doing so. these constraints, however, are not absolute; there is room for the prudent exercise of the local law advantage. having determined that the existence of the cacs does not preclude the sovereign from using the local-law advantage to facilitate other paths to a restructuring, we turn to a closer examination of the two options mentioned above: a maturity extension and the imposition of a withholding tax. a paper from (edelen et al.) , and two follow-ups from (cervantes et al. and cramer et al.) , suggest that there might be a strategy to unilaterally extend maturities of debt with especially low coupon rates. assuming this strategy is workable, the idea would be to ask the holders of local-law bonds with especially low coupons to voluntarily extend maturities. the addendum to that polite request would be that those who refused would have their maturities unilaterally extended by law. the provision that the aforementioned papers hone in on is article of the italian public debt consolidated act (the "debt act"). under article : [ the ministry] has the authority, within the annual limits established by the budgetary law, to issue framework decrees that allow the treasury to . . . proceed, in order to restructure the national and external public debt, to the reimbursement before maturity of bonds, to the transformation of maturities. that language seems clear on its face. it says that the ministry of economy and finance (the 'ministry') has the authority to issue decrees to allow the treasury to either redeem bonds at par (maybe because interest rates have dropped considerably since issuance) or transform (extend) maturities (maybe because interest rates have increased considerably since issuance). in both cases, investors lose and would presumably impose a reputational cost on the issuer. but the options to either reduce or extend the duration of a loan are familiar contractual provisions in the debt world (particularly the former ). one might question whether, if italy does hit turbulent times, this power to extend maturities will be of much value. after all, countries in deep sovereign debt crises typically need to impose principal haircuts as well as maturity extensions. the objection is a fair one. but, given the large amount of the italian debt that is held domestically and the negative impact on the economy that will likely be caused by imposing brutal principal haircuts to the holdings of domestic financial institutions, maturity extensions may be an attractive option for the government. this is especially so if a significant portion of the italian debt stock was incurred when borrowing rates were especially low (as is the case). under the structure of article , the treasury department receives the power to call bonds or extend maturities only if a framework decree authorizing such actions is issued by the ministry. these decrees are not law; they are administrative actions implementing legal authority. they are also of limited duration. each decree lasts for a year, no more. in prior years, they have been used to authorize the treasury to do both debt exchanges and redemptions. it was also under article 's structure that a framework decree was issued by the ministry to impose euro cacs starting in . let us say therefore that the italian government decides to issue a framework decree in to authorize the treasury, if need be, to extend the maturities of all italian local-law bonds by five years. there are at least two possible barriers to the treasury utilizing this authority. we take the potential barriers in turn. first, we consider article of the debt act. this is perhaps the most significant barrier to the use of article . it requires that 'payments of public debt [cannot be] reduced, paid late or subject to any special levy, not even in case of public necessity'. the question then is whether extending maturities or reducing them (as article seems to allow the treasury to do) violates the '[may not be] paid late' requirement. at first cut, it does not appear so, since that reading would reduce article to meaninglessness. that said, the argument could also be made that our reading of article reduces article to meaninglessness as well. and courts generally do not like to negate statutory provisions. the reality, therefore, is that if italy decides to go down this path, a court will likely have to decide which of the clauses to favour, while putting a gloss on the other. and italy's hope would have to be that the court chooses to interpret article in a manner that weakens it, such as by reading it to require simply that whatever the maturity date iswhether shortened, lengthened or kept the same-it has to be complied with. the foregoing is not implausible in the context of the euro cacs. the reason being that if article were to be interpreted to bar extensions of maturity, that would also negate crucial provisions of the cacs that, by italian law, explicitly allow for the sovereign to extend maturities of the bonds with euro cacs with a super majority approval of the creditors. the question for the italian government will be whether it wishes to go down this path of litigation. earlier we discussed the question of whether the existence of the euro cacs as a restructuring mechanism constrains the use of article 's power generally. now, we address the question of whether the treasury might use its article power in a specific bond that contains a euro cac as part of its contract terms. after all, if a crisis were to occur at any time over the next few years, there would be more italian local-law debt with cacs than without. the euro cacs have an explicit mechanism by which the maturities of the bonds can be extended, requiring a pre-specified vote of the creditors. our view is that, for those bonds, the right of the sovereign to unilaterally extend the maturities is dubious. the reason being that these bonds already have a term explicitly governing how maturities may be extended. but for the remaining debt-which we estimate in the years - to range between per cent and per cent of the total debt stock ( figure )-there are no cacs. for that latter subset, there is a strong argument that italy can use the power that it already has by law to unilaterally extend maturities. we should caution though that such a power is unlikely to be unlimited. if the sovereign, for example, tried to extend the maturities of its one-year bonds by years, there would likely be an immediate expropriation challenge. because the option of unilaterally extending maturities is likely to only work for locallaw bonds that do not contain cacs and the relief that italy would obtain would be limited compared to a more significant restructuring that reduces principal amounts as well as extending maturities, italy may wish to pursue other options presented in this article. however, if italy wanted to make this tool a more feasible option for a future restructuring, it could reduce the litigation risk associated with this option by proactively clarifying the apparent discrepancy between article and article . indeed, repealing article might be even better. another objection that we have heard informally from some in the italian government has to do with the ministry's annual decrees issued between and , which say that restructurings in those years will occur via 'mutual consent'. this guidance, we suspect, was a signal to the markets that restructurings would be done via a consultative process. there is no specification of what that consultative process would be, nor have we been unable to uncover evidence of the intent behind the words 'mutual consent'. but we have reason to think that 'mutual consent' does not mean that unanimous approval of creditors is required. there are two points worth considering here. given that bonds issued after contain euro cacs, "mutual consent" cannot possibly mean a requirement of unanimous creditor approval because that would contradict the explicit super majority requirements of the euro cacs for maturity extensions. further, the mutual consent requirement, whatever it means, comes out of an annual decree. it is up to the ministry, every year, to decide whether it wishes to use that language in its decrees. if it happens to be a year where the ministry wishes to give the treasury to power to unilaterally extend maturities, it can delete the 'mutual consent' language from that year's decree, as it has in many prior years. for discussions of the argument that the power of unilateral extension might apply to the bonds with cacs as well, see cervantes et al (n ); cramer et al (n ). we do not, however, think that an expropriation objection would work generally against all uses of the art power to unilaterally extend maturities. expropriation objections apply to a state taking away property rights that creditors have been granted (eg, the greek retrofit of ). however, art has been in place since at least. see cervantes et al (n ). that said, italy would be on stronger ground in the future if it issued a clarification of art 's powers earlier rather than later. inherent to sovereignty is the power to tax. bondholders who are owed moneys by a sovereign have, therefore, since time immemorial, worried about the sovereign deciding to impose a new tax on the payments it owes as a way to avoid payment. as a result, almost every international sovereign bond governed by the laws of new york or england contains what is called the tax gross up clause. this clause protects the bondholder against the sovereign deciding to levy an extra tax on the payments it owes on the bond as a way of doing a back door restructuring. most local-law-governed bonds, however, do not contain such a protection. and that is the case for all of italy's local-law bonds, except the small fraction of luxembourg-listed ones mentioned earlier. a simple strategy to deal with holdouts, therefore, is for the minister of finance to give a speech to local bankers in which she says that holdout bonds will be taxed so that the net (after tax) return will equal the payout on the new bonds. this was tried in jamaica's restructuring of local-law bonds in . the minister's threat was successful and he never in fact had to introduce the tax. the fly in the ointment is if the country has bilateral investment treaties and double taxation treaties that bar such actions against the citizens of other countries. an italian government considering such a strategy would need to consider the risk of future litigation under these treaties. indeed, it could reduce the risk of this litigation by being careful about the provisions it agrees to in any new treaties. taking steps today to reduce the future litigation risk associated with an exercise of italy's power to unilaterally extend maturities or impose a withholding tax on holdouts would improve italy's future restructuring options. but neither of these options provides italy with as much flexibility for significant debt relief as the option of retrofitting more effective cacs, described in the next section. governments, as we noted at the start, are generally reluctant to do anything that might signal to the outside world that they are concerned about the possibility of a financial crisis; even if everyone recognizes that it is a real possibility. so, 'do a lot', is not a strategy we expect to be embraced. nevertheless, let us assume for the purposes of this section that a sovereign such as italy would want to minimize the future costs of potential holdouts in a possible restructuring. buchheit explains how the clause works: 'any deduction as a result of the imposition of withholding taxes thus becomes the borrower's responsibility because the borrower's payment to the lender must [by virtue of the tax gross up clause] be topped up to compensate for the deduction.' lee c buchheit, how to negotiate eurocurrency loan agreements ( st edn ). we have found no mention of this technique in the sovereign debt literature. unsurprisingly, sovereign debt restructuring guru, lee buchheit, advised jamaica at the time. we have not delved into the complexities of italy's tax agreements with other countries. the aforementioned art could pose a barrier here as well, since it bars any 'special levy'. as mentioned earlier, clarifying that art was not meant to pose such a barrier in advance of a crisis might be advisable. theresa arnold, mitu gulati and ugo panizza • euro area sovereign debt in the era of operating under that assumption, we think that the strategy of enhancing the existing cacs in bonds that have them and retrofitting them in the debt that does not already have them is a good option for a number of reasons that we explain below. as noted earlier, all euro area sovereign bonds issued since with a maturity greater than one year already have cacs in them. but these cacs contain the requirement that a . per cent vote of the holders, in principal amount, be obtained for each bond in order to use the aggregation feature to modify all of the bonds containing the cacs. and this creates a vulnerability to holdout creditors. further, there is a whole set of debt instruments that are going to be even more vulnerable to holdouts, which are the bills with maturity under a year, the long-term bonds issued before , government guaranteed instruments, non-bond debt (eg, syndicated loans) and so on. the reason being that cacs were not made mandatory for these sovereign debt instruments. euro area sovereigns could have inserted them voluntarily; but, as far as we know, none did. realistically though, if the crisis is an especially serious one the government is going to need to restructure some of these instruments as well. thus far, we have tackled the easy question of whether the sovereign might use one of its other powers-such as its taxing authority-to engineer a debt reduction without running afoul of the mandatory cacs. the answer was yes. the trickier, albeit closely related, question is whether the sovereign can enhance the existing cacs in its bonds by using its local-law authority. by enhancing, we mean making changes to the existing model cacs provided by the european authorities to make the bonds even less vulnerable to holdout problems than they are now. in particular, two modifications that have already been the subject of debate among policy makers are: (a) introducing a mechanism by which the entire debt stock of a country can be modified at a single shot; for example, via a vote of per cent of all the holders across all the bonds, so long as some non-discrimination criterion is satisfied (the so-called single shot or single-limbed feature); and (b) an addendum to the voting rules for modifications that disenfranchises the ecb from having its votes counted (assuming that the ecb continues to hold to the position that it is legally constrained from voting). conventional wisdom in sovereign debt restructurings is that short maturity instruments (under a year), trade credits, and guaranteed debt, generally get paid in full. but that assumption only holds where the amount of debt in these instruments is small. in the recent barbados restructuring of - , the amount of short-maturity debt was so large that it had to be restructured. and, since those instruments had no cacs, they had to be retrofit into them in the same manner as was both changes to the existing euro cac structure correct for design flaws in the original version and enable the cacs to better achieve their goals of deterring holdouts while providing a clear and predictable mechanism by which creditors can participate in a burden sharing exercise. at the time the original euro cacs were designed, it was thought that a bond-by-bond vote requirement of . per cent would be enough to deter holdouts. and the implications of the ecb not being able to vote in a restructuring (or having to always vote no, regardless of the restructuring proposal) had not been fully considered. so, the question is whether an individual nation, such as italy, concerned about the possibility of a crisis, can act on its own via its 'local law' advantage to legislate improvements in its cacs consistent with the original goals of those cacs. there are two potential barriers here. first, for new bonds issued with the improved cacs, there is the question of whether deviating from the form mandated in violates european law. secondly, for already-issued bonds where these provisions are retrofit via legislative action, there is the potential for creditor lawsuits claiming an interference with property rights. we take the possibilities in turn. neither poses a significant barrier. a country including enhanced anti-holdout provisions in its new bonds potentially violates the instruction in the esm treaty that cacs be introduced in a manner such that their 'legal impact is identical'. the argument against italy being able to fix the design flaws in its cac would be that all european countries are required to have the same flaws until the european authorities decide it is time to fix those flaws for everyone. there is a certain formalist appeal to the foregoing. 'identical legal impact' arguably means the same in every way. but, the very manner in which these euro cacs were designed tells us that the creators could not have meant to use the term 'identical legal impact' in such a broad fashion. the more likely meaning of 'identical legal impact' is much narrower. that is, that every country would have an identical baseline of the same basic cac framework that would signal to holders of sovereign bonds that a debt restructuring was possible. and that one way (not the only way) by which this restructuring could be implemented would be via the basic euro cac. and everyone had to have that identical baseline. there was no prohibition, however, on the sovereign enhancing the operation of the cacs by including additional provisions to its debt contracts. one way to see the foregoing is to compare the operation of cacs in the italian foreign-law (new york law) versus local-law bonds. art ( ) of the esm treaty says: 'collective action clauses shall be included, as of january , in all new euro area government securities, with maturity above one year, in a way which ensures that their legal impact is identical'. some commentators do take this view. for example, klaus-albert bauer writes: 'a natural understanding of [esm art ] this clause would seem to prohibit not only the issue of bonds with non-conforming collective action clauses but also the later unilateral amendment of euro area cacs to suit a particular issuer's needs in times of crisis.' klaus-albert bauer, 'the euro area collective action clause-some questions and answers at ', in collective action clauses and the restructuring of sovereign debt (n ). theresa arnold, mitu gulati and ugo panizza • euro area sovereign debt in the era of the cacs in the italian foreign-law bonds, such as the one issued in october , are buttressed with all sorts of potent investor protections including acceleration clauses, cross default provisions, waivers of immunity and consents to jurisdiction. what this means is that cacs in the foreign-law bonds, if the bondholder has the votes to block a restructuring attempt, can be turned into a potent litigation weapon. assets can be seized, payments to other bondholders can be blocked and so on. conversely, in the local-law bonds that lack these protections, there is no weaponry for the holdout creditor other than the hope that the sovereign does not want to suffer the reputational cost of default. the point being that the issuer, by including a stand-alone cac in the local law bonds and not adding extra creditor protections, has an effective anti-holdout weapon, rather than the possible creditor weapon that the cac tends to be in the foreign law bonds. both the localand foreign-law bonds contain identical cacs, but they can potentially have radically different effects as a function of the other contractual bells and whistles that the issuer adds (or chooses not to add) to the baseline. and this was explicitly envisioned right from the start by the european authorities, who understood that sovereign bonds would be issued under both local and foreign law and that the sovereigns may want to include enhancements such as acceleration clauses or trustee structures. plus, the actual cacs for foreign-law bonds were designed differently in one crucial fashion right from the start-the governing law is a reserved matter that cannot easily be changed in the foreign-law bonds whereas it is not a reserved matter at all in the local-law bonds. that is a significant difference, as weidemaier ( ) points out. in other words, the cacs were designed such that they could not have identical impact (in its most literal sense) right from the start. given the foregoing, we think that the sensible interpretation of article ( )'s dictates is that every euro area nation needs to implement identical model cacs. but those serve as a baseline; ensuring that creditors all are forewarned that a restructuring of sovereign debt in the future is possible and that one method that might be used is the basic euro cac mechanism. nothing, however, stops the sovereign from either adding in more investor protections (making the cac a weaker tool from the sovereign's perspective) or adding improvements to the voting mechanism (making the cac a stronger tool from the sovereign's perspective) as long as the basic model cacs are kept in place. to reiterate, the euro cacs serve as an identical baseline for all euro area sovereigns. from a policy perspective, it is easy to see why the foregoing makes sense. euro area sovereigns vary vastly in terms of their economic strength and the types of markets they are able to tap. that means that they will likely need to offer different contractual rights to investors in order to borrow effectively; especially in a world where the european authorities have made it clear that they are not guaranteeing the payment of each individual sovereign's claims. to the extent a sovereign wants to prepare for a future crisis by experimenting with using enhanced restructuring tools that build on what the european authorities have already provided, and particularly if this is done in consultation with the european authorities, it is hard to see why those authorities would not wish that. the experimentation by different sovereigns with various enhancements can only serve to provide the authorities with helpful information. last but not least, there is the question of whether, even if there was a hypothetical violation of the article ( ) 'identical legal impact' dictate, there would be any practical impact. with the caveat that the matter of international treaty enforcement is beyond our expertise, our understanding is that violations of treaty obligations generally only give the signatories to the treaty the right to bring legal actions. the presumption is that there are no private rights of action. here, if the european authorities are happy with the actions of the individual state, there is not going to be any complaining; just the clinking of champagne glasses. if faced with a debt crisis situation and the need to protect against holdouts, italy will likely wish to retroactively add the two enhancements mentioned above-the option of using single-shot cacs and the disenfranchisement of the ecb-to all of its already-issued and outstanding local-law-governed debt. the legal barrier to making such a retroactive change, we believe, is non-trivial. the starting point in analysing this question has to be the recognition that most modern legal systems disfavour retroactive changes to contract rights. moreover, scepticism about the government's need to make such changes is likely to be especially heightened in contexts where the government is seeking to reduce its own debt obligations. to illustrate, let us say that the current euro cacs allow the issuer the following options: (a) if holders of per cent or more of an individual series of bonds (in principal amount) approve of a restructuring offer, that offer becomes binding on all holders. (b) in a cross-series modification, if holders of . per cent or more (in principal amount) approve of the restructuring offer, and per cent or more (in principal amount) of all the holders of the various series aggregated also approve of the offer, the offer is binding on all holders. to these existing options, let us now say that italy wishes to add a third and different option, using its local-law power. that option enhances the existing cacs by permitting, in the same fashion that is standard for bonds on the international (foreign-law) market, a single-shot restructuring option. (c) in a cross-series modification, if the restructuring offer satisfies the condition that the terms are uniformly applicable to all holders, and per cent or more of the holders of all the series approve the offer, it will be binding on all holders. let us say that italy wishes also to add in a provision to disenfranchise the ecb or any other supra-national institutions that might be mandatorily required to vote one way or the other on a restructuring plan without consideration of the merits of the situation. for bonds that were already issued, prior to the hypothetical italian debt crisis, with either no cacs or the first-generation euro cacs, these modifications will likely face legal challenge as a violation of property rights. both the italian constitution and european law provide protections for expropriations of property rights. article of the italian constitution protects against interference with property rights unless the action is for reasons of 'general interest' and the government makes 'provisions for compensation'. and article of the european convention on human rights is similarly protective with protections for the peaceful enjoyment of property except under conditions of 'public necessity'. in such situations, the interference with property rights needs to be proportional to the public need. the two questions to ask, therefore, are (i) whether the inclusion by italy of an additional option to the existing model euro cac and a provision disenfranchising the ecb, in order to better tackle a financial crisis, would be ruled as the kind of action that was in the general public interest and proportional to what was needed, and (ii) what compensation might be required, if there was a violation. the question of whether the retrofit inclusion of a single-shot cac would survive the kind of proportionality analysis that was done by courts in the aftermath of the greek restructuring has already been the subject of two excellent papers by manuelides ( ) and weidemaier ( ) . both suggest that a retrofit of a single-shot cac option would likely pass muster in the european courts. and although they do not analyse the this condition that the offer must be 'uniformly applicable' is basically a non-discrimination condition that was put in place in international bonds in when the single-shot cac restructuring option was added to the standard terms. question of adding in a provision that disenfranchises the ecb, we think that that would similarly be ruled to not be an undue interference with property rights. the key case that helps predict how the retrofit would fare is the european court of human rights' decision in mamatas and others v greece. others such as manuelides ( ) have parsed the implications of the various portions of the court's discussion and we will not repeat that analysis here. however, it is helpful to boil down the core of the court's analysis in the mamatas case and indeed a variety of other cases from the corporate context where corporations have put in place contractual modifications to help ameliorate a holdout problem. as we see it, the crucial question for the court will boil down to whether the sovereign has, in its use of the local-law advantage, acted opportunistically to take value away from the creditors and benefit itself. or, alternatively, whether it acted in the public interest in trying to fix flaws in the existing mechanism for restructurings so as to enable an orderly restructuring mechanism that benefits both the general public and the majority of creditors. assuming that the steps that italy takes are in the vein of correcting flaws in the existing restructuring mechanism because of the possibility of an imminent crisis and, moreover, that they are similar to those that the european authorities have indicated are in the works for implementation across europe anyway, it is hard to see how or why a court would rule the changes invalid. this is essentially what greece did with its cac retrofit in march . at the time of the greek restructuring in , european policy makers had already given the green light for a committee to design a cac suitable for inclusion in all euro area bonds. but that committee process took time, given that agreement from all the member states had to be obtained, and was not ready as of march , by which time greece was unable to wait any longer. therefore, greece's restructuring lawyers designed a cac that basically was an advance version of what would subsequently be designed at the european level. indeed, the experience with the use of the greek designed clause has been useful in informing discussions of what kind of clause was appropriate for euro-wide inclusion. more importantly for our purposes, it was important to the european court of human rights-in the context of its approval of greece's retrofit-that greece had retrofit the kind of provision that was standard. finally, there is the matter of damages. if it so happens that courts rule that italy has violated property rights protections, the question to ask is what the monetary damages would be. after all, if it turns out that the impact on the prices of the bonds from adding in features that improve their operation is an increase in the price of the bonds, then damages should be small (zero). and if damages are negligible, it should not matter if the courts find a technical legal violation. different legal systems analyse contract and expropriation damages differently. if viewed as a pure contract violation, the legal remedy in both civil and common law systems is what is called expectation damages; which is to give the aggrieved party the monetary amount that puts her back in the position she would have been had there not been a violation. and one way to measure that would be to look at the prices of italian sovereign bonds before and after the retrofit. based on the existing empirical work on the pricing impact of including cacs in sovereign bonds, it is safe to predict that the impact of the retrofit will either be to increase the market price of the bonds (after all, they are less subject to the cost of holdouts now) or to have no price impact at all. that is, damages would be zero. an alternative possibility would be for the court to ask what the value to the creditor would have been to be a holdout from the restructuring. we think it unlikely a court would go down this path, given how speculative the calculations would have to be. that is, unless the crisis has hit and the market begins pricing the holdout-friendly bonds at a premium to bonds that are easier to restructure. this may happen for two reasons. first, clauses that have to do with restructuring are likely to be more salient at a time of crisis and hence may be taken more seriously and even misinterpreted during these periods. in tranquil times, however, markets may end up barely noting such an innovation. secondly, if the sovereign takes action such as retrofitting cacs in the midst of a crisis, it is likely to be viewed as a signal that the sovereign's situation is worse than the market had anticipated. furthermore, there may be a price differential simply because turbulent times are characterized by high price volatility. assume that the price of a group of bonds happens to drop the day a retrofit is included in these bonds, a court may interpret this drop in price as linked to the inclusion of the retrofit even if this drop was purely driven by the high price volatility that characterizes turbulent periods. put simply, the greater the amount of daylight between when the retrofit is done and when the crisis hits, the smaller the likelihood of significant damages. the choice our article poses is between doing something and doing nothing. this choice can be better understood if we frame the problem in the same way as we would frame the decision of whether we should buy insurance to protect ourselves from a particular event that may or may not occur in the future. in this case, the uncertain event is the need to restructure one's sovereign debt. from the point of view of the borrowing country, the expected cost of a restructuring is p  c; where p is the probability that a debt restructuring will be needed and c is the political and economic cost of the restructuring. p and c, respectively, measure the probability and the cost of restructuring if the country does not do anything to protect itself. if the country buys insurance (ie, if it does something along the lines described above), it will pay an insurance premium s, it will need to restructure with probability ap, and, in case of restructuring, it will pay a cost c (with c <c, for the reasons explained above). hence, the expected costs if the country does something to protect itself will be: s þ apÂc . the insurance premium s captures the possible increase in the sovereign spread associated with doing something. this increase in spread could be due to either signalling or to moral hazard (or both). in the case of signalling, spreads may increase because, by doing something, the country reveals private information that signals that its debt situation is worse than what market participants thought. in the case of moral hazard, spreads may increase because the country now faces lower restructuring costs (remember c <c) and it is thus more likely to restructure its debt. moral hazard would hence be reflected in a higher probability of a debt restructuring: ap> p, where a captures the increase in the probability of restructuring associated with doing something. doing something will be convenient if the expected cost of implementing a reform is lower than the expected cost of doing nothing. that is, if: (s þ apÂc) < (pÂc). this condition can be written as: s < pðc À ac Þ theresa arnold, mitu gulati and ugo panizza • euro area sovereign debt in the era of we already discussed that c is likely to be substantially smaller than c. therefore, if the probability of a future restructuring in non-zero (ie, p > ), and if moral hazard is not very important (ie if a is not much larger than one) p(cÀac ), will be positive. but what do we know about s? the existing evidence suggests that s is likely to be very small, or even negative. for instance, multiple papers find that that the introduction of cac in european bonds was associated with a reduction (and not an increase) in yields: pointing to the idea that s could be negative (or, for sure, not positive and large). note that this is view also seems to be shared by the italian authorities. italy recently issued a dollar bond which included all sorts of creditor protections, but according to the director general for public debt of the italian treasury if the bond had been issued under local terms and priced in euros the yield would have been broadly in line with the current yield on italy's benchmark -year btp bonds. in fact, the spread over us treasuries of the italian dollar bonds ( basis points) was basically identical to the spread of italian btps over german bunds. taken together the evidence on bonds with cacs indicates that the market does not penalize bonds that are easier to restructure and anecdotal evidence on the recent italian bond issuance suggests that the market does not seem to reward bonds that have clauses that make them especially creditor friendly. this all suggests that s is likely to be very close to zero. but if s is close to zero, then the moral hazard problem (which contributes to s) is unlikely to be very important, making a close to one. hence, as long as the probability of restructuring is non-zero the condition that s< p(cÀac ) is likely to hold and the option of doing something now dominates that of doing nothing. in theory, that is. see giuseppe fonte, italy plans to issue more foreign currency bonds next year, reuters, october ( ) <https://www. cnbc.com/ / / /reuters-america-update- -italy-plans-to-issue-more-foreign-currency-bonds-next-year.html> the economics and law of sovereign debt and default we draw from mark weidemaier and yannis manuelides on this point. see mc weidemaier, restructuring italian (or other euro area) debt: do euro cacs constrain or expand the options road to nowhere: the legal and remedial futility of challenges to a restricted single-limb retrofit of local-law italian debt stock on private rights of actions under international treaties more generally, see paul b stephan for discussions, see buchheit and gulati restructuring sovereign debt under local law: are retrofit collective action clauses expropriatory' ( ) harv bus l rev this is one of the lessons of the famous gold clause cases of the s, where the us government used the local law advantage to abrogate the gold clauses in its bonds and in the bonds of private parties. see boudreau, ibid. for additional detail, see sebastian edwards, american default: the untold story of fdr, the supreme court and the battle over gold financial crises and constitutional compromise in the actual euro cacs, there are additional options and different voting thresholds and quorum requirements for when the votes are taken at a physical meeting of the holders versus in writing mitu gulati and ugo panizza • euro area sovereign debt in the era of covid- manuelides also analyses the important precedent of germany retrofitting cacs into its local corporate instruments some years prior. see manuelides (n ). numerous others have also analysed the implications of the litigation that followed the restructuring government debt under local law: the greek experience and implications for investor protection under european law the future of involuntary sovereign debt restructurings, mamatas and others v. greece and the protection of holdings of sovereign debt under the echr' ( ) cap mkts international arbitration and greek sovereign debt' ( ) oregon rev int'l l the greek bond haircut: public and private international law and european law limits to unilateral sovereign debt restructuring exit consents in sovereign bond exchanges for reflections on the design of the greek restructuring from its primary architect, see lee c buchheit, the greek debt restructuring of ', global restructuring rev mitu gulati and ugo panizza • euro area sovereign debt in the era of covid- the fact that the voting thresholds that greece saw in its retrofit cacs in were lower than those used in international bonds at the time did not trouble the courts (it had a simple % quorum and . % aggregate vote requirement). nor, for that matter, did the fact that greece used the single-shot cac that aggregated votes across all the series-a type of provision that is now standard we are assuming a scenario where the litigation gets resolved some years after the restructuring has been conducted. that puts the remedy of specific performance out of the realm of consideration since a court is unlikely to be willing this was the outcome in the gold clause case of the s (perry v united states) that involved a constitutional challenge to the us government changing the terms of its own bonds retroactively. see vanberg and gulati for a discussion of comparative contract law remedies, see, eg, luca ficetola, comparing remedies for contract violations in italian and english law but it is a possibility. see, eg, paolo colla et al pricing terms in sovereign debt contracts: a greek case study' ( ) cap mkts l hidden holdouts: contract arbitrageurs and the pricing of collective rights for the argument that whatever may make a debt restructuring process smoother is optimal ex post but not ex ante, see key: cord- - axsebya authors: lelli, davide; lavazza, antonio; prosperi, alice; sozzi, enrica; faccin, francesca; baioni, laura; trogu, tiziana; cavallari, gian luca; mauri, matteo; gibellini, anna maria; chiapponi, chiara; moreno, ana title: hypsugopoxvirus: a novel poxvirus isolated from hypsugo savii in italy date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: axsebya interest in bat-related viruses has increased considerably during the last decade, leading to the discovery of a rising number of new viruses in several bat species. poxviridae are a large, diverse family of dna viruses that can infect a wide range of vertebrates and invertebrates. to date, only a few documented detections of poxviruses have been described in bat populations on three different continents (america, africa, and australia). these viruses are phylogenetically dissimilar and have diverse clinical impacts on their hosts. herein, we report the isolation, nearly complete genome sequencing, and annotation of a novel poxvirus detected from an insectivorous bat (hypsugo savii) in northern italy. the virus is tentatively named hypsugopoxvirus (hypv) after the bat species from which it was isolated. the nearly complete genome size is , nt and it encodes genes. genome analyses suggest that hypv belongs to the chordopoxvirinae subfamily, with the highest nucleotide identity ( %) to eptesipoxvirus (eptv) detected from a microbat eptesicus fuscus in wa, usa, in . to date, hypv represents the first poxvirus detected in bats in europe; thus, its viral ecology and disease associations should be investigated further. poxviruses are dsdna viruses with large genomes ( to kb) that belong to the family poxviridae. the family is divided into the entomopoxvirinae and the chordopoxvirinae subfamilies of viruses, which infect insects and vertebrates, respectively. according to the international committee on taxonomy of viruses (ictv) release [ ] , genera have been created to classify chordopoxviruses (avipoxvirus, capripoxvirus, centapoxvirus, cervidpoxvirus, crocodylidpoxvirus, leporipoxvirus, molluscipoxvirus, orthopoxvirus, parapoxvirus, suipoxvirus, and yatapoxvirus), but other viruses remain unclassified and new genera are likely to be recognized in the future. poxviruses show a diverse host range, with some viruses having wide host tropism (e.g., orthopoxviruses) and thus being consequently associated with greater zoonotic risks [ ] , and others having strict host specificity. in recent decades, bats have been increasingly recognized as reservoirs of emerging viral infections, which has important ramifications for animal and public health [ ] . however, the majority of bat-borne viruses that can cause severe diseases in humans and other mammals, do not cause apparent clinical signs in bats. consequently, it has been assumed that bats may have a "special" relationship with viruses based on physiological, ecological, evolutionary, and/or immunological aspects, which allow them to act as special viral reservoirs with exaggerated viral richness [ ] [ ] [ ] [ ] . currently, four poxviruses from the microchiroptera and macrochiroptera suborders have been detected in bat populations on three continents (america, africa, and australia) [ ] . specifically, eptesipoxvirus (eptv) was isolated in north america in from eptesicus fuscus [ , ] ; eidolon helvum poxvirus (ehpv ) was detected in west africa in from eidolon helvum [ ] ; the pteropox virus (ptpv) was identified in northwestern australia in from pteropus scapulatus [ ] ; and a fourth poxvirus was also identified in south australia from miniopterus schreibersii bassanii in [ ] . it is remarkable that these viruses are phylogenetically divergent and are associated with variable clinical manifestations. virological investigations focused on poxviruses in bat populations may have a positive impact for future ecological studies of bat-pathogen interactions. moreover, from the perspective of the one health approach, bats could benefit from these studies, since european bat populations are currently undergoing a global decline that could be linked with so far overlooked viral infections. in this study, we report the isolation, nearly complete genomic sequencing, and annotation of a novel poxvirus detected from an insectivorous bat (hypsugo savii) in northern italy. the virus was tentatively named hypsugopoxvirus (hypv), according to the bat species from which it was isolated. phylogenetic analyses suggest that hypv belongs to the chordopoxvirinae subfamily, revealing the highest similarity ( %) with eptesipoxvirus (eptv) detected from the microbat eptesicus fuscus in wa, usa in , which is associated with bat necrosuppurative osteomyelitis in multiple joints. hypv is the first poxvirus detected in bats in europe and its viral ecology and disease associations should be investigated further. dead bats from different species were collected for virological investigations from wild animal rescue/rehabilitation centers in the context of a general surveillance project that has been implemented in northern italy since - , which focuses on the detection of emerging bat viruses [ ] [ ] [ ] . the bats were taxonomically identified based on their morphologic characteristics, according to the european bat identification keys [ ] . the carcasses were necropsied, and tissue samples were collected for further laboratory exams, particularly for viral detection and isolation. after necropsy, organ samples (lungs, heart, kidney, brain, and intestines) were mechanically homogenized in minimal essential medium ( g/ ml), which contained antibiotics. they were then centrifuged at g for min. samples were inoculated in confluent monolayers of vero and marc cells (african green monkey), incubated at • c with % co and observed daily for seven days to assess their cytopathic effects (cpes). in the absence of cpes, the cryolysates were sub-cultured twice onto fresh monolayers. cell culture supernatants showing cpe were partially purified by ultracentrifugation at , rpm for h (rotor tst kontron) through a % (w/w) sucrose cushion, and the pellet was re-suspended in pbs. this antigen was kept at − • c and then submitted for viral identification with the ngs approach and negative-staining electron microscopy (nsem) by using the airfuge (beckman instruments, palo alto, ca, usa) method [ ] . viral dna was extracted from µl of positive cell culture supernatants using a biosprint one-for-all vet kit (qiagen s.p.a., milan, italy). sequencing libraries were made with a nextera flex kit (illumina inc. san diego, ca, usa) in accordance with the manufacturer's instructions. libraries were sequenced on a miseq instrument (illumina inc. san diego, ca, usa) by using a miseq reagent kit v in a cycle paired-end run. data were assembled de novo by the clc genomic workbench v. (qiagen s.p.a., milan, italy). genome annotation and analysis was performed with tools from the bioinformatics suite developed at the viral bioinformatics resource centre [ ] . the genome annotation transfer utility (gatu) [ ] uses a reference genome to automatically annotate poxvirus genes with clear orthologs in the reference. other possible genes were presented to the annotator for further characterization and to make final annotation decisions. the case specifically concerned a juvenile hypsugo savii male that spontaneously died in a wildlife recovery center in valpredina, cenate sopra (bg), northern italy after several weeks of hospitalization. the sick bat was originally found alive on july , in telgate (bergamo province, northern italy) by a private citizen who brought it to the center. clinically, the bat had a humerus fracture, sensory depression and a lack of appetite but normal body mass. the death occurred days after admission to the center on september , ; then, the carcass was sent to the lab for necroscopy and further analyses. pathological lesions in the internal organs indicative of infectious diseases were not observed, but a soft bone callus due to pathological healing of the humerus fracture associated with osteomalacia and calcium deficiency was detected. a virus was isolated on marc cells inoculated with the organ pool composed of the bat's heart and lungs. the cpe occurred on the third day post-inoculation during the second passage and was characterized by a diffused degeneration of a monolayer with rounded cells floating in the culture medium ( figure a ,b). the cell culture supernatant showing cpe was submitted to the ngs in order to identify and characterize the unknown isolate. furthermore, nsem performed on the purified and concentrated antigen revealed the presence of viral particles that unequivocally morphologically resembled those belonging to the genus orthopoxvirus ( figure c ). the virus was tentatively named hypsugopoxvirus (hypv), according to the bat species from which it was isolated. table summarizes the basic information on the hypv identified in this study in comparison with all known poxviruses detected to date in bats worldwide. after ngs sequencing, the nearly complete viral genome of a poxvirus was obtained from one contig of , nucleotides originating from , reads with an average coverage of . . the nearly full genome sequence of the viral strain was determined and compared with those of other members of the poxviridae family available on genbank. for the nearly complete viral genome sequencing, blast analysis revealed the highest nucleotide identity ( %) to the eptesipoxvirus (eptv) strain "washington", a member of the chordopoxvirinae subfamily identified in microbats in the usa ( table ). the nearly complete genome sequence for hypv was submitted to genbank under accession number mk . [ , ] a conservative approach was taken for genome annotation to avoid over-annotating open reading frames (orfs) that were unlikely to represent functional genes. orfs less than codons or overlapping by more than % with well-characterized genes were not considered for annotation unless supported by other evidence. a total of genes were annotated for hypv, showing a percentage value of nt identity with its closest related virus eptv ranging from . % for the hypv- gene (serpin ) to % for the hypv- gene (vltf- ) ( table ) . when the seven conserved genes-rpo , rap , mrna capping enzyme large subunit, p a precursor, rpo , vetf-l, and dna primase-were considered individually, the value of nt similarity with eptv ranged from . % to . %. the above conserved genes that have been used for phylogenetic analysis in previous studies [ , ] are presented in bold in table . hypv showed nucleotide divergence from its closest relative, eptv. the smaller genome size with , nt encoding genes for hypv in comparison to , nt and genes for eptv, is likely due to the omission of the itrs from the analysis and therefore, is not possible to establish the exact length of its the viral genome. two orfs (hypv- and hypv- , table ), whose function is still unknown, appear to be unique to hypv. table . hypv genome annotation and nucleotide identities for each gene to the most similar strain eptesipoxvirus (eptv). the seven conserved genes used for phylogenetic analysis in previous studies [ , ] are presented in bold. ( - ) or "−" ( - ) ). the potential zoonotic risks associated with bats and their fascinating and special relationship with viruses have attracted the attention of many researchers worldwide. consequently, general and target surveillance on bat populations has increased in the last decade with the purpose of clarifying the genetic diversity of bat-associated viruses as well as acquiring comprehensive information on bat-pathogen interactions. in fact, viral disease prevention and biological conservation issues could both benefit from such research. virological surveillance of bat populations in italy is a relative novelty and has only recently been extensively applied, but almost immediately, a great heterogeneity of virus identifications has been observed. viruses belonging to several viral families, such as reoviridae [ ] , coronaviridae [ , [ ] [ ] [ ] [ ] [ ] , paramyxoviridae [ ] , rhabdoviridae [ , ] , and astroviridae [ ] , have been detected, allowing the identification of some novel/previously unknown viral agents. the results of the general surveillance of bats, which have been randomly applied so far as pilot virus discovery studies, may drive future activity to more specific longitudinal and target studies aimed at understanding the epidemiology of potential new pathogens. in this study, a novel poxvirus, hypv, was detected from the microbat hypsugo savii in italy. this likely represents the first poxvirus detection in bats in europe. in fact, only four poxviruses have been documented to date in bat populations worldwide, and these and these have diverse and somehow incomplete descriptions, with just some common aspects. firstly, ehpv was detected in with a high-prevalence in throat swabs from apparently healthy african megabats (eidolon helvum), and metagenomic analysis identified poxvirus sequences that were most closely related with molluscum contagiosum (mocv), a human-only pathogen [ ] . in the same year of , another bat poxvirus was incidentally detected in south australia during the investigation of an outbreak of parasitic skin disease in a population of the microbat species, miniopterus schreibersii bassanii. in one of the twenty-one bats examined, an independent (non-nematode-associated) lesion containing intracytoplasmic inclusion bodies indicative of poxvirus infection was observed, and this was confirmed with electron microscopy [ ] . between and , eptv was detected in adult big brown bats (eptesicus fuscus) with severe joint disease (tenosynovitis and osteoarthritis) at a wildlife center in northwestern united states. phylogenetic analysis revealed that eptesipoxvirus is most closely related to the cotia virus, a virus detected in sentinel suckling mice in sao paulo, brazil in [ , ] . ptpv was detected from an australian little red flying fox (pteropus scapulatus) that died following entrapment on a fence. post-mortem examination revealed multiple nodules on the wing membranes. phylogenetic analysis indicated that ptpv is not closely related to any other poxvirus isolated from bats or other species, and that it likely should be placed in a new genus [ ] . it is noteworthy that ptpv and ehpv were isolated from megabat hosts (pteropus scapulatus and eidolon helvum, respectively), whereas eptv and hypv were isolated from microbats (eptesicus fuscus and hypsugo savii, respectively). while ehpv was detected in apparently healthy bats, the other viruses were identified in sick bats and their association with the pathological condition was assumed. specifically, clinical symptoms of eptv in eptesicus fuscus manifested in the form of joint swelling and increased lethargy [ ] . ptpv-infected pteropus scapulatus presented vesicular to nodular skin lesions on the wing membranes that are typical of poxvirus infections [ ] . hypv was detected in a bat showing pathological healing of the humerus fracture associated with osteomalacia and calcium deficiency. neither symptom was directly linked to fatality and thus the capability of these viruses still needs to be ascertained, including the role of hypv in causing deadly disease in bats. the results of our study indicate that hypv presents the typical morphology of the orthopoxvirus genus and that it could be isolated in cell culture. indeed, its final identification was obtained by genomic characterization. the nearly complete genomic sequencing clearly demonstrated that hypv is a new virus that is distantly related to its closest known relative eptv (wa, usa, ) with a nucleotide identity of % (almost whole genome). indeed, the percentage value of the nt identity of hypv with eptv ranged from . % for the hypv- gene (serpin ) to % for the hypv- gene (vltf- ). regarding orfs annotation the hypv was shown to be defective in particular in the itr genes i.e., out of described in eptv, but this should be not a real structural defect but more likely due to the omission of the itrs from the analysis. on the contrary, two orfs, whose function is still unknown, appear to be unique to hypv. to conclude, a new poxvirus, hypv, was detected in bats in europe and its viral ecology and disease associations should be investigated further. author contributions: d.l. designed the study and wrote the manuscript; a.l. performed electron microscopy, participated in study coordination, and helped to draft the manuscript; c.c. and l.b. performed the next-generation sequencing and data analysis; a.m.g. and m.m. performed the sampling and data collection; g.l.c. performed the clinical investigations; a.p. and f.f. performed the necropsies and molecular tests; e.s. and t.t. were involved in the virological analysis and interpretation of the results; a.m. performed the molecular genetic studies and helped to draft the manuscript. all of the authors have read and approved the final manuscript. an increasing danger of zoonotic orthopoxvirus infections bats: important reservoir hosts of emerging virus mass extinctions, biodiversity and mitochondrial 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from sentinel mice in sao paulo biological characterization and next-generation genome sequencing of the unclassified cotia virus span (poxviridae) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: special thanks to anna tirelli, loredana zingarello, giovanni bozzoni and all technicians in the izsler virology section for their valuable technical work and support in virological analysis. the authors declare no conflict of interest. key: cord- - m voc authors: rovetta, alessandro; bhagavathula, akshaya srikanth title: modelling the epidemiological trend and behavior of covid- in italy date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: m voc as of march , , over , across the world, italy became the red hotspot for the covid- pandemic after china. with over , cases and deaths reported in the month of march in italy, it is necessary to stimulate epidemic trend to understand the behavior of covid- in italy. by s.e.i.r. simulation, we estimated the most representative epidemic parameters occurred from march to , , thus being able to evaluate the consistency of the containment rules and identify possible sars-cov- local mutations. our estimations are based on some assumptions and limitations exited. the current surge of covid- pandemic is devastating globally, with over , cases and more than deaths reported [ ] . in europe, covid- cases are most dramatically started to increase from the first week of march . of these, italy is grappling with the worst outbreak, with over , confirmed cases and around deaths by march , [ ] [ ] [ ] [ ] [ ] . this exponential increase in covid- positive cases in italy raised turmoil, and the government decree to a lockdown of the entire country [ ] . given the seriousness of the situation, it is absolutely necessary not only an immediate intervention but also a criterion for assessing its effectiveness. article on modeling epidemic transition of covid- have been published [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and based on the previous publication from china, south korea, iran, and japan presented the estimation of epidemic trends and transmission rates [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . through this research, we evaluated the consistency of the containment rules and identified possible sars-cov- local mutation using the s.e.i.r mathematical model. we used the most representative epidemic parameters that occurred during the first half of march to predict the trend of infections. therefore, to assess the effectiveness of the containment measures, it will signal the presence of a plausible evolutionary mutation. to do this, it will be sufficient to compare the general trend foreseen by the s.e.i.r. with the real italian population. to carry out this study, we used the most recent data, found in the scientific literature relating to covid- total and active cases, deaths, recoveries, and all epidemic parameters, have been . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint used. since the chinese situation has provided the most information, great statistical importance was given to the sars-cov- evolution in the hubei region, with the implicit assumption of "very significant local mutations" absence necessary for generalizing the reports through classical inference. the statistics collected on virus mortality were first divided by age group and then recalibrated on the italian demography, in order to be as representative as possible. to do this, we considered the most representative population of covid- cases reported between march, - , , the number of infected persons necessary to approach the required mortality rate was added to the correct values. to obtain a more realistic trend and assuming true the probable "non-relapse patients", and we applied s.e.i.r. model to predict the virus progress in italy. as a note, it is not possible to estimate the containment measures taken by the government. however, at the same time, assuming that only half of the population was susceptible to the virus precisely due to the above containment measures. thanks to the comparison between real and theoretical evolution, it is likely to estimate the presence of essential mutations and/or the limitation strategy effectiveness through similarities, anomalies, or substantial deviations from each other. we used s.e.i.r. differential equations and non-linear methods to resolve the gaps analytically. an interactive algorithm was developed using c++ software (version) to find a solution through a finite discretization method. the total population number has been considered constant because of the very low deaths/population ratio. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint by entering the initial values for the incubation time /σ, recovery time /γ, basic reproduction number r , and number of infected i on march , , the software prints the s.e.i.r. values day by day. the best epidemic parameters were estimated through continuous iteration until the "closest values to the real ones" were reached until march , . the number of initial incubates was calculated with the formula e =r ·i . after the reconstruction of the real data on covid- in italy in the period march , - , through the above methods, the best estimates obtained for the italian epidemic parameters are /σ = ( ± ) days, /γ = ( ± ) days, r = . ± %, i = ± %. the model predicts the following values for the next days (march , - ) with "d " . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . these values obviously refer to the population with the constant mortality rate of . %; however, the real number of deaths must be strictly less than the theoretical one expected or we will talk about mutation. thus, thanks to the results obtained from the application of the s.e.i.r. model we can foresee three possible scenarios: if, in italy, the real next-days trend of the total infected number will be lower than that shown in figure , we can assume the following events set out in order of probability: the containment measures adopted days ago are taking effect; sars-cov- has undergone a significant anti-evolutionary mutation. if, in italy, the real next-days trend of the total infected number will be equal than that shown in figure . we assume the following events set out in order of probability: the containment measures adopted days ago are not taking effect; the containment measures adopted days ago are taking effect and sars-cov- has undergone a significant evolutionary mutation. if, in italy, the real next-days trend of the total infected number will be higher than that shown in figure . we assume the following events set out in order of probability: the containment measures adopted days ago are not taking effect and sars-cov- has undergone a significant evolutionary mutation; the s.e.i.r model is no-more representative of the covid- italian case and we should utilize the s.e.i.r.s. model. even considering an admissible "theoretical-estimates statistical error" of ± % these values could not be accepted since the trend indicates a faster growth than that predicted by the model with a breakeven point between and march and a percentage difference in net growth. deepening the discussion and going to analyze the cases region by region, a very significant result turns out: with the exception of lombardy, the trend in the number of infected is very good given that · it seems unlikely that the virus, without significantly changing, could infect a patient again [ , ] [ ], . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint · the mortality rate in italy is too low to be significant in the short term [ ] , · young children appear to have a secondary role in the spread of the infection [ ] [ ], we will investigate the possible scenarios through s.e.i.r. model. using the discretization carried out in the previous study "mathematical-statistical modeling of covid- on the restricted population" [ ] , a new c++ software was created to print the values of the s.e.i.r. day by day until a specific date and/or situation ( ) . however, unlike the aforementioned study, the prediction period will be extended to a certain number of days in order to verify the effectiveness of the containment measures in italy. since no european or international standard for data collection has been established, one of the crucial points of this analysis is the interpretation of the information available. on february , in italy, a real "communicative turnaround" was announced so that only the most important symptomatic cases would be publicly counted from then on [ ] . since there do not appear to have been significant large-scale mutations and that a good amount of data is now available, we expect the who mortality data to be valid in italy as well [ ] [ table ]. the more recent study about sars-cov- mortality on symptomatic and asymptomatic covid- patients in the hubei region shows the following sars-cov- mortality per age-groups [ ] [ table ]. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org / . / . / since the italian population is distributed according to the following age-percentages [ ] [ ] [ table ] Σ i p i · dr i / = . % for this reason, it makes sense to conclude that the italian covid- death rate is exaggeratedly overestimated if we rely on ministry of health data. on the other side, the true-infect number (as the aforementioned "communicative turn" announced) must necessarily be higher. trying to respect the percentage just calculated we obtain the following table . citing the adapted system of non-linear differential equations and their discretization [ ] legend: . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint · /γ is the asymptomatic incubation time; · /σ is the recovery time; · r is the basic reproduction number; · s is the number of susceptible people; · e is the number of active exposed people (people in incubation); · i is the number of active infected people; · r is the number of recovered people (no longer infectable). · x n is the number of day-n "x-category" people; · x t is the number of table "x-category" people; significant discussion: in order for the supposed data to be representative of reality, we should admit the existence of many more cases than expected. all this is consistent both with the idea the infection had been present on the territory for some time, in the form of a less aggressive strain, than with the time course of the disease [ ] . the number of people healed is also in line with international percentages [ ]. validity grade: plausible and supported by empirical evidences. warning level: high. assumptions: /σ = days, /γ = days, r = . . . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint warning level: high. since test turned out to be the most likely, we focus on it. peak-day: . peak value: imax = . · people (almost million people); i < -day: (almost one year); . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . i = -day: . we can also fit the curve through a convenient gaussian f(x) = y + gauss(a,x ,σ) (x) function, useful for modeling the initial trend of the virus. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint the model is approximate as a direct consequence of the uncertainty of the data it was supposed to fit but it can act as a valid comparison for the identification of any sars-cov- genetic mutations as well as for the evaluation of the containment measures effectiveness. observed the comparison between the predictions of the s.e.i.r. and the actual trends highlighted above, as well as between the trends of lombardy and the other italian regions, there are valid reasons to assert that in lombardy, in particular in the cities of brescia, bergamo and milan, a very significant evolutionary mutation of the virus may be underway, which would require immediate containment. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint early phylogenetic estimate of the effective reproduction number of -ncov adjusted age-specific casefatality ratio during the covid- epidemic in hubei, china transmission potential of covid- in iran positive rt-pcr test results in patients recovered from covid- la giravolta comunicativa sul coronavirus, menotamponi e contare solo i casi gravi f. . . . mensana srls research and disclosure division, via moro aldo - who database hongbing song, daniel dajun zeng, estimating the effective reproduction number of the -ncov in china key: cord- - ebcuzd authors: liotta, giuseppe; marazzi, maria cristina; orlando, stefano; palombi, leonardo title: is social connectedness a risk factor for the spreading of covid- among older adults? the italian paradox date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ebcuzd italy was one of the first european countries affected by the new coronavirus (covid- ) pandemic, with over , infected people and close to , deaths, until march (st). the pandemic has hit especially hard because of the country's demographic structure, with a high percentage of older adults. the authors explore the possibility, recently aired in some studies, of extensive intergenerational contact as a possible determinant of the severity of the pandemic among the older italian adults. we analyzed several variables to test this hypothesis, such as the percentage of infected patients aged > years, available nursing home beds, covid- incidence rate, and the number of days from when the number of positive tests exceeded (epidemic maturity). we also included in the analysis mean household size and percentage of households comprising one person, in the region. paradoxically, the results are opposite of what was previously reported. the pandemic was more severe in regions with higher family fragmentation and increased availability of residential health facilities. between december , and march , , , cases and , deaths due to new coronavirus (covid- ) have been reported [ ] . as is well known, italy is one of the countries most affected by the pandemic, with , deaths and , cases recorded in the months of february and march [ ] . many have wondered why covid- has hit so hard in italy, but unfortunately, over the weeks it has become increasingly clear that other eu countries as well as the us are developing similar growth trends. recently some studies [ ] stated that age, in both local and national context, as well as the social connectedness of older and younger generations act as powerful determinants in the spread of pandemics. while there is a very clear association between the case fatality rate and age demographics (italy has the second oldest population worldwide and has the highest ageing index in europe [ ] with a value of . ), we wanted to test the hypothesis that the supposed closeness between younger and older generations in italian families may have played a major role in the pandemic spread. in a a a a a fact, dowd, et al. stated that "italy is also a country characterized by extensive intergenerational contacts which are supported by a high degree of residential proximity between adult children and their parents." [ ] . according to this observation, we expected to find a strong direct association between family size and the spread of covid- . conversely, social distance should be favored in cities and regions with a high number of residential structures for long term care, usually devoted to older adults. the study is based on the available population data from each italian administrative region. the sources of data used in this paper are the daily situation reports on covid- published by the italian ministry of health ( february- march), the national institute of statistics (istat) data set on households and population, and the th report generated by the non-self sufficiency network ( - ). in italy, people aged > are . millions, of which . % are men (mean age . ) and . % are women (mean age . ). several variables with plausible association with the spread of covid- among citizens aged > years have been taken into consideration. these include the percentage the italian population aged > years [ ] , covid- incidence rate, and number of days from when the number of confirmed cases exceeded (epidemic maturity) [ ] . additionally, we explored the relationship between the proportion of infected patients aged > years and social connectedness indicators, such as the percentage of family comprising one members and household size [ ] . finally, we considered the availability of beds in nursing homes [ ] since the pandemic severely impacted nursing home residents throughout the country caused by difficulties in social distancing in these settings as well as the well documented lack of personal protective equipment (ppe) during the first phase of the pandemic in italy. the population aged > years was chosen because they account for about % of the deaths due to covid- [ ] , while representing . % [ ] ) of the total population and about % [ ] of the nursing home residents. univariate (pearson correlation) and multivariate (linear regression) analyses were conducted to test the hypotheses. table reports the proportion of residents aged > years among the total number of covid- cases, according to the italian administrative regions, that ranges from . % for basilicata to . % for marche region [ ] . the deviation from the italian average ( . %) is + % for the highest value and - . % for the lowest. this cannot be explained by the different percentages of residents aged > years among the population that ranges from . to . %. the maturity of the epidemic (the number of days since the infection exceeded confirmed cases) ranges from to days according to region. this could partially explain the differences, even if other factors seem to be involved. the incidence rate among the general population ranged from . / , residents (valle d'aosta) to . / , residents (sicily and campania) and could explain the spread of the infection among the residents aged > years. the mean household size showed a negative correlation with the proportion of infected residents aged > years, whereas the percentage of households with one member and availability of nursing homes beds showed a positive correlation with the proportion of infected residents aged > years (table ) . multivariate analyses allowed the comparison of different models of the diffusion of the epidemic to the target population ( table ). the first model included the percentage of the general population aged > years and the measurements of the impact of the pandemic at population level. the incidence rate dropped from the stepwise model, showing that the duration of the circulation of the virus among the population, rather than the incidence rate, determined the increased proportion of infections among the population aged > years. the second step was to add variables related to social connectedness into the model. at this stage, the percentage of households with only one member was excluded, whereas the mean number of households was included in the model. finally, the percentage of nursing home beds in the total population was included in the model and the mean household size dropped out with a reduction of the adjusted r . this result prompted us to find a combination of the two variables that captured different aspects of a process that linked household size to the percentage of available nursing home beds. a combined index was set up (percentage of nursing home beds/mean household size) that was included in the final model with a slight but significant increase of the adjusted r . fig shows the linear relation between the model and outcome variable. the hypothesis of a relationship between social connectedness and spread of covid- was not confirmed by this study. the above mentioned analyses show the spread of the infection among the population aged > years was associated with the percentage of households comprising one member (even if it was statistically insignificant in the multivariate analyses), and inversely associated with the mean household size, the latter independently from the population age-structure and spread of covid- in the general population. the spread of covid- among the older adults was also independently associated with the available nursing homes beds. the model explains more than % of the variation of the proportion of infected patients aged > years. social relationships in italy have changed dramatically in the last years. the istat reported that, in , one third of italian families ( %) were composed of only one person and only . % had � members [ ] . if we include couples without children ( . %), more than half ( . %) of the million italian families comprise � people [ ] . the istat report stated that more than % of the population aged > years has no one to count on in case of need, and living alone has become the most common living arrangement, as it is all over in europe (about one third of households comprise only one member) [ ] . more than % of the population aged > years in italy is living alone [ ] , and in some regions, as in veneto-northern italy, this percentage increases to %, with about % of older adults that are not self-sufficient [ ] . more than % of the residents aged > years in lombardy, the most impacted region in italy, live alone; here, in the last years the household size decreased by about % [ ] , whereas in campania (southern italy with one of the lowest proportions of infected patients aged > years) it decreased by < % [ , ] . it is likely that in a situation of forced isolation, social relationships represent a powerful tool to reduce the risk of contagion, allowing community-dwelling older adults to receive some assistance (bringing food or drugs home, thus facilitating the older adults to accomplish with social distancing). this kind of support is increasing in many italian cities. the protective power of social connectedness emerged in many crises, mainly because of the increasing prevalence of bio-psycho-social frailty among older adults who have to face repeated "environmental" stresses, such as a pandemic or a heat wave. this was not the case in nursing homes that banned visitors since the early phase of the epidemic, even though this approach did not prevent the spread of the infection. nursing homes are dealing with the same problems as all closed communities, the struggle to ensure social distancing between people who need care, and the lack of ppe, as was the case at national level. in short, social distancing does not necessarily imply social isolation. similarly, social connectedness does not imply physical closeness (which is dangerous in an epidemic) with social contacts. italian nursing homes (called rsas-translated in english as social and health care facilities) house > , older adults in italy. a very recent cross-sectional survey conducted by iss [ ] is going to explore the spread of covid- in a sample of , rsas because of several micro epidemics took place in numerous facilities. the role played by residential care facilities in italy as well as in many other countries, due to their lack of preparedness to this kind of events, is progressively emerging [ , ] . in some cases public policies aimed at discharging covid- cases from hospitals to nursing homes in order to improve the capacity of the health systems to face the lack of hospital beds are suspected to have increased also the risk of infection among the nursing homes' hosts [ ] . there are some limitations in the present study. first, the study analyzed the proportion of cases in patients aged > years instead of analyzing age-specific infection or mortality rates according to regions, as these data are not retrievable from public sources at this stage of the infection. second, the quality of social connectedness parameters is limited, even though this is a limit of each study exploring social relationships based on routine data instead of gathering information ad hoc. finally, the number of performed tests, which varies enormously according to the region (ranging from . to . per , inhabitants) [ ] , could affect the proportion of confirmed cases. the policy at the regional level regarding testing varies from an approach aimed at identifying asymptomatic cases as much as possible (veneto) to one aimed at testing only the symptomatic in order to give priority to most severe cases. of course, the most severe cases are more prevalent among the very old, so the differences in the approach could lead to an overestimation of cases in patients aged > years in regions where the total number of tests is lower and the number of confirmed cases/number of performed tests ratio (nct/npt � ) is higher. indeed, this could be the case since the correlation between the nct/npt ratio and the proportion of cases in patients aged > years was robust ( . ; p = . ), but the multivariate model remained unchanged and the nct/npt ratio was not shown as a statistically significant variable. the association of social connectedness with the spread of covid- among older italian adults, hence older adult mortality rate, is not confirmed. paradoxically, it seems that the variables associated with social isolation are risk factors for increase in the proportion of cases in italian patients aged > years among the total number of cases. this is consistent with the observation that social relationships are a protective factor against increased mortality rates during a crisis impacting the frailest populations. nursing homes bed rate is one of the determinants of sars-cov- infection rate among the individuals aged> in italy. conceptualization: giuseppe liotta, maria cristina marazzi, stefano orlando, leonardo palombi. who. coronavirus disease (covid- ) situation report- demographic science aids in understanding the spread and fatality rates of covid- popolazione per età, sesso e stato civile aspects of daily life-households size l'assistenza agli anziani non autosufficienti in italia. il tempo delle risposte-sesto rapporto covid- , aggiornamento nazionale del / / guests of social and health care residential facilities: elderly guests by age class and type of need-reg covid- , aggiornamento nazionale del / / -appendice annuario statistico italiano annual report -the state of the nation annuario statistico italiano veneto . ultraottantenni vivono da soli la condizione degli anziani in lombardia-sintesi active ageing policy in campania (italy) mpra paper mortality associated with covid- outbreaks in care homes: early international evidence report on covid- and long-term care in italy: lessons learned from an absent crisis management. article in ltccovid.org, international long-term care policy network, cpec-lse competing interests: the authors have declared taht no competing interest exists. key: cord- - hmrt h authors: di martino, barbara; di profio, federica; melegari, irene; robetto, serena; di felice, elisabetta; orusa, riccardo; marsilio, fulvio title: molecular evidence of kobuviruses in free-ranging red foxes (vulpes vulpes) date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: hmrt h red foxes (vulpes vulpes) are susceptible to viral diseases of domestic carnivores. in this study, by screening rectal swabs collected from red foxes in italy, we identified kobuvirus rna in five samples. based on analysis of partial rdrp and full-length vp genes, all of the strains shared the highest identity with canine kobuviruses (cakvs) recently detected in the us, the uk and italy. these findings provide the first evidence of the circulation of these novel viruses in foxes. demonstrating that non-domestic carnivores are susceptible to kobuvirus infections. here we report the detection of kobu-like viruses in faecal samples obtained from italian red foxes (vulpes vulpes) that are genetically very similar to cakvs. between september and may , individual rectal swabs were collected from red foxes in northern italy (valle d'aosta and piemonte regions) submitted to the national reference center for wild animal diseases (cermas). the samples were obtained from animals of both genders ( females and males) found dead or shot during the regular hunting season. twelve foxes were under year of age, and were adults. fresh faecal samples were placed into sterile containers and stored at - °c until tested. rna was extracted from ll of % (wt/vol) faecal suspension using trizol ls (invitrogen, ltd, paisley, uk) procedure. the final rna pellet was resuspended in ll of rnase-free water and used directly in rt-pcr assays or stored at - °c. viral dna was extracted from the supernatants of faecal homogenates by boiling for min and chilling on ice as described previously [ ] . kobuvirus rna was detected by rt-pcr using a broadly reactive primer pair, univ-kobu-f/univ-kobu-r, which amplifies a -bp fragment of the d gene of all kobuviruses, following reaction conditions described previously [ ] . the samples were also tested by rt-pcr for canine coronaviruses (ccovs) [ ] and canine noroviruses (novs) [ , ] , and by pcr for canine parvovirus (cpv- ) [ ] . novel primers (vp rf-f, -gcgggcgaatcctt caac- , and vp rf-r, gcgacctttcggagcgcc- ) to amplify the complete vp gene were designed by visual inspection of an alignment containing the kobu-like sequences obtained in this study and the corresponding conserved regions of the cakvs and aivs available on the ncbi website. all of the amplicons were purified using a qiaquick gel extraction kit (qiagen gmbh, hilden, germany) and sequenced directly using bigdye terminator cycle chemistry and a dna analyzer (applied biosystems, foster city, ca). rt-pcr products for which no sequences were obtained by direct sequencing were cloned into the pcr . vector (invitrogen, ltd, paisley, uk). plasmid dna was purified using a miniprep kit (qiagen gmbh, hilden, germany); three clones per product were sequenced. basic local alignment search tool (blast; http://www.ncbi.nlm.nih.gov) and fasta (http://www. ebi.ac.uk/tools/sss/fasta/) with default values were used to find homologous hits. sequence editing and multiple alignments were performed with the bioedit software package, version . [ ] . a phylogenetic tree (neighbor joining and p-distance model) with bootstrap analysis ( , replicates) was constructed using the mega software package, version . [ ] . out of faecal specimens, five ( . %) were found to be positive for kobuvirus rna, and two ( . %) for ccov rna. canine novs and cpv- were not detected. kobuvirus was the single identified enteric virus in four specimens, while a mixed infection with both pathogens (ccov ? kobuvirus, n = ) was found in one sample. all of the samples were obtained from adult animals. none of the foxes that were positive for ccov or kobuvirus rna showed pathological lesions indicative of enteritis. by fasta and blast analysis of the partial d region amplified with the primers univ-kobu-f/univ-kobu-r, the five fox strains (genbank accession no. kf -kf ) shared . - . % nt sequence identity with each other and displayed the closest relatedness ( . - . % nt) with the cakvs previously found in the us, the uk and italy [ , , , ] . identity to feline kobuviruses [ ] ranged from . % to . %, while nt sequence identity to human aivs was . - . %. the full-length sequence of the vp gene was determined for each of the five kobuvirus strains (genbank accession no. kf -kf ). sequence analysis indicated that the fox kobuviruses were highly related to each other ( . - . % nt and . - % aa identities) and to the cakv strain uk/ (accession no. kc ) ( . - . % nt and - % aa identities) recently identified in a healthy dog in the uk [ ] . neighbor-joining phylogenetic analysis based on the vp sequences was carried out with a selection of strains representative of the genus kobuvirus, including murine kobuvirus [ ] , human aivs, wild-boar kobuvirus [ ] , the ferret kobuvirus strain mpkov [ ] , and the prototype strains porcine kobuvirus s- [ ] and bovine kobuvirus u [ ] . based on inspection of the tree (fig. ) , all of the fox kobuvirus sequences formed a tight cluster with the cakvs recently identified in the uk and in the us [ , , ] , sharing a common root with murine kobuvirus and human aivs. historical evidence shows that foxes are susceptible to viral diseases of domestic carnivores, including infections with canine distemper virus [ , ] , cpv- [ ] , canine adenoviruses [ , ] , ccov [ ] , and canine herpesvirus [ ] . in the present study, we detected kobuviruses in freeranging red foxes with a prevalence rate of . % ( / ). investigations based on the molecular analysis of the fulllength vp gene revealed that the five fox strains were most closely related to the cakvs ( . - % aa) recently identified in dogs [ , , ] . therefore, the fox kobuviruses detected in this study may be considered members of genotype cakv- within the species aichivirus a. in addition, this is the first study demonstrating the presence of ccovs in free-ranging red foxes in italy. although cakvs were found in both asymptomatic and diarrhoeic dogs, the pathogenic potential of these novel kobuviruses in carnivores remains to be elucidated. in this preliminary study, all of the cakv-like viruses detected were identified in adult foxes found dead or shot during the regular hunting season without apparent pathological enteric lesions. accordingly, it is unclear if these viruses may have a role in the aetiology of diarrhoea in foxes. future large-scale virologic investigations by screening faecal samples collected from both diarrhoeic and healthy animals are needed to assess the distribution and pathogenic potential of cakv-like viruses in foxes. in conclusion, this study provides direct evidence of the circulation of cakv-like viruses in red foxes, reinforcing the previous observations that kobuvirus infections are probably as frequent in wild animals as they are in domestic animals [ , ] . additional studies are warranted in order to determine the significance of these findings and to evaluate the epidemiological and clinical importance of these novel viruses in wild carnivores. furthermore, as fox kobuviruses are newly recognized viruses and information on their diversity is still limited, a definitive characterization should rely on sequence analysis of the complete genome. fig. neighbor-joining phylogenetic tree based on the vp gene ( bp) of the kobuvirus strains detected in red foxes. the tree was generated using the neighbor-joining method and p-distance correction, supplying a statistical support with bootstrapping of replicates. the scale bar indicates nucleotide substitutions per site. the markers indicate the sequences detected in this study ratification vote on taxonomic proposals to the international committee on taxonomy of viruses prevalence and genetic diversity of aichi virus strains in stool samples from community and hospitalized patients molecular epidemiology of canine adenovirus type and type in free-ranging red foxes (vulpes vulpes) in italy antigenic analysis of canine parvovirus strains isolated in italy phylogeny and prevalence of kobuviruses in dogs and cats in the uk detection and genetic characterization of feline kobuviruses a real-time pcr assay for rapid detection and quantitation of canine parvovirus type in the feces of dogs canine kobuviruses in diarrhoeic dogs in italy bioedit: a user-friendly biological sequence alignment and analysis program for windows / /nt aichi virus infection in elderly people in sweden aichi virus infection in children with acute gastroenteritis in finland characterization of a canine homolog of human aichi virus mega : integrated software for molecular evolutionary genetics analysis and sequence alignment viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses canine distemper epizootic among red foxes novel norovirus in dogs with diarrhea the fecal viral flora of wild rodents development of a nested pcr assay for the detection of canine coronavirus candidate new species of kobuvirus in porcine hosts complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus, a member of a new species in the genus kobuvirus, family picornaviridae detection of aichi virus shedding in a child with enteric and extraintestinal symptoms in hungary porcine kobuvirus in wild boars (sus scrofa) metagenomic analysis of the ferret fecal viral flora survey on viral pathogens in wild red foxes (vulpes vulpes) in germany with special emphasis on parvoviruses and analysis of a dna sequence from a red fox parvovirus rational optimization of generic primers used for norwalk-like virus detection by reverse transcriptase polymerase chain reaction isolation and characterization of a new species of kobuvirus associated with cattle isolation of cytopathic small round viruses with bs-c- cells from patients with gastroenteritis detection of canine coronaviruses genotype i and ii in raised canidae animals in china acknowledgments this work was financed by grants from the university of teramo, italy, and from the italian ministry of university and research. conflict of interest all authors declare that there are no financial or other relationships that might lead to a conflict of interest. all authors have seen and approved the manuscript and have contributed significantly to the work. key: cord- -vahd z authors: cesari, matteo; proietti, marco title: covid- in italy: ageism and decision-making in a pandemic date: - - journal: j am med dir assoc doi: . /j.jamda. . . sha: doc_id: cord_uid: vahd z nan the world health organization declared the covid- situation as a pandemic on march th, . to date, italy is the country after china at having been most severely hit by this humanitarian and public health tsunami. projections are even suggesting that the number of deaths due to sars-cov- in italy will continue to increase in the near future, leaving us the sad world record of casualties. what has happened in italy during these last few weeks? on february nd, a "red zone" was defined by the government to quarantine a group of several towns in the lombardy region, just a few hours after the diagnosis of the first case in italy. this area, where about , persons live, included codogno (where patient was identified), castiglione d'adda, and casalpusterlengo. on march th, the red zone was extended to the entire region of lombardy (about million people) and several surrounding provinces in a new attempt of preventing the uncontrolled diffusion of the virus to the rest of the country. the following day, the entire country was transformed into a "red zone". on march st, a complete lockdown of italy was ordered by the government as a drastic and unprecedented countermeasure against the coronavirus. behind this story of the italian crisis is the drama of a healthcare system close to collapse. the exponential increase of patients admitted to emergency departments with fever and/or respiratory symptoms resembled the mounting wave of a tsunami. it soon became evident how inadequate the availability of beds was to face the continuous flow of patients. the situation was aggravated by the need to isolate covid patients, given the high contagiousness of the virus. at the same time, intensive care units started to saturate, and the number of devices for ventilating patients suddenly appeared insufficient to address the growing demand. furthermore, healthcare professionals started falling sick (sometimes even dying) as consequence of their untiring willingness to serve the community, as well as the infrastructural unpreparedness for the enormity of the outbreak. our world was completely subverted by the emergency. no plans or protocols had the time to be tested and verified, at least on a large scale. the rapidity of the evolving scenario made it necessary to adopt easy and pragmatic solutions even for critical and delicate matters. not surprisingly, the usual, despicable age criterion started to be implicitly adopted in the decisional algorithm for the allocation of scarce resources to the mounting number of patients. it is noteworthy that during the early crisis, the società italiana di anestesia, analgesia, rianimazione e terapia intensiva (siaarti; italian society of anesthesia, analgesia, and intensive care) released clinical ethics recommendations for the allocation of treatment in exceptional resource-limited situations. the document mentions the word "age" twice, in two critical paragraphs. they read as follows: " . it might be needed to set an age limit for the admission to intensive care. it is not a mere choice related to values, but to spare resources that might be extremely scarce to those who have in primis the highest chance of survival and then to those who may have more years of life saved, in order to maximize benefits for the largest number of persons. . the presence of comorbidity and functional status must be carefully evaluated in addition to age. it is possible that a relatively short stay in healthy persons might potentially become longer and thus resource consuming over the healthcare system in case of persons with advanced age, frailty or severe comorbidity." it is important to consider that what the siaarti mentioned as a scenario of "extremely scarce" resources may correspond to the optimistic vision of the saturation that the lombardy region has been experiencing over the past weeks. persons with covid- often experience extremely rapid (and often unexpected) clinical changes, with sudden respiratory distress. clinicians often find themselves in the position of having to act quickly to move a patient from the acute care ward to the intensive care unit, to be placed on a ventilator. it is not rare to see that in - minutes, the patient turns from relatively stable to extremely critical. in this scenario, which is the risk factor for negative outcomes that is easier and quicker to obtain? of course, the patient's age… if we want to fight such an ageistic approach and replace the age criterion for the allocation of resources, we must have and propose a parameter more robust than age but equally easy-to-obtain, that can be used for critical and rapid decision-making. otherwise, geriatricians might be at risk of remaining too theoretical and disengaged from the real world. we must show that we understand why intensive care physicians are prioritizing the life of a year-old person over that of a year-old, and that this is the best decision. they have never been exposed to anything other than this approach. and the critical nature of the situation can further provide ground for justifying such arguable choices. "all is fair in love and war"-and we are indeed in war! the document referred by the siiarti recommendations was developed without the involvement of geriatricians. it discusses how to choose if a patient should undergo palliative versus intensive care. the criterion that is most frequently used is age. however, the most recent recommendations seem to create some formal openings to geriatric concepts that are traditionally ignored, and therefore, to reconsider basing decisions only on the number of years lived. it is true that age is always at the beginning considerations that drive decisions; everything is still strongly designed to lead toward the exclusion of older persons. at the same time, one should not underestimate the statement that "the presence of comorbidity and functional status must be carefully evaluated in addition to age". the sentence might appear superficial to those who do not understand it, perhaps because this is not typically the case. at the same time, the statement potentially draws a first line in the sand for the future. it is a starting point to help discriminate what should be done and what should not be done, between good clinical practice and pure malpractice. implementation of these principles into decisional algorithms should, we believe, be part of pandemic preparation everywhere. in settings where rationing of resources becomes a necessity and such preparation has not been made, medical staff or oversight organizations should implement ad hoc guidelines that incorporate key prognostic factors beyond age -most notably frailty, comorbidity, and functional status. , in this manner, a sentence about function and comorbidities in an ethics document underscores the need to operationalize the meaning of prognosis at advanced age, and acknowledges the critical role that function and comorbidity play in the aging individual. clinicians familiar with principles of geriatrics and gerontology could thus support the development of more contemporary recommendations by identifying valid, efficient ways of measuring comorbidities and function across different settings and specialties. we might suggest the use of simple tests and scales, such as the clinical frailty scale, or the assessment of mobility independence, that might optimally capture the pre-illness health status of the individual, mirroring his/her physiological reserve, and, by incorporating such tools into electronic records for rapid assessment, provide support for better clinical decisionmaking than the all-too-simplistic criterion of chronological age. we realize we might be too optimistic to think that ageism is going to soon be defeated among clinicians. age is still the first criterion mentioned. however, we get some hope reading that, unlike the past, it is not the only criterion being proposed. will comorbidities and functional status start to change how we think and act in times of crisis? it is probably still too early to see major changes. however, while continuing to push towards a less ageistic society and medical practice, we should take advantage of these openings that arise from non-geriatricians. these are indeed opportunities to build constructive exchanges. if the principles of geriatrics had been incorporated into pandemic planning before this crisis, perhaps we would today have more justification to counter the ageistic approach. while ageist attitudes cannot be justified, we who focus on the care of older persons must take some responsibility for what is not happening. we need to realize how much work we still have ahead of us in educating and reframing the thinking among our clinician colleagues and our society, and therefore roll up our sleeves and perhaps leave aside some of our ego. when we hear that the decision of using a ventilator for a person with respiratory distress is based on his/her birth date, we must admit our failure and realize how many problems modern medicine has -in particular, that without our input, modern medicine may be at risk of having lost the meaning and value of the human life. who director-general's opening remarks at the media briefing on covid- - clinical ethics recommendations for the allocation of intensive care treatments, in exceptional, resource-limited circumstances grandi insufficienze d'organo "end stage": cure intensive o cure palliative? documento condiviso per una pianificazione delle scelte di cura prognostic indices for older adults: a systematic review the central role of prognosis in clinical decision making frailty and multimorbidity: different ways of thinking about geriatrics evidence for the domains supporting the construct of intrinsic capacity a global clinical measure of fitness and frailty in elderly people a diagnosis of dismobility-giving mobility clinical visibility: a mobility working group recommendation a comparison of frailty indexes for the prediction of falls, disability, fractures, and mortality in older men prognostic indices for older adults: a systematic review the central role of prognosis in clinical decision making key: cord- - hm ua s authors: solarino, biagio; aricò, maurizio title: covid- pandemic and pediatric healthcare policy in italy: time for a change date: - - journal: pediatr rep doi: . /pr. . sha: doc_id: cord_uid: hm ua s the outbreak of coronavirus disease pandemic forced the italian government to act extreme measures that put the entire country under lockdown. the citizens experimented a radical transformation of their lives while the healthcare system started building a novel covid-system that quickly dissolved the former organization, included pediatric healthcare network. for a number of reasons covid- pandemic is polite with children; however, the fear of this unknown disease drove to develop dedicated pathways in the pediatric emergency departments for the suspected and diagnosed cases of covid- , together with their relatives. the combination of conflicting feelings resulted in an unexpected scenario: the number of accesses to the pediatric hospital or unit(s) sharply dropped to less than half. how do we explain this phenomenon? the authors believe that our health care system may learn a lesson from the catastrophe of covid, if we will be able to redesign our way of providing our cares to children. nothing should be taken for granted, because this might be a real “time for a change”. the outbreak of coronavirus disease pandemic forced the italian government to act extreme measures that put the entire country under lockdown. the citizens experimented a radical transformation of their lives while the healthcare system started building a novel covid-system that quickly dissolved the former organization, included pediatric healthcare network. for a number of reasons covid- pandemic is polite with children; however, the fear of this unknown disease drove to develop dedicated pathways in the pediatric emergency departments for the suspected and diagnosed cases of covid- , together with their relatives. the combination of conflicting feelings resulted in an unexpected scenario: the number of accesses to the pediatric hospital or unit(s) sharply dropped to less than half. how do we explain this phenomenon? the authors believe that our health care system may learn a lesson from the catastrophe of covid, if we will be able to redesign our way of providing our cares to children. nothing should be taken for granted, because this might be a real "time for a change". the outbreak of coronavirus disease (covid- ) pandemic induced a radical transformation in our lives. italy rapidly turned out to be one of the most heavily affected countries worldwide. thus, the top priority of the policymakers became to reduce the pandemic morbidity and mortality rates as much as possible. with the aim of reducing the r index to lower than . , the government followed the example set by china to impose "social distancing". this resulted in extreme measures that put the entire country under lockdown. the leading hypothesis was that an explosive spike of covid- dissemination would have been unbearable for the italian healthcare system, especially in terms of number of intensive care unit (icu) beds and facilities simultaneously available. thus, while the people was practically locked down at home (or at the site they happened to be), the healthcare system(s) started building a novel, parallel covid-system: many hospitals, usually the largest and more advances ones, were either converted into "covid- hospital" or split into a covid-area and a residual, minor non-covid area. with the double aim of reducing the circulation of patients in the hospital area, but also to spare human resources to throw into the covid activities, all outpatient practices were virtually abolished (or at least sharply reduced): programmed admission were postponed, and elective surgery was limited to very selected cases requiring urgent oncologic treatment, trauma or emergency. primary care visits were reduced, and soon thereafter prohibited, trying to support the patients with remote (phone, whatsapp or videocall) aid. the healthcare system we have been working in simply dissolved within days. for a number of reasons, not yet fully clarified, covid- pandemic is polite with children. symptoms caused by this coronavirus are similar to those regularly observed in winter season in children with common viral infections; the risk to develop serious complications is minimal, and the fatality rate is almost absent. yet, pediatric healthcare network was also disrupted. the fear of this unknown disease, and uncertainty about the presenting phenotype, drove the need to develop dedicated pathways, as to isolate suspected and diagnosed cases of covid- , together with their relatives. a combination of conflicting feelings resulted in an unexpected scenario: despite the rising fear for this new disease, the number of accesses to the pediatric emergency department (ped) sharply drop to less than half. social distancing measures, leading to closure of schools, parks, gyms and other social places, make the children to spend the entire day indoor, at home, thus reducing not only the risk to be infected by other children with common infectious agents, but also the risk of traumatic injuries. furthermore, the fear to be infected in the hospital waiting room has likely contributed to reduce the access to the ped. is this enough to explain this phenomenon? alternatively, is covid- pandemic disclosing the real need for hospital medical care in italy? current child healthcare system comprises three main levels of intervention: a) primary care; ) secondary care in pediatric hospital units; ) tertiary pediatric children's hospital. ed services are provided for free by the national health system, with only a small co-payment based on family income, which may vary on a regional basis. in a national survey of ped activities, longhi et al. underlined a significantly higher percentage of children hospitalized after accessing the ed in southern regions. these observations support the concept that the organization of our health care system, although universally accessible regardless the patient/family income, remains someway heterogeneous. covid- pandemic put under the spotlight some inappropriate ways of using it, which cause reduced efficiency and effectiveness. due to the process of institutional devolution, individual regions apply different policies. indeed, we have no more a single nhs, but rather over autonomous regional authorities. whether this was effective and convenient is today deeply challenged, or even rejected by those who strongly recommend its return to a unique nhs. devolution has been justified by the aim at economies and better administration by managers located closer to the activities. however, is it only a matter of money and resources invested? coming to pediatric health care, during covid pandemic the access to pediatric ed sharply dropped to one-half. this happened nationwide and within days. were those missing requests necessary or simply inappropriate? reasons may rest on both sides, the patient and the doctor. until the age of years, pediatric primary care is provided during the weekdays only by "family" pediatricians that work on private practice; between and years, one can choose to associate or substitute the pediatrician with a general practitioner. during the weekend, a pediatric ambulatory consulting service located in the hospitals provides primary, nonurgent care. this setting should cover any child need, unless an urgent or even emergency care is needed, requiring admission to eds, with a yellow or red code, in accordance to pediatric triage scale categorization. monitoring pediatric eds before covid pandemic had clearly documented frequent overcrowding, resulting from inappropriate accesses for children deserving only primary care treatment. unnecessary overcrowding in the ed not only increased the waiting time, but also induced frequent complaining by the families. minimal or low priority (white or green) codes may frequently happen to wait many hours before completing the visits and the exams prescribed (how appropriately?) even for minor disease/symptoms. sometimes complaining progressed even to assault the ed workers, both doctors and nurses. , in this environment, the relationship between doctor and patient/family slips down to a very poor quality, making it difficult for healthcare professional to provide respectful and sensitive care, and create the usual, expected relationship with patient/family. this may increase the risk of errors and unintended harm. as a reaction to this stressing (or even aggressive) atmosphere, despite moderate or minimal morbidity, eds pediatrician have frequently hospitalized these children, and prescribed unnecessary (and potentially damaging) imaging studies, in the well-known pattern of "defensive medicine". , furthermore, also in italy, like in many other developed countries, health workers (and even hospital administrators) are increasingly exposed to malpractice claims: thus, inappropriate hospitalizations may result from the attempt to defend from any parental claim. frequently the child caregiver reports insufficient or unsatisfactory phone contact with the family pediatrician, thus resulting in taking the child to the hospital. if so, the attempt to organize a primary care setting able to prevent the child/family from an unnecessary hospital access has failed. this was the pic-ture most frequently observed nationwide. in the era of pandemic, emergency regulation prevented family pediatricians from visiting children, thus limiting their role to remote counselling. yet, instead of further pushing the children toward the hospital, this was associated with a sharp reduction of the hospital access to ped. the parents quickly shifted to a conservative pattern, limiting hospital access to what is perceived as a real emergency. so, what? on one side, one can take the impression that most of unnecessary access have been spared. on the other side, we should consider that inappropriate self-triage made by the parents and driven by pandemic scare may occasionally expose some children to a delayed diagnosis and treatment of severe conditions. in our countries, the evolution of pandemic pushes each of us to think how to go beyond this tragic phase, and how to "reopen italy". this applies not only to economy and production, but also to public services including the health system. we should be able to invest this "still time" in designing a new model of for pediatric healthcare delivery. we need to be more effective in spending our huge resources, and in particular, to be more appropriate in the choice of the pathways for care of patients with different needs, from the healthy newborn to patients with rare disease and severe handicap, which may impact on the society even more than pediatric cancer. we should be able to rebuild the outpatient network, which may support preventive medicine, early diagnosis and rehabilitation. fancy technologies, becoming one of the clues to get out of covid pandemic, may become part of the health-care system, reducing undue transportation by increasing remote control of chronic patients. in conclusion, we should try to revisit covid- pandemic chronicle and decode the many messages this tragic "natural experiment" provides us. we have learned a lot on the mechanisms underlying the immune system by the tragedy of congenital, once invariably fatal, childhood immune deficiencies. comparably, our health care system may learn a lesson from the catastrophe of covid, if we will be able to redesign our way of providing our cares to children. nothing should be taken for granted, because this might be a real "time for a change". the italian health system and the covid- challenge feasibility of controlling covid- outbreaks by isolation of cases and contacts -ncov: polite with children! systematic review of covid- in children shows milder cases and a better prognosis than adults the child health care system in italy pediatric emergency room activities in italy: a national survey health decentralization at a deadend: towards new recovery plans for italian hospitals emergency department use by pediatric patients in lombardy region, italy: a population study health services for children in western europe addressing risks of violence against healthcare staff in emergency departments: the effects of job satisfaction and attachment style the piper weekend study. children's and adults satisfaction regarding pediatric pain in italian emergency department principles of pediatric patient safety: reducing harm due to medical care implementation of a ct scan practice guideline for pediatric trauma patients reduces unnecessary scans without impacting outcomes defensive practice is indefensible: how defensive medicine runs counter to the ethical and professional obligations of clinicians. med health care philos delayed access or provision of care in italy resulting from fear of covid- hospital efficiency: how to spend less maintaining quality? virtually perfect? telemedicine for covid- prevalence and outcomes of primary immunodeficiency in hospitalized children in the united states key: cord- - n pojb authors: zullo, federico title: some numerical observations about the covid- epidemic in italy date: - - journal: nan doi: nan sha: doc_id: cord_uid: n pojb we give some numerical observations on the total number of infected by the sars-cov- in italy. the analysis is based on a tanh formula involving two parameters. a polynomial correlation between the parameters gives an upper bound for the time of the peak of new infected. a numerical indicator of the temporal variability of the upper bound is introduced. the result and the possibility to extend the analysis to other countries are discussed in the conclusions. the coronavirus disease (covid- ) has been recognized in italy starting from st january , also if there are some evidences that the first cases started some time earlier [ ] . the spread of the disease started from the northern regions of italy, lombardy and veneto on the st of february : after about twenty positive cases, two small areas had been put in quarantine but, since then, the number of infected increased exponentially in this two districts. from the localities situated farthest south in lombardy the disease spread in closest regions, emilia-romagna above all, with the provinces of piacenza, parma, modena, rimini, but soon after also in other provinces of lombardy (e.g. in the province of bergamo). on the th of march a lockdown for the entire region of lombardy (with a population of about millions of inhabitants) and other fourteen provinces (population of about six millions of inhabitants) was imposed by the central government of italy and then extended, on the th of march, to the entire country. up to the rd of march, the total number of infected is , of which in the lombardy region. since the start of the epidemic in china, a certain number of studies appeared in the mathematical community about this subject: the description of the spatial or temporal diffusion of the infected in given regions [ ] , [ ] - [ ] , the transmission dynamics of the infection [ ] , the economic and financial consequences of the epidemic [ ] , the effect of atmospheric indicators on the spread of the virus [ ] , are only a fraction of the topics under investigation in these days. one of the simplest non-linear deterministic continuous (in time) model of epidemiology is the sir model, in which the overall population is divided in three disjoint classes: s is the number of susceptible individuals, i the number of infectious individuals and r the number of recovered individuals. albeit its non-linearity, the dynamic of the model is fairly uncomplicated and manageable from an analytical point of view, and displays very interesting properties such as the existence of an epidemic threshold (see e.g. [ ] ), making it a very reasonable model. in section ( ) the sir model is introduced and briefly discussed, in section ( ) we will analyze the data of the cumulative number of infected in italy on the base of two simple hypotheses. an upper bound for the timing of the peak of new number of infected is obtained. in the conclusions we will comment about the results and look for possible extensions. the sir model describes the evolution of the individuals in the susceptible, infectious or recovered classes with the following differential equations: the total population n = s + r + i is a conserved quantity from the dynamical point of view, meaning that there are only two independent variables in the set of equations ( ) . the characteristics of this model are well-known and the interested readers can look for example at the discussions in the classical books of braun [ ] and murrray [ ] . here we will make only few observations, relevant for the next sections. some authors do not include the denominator n on the right hand side of ( ), since it is a constant and can be absorbed by a re-definition of the parameter r. however, we will keep it: in this way it is evident the scaling property of this model: if the initial conditions (s , i , r ) are scaled by a common constant factor α, (and so the total population is scaled by a factor α), the solution is scaled by the same factor. some temporal properties of this model, like the time corresponding to a maximum in i (the time of the peak of the infected), do not depend on the scaling. this property is very useful, since the actual number of infected or susceptible (and then of recovered) is in general not known. the reasonable assumption that the same fraction (with respect to the total) of infected, susceptible and recovered individuals are known, gives the possibility, in this case, to compare the measured data with the properties that are scale-independent. the solution of the system ( ) can be written in terms of just one variable: if r(t) is known, i(t) can be obtained by the third equation and s(t) from the first one or from the constrain n = s(t) + i(t) + r(t). if the epidemic is not severe (the number r(t) can be considered small compared to the overall population), an explicit formula for the number of recovered can be obtained in terms of the hyperbolic tangent function. the functions reads as where we used the initial value r( ) = and the parameters (α, β, c) can be made explicit in terms of the parameters appearing in ( ) . the previous is one of the example of the so-called s-shaped epidemiological curve (with a "peaked" derivative, the function sech ) that universally describes an infection disease. the value of the number of infected can be obtained by derivation, i.e. i(t) = αβsech(βt − c) . when considering the cumulative number of infected, r + i, the contribution of sech is negligible on the tails, whereas it is more pronounced in correspondence of the maximum of sech , but it is however small if the value of the parameter β is less than one. in this case, the value of r + i is well approximated by a tanh formula like ( ), with a certain different value of c. this is what we will assume in the next section. it is clear that the description made in the previous section is very basic. however it has the advantage to be manageable and to incorporate the main properties of the sir model. it is not by chance that the first application of the sir model (the bombay plague of ) by kermack and mckendrick [ ] used precisely the tanh formula above. in the following we will base our analysis on two hypothesis: (i) we assume that the cumulative number of infected is described by a tanh model. this assumption is independent on the underlying dynamical model considered, but may be justified on the base of some of them (e.g. the sir model, as shown in the previous section). (ii) we assume that, whatever it is the underlying model describing the evolution of the number of infected, this model is scale invariant, in the sense specified in the previous section. the second hypothesis is fundamental since we are going to look at scale-independent quantities: even in the case the measured number of infected and recovered individuals are different from the actual values, it is possible to estimate these quantities. the cumulative total number of infected that will be considered in the next lines are those of entire italy territory. there are at least two reasons that suggested to not take regional or local data: the first one is that the epidemic started to spread across three different regions (lombardy, veneto and emilia-romagna) and there could not be a correspondence between the locality where a certain fraction of inhabitants reside and the region where this fraction was infected. this is also true at a national level, but the fraction is assumed to be smaller. the second reason is that a non negligible number of workers and students moved, just before the lockdown, from the regions in the north of italy to their regions of origin in the center and south of italy. the possibility that a non negligible flow of infected people passed from the north to other regions should be taken into consideration. by taking the entire national set of data we overpass the above issues. the data can be taken for example from italian protezione civile [ ] or from who [ ]. the cumulative total number of infected will be indicated by f n , with f = corresponding to the number of infected on st of february . the subscript n stays for the number of days from the starting of epidemic. these data will be opposed to the continuous formula the value of β will be taken to be constrained by the equation the function f ( ) then depends on two parameters, α and c. when necessary, to stress the dependence on these parameters, we will denote the function with f α,c (t). the cumulative final number of infected expected from formula ( ) is given by f ∞ = α( + tanh(c)). it is possible to estimate the parameters α and c by minimizing the difference between the actual and predicted number of cases, i.e. minimizing in order to have a reasonable minimum number of data, we start the analysis by taking n ≥ . the values of the parameters minimizing the sum s n are reported in table ( ) at the st day of epidemic the final number of infected is estimated to be , but this is only a lower bound since the values of α n and c n are increasing. a plot of f α ,c (t) and of the cumulative number of infected is reported in figure ( ) . a fundamental observation is that the function s n actually has a basin of depressed values, showed in detail in figure ( ) for a given value of n. this basin of minimum seems to indicate that there is a given function α(c) giving a family of tanh curves with clearly, by considering a number n of values of α n and c n to fit a k , k= ,..., , we will obtain a set of values {a k,n }. by fitting all the data available (i.e. by taking n = ), we get the following values for the coefficients a k : it is possible to get more terms in the sum ( ), but the cubic term is sufficient to get a formula accurate enough to what we are going to say. the plot of the fit is given in figure ( ), together with the values of the residuals α n − k= a k c k n , where the values a k are those given in equation ( ) . a comparison between the curve α(c) and the basin of minima for s n has been plotted in figure ( ): the red curve is the function ( ) with the black dots giving the actual values of (c n , α n ) in table ( ) . the function α(c) denotes a trend in the data that may be useful. if in the next days the values of the infection continue to rise, it is reasonable to expect that the values of α and c will be constrained closely by the same curve. clearly the model used here is rough, but it can give at least an idea about the future trend of the data. we are tacitly assuming that there will be no other cluster of infection around italy in the next days: the point will be discussed later. now we consider the function f in ( ) as a function of t and c alone, since the value of a is constrained by the curve ( ) . the plot of the derivative of this function (with respect to t) gives the time of the peak of infections. the plot is reported in figure ( ): we notice that the maximum of the derivative of the cumulative number of infected increases with n up to c ∼ . and then decreases by increasing c. this gives an upper bound for the peak of new number of infected (the point where the second derivative of f (t) ( ) is zero), given by days after the first infection. in the next days (at the moment of writing we are exactly at the th of march, exactly at days) the description above can be tested. the above analysis, despite using a rough function for the total number of infected, is able to give an upper bound for the time of the peak of new infected (around rd of march) thanks to the observation that the values of α n are, in a certain sense, not independent on the values of c n and are well described by a polynomial interpolation with linear coefficient. the hypothesis about the scale invariance of the underlying model (that, we repeat, not necessarily is represented by the sir model) is fundamental for the accuracy of the result. another underlying assumption is that the restrictive measures will be kept and observed in the next days and there will be no other clusters in the south of italy (in the sir model language, the values of s are below the epidemic threshold, see e.g. [ ] ). in the unfortunate case that there will be other clusters, it is possible to think at a substitution of the tanh curve by a combination of such functions: if there are two clusters of comparable magnitude, then we will have f (t) = α tanh(β t − c ) + α tanh(c ) + α tanh(β t − c ) + α tanh(c ) in a next paper other sets of data will be analyzed, whereas the above analysis will be updated if necessary. coronavirus and oil price crash differential equations and their applications: an introduction to applied mathematics early phylogenetic estimate of the effective reproduction number of sarscov analysis and forecast of covid- spreading in china weifeng lv: high temperature and high humidity reduce the transmission of covid- early dynamics of transmission and control of covid- : a mathematical modelling study a contribution to the mathematical theory of epidemics data analysis for the covid- early dynamics in northern italy data analysis for the covid- early dynamics in northern italy. the effect of first restrictive measures modelling and predicting the spatio-temporal spread of coronavirus disease (covid- ) in italy key: cord- -d q zkog authors: kumar, sumit; sharma, sandeep; kumari, nitu title: future of covid- in italy: a mathematical perspective date: - - journal: nan doi: nan sha: doc_id: cord_uid: d q zkog we have proposed an seir compartmental mathematical model. the prime objective of this study is to analyze and forecast the pandemic in italy for the upcoming months. the basic reproduction number has been calculated. based on the current situation in italy, in this paper, we will estimate the possible time for the end of the pandemic in the country. the impact of lockdown and rapid isolation on the spread of the pandemic are also discussed. further, we have studied four of the most pandemic affected regions in italy. using the proposed model, a prediction has been made about the duration of pandemic in these regions. the variation in the basic reproduction number corresponding to the sensitive parameters of the model is also examined. since its first appearance in wuhan (china), the severe acute respiratory syndrome coronavirus- disease (covid ) is spreading rapidly across the globe [ , ] . the number of patients are increasing exponentially and in some of the countries, thousands of people are losing their lives, almost every day, due to covid . the seriousness of the situation can be understood by the fact that the number of infected cases have crossed the . million mark and more than thousand confirmed deaths, across the globe, due to the disease as on th april [ ] . moreover, the outbreak has spread in more than countries [ ] . owing to the grievous condition, the world health organization first declared the covid as a public health emergency of international concern on january and later a pandemic on march [ ] . in fact, at this point of time, the covid seems to be unstoppable. different countries and health agencies are working together to find some robust method to prevent the ongoing wave, which now becomes a pandemic, of the disease. moreover, the ongoing outbreak forced various countries to implement the countrywide lock down to break the chain of transmission of the corona virus. this results in a huge loss in terms of economy and resources and at the same time created a chaotic situation worldwide. efforts have been made from different corners of the research community to understand the structure of the virus and the transmission mechanism of the disease. different studies analyze the role of social distancing (which is the only effective method available till date) in the prevention of covid . at the initial stage, it is believed that transmission of the disease took place through animal to human mode. but later it has been established that the direct transmission of the disease is also possible and is the primary reason for the acute transmission to various countries [ , , ] . the possibility of hospital related transmission was also explored and it was suspected to be the possible cause in % of patients [ ] . along with its high transmission efficiency, the high level of global travel contributed heavily to the spread of sars-cov- across the globe [ ] . the ongoing pandemic of covid- challenged many developed nations including germany, spain, usa, italy, france and several others and has caused catastrophic impacts on the lifestyle of the people of these countries. this is almost opposite to the pattern of infectious diseases observed to date. in the past, such an outbreak of pandemic generally took place in underdeveloped and developing countries. the african region which is the host of many infectious diseases has the lowest reported cases of covid- till date. italy recorded its first case of covid on february , , at lodi (lombardy) [ ] . in the next hours, the infected cases increased to [ ] . moreover, at the initial stage of the outbreak, italian data followed closely the exponential growth trend observed in hubei province, china. till date, italy is one of the countries that have faced grievous consequences of covid . till th april , italy has , , recorded cases and , deaths due to covid . in terms of reported cases, it is the third highest while it is on the top when it comes to the number of deaths across the globe. of the patients who died, . % were aged - years, . % were aged - years, . % were aged - years, and . % were aged - years. the male to female ratio is % to % with older median age for women ( . years for women vs . years for men) [ ] . moreover, the estimated mean age of those who lost their lives in italy was years [ ] . the covid outbreak completely disturbed the economic condition of italy. several family owned small sectors are suffering [ ] . the condition of italy surprised the research fraternity because italy stands in the top five countries in terms of medical facilities. the ongoing dismal scenario in italy has forced the government to admit that they do not have any control over the spread of the disease as well as do not know when the ongoing web of covid will stop. due to the catastrophic impacts of the covid- outbreak, efforts have been made to analyze the trend of the disease and predict the future of the epidemics [ , ] . the work carried out in [ ] predicts that in the absence of timely implementation of available medical resources, the authorities will not be able to control the outbreak of the disease. the study further concludes that together with the medical facilities peoples movement and social activities should be restricted immediately in order to curtail the burden of the covid- . the work carried out in [ ] , collected the day to day data and measured the possible similarity between italy and hubei province (china). further, the study also shows that the number of deaths increased almost five times as the available treatment facilities reached the limit. the limited availability of medical staff and facilities also delays the possible end of the covid- crisis from april to may . however, to the best of our knowledge so far no compartmental deterministic mathematical model has been developed and studied for covid- spread in italy. in the current work, we propose a compartmental mathe-matical model to study the case of italy. the proposed model incorporates four different compartments namely -susceptible, exposed, infected and recovered population. the present covid- has an incubation period ranges up to days. therefore, to make our model realistic, we include the exposed population along with the infected population, which certainly results in improved prediction. we collected the data of italy for covid available on the worldometers[ ] website. further we trained the proposed mathematical model using the data avilable till th april . using this improved mathematical model we study the impact of lockdown on the spread of covid in italy. also, we predict the possible end of the current outbreak of covid in italy. moreover, to make our predictions more realistic, we have trained and validated our model with covid- data of some the highly affected regions of italy. the proposed model describes the transmission mechanism of covid- . in the modelling process, we have divided the human population into four mutually exclusive compartments, namely, susceptible (s), exposed (e), infected (i) and recovered (r). the exposed class is the collection of those individuals which have just got infected and are not showing any symptoms of the disease, while the infected class individuals have clear symptoms of covid- . we further assume that a recovered individual will become immune to the infection. the flow chart of the model is presented in figure . based on the above assumptions, the model is governed by the following system of equations:- in the above model, n represents the total population. we assume that all the new recruiters joined the susceptible class at a constant rate a. β is the disease transmission rate from the infected individuals to susceptible individuals. we further assume that susceptible individuals once come into the contact of infected individual will not directly join the infected class. they first join the exposed class (e) and after certain period of time shows visible symptoms of the disease and enters into the infected class (i). exposed class individuals are assumed to be less infectious as compared to the infected class individuals. therefore, β represents the disease transmission rate for exposed individuals. clearly, β ≤ β. here, α is the rate at which exposed individuals join the infected class. α is the recovery rate of exposed individuals and α is the recovery rate of infected individuals. θ is the disease induced death rate. µ is the natural death rate. in this section, we check the mathematical feasibility of the proposed model. for this purpose, we check whether all the solutions of the proposed model will remain positive and bounded or not. our proposed model system involves human population. hence for the initial state, all the compartmental values are assumed to be non-negative. we consider the following initial condition for analysis: to show the epidemiological feasibility of the proposed model system , it is required that all the solutions remain non-negative. hence, in the following theorem we verify that all the solutions with non-negative initial condition will remain non-negative. the following theorem establishes the positivity of the solutions. theorem : the solution (s(t), e(t), i(t), r(t)) of the proposed model system is non-negative for all t ≥ with non-negative initial condition. proof : from the first equation of system ( ), we have now, integrating the equation on both sides on simplification we have, this gives where t ≥ is arbitrary. similarly we can show the non-negativity for compartments e, i and r. hence, the solution (s(t), e(t), i(t), r(t)) will remain positive for non-negative initial condition. in order to accurately predict the epidemic, the solutions of the mathematical model should be bounded. the following theorem guarantees the boundedness of the solutionsof the covid- model designed for italy. theorem : all solutions of the proposed model are bounded. proof : we need to show that (s(t), e(t), i(t), r(t)) is bounded for each value of t ≥ . from our model system we obtain: from here, we can conclude that similarly, we can show the boundedness of every compartment of the model. therefore feasible region for our model system will be as of now, italy is one of those countries which are highly effected by covid- . according to the data collected from [ ], the total number of covid- cases has crossed as on th april . there has been more then deaths in the country due to this epidemic. our proposed model aims to predict the future scenario of the covid- epidemic in italy by analyzing its present state in the country. in this section, we perform rigorous numerical simulations to get an insight of the epidemic in italy. the parameter values used for the simulation are provided in table we have considered the following initial condition to perform our analysis. this is taken on the basis of the officially reported data by [ ] as on st march . (s( ), e( ), i( ), r( )) = ( , , , ) according to the situation report- available on the official website of who[ ], the first two covid- positive cases in italy were reported on st january . both the infected individuals had travel history to the city of wuhan, china. this was the initial phase of the epidemic in the country. due to the lack of government concern on this matter, the epidemic started to grow slowly but significantly. by the end of february the number of cases reached to [ ] . we trained our model defined for italy for the month of march to check its accuracy with the official data available [ ] . it was observed that our model predicted the epidemic closely. figure it can be seen from the simulated results (see fig. ) that, the active cases will still increase till the end of april. the pandemic cases may start to decrease by the first week of may. the epidemic may hit its peak in italy between th april to rd may as shown in figure . also, according to our proposed model and if the conditions remains the same, the epidemic in the country may not last till the end of july . in this section, we will discuss the factors which can significantly control the spread of pandemic. the two major factors discussed here are (a) early lock down and (b) rapid isolation. on th march , the government of italy imposed a nationwide quarantine restricting the movement of the population except for necessity, work, and health circumstances, in response to the growing pandemic of covid- in the country. in this section, we will investigate the effect of early lock down on the spread of pandemic in italy. it is clear from figure that the epidemic could have been controlled at a very early stage if the government had imposed the lock down early in italy. figure shows three different scenarios of the epidemic in italy with different initial values of susceptible class. figure ( a) shows the current scenario of italy. however, if lock down would have been imposed prior to th march , the number of susceptible would have been significantly low. in figure ( b) , we assume the susceptible population to be . crores due to the lock down in the country. it can be seen from the plot that, it has not only significantly reduce the number of infections, but also caused the overall death of pandemic by th april, . also, figure ( c) indicate that the epidemic could have been eliminated by st march, if the susceptible population have been reduced to lakhs. covid- is a global pandemic which is spreading all across the globe. early research shows that the disease transmission rate from an infected individual to a susceptible is very high [ ] . the transmission rate can be reduced by isolating the infected individuals as quickly as possible. in this subsection, with the help of numerical simulations we will show the variations in infected population for different values of β, disease transmission rate from infected individual to susceptible individuals. figure shows various scenarios of the epidemic in italy in case disease transmission rate would have been timely controlled. a rapid isolation of infected population will lead to reduce the disease transmission rate, β. from figure , we see that as disease transmission rate, β is reduced from . to . , it not only decrease the active number of infections from to , but also the overall lifespan of pandemic reduced from july to april , . in this section, we will study and analyze the spread of the pandemic in different regions of italy. there are few regions which have become hotspots for the pandemic in the country. these regions include lombardia, emilia romagna, piemonte and veneto. in order to study the pandemic more precisely, we have assumed that the transmission rate in these regions varies from the generalized transmission rate of italy. this can be explained, as there is a visible population gap in these regions. also, we have assumed that by first week of march, whole population of these regions is susceptible. hence, we have assumed that there are no new individuals available for recruitments, hence a equals zero. for this purpose we have used the parameters values available in table . the disease transmission rate from exposed individuals is also reduced to . . all the remaining parameter values are considered to be same as given in table in figure , we study the case of lambardia region of italy. it is interesting to note that cases of infectives were highest in this region of the country. in sub-figure a, the proposed model is used for fitting the available data. in sub-figure b, we used our proposed model to predict the future of pandemic in lambardia. we see from the study that by mid july, the pandemic will die out in this region. figure shows the case study of emilia romagna region of italy. in figure ( a), we can see the accuracy of our proposed model. figure ( b) consist of the upcoming predicted scenario of the epidemic in this region. according to our proposed model the pandemic will last till mid july, in this particular region. figures and illustrate the behaviour of the pandemic in piemonte and veneto regions respectively. sub-figures ( a) and ( a) exhibit the authenticity of our proposed model with the officially reported covid- cases in these regions. the possible extinction of pandemic in these regions is shown in sub-figures ( a) and ( a). according to our proposed model, the pandemic in both of these regions will last till the last week of july, . also, it can be observed from sub-figure ( b) that the active infected cases in piemonte will not surpass . whereas, the active infected cases in veneto may hit the mark ( see sub-fig b) . shows the accuracy of our model with the officially reported data of these regions. also, from our simulations, one can conclude that the epidemic is yet to reach its peak. the basic reproduction number is one of the key parameter to examine the long term behaviour of an epidemic. it can be defined as the number of secondary cases produced by a single infected individual in its entire life span as infectious agent. we have used next-generation matrix technique explained in [ ] , to derive the expression of basic reproduction number r . the r takes the following expression we will analyse the variation in r for different values of the parameters involved in the model system. figure illustrates the simultaneous variation in the basic reproduction number for different values of corresponding parameters. the parameter values used are given in table . a seir type compartmental model is proposed to study the current scenario of covid- in italy. our proposed model accurately fits the officially available data of the pandemic in italy. also, it is concluded from the study that the pandemic may still grow in italy till th april, . the pandemic may hit the . lacks active cases mark (see fig. ) in italy. also, we have discussed how the lock down that was imposed on th march, was a good but a delayed decision of the government of italy. through simulations, we have shown that a rapid isolation of the infective individuals and early lock down in the country are two of the most efficient procedures to terminate the spread of covid- . as of now, the vaccination of covid- have not been discovered. hence, this research can also be beneficial for the countries which are in the initial stage of the pandemic, as our research describes two of the most effective procedures to counter the spread of the pandemic and its long term impact on the spread of disease. the expression of the basic reproduction number r is also derived. as our proposed model involves various parameters, we have shown the sensitivity of these parameters via numerical simulations. it is clear from the simulations (see fig. d ) that the transmission rates, β and β are the most sensitive parameters. the reproduction number can be minimized if we can reduce these two parameters. severe acute respiratory syndrome coronavirus (sars-cov- ) and corona virus disease- (covid- ): the epidemic and the challenges corcione, -novel coronavirus outbreak: a new challenge early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china novel wuhan ( -ncov) coronavirus the next big threat to global health? critical care utilization for the covid- outbreak in lombardy, italy: early experience and forecast during an emergency response covid- and italy: what next? covid- in italy: momentous decisions and many uncertainties predicting the ultimate outcome of the covid- outbreak in italy the reproductive number of covid- is higher compared to sars coronavirus the construction of nextgeneration matrices for compartmental epidemic models this research can be extended in various ways. one can refine the model by introducing new compartments in order to examine the epidemic more precisely. there are certain assumptions which we have made while constructing this model because of the limited data and short onset time. as more data will be available in the future, this model can be trained with more real data to increase its efficiency. key: cord- -rj z po authors: fontanella, marco m.; de maria, lucio; zanin, luca; saraceno, giorgio; terzi di bergamo, lodovico; servadei, franco; chaurasia, bipin; olivi, alessandro; vajkoczy, peter; schaller, karl; cappabianca, paolo; doglietto, francesco title: neurosurgical practice during the sars-cov- pandemic: a worldwide survey date: - - journal: world neurosurg doi: . /j.wneu. . . sha: doc_id: cord_uid: rj z po abstract background and objective the sars-cov- pandemic has consistently changed medical practice throughout specialties, regardless of their contribution in facing the disease itself. we surveyed neurosurgeons worldwide to investigate the situation they are experiencing. design and participants a -question, web-based survey was administered to neurosurgeons worldwide through the wfns and the neurosurgery cocktail from march to april , by web link or e-mail invitation. questions were divided into three subgroups: general information, health system organization, and institutional plans for the sars-cov- outbreak. collected data was initially elaborated using survey monkey® software. country specific data were extracted from the who website. statistical analysis was performed using r version . . . results of the respondents, most were from italy ( %), india ( %), and pakistan ( %). surgical activity was significantly reduced in most centers ( %) and dedicated in-hospital routes were created for sars-cov- patients ( %). patient screening was performed only when there were symptoms ( %) and not routinely before surgery ( %). the preferred methods included a nasopharyngeal swab and chest x-ray. health professionals were rarely screened ( %) and sometimes, even if sars-cov- positive, were asked to work if asymptomatic ( %). surgical planning was changed in most institutions ( %), while indications were modified for non-urgent procedures ( %) and remained unchanged for subarachnoid hemorrhages ( %). conclusions most neurosurgeons worldwide reported work reorganization and practices that respond to current international guidelines. differences in practice might be related to the perception of the pandemic and significant differences in the health systems. sharing data and experiences will be of paramount importance to address the present moment and challenges in the near future. we are in the midst of a pandemic caused by a novel coronavirus, sars-cov- , first detected in wuhan (china) in december . since then, covid- has spread quickly, with more than , , confirmed cases and more than , deaths on april , with countries involved worldwide. given the serious public health risk, medical practice has consistently changed during sars-cov- pandemic. the impact of the covid- outbreak might change in relation to the diffusion of the virus, as well as the health system of the individual country; furthermore, this pandemic is influencing different medical specialties in a variety of ways. most surgical subspecialties are not primarily involved in fighting the disease itself, but they must still change their organization, as most national and international societies suggest stopping all elective activity, maintaining only emergent and urgent procedures. [ ] [ ] [ ] [ ] [ ] [ ] [ ] neurosurgeons might feel fairly useless during the sars-cov- pandemic. however, international guidelines have been introduced calling for a tailored triage according to the degree of emergency, , , and we believe that sharing information about the organization of neurosurgical activity throughout the world might be helpful at this time. furthermore, it might be interesting to investigate whether there is any association between the level of infection and consequent country reorganization of the neurosurgical system, as well as the neurosurgeons' practice. we, therefore, conducted an online survey that was submitted to neurosurgeons worldwide between march and april , through the world federation of neurosurgical societies (wfns) and the neurosurgery cocktail. [ ] [ ] [ ] the questions were divided into three subgroups: . information on the country and its involvement by neurosurgeons were asked about their country of practice, its involvement by the pandemic, and duration of the emergency; . health system organization and screening for health professionals: national and regional measures adopted to face the outbreak were queried, as well as the screening rate and precautions undertaken for sars-cov- positive health professionals; . institutional plans for the sars-cov- outbreak: any special measures adopted for sars-cov- positive neurosurgical patients were investigated, i.e. their screening rate and method, any changes in surgical indications, planning and activity for oncologic procedures, non-emergency surgeries, and subarachnoid hemorrhages (sahs). most question were closed-ended, multiple choice. some allowed also an open answer (q , and -see supplementary material). the primary goal was to collect data on neurosurgeons' perceptions of the health emergency, the national/regional measures undertaken for health professionals and throughout neurosurgical departments, and the changes in neurosurgical indications, planning, and activity. the secondary aim was to investigate correlations between the data collected and the epidemiological scenario in each country. the third aim was to look into differences among regions, nations and territories along with possible causes and consequences of diversities. data were initially elaborated using survey monkey ® software online; country specific historical data were extracted from the who website. for open answers, the most recurring terms were rendered as a "word cloud", in which a population of words is represented with different sizes according to their frequency. survey's responses to q were first converted into estimates in days (< week: days; > week to < month: days; > month to < month: days; > months: days). pearson's correlation was used to estimate the correlation between the number of answers in the surveys with the number of new cases in each country and the duration of the pandemic. the latter was calculated as the difference in days from the first confirmed case to the last day of the survey. where a i is the observed number of answers registered for country i. for each country, the median of the differences between each survey's entry date and perception date was calculated. statistical analysis was performed using r version . . . on april , the survey was closed and responses had been collected. the skipping rate for each question ranged from % (q ) to % (q ). most responses ( %) were filled during only days in march (saturday , sunday , and tuesday ). a total of countries worldwide responded to the survey (fig. ) . most respondents were from italy ( %), followed by india ( %) and pakistan ( %). statistical analysis did not reveal a significant correlation between the incidence of disease and number of responses by country (fig. ). for the majority of respondents ( %), the nation was facing a sars-cov- outbreak. the duration of the health emergency was between week and month for most respondents ( %), between month and months for % respondents, shorter than week for % respondents, and longer than months for % respondents. figure shows correlations between disease activity during the survey and time-length perception of respondents from some countries. regarding the special measures adopted in neurosurgical departments to face the sars-cov- outbreak, in most cases there was a reduction of surgical activity without centralization ( %), while there was a centralization of surgery in high-volume centers in % cases and full closure of neurosurgical departments in %. no special measures to face the outbreak were reported in % of centers. figure b shows the categorizations of special measures undertaken by countries in relation to the incidence of disease. the overall reported screening rate of health professionals for sars-cov- was %; % respondents reported that sars-cov- positive health professionals were asked to keep working if asymptomatic. with respect to the precautions adopted worldwide for sars-cov- positive neurosurgical patients, in most cases hospitals reserved dedicated routes for them ( %), in % cases specific operating rooms were dedicated to sars-cov- patients, and in % cases neurosurgical units were reserved for sars-cov- patients. other respondents ( %) replied with open answers and among them, no special measures were usually undertaken. the overall screening rate for sars-cov- was % for symptomatic patients and only % for patients undergoing surgery. the preferred methods for screening was nasopharyngeal swab ( %), followed by chest ct scan ( %) and chest x-ray ( %). surgical planning was globally changed in most institutions ( %): only urgent or emergency procedures were performed in % cases; urgencies/emergencies and procedures that could not be postponed were performed in % cases. oncologic procedures were preserved in % cases. the resulting reduction rate in number of surgical procedures was > % for almost half of respondents ( %). surgical indications for sars-cov- patients were modified in % cases for pathologies such as chronic subdural hematomas (cshs) and tumors, while the modus operandi in treating aneurysmal sahs did not change in % of centers. this survey, dedicated to neurosurgery and sars-cov- worldwide, demonstrated a number of interesting findings. a high number of responses (n= ) was received, suggesting a relevant global impact of covid- on the neurosurgical community, even though it is a surgical specialty that is not primarily involved in fighting the disease. italy and india were the countries with the most respondents ( fig. ). this finding is independent of the incidence of disease, as shown in figure . conversely, the us was the country with least number of respondents in relation to the incidence of disease during the study period. although it might be tempting to relate the number of answers to perception of the health emergency, we should point out that the survey circulated widely among neurosurgeons, but we cannot state that the percentage of respondents (i.e. respondents/non respondents) was the same among the different nations. the same correlation was found with regards to the medical perception of disease activity (q ) in different countries, and only few respondents ( %) claimed their country was not facing the outbreak during the time period studied: among them, neurosurgeons from germany were probably the most "wrong", since their country had between to sars-cov patients during the study period (fig. a) . notably, reactions and perceptions of covid- impact on a country may be consistently driven by government actions, as happened in india and pakistan, where most strict lockdown measures were undertaken with respect to other world countries, , possibly influencing general and health professional awareness of the health emergency. nonetheless, the differences in number of responses might be due to a heterogeneity of the survey distributions among different countries. furthermore, the perception of the emergency might be related to the health system, with germany having the highest rate of icu beds/population. regarding the time-length perception of covid- , italian and iranian respondents perceived the start of the health emergencies much earlier than the actual one (fig. ) ; chinese neurosurgeons, instead, located the start of the health emergency almost at the inflection point of decrease in incidence rate, when the sars-cov- pandemic was about to reach the plateau phase (fig. ) . the perception corresponded well to reality among the other respondents. it is tempting to interpret these data as the consequence of the strain that physicians are experiencing in countries with the longest disease involvement at the moment of the study: some might perceive that the emergency is longer than reality due to the continuous stress, or because of media pressure about other countries (i.e. china and iran) experiencing the outbreak. on the other hand, accumulating evidence shows that sars-cov- might have been circulating in italy well before february st, thus explaining the significant outbreak that took place in lombardy in northern italy. others may perceive that the emergency is shorter than reality due to epidemiology for complex reasons: chinese people outside wuhan experienced the outbreak at a later stage of the epidemic and individuals emigrating from wuhan was the main infection source for other provinces, causing a rapid increase in case load when wuhan was already in the plateau phase; the general perception in china about the national involvement might have been in reality delayed. health system organization with respect to health system organization, the most frequent action undertaken globally was reduction of surgical activity without centralization ( %, yellow bars on fig. b ). significant high rates were registered in india ( %) and pakistan ( %). centralization of surgery to high-volume centers was reported in only % cases and italy was the country with the highest number of positive respondents ( %), followed by germany ( %) and india ( %). only % of respondents report that their country did not undertake any special measure. these data show how most countries acted according to international guidelines in the management of elective procedures. india and pakistan have been reported to be the world's best respondents to the sars-cov- pandemic, - thus reflecting high rates of neurosurgical activity reorganizations. neurosurgical centers should undertake national and regional measures to meet patients' needs with logistical capabilities, as reported by guidelines. interestingly, health reorganization may vary signficantly even within the same country. more than , positive cases were confirmed in italy by april with more than , in lombardy alone. in this region, which is still at the center of the health emergency in italy, neurosurgical departments were urgently reorganized and centralization of surgery in highvolume centers was decided. [ ] [ ] [ ] other italian regions are still facing the health emergency, but at lower levels with incidences that tend to decrease from the north to the south. the same incidence disproportion between regions within a country is clearly visible in even smaller european countries such as switzerland, where the cantons of vaud and geneva account for more than , cases each, while the canton of schaffhausen has not yet reached cases. these significant variations in a single country justify the different regional reorganizations. guidelines for risk assessment and management of exposure of healthcare workers vary according to the risk of sars-cov- infection (categorized as high or low) and recommend covid- testing only for workers at a high risk of infection. in this sense, the global attitude did not deviate significantly from recommendations, , as only % of respondents reported ongoing screening for health professionals, mainly from brazil ( %), mexico ( %), and germany ( %). a minority of respondents ( %) declared that sars-cov- positive health professionals kept working if asymptomatic and a large portion of these respondents were from italy ( %). indeed, no clear national guidelines are available for sars-cov- positive health professionals, resulting in heterogeneity of recommendations throughout the country. covid- positive italian health professionals have reached more than , , with more than deaths of physicians (most of them are general practitioners) and almost nurses. at spedali civili hospital, in brescia in northern italy, sars-cov- positive health professionals are not allowed to work and daily temperature screening procedures are undertaken at the hospital entrance for both health professionals and visitors. nonetheless, there is a general perception that health professionals might have been asymptomatic carriers of the disease. regarding precautions adopted worldwide for sars-cov- positive neurosurgical patients, the most widely undertaken measure globally was to reserve dedicated routes to sars-cov- patients ( %), while specific operating room and entire neurosurgical units were created in a minority of cases. some respondents ( %, mainly from austria, germany, and uk) reported not taking any special measures for sars-cov- patients. however, guidelines clearly state that sars-cov- positive patients should be cohorted in a separate location from sars-cov- negative patients and specific hospital policy for management of known or suspected sars-cov- positive patients in the operating room should be developed. [ ] [ ] concerning the screening of neurosurgical patients, facilities should use portable radiography when chest x-rays are considered necessary, thus avoiding the need to bring patients into radiography departments; chest ct scan has been recently reported to have a high sensitivity ( %) for covid- screening, but lower specificity and accuracy. , a recently published paper in jama analyzed the sensitivity of different rt-pcr screening sources demonstrating that bronchoalveolar lavage fluid is the most sensitive specimen ( %), followed by sputum ( %), nasal swab ( %), fibrobronchoscope brush biopsy ( %), pharyngeal swabs ( %), feces ( %), and blood ( %); the authors underline that multiple testing from different sites improve sensitivity and reduces false-negative results. most guidelines at present recommend a single upper respiratory nasopharyngeal swab for suspect cases. in this survey, most respondents referred that nasopharyngeal swab was the preferred method for screening ( %), followed by ct scan ( %), and chest x-ray ( %). some respondents indicated more than one screening method, especially those from italy ( %) and india ( %), where the most common combination was the nasopharyngeal swab with chest x-ray. the covid- outbreak had a relevant impact on surgical planning, with most respondents reporting a significant change in surgical activity in their institutions ( %). the majority ( %) performed only procedures that could not be postponed (i.e. tumors with evident mass effect) and/or urgent/emergency procedures, while in a few cases ( %) the entire neurosurgical department was closed. this obviously resulted in a significant reduction of the overall number of surgical procedures: most respondents claimed more than % reduction of surgical interventions. procrastinating elective procedures has been one of the crucial indications delivered by international societies - with many important aims: a. to contain the spread of sars-cov- , by reducing visits to hospitals by people with no urgent medical issue; b. to reduce the patient load on intensive care units with non-covid- patients; c. to reduce the possibility of treating asymptomatic sars-cov- patients, who would be at high risk of deteriorating due to the surgical stress and would increase the risk of infecting health professionals. surgical indications for sars-cov- non-emergency patients (i.e. cshs and tumors) have been modified in only % cases, while % neurosurgeons worldwide referred that their institutions continued operating on elective neurosurgical patients in the same way as the pre-outbreak era; international guidelines clearly state that non-emergency procedures should be delayed. [ ] [ ] [ ] [ ] [ ] studying correlations between incidence of disease and actions undertaken by various countries (fig. c) , middle-eastern nations (i.e. turkey, egypt, saudi arabia, etc.) were the most reactive to the health emergency, followed by european countries (i.e. italy, spain, austria, etc.), and the americas (i.e. us, mexico, brazil, etc.). as for aneurysmal sahs, most respondents ( %) did not change their indications and treatment (fig. d) . even if some of these findings might seem against guidelines, the word cloud resulting from the open answers puts "patient" at the center and sums up what international societies have been suggesting: "postpone surgery and be conservative as much as possible, delay elective procedures, but, as for emergency symptomatic patients, try to operate with all recommended precautions" (fig. ) . we must indeed stress that all medical efforts, institutional plans, and health system organization would be useless without the appropriate and recommended use of personal protective equipment (ppe). , , although india is the world's second most populous country, the incidence rate of sars-cov- infections has risen less than other countries since the beginning of the outbreak. the reason might be found in the earlier government actions that india undertook while the virus was spreading out from china. , , limits of the study our study has many limits. first, it is not an epidemiological study and does not allow drawing conclusions about the actual prevalence and incidence of the variables investigated. it does allow, though, to draw conclusions regarding the perception of neurosurgeons about the covid- health emergency with respect to the actual epidemiology data. second, although this survey spread out widely among neurosurgeons, respondents were mostly from italy, india and pakistan, while the rest of the world was represented with lesser numbers. heterogeneity of the survey's percentage of respondents (i.e. respondents/non-respondents) among different countries might have biased some responses. notwithstanding, this is the first survey conducted on the impact of covid- on the neurosurgical community and we believe that data from this study can help neurosurgeons and global health organizations to tackle this health emergency. sars-cov- pandemic has consistently changed medical practice, with an enormous impact on all specialties, regardless of their contribution in facing the disease itself. neurosurgeons worldwide have changed their surgical planning and activity, in most cases following national and international guidelines. dedicated routes were put in place for sars-cov- patients in most cases and surgical activity was limited to procedures that could not be postponed, resulting in an overall reduction of surgeries by more than %. the lockdown will be soon followed by the rebuilding phase, when delayed elective procedures will need to be performed, thus opening a new challenge that to be addressed, possibly by sharing current knowledge and experience worldwide. deviation for each country of registered number of answers to the survey from the expected value. blue bars indicate an excess of answers; red bars indicate a lack of answers. covid- ) covid- in neurosurgery news, guidelines and discussion forum covid- : recommendations for management of elective surgical procedures. american college of surgeons neurosurgery in the storm of covid- : suggestions from the lombardy region, italy (ex malo bonum) hub and spoke' lombardy neurosurgery group. may we deliver neuro-oncology in difficult times (e.g. covid- )? neurosurgery during the covid- pandemic: update from lombardy, northern italy. acta neurochir (wien) / société française de neurochirurgie world federation of neurosurgical societies telegram: join group chat the r project for statistical computing the coronavirus disease global pandemic: a neurosurgical treatment algorithm india is enforcing the harshest and most extensive covid- lockdown in the world the variability of critical care bed numbers in europe focolaio di infezione da nuovo coronavirus sars-cov- : la situazione in italia distribution of the covid- epidemic and correlation with population emigration from wuhan, china coronavirus disease (covid- pakistan's response to coronavirus among world's best, says who country head how india is responding to covid- : quarantine, travel limits and tests embed=y&:showvi zhome=no&:host_url=https% a% f% fpublic.tableau.com% f&:embed_code_version= &: tabs=no&:toolbar=yes&:animate_transition=yes&:display_static_image=no&:display_spinner= no&:display_overlay=yes&:display_count=yes&publish=yes&:loadorderid= . accessed covid- -raccomandazioni per gli operatori sanitari coronavirus in italia, morti altri sette medici: il totale sale a . la repubblica am i part of the cure or am i part of the disease? keeping coronavirus out when a doctor comes home maintaining trauma center access and care during the covid- pandemic: guidance document for trauma medical directors. american college of surgeons congress of neurological surgeons -cns.org chest ct features of covid- in correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases | radiology detection of sars-cov- in different types of clinical specimens guidance for wearing and removing personal protective equipment in healthcare settings for the care of patients with suspected or confirmed covid- guidance for health system contingency planning during widespread transmission of sars-cov- with high impact on healthcare covid- dashboard /profiles/julia-hollingsworth">julia h <a href="/profiles/brettmckeehan">brett mckeehan</a> and tara john. coronavirus live news and updates from around the world do you modify surgical indications if the patient is ncov positive for non-emergency surgery (e.g. chronic subdural hematoma, tumors?) you modify your modus operandi in treating aneurysmal subarachnoid hemorrhage? * abbreviations list sars-cov- : severe acute respiratory syndrome coronavirus wfns: world federation of neurosurgical societies who: world health organization ncov: novel coronavirus sahs: subarachnoid hemorrhages cshs: chronic subdural hematomas icu: intensive care unit rt-pcr: reverse transcription polymerase chain reaction survey administered to neurosurgeons through the wfns and the neurosurgery cocktail from march to april by web link or e-mail invitation.how have we changed our neurosurgical activity in the storm of -ncov pandemic?as neurosurgeons we might feel fairly useless during the -ncov pandemic. we think, though, that it is important to share information on the organization of the neurosurgical activity in this moment. your participation to this brief survey is highly appreciated. key: cord- -ccrxoya authors: peters, michael a. title: philosophy and pandemic in the postdigital era: foucault, agamben, Žižek date: - - journal: postdigit sci educ doi: . /s - - - sha: doc_id: cord_uid: ccrxoya nan computer-scientific, culturalwhich is pandemic. governments are nothing more than grim executioners, and taking it out on them seems more like a diversionary maneuver than a political reflection. (nancy ) in italy, the disaster struck because the government failed to act quickly enough or to pursue the right policies. as pisano et al. ( ) point out: 'in a matter of weeks (from february to march ), italy went from the discovery of the first official covid- case to a government decree that essentially prohibited all movements of people within the whole territory, and the closure of all non-essential business activities.' it is a different situation in the usa and in the uk where trump and johnson discounted the virus threat, instituted a huge bail-out for business, and talk freely and against their own medical advisors of getting back to work by easter. maybe, in italy, agamben's thesis applies. in other countries, the logic of government follows a different line based on propping up markets and the economy even at the risk and expense of large numbers of infections and deaths. the thesis must be able to take account of a nation's health infrastructure-a fact that differentiates between social democratic models of public health and market-based forms like the usa where there is no universal provision. trump wants to get everyone back to work as soon as possible and his government policies reflect this privileging of capital and the expense of labor. it seems likely that all the digital home-delivery companies and supermarkets that depend on cheap mostly black and latino labor that still able to operate in the usa will do so on the backs of low paid and temporary jobs. in the us state with its continual state vs federal tensions government operates differently than italy (or china, for that matter). the emphasis of the two trillion dollar 'coronavirus aid, relief, and economic security act' (cares act) (the senate of the united states ) is to keep american workers paid and employed, to provide assistance to workers' families and businesses, and to support the health care system, with an accent on public education and innovation prioritizing zoonotic animal drugs. title iv-'economic stabilization and assistance to severely distressed sectors of the united states economy' looks at emergency relief and taxpayer protections as well as debt guarantee and hiring flexibility. surely the form of governmentality represents another example of the ability to use reverse logic of neoliberalism to socialize any losses and privatize gains? the stimulus package of two trillion is the biggest in american history. john cassidy ( ) remarks 'as a comparison, the obama stimulus package that was passed in was about . per cent of g.d.p., or half as big.' it's too early to dismiss agamben's theory for it may well prove to be correct especially as the time of the us elections draw closely: it is entirely possible that trump will use 'state of emergency' to take exceptional government powers to declare a postponement for a year or two. in pandemic! covid- shakes the world, slavoj Žižek ( ) comments on the panic globally facing us in the times of covid- when 'we live in a moment when the greatest act of love is to stay distant from the object of your affection. when governments renowned for ruthless cuts in public spending can suddenly conjure up trillions. when toilet paper becomes a commodity as precious as diamonds.' he touches a chord when he writes with customary irony: an average consumer reason[s] in the following way: i know there is enough toilet paper and the rumor is false, but what if some people take this rumor seriously and, in a panic, start to buy excessive reserves of toilet paper, causing an actual shortage? so i better buy reserves myself. it is not even necessary to believe that some others take the rumor seriouslyit is enough to presuppose that some others believe that there are people who take the rumor seriouslythe effect is the same, namely the real lack of toilet paper in the stores. is something similar not going on in the uk and california today? (Žižek ) . toilet rolls are a prime example of bourgeoise western culture, a pinnacle of consumer capitalism that provides thousands of choices of quality, material, perfume, strength, decoration etc. indeed most of the world does not yet have western style toilets let alone specialty toilet paper. the who reports that in , only % of the global population ( . billion people) used a safely managed sanitation service and . billion people still do not have basic sanitation facilities such as toilets or latrines with many defecating 'in the open, for example in street gutters, behind bushes, or into open bodies of water' (world health organization ). the who makes the conclusion that poor sanitation is linked to transmission of pandemic diseases and reduces human well-being. if ever there is an indicative index of development, it would have to be closely connected with sanitation and the ability to manage human waste hygienically. no doubt in poor countries like india, middle eastern, and african countries where there is little or no national health infrastructure, we are about to witness the devastation of entire communities that will linger on well after conditions elsewhere improve and the global economy restarts. this will be the greatest generational setback for these countries. western panic buying of toilet rolls based on a viral rumor creates the problem of shortage, as i observed many times in new zealand supermarkets at the beginning of the lockdown when prime minister jacinda ardern made public announcements that there are no shortages of any supermarket item. people stood for a long time in queues that stretched around the isles jammed up against one another contravening social distancing design to preserve individual isolation. panic buying of toilet rolls in nz had increased % over last year (shaw ) . hoarding and panic buying are examples of herd behavior where conditions for a self-fulfilling prophecy operate to cause the shortage that most fear. consumer behavior theory mostly addresses 'single decision-makers faced with making economic choices in relative social isolation' rather than 'collective action such as fads and fashions, stock market movements, runs on nondurable goods, buying sprees, hoarding, and banking panics.' as strahle and bonfield ( ) go on to note '[p]anic, as historically conceived, has been represented as a polar case of collective disorganization …. clearly resting beyond the explanatory power of economic theories which depend on the rationality assumption.' i prefer to use the argument from 'cumulative collective irrationality' that contradicts the theory of efficient markets. christoph j. merdes in his dissertation on collective irrationality notes: collective (ir)rationality finds application in all areas of human social life, and a better understanding of the phenomena, the underlying processes and the evaluative standards could greatly improve our ability to organize everything from markets over democratic government to cooperative scientific inquiry and the social norms of everyday life. (merdes ) collective irrationality is an endemic feature of human life that has been around since the beginning of social life, predating capitalism but the market provides some classic examples and raises questions about the ability of the market to operate efficiently or rationally in times of disaster. when Žižek suggested 'that the coronavirus epidemics may give a new boost of life to communism' he has in mind what the world health organization is saying 'we should mobilize, coordinate, and so on. .. like, my god, this is a dangerous situation. they're saying this country lacks masks, respirators, and so on. we should treat this as a war. some kind of european coordination. .. maybe even wartime mobilization. it can be done, and it can even boost productivity.' he acknowledges that 'the strong approach to the crisis by the chinese state has workedor at least worked much better than what is now occurring in italy, the old authoritarian logic of communists in power also clearly demonstrated its limitations.' (Žižek ) . the covid- virus infection began in china and despite western skepticism about the number of confirmed cases, it seems that the number of new confirmed cases of the coronavirus originating within china as opposed to chinese returning home has stopped indicating an effective period of social confinement of some - months. as david cyranoski ( ) reports in nature: 'researchers are studying the effects of china's lockdowns to glean insights about controlling the viral pandemic.' it seems obvious that china's extreme lockdown has been successful in limiting the spread of the virus. the only problem was that the lockdown started too late and also that the free flow of important scientific information (and whistle blowing) was halted in the early stages. certainly the covid- pandemic provides an ideal philosophical and political experiment not possible except in speculative terms during normal times, and western governments have responded very differently from one another. in terms of political theory, the question is whether state-led policies work better in times of crisis and emergencies. panagiotis sotiris argues that the shift from the power of the sovereign as a right of life and death power to state guarantee of the population' health and productivity led to an expansion without precedent of all forms of state intervention and coercion. from compulsory vaccinations to bans on smoking in public spaces, the notion of biopolitics has been used in many instances as the key to understanding the political and ideological dimensions of health policies. (sotiris ) biopolitics is foucault's depiction of the administration of life and a territory whose population is its subject, an administration the aim of which is to create conditions for life for survival and increase and above all for putting life in order economically and politically. an aspect of this paradigm that is missing from foucault's analysis-he died before the genomics revolution got under way-is bioinformation, and the bioinformational paradigm where these two forces of new biology and information coalesce, overlap, and intermingle in the logic that drives bioinformatics and bioinformational capitalism that is self-renewing in the sense that it can change and renew the material basis for life and capital as well as program itself. the viral experiments of globalization, interconnectivity, and pandemic in the postdigital era, disastrous as it has been involving incalculable human suffering, at the same time provide the opportunity to raise some questions rather than embrace a theory dogmatically (see peters et al. ) . this is my theme of openness in philosophy that proceeds without too much dogma. these questions in no particular order may not be the best questions but they indicate a forward-looking experimental philosophy: & which political system works best at quarantine and social isolation-american individualism or chinese collectivism; democracy or one-party state; free-market or welfare state? the invention of an epidemic. the european journal of psychoanalysis the state of exception provoked by an unmotivated emergency the good, the bad, and the ugly in the two-trillion-dollar stimulus what china's coronavirus response can teach the rest of the world discipline & punish: the birth of the prison coronavirus and philosophers. the european journal of psychoanalysis collective irrationality: an agent-based approach. dissertation an der fakultät für philosophie viral exception. the european journal of psychoanalysis viral modernity? epidemics, infodemics, and the 'bioinformational' paradigm. educational philosophy and theory lessons from italy's response to coronavirus covid coronavirus: by the numbers -what kiwis have been panic buying against agamben: is a democratic biopolitics possible understanding consumer panic: a sociological perspective coronavirus aid, relief, and economic security act home/newsroom/fact sheets/detail/sanitation pandemic! covid- shakes the world key: cord- - rm za z authors: curtale, filippo; perrelli, fabrizio; mantovani, jessica; atti, marta ciofi degli; filia, antonietta; nicoletti, loredana; magurano, fabio; borgia, piero; di lallo, domenico title: description of two measles outbreaks in the lazio region, italy ( - ). importance of pockets of low vaccine coverage in sustaining the infection date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: rm za z background: despite the launch of the national plan for measles elimination, in italy, immunization coverage remains suboptimal and outbreaks continue to occur. two measles outbreaks, occurred in lazio region during - , were investigated to identify sources of infection, transmission routes, and assess operational implications for elimination of the disease. methods: data were obtained from several sources, the routine infectious diseases surveillance system, field epidemiological investigations, and molecular genotyping of virus by the national reference laboratory. results: overall cases were reported, sustained by two different stereotypes overlapping for few months. serotype d was likely imported from romania by a roma/sinti family and subsequently spread to the rest of the population. serotype b was responsible for the second outbreak which started in a secondary school. pockets of low vaccine coverage individuals (roma/sinti communities, high school students) facilitated the reintroduction of serotypes not endemic in italy and facilitated the measles infection to spread. conclusions: communities with low vaccine coverage represent a more serious public health threat than do sporadic susceptible individuals. the successful elimination of measles will require additional efforts to immunize low vaccine coverage population groups, including hard-to-reach individuals, adolescents, and young adults. an enhanced surveillance systems, which includes viral genotyping to document chains of transmission, is an essential tool for evaluating strategy to control and eliminate measles the world health organization regional office for europe (who/euro) has set as the target for elimination of measles in the region [ , ] . this objective has already been achieved in some states through routine immunization programmes which maintain high coverage using a two-dose measles-mumps-rubella (mmr) vaccine schedule [ ] . however, vaccination coverage still remains inadequate in several western european countries, including italy, and although mass vaccination has successfully lowered the incidence of the disease outbreaks continue to occur, often affecting communities with low vaccination coverage [ ] . in italy, measles vaccine (mv) was introduced in and routine administration of one dose of measles vaccine to all children aged ≥ months was recommended since . combined mmr vaccines became available in the early s and the two-dose schedule (first dose at - months and second dose at - or - years) was introduced in [ ] . however, despite the existence of national recommendations, responsibility for implementation of measles vaccination activities lies within each of italy's regions. this has contributed to non uniform coverage across regions, with lower rates observed mainly in central and southern regions (including lazio) with respect to northern regions [ ] . in the years - a large measles outbreak occurred in italy with over , estimated cases among children below years of age [ ] . following the outbreak, in november , all italian regions signed an agreement with the italian ministry of health to implement the "national plan for the elimination of measles and congenital rubella" [ ] . strategies include among others, improving routine mmr coverage among children below years of age, implementing supplementary vaccination activities for older children and adolescents (aged - years) and strengthening disease surveillance. following implementation of the elimination plan, national vaccination coverage for first mmr dose in children at two years of age increased from . % in , to . % in . during the same period of time, incidence of measles in italy decreased from . / , to < / , [ , ] . in the lazio region, the measles elimination plan succeeded in increasing coverage for the first dose of mmr among children at two years of age from . % in to . % in [ ] . during the same five year period, coverage among school-aged children ( - years of age) increased from % to % while coverage for the second dose at years of age increased from % to %. in and a historically low incidence of < case per , was reached in the lazio region, with approximately measles cases reported per year. however, sporadic cases continued to be reported, especially among susceptible adolescent and adults. pockets of low coverage were also present in specific areas and among emarginated and hard-to-reach populations (hrp), such as the roma/sinti population, and illegal immigrants [ ] . between june and august , two measles outbreaks occurred in the lazio region. the first outbreak (june-december ) initially involved the roma/sinti population, and subsequently spread to the rest of the population. the second outbreak overlapped with the first (october -august ) and affected mainly the italian adolescent and adult population. in this article we describe the two outbreaks and highlight the importance of pockets of low vaccine coverage in sustaining such outbreaks. data from the mandatory infectious diseases surveillance system, field epidemiological investigations, and molecular characterization of measles virus by the national reference laboratory are presented. lazio, one of italy's regions, has a population of . million people ( ), . million of which living in the urban area of the capital city of rome. it is divided into provinces (rome, rieti, viterbo, latina and frosinone) and local health units (lhus). the public health agency of the lazio region (agenzia di sanita' pubblica, asp), is responsible for surveillance of infectious diseases and immunisation coverage in the region. the asp monitored the two measles outbreaks and coordinated outbreak control activities in the lhus of the region. in italy, measles is a disease subject to mandatory notification, and according to the routine procedure, physicians must report suspected measles cases to their lhu within hours of diagnosis. the local health authorities then report confirmed measles cases to the asp monthly. at the beginning of the outbreaks this procedure was modified and physicians were asked to report suspected measles cases to both the local health authorities and asp offices within hours of diagnosis. personnel of the lhus performed epidemiological investigation of suspected cases including laboratory investigation and contact tracing. a suspected measles case was defined as a subject with fever (≥ °c), generalised rash and at least one of the following symptoms: cough, coryza, or conjunctivitis. a confirmed case was defined either as a laboratory confirmed case (in which measles-specific igm antibodies were present in serum or saliva samples or measles virus nucleic acid was detected in urine samples by pcr or as a case with an epidemiological link to a confirmed case. for each confirmed case, demographic data characteristics (including whether cases belonged to a roma/sinti community), vaccination history, date of onset of symptoms, and hospitalisation, were collected. information was conveyed to the asp, which discarded non confirmed cases from the database, eliminated redundant records, performed quality check and contacted the local health authorities for any missing information. to assess presence of indigenous transmission or sources of imported virus the data set was integrated with information provided by the national institute of health (istituto superiore di sanità, iss), which conducted viral molecular characterization from urine samples of measles cases, utilising the pcr technique [ ] . data analysis regarding the age distribution and immunization status of cases, as well as the percentage of cases requiring hospital admission was performed on the total number of cases reported in the two outbreaks. phylogenetic analysis based on the available partial nucleoprotein gene sequences of measles virus and tree reconstructions were performed with mega software version . [ ] . virus isolates and genotypes were designated according to the new official who nomenclature [ ] . data from confirmed cases was entered into microsoft excel and converted to sas version (sas institute inc., cary, nc) for analysis. the temporal and geographical distribution of cases, together with the age distribution, was calculated separately for the roma/sinti and rest of the population. the present study did not require approval from an ethics committee. laziosanità -the public health agency of the lazio region is the local government agency responsible for the collection of infectious disease notifications, hospital admission and discharge records and laboratory surveillance data. the management of these data for public health purposes does not require a patient's informed consent nor does it require any authorization regarding privacy laws. from june to the end of august , a total of cases were reported, of which in and in . the two outbreaks overlapped and not all cases were genotyped; therefore, it was not possible to determine the exact number of cases that occurred in each of the two outbreaks. overall, / cases occurred amongst the italian ethnic population and / amongst the roma/sinti population. seventy-eight cases ( %) were laboratory confirmed while the remaining were epi-linked. the virus serotype was identified for fifty-seven cases (serotype d : n = ; serotype b : n = ). the first reported case was an eight year-old roma child of romanian nationality, living in a settlement located in the outskirts of rome. the child was admitted to hospital with rash, fever, diarrhoea, conjunctivitis, rhinitis and otitis on june , reporting onset of symptoms on june . analysis of routine data allowed the identification of an additional case that had been notified one week previously, in rome by a different lhu. this case was an unvaccinated six-year-old roma child, also of romanian nationality, who developed symptoms on june . the child was not hospitalised and the source of his infection was not determined, since his parents refused to answer to questioning by the health officials and then moved away. he transmitted the infection to an eight-year-old child living in the same building, who developed symptoms on july and was hospitalised. in the last week of june two additional roma/sinti children, in two different health authorities, developed measles symptoms on june and june respectively. by the end of july in rome, an additional measles cases had been reported in various settlements of the roma/sinti community and cases among the rest of population. two cases among the italian ethnic population, who developed symptoms on and july, reported contact with roma/sinti patients with measles in a hospital waiting area, on and july in november , a cluster of six measles cases was reported amongst adolescents and young adults attending a professional school in the outskirts of rome (attack rate . %). the infection subsequently spread outside the school and cases continued to be reported amongst the italian ethnic population until the summer . this second outbreak reached a peak in march and was considered over only in august (figure ). during july-august three measles cases were notified per month, bringing the incidence level to that reported in the lazio region before the described outbreaks (≤ cases per month, or < / , population per year). the d genotype, grouped in two different clusters of common origin, was responsible for the first cases reported in the roma/sinti population and detected in several other cases, including the italian ethnic population, up to december . starting in october the b genotype was isolated in a contact of a case from the school outbreak, overlapping for some time with genotype d (figure ). the first outbreak (d ) started in the rm-b lhu (figure rome, where the outbreak in the professional school occurred. (figure ). overall, the median age of cases was years. children aged - years were the most affected age group, with one third ( / ) of cases reported belonging to this age group ( / , incidence for children < years). measles incidence in the total population during the two outbreaks was / , . when analysed separately, the age distribution of measles cases was different among the roma/sinti population (median age two years) with respect to the rest of the population (median age years). seventy percent ( / ) of roma/sinti cases occurred in children aged - years and over % ( / ) were aged below years. the age distribution of cases in the italian ethnic population was more evenly distributed among all age groups. only % of cases ( / ) were aged - years, and less than % ( / ) occurred in children below years of age ( figure ). none of the roma/sinti cases were vaccinated against measles, and only ( . %) cases from the italian ethnic population had received one dose of measles-containing vaccine. overall, over % ( / ) of cases required hospital admission. fifteen cases ( . %), of whom from the first outbreak and three from the second, reported a hospital contact with children affected by measles, either in the waiting area or after admission for a different condition. four of these contacts were healthcare professionals (two nurses and two physicians). out of the reported cases of nosocomial transmission, seven involved contacts with roma/sinti children, including the first two cases in the italian ethnic population. in response to the described clusters of measles cases, active tracing and vaccination of susceptible contacts was performed by local health authorities. a second dose of mmr vaccine was also offered to incompletely vaccinated contacts. in addition, mmr vaccine was offered to all susceptible or incompletely vaccinated children and adolescents attending any of the schools in which cases had been detected and to roma/sinti children up to years of age. vaccination sessions were conducted directly in the involved settlements; in total persons in seven settlements were vaccinated in august . isolation of cases and susceptible contacts (in hospital for roma/sinti patients and at home for other subjects) was recommended, local health authorities were urged to identify possible contacts of all suspected measles cases, and alert general practitioners, family paediatricians, and hospitals about the outbreaks. physicians were asked to report cases within hours of diagnosis. in addition, guidelines regarding respiratory isolation of patients with suspected measles, and vaccination of susceptible hospital staff were forwarded to all hospitals of the region. before the start of the / school year a meeting was organized by the asp with staff in charge of the measles elimination campaign and the public health departments of the health authorities. local media were also informed of the outbreaks. the two described outbreaks, which involved cases (incidence / , ) notified from june to august , represent the most serious episodes after the - measles outbreak [ ] . they confirmed that pockets of low vaccination coverage exist in some areas of the lazio region, particularly among roma/sinti communities and adolescents thanks to relatively high immunisation rates amongst new born children ( , %) [ ] and the work done by the local health authorities, conducting contact investigation of cases', vaccination of susceptible school and household contacts, and implementing isolation measures, the outbreaks did not affect the whole region and, in the city of rome, was mainly limited to a few peripheral districts (figure ). both outbreaks started in populations known to have low coverage, (roma/sinti community, and students of a secondary school). the subsequent spread to the rest of the population, at least for the first outbreak, was facilitated by nosocomial transmission. differences were found in the affected age groups among the roma/sinti and the rest of population. as expected in a susceptible population, the most affected age group in the roma/sinti population was the - year-old age group. conversely, in the italian ethnic population, which had a higher percentage of vaccinated subjects with respect to the roma/sinti population, especially among young children, most cases occurred in the - year-old age-group. molecular characterization of measles virus is an important surveillance tool for monitoring measles elimination [ , ] . in this case it was fundamental in tracing the origin of both outbreaks and showing that two distinct chains of transmission took place in the region. it is highly likely that the first outbreak, due to the d measles serotype, which is not endemic in italy [ ] , was imported from romania. in fact, first cases occurred in families of romanian nationality with family and social links in romania. in addition, the d sequence identified among the first cases in lazio was found to be identical to the d isolated in romania during the - outbreak [ ] in which over cases and deaths occurred [ ] . a d measles strain highly correlated with the one isolated in lazio caused a smaller outbreak in northern italy (south tyrol), between june and august [ ] . the first case was reported during the same week of the beginning of the first outbreak in lazio, and of cases belonged to the roma/sinti population. in south tyrol, a transit camp for roma/sinti travelling between italy and eastern europe appeared to be the entry point for the d measles genotype in italy (figure ) . between august and february other measles outbreaks linked to the lazio outbreak (d ) occurred in sardinia (italy) and barcelona (spain), both affecting mainly roma/sinti communities. in sardinia, nine cases, all aged below years and three of which laboratory confirmed, with genotype d isolated, were reported from a roma/sinti settlement in the town of alghero, between august and september [ ] . four of the children had travelled to rome from to august , to attend a funeral. in the barcelona region (spain) an outbreak occurred from october to february . the first case, a six-year-old girl of eastern european origin had attended a family gathering in italy with her mother where other guests may also have had measles. genotype d was identified [ ] confirming the link with the outbreak in lazio (figure ) . genotype d was no longer isolated after the end of , being replaced by b genotype starting in october . despite an accurate epidemiological investigation, the origin of this genotype was not identified. b is not considered endemic in italy and is most frequently detected in sub-saharan africa, although transmission of this virus within europe has been reported. the isolated b was similar, but not identical, to the strain circulating in uk in [ ] . this serotype was first identified in lazio and subsequently introduced in puglia (figure ) , a region of south italy, where it was responsible for an outbreak from november to january [ ] . the percentage of cases which required hospitalisation during the two outbreaks in lazio was high ( %). this can be partly explained by the well known underreporting of cases by general practitioners and paediatricians, as compared to hospital physicians. the number of cases requiring hospitalisation, especially during july-september , was sufficiently high to create problems to the hospital system. the inadequate number of isolation beds in hospitals may represent a serious problem in case of occurrence of an epidemic due to a more aggressive infective agent, such as sars or pandemic flu. measles nosocomial transmission has been recently documented in several other outbreaks in italy and other european countries [ ] [ ] [ ] [ ] and the public health importance of nosocomial measles transmission has been established in many situations. the cases reported in this paper represent only % of the total number of cases and therefore did not contribute significantly to the measles incidence during the outbreak. however, these cases most likely represent an underestimate of the real number of infections that occurred through hospital contacts and nosocomial transmission. it is likely that isolation measures and separate admission procedures were not always adopted in case of admission of a patient with signs and symptoms compatible with measles. in the first outbreak, nosocomial transmission may have been responsible for the spread of the infection from the roma/sinti to the italian population. also of concern is the fact that four healthcare professionals developed measles. as the incidence of measles declines, nosocomial transmission is likely to become an important source of infection and sustain the occurrence of outbreaks among non-immunised health staff and hospital contacts, representing a serious problem in the elimination of measles [ ] . strategies to minimize nosocomial spread of infection should become a priority for control and effectively implemented in the future. the described outbreaks highlight the threat represented by pockets of susceptible populations, even in the presence of good coverage levels in the overall population [ ] . these groups include hard-to-reach populations (hrp) such as the roma/sinti communities (estimated mmr coverage in italy below %,) [ ] , families who refuse vaccination for ideological or religious reasons, as reported in recent outbreaks amongst students in private religious schools and orthodox communities in europe and israel [ ] [ ] [ ] , and families objecting to having their children vaccinated out of concern for vaccinerelated adverse events [ ] [ ] [ ] . the risk for the community represented by hrp or organized groups of objectors should not be underestimated and represent a more serious treat than sporadic susceptible individuals. susceptible population groups may reintroduce indigenous measles virus transmission even in countries confirmed as disease-free or in a population with high immunization coverage [ ] facilitating the transmission of the disease to susceptible individuals still present in the region. efforts are needed to improve methods to identify areas with low coverage and to develop specific strategies which target susceptible groups. an improvement in health services delivery may be needed to reach roma/sinti communities and new immigrants. at the same time, more effective communication strategies should be defined to address subjects objecting to vaccination either for religious or other reasons, involving these subjects in a wider discussion on their responsibility toward the community. the implementation of catch up campaigns targeting adolescents and young adults should also be considered, with the additional objective of protecting women of childbearing age against rubella. world health organization regional office for europe: strategic plan for measles and congenital rubella infection in the european 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survey on childhood immunization coverage and reasons of non-compliance in lazio region high genetic diversity of measles virus, world health organization european region molecular characterization of measles viruses world health organization: update of the nomenclature for describing the genetic characteristics of wild-type measles virus: new genotypes and reference strains monitoring of measles elimination using molecular epidemiology the benefit of molecular characterization during a measles upsurge in denmark net: measles outbreak in romania measles hits romania: promed-mail cluster of measles cases in the roma/sinti population measles outbreak in the barcelona region of catalonia gentic characterization of measles virus strain isoltated during an epidemic cluster in puglia an outbreak of mealses including nosocomial transmission in abulia current measles outbreak in greece measles outbreak in the region of nosocomial outbreaks -a potential threat to the elimination of measles measles elimination target: the need to meet the specific risk group measles outbreak in a community with very low vaccine coverage, the netherlands. emerging infectious diseases parent du chatelet i, the investigation team: outbreak of measles in two private religious schools in bourgogne and nord-pas-de. calais regionas of france an outbreak of measles in an ultra orthodox jewish community in jerusalem, israel, -an in dept report measles outbreak in germany: over cases now reported in nordrhein westfalen measles in south-west germany imported from switzerland -a preliminary outbreak description implication of a measles outbreak in indiana for sustainable elimination of measles in the united states pre-publication history the pre-publication history for this paper can be accessed here submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we would like to thank all the health staff in the communicable diseases and immunization units of the lhu of lazio for the time and effort they have devoted to the control of the outbreaks and to assist asp in the data collection and analysis. special thanks to laura alecci (asp), for creating, maintaining, and updating the data-set, claudia fortuna and antonella marchi (mipi) for the laboratory work, and to paolo giorgi rossi (asp) for his help in finalizing the manuscript. authors' contributions fc coordinated the epidemiological data collection and the outbreaks' control measures, supervised the data analysis, and drafted the manuscript. fp supervised the local health units personnel and the implementation of public health measures. jm performed the statistical analysis and produced the graphs. mcda revised the results of data analysis and contributed in drafting the manuscript. af contributed to epidemiological data collection, participated in drafting and revising the manuscript. ln supervised the laboratory work and molecular genetic study. fm carried out the sequence alignment, the molecular genetic studies and contributed in drafting the manuscript. pb formulated the original study hypothesis and participated in the study design. ddl conceived the study, and participated in its design and coordination. all authors read and approved the final manuscript the authors declare that they have no competing interests. key: cord- - sgeraws authors: remuzzi, andrea; remuzzi, giuseppe title: covid- and italy: what next? date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: sgeraws the spread of severe acute respiratory syndrome coronavirus (sars-cov- ) has already taken on pandemic proportions, affecting over countries in a matter of weeks. a global response to prepare health systems worldwide is imperative. although containment measures in china have reduced new cases by more than %, this reduction is not the case elsewhere, and italy has been particularly affected. there is now grave concern regarding the italian national health system's capacity to effectively respond to the needs of patients who are infected and require intensive care for sars-cov- pneumonia. the percentage of patients in intensive care reported daily in italy between march and march , , has consistently been between % and % of patients who are actively infected. the number of patients infected since feb in italy closely follows an exponential trend. if this trend continues for more week, there will be infected patients. intensive care units will then be at maximum capacity; up to hospital beds will be needed by mid-april, . our analysis might help political leaders and health authorities to allocate enough resources, including personnel, beds, and intensive care facilities, to manage the situation in the next few days and weeks. if the italian outbreak follows a similar trend as in hubei province, china, the number of newly infected patients could start to decrease within – days, departing from the exponential trend. however, this cannot currently be predicted because of differences between social distancing measures and the capacity to quickly build dedicated facilities in china. according to nature, the spread of coronavirus disease (covid- ) is becoming unstoppable and has already reached the necessary epidemiological criteria for it to be declared a pandemic, having infected more than people in countries. therefore, a coordinated global response is desperately needed to prepare health systems to meet this unprecedented challenge. countries that have been unfortunate enough to have been exposed to this disease already have, paradoxically, very valuable lessons to pass on. although the containment measures implemented in china have-at least for the momentreduced new cases by more than %, this reduction is not the case in other countries, including italy and iran. italy has had confirmed cases according to the istituto superiore di sanità as of march , and deaths. only china has recorded more deaths due to this covid- outbreak. the mean age of those who died in italy was years and more than two-thirds of these patients had diabetes, cardiovascular diseases, or cancer, or were former smokers. it is therefore true that these patients had underlying health conditions, but it is also worth noting that they had acute respiratory distress syndrome (ards) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) pneumonia, needed respiratory support, and would not have died otherwise. of the patients who died, · % were aged - years, · % were aged - years, · % were aged - years, and · % were aged - years (those aged > years made up · %). the male to female ratio is % to % with an older median age for women ( · years for women vs · years for men). on march , , the italian government imple mented extraordinary measures to limit viral transmission-including restricting movement in the region of lombardythat intended to minimise the likelihood that people who are not infected come into contact with people who are infected. this decision is certainly courageous and important, but it is not enough. at present, our national health system's capacity to effectively respond to the needs of those who are already infected and require admission to an intensive care unit for ards, largely due to sars-cov- pneumonia, is a matter of grave concern. specifically, the percentage of patients admitted to intensive care units reported daily in italy, from march , up until march , was consistently between % and % of patients who were actively infected. in italy, we have approximately beds in intensive care units. of those, as of march , are already devoted to patients with sars-cov- infection, and in the near future this number will progressively increase to the point that thousands of beds will soon be occupied by patients with covid- . given that the mortality of patients who are critically ill with sars-cov- pneumonia is high and that the survival time of non-survivors is - weeks, the number of people infected in italy will probably impose a major strain on critical care facilities in our hospitals, some of which do not have adequate resources or staff to deal with this emergency. in the lombardy region, despite extraordinary efforts to restrict the movement of people at the expense of the italian economy, we are dealing with an even greater fear-that the number of patients who present to the emergency room will become much greater than the system can cope with. the number of intensive care beds necessary to give the maximum number of patients the chance to be treated will reach several thousand, but the exact number is still a matter of discussion among experts. health-care professionals have been working day and night since feb , and in doing so around % (n= ) of them have become infected, and some have died. lombardy is responding to the lack of beds for patients with covid- by sending patients who need intensive care but are not infected with covid- to hospitals outside of the region to contain the virus. we present the following predictions to prepare our political leaders-those who bear the greatest responsibility for national health systems and the government at the regional level, as well as local health authorities-for what is predicted to happen in the days and weeks to come. they can then implement measures regarding staff resources and hospital beds to meet the challenges of this difficult time. official numbers of infected people during the covid- virus outbreak in italy are indicative of the spread of the infection, and of the challenges that will be posed to italian hospitals and, in particular, intensive care facilities. the number of patients who are infected has been published daily since feb , . it is possible to fit the available data for the number of patients who are actively infected into an exponential model, as reported in figure a . the value of the exponent can be computed as r= · ( per day) and is consistent with the number of infected patients reported by the italian health ministry. the consistency between the exponential prediction and the reported data is very close up until day . if the increase in the number of infected patients follows this trend for the next week, there will be more than patients infected by march , as shown in figure b . on the basis of the exponential curve prediction, and the assumption that the duration of infection ranges from to days, it is possible to calculate that the basic reproduction number ranges from · to · . this number is similar to that reported for the initial phase of the infection outbreak in the city of wuhan, china and slightly higher than · , as reported by li and colleagues in a more recent report. the number of patients admitted to intensive care units increased similarly in italy, with an exponential trend up until march . the best fit of the data reported by the italian ministry for health can be obtained using the same exponent that best fits the number of patients figure a . the data available up until march show that the trend in the number of patients who will need admission to intensive care units will increase substan tially and relentlessly in the next few days. we can predict with quite a good degree of accuracy that this number will push the national health system to full capacity in a matter of days. considering that the number of available beds in intensive care units in italy is close to , and assuming that half of these beds can be used for patients with covid- , the system will be at maximum capacity, according to this prediction, by march , . this situation is difficult, given that the number of patients who will need to be admitted to the intensive care unit is predicted to further increase after that date, as shown in figure b . at this point, the most important question is whether the increase in the number of patients who are infected and those requiring intensive care admittance will continue to rise exponentially and for how long. if the change in the slope of the curve does not take place soon, the clinical and social problems will take on unmanageable dimensions, which are expected to have catastrophic results. the only way we can make such predictions is by comparing the trends in the data collected in the hubei region in china for covid- infection with that for the italian population. from the official report of the who-china joint mission on coronavirus disease , it is possible to derive the cumulative curve of the patients who are infected from the start of the data series. these data, as reported in figure , show that the initial phase of the infection outbreak followed the expected exponential trend, with the same exponent previously calculated for the number of italian patients who were infected. starting jan , the cumulative number of patients who were infected started to diverge from the exponential trend days later. if the italian outbreak follows a similar trend to that in china, we can suggest that the number of newly infected patients might start to decrease within - days from march . similarly, we can foresee that the cumulative curve of patients who are infected will peak days later, with the maximum load for clinical facilities for the treatment of these patients foreseen for that period. the most difficult prediction is the maximum number of infected patients that will be reached in italy and, most importantly, the maximum number of patients who will require intensive care unit admission. this prediction is of crucial importance to plan for new facilities in italian hospitals and to calculate the time period in which they need to be available. on the basis that the region of hubei in china has a slightly smaller population than italy (approximately million in hubei and million in italy), we tentatively assumed that the trend for the maximum number of patients who are actively infected would be similar in the two territories. in doing so, we cannot overlook the fact that the effect of travel restric tions on the spread of the covid- outbreak and the extraordinary community measures taken within and outside of wuhan are unlikely to be replicated else where. moreover, the current approach to these patients in lombardy implies non-pharmacological and pharmacological interventions, including antiretroviral medication, which might be different from the wuhan outbreak, and could distort the calculation. we also realise that there is heterogeneity in the transmission dynamics between the city of wuhan and elsewhere in the province, where the number of people who are infected remains lower. therefore, it might not be unrealistic to assume that what is going to happen in italy soon might mirror what happened in hubei. of course, it would have been more appropriate to directly compare greater wuhan ( million people) with the region of lombardy ( million people), the most seriously affected region in italy at the moment, but such data are not available. we do not currently have additional evidence we can take into consideration to make more robust assumptions regarding the exact number of patients who will be infected in the future days or weeks. on the basis of the available data, the number of infected patients reached approximately at the end of february, , in the hubei region, when the number of new cases decreased to almost zero. given that so far the percentage of patients requiring ards treatment is close to % for patients who are actively infected, at least in lombardy, we can assume that we will need approximately beds in intensive care units during the worst period of infection, which is expected to occur in about weeks from march . this is challenging for italy, as there are now just over intensive care beds in total. the aim now is to increase this number to safely meet urgent future needs. according to our prediction, we have only a few weeks to achieve this goal in terms of procuring personnel, technical equipment, and materials. these considerations might also apply to other european countries that could have similar numbers of patients infected and similar needs regarding intensive care admissions. since , italy has had the privilege of having a national health system (servizio sanitario nazionale), which was reshaped from - . its principles and organisation derive from the british national health service model, and it is based on three fundamental principles. the first principle is universality-all citizens have an equal right to access services provided by the national health system. the second is solidarity-every citizen contributes to financing the national health service based on their means, through progressive taxation. the third is uniformity-the quality of the services provided by the national health service to all citizens in all regions must be uniform. all individuals are supposed to pay for it as taxpayers, each person giving a little to receive a lot in return, if they become unwell. in theory, we are in a better position than many other countries to react to the current outbreak. however, an aggressive approach needs to be taken with patients who are critically ill with sars-cov- , often including ventilatory support. the system's capacity to respond to changing circumstances has been under enormous pressure, at least in the lombardy region, where two clusters have already emerged since feb . we predict that if the exponential trend continues for the next few days, more than hospital beds for patients in intensive care units will be needed in only week to treat ards caused by sars-cov- -pneumonia in italy. in the meantime, the government is preparing to pass legislation that will enable the health service to hire more doctors and nurses and to provide more ventilators to italian hospitals. these measures are a step in the right direction, but our model tells us that they need to be implemented urgently, in a matter of days. otherwise, a substantial number of unnecessary deaths will become inevitable. intensive care specialists are already considering denying life-saving care to the sickest and giving priority to those patients most likely to survive when deciding who to provide ventilation to. this attitude has already been criticised by the current president of the italian comitato di bioetica who, in a recent declaration to lay press stated that the constitution recognises the right of every individual to receive all necessary health care. they might not recognise that the reality is that intensive care wards are overflowing with patients and that covid- is not a benign disease. our doctors and nurses are modern heroes in an unexpected war against a difficult enemy. in the near future, they will have no choice. they will have to follow the same rules that health-care workers are left with in conflict and disaster zones. we hope that the present analysis will help political leaders and health authorities to move as quickly as they can to ensure that there are enough resources, including personnel, hospital beds, and intensive care facilities, for what is going to happen in the next few days and weeks. finally, our analysis tends to suggest that measures to reduce transmission should certainly be implemented, as our government did on march , by inhibiting people's movement and social activities, unless strictly required. rather than revising the schengen visa-free zone, the most effective way to contain this viral outbreak in european countries is probably to avoid close contact at the individual level and social meetings in each country. ar was responsible for data analysis and statistics, and writing of the manuscript. gr was responsible for data analysis and writing of the manuscript. we declare no competing interests. time to use the p-word? coronavirus enter dangerous new phase tourism flows and death rates suggest covid- is being under-reported real-time nowcast and forecast on the extent of the wuhan cov outbreak, domestic and international spread early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia joint mission coronavirus covid- global cases we thank kerstin mierke for writing assistance. key: cord- -cd qnp m authors: modenese, alberto; gobba, fabriziomaria title: increased risk of covid- -related deaths among general practitioners in italy date: - - journal: healthcare (basel) doi: . /healthcare sha: doc_id: cord_uid: cd qnp m to date, data on covid- -related death cases among physicians from different medical specialties are incomplete and scattered. in italy, available data highlight that general practitioners (gps) are, apparently, the most heavily affected group. indeed, they currently represent . % of the total covid- related death cases occurred among physicians, whereas they constitute about % of the total number of doctors. this high proportion is most likely the consequence of a work-related contagion happening especially during the first weeks of the epidemic, and persisting also in the following weeks, after the national lockdown. there are various reasons for these higher contagion rates: gps perform a lot of medical examinations daily, usually in close contact with patients. especially at the beginning of the epidemic, gps might have had scant information on the specific safety procedures for the prevention of sars-cov- transmission (e.g., there was limited knowledge on the possibility of contagions deriving from asymptomatic patients) and, moreover, the availability of personal protective equipment was insufficient. furthermore, the risk of infection is highly increased by the virus’ characteristics, like its survival for several hours/days on different surfaces and its persistence in the air after an aerosolization process, with possibilities to be transmitted over distances longer than two meters. following these observations, and considering the high cost in term of gps’ lives, the covid- pandemic will probably revolutionize the approach to patients in general practice. clear and effective guidelines are absolutely and urgently needed for the refinement of adequate measures to prevent sars-cov- infections among gps. healthcare workers (hcw) have a high occupational risk related to sars-cov- infection [ ] . italian data show that, currently, . % of all the diagnosed covid- cases ( , out of , diagnoses) occurred in hcw [ ] . this high risk is not unexpected, and was also observed during the sars and mers outbreaks, respectively in and . specific procedures for the protection of hcw have been proposed by authoritative organizations, such as the world health organization (who) [ ] , the european centre for disease prevention and control [ ] and the centre for disease control and prevention (cdc) [ ] . nevertheless, available data call into question the effectiveness of current preventive procedures and support the need for further development that, in the era of precision medicine, should be more tailored to the specific activities performed. in this context, an interesting aspect likely to provide useful insight is knowledge on variations in disease occurrence among physicians practicing specific medical specialties. significant differences are likely, for example, in the type and modality of contact with patients, in the environment where contacts occur, as well as in the medical procedures applied. a group with various specific peculiarities is certainly that of family physicians/general practitioners (gps), representing one of the front lines of the war against covid- . gps have been visiting an overwhelming number of patients, often directly at their homes, with scarce possibilities, if any, to control the work environment. especially during the first phases of the outbreak, they were unaware of the presence of sars-cov- -infected patients, with an incomplete knowledge of the risk, of the adequate preventive procedures to be applied and, possibly, also with an insufficient/inadequate availability of personal protective equipment (ppe) [ ] . data on specific covid- contagion among gps is currently scant, but, in italy, one of the countries with the highest number of covid- cases and mortality, at least some mortality data are available. in fact, the official webpage of the italian federation of the colleges of physicians (fnomceo) is publishing and updating daily a list of the italian physicians that have died as a consequence of sars-cov- infection, also reporting their medical specialty [ ] . as of the april , physicians had died. all physicians who died due to covid- are reported in this list, including both active and retired physicians. for this reason, in order to have a more reliable overview of the situation, we have excluded all cases of deaths which occurred in physicians over years old (private practice is common for some years after formal retirement, which usually happens between and years). after this exclusion, the number of physician deaths due to covid- which occurred in italy by the april is reduced to . during the following weeks of may (up to may ) another eight deaths were reported, of which one was a gp and seven were physicians with other medical specialties. by april , there were gps-by far the most represented medical specialty-contributing . % of the total number of covid- -related deaths among italian physicians ( figure ). gps are followed by dentists and surgeons, representing, respectively, . % and the . % of all deaths, and then by anesthetists and cardiologists, both contributing four cases, corresponding to . % ( figure ). the mean age of the physicians is similar in gps and other medical specialties ( . vs . y/o) and is lower compared to the mean age of those who have died in the general population ( . years [ ] ), supporting the specificity of the contagion in these groups. the first italian case of covid- in italy was diagnosed in the lombardy region on february, schools were closed on march, and since the second week of march the majority of work activities have been locked down, until may. the first cases of death among italian physicians were reported on the fnomceo website on march in two gps practicing in the lombardy region. nevertheless, it should not be excluded that some cases may have occurred even before that date. at the end of april, we were at the eighth week of lockdown in italy: figure shows the daily cumulative increase in death cases among physicians since the first case reported, reporting the trend of the death cases in these eight weeks of national lockdown. it should be noted that, up to the nd of april, gp deaths were more common than deaths reported in other medical specialties, reaching % at the beginning of april; after that, the rate remained at %. in the second half of april, the proportion reached the current estimate, . % (figure ). however, even if decreasing, this proportion is still relevantly higher, approximately three times greater than the overall ratio of gps to physicians practicing other medical specialties in italy, which can be estimated at around % according to the italian national institute of statistics (istat) [ ] , suggesting the persistence of a higher risk. this difference between the proportion of gps to other physicians (using currently available occupational data) and the deaths related to covid- indicates a relevantly high occupational risk for gps. nevertheless, further well-designed studies will be needed to collect more precise data reporting covid- prevalence, incidence and mortality rates among different medical specialties and hospital departments. such studies will allow the identification of specific occupational hazards and possible increased risk of infection and mortality among various categories of hcws. considering the currently available italian data, the trend in deaths suggests that the work-related contagion of gps was more critical during the first weeks of the epidemic in italy, with a slight decrease in more recent days, even if the proportion is still very high for gps. the relatively high number of death cases among gps deserves some examination. considering the sars-cov- incubation period and the period elapsed between symptom onset and death, it can be said that many of gps became infected in february or the first week of march, when in italy there was still a scant awareness of the risk related to covid- and, in general, risk perception was largely lower compared to the weeks after the national lockdown. moreover, february and early march in italy is a period of the year when gps perform a lot of medical examinations of patients with influenza-like symptoms. recent data have confirmed that sars-cov- salivary viral load is particularly high during the first week after symptom onset [ ] , and viral rna has been documented in throat swabs for more than days, with high titers in the saliva [ ] . furthermore, especially at the beginning of the epidemic, the exponential increase in cases over a few days did not give gps enough time to adopt adequate safety procedures to visit the patients and to be appropriately informed on the most effective ways to prevent covid- transmission. it should also be noted that the availability of personal protective equipment (ppe) was insufficient to allow the high number of visits requested, as recently reported in a survey from one of the italian regions with the highest incidence of sars-cov- infections, lombardy [ ] . another relevant point is that several studies documented sars-cov- infections in asymptomatic patients, i.e., persons not manifesting any symptoms inducing the suspicion of infection and, consequently, in no need of specific preventive measures [ ] . these persons may have contributed to the transmission of infection to gps, especially during the first weeks of the epidemic in italy. in addition, it has to be considered that a medical examination usually implies a short distance between the patient and the doctor, but, even with reduced direct contact and extensive use of protections, there are additional problems, such as (a) the documented persistence of the pathogen on the surfaces of the clinic for up to several hours or a few days, depending on the type of material [ ] and (b) recent evidence suggesting the possibility of viral transmission at distances greater than two meters, and its detection in the air up to three hours after aerosolization [ ] . in conclusion, according to the abovementioned observations, and especially considering the serious loss of life among italian gps, the covid- pandemic will probably revolutionize the approach to the patient in general practice [ , ] . the refining of adequate strategies and procedures to prevent covid- infection among italian gps, as well as in the rest of the world, is crucial, and clear and effective guidelines are absolutely and urgently needed. the way the general practice system is organized in italy may have played a role in the increased proportion of deaths among gps. even before of the covid- outbreak, several critical points were observed: most of gps are used to working single-handedly and, furthermore, there are difficulties in recruiting new workforces, making italian family doctors quite an aged working category, with the % older than y [ ] . on average, the departments of general practice in italy are open for less than h per week, and doctors ensure continuity of care for out-of-hours emergencies at patients' homes [ ] . after the covid- outbreak, many italian scientific societies of general practice, such as the italian federation of family doctors (federazione italiana medici di famiglia-fimmg) and the italian society of general medicine (società italiana di medicina generale, simg), produced practical indications and operational guides for gps in order to safely ensure care to their patients during the covid- epidemic, adopting appropriate preventive measures to preserve their health [ , ] . other important points to be stressed are the progressive activation, since the second half of march, in many italian regions, of special medical units for the assistance of covid- patients at home, called "usca" (unità speciali di continuità assistenziale), as prescribed by a national decree [ ] , and the promotion of telemedicine procedures, when possible [ ] . these new practices may have reduced the direct contact of gps with patients actively infected with sars-cov- . it is not yet clear whether the slight decrease in the proportion of gps among those physicians who died during the latest weeks of the epidemic in italy may be attributed to these improvements in targeted prevention, or is only a reflection of the attenuation of the epidemic in italy as a consequence of the lockdown. nevertheless, the high proportion of deaths among gps compared to other medical specialties, which persists, clearly indicates the need for further development of effective and, possibly, more tailored preventive measures. funding: this research received no external funding. the authors declare no conflict of interest. covid- : protecting worker health integrated surveillance of covid- in italy (ordinanza n. del / / ) may update infection prevention and control and preparedness for covid- in healthcare settings-second update information for healthcare professionals about coronavirus (covid- ) covid- : don't forget the impact on us family physicians case-fatality rate and characteristics of patients dying in relation to covid- in italy elaborazione di dati sul personale in attività nel sistema sanitario pubblico e privato (data analysis on active health personnel in the public and private health services) temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study persistent viral rna positivity during recovery period of a patient with sars-cov- infection clinician education and adoption of preventive measures for covid- : a survey of a convenience sample of general practitioners in lombardy asymptomatic transmission, the achilles' heel of current strategies to control covid- persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents airborne or droplet precautions for health workers treating covid- ? covid- : how coronavirus will change the face of general practice forever english and italian national health services: time for more patient-centered primary care? - (models for the management of suspected and confirmed covid- cases in the general practice) ed esperienze pratico-cliniche per la gestione del covid- (summary of good practices based on current knowledge and clinical experiences for the management of covid- : a practical guide for general practitioners) disposizioni urgenti per il potenziamento del servizio sanitario nazionale in relazione all'emergenza covid- (urgent measures for the strengthening of the national health covid- report n. / -interim indications for telemedicine assistance services during the covid- health emergency this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -jrhqh authors: borghese, a.; moioli, b. title: buffalo husbandry | mediterranean region date: - - journal: encyclopedia of dairy sciences doi: . /b - - - / - sha: doc_id: cord_uid: jrhqh nan the hotter part of the day and feeding roughage during the night. river buffaloes are susceptible to most diseases affecting cattle (table ) . compared with cattle, buffaloes show greater resistance to foot-and-mouth disease and brucellosis but have a higher incidence of parasitic diseases because of their wallowing habits. the dairy buffalo is as susceptible to mastitis as the dairy cow. bacteria causing mastitis, their treatment and control are similar to those for cattle (see mastitis pathogens: contagious pathogens; environmental pathogens). there is a high incidence of calf mortality caused by toxocara vitulorum, virulent strains of escherichia coli, rota and corona viruses. larvae of to. vitulorum are transmitted from the dam to the calf through the milk during the ®rst month of life. puerperal metritis and retained foetal membranes occur in the buffalo. the high incidence of metritis and other genital infections has been partly attributed to the unhygienic practice of dilating the vagina with either inserting objects or blowing air for stimulating milk letdown. high milk-producing river buffaloes are as susceptible to metabolic disorders as dairy cows. apparently, the aetiology is similar because affected buffaloes respond to therapy and control as for dairy cows. the buffaloes reared in the mediterranean region are the asian buffalo or water buffalo, i.e. bubalus bubalis. this species includes two types: ( ) the river type, with chromosomes, with an adult male weight ranging between and kg, and annual milk production of ± kg; and ( ) the swamp type, with chromosomes, with an adult male weight of ± kg, and annual milk production up to kg. while the major purpose of the river buffalo is milk, the swamp buffalo is reared mainly for draught. only % of the world buffalo population is reared in the mediterranean region, which includes a few countries of europe and the near east; these buffaloes are all of the river type. the number of buffaloes in the mediterranean region declined during the twentieth century for three reasons: holsteinization of dairy cows, mechanization and the poor market demand of buffalo products. signi®cant numbers of buffaloes (over ) are at present found only in italy, romania, egypt, turkey, azerbaijan, iraq and iran. in bulgaria the number of buffaloes has decreased to fewer than . in all these countries, buffaloes represent only a very small portion of total livestock, except in egypt, where buffaloes are more numerous than cattle. in the last years, because of the strong market demand for buffalo cheese and as a consequence of the`milk quotas' restriction imposed by the european union, the number of buffaloes has increased in italy. in egypt, iran and azerbaijan also, there is a preference for buffalo dairy products compared to cows' milk products. during the s, buffaloes have been imported to germany, the united kingdom and the netherlands, where a few milkproducing herds can be found. buffaloes of the mediterranean region differ phenotypically from country to country because of the different environment and management practices and because no exchange of genetic material between countries has occurred, except in bulgaria, where crossbreeding with the murrah breed was carried out by importing in a signi®cant number of animals from india. major morphological differences between the buffalo populations of different countries include: ( ) the variable size, ranging between a minimum of and kg liveweight for adult females and males, respectively, in egypt to a maximum of and kg in iraq, the most frequent weights being and kg; ( ) the shape of the horns; and ( ) the coat colour, from dark-grey and dark-brown to black, showing white spots in some cases. the main objective of breeding buffaloes in europe and the near east is milk production. everywhere in this region, their milk is sold at a higher price than cows' milk. average herd size (number of females of breeding age) is below eight in the whole region, except italy ( ), syria ( ) and iran ( ). the proportion of breeding females to total buffaloes is % in all countries, with a minimum of % in azerbaijan, where a dual-purpose line of buffaloes is maintained, and a maximum of % in italy, where males have no market. in countries where the majority of buffaloes are reared in very small herds there are also a few bigger private, cooperative or state herds (bulgaria, egypt and turkey) (figure ) . the number of calves produced per cow per year varies from . in egypt to . in azerbaijan and syria, with average of . . the age at ®rst calving averages months. in italy a good proportion of buffaloes calve at months of age, whereas in egypt and syria a high number are months or more at ®rst calving. in mediterranean countries, all herds have their own bull except in the areas with very small herds ( ± breedable buffaloes) in romania, bulgaria, egypt and turkey, where there are groups of bulls for breeding at village level. of®cial milk recording for the productivity of buffaloes is performed in egypt, italy, bulgaria, romania, the united kingdom, azerbaijan and iran. genetic evaluation of buffaloes is done in italy, bulgaria, romania, egypt, iran and azerbaijan. choice of breeding bulls/heifers is made on the basis of: . lactation yield (bulgaria, greece, azerbaijan). . lactation certi®cate (italy, egypt, iran). where milk recording is not practised, a better bull is judged on his appearance, size and strength. natural breeding stations exist in egypt and bulgaria. bulls in the breeding stations are provided by development programmes of the government or other agencies. due to dif®culties in oestrus detection and lack of organization, arti®cial insemination (ai) is still practised only to a very limited extent for buffaloes in the mediterranean region: in italy for . % of the buffaloes, in egypt and iran for . % and in romania for . %. in the large cooperative and state farms in bulgaria, ai is used on % of the buffaloes. in the other countries it is not used at all. the low use of ai has slowed down the implementation of national selection schemes for genetic improvement of milk productivity. research trials conducted in italy have indicated that arti®cial insemination is feasible and successful in buffaloes using the following technique to synchronize oestrus and boost fertility. a progesterone-releasing intravaginal device (prid) of silicon coils is inserted and left for days; on the th day injections of iu of ecg (equine chorionic gonadotrophin) and mg of luprostiol (prostaglandin f a ) are given; arti®cial insemination is performed at , and h after removal of the prid on day . the most common housing system is that referred to as`traditional', consisting of keeping buffaloes indoors at night and con®ned in fenced areas during the day (egypt, turkey, iraq, syria); in the favourable season they are allowed to graze during the day (romania, turkey and on some farms in italy). in the marshes in the southwest of iran, buffaloes are kept outdoors on pasture throughout the year, whereas in the northern areas, around the caspian sea, they are kept in barns in winter. lactating buffaloes are kept tied throughout the year in bulgaria, romania and azerbaijan. in italy, they are housed loose in paddocks all year, with the same modern systems used for dairy cows; one-third of italian buffaloes are also put on pasture in spring. one-third of iraqi buffaloes wallow in marshes all year, the water reaching a level halfway up their bodies. they swim far and wide to ®nd food and when the water is high, they stand on platforms made of papyrus, reeds and mud; on these platforms the farmers sometimes build huts to house the buffaloes; these platforms can be pushed to different places in the marshes. the length of lactation varies from to days, the most frequent being . average lactation milk yield is kg. italian buffaloes give the highest average yield (almost kg) (figure ) , while in turkey, average yield is below kg. in the iraqi marshes buffaloes produce no more than kg in days. the fat content of the milk throughout the lactation is over % in italy, turkey, azerbaijan and iraq, around % in bulgaria, romania and egypt, and less than % in iran. the average daily milk yield of river buffaloes shows very wide variability, depending on the breed, the country and especially the management and feeding system. it can range from to kg day À for poorly fed animals (grazing and fed byproducts) to kg day À in intensive management systems. in the large commercial herds in italy and bulgaria, buffaloes are machine-milked, twice a day. in the smaller herds elsewhere in the mediterranean, buffaloes are usually hand-milked, and often the calf is allowed to stand with its dam to assist letdown which otherwise may be slow. extensive management systems, as practised in europe and the near east, include grazing in the favourable seasons. in all cases, green forage`cutand-carry' ± composed of legumes varying from country to country ± concentrates and byproducts are the basic foodstuff. green forage and hay are made mainly of lucerne (alfalfa) in italy, bulgaria, romania and turkey and trifoliulm alexandrinum in egypt. the most common byproducts fed to buffaloes are brewers' grains in italy and bulgaria, sugar-beet pulp in italy, bulgaria and iran, cotton waste in egypt and azerbaijan, tomato peel in italy, apple-juice wastes in iran, sugar-cane wastes in egypt and iran, maize stalk and cobs in iran, egypt and romania and straw everywhere. in the iraqi marshes, when the buffaloes return at night to the¯oating islands where they live, they are fed green forage cut by the farmer during the day; this forage is composed of reeds, papyrus, various water plants, and rice hulls when available. in italy, dairy buffaloes are managed in the same intensive way as dairy cows, maintained in loose housing paddocks throughout the year. maize silage and grass silage are the main feeds. average yearly milk production for buffaloes in italy is kg although % of recorded buffaloes yield more than kg. an example of figure italian buffalo, in her ®fth lactation; she produced feeding schedules for high-yielding buffaloes is given in table . similar high-energy diets ( . ± . milk feed units (mfu) kg À dry matter) are used in italy for particular selected genotypes where highest milk yield is desired because of the high prices for buffalo milk and because such diets increase milk protein ( . ± . %) and milk fat ( ± %) resulting in higher cheese yields. buffalo pathologies are similar to those described for cattle. few diseases are peculiar to buffaloes. they tend to show more resistance to diseases than cattle, perhaps because of their adaptation to hot±humid climates. parasitic infections, particularly in developing countries, are very common in buffaloes. they include: gastrointestinal helminths (strongyloides, toxocara, moniezia, mammomonogamus) and coccidia (eimeria, giardia, cryptosporidium), liver parasites (fasciola), tick parasites (hyalomma, sarcoptes) blood parasites (theileria), all of which produce important economic losses in buffalo breeding. escherichia coli can cause gastroenteric pathologies in buffaloes, particularly in calves, associated with other bacteria (enterobacter, pseudomonas, klebsiella) or with coccidia or verminosis or virosis. respiratory diseases are caused by pasteurella, staphylococcus, streptococcus, escherichia coli, and can result in high mortality, if the animals are not treated with antibiotics. pasteurella multicida is responsible for haemorragic septicaemia, the most serious disease in buffaloes because of the high mortality particularly in tropical asian countries; however, it can be controlled with antibiotics and vaccines. tuberculosis, produced by mycobacterium, is a serious zoonosis, which could be eradicated after tuberculin diagnosis. brucellosis, caused by brucella, is another zoonosis that needs to be eradicated after serological diagnosis. it can cause serious disease in the human population and reproductive disorders and infertility in buffaloes. vaccination can be applied only in developing countries. leptospirosis is another zoonosis, produced by infection with leptospira from water sources contaminated by rodents. listeriosis, caused by listeria, produces meningoencephalitis, abortion and septicaemia: the source of the infection may be silage in the buffaloes' diet. chlamydia, rickettsia and johne's disease (caused by mycobacterium tuberculosis) are present even in developed countries also, and the evidence of mastitis incidence links them to dairy buffaloes. prophylaxis is very important to control some viral infections that could cause neonatal diarrhoea (rotavirus, coronavirus), particularly in intensive systems. bovine rhinotracheitis (ibr), bovine diarrhoea (bvd) and bovine herpesvirus (bhv) have all been diffused by animals in intensive buffalo farms. buffaloes are also susceptible to foot-and-mouth disease. buffaloes are affected by fungal infections, tumours and reproductive disorders including prolapse of the uterus. compared to that of cattle, buffalo milk is richer in fat ( ± . %) and protein ( ± %); it has a lower cholesterol content and higher tocopherol content. it is richer in calcium and phosphorus and has less sodium and potassium. peroxidase activity is ± times higher than in cows' milk so that it can be preserved longer; b-carotene is more completely transformed to retinol and therefore the milk looks whiter than cows' milk. the milk can be consumed in liquid form or processed into a wide range of products, either alone or mixed with milk from other livestock: fermented milks, butter, ghee, condensed and powdered milk, and cheeses are produced. many farms produce their own cheese and cream which they sell directly. classifying the types of the more common classi®cation of cheese is based on the type of coagulation: either enzymatic coagulation (by rennet), or acid coagulation (after natural acidi-®cation or by the action of lactic bacteria). most of the cheeses produced in the mediterranean area, including mozzarella, belong to the acid-enzymatic category, meaning that acid coagulation prevails. the demand for high-quality mozzarella in italy and the world has stimulated an increase in buffalo farming in italy and improved animal management techniques. mozzarella production is higher than all other cheeses and is forecast to increase still further. yoghurt is produced in bulgaria, romania and albania from either buffaloes', cows' or sheep's milk. in egypt, rayat is produced from natural acidi®cation of raw milk, after the removal of cream, with no addition of bacteria, and zabadi is produced industrially. raha is the fermented milk of iraq, produced either from whole or skim milk, and laban or khather are the fermented milks of syria, all of which are often produced from buffaloes' milk. creams also show variable techniques of production. in egypt, queshta mosakhana is the¯oating cream removed after boiling milk. gaymar in iraq is obtained both from spontaneaous¯oating or from spinning, in which case it is then pasteurized. in italy, cream from buffalo milk is obtained after spinning; it is then pasteurized. after thermoacid coagulation (citric acid) of cream, another dairy product is obtained, called mascarpone. quishada (made in syria) is obtained from raw or boiled milk; sometimes, this cream is heated to make it more concentrated. industrial butter is produced by churning of cream, often after pasteurization. the home-made product is obtained simply by churning acidi®ed milk. a peculiarity of buffalo butter is the colour, which is much whiter than cows' milk butter, due to the lack of carotenoids. ghee is obtained by boiling butter. it was very popular in egypt and was found very useful in the baking industry. in azerbaijan ghee is the only product obtained from buffalo milk. it is evident that the dairy products made from buffalo milk in the mediterranean area need to be more deeply studied; the variability of their technologies is an important part of global biodiversity. buffalo husbandry: asia. cheese: pasta-filata cheeses. concentrate feeds: byproduct feeds. dairy animals: water buffalo. dairy farm design and layout: building and yard design, warm climates. diseases of dairy animals fermented milks: middle east. milk: introduction. milk fat products: anhydrous milk fat ± butteroil, ghee; milk-fat based spreads. milking machines: principles and design. parasites, external: tick infestations. parasites, internal: gastrointestinal nematodes. further reading agricultural academy of bulgaria the water buffalo conception rate in synchronized and arti®cially inseminated buffalo cows in two different seasons under ®eld conditions proceedings of the th world buffalo congress proceedings of the international symposium on buffalo products proceedings of the international symposium on prospects for buffalo production in the mediterranean and middle east the husbandry and health of the domestic buffalo proceedings of the th world buffalo congress proceedings of the rd world buffalo congress key: cord- - pvk fqn authors: indini, alice; aschele, carlo; bruno, daniele; cavanna, luigi; clerico, mario; fiorentini, giammaria; fioretto, luisa; giordano, monica; montesarchio, vincenzo; ortega, cinzia; pinotti, graziella; scanni, alberto; zamagni, claudio; blasi, livio; grossi, francesco title: reorganization of medical oncology departments during covid- pandemic: a nationwide italian survey date: - - journal: eur j cancer doi: . /j.ejca. . . sha: doc_id: cord_uid: pvk fqn abstract the novel severe acute respiratory syndrome coronavirus- (sarscov- ) pandemic is a global health problem, which started to affect china by the end of year . in europe, italy has faced this novel disease entity (named covid- ) first and severely. covid- represents a significant hurdle for public health services and a potential harm for patients with cancer. the collegio italiano dei primari oncologi medici (cipomo) is an italian association of head physicians in oncology departments, which promotes working and research activities in oncology on a national basis. in the midst of the epidemic in italy, the cipomo promoted a national survey aiming to evaluate the impact of covid- on clinical activity of oncologists and the implementation of containment measures of covid- diffusion. overall, head physicians participated in this survey, with a homogeneous distribution on the national territory. results show that the following measures for oncologic patients have been promptly implemented through the whole country: use of protective devices, triage of patients accessing the hospital, delay of non-urgent visits, and use of telemedicine. results of this survey suggest that italian oncology departments have promptly set a proactive approach to the actual emergency. oncologists need to preserve the continuum of care of patients, as the benefit of ensuring a well-delivered anti-cancer treatment plan outweighs the risk of covid- infection. international cooperation is an important starting point, as heavily affected nations can serve as an example to find out ways to safely preserve health activity during pandemic. on march th , the world health organization (who) declared the novel severe acute respiratory syndrome coronavirus- (sars-cov- ) outbreak a pandemic [ ] . by the end of february , italy was experiencing the rapid spread of the virus, which started to affect the north of the country with a daily increase in the number of cases and consequent deaths [ ] . in italy, data regarding the diffusion of the novel coronavirus disease (covid- ), caused by sars-cov- confirmed its higher lethality compared to that observed in china and worldwide ( % vs . %) [ ] . following the chinese model, containment measures to reduce the risk of covid- in italy have been promptly activated and implemented. the first national decree, issued on march th , instituted a containment zone concerning the most affected areas of the country (the so-called red zone, which at that time included regions in the north of italy: lombardia, emilia romagna, and veneto). in the following days, a series of decrees have extended increasingly strict measures to the whole national territory. the main provisions included: forbidding all gatherings of people; restricting movements of people within and outside the hometown, except for circumstances of necessity; encouraging employees to work from home. in this circumstance, health workers cannot take any leave, and are asked to suspend all non-urgent activities. all planned surgeries are postponed, to give over intensive care beds to the treatment of patients with covid- , and hospitals had to create new intensive care places by converting operating and anesthetic rooms. this pandemic represents a significant harm for patients with cancer, who are at high risk of infections due to several predisposing factors [ ] . moreover, most treatment procedures in oncology cannot be delayed without compromising the efficacy of treatment itself. in italy, specific indications on oncologic patients' management were given on march th , mainly regarding caution measures to reduce the risk of infection (i.e. use of personal protective equipment, practice social distancing). while encouraging physicians to postpone follow up visits, indications were given to guarantee oncologic services even within the red zone, in order to maintain the continuum of care. at the present time, data regarding diffusion and management of covid- in oncologic patients are scarce [ , ] . such emergency has led italian oncologists to join forces, with the aim to find a way not to compromise the continuum of care of patients and to preserve safe everyday clinical here we present the results of this survey, providing an overview of covid- epidemic in italy and assessing potential interventions to overcome this critical situation. the survey includes a total of questions, which are divided in sections: the first section assesses the routine use of preventive measures (e.g. vaccinations) in oncologic patients; the second contains questions regarding covid- diffusion containment measures adopted before the enactment of national decrees in this regard; the third and last section assesses the diffusion of covid- in oncology units and its impact on working activity, after national decrees on containment measures were adopted. a complete original version of the survey is provided in supplementary material . the survey was launched online on march th , and closed on march th . figure results of section show that the overall tendency throughout italy is to perform preventive vaccination in oncologic patients ( table ). most vaccinated patients are either receiving active systemic treatment (chemotherapy, immune-therapy, and hormonal or targeted therapy), and/or present one or more predisposing risk factors (i.e. age ≥ years, cardiovascular and/or respiratory disease, chronic infections, diabetes, obesity, immune-suppressive therapies). the most widely adopted vaccination is for seasonal flu, however more than % of oncologists suggests also performing pneumococcal vaccination in those patients. seasonal flu cases are not usually reported by medical oncologists to the dedicated national registry, rather this is commonly a duty of family doctors. answers to the questions in section clearly show that, even if by the time of the survey covid- represented an emergency mainly in the north of italy, diagnostic measures for all patients accessing oncologic services were immediately activated in the whole country ( table ) . triage of patients included vital signs monitoring before entering the hospital (body temperature, spo , respiratory rate), but also questioning patients on the presence of symptoms during the days before the visit, and possible contacts with subjects affected by covid- or coming from high-risk areas. in more than % of cases, triage procedure was followed by preventive isolation and diagnostic work up of symptomatic patients, consisting in chest x-ray and rhino-pharyngeal swab to rule out the presence of sarscov- . after the very first reports of covid- in italy, measures to reduce hospital accesses for oncologic patients were taken almost throughout the country. such measures consisted mainly in delaying visits not considered to be urgent (i.e. patients in follow up after surgery and/or radiotherapy, and/or patients with breast cancer receiving adjuvant hormonal treatment after surgery), even more so if the patients presented risk factors (i.e. age > years, presence of comorbidities). alternative ways to get in touch with patients have been widely used: most patients underwent telephonic interviews with interpretation of laboratory and radiologic exams report, while in other cases family doctors were delegated to inspect the results of follow up exams. access to oncologic structures was likewise limited and/or denied for visitors and caregivers, either for outpatient visits, day hospital and ward admissions. with the enactment of the decree on march th and subsequent decree on march th , containment preventive measures became effective in the red zone before, and on the whole italian territory thereafter. however, by that time most oncologic units had already activated measures to contain accesses, under regional or internal (hospital medical direction) orders. as so, more than % of oncologic structures did not have to modify the measures they implemented to reduce the risk of infections, in view of the actual legislation. due to such measures, more than % of structures had reported a significant reduction in their routine activity, while % had only a negligible reduction and % had not substantially changed their activity. answers to the questions in section show that almost one third of oncologic structures had to employ their oncologists for guard duties in internal medicine ward and/or emergency department; in % of cases, guard duties in covid ward were included (table ). this percentage was understandably higher in the north of the country, reaching % and % of oncologists employed for internal medicine/emergency department and covid wards guard duties, respectively, in the red zone. twenty-four percent of italian oncology departments had at least one patient diagnosed with covid- , with a higher rate in the red zone ( %) and no diagnosis at all in the south of italy and in islands. examining reports from the red zone, it emerges that most patients accessed emergency room presenting with fever and/or respiratory symptoms ( %), while a significant proportion of patients was diagnosed after a triage procedure ( %) and/or a medical interview regarding possible contacts with subjects at risk ( %). results of this survey deserve several considerations. some effective points emerge from this survey, which partly have been also raised in the comment recently released by the european society for medical oncology (esmo) [ ] . first, oncologists face the need to preserve the continuum of care for most of their patients, as the benefit of ensuring a well-delivered anti-cancer treatment plan outweighs the risk of covid- infection. however, the risk from covid- exposure varies significantly from patient to patient, making treatment tailoring important now more than ever. second, physicians have to get used to a new working activity, which implies the use of tele-consultation services when feasible, and reducing the number of visits by means of customizing treatment delivery (three or two-weekly as opposed to weekly, oral or subcutaneous alternatives as opposed to intravenous administration). also, in view of a visit a telephonic "previous day" triage could help, in order to avoid the access of symptomatic patients to oncologic wards. third, protection of patients and physicians is paramount in order to keep providing the best service in a safe way. this last issue raises the question on how to optimize oncologic resources for covid- emergency. due to both clinical characteristics of oncologic patients, which are frail and at high risk for infections, and features of oncologic services, which often cannot be postponed, how can oncologist reasonably help in this emergency without compromising patients' continuum of care? to date, no clear indications have been provided to health care providers in oncology, making it difficult to create a common line of action. in conclusion, results of our survey suggest that italian oncology departments have promptly set a proactive approach to the actual emergency. together with indications provided by national decrees and internal dispositions, several individual initiatives have contributed to reorganize working activity in the actual condition. the medical community worldwide is facing a difficult situation, and oncologists in particular require several extra precautions to protect the patients first and their activity thereafter. international cooperation is an important starting point, as heavily affected nations can serve as an example to find out ways to safely preserve health activity during pandemic. the municipal health services in wuhan (china) report to the who a cluster of patients with pneumonia of unknown etiologic agent in the city of wuhan, in the chinese province of hubei. january th chinese authorities make a preliminary determination of a novel (or new) coronavirus (sarscov- ), as the causal agent of the severe acute respiratory syndrome, named covid- . chinese investigators conduct gene sequencing of the virus, using an isolate from one positive patient sample, making diagnostic tests promptly available worldwide. the italian ministry of health sets up a task force to coordinate interventions on the italian territory, together with international responsible institutions. a surveillance system for suspected cases is established. january th two chinese tourists hospitalized for respiratory tract infection, in rome, are the first confirmed cases of covid- detected in italy. regional health authorities implement measures to track contacts of the two subjects. all contacts resulted negative for covid- . italian government decides to interrupt all air connections with china. the who declares covid- diffusion in china a public health emergency. the italian council of ministers declares national public health emergency condition. february st the italian national institute of health confirms the first case of local transmission of covid- infection in a -year-old patient in codogno. february th the who raises the threat definition for covid- epidemic at a "high level" of threatening for the global health. march th - th a national decree institutes a containment zone concerning the most affected areas of the country, located in the north of italy (lombardia, emilia romagna, and veneto). the main provisions include: forbidding all gatherings of people; restricting movements of people within and outside the hometown, except for circumstances of necessity; encouraging employees to work from home. health workers cannot take any leave, and are asked to suspend all non-urgent activities; all planned surgeries are postponed, to give over intensive care beds to the treatment of patients with covid- . march th the italian ministry of health issues recommendations for oncologic and oncohematologic patients, providing protective measures for off-therapy patients and those receiving systemic treatment. oncologists are required to postpone follow up visits, in order to reduce patients' access to hospitals. patients with thoracic tumors and those who underwent pulmonary resection are to be considered a subgroup of high risk patients. march th the italian council of ministers urgently sets increasingly strict containment measures, to the whole national territory. the who declares the novel sarscov- outbreak a pandemic. march th three italian scientific associations (aiom, cipomo and comu) release an official document for the management of oncologic and onco-hematologic activities during covid- pandemic. the who declares europe is becoming the new epicenter of covid- pandemic. abbreviations: aiom, associazione italiana di oncologia medica; cipomo, collegio italiano dei primari oncologi medici ospedalieri; comu, collegio degli oncologi medici universitari; covid- , novel coronavirus disease; sarscov- , severe acute respiratory syndrome coronavirus- ; who, world health organization. the world health organization (who) website covid- in italy: momentous decisions and many uncertainties the world health organization (who) health emergency dashboard diagnosis, treatment, and prevention of influenza infection in oncology patients cancer patients in sars-cov- infection: a nationwide analysis in china sars-cov- transmission in patients with cancer at a tertiary care hospital in wuhan, china the authors they have no conflicts of interest to declare for the present paper. all authors have no conflict of interest to disclose. key: cord- - dvjaa authors: nan title: administrative boundaries and urban areas in italy: a perspective from scaling laws date: - - journal: landsc urban plan doi: . /j.landurbplan. . sha: doc_id: cord_uid: dvjaa delineating boundaries of urban areas is no easy task, due to the inherent complexity of the problem, heterogeneity of relevant data and little consensus on how to properly measure the results. any such delineation must eventually be cast onto administrative boundaries, an essential requirement for real-world applications. in the effort of relating administrative and alternative boundaries, we investigated in italy the validity of general scaling laws, such as the area-population relation, and proposed a practical application. relying on open data for population, settlements and road networks, we showed the extent to which scaling relations hold for different boundaries for urban areas, and how they compare to each other. we considered, beside italian municipalities, urban areas based on the idea of “natural cities”, obtained using head/tail breaks of areas related to human mobility as an explicit indicator of existence of a city. area-population data for administrative boundaries can be reconciled with scaling relations valid for both the world’s cities data and with those obtained from natural cities, provided an effective area is adopted in place of polygon planimetric area of municipalities. we eventually proposed an aggregation of administrative units using the empirical scaling relation as an objective function for accepting or rejecting pairwise fusion of boundaries. we suggest considering such a method, along with expert considerations, as an additional tool for real-world urban planning as seen from the very general perspective of seemingly abstract scaling laws. virtually all fields of science have investigated and identified scaling laws for many years. studies based on very general laws, such as the zipf's one regarding the rank-frequency distributions in physical and social phenomena following a power-law (zipf, ) , and gibrat's law, describing rates of growth of different phenomena such as private firms and cities (gibrat, ) , date back to the first half of the th century. similar relations where soon found to hold for a wide range of phenomena, and investigated in a substantially large body of literature. geo-spatial, human geography and urban sciences make no exception: examples of studies of city size distributions as power laws can be found as early as in (auerbach, ) . fang and yu ( ) recently compiled a review of more than , research papers on the topic of urban agglomeration studies. scaling relations regarding urban systems imply that there is no special size for a city, nor a special stage of development: scaling relations tend to be universal. a scaling relation for a given set of urban systems implies that all of those cities are a scaled version of one another, which makes their shape, size and expansion predictable (batty, ; bettencourt & west, ) . despite the large number of studies about universal properties of urban systems, and claims of potential applications for planning, though, practical applications seems to be hard to obtain, with notable exceptions (barthélemy, bordin, berestycki, & gribaudi, ; furtado, ) . most of the existing studies aim at showing the validity of scaling relations, through extensive data gathering from heterogeneous sources (bettencourt, lobo, helbing, kühnert, & west, ; jiang & liu, ; fang & yu, ; long, zhai, shen, & ye, ) and development of dedicated analytical and statistical methods (warton, wright, falster, & westoby, ; clauset, shalizi, & newman, ; taskinen & warton, ) . a notable effort has been devoted to the very delineation of urban boundaries, which is crucial to any further speculation about the properties of urban systems. boundaries determine not only the size but also the physical form of cities which, in turn, is deeply related to their inter-urban and intra-urban function. social implications of city forms stemming from plans and mobility within neighborhoods were discussed by park ( ) . a modern view of the relation between form and function was clearly stated by batty and longley ( ) , who recognized the fractal geometry of cities and identified them in mathematical terms with population density functions. more recently venables ( ) argued that the relation of form and function depends on the evolution in time of cities and on the economic interactions in which cities are involved during their development. this is a view of cities as complex entities characterized by emergent phenomena, whose shape is not well defined. the metrics to estimate urban forms are also object of debate (vanderhaegen & canters, ) . a number of different approaches to city boundary delineation exist in the literature, which we will review and discuss in section . nevertheless, in order to aim at any practical application of scientifically sound models one has to face the fact that, political or administrative boundaries exist in any country of the world and at different geographical scales. hence, existing political boundaries have to be considered for practical applications of scientific conclusions (hamilton & rae, ; thomas, jones, caruso, & gerber, ) , no matter what method is used to establish geographical boundaries of cities. in this paper, we assessed the extent to which planimetric areapopulation (a-p) scaling laws holds in italy, with respect to both urban boundaries and administrative subdivisions. we further applied scaling relations to propose a fusion of a large sub set of administrative boundaries on the very general grounds provided by a-p relations. we considered italian municipalities, the national administrative subdivision of finest granularity ( , polygons in year , see section ). next we considered one of the existing definitions of urban areas, based on the idea of "natural cities". they can be obtained from nodes of communication networks, as originally proposed by (rozenfeld, rybski, gabaix, & makse, ) and recently applied in a range of different study areas and data by jiang and jia ( ) and jiang and liu ( ) . such definition of cities, or urban areas, imply different measures of planimetric areas and population distributions with respect to the ones encompassed by administrative boundaries, which we describe in detail in section . to our knowledge, we performed the first delineation of modern city boundaries in italy, based on one of the methods existing in the literature, and calculated scaling exponents for a-p relations. a study of scaling relations in a few european countries, including italy, was previously performed by bettencourt and lobo, . bertuglia, bianchi, and mela, represents a notable reference to urban studies in italy, though not specifically focused on scaling relations but more in general on a science of cities, of the methods and tools used in their analyses, mostly within complexity and self-organization theories. we performed a selection of pairs of neighbouring municipalities and, using distance from the empirical a-p relation as an objective function, we accepted or rejected the fusion of the two municipalities, were the candidate mergers have aggregate population and area. mergers were constrained to be within the same higher-level administrative area, namely one the twenty italian regions. the results of the analysis outlined above are described in detail in section , and discussed in section . in particular, we discuss the similarities and differences between scaling relations obtained within municipalities' boundaries and within "natural cities", and we compare with two existing clustering of municipalities operated by various italian agencies. conclusions of this work are drawn in section . the complexity of phenomena occurring within cities, and contributing to their birth and expansion, have been investigated in many respects, since the early studies. power-law scaling relations are usually studied as a function of the size of cities, taken as their population. scaling was found to hold for city spatial extent (area), total length of networks (street, piping, wiring), economic and social indicators, and so on (bettencourt et al., ) . infrastructures, such as the total road surface and the total length of electrical cables, show a scaling exponent smaller than unity. on the other hand, quantities related to wealth creation and innovation, such as new patents, employment and gross product, are represented by power laws with an exponent larger than unity: super linear scaling signals that as the city size increases, such quantities must do so at an ever increasing rate in order to sustain system development. to make sense of urban scaling, bettencourt ( ) reported about a comprehensive mathematical framework based on a set of basic principles. the complex dynamics of spatial, infrastructural and social variables could be formalized defining a net urban output function. it was used as a measure of urban efficiency, and its maximization could distinguish between optimal and sub optimal cities. the approach was inspired by the explanation of universal scaling relations in plants (west, brown, & enquist, ) , based on resources being distributed through the system by branching networks. that was later generalized to other living systems (west, ) and other domains, including urban systems (west, ) . alternative explanations of urban scaling exists; for example, using cellular automata, wwhite and engelenhite and engelen ( ) and rybski, ros, and kropp ( ) simulated city size and fractality (both widely used to explain self-organized systems) within a percolation model. recently, liu, batty, wang, and corcoran ( ) studied several aspects of urban change and, most notably, advocated adoption of cellular automata-based models by politicians and practitioners. so far, no standardized international criteria exist for determining the boundaries of a city and often multiple boundary definitions are available for any given city (united nations, ) . as a matter of fact, united nations ( ) categorized urban areas in three nested levels, in order of increasing sizes: city proper, urban agglomeration and metropolitan area, corresponding to different areal and population sizes. other definitions exist in the literature and in practitioners work, often contrasting with the definitions above. for example, fang and yu ( ) consider an "urban agglomeration" as a system of "integrated cities", and surveyed , studies published starting from the beginning of th century, in which the same spatial organizations has been termed either as "megalopolis", "city group" and "city cluster". correspondingly, the delineation of cities has been performed in a number of different ways, relying on very heterogeneous data sources, even if no general consensus has been reached on how to delineate a city (masucci, arcaute, hatna, stanilov, & batty, ) . batty ( ) distinguished city delineation methods by three different criteria: (a) population/urbanization density, (b) interactions, described by different kinds of networks, and (c) geographical proximity/contiguity. we reviewed a sub set of them, given then large number of existing relevant studies, and adopted in this work one of the existing methods. differences in city delineation methods imply different levels of aggregation of variables in social, economic and spatial urban-related analyses. this is known as the modifiable areal unit problem (maup, openshaw, ; manley, ) , which may show up in any study associated with the use of data aggregated within geographical areas. it has been investigated in various fields of spatial analysis, such as landscape ecology (jelinski & wu, ) , natural hazards (alvioli et al., ; alvioli, guzzetti, & marchesini, ) and of course road traffic analysis (viegas, martinez, & silva, ) and census data analysis (flowerdew, ) . in this work, we did not explicitly consider maup effects, though grouping a varying number of municipalities into one single boundary clearly falls into that class of problems. one seemingly straightforward tool for city delineation, as an example of density-based methods above, is the use of a map of artificial surfaces, detected using remote sensing (lu, li, kuang, & moran, ) . one can assume that any impervious (or sealed) surface is part of an urban system (ma, omer, osaragi, sandberg, & jiang, ; thomas et al., ) . in fact impervious surfaces are both built-up and nonbuilt-up, and include a variety of objects that can be identified with human locations or activities (steidl, schleicher, & sannier, ) . impervious areas, though, come as raster layers, and the raster grid cells have to be clustered in order to obtain individual cities. this step introduces the additional difficulty of delineating boundaries between areas who might actually have relations, either spatial or regarding human activities: cities are difficult to delineate, and also difficult to study in isolation (bettencourt & west, ) . arcaute et al. ( ) studied data from uk census using different density thresholds for both population densities and commuting flow destinations. they looked at scaling laws of a large set of urban indicators, aggregated within the delineated cities. they found that most of the indicators exhibited a scaling exponent (cf. eq. ( )) very close to unity, thus apparently violating the expected universal regime of either a sub linear scaling for infrastructure and services, a super linear scaling regime for outcomes of social interactions (bettencourt, ) or a linear regime, in principle expected only for goods related to individual human needs . they explained the values of scaling exponents suggesting that two different regimes exist between cities confined within a domestic environment as opposed to those driving international dynamics. along a similar line, cottineau, hatna, arcaute, and batty ( ) performed a sensitivity analysis of scaling relations on the spatial aggregations of census units in france, aggregated in about , different ways by combining criteria that took into account density, commuting flows and population cutoffs. the general conclusion was that scaling estimations are subject to large variations, and that variations with respect to city delineation criteria are neither random nor universal for all the variables under study. as a consequence, even if cottineau et al. ( ) did not propose a "good" definition of city to study urban scaling, and they clarified that the methodological choice should be operated depending on the attribute under investigation. oliveira, furtado, andrade, and makse ( ) proposed a worldwide model to delineate city boundaries using a population density threshold and a distance threshold representing the mutual commuting distance between populated areas, singled out by a local clustering procedure. they used worldwide census data at about km resolution. they found that zipf's law for population holds for their cities, and proposed a list of the most populated cities in the world, according to their algorithm. street networks are undoubtedly proxies for human presence and activity. the study of networks and human mobility, for various purposes, is a field of research on its own and we will not attempt to review it here (barbosa et al., ) . it suffices to mention that studies of spatial networks and their topological properties are very closely related to urban studies (barthélemy & flammini, ) and network optimization is relevant for urban planning (durand, ; barthélemy & flammini, ) . masucci et al. ( ) used intersections of street networks and a parameter-free clustering technique to distinguish between urban streets and rural streets in the uk. clusters obtained from urban streets actually exhibited scaling behaviour, while rural ones did not, suggesting two distinct phases in the process of urbanization. non-physical networks also represent human activities and, thus, the existence of urban areas. for example, shutters et al. ( ) considered the interrelations among different types of occupations and how they eventually define an overall urban economy in six world's countries and showed scaling with city size. yin, soliman, yin, and wang ( ) considered citizen commute patterns obtained from online social network data to define anthropographic boundaries to delineate cities, finding good correspondence with administrative boundaries in the uk. yin et al. ( ) also noted that, while social networks data may have limitations and biases, they also have the important advantage of allowing time dependent and, at least in principle, real-time analyses (khodabandelou, gauthier, & fiore, ) . the relevance of non-physical networks, most importantly of digital communications, was also stressed by batty ( ) , who envisaged the next revolution dictating the shape of cities as the digital one, after the industrial and the electrical revolutions. eventually, as an example of a method probably falling in the category of proximity-based methods described by batty ( ) and jiang and liu ( ) described a technique for delineating city boundaries using street networks to single out individual blocks, defined by the portion of space within closed street loops. the collection of the resulting block areas were found to follow a log-normal (fat-tailed) distribution, characterized by a much larger number of small areas with respect to large areas. the mean value of the distribution was used to distinguish "large" from "small" areas. jiang and liu ( ) noted that a high smaller-to-large (about %- % to %- %) area ratio qualitatively signals the presence of scaling, which was also studied by means of power-law fits. in this work, we applied a variant of this datadriven, parameter-free method in italy. all of the data utilized in this work were either open data available from governmental agencies, data from volunteered geographic information projects or data otherwise available for free on the world wide web (www). we downloaded a vector layer containing polygons representing italian municipalities from the www site of the italian national institute for statistics (istat, ) . a database table associated with the vector layer contained data about population, aggregated at municipality level from census data surveyed in , the most recent one. we did not exploit the full resolution of population data at census section level because we are interested in a municipalitylevel analysis. the next-level political subdivision in italy is represented by provinces and regions. area, population and number of municipalities in individual italian regions are listed in table . from the istat www site we also downloaded an aggregation of municipalities operated by istat itself, based on census and commuting data, which goes under the name of sistemi locali del lavoro (local labour systems, sll). an additional aggregation of municipalities exists in italy, which was prepared with different methods and with different aims (that we do not discuss here), with respect to sll. the second aggregation goes under the name unioni di comuni (udc, literally associations of municipalities). we compared both sll and udc to the aggregation proposed in this work, in section . we downloaded a continental (european) imperviousness layer, available for various years from the copernicus land monitoring service (clms, ) . we considered the layer corresponding to year , the closest in time to the census data year, . the raster layer has m x m resolution and values of the raster cells give a measure of the degree of imperviousness, in percentage. in this work we were interested in any sealed surface i.e., with any imperviousness degree different from zero, as a first approximation. impervious areas included: housing areas, parking lots, railways close to other impervious areas, industrial and commercial areas, various paved and covered soil, and others. they did not include: inactive built-up areas, temporary coverage, sand, dump areas, cultivated areas, and others. we downloaded road network vector layers from the openstreetmap www site (osm, ) . from these layers we extracted the locations of street intersection to generate natural cities, as described in detail in section . . the total number of street segments in the data set is , , (including overlapping segments across neighbouring regions), amounting to , , intersections. eventually, to visually compare scaling relations in italy with independent area/population data for european and world cities, we downloaded data from the www site of the demographia world urban areas, (dwua, ). data were compiled applying a consistent definition of built-up urban areas, disregarding political boundaries that are generally associated with metropolitan areas or sub-national boundaries. this data set was not crucial since our methodology (section ) is not dependent on world's cities data, and neither are our results or conclusions. it was used in the log-log plots in fig. , for illustrative purposes. in the figure, world's cities data are grouped within ten individual countries, five in europe and five elsewhere. we discuss the plots in fig. in the next section. the ultimate aim of this work is to propose a practical application of long-known urban scaling relations, in particular of area-population relations, for the fusion of existing administrative boundaries. a concise outline of the methods used to achieve such a goal is as follows: • we considered municipalities, the finest administrative subdivision in italy, and investigated area-population relations within their boundaries. • we checked the validity of scaling relations using either the total area of municipalities, a, and a reduced (effective) area, a eff . • then, we focused on an independent method for the delineation of city boundaries available in the literature, namely the method of natural cities, based on the idea that street intersections are locations of human activities. • this objective (parameter-free) delineation of city boundaries was used to extract area-population relations, to be compared with the ones followed by the area and population of individual municipalities. • eventually, we singled out candidate municipality polygons for iterative pairwise mergers. mergers were accepted if the overall area and population within the aggregate polygons were favoured by the empirical a-p relation and overlapped a city, rejected otherwise. throughout this work, we commited to the idea that deviations from area-population scaling laws exhibited by some of the points representative of urban areas in the area-population plane (p a , ) measure how each city, or urban area alike, deviates from expectations based on its size (bettencourt, ; west, ; van raan, ) . in fact, a scaling relation of the form: implies that a city twice as large of another city, in terms of population p, is expected to cover a planimetric area = a a , with a and a the area of the smaller and larger city, respectively. establishing if the value of the scaling exponent is larger, smaller or consistent with unity is crucial to understand if data implies an diseconomy of scale ( < , sub linear scaling, or decreasing returns), an economy of scale ( > , super linear scaling, increasing returns), or constant returns to scale ( = ) (the expressions increasing, decreasing and constant returns are borrowed from economics jargon; here they simply refer to proportionality to city size, and have nothing to do with monetary measures). the basics of area-population relations are illustrated in fig. , which shows how well the supposedly existing scaling holds for world's cities data available on the www, not subjected to any kind of processing. the straight lines in fig. were obtained by linear regression of the log of area versus the log of population. in fact, taking the log of eq. ( ) one obtains a linear relation between the logarithms of area a and population p: )) of population data normalized to its average (p p / ), fitted as a log-normal (blue) and as a power-law (green). (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) with = log . the values of the exponents (slopes of the linear fits in log-log representation) for cities in europe, for europe altogether (five countries shown in the figure) and for the whole world (the whole set of , data points) are listed in the caption of fig. . the values of regression coefficients are also listed in the caption, along with the calculated uncertainties. bettencourt, and references therein reported similar fits to the ones obtained here. we also characterized the world's cities data set fitting the population data with power laws. to this end, we used the advanced package powerlaw, written by gillespie ( ) for the r programming language. one actually fits the cumulative density function extracted from data with a power-law. such procedure was inspired by the idea behind the zipf's law, a relationship between the sizes s of objects in a set and their rank r, defined as follows: where is the power-law exponent. in the case of cities, size is given by population. the powerlaw package builds a cdf and fits that both with power-law and with log-normal functional forms. it also automatically finds the minimum value under which the data have to be discarded for the fitting procedure to be reliable. we considered population divided by its average as main variable = x p p / . the cdf function reads as follows: which represents the probability of finding an object with size larger than a given size in a given set (clauset et al., ) . fits of the cdf of europe's city population are shown in the inset of fig. (a) , and in the inset of fig. (b) for world's cities. they show that data cannot be described by any of the fits in the whole p range. the power-law fit (green curves) seems to work well starting from the point where the cdfs obtained from data show a marked change in slope, while the lognormal fit (blue curves) seems to fit best the region of extremely large cities. oliveira et al. ( ) recently described a worldwide model to delineate city boundaries and reported about fits similar to the ones in the insets of fig. . our fits and the quoted ones share the relevant feature of a change in slope, which in our opinion makes the use power-law fits more difficult than a-p relations for the practical purpose that we aim to. universal validity of zipf-like power laws, and their usefulness for urban planning, was also questioned by cottineau ( ) . for these reasons, in the methodological part of this work, we did not use zipflike power-law fits of the cdfs to investigate administrative boundaries and urban areas in italy, but we rather used area-population relations as in eqs. ( ) and ( ). we will show that area-population scaling relations can be deduced from data on italian municipalities, provided area is taken into account in a proper way by selecting an effective area actually occupied by urbanization, in place of the entire area of municipalities. to investigate the existence of a scaling relation between area and population of italian municipalities, we first utilized the whole area encompassed by each municipality's administrative boundary (polygon) and raw population data, as provided by istat (see section ). fig. shows a log-log plot of area vs. population raw data (black dots), along with a linear fit (black curve). fits in this figure and throughout this work were performed by the fit function provided by the gnuplot . graphic software (williams et al., ) ; values of slope coefficient in eq. ( ) (the exponent of eq. ( )), and correlation coefficients r are listed in the figure's caption. we note that the slope of the fitted line is not nearly close to the slope of any urban a-p scaling relation; the fit for european cities in fig. is shown in green in the same plot for comparison. moreover, the small value of the r coefficient of the fit signals a correlation to be unlikely in this a-p data set. an imperviousness layer is a measure of how much the soil is sealed by artifacts and, as such, it can be considered as a direct observation of continued human presence. we used the data set obtained from copernicus www site (steidl et al., ) to constrain the area a of municipalities to their effective area occupied by urbanization, by simply cutting the imperviousness layer within the municipalities polygons in a gis. counting the number of m x m pixels within each polygon provides an estimate of urban area. we refer to that as a eff , in the following. beyond administrative boundaries, examples of city delineation from imperviousness maps are abundant in the literature (see e.g. ma et al., and references therein) . we used the imperviousness map only to calculate the effective area a eff occupied by urbanization in individual municipalities. these urbanization clusters, though, were still selected by administrative boundaries and never labelled as "cities". in fact, we decided to adopt a different approach, for the following reasons. typical approaches for city delineation from sealed surfaces are often based on numerical thresholds (number of cells, or density thresholds), to consider groups of grid cells as individual cities. introduction of numerical parameters represents a difficulty of the method. moreover, the raster map resolution represents an additional lower limit to the accuracy of a delineation performed from sealed surfaces. we performed preliminary city delineation (alvioli, ) , based on the imperviousness map, showing that (a) due to the above reasons, the points representing cities on the (p a , ) plane were distributed in a somewhat biased way, and (b) using such points for a linear a-p fit, we found the same slope as in the case obtained from the method of natural cities, described in the next section. thus, we preferred the latter as a parameter-free method, adopted in the remaining of this work. the definition of "natural cities" was inspired by the city clustering algorithm first proposed by rozenfeld et al. ( ) . the original algorithm starts from a collection of point-like populated sites, and performs an iterative clustering of sites within a given radius. iterations end when no points exist within the radius from each of the points in a cluster. the algorithm was applied by selecting streets nodes as starting points by jiang and jia ( ) , who performed city delineation with this approach in united states, finding that the distribution of sizes (i.e. number of street nodes) of the resulting city set neatly follow a zipf-like law. the algorithm was further generalized by using either city blocks as clustering domains (jiang & liu, ) , triangulated irregular network (tin) (jiang & miao, ) , or thiessen polygons (jiang, ) . in all of the variants of the original algorithm, street nodes were taken as a starting point of the city delineation procedure. in this work, we used street nodes obtained from the openstreetmap vector layer as a starting point. we generated a tin network from these points using the grass gis module v.delaunay, separately for the peninsular italy and the major islands, sicily and sardinia. all of the gis operations in this work were performed within grass gis, version . (neteler, bowman, landa, & metz, ) . we considered the area distribution of the triangles in the tin network, calculated its average value and checked that an imbalanced ratio existed within the three sub regions of italy for the number of areas above (smaller number) and below (larger number) the average value: namely, . for peninsular italy, . for sicily, and . for sardinia. jiang and liu ( ) found for comparable quantities in france, germany and uk the values of . , . and . for the ratio, respectively. an imbalanced ratio of large-to-small areas is the signature of a fattailed distribution, and of the possibility of splitting each of the three sets in a meaningful way using the natural breaks rule, discarding areas larger than average. actually, we discarded all of tin polygons with area above average, along with all of the polygons with area below average which were adjacent to a polygon with large area, following jiang and liu ( ) . this last step is a parameter-free replacement for utilizing a clustering radius to single out cities in this work, at variance with rozenfeld et al. ( ) . the idea of natural cities was further exploited in this work to devise a fusion algorithm for administrative boundaries. the aim of this paper is to propose a method for the fusion of administrative boundaries in italy at municipality level, driven by areapopulation scaling relations. the proposed procedure is grounded on our view of the empirical area-population scaling relation as a "performance" assessment of each individual datum in the data set (municipalities, in this case) as compared to expectations generated by the whole data set based on the individuals' size (population, in this case) (bettencourt, ; west, ; van raan, ; o'clery, curiel, & lora, ) . we argue that if a robust scaling relation as a function of municipalities' size (population) can be obtained, the vertical distance on the (p a , ) plane of an individual's representative point from the linear scaling law can be viewed as a measure of its "performance", in absolute value (i.e., considering equivalent both if it is above or below the linear fit). under this perspective, distance of a city's representative point from the scaling law represents a measure of how much the city is making a productive use of available resources. thus, we suggest that one can rank municipalities starting from the farthest from the scaling law. starting from the highest ranking, one can select all of the adjacent (surrounding) municipalities and try and find a candidate polygon to be merged with the central one, based on two requirements: (i) the aggregate areas and populations of the candidate mergers have a representative point in the (p a , ) plane which is closer to the scaling law than both the starting points, and (ii) the two candidates for the merger are connected by an urban area, as obtained by the method natural cities. if both the requirements are fulfilled, the two polygons are merged. the procedure can be iterated until all of the existing polygons have been checked against all of the possible mergers. the requirement (i) is the simplest one that one can devise for such a purpose; one could also adopt more involved measures for ranking performance with respect to an empirical scaling relation. the same goes for requirement (ii), because it seems reasonable that two municipalities can be considered for fusion into a single administrative unit if they share a common city, in a broad sense. the requirement is generic enough that it may be fulfilled by an urban agglomeration of any size, and the range of variation of city size is very large as it will be shown in the following. both requirements, and the whole procedure, are suited to be implemented in a gis with an automatic iterative procedure. the algorithm of the procedure for polygon fusion is described by the pseudo-code in the listing algorithm . lastly, we need to account for coarser administrative boundaries besides municipalities, namely provinces and regions. we disregarded provinces, which have themselves changed multiple times in italy in recent times. we rather focused on regions, of which italy is subdivided into twenty, and whose properties relevant for this work are listed in table . we restricted fusion between polygons of municipalities belonging to the same region, but still used the national scaling law as a measure to accept/reject mergers. in this section, we describe results obtained for the following four main points: . validity of area-population scaling relations for italian municipalities, described in sections . and . ; . delineation of urban areas (or cities) in italy, with a variation of the method of "natural cities" recently introduced in the literature, described in section . ; . pairwise fusion of a few adjacent polygons corresponding to italian municipalities, described in section . ; . comparison among the proposed aggregation and existing aggregations of municipalities in italy. we discuss the four points separately in the following. fig. shows area-population relations built with raw a and p data for italian municipalities (black dots/curve). the figure also shows the effect of constraining the area a of municipalities to their effective area a eff occupied by urbanization, in a broad sense (red dots/curve). in this work, we adopted any level of imperviousness (steidl et al., ) as an indicator of urbanization. we also plotted again data for the european cities (green dots/curve), whose fit nicely compares with the a eff -p result. we explicitly investigated the effect of different definitions of a and p. first, we studied the effect of modifying p with the inclusion of daily commuting data, vs. total a; the effect was negligible. then, we checked the effect of adding and removing pixels overlapping with roads to modify a eff . in fact, in the imperviousness layer roads may or may not be correctly identified so this step is not trivial. we found that adding missing roads reduces the slope of the scaling relation, which nevertheless still falls within the range proposed by bettencourt ( ) , while removing all of the roads again has negligible effect. eventually, we checked that the effect of modifying p using commuting flow data vs. the a eff plus roads areas in conjunction. this seemed the most accurate approximation we could devise, since people actually spend time on roads too. again, we found no substantial change with respect to the red curve in fig. , which did not account for commuting. we may deduce from fig. and from the considerations above that either using the only impervious area a eff , or a eff plus roads, produce a reasonable scaling relation against p, regardless of the use of modifications to p due to commuting data. we also note that the a eff -p relation, red line in fig. , compares well with european cities data. thus, we adopted the a eff -p linear fit as a baseline scaling relation in the pairwise fusion of municipalities. we performed delineation of urban areas by using the idea of "natural cities" built from the bottom-up using the tin network obtained from street nodes, as described in section . . thus, in this work we compare results of three different approximations of a-p relations, namely: (i) plain area a of municipalities vs. p, (ii) impervious (or effective) area of municipalities a eff vs. p, (iii) area from cities calculated with the natural cities method vs. the corresponding population. delineation of cities based on the "natural cities" method, resulted in a total of , urban agglomerations; additional numerical details are listed in table . fig. shows a map of cities delineated using the method of natural cities. we operated delaunay triangulation from street nodes independently for the three major geographically disjoint sub sets of italy, namely peninsular italy, sicily and sardinia. we found instructive to compare the three different approximations (i)-(iii) listed above from which we built the different a-p scaling relations. fig. shows results for sicily, to allow a larger-scale comparison. the comparison is still qualitative, but one can appreciate the differences. transition from approximation (i), fig. (a) , to approximation (ii), fig. (b) , clearly shows the difference in area measured by the corresponding scaling relations in fig. , black and red curves/dots, respectively, within municipality boundaries. when such boundaries are removed, as in approximation (iii) fig. (c) , areas are computed for the distinct clusters singled our by the natural city delineation method. fig. shows a quantitative assessment of a-p relations within the approximations (ii) and (iii). the figure shows separately data for peninsular italy, sicily and sardinia, where urban areas were delineated separately. the plot show a single linear fit, corresponding to aggregate data for the whole of italy. the linear fit goes into the direction of slightly decreasing the slope obtained in case (ii). data nicely distribute on the (p a , ) plane, clearly hinting to a linear correlation. some patterns seem to emerge, mostly as collinear structures to the overall linear fit in the sicily and sardinia sub sets, suggesting that some minor bias might occur in the case of delineation of natural cities in smaller areas. we used the natural cities delineation for the polygon-fusion procedure, the final aim of this work. the procedure for pairwise fusion of polygons representing municipalities is iterative. we started selecting the polygon whose representative point on the a-p plane is farthest in the vertical direction from the red curve in fig. , the empirical scaling relation for municipalities. fusion is pairwise, meaning that for each polygon we checked for another candidate polygon at a time to be merged with. this means the outcome of the procedure depends on the order of selection of both candidate polygons; nevertheless, for each checked polygon there were often only one or no acceptable mergers, making the dependence on the order of choice rather weak. the algorithm of the procedure for polygon fusion is described in detail by the pseudo-code in the listing algorithm . polygon fusion was restricted within individual regions in italy, a higher-order administrative subdivision, because mergers across different regions would be very unrealistic. the overall process was performed in parallel by gis procedures. each procedure goes to completion i.e., checks all of the existing polygons and performs the possible mergers according to the requirements described in section . , in a few hours. at the end of the run, we re-calculated the new fitting parameters , of eq. ( ), when the individual results obtained for each region were collected in one single map. the result for the new parameters of the national scaling law is substantially the same as for the parameters obtained from the original polygons. to visualize the effect of the fusion procedure, fig. shows a few examples among the few hundreds mergers operated by the proposed algorithm. in the figure, examples are shown of mergers between a point above and a point below the linear fit, and of examples of both points below the fit. examples of both points above the fit occur rarely, because the cumulative area being larger than both the original points would often cause violation of the requirement for the new point to be closer to the linear fit on the vertical (a) direction. the figure also shows three of the many mergers undergone by the largest municipality, including the city of rome, which after applying the proposed procedure would incorporate many neighbouring municipalities, as shown in fig. . fig. shows a pictorial representation of the performance of the original subdivision, consisting of polygons, and of the subdivision after the fusion procedure, consisting of polygons. the figure shows only new polygons of the proposed delineation, for clarity of presentation. the performance is measured as the vertical distance (in powers of ten) of each representative (p a , ) point from the empirical scaling law. note that, in this acceptation, the vertical distance represents the amount by which each a eff exceeds or falls behind the expectation suggested by the scaling law, given the municipality's population. at variance with the procedure itself, in which vertical distance was taken in absolute value, we have coloured each polygon in fig. in classes: a central one, in grey, four classes below the scaling law and four classes above, to visually distinguish points falling below or above the fit with different colour ramps. the below-above classes are symmetric, and were dictated by a data-driven classification using the head/tail break method with respect to the vertical distance of each point from the scaling law, in the log-log scale. table lists the number of polygons and the total area a (not a eff ) in the different classes. since we performed the procedure for the fusion of boundaries within administrative regions, we show in fig. the corresponding individual results. the figure includes a eff -p points for existing municipalities (labelled as "in") and the points for the new delineation ("out"), along with the initial and final slopes of the fits calculated within each region. the figure aims at illustrating differences between the various regions, but in the fusion procedure we always used the national scaling law. it is interesting to compare the proposed aggregation of municipalities with two existing ones, namely the sll and udc introduced in section . fig. (a) shows the a-p relation of sll (cyan) in which we used effective area for all of the data sets included in the plot. local labour systems, consisting of polygons, are substantially larger than municipalities, , polygons, and the fit to sll (cyan, dashed curve) has a less steep slope then both the fit to existing municipalities (grey, solid curve) and polygons after the mergers (brown, dotted line) suggested in this work. the udc layer contains modified polygons, while our new layer contains new polygons with respect to the existing municipalities layer. the total area of the "new" polygons in udc is , km ( "old" municipalities), which compares to the , km ( "old" municipalities) of the new subdivision proposed in this work. fig. (a) shows only the new points contained in udc (blue) and in the new delineation (brown), clearly highlighting that the former contains a bias towards larger areas with respect to the existing administrative boundaries (grey), failing to generate a corresponding increase in population within the merged polygons. similar considerations holds for the sll described above. in this work, by construction, each "new" polygon has to follow the general trend of the empirical area-population scaling law better than both the two original polygons. a chance for direct comparison is provided by official data in italy (istat, ), which reveal that the number of municipalities in italy in the last decades have decreased from , in , to , in . we applied the polygon fusion procedure to data, municipalities, because census data was available to us only for that year. fig. (b) shows that the points representative of the only new (aggregated) municipalities on the (p a , ) plane are distributed in a rather different way with respect to the points representative of mergers proposed in this work, deviating from the national scaling law. we stress that in fig. (b) the population used for the boundaries was from , because the data were not available to us at the time of writing. an area-population relation built with raw a and p data for italian municipalities, shown in fig. (black curve) , is far from any scaling law defined in the literature for world's cities (cf. fig. ) . a meaningful scaling law is found instead by restricting the area a of municipalities to their effective area a eff occupied by urbanization, in a broad sense. a similar quantity (namely, paved areas) was also investigated by bettencourt ( ) , therein referred to as a n . it was reported by the reviewed literature to scale with population with exponents in the range [ . , . ], which includes the range obtained in this work for municipalities' effective area, = ± . . . bettencourt ( ) also mentioned (in the paper's supplementary material) that up-to-date estimates of cities' paved area from remote sensing is desirable. we believe that the data sets used in this work, available from the copernicus programme homogeneously for the whole of europe at high resolution and with constant updates, should be considered for similar studies at continental level and for time-dependent analyses (clms, ) . the definition of criteria for the study of time evolution of urban systems was considered by different authors table for numerical evaluation of the results, fig. for comparison with administrative boundaries and fig. for a eff -p relations. statistics for the administrative regions labelled with three-letter codes are listed in table . map is in lambert azimuthal equal area (laea) projection, epsg: . ), and projections for the future until year also exist by authoritative organizations (united nations, ) . this is a relevant point, since the whole world's population is expected to live in cities of some form in the near future, though city size distributions are expected to experience little change (batty, ) . delineation of urban areas is relevant to considerations about the pandemic event, as the diffusion of an infection is clearly dependent on the spatial distribution of citizens at the national level and dynamics of social interactions within urban boundaries and across neighbouring cities. very recently, stier, berman, and bettencourt ( ) observed that growth rates of covid- in us cities revealed a power-law scaling relationship to city population. given that italy was among the most affected countries by the virus, delineation of urban areas in italy may acquire additional relevance in the present context and, most importantly, in the near future. very recently van raan ( ) studied the scaling of gross urban product (gup) versus population in denmark, germany and the fig. ; dots show the (p a , ) points for the three major disjoint geographical domains in italy. we compared to results for municipalities (black and red curves; = ± . . and = ± . . , respectively). in green the a-p fit for european cities, = ± . . . a comparison of the geographical distributions of the results, only for sicily, is in fig. . (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) fig. . pictorial representation of pairwise fusion of polygons, each represented by a grey dot. each colour represents a sample merger, among the few hundreds operated by algorithm . full circles correspond to the two original municipalities and empty squares to the polygon after the merger. the red, cerulean blue and orange groups show examples of mergers between a point above and a point below the linear fit (dashed line); the green and blue groups are example of both points below the fit. the brown, pink and cyan groups shows three of the many mergers undergone by the largest municipality (rome) which after the procedure would incorporate many neighbouring municipalities (see fig. ). (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) netherlands. like in this work, van raan ( ) considered urban areas, cities and municipalities. in particular, they distinguished urban and rural municipalities, finding substantially different scaling exponents, and concluded that urban regions consisting of a single municipality perform better than those including many municipalities. even if the scaling relation is different from the one considered here, i.e. gup versus p instead of a eff versus p, the two studies are clearly related, and the conclusions of van raan ( ) indirectly support the motivation and methods of the present work. in fact, both studies look at the performance of larger, coarser municipality boundaries with respect to smaller, finer boundaries, and both studies consider scaling relations, actually deviations from the empirical scaling relation, as performance indices. a sound assessment of area-population scaling for municipalities allowed to propose a criterion to merge municipalities in italy, driven scaling relations. the exercise of practical interest, in that official data in italy (istat, ) reveal that the number of municipalities changed by about units in the last thirty years; unofficial sources available on the www reveal that fusion of municipalities (comuni) started in . moreover, additional variation of such boundaries are planned for the near future. suppression or fusion of two or more municipalities into a new one were dictated by a number of different criteria, under the general aim of reducing the total number of administrative centers. we believe that the perspective from scaling laws proposed in this work can be useful to complete the criteria currently adopted for fusion of administrative boundaries, to obtain a comprehensive point of view. adoption of multiple criteria could avoid negative effects of individual criteria, when applied separately. for example, the only prescription of following an area-population scaling law could promote the practice of indiscriminate land use or urban sprawl, given that an administrative unit would be favoured if a large population corresponds to a large urban area. this could be avoided by the joint application of multiple criteria. the procedure for the fusion of administrative boundaries produced a new delineation consisting in units instead of the existing units. considering the empirical scaling relation of fig. as a metric for "performance" of each unit, the procedure reduced the total area of units classified as "less performing" from km to km (cf. table ), reduced the total area in intermediate classes from , km to , km , and increased the total area in the "most performing" class from , km to , km . we stress that we used the empirical a eff -p scaling law in the most straightforward way we could devise, to show the feasibility of the approach. an improved procedure could be implemented by means of additional criteria, for example applying considerations of economic table for results in terms of number of polygons and area in each class. maps are in laea projection, epsg: . the number of polygons and total area corresponding to the existing subdivision in municipalities in italy (old), and the proposed one (new). class corresponds to grey in fig. ; class , green; class , cyan; class , yellow; class , magenta; class , orange; class , cerulean blue; class , red; class , blue. number ( nature, or considering actual commuting flows in the commuting matrix described in section to single out candidate polygons for mergers. preliminary calculations aimed at checking the effects of simply relocating population from/to municipalities according to daily commuting flows showed to be of little relevance, as far as scaling laws are concerned (not shown here). the sll subdivision based on commuting flows, defined in italy by istat, provides a more sophisticated use of such information, independent on scaling considerations. fig. (a) shows a comparison of the a-p relations from our aggregation method and aggregations provided by sll and udc, highlighting rather substantial differences. regarding sll, fig. (a) clearly shows that the distribution of points in the (p a , ) plane is biased towards larger areas than the empirical scaling law, which was expected, given the different rationale and purpose of the sll subdivision. regarding udc, the differences are mainly due to the fact that both the considered existing methods do not take into account area and population simultaneously, the proposed delineation. the substantial difference is that the total population interested by udc amounts to , , citizens, while the corresponding figure for our new delineation is , , citizens (sll was delineated using different input data, so we do not report corresponding figures, here). this is clear indication that fusion of administrative boundaries performed in different ways may generate very different scenarios, population-wise. similar considerations holds for the current subdivision into municipal units, fig. (b) , where the new units involved , , citizens. a proper discussion should take into account both geographical boundaries and population data from , thought, while data in the figure correspond to population in . recently, an additional aggregation of municipalities was proposed by the italian civil protection department (dpc, ), going under the name of contesti territoriali (literally territorial contexts ct). this particular aggregation, while bearing a certain relationship with the abovementioned sll and udc, were developed for emergency planning and risk management. thus, the purpose of the aggregation proposed here is probably not directly comparable with the ct aggregation and was not explicitly considered in this work. a meaningful comparison with a proper discussion will be presented elsewhere. scaling laws for urban systems are deemed near-universal, because scaling of a specific indicator on city size slightly depends on geographical location and/or the criteria imposed for the very delineation of cities. nevertheless, once an empirical a-p law is established for a given country or otherwise homogeneous study area, it may be viewed as a general and robust measure of the "performance" of a city of a given size, in terms of expectations about its area (bettencourt, ; west, ) . we presented an analysis of italian urban areas in relation to existing administrative boundaries. we believe that the present work contributes to filling the existing gap in the literature, in which delineation of urban areas were not found for italy, allowing to distinguish the behaviour of different urban systems within europe. our results can be summarized in the following points: fig. . a eff -p relations for the individual administrative regions. in each panel, the three-letter code corresponds to table and fig. ; grey points and curves refer to existing municipalities' data and the corresponding fit (in), while coloured points and curves refer to the new delineation (out). the dotted curve is the a eff -p national fit, first shown in fig. as a red curve and referred to throughout this work. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) • area a for italian municipalities scales sub linearly with population p, with a slope smaller than the slope obtained for italian and european cities from data available on the www, and within the range reported for world's cities by bettencourt ( ) . scaling is not obtained for the total area, though, but for the effective built-up (actually, impervious -or sealed) area a eff (fig. ); • we obtained "natural cities" using natural breaks selection of geographical zones built from the nodes of the national street network (figs. and ; (jiang & liu, ; jiang & miao, ) ). such a delineation of cities in italy provides very similar a-p relations with respect to the one valid for effective area of municipalities (fig. ); • a reduction of the number of administrative units, at municipality level, could be performed using the a eff -p scaling law as a general criterion for pairwise fusion of neighbouring polygons (fig. ); • iterative application of the merging procedure produced a new aggregation, containing , units, as compared to the existing , units ( figs. and ) . units are larger than the existing ones, but their population still follows the empirical a-p scaling law, at variance with existing aggregations of municipalities in italy (udc, sll and the current administrative boundaries; fig. ). we stress that a data-driven method for delineating cities such as the definition of natural cities adopted in this work, in conjunction with the many different methods considered by e.g., cottineau et al. ( ) , may contribute in finding a comprehensive and objective delineation method. the proposed real-world application of scaling laws is an example of the possibilities offered by such universal relations, suggesting their use in conjunction with other (expert, political, social, economic) criteria to delineate boundaries that simultaneously comply with, or are obtained from reasoned aggregation of, existing administrative subdivisions. one of the advantages of pursuing a path like the one outlined here is that it also represents a bottom-up approach, based on the ideas of emergent phenomena from self-organization, which may reveal more effective than top-down, centralized approaches to planning (barthélemy et al., ) . inclusion of general principles such an empirical a-p scaling law into the delineation of administrative boundaries goes into the direction of pursuing a comprehensive science of cities, envisaged by batty ( batty ( , . a science of cities which takes into account complex behaviour and emergent phenomena, which form the basis of scaling laws, may lead to cities and administrative boundaries which are more resilient against environmental, socioeconomic, and political issues (meerow, newell, & stults, ) , and hopefully against natural disasters (united nations, ) . we further highlight the importance of exploiting open data, including satellite and volunteered data which, in conjunction with modern open source gis software with powerful processing capabilities, and with a multidisciplinary approach, may be relevant for urban and administrative planning. the methods presented in this work can be readily extended to any country and in particular to the whole of europe, where data from the same sources used here is already available. algorithm . pseudo-code structure of the algorithm, implemented in grass gis, for pairwise fusion of municipality polygons driven by the empirical aeff -p scaling relation as in eq. ( ) and represented by the red curve if fig. . the flag chk is set to at the beginning of the procedure for all of the polygons. cities referred to in this pseudo-code are natural cities described in sections . and . . fig. . comparision of a eff -p relations with existing aggregations of municipalities in italy. (a) the existing (grey) and proposed (brown) units; we show only new mergers proposed in this work, and compared to alternative delineations, namely the "unione di comuni" (udc, blue) and the "sistemi locali del lavoro" (sll, cyan). for udc, we show the only aggregations differing for one or more units (as for the mergers), while we show all of the sll points, which have much larger areas than all of the other delineations. (b) grey and brown as in (a), compared to the actual new municipalities in italy, in (black points); population data were from for all the data sets. 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b: planning and design bivariate line-fitting methods for allometry a general model for the structure and allometry of plant vascular systems life's universal scaling laws cellular automata and fractal urban form: a cellular modelling approach to the evolution of urban land-use patterns gnuplot . : an interactive plotting program depicting urban boundaries from a mobility network of spatial interactions: a case study of great britain with geo-located twitter data human behavior and the principle of least effort i am indebted with the whole geomorphology group of cnr irpi, perugia, especially with dr. ivan marchesini for introducing me to the world of gis processing. i also thank dr. bin jiang, faculty of engineering and sustainable development, university of gävle, sweden, for thorough discussions about the definition of natural cities, and dr. luca fondacci, department of engineering, university of perugia, italy, for interesting discussions about urban planning. this paper also benefited from constructive comments of three anonymous reviewers. key: cord- -wds e i authors: tejedor, santiago; cervi, laura; tusa, fernanda; portales, marta; zabotina, margarita title: information on the covid- pandemic in daily newspapers’ front pages: case study of spain and italy date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: wds e i spain and italy are amongst the european countries where the covid- pandemic has produced its major impact and where lockdown measures have been the harshest. this research aims at understanding how the corona crisis has been represented in spanish and italian media, focusing on reference newspapers. the study analyzes front pages of el país and el mundo in spain and italy’s corriere della sera and la repubblica, collecting news items and data evidences employing a mixed method (both qualitative and quantitative) based on content analysis and hemerographic analysis. results show a predominance of informative journalistic genres (especially brief and news), while the visual framing emerging from the photographic choice, tend to foster humanization through an emotional representation of the pandemic. politicians are the most represented actors, showing a high degree of politicization of the crisis. spain and italy, with , and , confirmed cases, respectively, are amongst the european countries where the covid- pandemic has produced its major impact and where lockdown measures have been the harshest. due to the reduced mobility and the imposed lockdown, the internet has proved to play a decisive role in terms of media consumption during the quarantine. social networks have occupied the first position among online platforms most frequently consulted by citizens. according to twitter, the information on the pandemic as well as the conversations related to the topic have caused a % boost in total active daily users, reaching the general level of million users per trimester. news check-up has experienced a prominent growth at that stage. specifically, the peak of media consumption coincided with the first measures of social distancing and has increased in correspondence with governmental communications. these data should be interpreted within the current crisis of journalism and the crisis of media credibility. a recent survey of countries by ipsos global advisor [ ] shows how citizens are rather skeptical towards the information they receive from the media, especially when it comes to online press. in spain, % of the surveyed trust in television, whereas % expressed their preference for traditional media, such as printed press. making reference to the intentions, the research claims that half of the total number of the surveyed believe that printed papers have "good intentions" as opposed to % considering that online newspapers and web pages are the ones with "the worst intentions" [ ] . based on the trust placed on the printed media-as the most credible and rigorous media-this research analyzes a total of front pages of the main daily newspapers in spain and italy ( each) . the research considers the daily newspaper's front page as a fundamental element that synthetizes and prioritizes the contents that the particular medium treats as the most important. at the same time, the front page maintains a direct relation to the digital version of the medium, somehow setting the agenda. in other words, the front page serves a privileged space for public identity construction [ ] . the study, carried out between february and april , collected pandemic-related news pieces and data evidences, aimed at answering the following research questions: • how has the covid- pandemic been covered on the front pages of spain and italy's main daily newspapers? • what types of journalistic genres have been used? • what types of political or social figures and institutions appear the most? • what role has been assigned to an image/photograph in the coronavirus-related information items of the front page? the covid- crisis has posed new challenges to journalism. media play a fundamental role in framing a crisis, since providing the right information from a reliable source is the key issue in this type of pandemic. the world health organization (who) has used the term "infodemic" to define the overabundance of information introduced by coronavirus and to warn the citizens against the risks caused by this information excess, that contain a great amount of hoaxes and rumors. as sylvie briand, director of infectious hazards management at who's health emergencies program notices, this phenomenon is not new "but the difference now with social media is that this phenomenon is amplified, it goes faster and further, like the viruses that travel with people and go faster and further". the role of social media in spreading misleading health information is not new [ ] , but the covid- crisis has shown the critical impact of this new information environment [ ] . many studies have focused on and are still focusing on how the disintermediated role of social media may foster misinformation: scholars studying iran [ ] and spain [ ] , stress how social media spread rumors, others [ ] try to analyze the structure of this infodemic, or concentrate on the effect of media exposure [ ] . within this social media euphoria, very few studies have focused on legacy media, intended as the mass media that predominated prior to the information age-particularly print media, radio broadcasting, and television-even if reality is showing that legacy media still plays an important role [ ] . a study [ ] noticed how cnn has recently anticipated a rumor about the possible lockdown of lombardy (a region in northern italy) to prevent the pandemic, publishing the news hours before the official communication from the italian prime minister. as a result, people overcrowded trains and airports to escape from lombardy toward the southern regions before the lockdown was in place, disrupting the government initiative, aimed to contain the epidemic and potentially increasing contagion. other literature [ ] stresses the importance of looking at mainstream media coverage pointing out the importance of a high quality scientific journalism [ ] . the analysis of the printed daily newspapers' front pages has been object of recurrent studies for the last years. starting with the classical works [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] up to the contemporary researches [ ] [ ] [ ] [ ] , various studies have dealt with content analysis of that essential element of the printed press [ , ] . other studies followed them, concentrating on the comparison between the front pages in printed and digital editions of a medium [ ] . as previously highlighted, daily newspaper's front pages are considered to be the most important page, displaying informative priorities and editorial position in relation to current issues [ ] . other studies [ ] single out three core elements of a daily newspaper's front page: headlines, or visual linguistic set; texts, or visual paralinguistic set and images, or visual non-linguistic set. in this context, the importance of media and information literacy, seen as the citizens' ability to access, use, assess, and create responsible and ethical content [ ] , has become crucial. media and information literacy refers to the vital role that information and media possess in the everyday life of a person, therefore this skill represents an indispensable component to exercise freedom of expression and information. in this vein, numerous studies [ ] [ ] [ ] [ ] stress the significance of a digital literacy development that would exceed studying merely technical or instrumental aspects to embrace the issues of the critical use of media. the research, based on previous studies [ ] , analyzes a total of news items extracted from front pages of the four main daily newspapers of spain and italy ( per country). el país and el mundo of spain alongside with corriere della sera and la repubblica of italy were chosen, based on their relevance and the availability of their front pages. the analysis has been carried out through the use of a template chart consisting of parameters and categories that were obtained mainly in inductive form. the study, possessing descriptive and explanatory character, employs a mixed method (both qualitative and quantitative) based on content analysis and complemented by direct observation and hemerographic analysis as the main techniques. the first technique focused on the analysis of various elements that constitute the front page designs by means of a template chart elaborated during the research process. subsequently, we implemented a hemerographic analysis of texts, headlines and images. the data were processed through descriptive statistics planning with spss software. the analytical chart has considered all the elements displayed in table . coronavirus-related information occupies % of the front pages. precisely, news items out of the total focus on topics related to the covid- pandemic. as for the main journalistic genres, (see figure ), we can observe brief as the most common. this journalistic genre, characterized by its conciseness and brevity, has been defined as brief, a summarized piece of news that solely reflects the most relevant data of the information, missing profound insight and context. the total of pandemic-related units possess the form of short pieces, that could oscillate between or lines. coronavirus-related information occupies % of the front pages. precisely, news items out of the total focus on topics related to the covid- pandemic. as for the main journalistic genres, (see figure ), we can observe brief as the most common. this journalistic genre, characterized by its conciseness and brevity, has been defined as brief, a summarized piece of news that solely reflects the most relevant data of the information, missing profound insight and context. the total of pandemic-related units possess the form of short pieces, that could oscillate between or lines. news occupies the second position in the list of types of the texts about coronavirus at the analyzed front pages. the informative approach towards, in other words, dominates the representation of the crisis. the effort of the daily newspapers to inform their readers on the characteristics, impact and spread of the virus has been detected. nonetheless, it is worth mentioning that opinion articles (with a total number of counted units) surpass other informative journalistic genres. moreover, the importance of the editorial photo with a total number of units, solely accompanied by the footnote, demonstrates a comprehensive approach to the topic through the communicative value of the image. the location of news items at the daily newspapers' front pages can be considered another element of high value when it comes to the detection of importance of each topic. in this sense the studies grant more value to the upper part and the right part from the reader's standpoint. the right part is the most valuable of the odd-numbered page and the left part is the most important for the even-numbered pages. concretely, projecting an imaginary v onto the open double page, the higher the position on the v, the more value the piece has (both in terms of editorial and advertising rates). accordingly, results show that news about coronavirus appear mostly located in the upper part, but in the left zone (see figure ). in this way, it is possible to point out that the newspapers place the news in an important area of their front pages. in addition, in second position, with a total of news items, is the upper right-hand area. in this way, it is possible to point out that the news have been progressively occupying the areas of greatest visual impact of the front page. however, this set of news items is very close to the news items on the pandemic that appear at the bottom of the front page, i.e., the one of least importance. a total of appear at the bottom left and at the bottom right. therefore, the distribution of the news on covid- between the two areas marked by the horizontal division of the first page (top/bottom) is very tight. news occupies the second position in the list of types of the texts about coronavirus at the analyzed front pages. the informative approach towards, in other words, dominates the representation of the crisis. the effort of the daily newspapers to inform their readers on the characteristics, impact and spread of the virus has been detected. nonetheless, it is worth mentioning that opinion articles (with a total number of counted units) surpass other informative journalistic genres. moreover, the importance of the editorial photo with a total number of units, solely accompanied by the footnote, demonstrates a comprehensive approach to the topic through the communicative value of the image. the location of news items at the daily newspapers' front pages can be considered another element of high value when it comes to the detection of importance of each topic. in this sense the studies grant more value to the upper part and the right part from the reader's standpoint. the right part is the most valuable of the odd-numbered page and the left part is the most important for the even-numbered pages. concretely, projecting an imaginary v onto the open double page, the higher the position on the v, the more value the piece has (both in terms of editorial and advertising rates). accordingly, results show that news about coronavirus appear mostly located in the upper part, but in the left zone (see figure ). in this way, it is possible to point out that the newspapers place the news in an important area of their front pages. in addition, in second position, with a total of news items, is the upper right-hand area. in this way, it is possible to point out that the news have been progressively occupying the areas of greatest visual impact of the front page. however, this set of news items is very close to the news items on the pandemic that appear at the bottom of the front page, i.e., the one of least importance. a total of appear at the bottom left and at the bottom right. therefore, the distribution of the news on covid- between the two areas marked by the horizontal division of the first page (top/bottom) is very tight. figure displays the main entities mentioned in the stories, that is to say institution or entities most recurrently mentioned or displayed. of the information, % mentions geographical scenarios (europe, madrid, milan, etc.). in this sense, there is a tendency to depersonalize the information and to extrapolate it to wider scenarios or territories. this aspect is important insofar as the subject is the element of the sentence that carries out the action contained in it. in % of the cases the front page news referred to national entities of non-political nature (the hospital, the emergency unit, the laboratory, the intensive care unit, the sports center, the cultural center, etc.). in particular, there is a notable reference to entities linked to hospitals and healthcare scenarios. national political entities (government, trade unions, spokespersons, minister of health, etc.) occupy the third position in the rank of entities linked to the news, with %. political entities from abroad (the who, the european union, the european parliament, etc.), with %, and non-political entities from abroad (especially universities, research groups or the media), with %, respectively, completed the list of entities. figure displays the main entities mentioned in the stories, that is to say institution or entities most recurrently mentioned or displayed. of the information, % mentions geographical scenarios (europe, madrid, milan, etc.). in this sense, there is a tendency to depersonalize the information and to extrapolate it to wider scenarios or territories. this aspect is important insofar as the subject is the element of the sentence that carries out the action contained in it. in % of the cases the front page news referred to national entities of non-political nature (the hospital, the emergency unit, the laboratory, the intensive care unit, the sports center, the cultural center, etc.). figure displays the main entities mentioned in the stories, that is to say institution or entities most recurrently mentioned or displayed. of the information, % mentions geographical scenarios (europe, madrid, milan, etc.). in this sense, there is a tendency to depersonalize the information and to extrapolate it to wider scenarios or territories. this aspect is important insofar as the subject is the element of the sentence that carries out the action contained in it. in % of the cases the front page news referred to national entities of non-political nature (the hospital, the emergency unit, the laboratory, the intensive care unit, the sports center, the cultural center, etc.). in particular, there is a notable reference to entities linked to hospitals and healthcare scenarios. national political entities (government, trade unions, spokespersons, minister of health, etc.) occupy the third position in the rank of entities linked to the news, with %. political entities from abroad (the who, the european union, the european parliament, etc.), with %, and non-political entities from abroad (especially universities, research groups or the media), with %, respectively, completed the list of entities. in particular, there is a notable reference to entities linked to hospitals and healthcare scenarios. national political entities (government, trade unions, spokespersons, minister of health, etc.) occupy the third position in the rank of entities linked to the news, with %. political entities from abroad (the who, the european union, the european parliament, etc.), with %, and non-political entities from abroad (especially universities, research groups or the media), with %, respectively, completed the list of entities. figure details which kind of people are mostly mentioned within the main characters in the information on covid- . interestingly, national political figures are the most numerous group with % of the total, stressing how the crisis is highly politicized. in second place, we find anonymous citizens who are protagonists in % of news items on the front page. if political figures normally make it to the front pages, within the covid- crisis, anonymous people have become co-protagonists of the front pages. public figures from different countries (with %) making statements about the pandemic outnumbered those affected by or suffering from the virus (with %) and international political figures (with %). finally, health personnel, who have generated important recognition and ovations, have only been the protagonists of % of the front page news about the virus, while researchers and scientists (with %) occupy the last place in percentage of presence in the front pages. headlines are also of great importance. their location on the page, and the type and size of the title contributes to underlining the importance of the information among the set of pieces selected to appear on that page. in relation to this (see figure ), the study has identified a predominance ( ) of appellative headlines, focused on drawing the reader's attention. for this reason, the headlines tend to be non-verbal and have very atomized structures that seek to convey to the reader news about a subject he or she already knows. as an example, figure shows the headlines from italian la repubblica: "tutti a casa" (everybody is at home) and "chiude l'italia" (italy closes) to announce the lockdown measures. headlines are also of great importance. their location on the page, and the type and size of the title contributes to underlining the importance of the information among the set of pieces selected to appear on that page. in relation to this (see figure ), the study has identified a predominance ( ) of appellative headlines, focused on drawing the reader's attention. for this reason, the headlines tend to be non-verbal and have very atomized structures that seek to convey to the reader news about a subject he or she already knows. as an example, figure shows the headlines from italian la repubblica: "tutti a casa" (everybody is at home) and "chiude l'italia" (italy closes) to announce the lockdown measures. headlines are also of great importance. their location on the page, and the type and size of the title contributes to underlining the importance of the information among the set of pieces selected to appear on that page. in relation to this (see figure ), the study has identified a predominance ( ) of appellative headlines, focused on drawing the reader's attention. for this reason, the headlines tend to be non-verbal and have very atomized structures that seek to convey to the reader news about a subject he or she already knows. as an example, figure shows the headlines from italian la repubblica: "tutti a casa" (everybody is at home) and "chiude l'italia" (italy closes) to announce the lockdown measures. and generally opt for the structure of subject, verb and predicate, enunciating a topic related to the pandemic trying to answer the "what" and the "who" of such information. the expressive ones, which have an evocative function on an event known to the reader, are the scarcest (only have been counted). this reduced number emphasizes the existence of a commitment among the media analyzed to avoid sensationalist headlines or those that seek only to externalize moods. another classification of headlines focuses on speech acts. in relation to these, as shown by figure , the headlines with textual quotations-which reproduce, between quotation marks, the declaration of one of the protagonists of the information-predominate ( in total). the majority presence of this type of headlines denotes an interest of the media in presenting the information from a personalized standpoint, thus, bringing the stories closer to the subjects that have generated them. indirectly quoted headlines ( ) and partially direct headlines ( ) accumulate a smaller number of cases. by analyzing the types of verbs used (see figure ), a predominance of strong interpretative verbs, characterized by highlighting the intensity of an action, is detected ( in total), followed by weak interpretive verbs (a total of ) which, although with less intensity, denote a willingness of the journalist to give more intensity to an action. the narrative ones, which are more neutral, add up to the continuity of the news about covid- on the front pages of the media, both in italy and spain, justifies this tendency towards headlines of an appellative nature that allude to facts that are familiar to the citizens. the informative headlines, with a total of units, are the most conventional and generally opt for the structure of subject, verb and predicate, enunciating a topic related to the pandemic trying to answer the "what" and the "who" of such information. the expressive ones, which have an evocative function on an event known to the reader, are the scarcest (only have been counted). this reduced number emphasizes the existence of a commitment among the media analyzed to avoid sensationalist headlines or those that seek only to externalize moods. another classification of headlines focuses on speech acts. in relation to these, as shown by figure , the headlines with textual quotations-which reproduce, between quotation marks, the declaration of one of the protagonists of the information-predominate ( in total). the majority presence of this type of headlines denotes an interest of the media in presenting the information from a personalized standpoint, thus, bringing the stories closer to the subjects that have generated them. indirectly quoted headlines ( ) and partially direct headlines ( ) accumulate a smaller number of cases. and generally opt for the structure of subject, verb and predicate, enunciating a topic related to the pandemic trying to answer the "what" and the "who" of such information. the expressive ones, which have an evocative function on an event known to the reader, are the scarcest (only have been counted). this reduced number emphasizes the existence of a commitment among the media analyzed to avoid sensationalist headlines or those that seek only to externalize moods. another classification of headlines focuses on speech acts. in relation to these, as shown by figure , the headlines with textual quotations-which reproduce, between quotation marks, the declaration of one of the protagonists of the information-predominate ( in total). the majority presence of this type of headlines denotes an interest of the media in presenting the information from a personalized standpoint, thus, bringing the stories closer to the subjects that have generated them. indirectly quoted headlines ( ) and partially direct headlines ( ) accumulate a smaller number of cases. by analyzing the types of verbs used (see figure ), a predominance of strong interpretative verbs, characterized by highlighting the intensity of an action, is detected ( in total), followed by weak interpretive verbs (a total of ) which, although with less intensity, denote a willingness of the journalist to give more intensity to an action. the narrative ones, which are more neutral, add up to by analyzing the types of verbs used (see figure ), a predominance of strong interpretative verbs, characterized by highlighting the intensity of an action, is detected ( in total), followed by weak interpretive verbs (a total of ) which, although with less intensity, denote a willingness of the journalist to give more intensity to an action. the narrative ones, which are more neutral, add up to a total of ; while the perlocutionary ones, which incorporate an effect that is intended to be achieved by means of an action, are the least numerous, with units counted. a total of ; while the perlocutionary ones, which incorporate an effect that is intended to be achieved by means of an action, are the least numerous, with units counted. out of all the pieces about the covid- crisis, , that is to say % of the total content on the pandemic, has some kind of photographic accompaniment. only are in black and white, and in have an artistic quality. the reduced number of photographs on the pandemic on the covers is striking, although it could be justified by the difficulty of obtaining images or doing so from a variety of themes that would allow for dynamism and renovation of the covers. regarding the characters that appear in the photographs, public figures such as pope francis, or celebrities, like sportsmen or writers have taken over the front pages ( . %). the second leading role is played by anonymous citizens who appear in everyday scenes, with a total of %, and national politicians account for %. only % have images of people affected by or patients who have contracted coronavirus. international political figures account for % of the total. finally, health personnel only appear in % of the photographs and scientists and researchers in %. the visual framing also shows a certain level of both spectacularization (with the presence of celebrities) and politicization of the crisis, while health workers and scientists that actually are on the frontline of the fight against the virus are less visible. by comparing italian and spanish news outlets, we can observe how covid- occupies the majority of the information in both countries. nonetheless, while in spain it occupies % of the front page; in italy covid- related pieces cover a striking % of the information (see figure ). italy was the first european country severely hit by the pandemic, so it makes sense to state that this unpleasant surprise somehow engulfed media attention. with regard to the information that presents a predominance of numeric data, the number of pieces is very low in both countries. spain, with %, and italy, with %, reinforce the scarcity of information focused only on figures or percentages. this might seem surprising, due to the overwhelming amount of data information (statistics, evolution of case numbers, etc.) we have received during the pandemic, nonetheless it confirms the interpretative role assumed by the printed press: while on line media can offer on line updating, the printed press offers a more interpretative vision of facts. figure displays the main entities portrayed in the information. geographical names, that is to say cities or regions, are the most numerous in the information on covid- ( % in italy and % in spain), followed by national institutions not linked to politics, which, with % in italy and % in spain, show the prominence that this type of institution has acquired in the framework of this crisis. political institutions are those that occupy the third place with % of the total in italy and % out of all the pieces about the covid- crisis, , that is to say % of the total content on the pandemic, has some kind of photographic accompaniment. only are in black and white, and in have an artistic quality. the reduced number of photographs on the pandemic on the covers is striking, although it could be justified by the difficulty of obtaining images or doing so from a variety of themes that would allow for dynamism and renovation of the covers. regarding the characters that appear in the photographs, public figures such as pope francis, or celebrities, like sportsmen or writers have taken over the front pages ( . %). the second leading role is played by anonymous citizens who appear in everyday scenes, with a total of %, and national politicians account for %. only % have images of people affected by or patients who have contracted coronavirus. international political figures account for % of the total. finally, health personnel only appear in % of the photographs and scientists and researchers in %. the visual framing also shows a certain level of both spectacularization (with the presence of celebrities) and politicization of the crisis, while health workers and scientists that actually are on the frontline of the fight against the virus are less visible. by comparing italian and spanish news outlets, we can observe how covid- occupies the majority of the information in both countries. nonetheless, while in spain it occupies % of the front page; in italy covid- related pieces cover a striking % of the information (see figure ). italy was the first european country severely hit by the pandemic, so it makes sense to state that this unpleasant surprise somehow engulfed media attention. int. j. environ. res. public health , , x for peer review of the characters that appear in the information correspond to very diverse profiles. national political figures are the most numerous, with % in italy and % in spain (see figure ). this aspect contrasts with the reduced presence of institutions, as mentioned above. thus, it is possible to with regard to the information that presents a predominance of numeric data, the number of pieces is very low in both countries. spain, with %, and italy, with %, reinforce the scarcity of information focused only on figures or percentages. this might seem surprising, due to the overwhelming amount of data information (statistics, evolution of case numbers, etc.) we have received during the pandemic, nonetheless it confirms the interpretative role assumed by the printed press: while on line media can offer on line updating, the printed press offers a more interpretative vision of facts. figure displays the main entities portrayed in the information. geographical names, that is to say cities or regions, are the most numerous in the information on covid- ( % in italy and % in spain), followed by national institutions not linked to politics, which, with % in italy and % in spain, show the prominence that this type of institution has acquired in the framework of this crisis. political institutions are those that occupy the third place with % of the total in italy and % in spain. the characters that appear in the information correspond to very diverse profiles. national political figures are the most numerous, with % in italy and % in spain (see figure ). this aspect contrasts with the reduced presence of institutions, as mentioned above. thus, it is possible to point out that politics is personalized as party politics through the representation of figures of the different parties of the country that complies with the seminal findings of hallin and mancini [ ] . the characters that appear in the information correspond to very diverse profiles. national political figures are the most numerous, with % in italy and % in spain (see figure ). this aspect contrasts with the reduced presence of institutions, as mentioned above. thus, it is possible to [ ] . in italy, citizens account for % of the total number of items; in spain, they account for only %. these differences are equally visible in the presence of researchers or scientists, which in italy is % and in spain reaches %. the main characters that appear in the photographs on the covers, displayed by figure , show important differences between the two countries. celebrities or public figures are the ones that absorb the most attention, with % of the total in italy and % in spain. this aspect emphasizes the importance given to this type of profile in the information and awareness of the pandemic. citizens, with % in italy and % in spain, would be in second place. there is, therefore, a prominent role in italy, citizens account for % of the total number of items; in spain, they account for only %. these differences are equally visible in the presence of researchers or scientists, which in italy is % and in spain reaches %. the main characters that appear in the photographs on the covers, displayed by figure , show important differences between the two countries. celebrities or public figures are the ones that absorb the most attention, with % of the total in italy and % in spain. this aspect emphasizes the importance given to this type of profile in the information and awareness of the pandemic. citizens, with % in italy and % in spain, would be in second place. there is, therefore, a prominent role for anonymous people. national politicians, with % and %, respectively, would be in third place. patients (with % and %) and researchers or scientists (with % and %) hardly appear in the cover images. even if health centers are not the most prominent settings in pictures, we observed how, when looking at the physical spaces represented in the news, summed up in figure , we can observe similarities and differences. first, while italian newspapers offer an emotional representation of the crisis by granting an enormous importance to the representation of empty spaces (such as squares or symbolical touristic spots, like the trevi fountain in rome, that in a normal situation would be crowded), spanish news outlets completely avoid this option, that only account for % of the total pictures. in both cases, however, urban spaces are the most recurrent (with % in italy and % in spain). in addition, although their importance is not prominent, the spaces related to political life (congress, etc.) with % in italy and % in spain, have a significant presence in the cover photographs. health centers or health camps, with % and %, are other places that appear next to citizens' homes (with % and %, respectively). even if health centers are not the most prominent settings in pictures, we observed how, when portrayed, these spaces are emotionally charged to dramatize the tragedy. figure shows two examples of spanish newspapers el mundo y el país showing coffins of victims in the middle of the crisis. even if health centers are not the most prominent settings in pictures, we observed how, when portrayed, these spaces are emotionally charged to dramatize the tragedy. figure the covid- crisis has been a shocking reality that took most countries by surprise. italy and spain have been amongst the first in europe to be hit by the pandemic. thus, observing media behavior is especially interesting. to answer our research questions, we can state that the covid- crisis has been covered mainly in an informative way: the analysis of the two main newspapers in spain and italy allows to observe a predominance of informative journalistic genres. in particular, the predominant genre is the brief, short news items, which lack contextual information, and do not offer in depth information to the readership. however, the choice of images, that is to say the visual framing of the stories, seems to suggest an emotional turn. in other words, even if the predominance of informative genres, together with the the covid- crisis has been a shocking reality that took most countries by surprise. italy and spain have been amongst the first in europe to be hit by the pandemic. thus, observing media behavior is especially interesting. to answer our research questions, we can state that the covid- crisis has been covered mainly in an informative way: the analysis of the two main newspapers in spain and italy allows to observe a predominance of informative journalistic genres. in particular, the predominant genre is the brief, short news items, which lack contextual information, and do not offer in depth information to the readership. however, the choice of images, that is to say the visual framing of the stories, seems to suggest an emotional turn. in other words, even if the predominance of informative genres, together with the avoidance of openly emotional headlines might suggest that the analyzed newspapers have avoided sensationalism, both the mentioned visual framing and the increased presence of anonymous citizens and celebrities among the subjects, can be interpreted as an attempt to humanize the information pieces, emotionally charging them. in particular, it is important to stress out the high level of politicization of the crisis: politicians have been the most recurrent actors both in the information and in the pictures. this, as seen, seems contradictory to the scarce presence of institutions. this result, however, should not be of surprise since, as already pointed out by hallin and mancini [ ] , both spain and italy belong to the polarized pluralist model, in which party politics is predominant to institutional politics. moreover, both countries are characterized by high intensity political polarization, therefore the management of the crisis has been the source of harsh political conflicts between government and opposition, that has been reflected by the media. concretely, spanish media outlets are the ones that give a more political vision of the crisis. accordingly, we observed a trend to objectify the different news actions and events through the use of geographical entities. the use of physical enclaves (italy, spain, milan or madrid, for example) lead to a simplification or generalization of reality that can bias the reading and interpretation of what has happened. in the same vein, it is important to stress that both health personnel and researchers directly involved in the fight against the virus, have a negligible presence both in pictures and information. in conclusion, we can sum up that the protagonists of the pandemic are not those affected, or involved in the fight, but rather anonymous citizens, and especially celebrities and politicians. these results cannot be discussed without taking into consideration the general framework of the social responsibility theory. as pointed out by mcquail in his seminal work [ ] , media should accept and fulfil certain obligations to the society and should meet high professional standards of accuracy, truth, objectivity and balance. therefore, journalists and professionals should be accountable to the society reflecting and respecting diversity, pluralism as well as diverse points of view and rights of reply. applying these criteria to the specific field of health communication, defined by sixsmith et al. [ ] as the study and use of communication strategies to inform and influence individual and community decision that enhance health, encompassing health promotion, health protection, disease prevention and treatment, we see how media are pivotal to the overall achievement of the objectives and aims of public health. in this sense, media practitioners and their organizations should be in charge of delivering rigorous health information, aimed at creating awareness about people's health, to prevent diseases and encourage healthy living. one of the main requirements of good health journalism [ ] , thus, is to present evidence-based news with proper perspective, and without giving rise to sensationalism or alarm. as pointed out by many studies [ , ] journalism training, in order to cope with these new challenges, should lay special emphasis on these aspects, providing not only specific health journalism training, but developing specific media and information literacy training devoted to health issues. acknowledging the geographical limitations, our study allows a series of conclusions to be drawn, which, from a diagnostic perspective, may help both scholars and journalism practitioners and deepen on the behavior and reaction capacity of newspapers in front of important and tragic events such as planetary pandemic. from a scholarly perspective, our work is embedded in a stream of literature that considers media to play a crucial role in framing public debates and shaping public perceptions by selecting which issues are reported and how they are represented [ ] [ ] [ ] [ ] . even if, as said, our results are limited to spain and italy, we have shown that printed newspapers avoid the massive use of data or percentages, leaving live updates to on line media, concentrating on more informative and interpretative pieces. this, on the one hand suggest they still play a crucial role in molding public opinion by offering more interpretative content [ ] , on the other they directly and indirectly interact with digital media, in charge of giving more live information. for this reason, our results suggest that legacy media should still be examined to see how they influence/are influenced in their interaction with online media. in addition, we have pointed how in both countries the pandemic has been highly politicized. this result, stressing out the salience of the political factor in representing the pandemic, underlines the need for more comparative research, analyzing media portrayal in different contexts and how different media, embedded in different political and cultural contexts, have reacted. in particular, the current corona crisis, having a global reach and effect, could be an ideal occasion to compare media behavior in different countries to observe the existence of similarities and differences, and to which extent different political cultures and political systems modify media reactions to a pandemic, following and proving hallin and mancini's model [ ] . in addition, even if reference newspapers seem to opt for an informative approach, their visual framing and the choice of images (i.e., empty places) emotionally charges the information. in this sense, besides the need for more comparative research to prove that this is a global trend, from a practical standpoint, our results align with the findings of previous studies [ , ] , stressing the need to promote media and information literacy, not only among citizens, but also among media professionals. as demonstrated, both the predominance of short news items, which lack contextual information, and the visual framing can make the process of processing the essence of information messages difficult, making the susceptibility of many people to misinformation as risky as susceptibility to the virus itself. thus, as previously pointed out, in order to achieve rigorous and responsible health information, journalism training should not only provide specific health journalism training, rather media and information literacy skills should be developed in this field. in the same vein, media and information literacy campaigns geared towards citizenship should focus specifically on health issues, since, as the current crisis has showed, health is one of the most sensitive topic when it comes to quality information to avoid the risk of misinformation. accordingly, the current corona crisis underlines the necessity of a reform of science communication. this pandemic has underlined that the media often does not offer rigorous scientific information, prioritizing a (possibly) misleading humanization of news. first, media professionals should be trained and help to implement fact-checking functions, particularly to debunk fake news, misinformation and disinformation on health subjects. moreover, a renewed cooperation and a greater communication between media, health experts, academia and policy makers is essential for improving quality of health news. for this, academic institutions, health bodies, and organizations engaged in scientific and medical research need to improve their communication with media, understanding the need to explain research findings, policies and trends to media professionals, who are in charge of "translating" and diffusing them to citizens. on the other side, media should rely on competent scholars from a wide range of disciplines, interacting, assessing and dialoguing with journalists in order to provide readers, and ultimately citizenship, with a better understanding of science-related issues such as a pandemic. la prensa sensacionalista y los sectores populares systematic literature review on the spread of health-related misinformation on social media effects of health information dissemination on user follows and likes during covid- outbreak in china: data and content analysis covid- related misinformation on social media: a qualitative study from iran social networks' engagement during the covid- pandemic in spain: 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control of communicable diseases in europe: current evidence, practice and future developments health communication: the responsibility of the media in nigeria. spec tejedor calvo, s. analysis of journalism and communication studies in europe's top ranked universities: competencies, aims and courses análisis de los estudios de periodismo y comunicación en las principales universidades del mundo. competencias, objetivos y asignaturas this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -s hf bo authors: palladino, raffaele; bollon, jordy; ragazzoni, luca; barone-adesi, francesco title: excess deaths and hospital admissions for covid- due to a late implementation of the lockdown in italy date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: s hf bo in italy, the covid- epidemic curve started to flatten when the health system had already exceeded its capacity, raising concerns that the lockdown was indeed delayed. the aim of this study was to evaluate the health effects of late implementation of the lockdown in italy. using national data on the daily number of covid- cases, we first estimated the effect of the lockdown, employing an interrupted time series analysis. second, we evaluated the effect of an early lockdown on the trend of new cases, creating a counterfactual scenario where the intervention was implemented one week in advance. we then predicted the corresponding number of intensive care unit (icu) admissions, non-icu admissions, and deaths. finally, we compared results under the actual and counterfactual scenarios. an early implementation of the lockdown would have avoided about , covid- cases, , non-icu admissions, , icu admissions, and , deaths, corresponding to % ( %ci: % to %), % ( %ci: % to %), % ( %ci: % to %), and % ( %ci: % to %) reduction, respectively. we found that the late implementation of the lockdown in italy was responsible for a substantial proportion of hospital admissions and deaths associated with the covid- pandemic. in early january a novel strain of coronavirus, sars-cov- , a virus which follows a human-tohuman transmission, was identified in the hubei province of china as the causative agent for a new disease later defined as coronavirus disease (covid- ), a respiratory disease which is often characterized by influenza-like symptoms but which can also evolve ( - % of the cases) into acute respiratory distress syndrome, or even sepsis, and multi-organ failure which might lead to death [ ] . starting from an outbreak in china, the scale of the emergency has rapidly grown globally, leading the world health organization (who) to declare the pandemic status on march th, when many countries had already introduced unprecedented physical distancing and containment measures to various extents [ ] . as of may th, almost six million of covid- cases and , deaths have been recorded worldwide [ ] . the effect of containment measures in curbing the covid- epidemic varied among countries [ ] [ ] [ ] [ ] [ ] [ ] . while a combination of stringent policies together with wide early-phase testing coverage and effective contact tracing strategies was effective in halting the covid- epidemic in countries such as mainland china, hong kong, and south korea, in others the epidemic slowed only recently [ , , , ] . factors explaining differences in time patterns might be found in the readiness of government responses and in the degree of compliance of the population to the implemented policies [ ] [ ] [ ] [ ] [ ] . italy, which has passed , confirmed cases and , deaths [ ] , is one of the most affected countries in the world so far and the first in europe where the public health emergency rapidly escalated at the national level. on march th, the government ordered a national lockdown, a measure including: (a) strict home confinement of the entire population; (b) closure of all non-essential commercial activities; (c) mobility restrictions related to the involved municipalities [ ] . the lockdown remained in place until may rd, when a slowdown of the epidemic in the different italian regions allowed its release [ ] . compared with china, italy introduced containment measures later in the course of the national epidemic, about one month after the first covid- case was reported in the country. italy's lockdown was enforced days after the one in hubei, when normalizing for the time when the outbreak hit cases in both countries [ ] . this prompted a debate, in italy and abroad, on the causes of such a delay and on how many covid- cases could have been avoided, had the lockdown been implemented earlier [ ] . a formal investigation into possible government mismanagement of the covid- crisis is currently ongoing [ ] . the aim of this study was to evaluate the health effects of late implementation of the lockdown in italy. for this reason, we estimated the number of deaths and hospital admissions for covid- that would have occurred if the lockdown had been implemented one week earlier than it was actually enforced. in the present analysis we used data on the daily number of covid- cases, hospitalized patients, and deaths recorded in italy from february th, the first day national data were made available, to may rd, the last day of implementation of the national lockdown. figures were provided by the official website of the italian department of civil protection [ ] . first, we evaluated the effect of the italian lockdown using interrupted time series (its) analysis. we modeled the time-series of daily new cases, y t , using the following quasi-poisson regression model, accounting for the possible overdispersion of data: where t is the time elapsed since the start of the study; t is the time elapsed since the implementation of lockdown (set to before the lockdown); x is a dummy variable indicating the pre-lockdown period (coded ) or the post-lockdown period (coded ); y is the logarithm of the number of new cases at time t; α is the intercept of the model; β represents the trend of new cases before the lockdown; β is the step change following the lockdown; β is the slope change following the lockdown; and e t is the error term of the model. preliminary analysis of the data suggested that no adjustment was required for autocorrelation of the error terms e t . we also assumed a two-week lag between the implementation of the lockdown (march th) and the start of its effects (march rd), to take into account the covid- incubation period and the diagnostic delay after symptoms onset [ ] . second, we evaluated the effect of an early lockdown on the trend of new cases, creating a counterfactual scenario where the lockdown was implemented one week in advance (i.e., on march nd instead of march th). third, based on the expected number of new cases, we predicted the corresponding number of intensive care unit (icu) admissions, non-icu admissions, and deaths, using a previously published mathematical model [ ] . briefly, the model simulates the progress of infected individuals between different compartments during the course of an epidemic: isolated at home, admitted in a non-icu ward, admitted in icu, recovered, dead. finally, we compared the number of hospital admissions and deaths under the actual and counterfactual scenarios. all the analyses were performed using the r software (r core team ( ). r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. url http://www.r-project.org/). from february th to may rd, , cases of covid- were observed in italy. there was an exponential increase in the number of new covid- cases until march nd, followed by a sharp reduction (table ; figure ). table reports estimated coefficients, while related predictions are plotted in figure together with the expected number of new cases under the counterfactual scenario. on may rd, the number of new cases under the counterfactual scenario was less than half than that estimated under the observed scenario. and deaths under the actual and counterfactual scenarios. all the analyses were performed using the r software (r core team ( ). r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. url http://www.r-project.org/). from february th to may rd, , cases of covid- were observed in italy. there was an exponential increase in the number of new covid- cases until march nd, followed by a sharp reduction (table ; figure ). table reports estimated coefficients, while related predictions are plotted in figure together with the expected number of new cases under the counterfactual scenario. on may rd, the number of new cases under the counterfactual scenario was less than half than that estimated under the observed scenario. figure shows differences in the total number of cases, non-icu admissions, icu admissions, and deaths under the two scenarios. the plots show that an early implementation of the lockdown would have averted about , covid- cases, , non-icu admissions, , icu admissions, and , deaths. on the relative scale, this corresponds to a reduction of % ( %ci: % to %), % ( %ci: % to %), % ( %ci: % to %) and % ( %ci: % to %), respectively (table ). figure shows differences in the total number of cases, non-icu admissions, icu admissions, and deaths under the two scenarios. the plots show that an early implementation of the lockdown would have averted about , covid- cases, , non-icu admissions, , icu admissions, and , deaths. on the relative scale, this corresponds to a reduction of % ( %ci: % to %), % ( %ci: % to %), % ( %ci: % to %) and % ( %ci: % to %), respectively (table ). moreover, the maximum hospital demand would have been much lower under the counterfactual scenario. the peak number of non-icu admissions would have been , rather than , (− %; % ci: − % to − %). a similar reduction would be expected for icu admissions as well ( vs beds; − %, %ci: − % to − %). moreover, the maximum hospital demand would have been much lower under the counterfactual scenario. the peak number of non-icu admissions would have been , rather than , (− %; % ci: − % to − %). a similar reduction would be expected for icu admissions as well ( vs. beds; − %, %ci: − % to − %). in italy, the covid- pandemic led to the implementation of containment measures at the highest level, with a national lockdown enforced on march th, . despite this, by the time the epidemic curve started to flatten, the health system had already exceeded its capacity in different areas of the country, raising concerns that the public health response was indeed delayed. we found that if restrictive measures had been enforced one week earlier, this would have had a significant impact on the evolution of the epidemic in terms of hospital admissions and deaths. by may rd, we estimated that there would have been a % reduction of covid- cases and % of confirmed deaths would have been averted. the covid- pandemic is threatening public health preparedness and medical response capacity globally. our findings add to a growing body of evidence supporting the need for rapid responses to contain the current covid- pandemic and similar threats that could occur in the future [ , , ] . besides italy, other european countries profoundly impacted by the pandemic such as spain, france, and the uk, as well as the us, also hesitated to enforce containment measures in a timely manner [ ] , with a consequent health, economic, and societal impact that still needs to be fully assessed. lack of collaboration between national health systems, as well as delayed communication by international organizations might be some of the factors explaining the late response to the emergency. public health intelligence at both the international and national level should identify all barriers and challenges associated with the current pandemic to improve response in the future. this is particularly necessary in this phase of the pandemic, as a possible second wave of infections is expected in the next months. as most european countries are gradually lifting restrictions, there is a need to enhance the existing surveillance systems and develop strategies for timely reactions to a new increase in the number of infections. to our knowledge, this is the first study assessing the impact of the delay in the implementation of containment measures on the spread of covid- epidemic, and the associated burden on the health system. however, several caveats merit discussion. first, analyses were conducted using publicly available data on confirmed cases, which did not account for the proportion of undetected cases, estimated to be high in italy, especially in the regions more affected by the epidemic [ ] . this means that, on the absolute scale, our estimates should be regarded as conservative. on the other hand, assuming that the timing of the lockdown is not associated with the detection rate, which seems plausible, the relative estimates provided are expected to be unbiased. second, we did not take into account how the different hospital demand under the two scenarios affected the treatment of critical patients. in the actual scenario, hospitals in the worst-hit areas often exceeded their capacity and experienced ventilator shortages [ , ] . this affected their capacity to deliver effective care to all critical patients. on the other hand, under the counterfactual scenario the maximum hospital demand would have been about % lower. for this reason, we probably underestimated the positive effects of an early lockdown in terms of reduced icu admissions and deaths. the covid- pandemic has been requiring unanticipated and extraordinary containment measures, which has raised concerns about public health preparedness of health systems globally. the late implementation of the lockdown in italy was responsible for a substantial proportion of hospital admissions and deaths associated with the covid- pandemic. understanding factors contributing to such a delayed response is fundamental to strengthen public health preparedness and timing in response capacity. clinical characteristics of coronavirus disease in china modelling the covid- epidemic and implementation of population-wide interventions in italy impact assessment of non-pharmaceutical interventions against coronavirus disease and influenza in hong kong: an observational study interventions to mitigate early spread of sars-cov- in singapore: a modelling study the effect of human mobility and control measures on the covid- epidemic in china sars-cov- and europe: timing of containment measures for outbreak control association of public health interventions with the epidemiology of the covid- outbreak in wuhan, china department of civil protection emergenza coronavirus: la risposta nazionale the guardian italians struggle with 'surreal' lockdown as coronavirus cases rise covid- epidemic in italy: evolution, projections and impact of government measures the new york times italy's virus shutdown came too late. what happens now? available online npr prosecutors question italy's top leaders over coronavirus response report of the who-china joint mission on coronavirus disease (covid- ) predicted effects of stopping covid- lockdown on italian hospital demand report -estimating the number of infections and the impact of non-pharmaceutical interventions on covid- in european countries key: cord- - epvo authors: martellucci, cecilia acuti; sah, ranjit; rabaan, ali a.; dhama, kuldeep; casalone, cristina; arteaga-livias, kovy; sawano, toyoaki; ozaki, akihiko; bhandari, divya; higuchi, asaka; kotera, yasuhiro; fathah, zareena; roy, namrata; ur rahman, mohammed ateeq; tanimoto, tetsuya; rodriguez-morales, alfonso j. title: changes in the spatial distribution of covid- incidence in italy using gis-based maps date: - - journal: ann clin microbiol antimicrob doi: . /s - - -z sha: doc_id: cord_uid: epvo nan massive spreading of the pandemic coronavirus disease (covid- ) in different continents [ , ] , have been observed [ ] . analyses mostly focused on the number of cases per country and administrative levels, multiple times without considering the relevance of the incidence rates. these help to see the concentration of disease among the population in terms of cases per , inhabitants. even more, using geographical information systems (gis)-based maps, stakeholders may rapidly analyze changes in the epidemiological situation [ ] [ ] [ ] [ ] . although the epidemic of covid- caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) started in italy on january , , no reports on the use of gis-based maps have been published to analyze the distinct differences in incidence rates across its regions and provinces during the last months. for these reasons, we have developed epidemiological maps of incidence rates using official populations, by regions ( st administrative level of the country) and provinces ( nd administrative level), for covid- in italy using gis. surveillance cases data of the cumulative number at march , april , and june , , officially reported by the italian health authorities were used to estimate the cumulated incidence rates on those dates using reference population data on sars-cov- confirmed infections (cases/ , pop) and to develop the maps by regions and provinces, using the gis software kosmo ® . , as performed in previous related studies [ , ] . starting on march , , the region of lombardy, together with additional northern and central provinces, in piedmont, emilia-romagna, veneto, and marche, they were put under lockdown. on march , , the government extended the lockdown measures to the whole country. up to march , , after days of epidemics, , cases of covid- were reported in the country, for a cumulated rate of . cases/ , population, reaching , cases during april , , for a rate of . , and , cases during june , , for a rate of . . all the regions of the country have been affected, with rates ranging from . (calabria) to . cases/ , population (aosta valley/vallée d' aoste) (june , ) ( fig. ) . higher diversity is found in provinces, where incidence rates ranged from . (sud sardegna, sardinia) to . (cremona, lombardy) (june , ) ( table ) . at lombardy are located five of the top ten provinces with higher incidence rates fig. ) , with considerable increases and changes from march , , to june , , in approximately months (fig. , table ). cremona (lombardy), piacenza (emilia-romagna), and lodi (lombardy) have become in the geographic core of the cumulated incidence rate of covid- in the north of the country and italy (fig. ) . from the gis-based maps, it is clear that spreading in the country is occurring from north regions and provinces such as lombardy. on march , , most of the southern regions were not affected (fig. ) , but approximately a month later, all of them reported covid- cases (fig. ) , including the insular regions of sicily and sardinia. while the change between april and june , has been %, there is still a concern in the country, mainly because, in this time, the number of deaths has reached over , deaths ( . %). italy reached the top of countries with the highest number of reported covid- cases, now is the ninth country in cumulated cases. it is the fourth in the european region, after russia, the united kingdom, and spain. italy represented one of the most significant sources of imported cases for other continents, as is the case of latin america, that received their first cases from milan, lombardy [ ] [ ] [ ] . patient of italy (it was not possible to find patient ) was discovered on february , , when a -yearold man from the city of codogno had shown up at the hospital. since that date, two large clusters of outbreaks have spread first in northern italy, later all over the country (fig. ) [ ] . cases are multiplying, and the national healthcare system is collapsing [ ] [ ] [ ] . many regions are increasing intensive care beds, revolutionizing entire hospital wards. in italy, the coordination of the swabs is managed regionally. once the epidemic began, for example, the veneto region started immediately with active surveillance, i.e., on asymptomatic, and this contained the spread of the virus compared to other northern regions. the health system is indeed regionalized, and dispositions of the ministry of health are translated into multiple regional decrees and regulations, often at different timings [ ] . many regions, for example, adopted evolving criteria for testing and diagnosis, according to dispositions from the central government, but also to test capacity, which was heavily reliant on the availability of reagents. samples were collected either in healthcare facilities, in provisional collection points, or even in people's houses, depending on the region and the phase of the pandemic. the epitome of these differences is the two most heavily affected regions, lombardy and veneto. lombardy hospitalized, even cases with relatively modest symptoms, causing numerous nosocomial outbreaks ( % of infections were among health professionals until march). veneto, instead, deployed widespread testing since the beginning, maintaining disease management as much as possible at the primary health care level [ ] . the healthcare workers are facing covid- pulling h shifts in critical situations with minimal to nonexistent personal protective equipment (ppe) [ ] [ ] [ ] . lacking ppe led both many healthcare workers to become covid- positive ( ), and to the death of several doctors ( ; about % of the total cases; march , ) [ ] . as observed in the gis-based maps, the covid- spreading in the country has been significant and moving from north to south across the time, with provinces reaching more than cases per , inhabitants ( fig. ) [ ] [ ] [ ] . differences in the incidence by regions would be related to different social and economic factors. such as people who travel abroad, for whom there is a sharp difference between northern regions (about % of travelers) and central and southern regions (about %). or net income at the household level, which is ranging from , € in the north-east to , € in the south [ ] . additionally, as has been recently suggested, climatic conditions could also influence the transmission of sars-cov- [ ] . testing capacity increased over time. while some degree of ascertainment bias is inevitable, the number of swabs performed nationally stabilized around , per day in mid-april when the peak in the number of active cases was registered (fig. ) . from that point onwards, daily cases only decreased, consistently with the impact of the lockdown imposed in early march. besides, and most importantly, while it is true that the epidemic might have gone undetected for some time before case was discovered in codogno, the growth in icu beds demand for subjects with respiratory failure that ensued in the following weeks is most likely explained by a substantial increase of cases. considering the limitations of diagnostics and the asymptomatic cases, these figures would be many times more. further characterization studies should include multiple gis-based maps with other variables at the regions and provinces levels such as deaths, hospitalizations, and icu rates per population to understand better the critical situation of the country and its administrative levels. going global-travel and the novel coronavirus history is repeating itself, a probable zoonotic spillover as a cause of an epidemic: the case of novel coronavirus coronavirus disease -covid- the first novel coronavirus case in nepal an interactive web-based dashboard to track covid- in real time mapping the changes on incidence, case fatality rates and recovery proportion of covid- in afghanistan using geographical information systems mapping the incidence of the covid- hotspot in iran-implications for travellers covid- in latin america: the implications of the first confirmed case in brazil lancovid: clinical features of the first cases and a cluster of coronavirus disease (covid- ) in bolivia imported from italy and spain successful recovery of covid- pneumonia in a patient from colombia after receiving chloroquine and clarithromycin covid- is not just a flu learn from italy and act now coronavirus disease (covid- ) in italy case-fatality rate and characteristics of patients dying in relation to covid- in italy covid- and italy: what next? what other countries can learn from italy during the covid- pandemic istat data warehouse. the temperature and regional climate effects on communitarian covid- contagion in mexico throughout phase publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations none.authors' contributions ajrm, conceptualization; data curation; formal analysis; methodology; software; writing-original draft; writing-review and editing. cam, data curation; formal analysis; methodology; writing-review and editing. rs, aar, kd, key: cord- -hop fxg authors: dattoli, giuseppe; palma, emanuele di; licciardi, silvia; sabia, elio title: on the evolution of covid- in italy: a follow up note date: - - journal: nan doi: nan sha: doc_id: cord_uid: hop fxg in a previous note we made an analysis of the spreading of the covid disease in italy. we used a model based on the logistic and hubbert functions, the analysis we exploited has shown limited usefulness in terms of predictions and failed in fixing fundamental indications like the point of inflection of the disease growth. in this note we elaborate on the previous model, using multi-logistic models and attempt a more realistic analysis. the covid- pandemic disease is bringing elements of novelty baffling for politicians, m d's and epidemic analyzers. it has already been stressed that, in absence of any anti-viral strategy, the only defense towards the spreading of the illness is the nation lockdown, a policy difficult to implement in italy. it has undergone different phases and lack of effective decisions, while the infection was raging in italy and attacking the rest of europe. the public health structures have suffered from an un-precedent stress in terms of people to care and of casualties. regarding this last point, in italy the percentage of deaths seems to be larger than abroad, but this might be result of absence of an accurate sampling of the positive cases. the lack of informations on the infecting capabilities of the virus and other uncertainties associated with a clear understanding of how the infection developed during the early stages of its spreading and a poor knowledge on the real entity of the "submerged" cases as well, made any attempt to fix the peak of the distribution of the infected/day denied by the facts themselves. in a previous note, by the present group of authors [ ] , two paradigmatic tools have been exploited to study the evolution of the illness: a) the logistic function [ , , ] (lf ) (fig. ) describing the evolution of a given population n (τ ) of n individuals at τ = (in the present case infected people) in an environment with carrying capacity k and growth rate r, is specified by where τ is the time, measured in some units to be specified. b) the hubbert curve [ ] (hc), namely the derivative of the lf , yielding the number of infections per unit time, i.e. it is a bell shaped curve ( fig. ) with the maximum located at in correspondence of which the infected rate is corresponding to a total number of infected the officially presented data on march upset this "reassuring" scenario and modified fig. a as reported in fig. b . the latter being characterized by an apparently anomalous behavior, dominated by an increase which mocks any every forecast based on a simple logistic model. as already stressed in ref. [ ] , the analysis of the data at national territory level had been developed with the bias that the barycenter of illness was shifted towards the north of italy. what was going to happen in those days has been that the cases from the rest of italy were surpassing those in lombardia (see fig. ). this imposes a new scenario in terms of statistical analysis as discussed in the forthcoming sections. in ref. [ ] we underscored the possibility that the logistic model might be not suitable for a description at national level in view of various in-homogeneities of the distribution of the infection and for the delay in the propagation, presumably also mediated by the massive transfer of people from north to south of italy. before considering a more elaborated point of view, we consider the data from "regione lombardia" only, where we have reported the relevant logistic curve (fig. ). it should be noted that the curves are relevant to sum of casualties and infected. the fit of the hubbert with % of confidence band is given in fig. , which displays three possible scenarios for the behavior of the infected and deceased/day. according to the previous forecasting, the peak should be reached in the next days. the lower curve predicts a peak by the end of march. in order to extend the analysis to the national territory, we have elaborated a different strategy using the bi-logistic analysis. we have therefore considered the incoherent sum of two logistics [ , ] . they are characterized by different growth rates and carrying capacities. the time differences τ − τ represents the time lag between the starting point of the two evolutions in fig. we have reported the results of a parameters fit (n ,i , r i , τ i ), i = , , and assuming a lag time of days, corresponding to the difference in time between the (official) start of the disease and the crossing time between the two curves in fig. . the fit displays almost equivalent n and k, but different growth rates. regarding the associated hubbert curve we obtain a plot exhibiting two peaks, with a delay between the two. this is a possible scenario, albeit questionable since it assumes that the rest of italy starts to contribute to the counting after a significant time lag. an alternative strategy is that summarized in figs. and , in which we have treated lombardia and the rest of italy as separated entities. the fit has been done using two independent logistics which have been summed "incoherently", thus getting two distinct hubbert curves and the relevant sum, exhibiting the peak in the next few days. in this follow up we have exploited a larger number of data on covid spreading and evolution in italy, to gain a more accurate scenario on the present status and how it may evolve. regarding this last point, many caveats are in order mainly with the understanding of the consistency of the submerged positives and how they may the data from the single italian regions may be, within this respect, instructive. in figs. - - we have reported the hubbert curves of a selected sample of regions . the plots display an almost coherent scenario with a slow decrease of the emergency in the next months (may). it is worth noting that regarding some regions (for example basilicata and sicilia) the situation is still evolving. the available data do not allow a reliable analysis in terms of logistic and hubbert curves (the % confidence interval is extremely wide) and no peak emergency can be foreseen. this forecast may be even optimistic and new outbreak of infections, which may spontaneously germinate if restrictions are not properly followed or if not surveyed cases will emerge as acute diseases. data from "protezione civile" https://github.com/pcm-dpc/covid- /blob/master/dati-regioni/dpc-covid -ita-regioni.csv . a final element of discussion comes from fig. where we have reported the worldwide and italian evolution of the covid cases/day , a kind of bi-logistic pattern is evident, which supports the ideas put forward in this and in the previous note. what we have attempted here is a little more than the picture of the situation, the lesson we may learn from the present pandemia is important but will be completely understood when not only italian but the worldwide pattern will be clarified. probably long time after the end of emergency. a note on the evolution of covid- in italy the origin of logistic regression logistic equation, from mathworld-a wolfram web resource quasi exact solution of the fisher equation hubbert's peak: the impending world oil shortage, published by a primer on logistic growth and substitution:the mathematics of the loglet lab software the work of dr. s. licciardi was supported by an enea research center individual fellowship.the authors express their sincere appreciation to dr. ada a. dattoli for her help in understanding the biological basis of the infection. key: cord- -mapfi f authors: chintalapudi, nalini; battineni, gopi; amenta, francesco title: covid- virus outbreak forecasting of registered and recovered cases after sixty day lockdown in italy: a data driven model approach date: - - journal: j microbiol immunol infect doi: . /j.jmii. . . sha: doc_id: cord_uid: mapfi f background: till march , , covid- cases were confirmed in italy including , deaths which explains how worst the epidemic has affected the country. after the announcement of lockdown in italy on march , situation was becoming stable since last days of march. in view of this, it is important to forecast the covid- evaluation of italy condition and the possible effects, if this lock down could continue for another days. methods: covid- infected patient data has extracted from the italian health ministry website includes registered and recovered cases from mid february to end march. adoption of seasonal arima forecasting package with r statistical model was done. results: predictions were done with . % of accuracy for registered case models and . % of accuracy for recovered case models. the forecasting of infected patients could be reach the value of , , and recovered cases could be registered value of , at end of may. conclusions: this study highlights the importance of country lockdown and self isolation in control the disease transmissibility among italian population through data driven model analysis. our findings suggest that nearly % decrement of registered cases and % growth of recovered cases will be possible. in the last weeks of , when the world was ready to welcome , many local hospitals in wuhan, china, were reported unusual number of patients who comes with severe pneumonia without knowing cause and not responds to any kind of vaccine or medicine. besides, these cases were further increased because of human to human transmission, and doctors confirmed that this unknown disease had similar epidemic of severe acute respiratory syndrome (sars) in and the agent causing this disease was recognized as a corona virus. sooner or later world health organization (who) named this virus as novel corona virus(ncov- ) or covid- . by early january , about suspected cases were identified in province of wuhan. at the beginning, the disease started as a local epidemic of china, but subsequently it quickly escalated all over the world, being transmitted by international travelers. at the present, there is no any scientific evidence for where it has originated. currently it is confirmed as a global pandemic and dozens of western countries are alarmed by this severe outbreak of corona virus. today ( march ), , covid- confirmed cases are including of , deaths were reported worldwide. more than countries had been affected, with major outbreaks in the united states (us), italy, spain, china, iran, france, and others. we can imagine the gravity of this pandemic situation by looking into these facts. in italy, death toll from corona virus jumped over , deaths since end of february and is still ongoing, whereas the number of infected cases from usa surpass about more than half million population. due to easy spreading of covid- , most national governments including italy announced lock down and people are not allowed to come out from their homes. as of this, nearly . billion global population went into self isolation. the world health organization (who) confirms that the incubation period (i.e., time elapse between exposure of pathogenic organism to symptom first appearance) of covid- outbreak is days. the basic reproduction number "r naught" or r is a contagiousness indicator or infectious transmissibility of parasite agents. in epidemic sciences and health literature r is highly encountered to understand a slow outbreak of disease. for instance, if r is equal to one, this means that average person who got disease could transmit over single individual. according to who, r for covid- is confirmed around . e . . recent modeling of r from italy confirmed by lombardy researchers at early outbreak in between . to . . lombardy region is considered as epicenter of corona virus outbreak in italy. , most people were died here than anywhere else in the world and later virus spread all over country with more than , confirmed cases. on march , the italian prime minister mr. g conte had given announcement of imposed national quarantine, restricting the people movement unless for health emergency or unavoidable work needs. statistics become consciously optimistic and daily number of new registered are becoming constantly stable since last week of march. however, because of both human-to-human and asymptomatic transmission of covid- , it is important to understand virus reproduction cases after this italy lock down. therefore, we developed a data driven model to forecast covid- outbreak daily registered cases and recovered cases, also estimated the chance of low infected patient cases for next days of italy quarantine. patient data were obtained from the official website of the italian health ministry (http://www.salute.gov.it/ nuovocoronavirus) that reports latest information of covid- infection in italy. the data model development was done based on the update of march . patient data consisted of three groups, namely registered cases, recovered cases and death cases. in this study, we excluded the death cases information and forecasted possible number of register and recovered cases in next two months. rather than observing entire data, we only considered observation from february because after the first two cases registered on january , no more epidemic was reported in italy till mid of february. fig. is the plot of total number of registered and recovered cases trend varied on daily basis. r is one of the tools that has relevant importance for epidemiologists, and had quick search function can enable users to get many r libraries devoted to outbreak management and analysis. auto-regressive integrated moving average (arima) and specified by three orderly parameters: (p, d, q); where 'p' is an auto aggressive referred to use of ancient values in model,'d' is the difference degree of integrated i(d) component, and 'q' is model error which is combination of last error terms e t . by summing above parameters with non-seasonal arima model can be written as linear equation mentioned in equation ( ). the model equation above mentioned as assumed to be a non-seasonal series. in this study, model specified by two sets of parameter order: (p, d, q) and (p, d, q) m (i.e., describes the seasonal component of m time intervals). the mathematical equations of arima model were explained in appendix section. to calculate the covid- re-production cases among italy patients, we imported 'autoarima' packages in r. after model exported, simple time series analysis was conducted to understand trends of corona epidemic in italy. the data available from italian health ministry website is obtained as day-to-day statistics. past -days patient data were recorded on excel sheet. the command read_excel ("data") was used to read the excel sheet. when working with time series in r, the data were converted in a time series (ts) for the number of registered cases per day from february to march mentioned as: days patient data from february (i.e., where serious outbreak was about to originated) to march with one day frequency was considered (fig. ) . the plots revealed that the trend in case registered at italian hospitals was going upwards and peak number of corona cases was registered in the last two weeks of march (fig. ) . this might be caused because of most people are traveled to home lands through public transports before lockdown was officially announced. through this migration of people, virus could spreads through and expose the symptoms on or after incubation period. in view of this, we conducted simple forecasting of covid cases if the same trend has been continued for two months. we applied 'autoarima' package in r to evaluate the values of (p, d, q) and forecaste the reproduction of infected cases. two arima models of covid- daily registered and recovered cases were designed. the possible residuals for these two models to understand the case variance were plotted and statistical analysis was performed using 'r' version . . . for data fitting in arima model to develop a model for covid- for both registered and recovered cases, we performed the commands mentioned below. install. packages("forecast") library(forecast) library(readxl) worldcovid <-read_excel("italycovid .xlsx") view(worldcovid ) tsworldcovid <-ts (italycovid $'daily registered cases', frequency z ,start z c( / / , )) tsworldcovid <-ts (italycovid $'daily recovered cases', frequency z ,start z c( / / , )) plot(tsworldcovid ) the -days covid- forecasting graphs of register along recovery cases (fig. ) , and normalized qq plots were computed (fig. ) . table presents the model outcomes and accuracy parameters. the probability of new positive cases and recovered cases in italy for next two months based on available data were computed. it is evident from fig. , the day forecasting of infected cases might rise in between the range of , e , , and recovered cases could increased in between the range of , e , with ci of e %. the regressive distribution of patient cases while two plots had observed to estimate the fitting accuracy. the model validation was assessed by prediction errors. based on the arima model accuracy evolution of covid- italian epidemic data on mentioned time period, we considered mean absolute prediction error (mape) parameter. the accuracy (acc) is defined in equation ( ); acc % z -mape * ( ). the models of arima( , , ) registered, and arima( , , ) recovered cases are validated with an accuracy of . %, . % respectivly. we used existed covid- epidemic data of italian patients to evaluate the probability of infected and recovered pateint number after having -day country lockdown. simple automatic forecasting package (autoarima) of 'r' was applied to conduct predictive modelling. our data driven model analysis highlights the necessity of country lockdown and self isolation to control disease transmissibility among italian population at the moment. at the present, italy is becoming the worst epidemic corona outbreak center. on march , towns in north italy announced quarantine after result of deaths and positive cases. unfortunately, in consequence of many italian citizens continued their daily life routine irrespective of outbreak results epidemic spread all over the country. after about one week, the italian government announced more than positive cases with deaths. on march the italian prime minister announced country lock down and strictly passed regulations to close malls, educational institutions, and sport events in order to stop infection among the other citizens. as mentioned, due to extreme characteristic of covid- is not expose the immediate symptoms while in the incubation time. after italy's lockdown, government officials make sure that people were at home. all national administration websites encourages companies to offer free online services. educational institutions and universities involved elearning methods, any data or publications on covid- made available for free to general public. covid- response team also conducting screening tests for domicile or long stay in high hit areas like north italy provinces. hospitals and medical centers are successfully handling patient flow to local hospitals and addresses individual issues about bed facilities, overcrowding in emergency departments, and patient transfer to other specialized facilities. all these critical circumstances were considered to understand what exactly happened in between the period of lockdown ( march ) announcement and incubation period (possibly march ). it can be observed in the fig. , the residual plot of positive covid- cases during the given period. from the plot, it is clear that the first two weeks trend seems normal and after march , a huge spike in case variance can be observed (i.e., to th days after quarantine had begun). one positive sign of this covid- epidemic in italy is after having established isolation, there is a significant growth of recovered case number, particular with last weeks of march (fig. ) . this could be because of the increased availability of medical devices, medications and health professionals in the most affected areas that might affect lowering of pandemic rates. at present, the italian citizens are also taking more preventive measures and maintaining social distancing to control speed of infection. as a result, disease transmission is expected to be reduced in the near future. preliminary results of this study suggest that if italian government and citizens could continue to be quarantined for another two months there could be chance of low tendency rate in infective cases. predictions mentioned that another , infected cases might be the registered in days which is lower than last days. arima models can forecast the simple up and downs and more predictive than regressive models without change in the overall trend. it is because arima can only look back the data of dependent variables (i.e, registered and recovered cases). this represents a primary limitation of this study. secondly, due to unwillingness to join in hospital, some confirmed cases are not ready to inform the medical authorities. this measure could affect the natural transmission of disease to family members which will also affect the study outcomes. finally, used data was retrieved from official italian health ministry websites, if any delay or mismatch of data reporting could results incorrect forecasting. covid- is a severe pandemic that all countries are facing. this results about half of the global population went into lockdown. at the present, italy is facing serious epidemic of positive and mortality rates. we estimated an increase in the size of registered cases and recovered case number population if the present lockdown could continue for another two months. results of this study indicate that nearly % of decay in positive cases and % of growth in recovered cases could be possible. in addition, present government taking some serious contaminant measures such as suspending training sessions of sports persons, professionals, and non professionals. all emergency issues remained same including to prohibit natural persons to move with public and private means of transport. advertising of prevention measures such as hand washing, mask wearing and disinfection was done continuously through national media which is largely influences the reproductive number of corona virus cases. the future of covid- diffusion in italy will largely depend on government regulations and motivation to carry self isolation of individual citizens. nc: data analysis, methods, results and study design; gb: manuscript preparation and statistical analysis; fa: final revision and study approval. distribution of the covid- epidemic and correlation with population emigration from wuhan, china severe acute respiratory syndrome (sars) which countries are under lockdown -and is it working complexity of the basic reproduction number (r ) critical care utilization for the covid- outbreak in lombardy correspondence estimation of covid- outbreak size in italy distance measures for effective clustering of arima time-series variations of qeq plots: the power of our eyes! arima models. in: time series: a data analysis approach using r time series forecasting using a hybrid arima and neural network model what can europe learn? j world news j the guardian the response of milan's emergency medical system to the covid- outbreak in italy forecasting with limited data: combining arima and diffusion models time series modelling of water resources and environmental systems this work was supported by institutional funding of the university of camerino, italy. dr nalini chintalapudi and dr gopi battineni were recipients of phd bursaries from the university of camerino. no author does not have any conflicts of interest. autoregressive integrated moving average (arima) model is aims to capture the auto correlation in the series modeling, and generally to do forecasting.arima model can completely be summarized by three parameters; p: the number of autoregressive terms, d: number of non seasonal differences, and q: number of moving terms. these three parameters (p, d, q) can used to define arima models, thus alternatively it is called as 'arima (p, d, q)' model. there are two types of models in arima such as generalized random walk modes (i.e., well tuned to discard all residual correlations) and generalized exponential smoothing modes (i.e., which can incorporate the long term trends and seasonality).the mathematical definitions are well explained below. if we consider 'b' is back shift operator which causes the observation that multiplies to be backward shifting in time by interval.for any time series z at any period t is considered as bz t z z tÀ , and for n powers of b : b n z t z zt À n arima is joint model of two individual models (autoregressive ar (p) and model average ma (q) ) is integrated by difference variable i(d). in arima models non-stationary time series is defined stationary by application of finite difference in data points.the general multiplicative arima/sarima framework can be written:where b is backshift operator, and f p ðb s Þ z À f b s À f b s À :::::::: À f p b ps ð Þ key: cord- -zvvaq p authors: galeazzi, alessandro; cinelli, matteo; bonaccorsi, giovanni; pierri, francesco; schmidt, ana lucia; scala, antonio; pammolli, fabio; quattrociocchi, walter title: human mobility in response to covid- in france, italy and uk date: - - journal: nan doi: nan sha: doc_id: cord_uid: zvvaq p the policies implemented to hinder the covid- outbreak represent one of the largest critical events in history. the understanding of this process is fundamental for crafting and tailoring post-disaster relief. in this work we perform a massive data analysis, through geolocalized data from m facebook users, on how such a stress affected mobility patterns in france, italy and uk. we find that the general reduction of the overall efficiency in the network of movements is accompanied by geographical fragmentation with a massive reduction of long-range connections. the impact, however, differs among nations according to their initial mobility structure. indeed, we find that the mobility network after the lockdown is more concentrated in the case of france and uk and more distributed in italy. such a process can be approximated through percolation to quantify the substantial impact of the lockdown. the pandemic outbreak of the covid- virus has resulted in a unprecedented global health crisis with high fatality rates and heavy stress to national health systems [ , ] and to the economic and social structure of countries [ , ] . as a result, an impressive effort is being exerted to understand on one side the epidemiological features of the outbreak [ ] [ ] [ ] [ ] and on the other side its economic consequences [ ] [ ] [ ] . the majority of countries governments have responded with non-pharmaceutical interventions (npi) aimed at reducing the mobility of citizens to decrease the rate of contagion [ ] . this calls for a better understanding of the patterns of human mobility during emergencies and in the immediate post-disaster relief. indeed the study of mobility habits is a foundational instance for several issues ranging from traffic forecasting, up to virus spreading and urban planning [ ] [ ] [ ] [ ] . however, a quantitative assessment of its statistical properties at different geographical scales remains elusive [ ] [ ] [ ] [ ] [ ] . the availability of rich datasets on mobility of individuals, coupled with the urgency of the current situation, has fostered the collaboration between tech giants, such as facebook and google, institutions and scholars [ , [ ] [ ] [ ] . along this path, the present work builds upon a collection of data from social network users and addresses the dynamics of spatial redistribution of individuals as a response to mobility restrictions applied to limit the disease outbreak. we perform a massive analysis on aggregated and de-identified data provided by facebook through its disease prevention movement maps [ ] to compare the * a.galeazzi @unibs.it † antonio.scala.phys@gmail.com ‡ w.quattrociocchi@unive.it effects of lockdown measures applied in france, italy and uk in response to covid- . the overall dataset spans over month of observations and accounts for movements of over m people. we model countries as networks of mobility flows and we find that restrictions elicit a transition toward local/short run connections, thus causing a loss in the network efficiency. our result mirrors previous results in the literature which found that critical phenomena are an intrinsic feature of mobility networks, leading to transition between isolated short-range flows and collective long range flows [ ] . moreover we contribute to the literature on mobility disruption during critical events [ ] [ ] [ ] [ ] [ ] by studying the effect of movement limitations across the whole territory of the countries in our dataset, with a geographic scope which is unparalleled in the literature. we provide a model that simulates the effects of movement restrictions, finding that transitions can be approximated by means of different percolation strategies. the general reduction of the overall efficiency in the network of movements is accompanied by geographical fragmentation with a massive reduction of long-range connections. however, the effect changes according to the starting mobility structure of each nation. in particular, we find that the network of uk and france after the lockdown is more concentrated while in italy is more distributed. we conclude the paper by showing how the the effect of restrictions in italy, uk and france can be approximated through percolation analysis. furthermore, our analysis reveals several interesting features. first, the three countries display differentiated mobility patterns that reflect the structural diversity in their underlying infrastructure: more centralized around their capital cities in the case of france and uk and more clustered in the case of italy. such infrastructural characteristics, together with different responses to national lockdown, contributed to the emergence of very distinctive configurations in terms of arxiv: . v [cs.si] may residual mobility patterns. france has one big cluster centered in paris and many smaller centers that disconnect as soon as the percolation process begins, italy exhibits four interconnected clusters, centered approximately in napoli, roma, milano and torino, that remain interconnected over time thus showing a high persistence and resilience. finally, uk has one cluster centered around london, but most of england exhibits a higher persistence with respect to france and italy, thus suggesting the presence of a more capillary network structure. the understanding of the different resilience features of national mobility networks is fundamental to craft and tailor specific release policies and to smooth the economic impact of lockdown. indeed, the correlation among mobility, disease spreading and economic prosperity is crucial both in emergency scenarios and in ordinary times, since the different resilience of mobility networks could be both a predictor of the severity of future systemic crises and a guide to improve the economic and social impact of policies. we represent national mobility networks as weighted directed graphs, based on movement maps made available by facebook through their data for good program [ ] (see materials and methods for further details). nodes correspond to municipalities and edges are weighted with the amount of traffic between two municipalities. we first aggregate mobility flows in two symmetric disjoint windows before and after the day of lockdown (see materials and methods), as shown in panels (a to f) of figure . by comparing the mobility network during the pre-lockdown phase (panels a-b-c of figure ) to the mobility network during the post-lockdown phase, we note a significant reduction of the overall connectivity. however, we notice that mobility restrictions have a higher impact on the connectivity of france, whereas they yields more limited effects in the other two countries. in fact, uk and italy show a reduction of % and % in the size of the largest weakly connected component (lwcc, that is, the maximal subgraph of a network in which any two vertices are interconnected through an undirected path), whereas france exhibits a reduction of almost %. such different impact on the lwcc across countries may depend on several co-existing factors, among which we can list the structural features of the underlying networks and the population density, that is , /km for italy, . /km for uk and /km for france. in panels g-h-i of figure , we further characterize daily connectivity patterns by computing the number of weakly connected components (no. wcc) and the size of the lwcc of the mobility networks. in all cases, we observe a decreasing trend on the size of lwcc and an increase of the number of wccs. it is worth noticing that these trends are present even before the lockdown dates, but they reach a steady state only during the days after the intervention. hence, the number of wccs and the size of the lwcc well capture the response of the countries to the lockdown. in the case of france (where the lwcc is paris-centric), we notice a strong fragmentation of the network from the beginning: in fact, the number of wccs is larger than the size of the lwcc, signaling that the mobility is well distributed along the whole country. however, paris remains connected by long-range connections to the remaining most active areas of bordeaux, toulouse, marseille and lyon. in the case of italy (where the lwcc contains all the main italian cities, i.e. napoli, roma, milano and torino), the lockdown enhances the importance of local mobility. notice that, while in the centre and the south of italy mobility remains localised mainly at regional level (in fig. e one can notice sicilia, campania, lazio and toscana), the lockdown reveals that northern italy is more interconnected, showing a clustered mobility for the main industrial regions, i.e. piemonte, lombardia, veneto and emilia-romagna. the uk is clearly london-centric: the size of the lwcc remains higher than the number of wccs, with strong local mobility patterns only in the bristol area and in the manchester-liverpool area. hence, the situation in terms of number of wccs and the size of the lwcc reflects the different underlying structure of the three countries: france with a huge hub in paris that is star-connected via long-range links to the local city-centered areas, italy with mobility distributed mostly over the center-northern region, and uk that appears as an extension of london, whose mobility network remains pervasive even after the lockdown. we further investigate the effect of lockdown focusing on the global efficiency [ ] of mobility networks, as shown in figure . the global efficiency is a measure that quantifies how optimal is the information flow in a network (further details are reported in materials and methods). we notice a decreasing trend of the efficiency in the period before national lockdown and a steady state in the days after the intervention (figure ), which is consistent with the observed decrease in global connectivity ( figure ). since the network efficiency is a measure that condensates information related to both clustering (i.e. connectedness of neighbours) and small world effect (i.e. presence of long-range connections that act as shortcuts), the trends observed in figure (top panels) well describe the effect of an ongoing shock that cuts both long range connections and the overall cohesiveness. moreover, the three countries experience different amounts of decentralization as a consequence of the lockdown. in order to capture the differences among them in more detail, we measure the heterogeneity of nodes in terms of their contribution to the fig. : outlook on national mobility networks for france, italy and uk during covid- pandemic. panels (a to f) show the largest weakly connected components (lwcc) of national mobility networks built on two disjoint symmetric windows: respectively weeks before (panels a-b-c) and weeks after (panels d-e-f) the day of national lockdown. the lockdown dates is march th for france; march th for italy; march th for uk. bright dots represent municipalities that belong to the lwcc. we observe the following reductions in terms of nodes that disappear from the main cluster. france: from , to , nodes. italy: from , to , nodes. uk: from , to nodes. panels (g-h-i) show the temporal evolution of daily connectivity for national mobility networks of municipalities, in terms of number of weakly connected components (no. wcc) and size of the lwcc. we visualize trends by means of a loess regression (dashed lines with % confidence intervals shaded in grey) and highlight lockdown and week-end days with vertical red lines, respectively solid and dashed. global efficiency using the gini index [ ] (see materials and methods). we first observe that, in correspondence of the initial decrease of global efficiency, the gini index displays an increasing trend that becomes steady after the lockdown date ( figure ). the observed trend means that the contribution of nodes to the global efficiency becomes more and more heterogeneous over time, until it reaches a steady state. this result indicates that the progressive disruption has very heterogeneous effects on the single nodes, suggesting that policies should be carefully tailored to avoid enhancing unequal treatments of different areas: as an example, in italy it has been observed that the lockdown could enhance economic disparities [ ] . such an effect could be even more pronounced in france, that shows a higher level of dishomogeneity in connectivity both at the municipal and at the provincial level when compared to italy. indeed, nodes that get disconnected are more likely to remain isolated as compared to a country such as italy that is based on a more distributed mobility network conversely, uk seems to have a different response to the shock distributing it more evenly among its nodes (refer to si for further details). (normalized by its maximum value during the period of observation), for the mobility network of municipalities. we visualize trends using a loess regression (dashed lines with % confidence intervals shaded in grey) and highlight lockdown dates using a solid vertical line. bottom panels display the temporal evolution of the gini index of the nodal efficiency. the gini index is used as measure of heterogeneity and it is computed considering the nodal contributions to global network efficiency.overall, we observe an increase of the gini index indicating an increasing heterogeneity over time. the drivers behind the empirical results reported in previous sections can be investigated by modeling the process that led to the disruption of mobility networks. therefore, in order to model the lockdown effect, we per-form an analysis based on percolation theory [ ] on the aggregated graph related to the period before the national lockdown. we assume that such a graph is a proxy for the structure of the mobility network in standard conditions (see materials and methods). we implement bond percolation on the aggregated networks by iteratively deleting edges following an increasing (respectively decreasing) weight order. during the process of network dismantling, we keep track of measures related to both cohesiveness and distance, namely the lwcc size, the global efficiency and the node persistence. in more detail, the node persistence measures the extent to which a node remains connected to the lwcc, that is, how much the node is resilient to percolation (see materials and methods). the top row of figure shows the results of the percolation process in terms of node persistence, carried out by removing edges in increasing weight order for france, italy and uk. to each node we assign an area on the map calculated by means of voronoi tessellation [ ] and colored according to node persistence (see materials and methods). the empirical evidence displayed in previous sections finds support in the percolation results. comparing the three cases, the differences in the network structure among countries clearly emerge: while france has one big cluster centered in paris and many smaller centers that disconnect as soon as the process proceeds, italy exhibits four interconnected clusters, centered approximately in napoli, roma, milano and torino (that roughly correspond to the high speed train lines), that remain interconnected thus showing a high persistence. conversely, uk has one cluster centered around london, but most of england exhibits a higher persistence with respect to france and italy, thus suggesting the presence of a more capillary network structure. surprisingly, deleting stronger edges first does not disconnect any of the three networks as fast as deleting weaker edges. this effect can be explained by assuming a "rich club" structure, where links corresponding to higher mobility flows are concentrated around core regions. indeed, the largest fragility of the mobility network toward the deletion of weaker edges hints at a core-periphery structure. to provide further insights regarding the different effects of the percolation process on each of the three countries, in the middle row figure we compare the trends of lwcc size and global efficiency throughout the percolation process. we first notice that the decay of the lwcc size differs depending both on country and edge removal strategy: removing edges sorted by decreasing weight seems to affect less the decay of the lwcc size than removing edges sorted by increasing weight, for all countries. however, we notice some differences in the decay of lwcc size among the three nations in the increasing case: while france exhibits an almost linear trend, uk shows a significant drop only after a notable amount of connections is removed. finally, italy seems to be in a intermediate condition, showing an almost linear decay interspersed fig. : results of the percolation process in terms of node persistence and edge removal strategies . percolation process is performed by iterating a cutting procedure on edges according to their weights calculated in the whole period before lockdown. top panels: node persistence during the percolation process. node persistence is defined as the number of iterations a node remains connected to the lwcc over the maximum number of iterations before the network is completely disconnected; the more persistent the node, the brighter the color. notice that we are defining persistence upon deleting edges by their increasing strength: hence, brighter nodes are not only the last to be disconnected, but are also those embedded in stronger mobility flows. central panels: size of the lwcc (largest weakly connected component) as a function of the residual edges. green curves correspond to deleting edges from the strongest to the weakest, while blue curves correspond to deleting edges according to their increasing weights. bottom panels: variation of global network efficiency obtained deleting edges by increasing (green curves) or decreasing (blue curves) strength. in both the central and the bottom panels, we plot the "empirical" values of lwcc and of the global efficiency for the mobility networks calculated aggregating flows in the weeks before (circles), during (triangles) and after (diamonds) the lockdown date. notice that, while lockdown has cut out the peripheries of the networks (central panels, but see also panels d-e-f of figure ), the effect of the reduced mobility also severely affects the network efficiency (lower panels). with steps. the bottom row of figure displays the normalized global efficiency as a function of the residual edge percentage. also in this case, france has a smoother decay with respect to italy and uk, especially for what concerns the percolation process based on increasing weight sorting. moreover, fixed the percentage of residual edges, uk has a higher efficiency when we consider the increasing percolation case. however, such a relationship changes when the decreasing percolation strategy is taken into account: in this case uk has a somewhat steeper decay of global efficiency, suggesting the presence of a higher number of high weight connections among nodes. again, we observe that core regions (the ones where strong links are concentrated, i.e. the most persistent regions in the upper panels) are the most relevant: in fact, the steepest decrease in efficiency happens when deleting strongest edges first. on the other hand, periphery regions only mildly contribute to the network efficiency, as demonstrated from the slowest decrease upon cutting weakest edges first. in order to shed light on the impact of the lockdown on the network edges, we report the trends followed by empirical networks superimposing them over the results of the percolation process. each point in the curve corresponds to a certain statistic computed on a mobility network aggregated over one week (see materials and methods). the mobility networks are divided into three categories based on the period taken into account: the weeks before the lockdown, the week in which the lockdown happened and the weeks after the lockdown. we note that the residual amount of edges decreases over time and it differs from country to country. france displays the lowest percentage of residual edges, while italy and uk tend to retain a higher percentage of their edges. moreover, we notice how italy and uk strongly differ in terms of the evolution of lwcc size: although they loose roughly the same fraction of edges, there is a strong difference in terms of the amount of connected nodes. in spite of such differences, the increasing percolation strategy is able to approximate the reduction in terms lwcc size quite accurately for all nations. the case of the normalized global efficiency is somewhat different: all the countries seems to have a similar loss proportionally to their initial efficiency. additionally, the trend of the empirical curves is closer to that of the decreasing percolation strategy. the major difference observed in the middle and bottom rows of figure is based on the fact that the considered empirical statistics follow opposite link removal strategies. this effect is due in part to the nature of the two measures; the former more related to the density and to the structure of connections and the second more related to their weight. in any case, despite eventual differences related to the network measures that we take into into account, it is interesting to observe how the lockdown cannot be modeled by a removal strategy based only on edge weights. rather, it is the result of a joint effect deriving from the removal of links based both on their structural importance and weight. the covid- pandemic is testing the structural strength of our global society. most national governments have simultaneously reacted to the contagion by applying mobility restrictions to contain the disease outbreak. the resulting disruption is similar to that caused by a natural disaster, but the effect is on a global scale. for this reason, in this work we analyze the effects of the lockdown on three different national mobility systems, the french, italian and british, by means of a m users dataset from facebook. we provide a structural analysis of the mobility network for each nation and quantify the effect of mobility restrictions applied to hinder covid- outbreak by means of percolation analysis. we find that lockdown mainly affects national smallwordness i.e., strong reduction of long-range connections in favor of local paths. our analysis suggests that the national resilience to massive stress differs and depends upon the inner connectivity structure. indeed, the three countries display very different mobility patterns that reflect the diversity in their underlying infrastructure, more concentrated in the case of france and uk and more distributed in the case of italy. our results may provide insights on the interplay between mobility patterns and structural dependencies in the network of movements at national level and may help design better transportation networks, improve system efficiency during natural catastrophes and contain epidemics spreading. we analyzed human mobility leveraging data provided by facebook through its data for good program [ ] . the platform provides movement maps which are based on de-identified and aggregated information of facebook users who enabled their geo-positioning. movements across administrative regions (i.e. municipalities in our case) are aggregated with a -hours frequency, and describe the amount of traffic flowing between two municipalities in a given time window. similar to data analyzed in recent research on mobility restrictions applied in china [ , ] , facebook does not really provide the number of people moving between two locations but rather an index, constructed with proprietary method to ensure privacy and anonymization, that highly correlates with real movements [ ] . we collected data relative to mobility in italy, france and united kingdom until april th, with different starting times depending on the availability of facebook maps (respectively for the sake of our analysis we represent mobility flows using a weighted directed graph where nodes are municipalities and edges are weighted based on the amount of traffic flowing between two locations. to represent mobility networks before/during lockdown we aggregate daily traffic on windows of - - days (respectively for france, uk and italy) before/after the day of intervention depending on the availability of data. from these data, we build several graphs for each country. we consider an oriented daily graph at municipality levels, where there can be only one edge per day from municipality a to municipality b, whose weight is the daily sum of all edges from a to b. the same procedure is applied for building aggregate networks at different time scales such as weekly graphs of pre/post lockdown graphs. the efficiency is a global network measure that combines the information deriving from the network cohesiveness and the distance among the nodes. it measures how efficiently information is exchanged over the network [ ] and it can be defined as the average of nodal efficiencies eij among couples of vertices of the network. given a weighted network g(v, e) with n = |v | nodes and m = |e| edges, the connections of g are represented by the weighted adjacency matrix w with elements {wij} where wij ≥ ∀ i, j. the global efficiency can be written by means of the following expression: where dij is the distance between two generic nodes i and j, defined as the length of the shortest path among such nodes. the shortest path length dij is the smallest sum of the weights wij throughout all the possible paths in the network from i to j. when node i and j cannot be connected by any path then dij = +∞ and eij = . following the methodology of [ ] , the global efficiency e glob (g) is normalized in order to assume maximum value e(g) = in the case of perfect efficiency. in such a setting the nodal efficiency, i.e. the contribution of each node to the global efficiency, can be simply written as: beside the geographical distance between two nodes of the graphs, a proximity can also be defined considering that two locations are closer if many movements happen between them. to compute network efficiency in our case we use the reciprocal of weights on links to obtain the shortest path distance among couples of nodes. the gini index is a classic example of a synthetic indicator used for measuring inequality of social and economic conditions. the gini index can be defined starting from the gini absolute mean difference ∆ [ ] of a generic vector y with n elements, that can be written as: the relative mean difference is consequently defined as ∆/µy where µy = n − n i= yi. thus, the relative mean difference equals the absolute mean difference divided by the mean of the vector y. the gini index g is one-half of the gini relative mean difference. g = ∆ µy ( ) values of g ∼ signal that the considered vector displays high inequality in the distribution of its entries, while values of g ∼ signal a tendency towards equality. to measure the extent to which a node resists to percolation process, we define the following quantity as node persistence. consider a graph g with nodes v ={v , . . . , v k } and edges e={e , . . . , e h }. suppose that the edges weights assume values in the set w = [w , . . . , wn+ ] with w < w < · · · < wn+ , and suppose that we run a percolation process that consists of deleting edges in increasing order, that is, in the first iteration we delete all the edges with weight less or equal than w , in the second iteration we delete all the edges with weights less or equal than w and so on until the last iteration, n + , where we delete all the edges in the network. notice that the percolation process can be performed similarly in the case edges are removed following a decreasing weight order. thus, at iteration i, we delete all the edges with weight less or equal than wi. consider now a node vj ∈ v . at each step of the process, vj may or may not be part of the lwcc of the network g. clearly, if vj does not belong to the lwcc of g at step i, it will not be in the lwcc of g at step i + , i + , . . . , n + . defining mv j as the maximum number of iterations i such that vj belongs to the lwcc of g, the persistence of node vj is defined as: that is, if the whole process takes n + iterations to disconnect the whole network, the persistence of a node vj is defined as the maximum number of iterations for which vj is connected to the lwcc over the maximum number of iteration for which there are connected nodes in the network. population mobility reductions during covid- epidemic in france under lockdown the effect of travel restrictions on the spread of the novel 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of state-level policies on social distancing in the united states using aggregated mobility data during the covid- pandemic democracy and mobility: a preliminary analysis of global adherence to non-pharmaceutical interventions for a preliminary analysis of global adherence to non-pharmaceutical interventions for covid- economic and social consequences of human mobility restrictions under covid- facebook disaster maps: aggregate insights for crisis response and recovery percolation transition in dynamical traffic network with evolving critical bottlenecks collective response of human populations to large-scale emergencies predictability of population displacement after the haiti earthquake quantifying human mobility perturbation and resilience in hurricane sandy patterns and limitations of urban human mobility resilience under the influence of multiple types of natural disaster leveraging twitter to gauge evacuation compliance: spatiotemporal analysis of hurricane matthew efficient behavior of small-world networks measurement of inequality of incomes percolation theory. reports on progress in physics nouvelles applications des paramètres continusà la théorie des formes quadratiques. deuxième mémoire. recherches sur les parallélloèdres primitifs theadvanced theory of statistics key: cord- -b f wtfn authors: caldarelli, guido; nicola, rocco de; petrocchi, marinella; pratelli, manuel; saracco, fabio title: analysis of online misinformation during the peak of the covid- pandemics in italy date: - - journal: nan doi: nan sha: doc_id: cord_uid: b f wtfn during the covid- pandemics, we also experience another dangerous pandemics based on misinformation. narratives disconnected from fact-checking on the origin and cure of the disease intertwined with pre-existing political fights. we collect a database on twitter posts and analyse the topology of the networks of retweeters (users broadcasting again the same elementary piece of information, or tweet) and validate its structure with methods of statistical physics of networks. furthermore, by using commonly available fact checking software, we assess the reputation of the pieces of news exchanged. by using a combination of theoretical and practical weapons, we are able to track down the flow of misinformation in a snapshot of the twitter ecosystem. thanks to the presence of verified users, we can also assign a polarization to the network nodes (users) and see the impact of low-quality information producers and spreaders in the twitter ecosystem. propaganda and disinformation have a history as long as mankind, and the phenomenon becomes particularly strong in difficult times, such as wars and natural disasters. the advent of the internet and social media has amplified and made faster the spread of biased and false news, and made targeting specific segments of the population possible [ ] . for this reason the vice-president of the european commission with responsibility for policies on values and transparency, vȇra yourová, announced, beginning of june , a european democracy action plan, expected by the end of , in which web platforms admins will be called for greater accountability and transparency, since 'everything cannot be allowed online' [ ] . manufacturers and spreaders of online disinformation have been particularly active also during the covid- pandemic period (e.g., writing about bill gates role in the pandemics or about masks killing children [ , ] ). this, alongside the real pandemics [ ] , has led to the emergence of a new virtual disease: covid- infodemics. in this paper, we shall consider the situation in italy, one of the most affected countries in europe, where the virus struck in a devastating way between the end of february and the end of april [ ] . in such a sad and uncertain time, propaganda [ ] in italy, since the beginning of the pandemics and at time of writing, almost k persons have contracted the covid- virus: of these, more than k have died. source: http://www.protezionecivile.gov.it/. accessed september , . has worked hard: one of the most followed fake news was published by sputnik italia receiving , likes, shares and comments on the most popular social media. 'the article falsely claimed that poland had not allowed a russian plane with humanitarian aid and a team of doctors headed to italy to fly over its airspace', the ec vice-president yourová said. actually, the studies regarding dis/mis/information diffusion on social media seldom analyse its effective impact. in the exchange of messages on online platforms, a great amount of interactions do not carry any relevant information for the understanding of the phenomenon: as an example, randomly retweeting viral posts does not contribute to insights on the sharing activity of the account. for determining dis/misinformation propagation two main weapons can be used, the analysis of the content (semantic approach) and the analysis of the communities sharing the same piece of information (topological approach). while the content of a message can be analysed on its own, the presence of some troublesome structure in the pattern of news producer and spreaders (i.e., in the topology of contacts) can be detected only trough dedicated instruments. indeed, for real in-depth analyses, the properties of the real system should be compared with a proper null model. recently, entropy-based null models have been successfully employed to filter out random noise from complex networks and focus the attention on non trivial contributions [ , ] . essentially, the method consists in defining a 'network benchmark' that has some of the (topological) properties of the real system, but is completely random for all the rest. then, every observation that does not agree with the model, i.e., cannot be explained by the topological properties of the benchmark, carries non trivial information. notably, being based on the shannon entropy, the benchmark is unbiased by definition. in the present paper, using entropy-based null-models, we analyse a tweet corpus related to the italian debate on covid- during the two months of maximum crisis in italy. after cleaning the system from the random noise, by using the entropy-based null-model as a filter, we have been able to highlight different communities. interestingly enough, these groups, beside including several official accounts of ministries, health institutions, and -online and offline -newspapers and newscasts, encompass four main political groups. while at first sight this may sound surprising -the pandemic debate was more on a scientific than on a political ground, at least in the very first phase of its abrupt diffusion -, it might be due to pre-existing echo chambers [ ] . the four political groups are found to perform completely different activities on the platform, to interact differently from each other, and to post and share reputable and non reputable sources of information with great differences in the number of their occurrences. in particular, the accounts from the right wing community interact, mainly in terms of retweets, with the same accounts who interact with the mainstream media. this is probably due to the strong visibility given by the mainstream media to the leaders of that community. moreover, the right wing community is more numerous and more active, even relatively to the number of accounts involved, than the other communities. interestingly enough, newly formed political parties, as the one of the former italian prime minister matteo renzi, quickly imposed their presence on twitter and on the online political debate, with a strong activity. furthermore, the different political parties use different sources for getting information on the spreading on the pandemics. to detect the impact of dis/misinformation in the debate, we consider the news sources shared among the accounts of the various groups. with a hybrid annotation approach, based on independent fact checking organisations and human annotation, we categorised such sources as reputable and non reputable (in terms of credibility of the published news and the transparency of the sources). notably, we experienced that a group of accounts spread information from non reputable sources with a frequency almost times higher than that of the other political groups. and we are afraid that, due to the extent of the online activity of the members of this community, the spreading of such a volume of non reputable news could deceit public opinion. we collected circa . m tweets in italian language, from february st to april th [ ] . details about the political situation in italy during the period of data collection can be found in the supplementary material, section . : 'evolution of the covid- pandemics in italy'. the data collection was keyword-based, with keywords related the covid- pandemics. twitter's streaming api returns any tweet containing the keyword(s) in the text of the tweet, as well as in its metadata. it is worth noting that it is not always necessary to have each permutation of a specific keyword in the tracking list. for example, the keyword 'covid' will return tweets that contain both 'covid ' and 'covid- '. table lists a subset of the considered keywords and hashtags. there are some hashtags that overlap due to the fact that an included keyword is a sub-string of another one, but we included both for completeness. the left panel of fig. shows the network obtained by following the projection procedure described in section . . the network resulting from the projection procedure will be called, in the rest of the paper, validated network. the term validated should not be confused with the term verified, which instead denotes a twitter user who has passed the formal authentication procedure by the social platform. in order to get the community of verified twitter users, we applied the louvain algorithm [ ] to the data in the validated network. such an algorithm, despite being one of the most popular, is also known to be order dependent [ ] . to get rid of this bias, we apply it iteratively n times (n being the number of the nodes) after reshuffling the order of the nodes. finally, we select the partition with the highest modularity. the network presents a strong community structure, composed by four main subgraphs. when analysing the emerging communities, we find that they correspond to right wing parties and media (in steel blue) center left wing (dark red) stars movement (m s ), in dark orange institutional accounts (in sky blue) details about the political situation in italy during the period of data collection can be found in the supplementary material, section . : 'italian political situation during the covid- pandemics'. this partition in four subgroups, once examined in more details, presents a richer substructure, described in the right panel of fig. . starting from the center-left wing, we can find a darker red community, including various ngos and various left oriented journalists, vips and pundits. a slightly lighter red sub-community turns out to be composed by the main politicians of the italian democratic party (pd), as well as by representatives from the european parliament (italian and others) and some eu commissioners. the violet red group is mostly composed by the representatives of italia viva, a new party founded by the former italian prime minister matteo renzi (december -february ). in golden red we can find the subcommunity of catholic and vatican groups. finally the dark violet red and light tomato subcommunities consist mainly of journalists. in turn, also the orange (m s) community shows a clear partition in substructures. in particular, the dark orange subcommunity contains the accounts of politicians, parliament representatives and ministers of the m s and journalists. in aquamarine, we can find the official accounts of some private and public, national and international, health institutes. finally, in the light slate blue subcommunity we can find various italian ministers as well as the italian police and army forces. similar considerations apply to the steel blue community. in steel blue, the subcommunity of center right and right wing parties (as forza italia, lega and fratelli d'italia). in the following, this subcommunity is going to be called as fi-l-fdi, recalling the initials of the political parties contributing to this group. the sky blue subcommunity includes the national federations of various sports, the official accounts of athletes and sport players (mostly soccer) and their teams. the teal subcommunity contains the main italian news agencies. in this subcommunity there are also the accounts of many universities. the firebrick subcommunity contains accounts related to the as roma football club; analogously in dark red official accounts of ac milan and its players. the slate blue subcommunity is mainly composed by the official accounts of radio and tv programs of mediaset, the main private italian broadcasting company. finally, the sky blue community is mainly composed by italian embassies around the world. for the sake of completeness, a more detailed description of the composition of the subcommunities in the right panel of figure is reported in the supplementary material, section . : 'composition of the subcommunities in the validated network of verified twitter users'. here, we report a series of analyses related to the domain names, hereafter simply called domains, that mostly appear in all the tweets of the validated network of verified users. the domains have been tagged according to their degree of credibility and transparency, as indicated by the independent software toolkit newsguard https://www.newsguardtech.com/. the details of this procedure are reported below. as a first step, we considered the network of verified accounts, whose communities and sub-communities are shown in fig. . on this topology, we labelled all domains that had been shared at least times (between tweets and retweets). table shows the tags associated to the domains. in the rest of the paper, we shall be interested in quantifying reliability of news sources publishing during the period of interest. thus, for our analysis, we will not consider those sources corresponding to social networks, marketplaces, search engines, institutional sites, etc. tags r, ∼ r and nr in table are used only for news sites, be them newspapers, magazines, tv or radio social channels, and they stand for reputable, quasi reputable, not reputable, respectively. label unc is assigned to those domains with less than occurrences in ours tweets and rewteets dataset. in fact, the labeling procedure is a hybrid one. as mentioned above, we relied on newsguard, a plugin resulting from the joint effort of journalists and software table tags used for labeling the domains developers aiming at evaluating news sites according to nine criteria concerning credibility and transparency. for evaluating the credibility level, the metrics consider whether the news source regularly publishes false news, does not distinguish between facts and opinions, does not correct a wrongly reported news. for transparency, instead, the tool takes into account whether owners, founders or authors of the news source are publicly known; and whether advertisements are easily recognizable [ ] . after combining the individual scores obtained out of the nine criteria, the plugin associates to a news source a score from to , where is the minimum score for the source to be considered reliable. when reporting the results, the plugin provides details about the criteria which passed the test and those that did not. in order to have a sort of no-man's land and not to be too abrupt in the transition between reputability and non-reputability, when the score was between and , we considered the source to be quasi reputable, ∼r. it is worth noting that not all the domains in the dataset under investigation were evaluated by newsguard at the time of our analysis. for those not evaluated automatically, the annotation was made by three tech-savvy researchers, who assessed the domains by using the same criteria as newsguard. table gives statistics about number and kind of tweets (tw = pure tweet; rt = retweet), the number of url and distinct url (dist url), the number of domains and users in the validated network of verified users. we clarify what we mean by these terms with an example: a domain for us corresponds to the so-called 'second-level domain' name [ ] , i.e., the name directly to the left of .com, .net, and any other top-level domains. for instance, repubblica.it, corriere.it, nytimes.com are considered domains by us. instead, the url maintains here its standard definition [ ] and an example is http://www.example.com/index.html. table shows the outcome of the domains annotation, according to the scores of newsguard or to those assigned by the three annotators, when scores were no available from newsguard. at a first glance, the majority of the news domains belong to the reputable category. the second highest percentage is the one of the untagged domains -unc. in fact, in our dataset there are many domains that occur only few times once. for example, there are domains that appear in the datasets only once. fig. shows the trend of the number of tweets and retweets, containing urls, posted by the verified users of the validated projection during the period of data [ ] newsguard rating process: https://www.newsguardtech.com/ratings/rating-process-criteria/ [ ] https://en.wikipedia.org/wiki/domain_name [ ] table annotation results over all the domains in the whole dataset -validated network of verified users. in [ ] . going on with the analysis, table shows the percentage of the different types of domains for the communities identified in the left plot of fig. . it is worth observing that the steel blue community (both politicians and media) is the most active one, even if it is not the most represented: the number of users is lower than the one of the center left community (the biggest one, in terms of numbers), but the number of their posts containing a valid url is almost the double of that of the second more active community. interestingly, the activity of the verified users of the steel blue community is more focused on content production of (see the only tweets sub-table) than in sharing (see the only retweets sub-table). in fact, retweets represent almost . % of all posts from the media and the right wing community, while in the case of the center-left community it is . %. this effect is observable even in the average only tweets post per verified user: a right-wing user and a media user have an average of . original posts, against . for center-left-wing users. these numbers are probably due to the presence in the former community of the italian most accessed media. they tend to spread their (original) pieces of news on the twitter platform. interestingly, the presence of urls from a non reputable source in the steel blue community is more than times higher than the second score in the same field in the case of original tweets (only tweets). it is worth noting that, for the case of the dark orange and sky blue communities, which are smaller both in terms of users and number of posts, the presence of non classified sources is quite strong (it represents nearly % of retweeted posts for both the communities), as it is the frequency of posts linking to social network contents. interestingly enough, the verified users of both groups seem to focus slightly more on the same domains: there are, on average, . and . posts for each url domain respectively for the dark orange and sky blue communities, and, on average, . and . posts for the steel blue and the dark red communities. the right plot in fig. report a fine grained division of communities: the four largest communities have been further divided into sub-communities, as mentioned in subsection . . here, we focus on the urls shared in the purely political sub-communities in table . broadly speaking, we examine the contribution of the different political parties, as represented on twitter, to the spread of mis/disinformation and propaganda. table clearly shows how the vast majority of the news coming from sources considered scarce or non reputable are tweeted and retweeted by the steel blue political sub-community (fi-l-fdi). notably, the percentage of non reputable sources shared by the fi-l-fdi accounts is more than times the percentage of their community (the steel blue one) and it is more than times the second community in the nr ratio ranking. for all the political sub-communities the incidence of social network links is much higher than in their original communities. looking at table , even if the number of users in each political sub-community is much smaller, some peculiar behaviours can be still be observed. again, the center-right and right wing parties, while representing the least represented ones in terms of users, are much more active than the other groups: each (verified) user is responsible, on average of almost . messages, while the average is . , . and . for m s, iv and pd, respectively. it is worth noticing that italia viva, while being a recently founded party, is very active; moreover, for them the frequency of quasi reputable sources is quite high, especially in the case of only tweets posts. the impact of uncategorized sources is almost constant for all communities in the retweeting activity, while it is particularly strong for the m s. finally, the posts by the center left communities (i.e., italia viva and the democratic party) tend to have more than one url. specifically, every post containing at least a url, has, on average, . and . urls respectively, against the . of movimento stelle and . for the center-right and right wing parties. to conclude the analysis on the validated network of verified users, we report statistics about the most diffused hashtags in the political sub-communities. fig. focuses on wordclouds, while fig. reports the data under an histograms form. actually, from the various hashtags we can derive important information regarding the communications of the various political discursive communities and their position towards the management of the pandemics. first, it has to be noticed that the m s is the greatest user of hashtags: their two most used hashtags have been used almost twice the most used hashtags used by the pd, for instance. this heavy usage is probably due to the presence in this community of journalists and of the official account of il fatto quotidiano, a newspaper explicitly supporting the m s: indeed, the first two hashtags are "#ilfattoquotidiano" and "#edicola" (kiosk, in italian). it is interesting to see the relative importance of hashtags intended to encourage the population during the lockdown: it is the case of "#celafaremo" (we will make it), "#iorestoacasa" (i am staying home), "#fermiamoloinsieme" (let's stop it together ): "#iorestoacasa" is present in every community, but it ranks th in the m s verified user community, th in the fi-l-fdi community, nd in the italia viva community and th in the pd one. remarkably, "#celafaremo" is present only in the m s group, as "#fermiamoloinsieme" can be found in the top hashtags only in the center-right and right wing cluster. the pd, being present in various european institutions, mentions more european related hashtags ("#europeicontrocovid ", europeans against covid- ), in order to ask for a common reaction of the eu. the center-right and right wing community has other hashtags as "#forzalombardia" (go, lombardy! ), ranking the nd, and "#fermiamoloinsieme", ranking th. what is, nevertheless, astonishing, is the presence among the most used hashtags of all communities of the name of politicians from the same group ('interestingly '#salvini" is the first used hashtag in the center right and right wing community, even if he did not perform any duty in the government), tv programs ("#mattino ", "#lavitaindiretta", "#ctcf", "#dimartedì"), as if the main usage of hashtags is to promote the appearance of politicians in tv programs. finally, the hashtags used by fi-l-fdi are mainly used to criticise the actions of the government, e.g., "#contedimettiti" (conte, resign! ). fig. shows the structure of the directed validated projection of the retweet activity network, as outcome of the procedure recalled in section of the supplementary material. as mentioned in section of the supplementary material, the affiliation of unverified users has been determined using the tags obtained by the validated projected network of the verified users, as immutable label for the label propagation of [ ] . after label propagation, the representation of the political communities in the validated retweet network changes dramatically with respect to the case of the network of verified users: the center-right and right wing community is the most represented community in the whole network, with users (representing . % of all the users in the validated network), followed by italia viva users with accounts ( . % of all the accounts in the validated network). the impact of m s and pd is much more limited, with, respectively, and accounts. it is worth noting that this result is unexpected, due to the recent formation of italia viva. as in our previous study targeting the online propaganda [ ] , we observe that the most effective users in term of hub score [ ] are almost exclusively from the center-right and right wing party: considering the first hubs, only are not from this group. interestingly, out of these are verified users: roberto burioni, one of the most famous italian virologists, ranking nd, agenzia ansa, a popular italian news agency, ranking st, and tgcom , the popular newscast of a private tv channel, ranking rd. the fourth account is an online news website, ranking th: this is a not verified account which belongs to a not political community. remarkably, in the top hubs we find of the top hubs already found when considered the online debate on migrations from northern africa to italy [ ] : in particular, a journalist of a neo-fascist online newspaper (non verified user), an extreme right activist (non verified user) and the leader of fratelli d'italia giorgia meloni (verified user), who ranks rd in the hub score. matteo salvini (verified user), who was the first hub in [ ] , ranks th, surpassed by his party partner claudio borghi, ranking th. the first hub in the present network is an extreme right activist, posting videos against african migrants to italy and accusing them to be responsible of the contagion and of violating lockdown measures. table shows the annotation results of all the domains tweeted and retweeted by users in the directed validated network. the numbers are much higher than those shown in table , but the trend confirms the previous results. the majority of urls traceable to news sources are considered reputable. the number of unclassified domains is higher too. in fact, in this case, the annotation was made considering the domains occurring at least times. table annotation results over all the domains -directed validated network table reports statistics about posts, urls, distinct urls, users and verified users in the directed validated network. noticeably, by comparing these numbers with those of table , reporting statistics about the validated network of verified users, we can see that here the number of retweets is much more higher, and the trend is the opposite: verified users tend to tweet more than retweet ( vs ), while users in the directed validated network, which comprehends also non verified users, have a number of retweets . times higher than the number of their tweets. fig. shows the trend of the number of tweets containing urls over the period of data collection. since we are analysing a bigger network than the one considered in section . , we have numbers that are one order of magnitude greater than those shown in fig. ; the highest peak, after the discovery of the first cases in lombardy, corresponds to more than , posts containing urls, whereas the analogous peak in fig. corresponds to , posts. apart from the order of magnitudes, the two plots feature similar trends: higher traffic before the beginning of the italian lockdown, and a settling down as the quarantine went on [ ] . table shows the core of our analysis, that is, the distribution of reputable and non reputable news sources in the direct validated network, consisting of both verified and non-verified users. again, we focus directly on the political sub-communities identified in the previous subsection. two of the sub-communities are part of the center-left wing community, one is associated to the stars movement, the remaining one represents center-right and right wing communities. in line with previous results on the validated network of verified users, the table clearly shows how the vast majority of the news coming from sources considered scarce or non reputable are tweeted and retweeted by the center-right and right wing communities; % of the domains tagged as nr are shared by them. as shown in table , the activity of fi-l-fdi users is again extremely high: on average there are . retweets per account in this community, against the . of m s, the . of iv and the . of pd. the right wing contribution to the debate is extremely high, even in absolute numbers, due to the the large number of users in this community. it is worth mentioning that the frequency of non reputable sources in this community is really high (at about % of the urls in the only tweets) and comparable with that of the reputable ones (see table , only [ ] the low peaks for february and march are due to an interruption in the data collection, caused by a connection breakdown. table domains annotation per political sub-communities -directed validated network tweets). in the other sub-communities, pd users are more focused on un-categorised sources, while users from both italia viva and movimento stelle are mostly tweeting and retweeting reputable news sources. and users, but also in absolute numbers: out of the over m tweets, more than k tweets refer to a nr url. actually, the political competition still shines through the hashtag usage even for the other communities: it is the case, for instance, of italia viva. in the top hashtags we can find '#salvini', '#lega', but also '#papeete' [ ] , '#salvinisciacallo' (salvini jackal ) and '#salvinimmmerda' (salvini asshole). on the other hand, in italia viva hashtags supporting the population during the lockdown are used: '#iorestoacasa', '#restoacasa' (i am staying home), '#restiamoacasa' (let's stay home). criticisms towards the management of lombardy health system during the pandemics can be deduced from the hashtag '#commissariamtelalombardia' (put lombardy under receivership) and '#fontana' (the lega administrator of the lombardy region). movimento stelle has the name of the main leader of the opposition '#salvini', as first hashtag and supports criticisms to the lombardy administration with the hashtags '#fontanadimettiti' (fontana, resign! ) and '#gallera', the health and welfare minister of the lombardy region, considered the main responsible for the bad management of the pandemics. nevertheless, it is possible to highlight even some hashtags encouraging the population during the lock down, as the above mentioned '#iorestoacasa', '#restoacasa' and '#restiamoacasa'. it is worth mentioning that the government measures, and the corresponding m s campaigns, are accompanied specific hashtags: '#curaitalia' is the name of one of the decree of the prime minister to inject liquidity in the italian economy, '#acquistaitaliano' (buy italian products! ), instead, advertise italian products to support the national economy. as a final task, over the whole set of tweets produced or shared by the users in the directed validated network, we counted the number of times a message containing a url was shared by users belonging to different political communities, although without considering the semantics of the tweets. namely, we ignored whether the urls were shared to support or to oppose the presented arguments. table shows the most tweeted (and retweeted) nr domains shared by the political communities presented in table , the number of occurrences is reported next to each domain. the first nr domains for fi-l-fdi in table are related to the right, extreme right and neo-fascist propaganda, as it is the case of imolaoggi.it, ilprimatonazionale.it and voxnews.info, recognised as disinformation websites by newsguard and by the two main italian debunker websites, bufale.net and butac.it. as shown in the table, some domains, although in different number of occurrences, are present under more than one column, thus shared by users close to different political communities. this could mean, for some subgroups of the community, a retweet with the aim of supporting the opinions expressed in the original tweets. however, since the semantics of the posts in which these domains are present were not investigated, the retweets of the links by more than one political community could be due to contrast, and not to support, the opinions present in the original posts. despite the fact that the results were achieved for a specific country, we believe that the applied methodology is of general interest, being able to show trends and peculiarities whenever information is exchanged on social networks. in particular, when analysing the outcome of our investigation, some features attracted our attention: persistence of clusters wrt different discussion topics: in caldarelli et al. [ ] , we focused on tweets concerned with immigration, an issue that has been central in the italian political debate for years. here, we discovered that the clusters and the echo chambers that have been detected when analysing tweets about immigration are almost the same as those singled out when considering discussions concerned with covid- . this may seem surprising, because a discussion about covid- may not be exclusively political, but also medical, social, economic, etc.. from this we can argue that the clusters are political in nature and, even when the topic of discussion changes, users remain in their cluster on twitter. (indeed, journalists and politicians use twitter for information and political propaganda, respectively). the reasons political polarisation and political vision of the world affect so strongly also the analysis of what should be an objective phenomenon is still an intriguing question. persistence of online behavioral characteristics of clusters: we found that the most active, lively and penetrating online communities in the online debate on covid- are the same found in [ ] , formed in a almost purely political debate such as the one represented by the right of migrants to land on the italian territory. (dis)similarities amongst offline and online behaviours of members and voters of parties: maybe less surprisingly, the political habits is also reflected in the degree of participation to the online discussions. in particular, among the parties in the centre-left-wing side, a small party (italia viva) shows a much more effective social presence than the larger party of the italian centre-left-wing (partito democratico), which has many more active members and more parliamentary representation. more generally, there is a significant difference in social presence among the different political parties, and the amount of activity is not at all proportional to the size of the parties in terms of members and voters. spread of non reputable news sources: in the online debate about covid- , many links to non reputable (defined such by newsguard, a toolkit ranking news website based on criteria of transparency and credibility, led by veteran journalists and news entrepreneurs) news sources are posted and shared. kind and occurrences of the urls vary with respect to the corresponding political community. furthermore, some of the communities are characterised by a small number of verified users that corresponds to a very large number of acolytes which are (on their turn) very active, three times as much as the acolytes of the opposite communities in the partition. in particular, when considering the amount of retweets from poorly reputable news sites, one of the communities is by far (one order of magnitude) much more active than the others. as noted already in our previous publication [ ] , this extra activity could be explained by a more skilled use of the systems of propaganda -in that case a massive use of bot accounts and a targeted activity against migrants (as resulted from the analysis of the hub list). our work could help in steering the online political discussion around covid- towards an investigation on reputable information, while providing a clear indication of the political inclination of those participating in the debates. more generally, we hope that our work will contribute to finding appropriate strategies to fight online misinformation. while not completely unexpected, it is striking to see how political polarisation affects also the covid- debate, giving rise to on-line communities of users that, for number and structure, almost closely correspond to their political affiliations. this section recaps the methodology through which we have obtained the communities of verified users (see section . ). this methodology has been designed in saracco et al. [ ] and applied in the field of social networks for the first time in [ , ] . for the sake of completeness, the supplementary material, section , recaps the methodology through which we have obtained the validated retweet activity network shown in section . . in section of the supplementary material, the detection of the affiliation of unverified users is described. in the supplementary material, the interested reader will also find additional details about ) the definition of the null models (section ); ) a comparison among various label propagation for the political affiliation of unverified users (section ); and ) a brief state of the art on fact checking organizations and literature on false news detection (section ). many results in the analysis of online social networks (osn) shows that users are highly clustered in group of opinions [ , - , , , ] ; indeed those groups have some peculiar behaviours, as the echo chamber effects [ , ] . following the example of references [ , ] , we are making use of this users' clustering in order to detect discursive community, i.e. groups of users interacting among themselves by retweeting on the same (covid-related) subjects. remarkably, our procedure does not follow the analysis of the text shared by the various users, but is simply related on the retweeting activity among users. in the present subsection we will examine how the discursive community of verified twitter users can be extracted. on twitter there are two distinct categories of accounts: verified and unverified users. verified users have a thick close to the screen name: the platform itself, upon request from the user, has a procedure to check the authenticity of the account. verified accounts are owned by politicians, journalists or vips in general, as well as the official accounts of ministers, newspapers, newscasts, companies and so on; for those kind of users, the verification procedure guarantees the identity of their account and reduce the risk of malicious accounts tweeting in their name. non verified accounts are for standard users: in this second case, we cannot trust any information provided by the users. the information carried by verified users has been studied extensively in order to have a sort of anchor for the related discussion [ , , , , ] to detect the political orientation we consider the bipartite network represented by verified (on one layer) and unverified (on the other layer) accounts: a link is connecting the verified user v with the unverified one u if at least one time v was retweeted by u, or viceversa. to extract the similarity of users, we compare the commonalities with a bipartite entropy-based null-model, the bipartite configuration model (bicm [ ] ). the rationale is that two verified users that share many links to same unverified accounts probably have similar visions, as perceived by the audience of unverified accounts. we then apply the method of [ ] , graphically depicted in fig. , in order to get a statistically validated projection of the bipartite network of verified and unverified users. in a nutshell, the idea is to compare the amount of common linkage measured on the real network with the expectations of an entropy-based null model fixing (on average) the degree sequence: if the associated p-value is so low that the overlaps cannot be explained by the model, i.e. such that it is not compatible with the degree sequence expectations, they carry non trivial information and we project the related information on the (monopartite) projection of verified users. the interested reader can find the technical details about this validated projection in [ ] and in the supplementary information. the data that support the findings of this study are available from twitter, but restrictions apply to the availability of these data, which were used under license italian socio-political situation during the period of data collection in the present subsection we present some crucial facts for the understanding of the social context in which our analysis is set. this subsection is divided into two parts: the contagion evolution and the political situation. these two aspects are closely related. a first covid- outbreak was detected in codogno, lodi, lombardy region, on february, th [ ] . in the very next day, two cases were detected in vò, padua, veneto region. on february, th, in order to contain the contagions, the national government decided to put in quarantine municipalities, in the area around lodi and vò, near padua [ ] . nevertheless, the number of contagions raised to , hitting different regions; one of the infected person in vò died, representing the first registered italian covid- victim [ ] . on february, th there were already confirmed cases in italy. the first lockdown should have lasted until the th of march, but due to the still increasing number of contagions in northern italy, the italian prime minister giuseppe conte intended to extend the quarantine zone to almost all the northern italy on sunday, march th [ ] : travel to and from the quarantine zone were limited to case of extreme urgency. a draft of the decree announcing the expansion of the quarantine area appeared on the website of the italian newspaper corriere della sera on the late evening of saturday, th, causing some panic in the interested areas [ ] : around people, living in milan, but coming from southern regions, took trains and planes to reach their place of [ ] prima lodi, ""paziente ", il merito della diagnosi va diviso... per due", th june [ ] italian gazzetta ufficiale, "decreto-legge febbraio , n. ". the date is intended to be the very first day of validity of the decree. [ ] il fatto quotidiano, "coronavirus,è morto il enne ricoverato nel padovano. contagiati in lombardia, un altro in veneto", nd february . [ ] bbc news, "coronavirus: northern italy quarantines million people", th march " [ ] the guardian, "leaked coronavirus plan to quarantine m sparks chaos in italy", th march origins [ ] [ ] . in any case, the new quarantine zone covered the entire lombardy and partially other regions. remarkably, close to bergamo, lombardy region, a new outbreak was discovered and the possibility of defining a new quarantine area on march th was considered: this opportunity was later abandoned, due to the new northern italy quarantine zone of the following days. this delay seems to have caused a strong increase in the number of contagions, making the bergamo area the most affected one, in percentage, of the entire country [ ] ; at time of writing, there are investigations regarding the responsibility of this choice. on march, th, the lockdown was extended to the whole country, resulting in the first country in the world to decide for national quarantine [ ] . travels were restricted to emergency reason or to work; all business activities that were not considered as essentials, as pharmacies and supermarkets, had to be closed. until the st of march lockdown measures became progressively stricter all over the country. starting from the th of april, some retails activities as children clothing shops, reopened. a first fall in the number of deaths was observed on the th of april [ ] . a limited reopening started with the so-called "fase " (phase ) on the th of may [ ] . from the very first days of march, the limited capacity of the intensive care departments to take care of covid-infected patients, took to the necessity of a re-organization of italian hospitals, leading, e.g., to the opening of new intensive care departments [ ] . moreover, new communication forms with the relatives of the patients were proposed, new criteria for the intubating patients were developed, and, in the extreme crisis, in the most infected cases, the emergency management took to give priority to the hospitalisation to patients with a higher probability to recover [ ] . outbreaks were mainly present in hospitals [ ] . unfortunately, healthcare workers were contaminated by the covid [ ] . this contagion resulted in a relative high number of fatalities: by the nd of april, covid deaths were registered among doctors. due to the pressure on the intensive care capacity, even the healthcare personnel was subject to extreme stress, especially in the most affected zones [ ] . on august th, , the leader of lega, the main italian right wing party, announced to negate the support to the government of giuseppe conte, which was formed after a post-election coalition between the renzi formed a new center-left party, italia viva (italy alive, iv), due to some discord with pd; despite the scission, italia viva continued to support the actual government, having some of its representatives among the ministers and undersecretaries, but often marking its distance respect to both pd and m s. due to the great impact that matteo salvini and giorgia meloni -leader of fratelli d'italia, a right wing party-have on social media, they started a massive campaign against the government the day after its inauguration. the regions of lombardy, veneto, piedmont and emilia-romagna experienced the highest number of contagions during the pandemics; among those, the former are administrated by the right and center-right wing parties, the fourth one by the pd. the disagreement in the management of the pandemics between regions and the central government was the occasion to exacerbate the political debate (in italy, regions have a quite wide autonomy for healthcare). the regions administrated by the right wing parties criticised the centrality of the decisions regarding the lock down, while the national government criticises the health management (in lombardy the healthcare system has a peculiar organisation, in which the private sector is supported by public funding) and its non effective measure to reduce the number of contagions. the debate was ridden even at a national level: the opposition criticized the financial origin of the support to the various economic sectors. moreover, the role of the european union in providing funding to recover italian economics after the pandemics was debated. here, we detail the composition of the communities shown in figure of the main text. we remind the reader that, after applying the leuven algorithm to the validated network of verified twitter users, we could observe main communities, that correspond to right wing parties and media (in steel blue) center left wing (dark red) stars movement (m s ), in dark orange institutional accounts (in sky blue) starting from the center-left wing, we can find a darker red community, including various ngos (the italian chapters of unicef, medecins sans frontieres, action aid, emergency, save the children, etc.), various left oriented journalists, vips and pundits [ ] . finally, we can find in this group political movements (' sardine') and politicians on the left of pd (as beppe civati, pietro grasso, ignazio marino) or on the left current of the pd (laura boldrini, michele emiliano, stefano bonaccini). a slightly lighter red sub-community turns out to be composed by the main politicians of the italian democratic party (pd), as well as by representatives from the european parliament (italian and others) and some eu commissioners. the violet red group is mostly composed by the representatives of the newly founded italia viva, by the former italian prime minister matteo renzi (december -february ) and former secretary of pd. in golden red we can find the subcommunity of catholic and vatican groups. finally the dark violet red and light tomato subcommunities are composed mainly by journalists. interestingly enough, the dark violet red contains also accounts related to the city of milan (the major, the municipality, the public services account) and to the spoke person of the chinese minister of foreign affair. in turn, also the orange (m s) community shows a clear partition in substructures. in particular, the dark orange subcommunity contains the accounts of politicians, parliament representatives and ministers of the m s and journalists and the official account of il fatto quotidiano, a newspaper supporting the movement stars. interestingly, since one of the main leaders of the movement, luigi di maio, is also the italian minister of foreign affairs, we can find in this subcommunity also the accounts of several italian embassies around the world, as well as the account of the italian representatives at nato, ocse and oas. in aquamarine, we can find the official accounts of some private and public, national and international, health institutes (as the italian istituto superiore di sanità, literally the italian national institute of health, the world health organization, the fondazione veronesi) the minister of health roberto speranza, and some foreign embassies in italy. finally, in the light slate blue subcommunity we can find various italian ministers as well as the italian police and army forces. similar considerations apply to the steel blue community. in steel blue, the subcommunity of center right and right wing parties (as forza italia, lega and fratelli d'italia). the presidents of the regions of lombardy, veneto and liguria, administrated by center right and right wing parties, can be found here. (in the following this subcommunity is going to be called as fi-l-fdi, recalling the initials of the political parties contributing to this group.) the sky blue subcommunity includes the national federations of various sports, the official accounts of athletes and sport players (mostly soccer) and their teams, as well as sport journals, newscasts and journalists. the teal subcommunity contains the main italian news agencies, some of the main national and local newspapers, [ ] as the cartoonists makkox and vauro, the singers marracash, frankiehinrg, ligabue and emphil volo vocal band, and journalists from repubblica (ezio mauro, carlo verdelli, massimo giannini), from la tv channel (ricardo formigli, diego bianchi). newscasts and their journalists. in this subcommunity there are also the accounts of many universities; interestingly enough, it includes also the all the local public service local newscasts. the firebrick subcommunity contains accounts related to the as roma football club; analogously in dark red official accounts of ac milan and its players. the slate blue subcommunity is mainly composed by the official accounts of radio and tv programs of mediaset, the main private italian broadcasting company, together with singers and musicians. other smaller subcommunities includes other sport federations, and sports pundits. finally, the sky blue community is mainly composed by italian embassies around the world. the navy subpartition contains also the official accounts of the president of the republic, the italian minister of defense and the one of the commissioner for economy at eu and former prime minister, paolo gentiloni. in the study of every phenomenon, it is of utmost importance to distinguish the relevant information from the noise. here, we remind a framework to obtain a validated monopartite retweet network of users: the validation accounts the information carried by not only the activity of the users, but also by the virality of their messages. we represented pictorially the method in fig. . we define a directed bipartite network in which one layer is composed by accounts and the other one by the tweets. an arrow connecting a user u to a tweet t represents the u writing the message t. the arrow in the opposite direction means that the user u is retweeting the message t. to filter out the random noise from this network, we make use of the directed version of the bicm, i.e. the bipartite directed configuration model (bidcm [ ] ). the projection procedure is then, analogous to the one presented in the previous subsection: it is pictorially displayed in the fig. . briefly, consider the couple of users u and u and consider the number of message written by u and shared u . then, calculate which is the distribution of the same measure according with the bidcm: if the related p-value is statistically significant, i.e. if the number of u 's tweets shared by u is much more than expected by the bidcm, we project a (directed) link from u to u . summarising, the comparison of the observation on the real network with the bidcm permits to uncover all contributions that cannot originate from the constraints of the null-model. using the technique described in subsection . of the main text, we are able to assign to almost all verified users a community, based on the perception of the unverified users. due to the fact that the identity of verified users are checked by twitter, we have the possibility of controlling our groups. indeed, as we will show in the following, the network obtained via the bipartite projection provides a reliable description regarding the closeness of opinions and role in the social debate. how can we use this information in order to infer the orientation of non verified users? in the reference [ ] we used the tags obtained for both verified and unverified users in the bipartite network described in subsection . of the main real network c) e) figure schematic representation of the projection procedure for bipartite directed network. a) an example of a real directed bipartite network. for the actual application, the two layers represent twitter accounts (turquoise) and posts (gray). a link from a turquoise node to a gray one represents that the post has been written by the user; a link in the opposite direction represents a retweet by the considered account. b) the bipartite directed configuration model (bidcm) ensemble is defined. the ensemble includes all the link realisations, once the number of nodes per layer has been fixed. c) we focus our attention on nodes i and j and count the number of directed common neighbours (in magenta both the nodes and the links to their common neighbours), i.e., the number of posts written by i and retweeted by j. subsequently, d) we compare this measure on the real network with the one on the ensemble: if this overlap is statistically significant with respect to the bidcm, e) we have a link from i to j in the projected network. text and propagated those labels accross the network. in a recent analysis, we observed that other approaches are more stable [ ] : in the present manuscript we make use of the most stable algorithm. we use the label propagation as proposed in [ ] on the directed validated network. indeed, the validated directed network in the present appendix we remind the main steps for the definition of an entropy based null model; the interested reader can refer to the review [ ] . we start by revising the bipartite configuration model [ ] , that has been used for detecting the network of similarities of verified users. we are then going to examine the extension of this model to bipartite directed networks [ ] . finally, we present the general methodology to project the information contained in a -directed or undirected-bipartite network, as developed in [ ] . let us consider a bipartite network g * bi , in which the two layers are l and Γ. define g bi the ensemble of all possible graphs with the same number of nodes per layer as in g * bi . it is possible to define the entropy related to the ensemble as [ ] : where p (g bi ) is the probability associated to the instance g bi . now we want to obtain the maximum entropy configuration, constraining some relevant topological information regarding the system. for the bipartite representation of verified and unverified user, a crucial ingredient is the degree sequence, since it is a proxy of the number of interactions (i.e. tweets and retweets) with the other class of accounts. thus in the present manuscript we focus on the degree sequence. let us then maximise the entropy ( ), constraining the average over the ensemble of the degree sequence. it can be shown, [ ] , that the probability distribution over the ensemble is where m iα represent the entries of the biadjacency matrix describing the bipartite network under consideration and p iα is the probability of observing a link between the nodes i ∈ l and α ∈ Γ. the probability p iα can be expressed in terms of the lagrangian multipliers x and y for nodes on l and Γ layers, respectively, as in order to obtain the values of x and y that maximize the likelihood to observe the real network, we need to impose the following conditions [ , ]        where the * indicates quantities measured on the real network. actually, the real network is sparse: the bipartite network of verified and unverified users has a connectance ρ . × − . in this case the formula ( ) can be safely approximated with the chung-lu configuration model, i.e. where m is the total number of links in the bipartite network. in the present subsection we will consider the case of the extension of the bicm to direct bipartite networks and highlight the peculiarities of the network under analysis in this representation. the adjancency matrix describing a direct bipartite network of layers l and Γ has a peculiar block structure, once nodes are order by layer membership (here the nodes on l layer first): where the o blocks represent null matrices (indeed they describe links connecting nodes inside the same layer: by construction they are exactly zero) and m and n are non zero blocks, describing links connecting nodes on layer l with those on layer Γ and viceversa. in general m = n, otherwise the network is not distinguishable from an undirected one. we can perform the same machinery of the section above, but for the extension of the degree sequence to a directed degree sequence, i.e. considering the in-and out-degrees for nodes on the layer l, (here m iα and n iα represent respectively the entry of matrices m and n) and for nodes on the layer Γ, the definition of the bipartite directed configuration model (bidcm, [ ] ), i.e. the extension of the bicm above, follows closely the same steps described in the previous subsection. interestingly enough, the probabilities relative to the presence of links from l to Γ are independent on the probabilities relative to the presence of links from Γ to l. if q iα is the probability of observing a link from node i to node α and q iα the probability of observing a link in the opposite direction, we have where x out i and x in i are the lagrangian multipliers relative to the node i ∈ l, respectively for the out-and the in-degrees, and y out α and y in α are the analogous for α ∈ Γ. in the present application we have some simplifications: the bipartite directed network representation describes users (on one layer) writing and retweeting posts (on the other layer). if users are on the layer l and posts on the opposite layer and m iα represents the user i writing the post α, then k in α = ∀α ∈ Γ, since each message cannot have more than an author. notice that, since our constraints are conserved on average, we are considering, in the ensemble of all possible realisations even instances in which k in α > or k in α = , or, otherwise stated, non physical; nevertheless the average is constrained to the right value, i.e. . the fact that k in α is the same for every α allows for a great simplification of the probability per link on m: where n Γ is the total number of nodes on the Γ layer. the simplification in ( ) is extremely helpful in the projected validation of the bipartite directed network [ ] . the information contained in a bipartite -directed or undirected-network, can be projected onto one of the two layers. the rationale is to obtain a monopartite network encoding the non trivial interactions among the two layers of the original bipartite network. the method is pretty general, once we have a null model in which probabilities per link are independent, as it is the case of both bicm and bidcm [ ] . the first step is represented by the definition of a bipartite motif that may capture the non trivial similarity (in the case of an undirected bipartite network) or flux of information (in the case of a directed bipartite network). this quantity can be captured by the number of v −motifs between users i and j [ , ] , or by its direct extension (note that v ij = v ji ). we compare the abundance of these motifs with the null models defined above: all motifs that cannot be explained by the null model, i.e. whose p-value are statistically significance, are validated into the projection on one of the layers [ ] . in order to assess the statistically significance of the observed motifs, we calculate the distribution associated to the various motifs. for instance, the expected value for the number of v-motifs connecting i and j in an undirected bipartite network is where p iα s are the probability of the bicm. analogously, where in the last step we use the simplification of ( ) [ ] . in both the direct and the undirect case, the distribution of the v-motifs or of the directed extensions is poisson binomial one, i.e. a binomial distribution in which each event shows a different probability. in the present case, due to the sparsity of the analysed networks, we can safely approximate the poisson-binomial distribution with a poisson one [ ] . in order to state the statistical significance of the observed value, we calculate the related p-values according to the relative null-models. once we have a p-value for every detected v-motif, the related statistical significance can be established through the false discovery rate (fdr) procedure [ ] . respect to other multiple test hypothesis, fdr controls the number of false positives. in our case, all rejected hypotheses identify the amount of v-motifs that cannot be explained only by the ingredients of the null model and thus carry non trivial information regarding the systems. in this sense, the validated projected network includes a link for every rejected hypothesis, connecting the nodes involved in the related motifs. in the main text, we solved the problem of assigning the orientation to all relevant users in the validated retweet network via a label propagation. the approach is similar, but different to the one proposed in [ ] , the differences being in the starting labels, in the label propagation algorithm and in the network used. in this section we will revise the method employed in the present article, as compared it to the one in [ ] and evaluate the deviations from other approaches. first step of our methodology is to extract the polarisation of verified users from the bipartite network, as described in section . of the main text, in order to use it as seed labels in the label propagation. in reference [ ] , a measure of the "adherence" of the unverified users towards the various communities of verified users was used in order to infer their orientation, following the approach in [ ] , in turn based on the polarisation index defined in [ ] . this approach was extremely performing when practically all unverified users interact at least once with verified one, as in [ ] . while still having good performances in a different dataset as the one studied in [ ] , we observed isolated deviations: it was the case of users with frequent interactions with other unverified accounts of the same (political) orientation, randomly retweeting a different discursive community verified user. in this case, focusing just on the interaction with verified accounts, those nodes were assigned a wrong orientation. the labels for the polarisation of the unverified users defined [ ] were subsequently used as seed labels in the label propagation. due to the possibility described above of assigning wrongly labels to unverified accounts, in the present paper, we consider only the tags of verified users, since they pass a strict validation procedure and are more stable. in order to compare the results obtained with the various approaches, we calculated the variation of information (vi, [ ] ). v i considers exactly the different in information contents captured by two different partition, as consider by the shannon entropy. results are reported in the matrix in figure for the th of february (results are similar for other days). even when using the weighted retweet network as "exact" result, the partition found by the label propagation of our approach has a little loss of information, comparable with the one of using an unweighted approach. indeed, the results found by the various community detection algorithms show little agreement with the label propagation ones. nevertheless, we still prefer the label propagation procedure, since the validated projection on the layer of verified users is theoretically sound and has a non trivial interpretation. the main result of this work quantifies the level of diffusion on twitter of news published by sources considered scarcely reputable. academy, governments, and news agencies are working hard to classify information sources according to criteria of credibility and transparency of published news. this is the case, for example, of newsguard, which we used for the tagging of the most frequent domains in the direct validated network obtained according to the methodology presented in the previous sections. as introduced in subsection . of the main text, the newsguard browser extension and mobile app [ ] offers a reliability result for the most popular newspapers in the world, summarizing with a numerical score the level of credibility and journalistic transparency of the newspaper. with the same philosophy, but oriented towards us politics, the fact-checking site politifact.com reports with a 'truth meter' the degree of truthfulness of original claims made by politicians, candidates, their staffs, and, more, in general, protagonists of us politics. one of the eldest fact-checking websites dates back to : snopes.com, in addition to political figures, is a fact-checker for hoaxes and urban legends. generally speaking, a fact-checking site has behind it a multitude of editors and journalists who, with a great deal of energy, manually check the reliability of a news, or of the publisher of that news, by evaluating criteria such as, e.g., the tendency to correct errors, the nature of the newspaper's finances, and if there is a clear differentiation between opinions and facts. thus, it is worth noting that recent attempts tried to automatically find articles worthy of being fact-checked. for example, work in [ ] uses a supervised classifier, based on an ensemble of neural networks and support vector machines, to figure out which politicians' claims need to be debunked, and which have already been debunked. despite the tremendous effort of stakeholders to keep the fact-checking sites up to date and functioning, disinformation resists debunking due to a combination of factors. there are psychological aspects, like the quest for belonging to a community and getting reassuring answers, the adherence to one's viewpoint, a native reluctance to change opinion [ , ] , the formation of echo chambers [ ] , where people polarize their opinions as they are insulated from contrary perspectives: these are key factors for people to contribute to the success of disinformation spreading [ , ] . moreover, researchers demonstrate how the spreading of false news is strategically supported by the massive and organized use of trolls and bots [ ] . despite the need to educate the user to a conscious fruition of online information through means also different from those represented by technological solutions, there are a series of promising works that exploit classifiers based on machine learning or on deep learning to tag a news as credible or not. one interesting approach is based on the analysis of spreading patterns on social platforms. monti et al. recently provide a deep learning framework for detection of fake news cascades [ ] . a ground truth is acquired by following the example by vosoughi et al. [ ] collecting twitter cascades of verified false and true rumors. employing a novel deep learning paradigm for graph-based structures, cascades [ ] https://www.newsguardtech.com/ are classified based on user profile, user activity, network and spreading, and content. the main result of the work is that 'a few hours of propagation are sufficient to distinguish false news from true news with high accuracy'. this result has been confirmed by other studies too. work in [ ] , by zhao et al. examine diffusion cascades on weibo and twitter: focusing on topological properties, such as the number of hops from the source and the heterogeneity of the network, the authors demonstrate that networks in which fake news are diffused feature characteristics really different from those diffusing genuine information. diffusion networks investigation appear to be a definitive path to follow for fake news detection. this is also confirmed by pierri et al. 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material guido caldarelli , , * † , rocco de nicola † , marinella petrocchi † , manuel pratelli † and fabio saracco † there is another difference in the label propagation used here against the one in [ ] : in the present paper we used the label propagation of [ ] , while the one in [ ] was quite home-made. as in reference [ ] , the seed labels of [ ] are fixed, i.e. are not allowed to change [ ] . the main difference is that, in case of a draw, among the labels of the first neighbours, in [ ] a tie is removed randomly, while in the algorithm of [ ] the label is not assigned and goes into a new run, with the newly assigned labels. moreover, the updated of labels in [ ] is asynchronous, while it is synchronous in [ ] . we opted for the one in [ ] for being actually a standard in the label propagation algorithms, being stable, more studied, and faster [ ] . finally, differently from the procedure in [ ] , we applied the label propagation not to the entire (undirected version of the) retweet network, but on the (undirected version of the) validated one. (the intent of choosing the undirected version is that in both case in which a generic account is significantly retweeting or being retweeted by another one, they do probably share some vision of the phenomena under analysis, thus we are not interested in the direction of the links, in this situation.) the rationale in using the validated network is to reduce the calculation time (due to the dimensions of the dataset), while obtaining an accurate result. while the previous differences from the procedure of [ ] are dictated by conservativeness (the choice of the seed labels) or by the adherence to a standard (the choice of [ ] ), this last one may be debatable: why choosing the validated network should return "better" results than the ones calculated on the entire retweet network? we consider the case of a single day (in order to reduce the calculation time) and studied different approaches: a louvain community detection [ ] on the undirected version of the validated network of retweets; a louvain community detection on the undirected version of the unweighted retweet network; a louvain community detection on the undirected version of the weighted retweet network, in which the weights are the number of retweets from user to user; a label propagation a la raghavan et al. [ ] on the directed validated network of retweets; a label propagation a la raghavan et al. on the (unweighted) retweet network; a label propagation a la raghavan et al. on the weighted retweet network, the weights being the number of retweets from user to user. actually, due to the order dependence of louvain [ ] , we run several times the louvain algorithm after reshuffling the order of the nodes, taking the partition in communities that maximise the modularity. similarly, the label propagation of [ ] has a certain level of randomness: we run it several times and choose the most frequent label assignment for every node. key: cord- -ff ex authors: ricoca peixoto, vasco; nunes, carla; abrantes, alexandre title: epidemic surveillance of covid- : considering uncertainty and under-ascertainment date: - - journal: nan doi: . / sha: doc_id: cord_uid: ff ex epidemic surveillance is a fundamental part of public health practice. addressing under-ascertainment of cases is relevant in most surveillance systems, especially in pandemics of new diseases with a large spectrum of clinical presentations as it may influence timings of policy implementation and public risk perception. from this perspective, this article presents and discusses early evidence on under-ascertainment of covid- and its motifs, options for surveillance, and reflections around their importance to tailor public health measures. in the case of covid- , systematically addressing and estimating under-ascertainment of cases is essential to tailor timely public health measures, and communicating these findings is of the utmost importance for policy making and public perception. vigilância epidemiológica covid- · subindetificação · subdeteção · sensibilidade sistemas de vigilância epidemiológica a vigilância epidemiológica é uma parte fundamental da prática de saúde pública. considerar e avaliar a sub-deteção de casos é relevante na maioria dos sistemas de vigilância, especialmente em pandemias de novas doenças com um amplo espectro de apresentações clínicas, porque pode influenciar os momentos e de decisões em políticas de saúde e a perceção de risco da população. este artigo apresenta e discute evidência inicial sobre a sub-deteção do covid- e os seus motivos, opções de vigilância e reflexões sobre sua importância para informar medidas de saúde pública. no caso do covid- , abordar e estimar sistematicamente a su-deteção de casos é essencial para adequar as medidas de saúde pública, e comunicar essas achados é de extrema importância para a formulação de políticas. epidemic surveillance is a critical component of public health practice. it gives us pictures of reality, informs policy and decision making, gauges health service demand, and feeds forecasts and models. however, surveillance is never perfect and diseases that present with a high proportion of mild, pauci-symptomatic, or subclinical cases can be hard to detect in most indicator-based surveillance systems and become harder to contain [ ] . surveillance pyramids can take different formats and proportions of undetected cases (often mild or asymptomatic). the scientific community is making efforts to address uncertainty about where to draw the lines separating different clinical manifestations and between ascertained and unascertained cases. covid- surveillance is challenging because mild and subclinical cases may not seek health services, cases are advised to avoid health care unless necessary, and testing capacity may be limited (fig. ). in the case of covid- , addressing and estimating under-ascertainment of cases is essential to tailor public health measures. communicating these findings is of the utmost importance for evidence-based policy making and community engagement. in europe in late february there was a fast rise in the number of confirmed cases in italy, and the probability of importation of cases of covid- may have been high in europe until italy stopped movement from northern italy and later from the rest of the country. the rapid rise and characteristics of confirmed cases in northern italy indicated an earlier important undetected community transmission, especially among mild cases. as such, it is possible that in late february many cases from northern italy (and possibly from other european areas) were exported all over europe. these cases may have given rise to mostly undetected transmission chains that remained active in the next months, justifying early enhanced surveillance and control measures that were taken by different governments to buy time for preparedness. the generalized under-detection of imported cases has been confirmed in a study in singapore, which estimated that the overall ability to detect imported cases was % ( % hpdi: - %) of singapore's capacity. in high-quality surveillance locations (based on the global health security index) it was % ( % hpdi: - %) [ ] . in line with these findings, a report by imperial college london estimated that / of covid- cases exported from mainland china remained undetected worldwide (until february ), resulting in several undetected transmission chains. they found countries in europe had relatively low sensitivity for imported cases [ ] . a relevant part of the fast growth in detected cases in italy and other countries could be related to undetected transmission chains from early introduced cases and accumulated a large pool of infected cases. it is likely that a high proportion of imported cases all around europe have not been found, and isolation, contact tracing, and quarantine could not be applied considering the who suspect case definition (as of february , ). although many countries advised travellers returning from certain areas to report (call a health line or medical doctor) if they had symptoms, it is possible that people with mild symptoms may not have followed this advice. consequently, many cases that might have followed from this would not be tested because they lacked a clear epidemiological link (fig. ) . in fact, several articles suggest a major under-ascertainment/under-reporting of total cases in different countries. one study modelled the real dimension of the epidemic in italy from international case exportations and estimated a real size of , cases ( % ci: , - , ), compared to a case count of , on february , , suggesting an under-ascertainment of % ( - %) of cases. in sensitivity analyses, the range of variation was from , to , cases (implying the identification of - % of cases) [ ] . publicly available epidemiological data for hubei, from january to february , , revealed a significant decline in the fatality rate with time. as testing becomes widespread, the detection of milder cases that would not seek health care in the early stages will result in a lower case fatality rate [ ] . another study used data from air travel and cases imported from iran in other middle eastern countries and estimated that there were , ( % ci: , - , ) covid- cases in iran as of february , which meant only . % of all cases had been reported [ ] . other models used reported infection within china, in conjunction with mobility data, a networked dynamic metapopulation model, and bayesian inference, and estimated that % of all infections were undocumented port j public health doi: . / ( % ci: - %) prior to january, , travel restrictions and that undocumented infections were the infection source for % of documented cases [ ] . adding to this we should consider specific surveillance system delays from infection and symptom onset to reporting. we should ask ourselves whether different choic-es at different timings would be made at an individual and policy level if we only looked to the confirmed cases. modelled estimations of total infected and counts of suspect/possible cases that were not laboratory confirmed should be considered and communicated together with uncertainty in model assumptions (fig. ) . some countries initiated earlier and wider testing than others [ ] . some started earlier to test admitted cases of bilateral pneumonia without other aetiology (allowing for earlier consideration of community transmission), and some tested contacts even if asymptomatic. criteria for testing in earlier stages was variable and resulted in very different testing rates [ ] . the different testing strategies and consequent different levels of under-ascertainment may account for some of the differences in the fatality rate found in different countries in europe such as italy and germany ( and . %, respectively) [ ] , though we should also consider the impact of overload of the health systems, characteristics of the specific surveillance system, and population demographics and behaviour. there are various options for surveillance to improve case detection or make estimations (table ) . limited capacity for testing may play a role in testing strategies, forcing some countries to prioritize testing, but it makes discussion and research around under-ascertainment even more relevant in those contexts. underascertainment in surveillance should make us consider an under-ascertained need for specific early measures (for example of social distancing) but also, in later stages, that a much larger part of the population could be immune and social distancing strategies could change, which also raises the importance of serologic surveys in those circumstances to confirm these estimates. one document of the european centre of disease control (ecdc) [ ] reports that "the detection of co-vid- cases and/or deaths outside of known chains of transmission is a strong signal that social distancing mea-sures should be considered." however, with restrictive testing strategies, these signals may be missed. the document states that "the early, decisive, rapid, coordinated and comprehensive implementation of closures and quarantines is likely to be more effective in slowing the spread of the virus than a delayed implementation" and that decisions "will very rarely be purely evidence-based" as social and political considerations will also need to be taken into account. evidence is always incomplete, especially in new pandemics. there is evidence of the effectiveness of public health measures such as travel bans, movement restrictions, and social distancing. a study used a seir (susceptible-exposed-infectious-removed) model to estimate epidemiological parameters before the implementation of measures in wuhan. if these measures had been initiated , , or weeks earlier in china, cases could have been reduced by , , and %, respectively, together with a significant reduction in the number of affected areas [ ] . another study reports that the effective reproduction number in wuhan fell from . ( % ci: . - . ) week before the introduction of restrictions on january , , to . ( . - . ) week later and calculated that in sites with wuhan-like transmission potential in early january, when there were only cases introduced, there is more than a % chance of the infection settling in this population [ ] . other simulation [ ] suggests that to control most outbreaks, for r (number of cases directly generated by one case in a population where all individuals are susceptible to infection) of - , more than % of contacts had to be identified. in most scenarios, highly effective case identification and isolation of contacts could control a new outbreak of covid- within months. however, importantly, the probability of control decreases with delays in the identification of cases and contacts, increased time from onset of symptoms to isolation, fewer cases identified, and increased transmission before symptoms. the oxford covid- government response tracker [ ] describes variation in government responses and explores whether rising stringency of response affects the rate of infection. they calculate a stringency-risk ratio but warn that recorded cases partly depend on how much testing is done, which is likely to co-vary with the stringency of the government's response. regular updated results will be available for anyone. a recent study by the imperial college covid- response team shows that in the uk and us context, suppression requires a combination of social distancing of the entire population, home isolation of cases, and household quarantine of their family members, that may need school and university closures and reports other strategies that may involve social distancing of those > years old. however, the study predicts that transmission will quickly rebound if interventions are relaxed and suggests that intermittent social distancing -triggered by trends in disease surveillance -may allow interventions to be relaxed temporarily [ ] . this strategy needs robust surveillance systems that allow for acceptable sensitivity to timely decide on interventions. we believe these triggers must consider evidence and debate around surveillance system sensitivity and under-ascertainment. as suggested by another imperial college report [ ] , this would be relevant to produce early evidence when exiting the co-vid- social distancing policy after achieving containment. due to different surveillance systems, different measures at different timings, different levels of stringency [ ] , and different demographic and social dynamics, the outcomes within europe may be heterogeneous. undetected introduction of imported cases and many mild undetected cases making up those transmission chains may warrant early measures. by looking at the death epidemic curves (likely less sensitive to variations in surveillance system sensitivity) of different countries we see differences that can be related to earlier detection and implementation of measures (fig. ) . notably, south korea has managed to keep new deaths by covid- low despite being in a relatively advanced stage of the epidemic and is one of the countries with a higher testing rate [ ] . an early study [ ] estimated the transmission potential of covid- in the country and suggested early sustained disease transmission in the region which contributed to the rapid early implementation of social distancing measures to contain the outbreak [ ] . considering uncertainty and under-ascertainment is critical for decision when cases could possibly mean , . delay from exposure and infection to reporting must also be considered. there is at this stage high uncertainty about the behaviour of the disease in europe, the effectiveness of measures in different contexts, and the timing for their implementation. often, measures should be implemented early with imperfect scientific knowledge. on the other hand, estimated total infection numbers can be important to justify measures for public opinion, engage them in preventive action, and reinforce recommendations for those who are tested positive as well as to inform decisions on exiting social distancing policies. more robust models will be produced soon, but models make assumptions based on surveillance data and other sources with different levels of uncertainty. under-ascertainment should be considered, and the best available information and data should be included and pursued by improving surveillance strategies to reduce or better estimate under-ascertainment. with more epidemiological evidence, namely greater certainty in relation to the proportion of asymptomatic infections and their risk of transmission, different surveillance system sensitivity, effective r in different contexts and effectiveness of containment measures and with the results of the first few x cases ffx investigation proposed by who [ ] for covid- models will better take into account underascertainment in different countries. understanding the extent of transmission in different regions can help policy makers tailor their response. acknowledging that "we can't fight a pandemic blindfolded," on march , , the who urged countries to test. under-ascertainment may be of relevance for cov-id- policy. there is uncertainty on this subject and in the studies mentioned in this article and presented reflections. however, it is the role of the scientific community in public health and epidemiology not only to communicate what is certain and based in robust established science, but also of what is uncertain, when it may be of relevance for decision making and can be further researched, discussed, and shared. as in most infectious disease surveillance, under-ascertainment can be a relevant piece of the puzzle for covid- science, policy making, and public opinion and should be systematically addressed and communicated. a novel coronavirus emerging in china: key questions for impact assessment quantifying bias of covid- prevalence and severity estimates in wuhan, china that depend on reported cases in international travelers who collaborating centre for infectious disease modelling; mrc centre for global infectious disease analysis abdul latif jameel institute for disease and emergency analytics relative sensitivity of international surveillance estimation of covid- outbreak size in italy based on international case exportations siettos c. data-based analysis, modelling and forecasting of the novel coronavirus preliminary estimation of the novel coronavirus disease [covid- ] cases in iran: a modelling analysis based on overseas cases and air travel data. medrxiv. . preprint substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov our world in data. how many tests for co-vid- are being performed around the world? world health organization. coronavirus disease european centre for disease prevention and control. considerations relating to social distancing measures in response to the covid- epidemic effect of non-pharmaceutical interventions for containing the co-vid- outbreak: an observational and modelling study centre for mathematical modelling of infectious diseases co-vid- working group. early dynamics of transmission and control of covid- : a mathematical modelling study feasibility of controlling covid- outbreaks by isolation of cases and contacts variation in government responses to cov-id- : version . . oxford: blavatnik school of government abdul latif jameel institute for disease and emergency analytics. imperial college london. impact of non-pharmaceutical interventions [npis] to reduce covid- mortality and healthcare demand report : evidence of initial success for china exiting covid- social distancing policy after achieving containment imperial college covid- response team transmission potential and severity of co-vid- in south korea the first few x [ffx] cases and contact investigation protocol for -novel coronavirus [ -ncov] infection the authors thank daniel thomas, sonia bounder, paula blomquist, and robert withaker for commenting on early drafts and aurora peixoto for artwork. the authors have no conflicts of interest to declare. the authors have no funding sources to declare. all authors contributed in a relevant way to the literature review and writing of the article. key: cord- -vgr ht a authors: wang, tianbing; wu, yanqiu; lau, johnson yiu-nam; yu, yingqi; liu, liyu; li, jing; zhang, kang; tong, weiwei; jiang, baoguo title: a four-compartment model for the covid- infection—implications on infection kinetics, control measures, and lockdown exit strategies date: - - journal: precis clin med doi: . /pcmedi/pbaa sha: doc_id: cord_uid: vgr ht a objective: to analyse the impact and repercussions of the surge in healthcare demand in response to the covid- pandemic, assess the potential effectiveness of various infection/disease control measures, and make projections on the best approach to exit from the current lockdown. design: a four-compartment model was constructed for sars-cov- infection based on the wuhan data and validated with data collected in italy, the uk, and the us. the model captures the effectiveness of various disease suppression measures in three modifiable factors: (a) the per capita contact rate (β) that can be lowered by means of social distancing, (b) infection probability upon contacting infectious individuals that can be lowered by wearing facemasks, personal hygiene, etc., and (c) the population of infectious individuals in contact with the susceptible population, which can be lowered by quarantine. the model was used to make projections on the best approach to exit from the current lockdown. results: the model was applied to evaluate the epidemiological data and hospital burden in italy, the uk, and the us. the control measures were identified as the key drivers for the observed epidemiological data through sensitivity analyses. analysing the different lockdown exit strategies showed that a lockdown exit strategy with a combination of social separation/general facemask use may work, but this needs to be supported by intense monitoring which would allow re-introduction/tightening of the control measures if the number of new infected subjects increases again. conclusions and relevance: governments should act early in a swift and decisive manner for containment policies. any lockdown exit will need to be monitored closely, with regards to the potential of lockdown reimplementation. this mathematical model provides a framework for major pandemics in the future. the novel coronavirus (sars-cov- ) and the infectionrelated disease (covid- ) were declared a public health emergency of international concern by the world health organization in early , and have since grown into a pandemic. , covid- has created an unprecedent global health problem, for which most healthcare systems were not well prepared. policies such as case isolation, social distancing, travel restriction, and quarantine represent the key measures adopted by various governments to control the outbreak. [ ] [ ] [ ] [ ] however, such measures also carry significant impact to individual psychological well-being and social/economic costs. many epidemiological models [ ] [ ] [ ] [ ] have been proposed to describe the dynamics of the transmission and simulate the course of the outbreak. however, few studies have assessed the impact of the effectiveness of various measures in the control of viral spread. a four-compartment model was established to describe the sars-cov- infection, assess the potential effectiveness of various infection control measures, and make projections on the best approach to exit lockdown. the population is divided into the following states: susceptible subjects (s), had close contacts (c, those exposed to infected subjects/pathogen but not necessarily infected), latent (e, infected and infectious but asymptomatic), infected (i; and symptomatic), recovered (v), and dead (d) ( fig. and supplementary data). the transmissibility of sars-cov- is modelled by two separate parameters-the social transmissibility factor β, which measures the probability of having close contact with infectious subjects, and the pathologic transmissibility σ , which measures the probability of an individual developing covid- upon contact with the pathogen. the model also allows a predetermined portion of infected individuals to stay latent for the entire incubation period and then move directly to the removed states (recovered or deceased) while bypassing the infected (i) compartment. the model was established based on demographic and covid- epidemiological data in wuhan. data from italy, the united kingdom (uk), and the united states (us) fit well with our model, assuming that these countries were affected by multi-sources at around the same time. β in the community was estimated separately. all other parameters were set to the estimated parameters from wuhan data before january . the four-compartment model and the validation in our four-compartment susceptible-quarantined-infected-removed (sqir) model, the transmissibility of covid- is modelled by two factors, the per capita contact rate (β, social interaction factor, when multiplied by the ratio of infectious individuals in the population, describes the probability of a subject moving from status s to status c), and infection rate upon contact (σ , the viral transmission factor, the probability of a subject moving from status c to status e). together with the quarantine rates (κ c and κ e ), they make up the parameters that can be modified by public health policies to suppress the outbreak. all other parameters in the model are pathogenic/viral characteristics that would not be affected significantly by non-pharmaceutical interventions. the progression rates from latent to infected and from infected to recovered were based on published estimates of . and . , respectively, which should remain relatively constant throughout the outbreak. [ ] [ ] [ ] the natural infection probability upon contact was set at . . the rate of covid- -related death of all hospitalized cases was set at . %. our model was calibrated using laboratoryconfirmed covid- cases in wuhan, china between december and march (fig. a) . the date of the first human covid- latent infection (d ) was set as december (assuming days before the first symptom of patient zero). the estimation of β was . under normal social circumstance, and . after lockdown, and σ was estimated to be . in the second and third periods, representing roughly a -fold decrease from . during the first period possibly related to the stringent compulsory facemask use policy. local government estimated that the effective quarantine rate after january was from % to %. in italy, our model fits, assuming there were four effective sources in italy at around that time (fig. b ). the estimated β was . before social distancing (slightly lower than wuhan, a metropolitan area densely populated). after nationwide lockdown, β was reduced to the population is divided into the following states: susceptible subject(s) (s), had close contact(s) (c, those that were exposed to the infected subjects/pathogen but not necessarily infected), latent (e, infected and infectious but asymptomatic), infected (i; and symptomatic), recovered (v), and dead (d). c m is the portion of the contact cases that are missed by contact tracing and will not be quarantined. individuals in states c, c m , and c q will progress to their respective latent groups e, e m (by contact tracing), and e q (quarantined). after the onset of symptoms, latent individuals will enter the infectious status i, and i q denoting the infected population treated in isolation wards. it was assumed that when the infected subjects have recovered, they will acquire immunity that does not wane during the timeframe of the analysis (i.e. of this season). . (starting march) and further reduced to . (starting march), suggesting that the stricter lockdown in italy achieved the same effect on β as in wuhan. our model assumed % effective facemask use compared to wuhan (σ = . ). for the uk, our model estimated that the number of effective sources was and β was . ( fig. c ). nationwide lockdown was implemented on march (β = . , σ = . ) and strengthened on march (β = . , σ = . ). for the us, based on cdc data ( days before the first illness onset), the estimated number of effective sources was (fig. c) . note that the effective number of sources might come down to six, assuming the infection arrived in the us in mid-january . the recent report that the sars-cov- in washington state had the same genotype as wuhan, whereas northeast us had predominately the genotypes related to europe was consistent with our projection. the estimates of β before and after the 'shelter in place' order were . and . , respectively, indicating that us compliance was within reasonable limits and the big jump in numbers was likely related to the multiple sources of virus arriving in the us at the same time. this model projected that implementation of case isolation/quarantine is an important measure to control this pandemic. in wuhan, combining social distancing and compulsory facemask use capped the growth rate of infected cases per day, but not enough to reverse the trend. adding contact tracing and quarantine (and other measures including general use of facemask) days after d completely curbed the outbreak in days. over the entire course, individuals ( . % of the population) were infected. the model projected that if no quarantine was taken and infection allowed to spread until herd immunity established, individuals ( . % of the wuhan population) would be infected (including crosses represent the cumulative numbers of cases observed. curves represent the model fitted to the observed data using mle. insets: the observed number of cases by date of illness onset (crosses) and the fitted curve in logarithmic scale. in wuhan, hubei, four distinct periods were defined: (a) before january before major public health interventions, (b) between january and january, when there was a travel ban and cancellation of social gatherings [which would lower per capita contact rates (β)] and compulsory facemask use [which would lower the infection rate upon contact (σ )], (c) between january and february, when quarantine was in place, and (d) between february and april. in italy, three distinct periods were defined: (a) from january ( days before the first covid- confirmed in rome) to march when the nationwide lockdown was implemented; (b) from march to march; and (c) after march when stricter lockdown policies were implemented that halted all nonessential operations. in the uk, two periods were defined before and after the nationwide lockdown was implemented on march. in the us, two periods were defined before and after the 'shelter in place' order in the san francisco area on march. being asymptomatic) at the end of year. for comparison, the annual culminative attack rates of two common human coronaviruses e and oc were . % and . %, respectively. to determine the sensitivity of each parameter, they were evaluated/matched to the observed outbreak data (supplementary table and supplementary fig. ). only the control measures were found to significantly affect the outcome. assuming that %- % of all hospitalized individuals needed critical care, , , the model estimated that the uk's need for hospital beds would plateau after april at around and critical care beds around , close to the estimates given by nhs england. for the us, the need for hospital beds and critical care units is still growing. our model estimated that on april , the us needed hospital beds ( per people) and around critical care beds, and by may (as predicted by our model on april ), the us would need an additional hospital beds ( per people) and around critical care beds. as of april, cdc reported that the overall cumulative hospitalization rate was . per people. by the world bank's estimation, the us currently has around million hospital beds and critical care beds, and, therefore, the covid- pandemic poses a heavy burden on the us healthcare system (the need for % more inpatient and % more critical care beds). the fact that covid- is very concentrated in new york city/new jersey suggests even higher projected needs there. the quarantine starting time after d was identified and the effective quarantine rate had the most impact to the outcome ( supplementary fig. ). sensitivity analysis of the quarantine rate of asymptomatic infected subjects (κ e ) showed a clear breakpoint between % and %. quarantine rates lower than % would lead to a completely uncontrollable outbreak. the impact of a delayed quarantine on the effectiveness of infection control was also significant (supplementary fig. ) . by reducing the duration between d and the start date of quarantine measures (assuming % quarantine rate) from to weeks reduced the overall attack rate from . % to . %. if quarantine measures started after weeks, these measures would not control the outbreak. we evaluated the impact of lockdown by estimating the projected equivalence of lockdown/social distancing to quarantine ( supplementary fig. a) . a stringent lockdown reduced the average social contact by > -fold, equivalent to % effective quarantine rate. however, the breakpoint of lockdown (i.e. the β that can control the outbreak on its own) was between . and , much lower than the estimated β achieved by lockdown in italy, the uk, and the us. thus, the effectiveness of such policy in these countries was reduced as observed. in wuhan, compulsory facemask use reduced σ by %, equivalent to % effective quarantine rate (supplementary fig. b ). the breakpoint of general facemask use was beyond % (around %), indicating that general facemask use alone would be insufficient for complete control of the outbreak . this effect was witnessed in wuhan during the second period ( - january). only after compulsory facemask use was combined with lockdown and quarantine in the third and fourth period (after january) did the number of new cases show a sharp downward trend ( fig. a, inset) . the combined effect of lockdown and general facemask use given different ranges of hospitalization ( %- %) is given in fig. . during the initial surge of the covid- outbreak in italy/uk/us, no disease control policy was in place (β = , σ = . ) and hospitalization rate (κ i ) was close to %, with the number of new cases per day doubling every days. both lockdown and general facemask use could reduce the growth rate of new infections; and when combined give the best overall effect. for lockdown to be effective, β needs to be reduced from to < . . currently in italy/uk/us, σ is estimated to decrease only slightly from . to . . with general facemask use at % compliance rate (σ = . ), β will need to be < . to reduce the number of new infections. this level of social distancing was achieved during lockdown in italy (estimated β = . ) and in the uk (estimated β = . ), but not yet in the us (estimated β = . ). with general facemask use (σ = . ), β will only need to be . , which is slightly lower than the current us estimate (β = . ). if stricter lockdown and facemask use are implemented together (β = . , σ = . ), the number of new cases would reduce by half every days, even without quarantine. higher hospitalization rate puts more cases under isolation and thus may ease the need for general facemask use. at % and % hospitalization rate, strict lockdown and compulsory facemask use (β = . , σ = . ) would reduce new infection by half in days and days, respectively. currently, most government advisers recommend continuation of lockdown till the outbreak is suppressed to an acceptable level ('wuhan approach'), a relatively safe approach. however, some governments are considering an exit to balance the sociopsychological impact of a long lockdown and the huge impact on economy. in italy, for lockdown to continue till zero new infection, it would have to continue until january (fig. a) . for the uk, it would not be until before the number of new infections dropped to zero (fig. b) . the us is still not seeing a firm plateau in the number of new cases and thus it may be even longer until the projection of zero new infection (fig. c) . a more balanced approach would be to reduce the number of new infections to a considerably low level, then relax the infection control policies measures in a controlled fashion with intensive monitoring. based on our projection, either strict quarantine of contacts, or a combination of both a relatively strict lockdown and general facemask use may be sufficient. it is important to note that the latter approach would need to be used in combination because of (a) the non-linear nature of the effectiveness, and (b) the existence of breakpoints as described above. we predict that in italy, a quarantine policy with an effective quarantine rate of % starting on may ( weeks after d ) in addition to the current implemented lockdown would reduce the incidence of new infections exponentially, reaching ≤ in just days ( june) and zero in days ( july). the same infection suppression effect can also be achieved with no active quarantine, but by using the same lockdown as at present, and mandate compulsory facemask use (fig. a) . in the uk, lockdown and general facemask use starting on may would bring the incidence of new infections to under in days ( july) and to zero in days ( september) (fig. b) . this is almost equivalent to an effective quarantine rate of %. an effective quarantine rate of % would bring the incidence of new infections to under in days ( june) and to zero in days ( august). in the us, general facemask use starting on may would bring the incidence of new infections to under in days, which is equivalent to a quarantine effective rate of %. if the government can achieve % effective quarantine rate, the incidence of new infections will drop to around in days ( july). implementing a monitoring-based, data-driven lockdown exit strategy is essential to sustain the containment of covid- . based on our model, loosening the quarantine too early while there are still a significant number of latent cases may lead to an uncontrollable second outbreak. with daily active monitoring of new infection numbers, it is possible to adjust the infection control policies to maintain new infections within a band trending downwards. as discussed, lockdown should only be loosened to social distancing (β = . ) and general facemask use when daily new infections are reduced to a very low number (e.g. cases) and re-implemented to an aggressive lockdown (β = . , similar to the lockdown in italy) with general facemask use when daily new infections is rising (e.g. ≥ cases). this approach will provide the time for vaccine, drugs, or other pharmaceutical interventions to catch up while allowing economical activities to be less uninterrupted in regions with low numbers of new infections. for italy (fig. d) , if the quarantine is lifted after daily new infections drops to < (june ) with all infection suppression policies currently in place (β = . , σ = . ), the number of new infections would quickly return to exponential growth. even with general facemask use (β = . , σ = . ), a second wave of outbreak is still inevitable. thus, the quarantine should last till the incidence of new infections is close to zero (on july), plus a -week wash out period (the estimated latent period) before implementing exit. in the uk, if quarantine is lifted after daily new infections drops to < ( july) along with all current infection suppression policies (β = . , σ = . ), it would only take days ( august) for the incidence of new infections to reach . however, an extended lockdown and quarantine that lasts days (until september to eliminate new incidences) would also be less plausible economically and politically. thus, we recommend that after the outbreak is suppressed to an acceptable level, restrictions can be relaxed gradually to keep the infection under control, allowing economic activities to recover. various level of social distancing and facemask use and data-driven lockdown policies are considered, including: disease suppression measures completely lifted (β = β , σ = . ), social distancing continues to apply (β = . , σ = . ), stringent lockdown continues to apply (β = . , σ = . ), social distancing and general facemask use continues to apply (β = . , σ = . ), compulsory facemask use continues to apply (β = β , σ = . ). data-driven lockdowns are also plotted: loosening of social distancing (β = . , σ = . ) and completely loosened (β = β , σ = . ). β : per capita contact (β) rate of the normal social distance in each country. this model can be modified to adapt to different adjusted scenarios. in the us, to effectively reduce the size of the outbreak, quarantine should remain in effect at > % rate. with quarantine, the number of daily new cases is projected to be < by july. afterwards, a monitoringbased, data-driven approach can be implemented. the same level of lockdown or social distancing plus general facemask use should continue. it should be noted that a full cycle of loosening and re-implementation would span days if during the loosening period, the infection control policies were completely lifted (β = . ), or days if loosened to social distancing (β = . ). in this study a four-compartment mathematical model was established for the sars-cov- infection, which could be useful in the policy decision-making process. second, our model suggested that italy, the uk, and usa likely had multiple sources of infections to account for the observed early sharp rise in the number of infected subjects. third, effective and early implementation of quarantine were the two most important factors for control of the outbreak. fourth, the relative contributions of quarantine, lockdown, social distancing, and the general facemask use were estimated. finally, different strategies for lockdown exit were evaluated and challenges identified. our model allows examination of the issues unique to sars-cov- infection, the highly infectious nature of this pathogen, the potential of this infection to overwhelm the healthcare system, and the alternative containment strategy implemented for this pandemic. the sensitivity analyses showed that, in the time from the index patient to control measures and effective quarantine, measures were most effective when the majority of the infected carriers, mostly asymptomatic - , were put under quarantine. our data support the latest recommendation from the us cdc on the use of facemasks in public during this pandemic. once the outbreak is under control, quarantine can be lifted through a data-driven, monitoring-based dynamic disease mitigation policy. thus, general availability of a rapid viral diagnostic test is critical. a real challenge will be the potential to have both sars-cov- and other upper respiratory viral pathogens prevalent at the same time (e.g. this winter) when this pandemic is still ongoing. if this occurs, our model will require adjustment for two pathogens simultaneously. our data highlight the importance for governments to act swiftly and decisively for any containment policies. also, any lockdown exit must be monitored closely with the potential for lockdown reimplementation. this four-compartment mathematical model describes sars-cov- infection, can be adjusted to reflect local transmission characteristics and public health capabilities, can help to determine the optimal local disease suppression strategy, and can help when making projections for the best local lockdown exit strategy. a novel coronavirus emerging in china -key questions for impact assessment a novel coronavirus outbreak of global health concern responding to covid- -a once-in-a-century pandemic? sars outbreaks in ontario, hong kong and singapore: the role of diagnosis and isolation as a control mechanism first-wave covid- transmissibility and severity in china outside hubei after control measures, and second-wave 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an observational cohort study clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china markov chain monte carlo: an introduction for epidemiologists association of public health interventions with the epidemiology of the covid- outbreak in wuhan, china spread and dynamics of the covid- epidemic in italy: effects of emergency containment measures first case of novel coronavirus in the united states phylogenetic network analysis of sars-cov- genomes clinical impact of human coronaviruses e and oc infection in diverse adult populations a methodology for performing global uncertainty and sensitivity analysis in systems biology clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study critical care utilization for the covid- outbreak in lombardy, italy: early experience and forecast during an emergency response. jama .online ahead of print transmission of -ncov infection from an asymptomatic contact in germany presumed asymptomatic carrier transmission of covid- epidemiological characteristics of pediatric patients with coronavirus disease in china. pediatrics . online ahead of print estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship estimation of the asymptomatic ratio of novel coronavirus infections mass gathering events and reducing further global spread of covid- : a political and public health dilemma we thank the volunteers who curated the number of cases in wuhan, us, uk, and italy. we thank members of lau, zhang, tong, and jiang groups for their assistance and discussions. supplementary data are available at pcmedi online. t. w., y. w., y. y., l. l., j. l., k. z., w. t., and b. j. collected and analysed the data. j.y.-n. l., developed the strategic approaches used for the analyses and helped to write the manuscript. t. w., k. z., w. t., and b. j. conceived and supervised the project and wrote the manuscript. all authors discussed the results and reviewed the manuscript. none declared. key: cord- - mossbr authors: andrle, michal; hebous, shafik; kangur, alvar; raissi, mehdi title: italy: toward a growth-friendly fiscal reform date: - - journal: econ polit doi: . /s - - - sha: doc_id: cord_uid: mossbr published in late , the italian medium-term fiscal plan aims to achieve structural balance by , although concrete, high-quality measures to meet the target are yet to be specified. this paper seeks to contribute to the discussion by ( ) assessing spending patterns to identify areas for savings; ( ) evaluating the pension system; ( ) analyzing the scope for revenue rebalancing; and ( ) putting forward a package of spending cuts and tax rebalancing that is growth friendly and inclusive, could have limited near-term output costs, and would achieve a notable reduction in public debt over the medium term. such a package could help the authorities balance the need to bring down public debt and, thus, reduce vulnerabilities while supporting the economic recovery. italy has been struggling with low economic growth since the s. productivity growth has been persistently anemic and has lagged that of the euro area-see anderson and raissi ( ) , andrle et al. ( ) , bugamelli and lotti ( ) and the references therein. held back by long-standing rigidities, the economy failed to take advantage of euro accession to modernize its institutions or adapt to a changing global trade and technological landscape. pellegrino and zingales ( ) attribute the weak productivity growth in italy to the lack of meritocracy in the selection and rewarding of managers, and the inability of firms to take full advantage of the information and communication technology revolution. akcigit et al. ( ) argue that political connections adversely affected firm dynamics, innovation, and creative destruction in italy, weighing on the economic growth. moreover, wage growth outpaced productivity growth, contributing to high structural unemployment; see kangur ( ) . easy access to finance pre-crisis boosted demand, but the double-dip recession earlier this decade and the subsequent tightening of credit conditions set italy back further; see doerr et al. ( ) , and mohaddes et al. ( ) . implementing structural reforms are, thus, of greatest importance. high public debt and an inadequate composition of fiscal policy have also contributed to italy's underperformance. public debt, above % of gdp and the second highest in europe, has been a perennial source of vulnerability-see reinhart and rogoff ( ) and chudik et al. ( chudik et al. ( , who consider the consequences of high and rising public debt on economic growth. italy has run primary fiscal surpluses that on average were higher than its euro area peers, but these were insufficient to lower debt and secure stability. the quality of fiscal policy has also insufficiently supported inclusive growth. the burden of high taxes has fallen on labor, public investment has been squeezed, and social benefits have centered on generous pensions. aiming for an adequate primary surplus to reduce debt, underpinned by high quality pro-growth and inclusive measures, is thus also important. this paper contributes to the debate by identifying growth-friendly and inclusive options for achieving fiscal balance that could potentially put italy's public debt on a firm downward path. firstly, it discusses public spending trends and composition. an analysis of spending over the past two decades reveals that: ( ) in the decade following euro accession, spending grew faster than potential output, owing in large part to the rapid growth of pensions; ( ) since the global financial crisis, spending has been broadly controlled, mainly through a freeze on hiring and wages and cuts in capital spending. pension spending though has continued to rise; ( ) despite the recent spending control, the pre-crisis spending excesses have not been reversed; and ( ) achieving sizable and durable expenditure savings may require lowering the large pension spending. improving the efficiency of health spending in some geographical areas could also lead to some savings. secondly, the paper analyzes the italian pension system. over half of current primary spending is social benefit expenditure, which is dominated by pension spending. at around % of gdp, pension spending in italy is the second highest in the euro area after greece. the italian authorities have legislated several reforms in the past. however, before the full effect of these reforms is evident over the very long run, fiscal pressures are likely to persist and weigh on italy's goal of achieving and maintaining a balanced budget. the second part of this paper finds that: ( ) despite past reforms, there remain generous parts of the system where italy is an outlier, pointing to areas of potential savings; and ( ) pension projections rest on optimistic assumptions of (a) employment, specifically that italy will go from having among the highest to very low unemployment rates; and (b) italy will maintain much higher real gdp growth rates for decades to come than has been its experience and policy settings. relaxing these assumptions implies a notable rise in projected spending over the coming decades until the full benefits of past reforms become evident. thirdly, drawing lessons from the literature and cross-country experience, it discusses options for revenue rebalancing in italy. the tax system is characterized by a high tax wedge, a relatively narrow tax base, and significant tax arrears. a fiscal devaluation strategy-a shift from taxing productive factors to taxing consumption and property-reveals the scope to ( ) decrease the tax wedge significantly; ( ) reduce value-added tax gaps (both compliance and policy), by harmonizing the reduced vat rates and improving the tax collection performance; ( ) rationalize tax expenditures; and ( ) raise revenues by re-introducing a modern property tax on primary residences. fourthly, it simulates, using the imf's global integrated monetary and fiscal (gimf) model, the impact of a growth-friendly and inclusive mix of spending and revenue measures along a gradual fiscal consolidation path that puts italy's debt-to-gdp ratio on a firm downward trajectory-see kumhof et al. ( ) and anderson et al. ( ) for modeling details. the model simulations show that a revenue-neutral and less distortionary tax reform, alongside current spending cuts and capital spending increases, can generate sizable output gains and a sustainably lower public debt ratio over the medium to long term. short-term output costs of this fiscal package, if implemented credibly, are likely limited. the rest of the paper is organized as follows. section discusses italy's public spending trends and composition. section analyzes the pension system and identifies certain areas that can be improved. section considers some options for revenue rebalancing. section conducts a simulation exercise to assess the macroeconomic effects of a growth-friendly mix of spending and revenue measures. section concludes. over the past two decades, primary spending in italy has grown faster than potential output. this was particularly the case in the years after euro accession. from to , italy's nominal current primary expenditure grew faster than the euro area average, and well above the country's average nominal potential growth-driven mainly by social benefit spending (primarily pensions), intermediate consumption (goods and services), and wages (in general services, defense and health). capital spending rose in line with that of the euro area average. from to , however, italy's nominal current primary expenditure grew at . % per year on average, below the euro area average of . %. the deceleration after the global financial crisis was driven mainly by the decline in the public sector wage bill-reflecting the freezing of nominal wages from to and a reduction in the number of public sector employees from . million in to around . million people in ; and a severe cut in capital expenditure, which declined by about % in nominal terms between and . nevertheless, even with these exceptional measures, total primary spending grew above the country's average nominal potential gdp growth over this period. italy has been unable to reverse its past overspending (especially those related to the pre-crisis period). the high cost of servicing public debt implies total public expenditure in italy about % of gdp above the euro area average (at . % of gdp versus . %). interest on debt ( . % of gdp in ) absorbs more resources than spending on education ( % of gdp), and is over ½ times as much as on defense ( . % of gdp). social benefits have dominated all other categories of spending, rising by about % cumulatively from to and by a further % since then. it constitutes half of total primary spending, up from % at the time of euro accession. the bulk of social benefits spending is in pensions (see sect. ), reflecting both a high share of elderly population and generous pension benefits. however, non-pension social benefit spending in italy is low, fragmented, and poorly targeted in comparison to other eu countries. the latter is evidenced in the disproportionately low share of social transfers accruing to the low-income working age population. there is also a higher reliance on intra-family transfers for social assistance, even as there is underspending related to social inclusion, family/child benefits, and housing relative to the euro area average. a decomposition of spending-using standard economic and functional classifications at the general government levels ( table )-reveals that, apart from pensions: ( ) interest payments exceed the euro area average by . % of gdp, given italy's high stock of public debt. ( ) other areas of overspending include intermediate consumption spending (primarily on goods and services) in the health sector; compensation of employees in defense, public order and safety, and health; subsidies in the economic affairs sector; and capital transfers in general services and economic affairs. ( ) it is notable that although overall public health spending in italy is in line with the euro area average, the bulk of it is for compensation of employees and intermediate consumption, in contrast with the euro area average where the share of spending on these two items is significantly lower and that of expenditure on provision of other social benefits is % higher. this points to room for potential efficiency savings, especially in some regions. medeiros and schwierz ( ) table italy and euro area: general government spending, - , and highlight regional differences and show that the output of public spending is lower in southern regions based on health-related variables, such as life expectancy at the age of . ( ) the main areas where italy underspends is in education (i.e., in the provision of goods and services and in total compensation). the public education expenditure gap is especially concentrated at the tertiary level, as highlighted in oecd ( a, b) . as for economic classification, underspending is largely in gross capital formation (fig. ) . there is, therefore, room to improve the spending mix to make it more growth friendly and inclusive. the above simple presentation indicates that rising social spending (primarily pensions) has crowded out spending in areas such as education and capital spending. achieving a more growth-friendly and inclusive spending policy mix, while making space to achieve the medium-term objective, will likely require rationalizing total social benefit spending; rationalization of non-pension social benefit spending; better efficiency in health spending in some regions; and reallocation of spending toward capital spending and education, while also improving the efficiency of outcomes in both areas. protection of the vulnerable could be further improved through complementary measures such as more intense use of active labor market policies and a modern social safety net; see imf ( ) for a proposal. following sharp cuts in capital spending and with the wage bill/gdp at its lowest in two decades, rationalizing social benefits spending appears unavoidable. in recent years, the authorities have pursued a strategy of notably cutting capital spending and curtailing the wage bill, which at . % of gdp is at its lowest level in several years. this strategy may be close to its limit, however, and may be neither sustainable nor desirable. there is a need for public investment to support stronger, sustained growth. moreover, as a share of total employment, public sector employment is below the euro area average; the age structure of public employees is titled toward older workers, implying the need to refresh the skill mix without reducing the headcount further (there have been recent announcements for hiring sizable numbers of new staff, in education and local offices); and, after years of wage freezes, wage increases are planned. this suggests limited room, if any, for further cuts in the overall wage bill or in capital spending, going forward, and thus for little alternative but to tackle the sizable social benefits spending (fig. ) . since , the italian pension system has undergone multiple reforms. these include pro-rata replacement of the old defined benefit (db) scheme with a notional defined contribution (ndc) scheme ( , tightening of eligibility requirements ( , , , , , ) , alignment of the statutory retirement age of women with that for men ( , ) , and indexation of the retirement age to life expectancy. the transition from the old db system to an ndc scheme divides pension beneficiaries into two categories: ( ) those with at least years of contributions by end- will largely maintain the db formula. for this group, the old pension rules are grandfathered for contributions accumulated until . for contributions accrued after , the ndc scheme applies. ( ) those with less than years of contributions by end- are subject to a pro-rated scheme. for this group, contributions accumulated up to will be subject to the db formula, whereas contributions accumulated after will be subject to the ndc scheme. the average contribution period in italy for new pensions is about years (expected to increase to years) and life expectancy at is about years. thus, by about , all new retirees entering the pension system will be fully subject to the ndc formula, whereas by about , the old db should be fully phased out also from the stock of existing retirees. eligibility requirements have been tightened considerably in a series of reforms, notwithstanding repeated attempts to weaken them. both statutory and early retirement ages are set to increase further over time as part of the 'fornero' reform (l. / ). currently, the statutory retirement age (sra) is years and months. early retirement is allowed regardless of age based on minimum years of service of - years. under ndc, workers may retire up to years earlier than the sra with minimum years of contributions and a pension of at least € per month. from onwards, the eligibility requirements are linked to changes in life expectancy at (every years up to and years starting from ). occasionally, pathways to early retirements were eased or implementation of stricter rules were postponed (e.g., the and budgets). the experimental "quota " early retirement rule, introduced in , created a discontinuity in the retirement age that needs to be addressed. in general, special treatments and incentives for early withdrawal from the labor market should be avoided in both db and ndc schemes (see also imf ). following the 'fornero' reform, the pension system ( ) adopts an actuarial computation of pension benefits using an implicit rate based on the accrued contributions, and automatically adjusted to mortality developments; and ( ) introduces periodic increases in all eligibility requirements in line with longevity developments. according to the quota rule, workers who are at least years of age with a minimum years of contributions are eligible for early retirement during - . women who are at least years of age with a minimum years of contributions are also eligible. automatic adjustments of the statutory retirement age to life expectancy were canceled for - . the existing db scheme is overly generous on many accounts: ( ) it uses a weighted average accrual rate of % (mef ) that is multiplied by the years of contributions and the reference wage (pensionable earnings) to obtain the monthly pension benefit. an accrual rate of % is high by international comparison, compared to about . - . % in the eu/euro area. ( ) for insurance years before , the reference wage is defined as the last monthly wage for civil servants or an average wage of the last - years in the private sector, based on different sources and occupations. for contribution years after , the number of annual wages involved in the calculation increases gradually until it covers the last years for employees and the last years for the self-employed. but the periods over which pensionable earnings are calculated are still too short and tend to inflate the pension benefits of the db scheme. on the other hand, the ndc (by definition) covers total lifetime contributions. under the db scheme, the early retirement penalty is % at the age of , % at the age of , and a further p.p. for each year below . these penalties are rather lenient- queisser and whitehouse ( ) calculate that, for italy, the actuarially neutral reduction in benefits for each year of early retirement is in the order of . %. as a result, the replacement rates under the current db/mixed scheme are high compared to other countries and place the adjustment burden disproportionately on future retirees (chart). the difference from the euro area average, according to european commission ( ) , is around % points. the simplest solution would be to reduce spending in db/mixed schemes equivalent to the thirteenth pension payment (i.e., the christmas bonus) that would constitute a . % cut in average pensions of the db component. another option that would improve intergenerational fairness is to recalibrate existing pensions based on the steady-state ndc formula or equivalent accrual rates. regarding the ndc scheme, although it can screen out many past excesses, the annuity factor is based on a too high internal rate of return (irr). in a "pure" ndc, the irr should be chosen to ensure actuarial balance between the system-wide assets and liabilities ("annex"). in steady state, the irr converges to the rate of economic growth. while in the italian ndc the irr that credits the notional capital each period is the moving average of nominal gdp growth over the past years, the discount rate used to derive the annuity factor, defined as the ratio of the irr to a rate of inflation indexing, is set at a rate of . %, based on an expected longrun real growth rate. absent comprehensive and decisive structural reforms, such a real rate of return is considerably above italy's current growth potential. in the italian pension system, the adjustment to macro-demographic conditions (such as the periodic revisions in the transformation coefficient) affect future generations of retirees only, leaving current retirees unaffected. the irr that credits the notional capital is linked to past performance. it would therefore be important to introduce an automatic adjustment (or sustainability) factor that links current pension payments to a measure of a long-term actuarial balance to shield against unforeseen shocks and improve intergenerational equity (see barr and diamond, , for a discussion on such a "break" mechanism in sweden). at around ¾% of gdp, spending on survivor pensions is the highest in the europe. according to eurostat, the average monthly survivors benefit per inhabitant (at constant prices) in italy was € compared to about € in the euro area in -the second highest in the euro area after luxembourg and the third highest in europe after luxembourg and denmark. similarly, survivor pensions in italy have very wide coverage: the number of survivor pensions forms about % of total pensions in italy and is much higher compared to about % in the eu on average. the eligibility for a surviving spouse in italy does not appear to be constrained by an age limit, the absence of which can also dis-incentivize return to the labor market, especially for women. survivor pension payments to family members other than surviving spouse or orphans should be strictly limited. revenues collected from the self-employed could be increased. at %, the pension contribution rates on wage earners are high. of the contribution rates on wage earners, about one-third is borne by the employee and two-thirds by the employer. for the self-employed and farmers, the contribution rate in was . %, set to increase to % by . one explanation for the difference in the contribution rates for employees and self-employed relates to differences in the gross base: for the selfemployed, the gross contribution base includes all contributions whereas for workers only one-third (the employees' share). however, even then, the "neutral" contribution rate for the self-employed should be at least %. the self-employed in italy exhibit below average revenue productivity compared to their peers in the euro area. this is indicative of the need to further harmonize the contribution rates as well as strengthen collection and payment compliance. the tax burden on pensions is favorable to retirees. according to the oecd ( ), both the gross and net replacement rates in italy are on average about % points higher than for the oecd average retiree. the extension of the non-taxable area for retirees, as was the case in the budget, will further widen the gap. compared to wage earners, retirees in italy are subject to preferential tax treatment in terms of a higher tax-free allowance and full exemption on health contributions on pensions. compared to retirees in other oecd countries, italy offers tax relief on pension income from private schemes. deviations from general rules and partial reform reversals can also undermine past reforms. for example, the provisions in the budget for an annual fourteenth pension payment to low-income persons and higher tax-free thresholds for pensioners are costly and depart from actuarial fairness. moreover, in january , early retirement rules were eased notably. workers who are at least years of age with a minimum years of contributions have become eligible for early retirement. women who are at least years of age with a minimum years of contributions are also eligible. moreover, automatic adjustments of the statutory retirement age to life expectancy were canceled for - . such a reversal of past pension reforms would raise the number of pensioners, lower labor force participation and potential growth, and add to an already high pension bill. although the ndc in the long run is expected to reduce pension spending, by itself it is not enough to deal with italy's fiscal problems. according to oecd ( a, b), future gross replacement rates in italy would remain one of the highest in the oecd (text chart) with both gross and the net replacement rates about - % points above the oecd average, depending on the average pensionable wage. similarly, european commission ( ) projects italy's pension spending to remain more than % of gdp above the average of other european countries. this is partly due to many features described above, including high discount factor, survivor pensions, and transformation coefficient for women with children, but also due to the very high pension contribution rate of %. rapid aging will also put strong pressure on spending on health and long-term care that, according to european commission ( ) , is expected to increase by about ½% of gdp by (net of lower spending on education). in the latest report by the department of the state accountant general (rga) of the ministry of economy and finance, long-term pension spending is projected to remain subdued, supported by the implementation of the above-mentioned past pension reforms and strong recovery in employment and productivity. according to the rga ( ) projections, pension spending as a ratio of gdp is expected to increase from . % in to just above % in and decline afterwards, reaching . % by . based on these findings, the pension system and overall public debt are understood to be sustainable over the long term, and it is argued that italy is in much better stead than many other euro area members that have still to come to terms with age-related spending. there are several offsetting factors that contribute to these projections. firstly, the rga notes that adverse demographic trends as captured by rising old-age dependency are the main drivers of future pension increases, adding about ½% of gdp to pension spending by . secondly, over the next decade, until , the benefit rate-the ratio of average pensions to gdp per worker-is expected to increase pension spending, owing to the generosity of the old though declining db component compared to low productivity growth. thereafter, the share of retirees under the ndc scheme is projected to become sufficiently large to dominate the more generous older db scheme, settling the benefit rate on a modest downward trend. thirdly, the strongest savings in the rga's projections stem from a sizeable pick-up in the employment rate, with a notable increase in labor force participation and a substantial decrease in unemployment, as well as from reforms to restrict early retirement and extend retirement ages (eligibility rate)-reducing pension spending by about ½ and % of gdp over the long-run, respectively. with the unemployment rate reaching as low as . % of gdp by (and remaining steady afterwards), italy is expected to move from one of the worst to among the best performers in the labor market. relaxing some of the optimistic demographic and macroeconomic assumptions suggest spending would be notably higher (fig. ) . the simulation results indicate that for the rga ( ) projections to materialize, the ndc system must cut average pensions of future retirees further by about ½% of gdp (or by more than by %). the increase in the employment rate for the - age bracket in rga ( ) appears optimistic, based on current policy settings. it increased from % in to ½% in , driven largely by a decline in the unemployment rate to about . % by . however, italy's long-run average unemployment rate has been around ½%. assuming italy's unemployment rate settles at %, which implies an increase in the employment rate to about % in the long-term, the total pension spending increases by ½% of gdp by (solid red lines in fig. ) . with strong employment recovery, the authorities are also expecting per capita real gdp and real labor productivity to grow at around ¾% annually, far above what has been observed for the last few decades. such projections appear very optimistic. lower tfp growth would lower gdp growth immediately but would impact pension benefits slowly-through wages that pass through to lower contributions and thus lower notional stock of pension capital. according to the rga ( ), . % points lower labor productivity growth would lead to about . % of gdp higher pension spending in both and , whereas . % points lower tfp alone would increase the pension spending to gdp ratio by . - . % of gdp, respectively. in response to a permanent negative labor productivity shock (of about ½% points per year), our simulations suggest that pension spending would be about % of gdp higher in both and (red long dash lines in fig. ) . the population projections by the united nations population division point to more rapid aging in italy compared to demographic projections with base year recently published by istat, increasing the long-run pension spending further by about % of gdp at peak (red dotted lines in fig. ). the rga ( ) also reports an additional set of pension projections based on the ec-epc (awg) assumptions (with a steady state unemployment rate of about ½% as well as a faster achievement of % tfp growth by ). the result is an increase in pension spending by about % of gdp at peak (blue long dash lines in fig. ) when compared to the national scenario. in sum, taking more prudent assumptions for the employment rate, productivity growth, and demographics, which are closer to the historical record and based on current policy settings, pension spending as a percent of gdp is projected to reach . in (about % of gdp above the rga's baseline projection for ) before decline to . in (about ½% of gdp higher than rga's baseline projection for ). consideration should be given to enacting measures that would yield savings in the near term and secure savings over the medium term, consistent with current policy settings. near-term savings come from addressing the excessive generosity and lack of actuarial fairness in the db and mixed schemes, and several options to a temporary negative labor productivity shock of the same size (over the period - ) though would result in a . % of gdp higher pension spending between and before the impact of the shock fades away. cross-country as well as italian experience suggest that pension reforms may face political opposition (e.g., france) or sometimes can run into institutional and legal boundaries, often manifested in constitutional principles such as equality and proportionality. an example is whether pension measures should apply to new retirees (i.e., on a flow basis) or to existing pensioners (i.e., impacting the stock). in some institutional settings pension measures can often be best implemented in a context of a coherent systemwide reform rather than in a piecemeal or ad hoc manner, affecting only certain categories of pensioners. it can also be easier-both from institutional and political perspectives-to implement pension reforms if they are based on well-established system-wide principles such as equalization of internal rates of return or actuarial fairness. for example, kohli and arza ( ) argue that reforms that involve stronger link between contributions and benefits may be more legitimate and easier to implement since, when compared to retrenchments or stand-alone parametric changes, these appeal to shared values and norms. pension reforms should also be clearly justified and accompanied by socio-economic and legal assessments. every effort should be done to clarify reform needs, that, inter alia, can involve improving system-wide equity or overall fiscal sustainability. this end are outlined below. these could go toward creating the room for achieving higher primary surpluses that italy needs to put public debt on a firm downward trajectory as well as to improve intra-generational equity by shifting the adjustment on retirees who thus far have been relatively better off. longer-term savings come from using more realistic (or conservative parameters) that guide long-term pension benefit calculations as well as ensuring actuarial balance. our results show that there is merit in: ( ) eliminating the fourteenth pension payment fully and the thirteenth payment with an equivalent reduction in annual benefits for all retirees in the db and mixed schemes. support for the most vulnerable-a justification provided for the introduction of the fourteenth pension payment to low income retirees in the budget-could be achieved through the social safety net, in particular a well-designed guaranteed minimum income scheme; ( ) introducing an age limit for a surviving spouse and limit any payments to relatives other than surviving spouse or orphan. this would restrict eligibility for a survivor pension, reduce spending, and incentivize labor force participation; ( ) recalibrating existing pensions based on the steady-state ndc formula or equivalent parameters for accrual rates and/or pensionable earnings. this would serve to reduce short-to medium-run pension spending by reducing benefits to those who have benefited from the generous db scheme. it will not affect the long-run steady-state spending (given by the ndc); ( ) harmonizing (effective) contribution rates of self-employed with those of wage earners. lower contribution rates for the self-employed constitutes preferential treatment. although from a system-wide point of view lower contribution rates in the ndc eventually translate into lower pension benefits, it reduces the financing available to the pension system in the payg system and is a source of unfairness. moreover, ( ) an option for reducing the high labor tax wedge-as part of a fiscal devaluation strategy-while lowering long-run replacement rates can rest on lowering employers' pension contributions. this not only reduces the tax wedge on labor for current workers, but also translates into lower future pension spending via the ndc scheme. however, this is not the first-best policy choice from the point of view of a fiscal devaluation when there is a tight (and actuarially fair) link between contributions and benefits that can be imposed by the ndc, since in that case pension contributions are effectively deferred savings that are less distortionary than other contributions (e.g., health) that are more redistributive in nature. this option could be considered if future pension spending cannot be reduced by other means. there is also value in: ( ) subjecting pension benefits to health contributions and realign the tax-free threshold with wage earners. retirees should not be burdened with pension or unemployment contributions, although they are relatively more frequent consumers of health services and therefore should pay health contributions. consideration should also be given to reversing the higher tax-free threshold for retirees introduced in the budget; and ( ) adjusting the ndc discount factor to reflect realistic growth potential and introduce an automatic adjustment mechanism that links pension spending to the long-term actuarial balance (as, for example, done in sweden, canada, and germany). the main purpose of such a mechanism is to allow for automatic adjustments in current pension payments as a response to permanent shocks, thus helping to keep the pension system solvent without a possible need to increase payroll taxes (that in turn would lead to increases in future benefits). the discount factor currently fixed at . % annually is well above the italy's long-term growth potential based on current policy settings. a key implication of the above simulations is that italy needs to pursue comprehensive growth-enhancing reforms as a matter of urgency to reduce nominal wage rigidities and increase productivity and long-run employment rates. in the absence of such reforms that will take time to yield gains and reduce existing imbalances, even the self-adjusting ndc cannot ensure the sustainability of the pension system and public debt. it would, therefore, be prudent to set the safeguards as well as the system-wide parameters to be in line with the economy's potential under current policies rather than the stronger growth rates assumed in the rga ( ) projections. such an approach would reduce the risk of needing to take painful, large adjustments over a short time and thus reduce policy uncertainty. the italian tax system has many aspects of a dual income tax (dit) regime. it applies a flat tax rate of % on capital income (dividends, interest income, and capital gains on securities), and % on rental income. labor income is subject to a progressive scale with a starting rate on the first earned euro of % and a top tax rate of % for income exceeding € , (the personal income tax is known as "irpef"). the corporate income tax (cit) rate, the so-called "ires," stands at %, but a surcharge of . % is imposed on financial and insurance companies. in addition to the ires, there is a "regional production tax"-an origin-based valueadded tax known as the irap-imposed as a fixed rate of . % on the net value of production. however, tax rates remain high and are applied on a relatively narrow base. total government revenues-at . % of gdp-compare favorably with the eu average of % (table ) . out of this, total tax revenues of . % of gdp in also compare favorably with revenues in the region. about high tax rates, it should be noted that the labor tax wedge is high-e.g., for a single person earning an average income it is . %, well above the oecd average of . %. this pattern is observed across levels of income and types of the essence of a dit regime is to tax capital at a low single rate and labor income under a progressive schedule. the % flat rate applies in the case of non-qualified shareholding. if certain thresholds' requirements are met, then . % of the (qualified shareholding) capital gains or dividends are subject to the progressive personal income tax scale. a reduced rate of . % is applied to the share of capital income deriving from state securities; and a tobin tax exists on financial transactions and stamp duties, consisting of taxes on stock of financial assets rather than incomes. ten percent of the irap paid during a year can be deducted from the ires. the labor cost for openended employees can be deducted from the irap tax base; there is a possibility for regions to reduce up to zero the tax rate of . % or increase it by up to . pp. the tax wedge is sum of taxes and sscs paid by employees and sscs paid by employers, minus family received benefits. the average tax wedge is the tax wedge divided by the total cost of labor for the employer. this measure can be computed at various levels of income and types of households (singles, couples, with or without children). the authorities plan to reduce the labor tax wedge by . - . % of gdp in - . this is to be implemented by extending the national income tax bonus (from € per month to € per month) in and potentially folded into a planned tax reform in . households. the ratio of the social security contributions (ssc) to gdp is . %, which is % points higher than the eu average. the share of personal income tax (pit) in total taxes is among the highest in the eu at %. the cit to gdp ratio is about %, well below the eu average of . %, even though the cit rate is significantly higher than the current eu simple cit average (excluding italy) of . %. with the irap, italian companies are taxed at an even higher rate. the standard vat rate is % compared to an eu average standard vat rate of about . %. about the relatively narrow base, it should be noted that tax expenditures are quite large, estimated by tyson ( ) at . % of gdp, and by the "commissione marè" report on tax expenditures at . % of gdp. a recent report from the ministry of economy and finance (mef) identifies measures of this kind on a legal basis. italy has one of the weakest performing vat systems in the eu, reflecting the presence of policy as well as compliance gaps. the vat c-efficiency-an indicator of the departure of the vat from a perfectly enforced tax levied at a uniform rate on all consumption-at about % is well below the eu average. combining this with a compliance gap of about %, as estimated by ec to be the fifth highest in eu, implies a policy gap of about % (the second highest in eu). moreover, the cit revenue productivity is only . % compared to the eu average of . %. alternatively, the implicit tax rate on corporate income in italy was . % in (the latest year available), as compared to . % for spain and . % for the uk. finally, tax evasion is very high. on average and over the period - , the amount of to reduce the labor tax wedge, italy has adopted several measures, including ssc exemptions, the € pit reduction, and deduction from the irap tax base of the labor cost of hires with permanent contracts. see keen ( ) for a detailed discussion of the c-efficiency measure. the policy gap can be further decomposed into those arising from exceptions and rate dispersion. a "fiscal devaluation" (see de mooij and keen ), support for investment, and a tax administration reform would make for a growth-friendly strategy. specifically, shifting the tax burden from labor income to less distortive tax bases would include (a) lowering employers' ssc rate to closer to the eu average; (b) using well-designed targeted instruments to increase labor supply; (c) introducing a modern property tax on primary residences and updating cadastral values; and lowering the vat policy and compliance gaps, e.g., by harmonizing the reduced vat rates, reducing the range of items subjected to reduced rates or exemptions, and considering a moderate increase in the standard vat rate; (d) eliminating inefficient tax expenditures (e.g., abolishing the mortgage interest tax credit); and (e) strengthening capital gains taxation by ensuring italy's right in the domestic law to tax capital gains from offshore indirect transfers of assets. investment could be encouraged through more effective, efficient, and credible tax provisions, building on measures such as adoption of an allowance for corporate equity (ace) regime since and several internationally-required anti-taxavoidance provisions. specifically, this strategy would include (a) streamlining targeted tax incentives to encourage innovation and r&d investment; (b) improving the design of ace, e.g., by providing a higher ace rate for start-ups; (c) abolishing the intellectual property (ip) box regime; and (d) improving the overall investment climate by addressing uncertainty in tax matters that dampen taxpayers' confidence and investment, e.g., by making the r&d tax credit permanent and credibly announcing the non-extension of enhanced depreciation. reforming tax administration, includes restoring autonomy to fiscal agencies, strengthening enforcement, relaxing legal constraints to tackle tax debt, and bringing instalment arrangements in line with international best practice. a "fiscal devaluation" is a revenue-neutral shift in the tax structure (e.g., from employers' social security contributions toward value-added and property taxes) with positive effects on output. reducing employers' sscs can stimulate labor demand in the short term. given wage rigidities and being in a monetary union with major trading partners, cutting employers' sscs can reduce labor costs (and producer prices, including those of exports) as well as increase labor demand in the short term. the resulting favorable effect on the trade balance could be temporary though, if nominal wages eventually adjust to fully offset the cut. however, the impact on employment and output may be longer lasting with a shift in the tax burden toward non-labor income (vat and property taxes) that is also less distortionary. moderately increasing the share of employees' sscs in total could, under certain conditions, partially finance the cut in the employers' ssc and ensure a stable stream of funding. changing the composition of the sscs by adjusting the employees' ssc share would address the risk of using general revenues to finance pension and social security obligations. however, there could be a negative effect on wages and labor supply, and thus the measure could best be introduced should the government decide to reduce personal income taxes and be complemented with other targeted measures. better use of targeted measures toward increasing labor supply, especially of low-income earners, is recommended. italy has the lowest labor supply of married women among eu countries. this is in part driven by a tax credit for non-working spouses that discourages their labor supply. a better design is to replace this with a tax credit if both spouses are employed (called working family, or in-work, tax credit), which can be increasing with the number of children (as, e.g., in the uk and the us). available evidence suggests that adopting in-work tax credits for low income earners, within a revenue-neutral reform, can have sizable impacts on the female labor-force participation and aggregate employment (saez et al. ; de mooij ) . for italy, colonna and marcassa ( ) find that replacing the dependent-spouse tax credit with an in-work tax credit increases married women participation rate by % points. however, although in-work tax credit alleviates the tax burden at the extensive margin, there is a risk of increasing distortion at the intensive margin of labor supply, which can be mitigated through an appropriate design of the in-work credit. re-introduction of a property tax on primary residences is a vital element of a modern tax system in italy. the municipality property tax (known as "imu") and the municipality tax on local services ("tasi") for primary residences were abolished in , owing to their unpopularity. the property tax is an efficient instrument and can raise significant revenues. in , recurrent taxes on immovable property raised . % of gdp in italy. even if taxes on primary residence were reintroduced, to fully exploit the potential of the property tax, it is essential to reform the cadastral system and update the cadastral declared value of the property on the rogito (deed of sale). using municipal property taxes to finance local governments enables the central government to reduce transfers to local governments and free up resources to fund the lowering of employers' sscs. lowering the vat policy gap by harmonizing the reduced vat rates can raise significant revenues. the vat compliance gap as of was € . billion, about % of total vat liability ( . % of gdp), significantly higher than the eu- average ( . %). halving this gap, while maintaining all tax rates unchanged, would increase revenues by . % of gdp. moreover, based on eu ( ), fully closing the policy gap, i.e., if no vat reduced rates and exemptions were applied, would enable italy to increase its vat revenue by an additional %. this estimate, however, is based on a full compliance scenario. adopting a lower number of reduced rates could be an intermediate step toward lowering this policy gap. decreasing the range of items subjected to reduced rates or exemptions is also important for lowering the policy gap. for instance, instead of exempting taxi services from the vat, they can be subject to the reduced rate; however, if taxis pay vat on their inputs, a careful analysis is needed to assess the revenue impact. tax arrears are at an alarmingly high level reaching € billion (as of ). toro et al. ( ) suggest that a significant amount of arrears is not collectable (e.g., because % of debtors are out of business or bankrupt and % relate to cases where enforcement actions were taken but did not result in actual collection) calling for effective write-off arrangements. recurrent tax concessions undermine voluntary compliance culture and the effectiveness of tax administration. about € billion of tax arrears is deemed recoverable. enforcement actions are critical that could be supported with timely filing, modern payment arrangements, and relaxing legal constraints. italy embraces a large set of tax credits in part reflecting income redistribution mechanisms. for example, within the personal income tax, there are tax credits for income sources, an € per month bonus, and family tax credits (including for dependent spouses and children). other allowances/deductions within the tax structure include substitute tax on capital income, ace allowances and participation exemption, reduced vat rates and compulsory payroll tax deductions. the largest item is the tax credits for income source. this item reflects the fact that the first bracket of income (from zero to € , ) is subject to a tax rate of %. these tax credits are reduced progressively until, at income levels above € , , they no longer apply. thus, this tax credit is warranted for redistribution. however, some other tax expenditures within the direct income tax should be revisited and could be gradually eliminated, including: ( ) mortgage interest tax credit. the tax credit is equal to % of the mortgage interest payments. the upper limit of this tax credit is € . since capital gains on primary residence in italy are exempt from the capital gains tax, and high household debt could be associated with stability risks (imf a), the mortgage interest tax credit should be phased out or at the very least its generosity should be lowered; and ( ) tax credit for medical expenses. this tax credit is equal to % of medical expenses exceeding € . . yet, redistribution motives in this area can be better-targeted using government expenditures, and furthermore, currently, health services are either subject to a reduced vat rate or exempted from the vat in italy. a simple and certain business taxation strategy is recommended that relies on two key elements: ( ) innovation and allowance for corporate equity, and ( ) removal of inefficient incentives. note that in the presence of ace, changes to statutory cit several measures were introduced in recent years to reduce the vat gap. examples include optional electronic invoicing, more frequent vat invoice transmissions, and split-payment and reverse-charge mechanisms. a analysis of the tax credit for medical expenses based on tax returns statistics show that the beneficiaries, mostly with incomes below € , , are . million for a total amount of € . billion of expenses. rates are less likely to impact investment decisions. the extent to which the revenue loss of the recent cit rate cut from . to % can be compensated by increased investment and growth depends on profit shifting and location choice. the lower cit rate can reduce incentives for profit shifting. however, this aspect is unlikely to be significant because, italy has agreed to comply with the atad and the g / beps minimum standards. these anti-avoidance measures help safeguard against profit shifting. the neutrality of the cit regarding ace means that any impact on investment will come in effect from changed location by multinational companies, but the location decision depends on several other tax and non-tax factors (including labor regulations and labor supply). firms that would have invested anyway would also benefit from the rate cut, adding to the fiscal cost but without benefit. ace contributes to very low, perhaps even negative, marginal effective tax rates, thereby positively impacting investment. effective tax rates summarize the impact of major elements of the tax base, such as depreciation allowances and the ace, along with the rate of tax itself. in theory, the marginal effective tax rate (metr)a measure of the tax burden on an investment that just yields the required rate of viable return-is zero in italy because the ace does not tax normal return. in the text figure, taken from the oxford center for business taxation, the metr is negative, suggesting that the marginal investment receives a subsidy in italy (although these calculations must be interpreted with caution as the negative rate is driven by strong assumptions underlying the calculations). another measure is the effective average tax rate (eatr), which is important for multinational companies' location choice for new affiliates (it measures the proportion of the present value of pre-tax profit that would be taken in tax). the eatr in italy compares favorably to several eu member states including spain, france, germany, and portugal. the impact of the ace on investment could be enhanced, for instance, by providing a higher ace rate for small businesses (perhaps contingent on an age requirement), re-linking the rate to government bond yields, and a premium to reflect risks, and introducing a minimum rate of - % in line with the eu common consolidated corporate tax base (ccctb) proposal to enhance tax certainty. well-designed r&d tax incentives can have a sizable impact on productivity. taxation can incentivize private r&d activities through the input side-in the form of an r&d tax credit or deduction-or the output side in the form of a reduced tax rate on ip income ("ip box"). while italy has measures on both sides, the former measures are more efficient. empirical evidence suggests that one euro spent by the government on r&d tax incentives, on average, increases domestic private r&d by one euro, whereas one euro spent on an ip box can, at best, increase r&d by less than one euro (imf b; dumont ) . bloom et al. ( ) estimate that a % reduction in the cost of r&d increases the level of r&d by about % in the short run and % in the long run. griffith et al. ( ) estimate that ip regimes have resulted in lower revenues from ip in the benelux countries and the uk not all eu countries adopt an ip box, while the tax rates for those that apply an ip regime are shown in the text figure. italy exempts % of qualified ip income from taxation, and taxes the within the supplementary budget, the base of the ace tax deduction was changed from "the increase in equity since " to "the increase in equity in the last years before the tax year considered". the strategy followed by italy to scale-up investment and enhance productivity includes: ( ) tax credits for r&d investments; ( ) accelerated depreciations, such as super and hyper-amortizations; ( ) subsides to smes to repay loans and agreements with banks to promote access to credit, as envisaged by the so-called nuova sabatini law; ( ) specific credits and crowdfunding for start-up and smes; ( ) tax allowances, such as ace; ( ) state guarantees on loans of smes; ( ) a reduced tax rate on incomes from the direct use or license for ip incomes (the so-called patent box); and ( ) targeted incentives to innovative start-ups. remaining % of that income at the statutory cit rate of % implying an effective tax rate of about %. in general, there are some concerns with ip boxes. the ip tax relief: ( ) rewards only success. successful r&d outputs are a function of many non-r&d related inputs (including management) that are not characterized with market failure. ip regimes may discriminate against potentially important r&d activities that may not be successful quickly. ( ) it is proportional to the amount of qualifying ip income, and not connected to the level of r&d expenditure. that is, two patents may generate the same income, thereby receiving the same benefits from the ip regime, even if they have different levels of r&d input. ( ) it cannot perfectly target the location of r&d. there is a distinction between the legal ownership of patents (and knowhow assets) and the location of r&d activities that led to the patents. ip boxes can influence the location of the legal ownership of the know-how assets (within the multinational group) with little effect on domestic r&d investments. essentially, large enterprises particularly in the manufacturing sector benefit the most from this scheme. options to streamline the existing r&d and investment incentives include: ( ) abolishing the ip box regime. the october european ccctb proposal envisages a super deduction for r&d expenditures. if implemented, the ccctb would phase out ip regimes. the supplementary budget attempts to harmonize the patent box regime to oecd standards. ( ) making the tax credit for r&d expenses permanent. ( ) credibly announcing that temporary super depreciation rules will not be renewed (starting from a given date). ( ) periodically assessing the effectiveness of the allowances for investment in innovative startups. potentially, this measure should not be size-based and apply only to startups. frequent changes to tax policy and administration, and excessive use of temporary provisions can be an important source of uncertainty (imf/oecd ). the frequency of tax changes in italy is high compared to other g countries, and introducing or renewing temporary measures with varying conditions is prevalent. temporary measures generate uncertainty when their expiry date is either unclear or not credible. such uncertainty risks creating a hold-up problem, as firms defer investment until the uncertainty is resolved. the imf's global integrated monetary and fiscal model (gimf) is used to illustrate the effects of the above-mentioned fiscal package. gimf is a multi-country structural dynamic general equilibrium model featuring italy, the rest of the euro area, and the rest of the world. it links the behavior of households, firms, and government sector within and among countries. the model has a consistent system of national accounting and stock-flow budget constraints for all sectors, including the government. the model belongs to exogenous-growth types of models, meaning that the long-term growth of output is exogenous. hence, all fiscal or structural measures may change only the structure of the economy, possibly increasing permanently the level of real output per capita; never long-term growth. the household sector consists of forward-looking optimizing households, as well as liquidity-constrained households who spend all their available income every period. the forward-looking households are modeled as overlapping generations (olg) with finite lives, following the blanchard-weil-yaari framework. the presence of olg households breaks the ricardian equivalence and is important for realistic results of fiscal policy in both the short and long run. households gain utility from consumption and disutility from labor effort, they consume traded and nontraded services and goods, receive labor income, transfers from the government, dividends from corporations, and pay taxes-income, consumption, and lump-sum taxes. firms produce intermediate and final goods using labor and capital inputs, accumulate capital, and import or export their production. firms pay taxes from corporate income. monetary policy in the euro area and rest of the world regions follows an inflation-forecast targeting rule to set policy interest rates. italy is a member of the euro area. government collects tax revenues (consumption, labor income, capital income, and lump-sum taxes) and spends them on government consumption, investment, and transfers to households. governments target specific debt-to-gdp (and thus deficitto-gdp) ratios and use a mix of instruments to achieve it. the government's commitment to sustainable public finance is credible for firms and households, who hold the stock of government bonds. the scenario modeled assumes a permanent fiscal consolidation of about ½% of gdp (in the structural primary balance) per year for years with the aim of achieving a small structural surplus in the medium term, supported by a pro-growth mix of revenue and expenditure reforms, and is compared to a trend or no-policy-change baseline. two types of growth-friendly revenue and spending measures are considered along the envisaged fiscal consolidation path: shifting taxation from direct to indirect taxes, and lowering expenditure and shifting its composition from transfers to investment. on the revenue side, a lower labor tax wedge ( . % of gdp) is offset by higher vat collections from reduced policy and compliance gaps ( % of gdp) and introducing a modern property tax on primary residences ( . % of gdp). on the expenditure side, spending on public consumption is lowered by . % of gdp, while productive public investment spending is increased by . % of gdp. the remaining portion of the fiscal consolidation, . % of gdp, is implemented via reduced social transfers. in the model-based analysis, it is assumed that higher public investment spending and an associated higher level of government capital exert positive spillovers on private sector productivity. finally, the marginal cost of borrowing, i.e., at issuance, is assumed to increase in the medium term from its average of . %. the -year italian government bond spreads vis-à-vis german bunds are assumed to decline gradually from an average of basis points in to basis points in , which is the average level of spreads over the past years. spreads are mainly driven by the evolving interest rate changes in italy as german -year bund yields are expected to remain low over the medium term. interest-rate spreads are expected to decline further in the long-term despite monetary policy normalization-given the quality of simulated fiscal adjustment and the gdp path in italy. the policy package would result in an output increase of around % and a lower debt-to-gdp ratio of around % points in a decade (fig. ) . the increase in output is even larger in the long run (around ½% higher than the baseline) while the debt-to-gdp ratio is more than % points lower than the baseline. for example, starting from % of gdp in , debt-to-gdp ratio would decline to % in years and gdp growth would be . % points higher per year. the positive response of the economy is a result of a less distortionary new tax structure, with lower labor tax wedges, and of the more productive spending, namely on public investment, and lower debt-service costs (implementation of this package would bolster investor confidence). lower taxes on capital induce firms to increase investment and raise their desired level for the private capital stock. lower labor income taxes encourage households to provide more labor. the net effect of lower income taxes and higher lump-sum as well as value-added taxes is positive on private consumption in the long term. the revenue-neutral tax reform on its own-with no change in the debt-to-gdp ratio-would result in higher private consumption and output owing to the economy moving towards less distortionary sources of taxation. the increase in productive public investment, lower public consumption and social transfers result in further output gains in the long run. the productive public spending stimulates private capital accumulation and the lower deficit and debt ratios result in significant savings on debt-service costs. in the short run, before the benefits of more productive investment and of lower debt fully materialize, the reduced social transfers and public consumption dampen somewhat private consumption and output. short-term costs though are quite modest and are traded for significant longer-term benefits of permanently higher private consumption and output. the fiscal consolidation/composition shift scenario assumes that the measures are gradually phased in over the period of years (½ of gdp per year). if the announcement of the reform is fully understood by firms and households and fully believed, the short-term costs are even smaller than in the case when the general public considers permanent only the measures implemented in the given year and in the past, but do not believe that future reforms will be implemented. when households and firms believe the whole path of fiscal reforms they invest more from the outset and reap the long-term benefits of the fiscal consolidation sooner. overall, the high quality of fiscal adjustment enhances the confidence about debt sustainability, hence, lowering the interest costs and offsetting the contractionary impact of a moderately-gradual fiscal tightening (see blanchard and other on contractionary fiscal expansions in italy). this contrasts with the italian experience during the european debt crisis for three reasons: ( ) the assumed adjustment in our simulations is more gradual; ( ) the quality of fiscal adjustment is improved; ( ) and fiscal multipliers are likely smaller because the output gap is narrower, and the public debt is higher. this paper contributed to the fiscal policy discussions in italy by: ( ) assessing spending patterns from a long-term perspective to identify areas for savings; ( ) evaluating the pension system; ( ) analyzing the scope for revenue rebalancing; and ( ) outlining a package of spending cuts and tax rebalancing that is growth friendly and inclusive. we showed that a comprehensive fiscal reform package predicated on a revenue-neutral and less distortionary tax reform, alongside current spending cuts and capital spending increases, can generate sizable output gains and a sustainably lower public debt ratio over the medium to long term. short-term output costs of this fiscal package, if implemented credibly, are likely limited. total liabilities are the present value of the sum of workers accumulated capital (k) and pensioners' annuity (na). total assets are the present value of the stream of future contributions (plus technical reserves). in practice, this true irr is only known ex post. however, it must be parameterized ex ante (to calculate the annuity) possibly based on the growth rate of the wage bill: where n is the growth rate of labor force (population) and g is the productivity growth. it is clear from eqs. ( ) and ( ) that the structure of the two systems is similar. when the rate of valorization in the db and the internal rate of return in the ndc system are equal (i.e., u = n) and the accrual rate (a) is equal the ratio of contribution rate to the annuity factor (c/g), the systems can, in fact, be identical. the main differences relate to the manner the schemes react to shocks and in available policy instruments to counter these shocks. in the ndc, pension benefits adjust automatically to shocks like a sudden decline in fertility (lower contributions) or an increase in life expectancy (that determines the annuity factor g). this is not to say that the db system cannot cope with such shocks; this could be achieved by linking the formulae or retirement ages to life expectancy for example. discretionary adjustments differ in terms of parameters that policy makers can control. examples of these in the ndc are irr computation rules, minimum retirement age, life expectancy tables, and methods to calculate annuity. in the db scheme, many such parameters are absent or non-discretionary; instead, policy-makers can control, inter alia, accrual rates or the way pensionable earnings are calculated. connecting to power: political connections, innovation, and firm dynamics. nber working papers getting to know gimf: the simulation properties of the global integrated monetary and fiscal model corporate indebtedness and low productivity growth of italian firms italy: quantifying the benefits of a comprehensive reform package l'efficienza della spesa per infrastrutture improving sweden's automatic pension adjustment mechanism do r&d tax credits work? evidence from a panel of countries - productivity growth in italy: a tale of a slow-motion change. bank of italy debt, inflation and growth robust estimation of long-run effects in dynamic panel data models. globalization and monetary policy institute working paper is there a debt-threshold effect on output growth? taxation and female labor supply in italy reinventing the dutch tax-benefit system: exploring the frontier of the equityefficiency trade-off fiscal devaluation" and fiscal consolidation: the vat in troubled times credit-supply shocks and firm productivity in italy evaluation of federal tax incentives for private r&d in belgium: an update the ageing report. economic and budgetary projections for the eu member states ownership of intellectual property and corporate taxation policy uncertainty and corporate investment italy's fiscal sustainability revisited tax policy, leverage and macroeconomic stability. imf policy paper, october (washington) fiscal policies for innovation and growth. fiscal monitor reforming italy's social welfare system and lowering taxes on labor: some considerations competitiveness and wage bargaining reform in italy the anatomy of the vat the political economy of pension reform in europe the global integrated monetary fiscal model (gimf)-theoretical structure efficiency estimates of health care systems in the eu -round of epc-wga projections-italy's fiche on pensions can italy grow out of its npl overhang? a panel threshold analysis economic survey of italy diagnosing the italian disease neutral or fair? actuarial concepts and pension-system design growth in a time of debt le tendenze di medio lungo periodo del sistema pensionistico e socio-sanitario. department of the state accountant general, ministry of economy and finance le tendenze di medio lungo periodo del sistema pensionistico e socio-sanitario. department of the state accountant general, ministry of economy and finance optimal income transfer programs: intensive versus extensive labor supply responses the rate of return of pay-as-you-go pension systems: a more exact consumption-loan model of interest social security programs throughout the world: europe enhancing governance and effectiveness of fiscal agencies reforming tax expenditures in italy: what, why, and how? imf working paper series acknowledgements we thank romain duval, rishi goyal, zsoka koczan, and roberto piazza as well as the italian authorities for helpful discussions and comments. this paper was written before the covid- pandemic. the views expressed here are those of the authors and do not necessarily represent the views of the imf, its executive board, or imf management. the italian pension system is currently prorated over the defined benefit (db) and notional defined contribution (ndc) schemes. as the names imply, a db pension plan provides a specified payment amount in retirement, while an ndc plan allows employees and employers to contribute and invest funds over time to save for retirement. key features of pure db and ndc pension schemes are explained below: the db system rests on four key parameters: ( ) the accrual rate (a), that is the pension entitlement for a full year's coverage as a share of earnings; ( ) a measure of earnings (w) that usually is lifetime average earnings; ( ) valorization factor (u), that is, the way how the earnings of earlier years are adjusted to reflect changes in standards of living between the year of retirement and these earlier years; and ( ) the retirement age (t). the benefit is then defined as: in the ndc system, each individual's contributions, (c), accumulate into a notional capital (in individualized accounts) that by end of any period (t) is:where ρ is the notional interest rate or the internal rate of return (irr). in computing the annuity (a series of equal payments) at retirement, the accumulated capital stock is divided by the annuity factor (g) that in turn is a function of life expectancy (le) at retirement and the irr:the irr in the pure ndc scheme is chosen to equalize the present value of system assets (a) with the present value of system liabilities (l), or:( ) ( + ) t−t , key: cord- -v r ek a authors: di lorenzo, giuseppe; di trolio, rossella title: coronavirus disease (covid- ) in italy: analysis of risk factors and proposed remedial measures date: - - journal: front med (lausanne) doi: . /fmed. . sha: doc_id: cord_uid: v r ek a nan until march , , there were ∼ , confirmed cases of coronavirus disease in italy, with , in-patients, , patients in intensive care units (icus), , positive in home isolation, and , deaths, according to the italian civil protection bulletin . italy currently has the highest covid- mortality rate worldwide, even compared to the people's republic of china where the number of covid- deaths totaled over , cases, including potential re-infections. globally, there are ∼ , cases and , deaths due to covid- . according to the world health organization (who) as of march , , the number of covid- positive cases in spain is increasing, with , infected cases and , deaths. in the united states, in the span of a few days, there were , cases and , deaths due to covid- ; germany has , confirmed cases, and france has , cases with deaths . after a short respite with , covid- cases and only deaths, the infection has resurged and the number of confirmed cases are continuously increasing in south korea . an analysis of the data in the daily updates communicated by the civil protection, showed that most of the covid- cases and deaths are limited to northern italy-especially lombardy, emilia romagna, veneto, and piedmont-with the numbers fortunately decreasing toward central and southern italy, and very few cases documented in basilicata . the most severely affected regions are also the regions where healthcare services have always been considered excellent; the hospitals of lombardy and veneto are the italian centers of excellence with regard to standard protocols and management for many diseases, especially neoplastic conditions, and there was a high rate of passive migration of patients from the south to northern hospitals. the lombardy region has a higher number of intensive care and resuscitation beds compared to southern italy; unfortunately, these places are fast running out of hospital beds and facing challenges in the provision of primary care for conditions other than covid- , necessitating the transfer of numerous patients to other regions . the situation would probably have been considerably worse if the regions of southern italy had the highest number of covid- cases. these numbers confirm the fact that we are facing a pandemic, which was declared by the who a few days ago. the lethality rate is determined as follows: the number of deaths due to covid- divided by the total number of confirmed coronavirus cases. in italy, the lethality rate is %, which peaks in lombardy (> %), whereas the lethality rate in wuhan was . % and remained < % in the rest of the people's republic of china . an initial rationale for the higher lethality rate could be the high average age of the italian population when compared to, for example, the people's republic of china and the republic of korea; in the latter, the majority of confirmed covid- cases are young women ( %), with % of positive cases in the age range of - years. the average age of those dying in italy is years, and more than % were men . another explanation for the higher lethality is the presence of other pathologies and the comorbidities of the elderly population. based on research by the who, a report of the who china joint mission on coronavirus disease , published in february , reported that patients without other comorbidities have mortality rates of . %, compared to covid- patients with other diseases that compromise their health condition and result in higher mortality rates, which were , , and . % for those with cardiovascular disease, diabetes, and cancer, respectively . in italy, data from the istituto superiore di sanità (iss) indicates that % of the patients who died had no other disease, % had disease, % had disease, and % had or more conditions. the most common chronic preexisting disease in the patients who died was arterial hypertension ( %), followed by ischemic heart disease ( %), atrial fibrillation ( %), and active cancer within the previous years ( %) . another cause for the higher lethality rate may be that italy had a higher number of infected individuals who were asymptomatic and infected others. as recently reported by li et al., the transmission rate from unreported infections was % of the rate of reported infections, and un-reported infections resulted in % of reported cases ( ). therefore, for each positive covid- case, there are ∼ - undetected cases; thus, the actual number of covid- cases could be up to times higher, and recalculation of the mortality rates on this basis would cause the actual national mortality rate of covid- to decrease approximately to the mortality rates of covid- in the people's republic of china. in lombardy, there is a considerable amount of business travel and many people work in hospitals, which could have amplified the infection spread. in fact, doctors and nurses constitute the most infected occupational categories. moreover, at the beginning of the epidemic in lombardy, especially in bergamo, many patients had visited general practitioners who had no experience with the new virus. https://www.who.int/docs/default-source/coronaviruse/who-china-jointmission-on-covid- -final-report.pdf (accessed march , ). several of these doctors have been infected and have, unfortunately, died. it also cannot be ignored that the elderly in italy have frequent contact with their children and often take care of grandchildren. the percentage of people between the age of and years who live with their parents is up to %, which is much higher than in other countries. adult children and grandchildren, who are often asymptomatic, would have infected their elderly parents. what remedial steps can be undertaken? ) prevention. certainly, as has been reiterated by the italian government several times in the previous weeks, it is necessary to limit the infection spread by not going out unless for work. on march , the prime minister, giuseppe conte, announced the closure of all non-essential production activities. however, activities essential to guarantee essential goods and services continue to remain operational. the minister of health has passed a new ordinance that increases the restrictions imposed on citizens wherein outdoor activities and visits to parks and gardens are prohibited . furthermore, restaurants and bars had already been closed until the end of march . some regions of southern italy have passed ordinances that prohibit, with immediate effect and until april , , any movement of persons entering and leaving these regions. for example, one can only enter or leave the calabria and campania regions for journeys deriving from verified essential requirements related to the provision of essential services or for serious health reasons. in light of the potential exposure to infection, an immediate measure of a -day quarantine will apply for those who violate these restrictions . ) increase icu beds and create new hospitals. for this, the president of the campania region, vincenzo de luca, worried about the deterioration in southern italy, has announced the forthcoming construction of two new modular hospitals in napoli and caserta. they will be a sort of "field" hospital, consisting of containers and blocks which will form the body of the hospitals, with added beds; other intensive care places were obtained by reconverting hospitals, in some cases ones that were previously closed. three thousand additional beds will be recovered from private clinics . the ministries of internal affairs and foreign affairs have announced that ∼ million masks and mechanical ventilators have been procured from many countries . ) increased testing for asymptomatic people, particularly those exposed on the frontlines such as doctors and nurses. the mortality rate in italy is higher because asymptomatic cases are not being tested and isolated. at the beginning of the epidemic, there was misinformation that asymptomatic cases did not transmit the virus. this statement is certainly incorrect, and the recognition of asymptomatic/mildly symptomatic cases could decrease the number of infections ( ). numerous regions of central and southern italy have communicated a decision to screen doctors and nurses even if they are asymptomatic , . according to the campania region official press release n • on th march, , referring to decree n • on th march, , serological tests will also be done on patients in pretriage. serological tests are quick qualitative tests which research antibody igm or igg anti corona virus' antigens . ) completely ban smoking. given that the most serious outcome of covid- is pneumonia, the number of deaths could reflect the presence of fine dust in the air, especially in lombardy, and the state of the average italian lungs, which are damaged by cigarette smoke. the fact that more men have died may be attributed to a smoking habit, and those who smoke are more likely to become seriously ill with covid- . ) protect and monitor patients with comorbidities: these patients are at a greater risk of infection with severe acute respiratory syndrome coronavirus (sars-cov- ), and cancer patients should consider postponing adjuvant treatments, elective surgical interventions, or follow-up visits where possible . to date, there is no vaccine for covid- , and it might take several months for any new vaccine to be developed. ) therapies. various drugs have been used, including antivirals (e.g., favipiravir, arbidol, remdesivir) and antimalarials (e.g., chloroquine) ( ), or tocilizumab in patients with high levels of interleukin (il ) and extensive bilateral pulmonary lesions or severe symptoms. the italian drug agency (aifa) has announced the authorization of the tocivid- study, which will assess the efficacy and safety of tocilizumab in the treatment of pneumonia in covid- . the above-mentioned study will evaluate the impact of tocilizumab (approved for rheumatoid arthritis), which has recently been reported to have conferred possible benefits on patients treated by dr. paolo ascierto. in the tocivid- trial, hospitalized covid- patients with pneumonia with early signs of respiratory failure or who were intubated and placed on ventilatory treatment within the previous h will be treated with tocilizumab . aifa has also authorized additional trials: the first trial is the combination of emapalumab, monoclonal antibody anti-gamma interferon with anakinra, an il- antagonist; in the second trial sarilumab, an il antagonist, will be used . in vitro studies have shown that nitric oxide (no) inhibits the replication of sars-cov- . recently, the u.s. food and drug administration (fda) has granted bellerophon therapeutics an expanded access to enable the use of inhalational no delivery systems in covid- patients . furthermore, tests that are faster than the current nasopharyngeal swab or serum tests would be desirable, especially in the asymptomatic population. recently, the us fda has approved the first coronavirus diagnostic test that can be conducted entirely at the point of care. the test will deliver results within min, which is much faster than current tests that require a sample to be sent to a centralized laboratory, which can take days for results to be reported . unfortunately, in recent years, investments in healthcare and research have been limited in italy. public healthcare expenditure in represented . % of the gross domestic product (pil), which is much lower than that of other countries such as france and germany; many italians have turned to the private sector (+ %) from the public sector in the last years. however, this is not the time for recriminations and airing of political differences. the battle ahead is long and will not end in a few weeks. the lifestyle and habits of italians have changed. to date, ∼ , icu beds are occupied, and, as reported by remuzzi and remuzzi ( ) , up to hospital beds will be needed by april ; therefore, all regions must prepare or secure additional beds. we must absolutely avoid a collapse of the healthcare system and having to choose who to cure and who to let die. at the moment, the best weapon against covid- is strict adherence to the rules. among other things, one should avoid social assembly, maintain interpersonal distances of at least m, possibly m, and thoroughly wash their hands often. substantial undocumentedinfectionfacilitates the rapiddissemination of novelcoronavirus (sars-cov ) discovering drugs to treat coronavirus disease (covid- ) covid and italy. what next? all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © di lorenzo and di trolio. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -fsrdu tq authors: allieta, m.; allieta, a.; rossi sebastiano, d. title: covid- outbreak in italy: estimation of reproduction numbers over two months toward the phase date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: fsrdu tq after two months from the first case in covid- outbreak, italy counts more than , confirmed positive cases. from the beginning of april , the nationwide lockdown started to show early effects by reducing the total cumulative incidence reached by the epidemic wave. this allows the government to program the measures to loosen lockdown restrictions for the so called "phase ". here we provided the reproduction number estimation both in space and in time from february th to april th, across two months into the epidemic. our estimates suggest basic reproduction number averaged over all the regions of . , confirming that epidemiological figures of the sars-cov- epidemic in italy are higher than those observed at the early stage of wuhan (china) outbreak. based on the sars-cov- transmission dynamics reported here, we gave a quantitative evaluation of the efficiency of the government measures to low the reproduction number under the unity (control regime). we estimated that among the worst hit regions in italy, lombardy reached the control regime on march nd followed by emilia-romagna (march th), veneto (march th) and piemonte (march th). overall, we found that the mean value of time to reach the control regime in all the country is about days from the february th and about days from the first day of nationwide lockdown (march th). finally, we highlighted the interplay between the reproduction number and two demographic indices in order to probe the "state of activity" of the epidemic for each italian region in the control regime. we believe that this approach can provide a tool in the management of "phase ", potentially helping in challenging decision to continue, ease or tighten up restrictions. after the first covid- case was diagnosed in lombardy, italy, on february th, , [ ] the novel coronavirus rapidly spread across the country leading to a dramatic spike in the number of new positive cases and deaths. to minimize the likelihood that people who were not infected come into contact with people who had contracted the disease, the italian government imposed a series of progressively more strict social distancing measures which culminated in a national lock-down announced on march th, . [ ] around two months from the first case and more than , confirmed positive cases later, from the beginning of april, the effect of the nationwide lockdown started to achieve some level of success and the number of new infections began to smoothly decrease. these early signs of a slowdown of the covid- pandemic in italy provide a comforting picture of the outbreak's stabilization which is driving the government to periodically review its lockdown measures in view of the so called "phase ", i.e. the period during which citizens will have to live together with the virus as of all the industrial sector, including the nonessential economic activities, will start to reopen. however, since the regional differences in the number of new positive cases has been reported to be huge, with the northern regions of italy (namely lombardia) being most affected, the establishment of the proper precautions to plan the "phase " is a truly complicated task. the planned restrictions and permissions that will be applied could thus vary from region to region. in this context, the systematical estimation of key epidemiological parameters, for each region can provide insight into the speed at which the disease had spread and will give a useful tool to figure out if a differential approach at the regional level on the measures to apply for "phase " is feasible to keep down the transmission of sars-cov . at the beginning of epidemic and during the lockdown phase, italian government and the mainstream of the local and national mass media have been emphasized the relevance of the basic reproduction number (r ) , i.e. the average number of secondary cases generated by a single primary case in a theoretically fully susceptible ( %) population, as the most important and informative parameter to monitor the epidemic trends. obviously, r has an undoubted relevance since when r > the infection may spread in the population and more r is large and deeper would be the interventions needed to control the epidemic. on the other hand, if r < , on average the infectious individual infects less than one person and the epidemic falls in a so called "control regime" where it will not be sustained, and it will die out. nevertheless, r is not the only parameter that affect the impact and the spreading of all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint the disease over a population which may largely result even from several demographic and epidemiological factors. in this communication, we provided an estimation of the basic reproduction number r for all the italian regions by the cumulative confirmed covid- cases continuously updated and made public at the website of dipartimento della protezione civile. [ ] in addition, we estimated the time dependent reproduction number rt, which is the average number of secondary cases generated by an infectious individual at time t. we linked rt related to the last date of our period of observation (april th, ), with two demographic and epidemiologic indices in a simple three-dimensional array in order to highlight the "state of activity" of the epidemic for each italian region. we provide a useful tool in the management of "phase ", potentially helping in challenging decision to continue, ease or tighten up restrictions. the official demographic data of the resident population, the surface and the population density updated on january st, , for each italian region and italy were taken from the italian national institute of statistics (istituto nazionale di statistica, istat) and reported in the official data of covid- epidemic in italy was taken from the task force of the dipartimento della protezione civile. cumulative data are available at various aggregation levels, namely national, regional and provincial and are accessible on github. [ ] data for the analysis were considered from february th to april th, . in this period, we collected the daily cumulative number of confirmed positive cases (n), the number of "active" confirmed positive cases (na), i.e., the number of infected people living not recovered from covid- , and the "density of infected people" (da), calculated as na/surface and expressed like the population density as number of persons/km , for each italian region and italy. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. to obtain the estimation of reproduction number we use the maximum likelihood estimation (ml) method which assumes that the number of secondary cases caused by an index case is poisson distributed with an expected value r. given then observation of (n , n ,..., nt) incident cases over consecutive time units, r is estimated by maximizing the following loglikelihood function [ ] : i is the distribution of the generation time corresponding to the distribution of the serial interval, i.e. the time between when a person gets infected and when they subsequently infect another other people, calculated at time i within the assumption that the incubation period does not change over the course of the epidemic [ ] . we consider that the distribution of the serial interval was expected to follow a gamma distribution with mean (±sd) of . ± . days as reported by the imperial college covid- response team [ ] . we note that this value agrees very well with gamma distribution with mean . days ( % ci, . to ) recently determined from the analysis of observations of individual serial intervals in clusters in lombardia (italy) [ ] . to estimate r = r , the ll(r) function must be calculated over a period where epidemic curves showed exponential growth. as a first guess, to select this time window we used the simple procedure described by obadia et al. [ ] in brief, we computed the function over a range of possibile time periods by determining the deviance r statistic for each iteration. largest r corresponds to the time window over which the ml model best described data. to evaluate the time dependent reproduction number rt we adopted the method developed by wallinga and teunis. [ ] the transmission probability (pij) of individual i being infected by individual j at ti, tj onsets, respectively, can be described mathematically as: [ ] the net reproduction number rj is then then sum of all pij involving j as the infector = ∑ and it can be averaged over all cases with same date of onset as all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint finally, since rt are computed by averaging over all transmission networks compatible with observed incidence data, no assumption is made about the time dependence of the epidemic unlike, for example the exponential growth in the well-known bayesian approach. [ ] , [ ] we believe, hence, that this model is particularly suitable to estimate the reproduction number in the post-peak period where the transmission is expected to decrease. all the above data analyses were performed using the r package [ ] as implemented in statistical software r. [ ] .results infection in italy shows that the exponential growth period may take place during the first - day from the national epidemic onset (february th , ). table s -s (supporting information) show demographic and epidemiological data, respectively. as it is widely known, table s shows that covid- epidemic affected (and is affecting) harder the northern italian regions, with n= and na= on april th, i.e. more than % of the cases of the country (with , % of the italian resident population), if we aggregate epidemiological and demographic data of the northern regions (lombardia, piemonte, veneto, emilia romagna, liguria, valle d'aosta, trentino-alto adige) plus marche and toscana regions. furthermore, in lombardia region epidemic had a huge spread, with n= and na= on april th, i.e. more than one third of the cases of the country (with . % of the italian resident population). in the top of the panels of figures s -s (supporting information) we reported the incidence data for all the regions plus italy. initial inspection of the datasets shows again that the exponential growth period may take place during the first - day from the relative all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint epidemic onset. it should be noted that for the evaluation of r in the initial outbreak stage, we considered data from february th , up to march th , since data in a wider range can be affected by the national lockdown on march th . in figure (a) we showed r values obtained for sars-cov- in all the regions and in italy. table reports the same data represented in figure (a), compared with those obtained by riccardo et al., [ ] d'arienzo et al., [ ] distante et al. [ ] . according to our ml estimation, (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. in this work, we analyzed the time evolution of incidence of the sars-cov- epidemic for two months from onset, february th to april th , in all the italian regions. we estimated the basic reproduction number (r ), by using the ml method in the early stage of the epidemic. in addition, we determined time evolution of this parameter across the two months of the observational period. finally, we linked rt, with two indices, the population density and da, the latter representing the density of infected people in a region as recorded on april th . firstly, we point out that these data can be considered only an approximation of the actual epidemic dynamics. indeed, the reported number of cases strictly depends on the number of swabs that are used for covid- testing and can be biased by several factors like underreporting, delays in recording as well as errors in classification of cases. [ ] therefore, large data noise is general observed, especially at the regional level, which requires a careful inspection of the epidemic curve as well as data smoothing in order to avoid unrealistic reproduction number estimation. as described in the results, for the evaluation of r in the initial outbreak stage, we considered data from february th , up to march th . data in a wider range can be affected by the national lockdown on march th . this period agrees well with previous investigation where the same time window has been assumed as the infection period to determine r for the whole italy. [ ] taking these preliminary considerations into account, our result of r = . for italy is highly consistent with values obtained by fitting the exponential growth rate of the infection across a -month period. [ ] similar conclusion has been drawn for northern regions transmission dynamics and the same results were found for the southern regions. [ ] in another work, riccardo f. et al. [ ] reported r ranging from . to . for six selected italian regions (lombardia, veneto, emilia-romagna, toscana, lazio, puglia). despite these values are lower than r obtained here, a variability of ~ . for most of the regions is thus confirmed independently of geographical location. again, gatto et al. [ ] , while including additional parameters like mobility and the spatial distribution of communities, determined a comparable initial generalized reproduction number r = . . overall, these data support the idea that epidemiological figures of the sars-cov- epidemic in italy are slightly higher than those observed at the early stage of outbreak in wuhan (china). [ ] the initial large values observed resulted from a sudden increase of independent first reported infections which in many cases can be related to the so called "super-spreading" all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint events. indeed, as observed for sars outbreak, [ ] in the early stage of the epidemic the time dependence of rt shows a fluctuating pattern characterized by wide confidence interval raised by the initial low number of cases used in the calculations. in this context, the superspreading events cannot be necessarily triggered by a single infector, but it can be related to few people which are perpetuating an epidemic in the susceptible population. [ ] here we observed that most of the regions have faced "super-spread events" in the early stage of epidemic. and significant is the observation of such event in southern regions after the early stages, the rt showed a decreasing trend which is likely to be affected by the temporal depletion of susceptible individuals (intrinsic factors) and by the implementation of control measures (extrinsic factors). [ ] both these factors slow down the growth rate of incidence and deeply affect the shape and time scaling of the epidemic peak driving rt to fall below . [ ] we found that the mean value of time to reach the control regime is about days from the (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . although the italian government's restrictive measures have proven to be of considerable utility in preventing even more devastating effects from the epidemic, the challenge in tackling "phase " appears even more demanding. in this line, obtaining simple and effective indices to evaluate the state of activity of the epidemic seems mandatory: if the rt index remains essential for understanding the trend in a given area, however it is not the only parameter to account for. briefly, if we consider two areas with the same rt, that of the two that has a population density and a higher percentage of infected people must be considered more at risk, monitored more carefully and potentially the target of more timely restrictive measures. the population density of a given area is clearly an rt-independent risk factor for the development of an epidemic that spreads through human infection, although the population density of the different italian regions may not be truly representative of the distribution of the population. urban areas and in particular metropolitan areas (rome, milan, naples) have a population density higher than the regional one. furthermore, due to the peculiar italian orography, some regions (for example liguria, valle d'aosta, trentino-alto adige) concentrate the population in a "habitable" area much less large than the total surface. while admitting its arbitrariness, da (ie the number of infected people per km ) is in some way a representative parameter of how much the epidemic was active in the previous period and, above all, what is the generic risk of "meeting" a subject affection in a given area. therefore, we suggest to associate a combined use of rt with da and population density to evaluate the epidemic risk of a specific area, in our case of the italian regions. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint caption: ( + ) date of epidemic onset february th ; (*) the original incidence data related to trento and bolzano were merged into a single region called trentino-alto adige resulting in a geographical disaggregation of italy into regions. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. caption: official data from the dipartimento della protezione civile available at https://github.com/pcm-dpc/covid - ; (*) the original data related to trento and bolzano were merged into a single region called trentino-alto adige resulting in a geographical disaggregation of italy into regions; n = aggregate number of infected people, na = number of active infected people, da = density of active infected people. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint figure s . daily incidence as numbers of new cases from february to april for covid- outbreaks (upper panel) and the corresponding time dependent reproduction number (rt) (lower panel) for the following italian regions: (a) abruzzo, (b) basilicata, (c) calabria, (d) campania, (e) emilia-romagna, (f) friuli v. g., (g) lazio. in upper panels vertical bars are the incidence data whereas in lower panels black dots are the rt mean values accompanying by grey vertical lines standing for % confidence intervals. in the same panel the horizontal solid line indicates the threshold value r = , above which an epidemic will spread and below which the epidemic is controlled. days are listed from the onset february th , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint figure s . daily incidence as numbers of new cases from february to april for covid- outbreaks (upper panel) and the corresponding time dependent reproduction number (rt) (lower panel) for the following italian regions: (a) liguria, (b) lombardia, (c) marche, (d) molise, (e) piemonte, (f) puglia, (g) sardegna, in upper panels vertical bars are the incidence data whereas in lower panels black dots are the rt mean values accompanying by grey vertical lines standing for % confidence intervals. in the same panel the horizontal solid line indicates the threshold value r = , above which an epidemic will spread and below which the epidemic is controlled. days are listed from the onset february th , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . in upper panels vertical bars are the incidence data whereas in lower panels black dots are the rt mean values accompanying by grey vertical lines standing for % confidence intervals. in the same panel the horizontal solid line indicates the threshold value r = , above which an epidemic will spread and below which the epidemic is controlled. days are listed from the onset february th , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint covid- : preparedness, decentralization, and the hunt for patient zero covid- working group, epidemiological characteristics of covid- cases in italy and estimates of the reproductive numbers one month into the epidemic the r package: a toolbox to estimate reproduction numbers for epidemic outbreaks estimation in emerging epidemics: biases and remedies estimating the number of infections and the impact of non-pharmaceutical interventions on covid- in european countries the early phase of the covid- outbreak in lombardy, arxiv different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures r: a language and environment for statistical computing. r foundation for statistical computing assessment of the sars-cov- basic reproduction number, r , based on the early phase of covid- outbreak in italy covid- outbreak progression in italian regions: approaching the peak by the end of march in northern italy and first week of april in southern italy int no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity guida alla lettura e all'interpretazione dei dati covid- covid- and italy: what next? lancet ( ) spread and dynamics of the covid- epidemic in italy: effects of emergency containment measures preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak the effective reproduction number as a prelude to statistical estimation of time-dependent epidemic trends no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted key: cord- -bygcyxpz authors: manes, gianpiero; repici, alessandro; radaelli, franco; bezzio, cristina; colombo, matteo; saibeni, simone title: planning phase two for endoscopic units in northern italy after the covid- lockdown: an exit strategy with a lot of critical issues and a few opportunities date: - - journal: dig liver dis doi: . /j.dld. . . sha: doc_id: cord_uid: bygcyxpz nan since the beginning of may , italy will gradually emerge from the novel coronavirus (covid- ), lockdown, which began on march . with the deceleration of the pandemic across the country, disease control measures have been eased through a well-structured plan and italy is now in the so-called "phase two" of lockdown, moving toward an eventual return to normalcy. after a pause for all but urgent procedures, endoscopy departments (eds) are converting back to their pre-covid- configurations and are returning to carrying out elective endoscopic procedures [ ] [ ] [ ] . however, safely recommencing outpatient activity raises several critical issues, such as the risk of exposure to infection for healthcare personnel and patients, the reduced availability of staff, the presence of infrastructural barriers and the lack of a clear policy regarding the timely rescheduling of cancelled or postponed endoscopies. we conducted a survey among the directors of eds in high-risk areas of northern italy with the aim of investigating the barriers and strategies to safely resume elective endoscopy activity in phase two of the lockdown. the study was conducted between april th - th , (two weeks before the start of "phase-two"). a total of eds, which participated in a previous survey on the covid- outbreak in italy ( ), received by e-mail a structured questionnaire, which consisted of sections (table ). the first section focused on the organizational characteristics of eds before and after the outbreak, in order to assess the impact of the pandemic on eds. the second section explored the availability of specialist staff and personal protective equipment (ppe) in the eds at the start of phase two. in the third section, the directors of eds were asked to foresee the endoscopy workload they would realistically estimate as achievable, according to local resources, in phase two (from may to july ), and to indicate strategies to optimize endoscopic activity in this reference period. of the eds invited, ( . %) completed the questionnaire. the median interval time from construction or last renovation of eds was . ( . - . ) (range - ) years. the main characteristics of eds in the pre-covid- period are shown in table . due to the covid- outbreak, ( . %) centres had their normal endoscopic activities reduced by - % and the remaining ( . %) by - %. overall, endoscopists (range - /ed), nurses (range - /ed) and health assistants (range - /ed) had been working in eds in the pre-covid- period. at the end of april, / ( . %) endoscopists, / ( . %) nurses, and / ( %) health assistants were not available due to covid- infection ( , and respectively), or reallocated to other units ( , and respectively) ( table ) . of endoscopy rooms used in the pre-covid period, ( %) in eds were not available, since they were either converted to covid- -care areas (n= ) or devoted to endoscopic procedures in covid- positive patients (n= ). regarding structural characteristics, ( . %) centres had either the waiting area ( / , . %) or the recovery area ( / , . %) or both ( / , . %) that were too small to guarantee sufficient distancing between patients or caregivers/escorts at the pre-crisis workload; ( . %) centres could not guarantee a "infected patients pathway" separated by "non-infected" areas; ( %) were lacking at least one negativepressure room; ( . %) did not even guarantee the separated dirty/clean pathways for endoscopes. in this phase of the pandemic, ppe shortage represented a critical issue for a minority ( - . %-) of centres. in general, only ( %) eds reported that they were able to immediately resume elective endoscopic activity at the pre-crisis volume, with respect of the safety protocols. when asked to foresee which increase in the endoscopic workload would have been bearable in the upcoming months, according to the services resources, the majority of the directors ( / , %) envisioned as realistic a workload increase up to % for the month of may as compared to the actual one. for the month of june, this figure was up to % and % in centres ( . %) and in ( . %), respectively. for the month of july, the majority of centres ( / , . %) envisioned a workload increase of at least % (n= ). returning to the pre-crisis workload by the end of july, september and october was judged as a realistic goal by ( . %), ( . %), and ( . %) participants, respectively ( figure ). all participants agreed that, once completely returned to elective endoscopy, the chance of overcrowding would be very high, due to the very large number of postponed cases that need to be rescheduled. however, only of them reported they were confident in being able to significantly increase (at least %) their activity with respect to the pre-covid- volume; conversely, the majority of participants realistically declared no ( centres) or minimal ( - %) increases in their activities to be possible. endoscopists suggested that implementing the application of guidelines (n= ), organizing a direct-line with general practitioners (gp) for triaging and scheduling/rescheduling patients (n= ), and promoting telemedicine and virtual visits (n= ) could be strategies to optimize endoscopic activity and promote a more rational use of resources. only % of those interviewed were pessimistic, fearing that the covid- crisis would have not brought any changes and improvements in ed organization. all centres claimed their availability to immediately restart screening activity at the pre-crisis volumes, but of them suggested to replace pre-and postcolonoscopy visits by telemedicine. the present survey demonstrates that there are several barriers preventing eds in northern italy to safely manage elective endoscopy activity in phase two of the pandemic. professional societies have issued guidelines to safely return to elective procedures , . these guidelines call for a pre-procedure screening of patients to assess risk of transmission, combining it with pcr-based testing prior the procedure, the use of ppe, and policies to facilitate social distancing for patients and visitors in the waiting and recovery rooms, restrictions on accompanying visitors, and distancing the procedure start times , . presently, pcr testing prior to the procedure is not available in italy. thus, social distancing and the appropriate use of ppe remain critical issues. unfortunately, despite % of eds had been recently (< years) built or renovated, waiting and recovery areas are inadequate in guaranteeing distancing among individuals in several services, and most units are not able to ensure high flows of patients, personnel and equipment. other issues hinder a prompt restart of elective endoscopy: endoscopy staff is still lacking in many endoscopy services, since physicians and nurses have been either infected or are still reallocated to other departments; some eds rooms are still unavailable due to their conversion to the management of covid- infected patients. indeed, most participants agreed that re-opening eds should be accomplished very slowly, with a limited increase in the number of procedures over the upcoming months and that it was unrealistic to return to pre-crisis workload in the next three months. phase two will be challenging: effort should be made to avoid overload of eds, but several procedures that were cancelled or postponed during the outbreak need to be rescheduled. there is, thus, an absolute need to redesign the organization models of eds and their interaction with territorial services. the italian "open access" system has several advantages (eliminating unnecessary pre-endoscopic office-based consultations), but it has also generated a high level of inappropriateness and misuse of resources , . from now on, a clear and thoughtful policy regarding the timely scheduling/rescheduling of endoscopy procedures according to their priority will be required. this can be achieved only on a case-by-case basis; priority should be given to patients for whom even a short delay would significantly alter the patient's prognosis. due to the great uncertainty about the duration of the pandemic , a further category of patients who should not be postponed are those who do not have life-threatening conditions but for whom treatment should not be indefinitely delayed until the end of the pandemic, such as colorectal cancer screenings. eds should, thus, strongly consider further postponing elective procedures or their cancellation, if inappropriate by reviewing and categorizing the procedure lists by both prescriber physicians and endoscopists. this scenario inevitably implies, at least temporarily, the shift from an open access endoscopy to a filtered access . a prioritization model for referrals has already been tested in italy , , but this process implies a close interaction between gps and specialists and requires a lot of time. telemedicine, as highlighted by our survey, could represent a useful tool to fill the gap between gps and specialists. telemedicine and virtual visits have never been performed in endoscopy units in italy but are advisable to promote the appropriate use of resources. measures for the return to routine endoscopy during the pandemic have been suggested by gastroenterological societies , and experts [ ] [ ] [ ] [ ] , but local applicability has never been evaluated. the present survey has been conducted in italy, but has provided data that are likely generalizable to most eds in the western countries. crisis periods, like wars, are usually followed by great technological and social evolution. nearly all endoscopists have agreed that the covid- pandemic may represent a great opportunity to remodel and rationalize the eds processes. nothing will be the same again and what we are organizing, planning and changing today will likely represent the basis of our work tomorrow: this opportunity has to be exploited to the best of our possibilities. reason for non-availability decreto del presidente del consiglio dei ministri - aprile -gazzetta ufficiale della repubblica italina in internet gastroenterology department operational reorganisation at the time of covid- outbreak: an italian and chinese experience italian gi-covid working group. endoscopy units and the covid- outbreak: a multi-center experience from italy position statement on gastrointestinal endoscopy and the covid- pandemic -european society of aga institute rapid recommendations for gastrointestinal procedures during the covid- pandemic endoscopy activity and covid- : bsg and jag guidance appropriateness of upper-gi endoscopy: an italian survey on behalf of the italian society of digestive endoscopy the asge guidelines for the appropriate use of colonoscopy in an open access system modelling the covid- pandemic and implementation of population-wide interventions in italy lay-off of endoscopy services for the covid- pandemic: how can we resume the practice of routine cases? gastroenterology priority and appropriateness of upper endoscopy out-patient referrals: two-period comparison in an open-access unit. dig liver dis improving the appropriateness of referrals and waiting times for endoscopic procedures preventing the spread of covid- in digestive endoscopy during the resuming period: meticulous execution of screening procedures covid- in endoscopy: time to do more? gastrointest endosc covid- : how to select patients for endoscopy and how to reschedule the procedures? endosc int open a proposal for the return to routine endoscopy during the covid- pandemic declaration of interests: authors declare that they do not have any conflict of interest contributors: gm, ar, and fr planned and designed the survey; gm and mc collected and analyzed data drafted the manuscript and figures. ss and cb, provided critical appraisal of the manuscript. ss and cb revised the manuscript only reducing the number of procedures is the waiting room of your endoscopic unit suitable to ensure adequate distance between patients/relatives/caregivers? a. yes b. no c. only reducing the number of procedures is the recovery room of your endoscopic unit suitable to ensure adequate distance between patients? a. yes b. no c. only reducing the number of procedures do you fear any shortage of ppes in your endoscopic unit after resuming the endoscopic activity? a. yes b. no perspective and proposal for resuming the endoscopic activity what would you suggest to restart safely and affectively the endoscopic activity? in your opinion your opinion, which increase in the endoscopic activity can be achieved in the month of july? a. % (the activity remains as today) b. % c. % d. % e. % return to the pre-covid- activity in your opinion, when will your endoscopic unit return to the pre-covid- activity? once completely re-opened % when could the crc screening activity restart in your endoscopic unit? a. immediately (in the month of may) at the pre-covid- volume b. immediately (in the month of may) at a reduced rate c. i would wait to restart the screening activity in your opinion key: cord- - unrcb f authors: gaeta, giuseppe title: social distancing versus early detection and contacts tracing in epidemic management date: - - journal: chaos solitons fractals doi: . /j.chaos. . sha: doc_id: cord_uid: unrcb f different countries – and sometimes different regions within the same countries – have adopted different strategies in trying to contain the ongoing covid- epidemic; these mix in variable parts social confinement, early detection and contact tracing. in this paper we discuss the different effects of these ingredients on the epidemic dynamics; the discussion is conducted with the help of two simple models, i.e. the classical sir model and the recently introduced variant a-sir (arxiv: . ) which takes into account the presence of a large set of asymptomatic infectives. different countries are tackling the ongoing covid- epidemics with different strategies. awaiting for a vaccine to be available, the three tools at our disposal are contact tracing, early detection and social distancing . these are not mutually exclusive, and in fact they are used together, but the accent may be more on one or the other. within the framework of classical sir [ ] [ ] [ ] [ ] [ ] and sir-type models, one could say (see below for details) that these strategies aim at changing one or the other of the basic parameters in the model. in this note we want to study -within this class of modelswhat are the consequences of acting in these different ways. we are interested not only in the peak of the epidemics, but also in its duration. in fact, it is everybody's experience in these days that social distancing -with its consequence of stopping all kind of economic activities -has a deep impact on our life, and in the long run is producing impoverishment and thus a decline in living conditions of a large part of population. in the present study we will not specially focus on covid, but discuss the matter in general terms and by means of generalpurpose models. our examples and numerical computations will however use data and parameters applying to (the early phase of) the current covid epidemic in northern italy, in order to have realistic examples and figures; we will thus use data and parameters arising e-mail address: giuseppe.gaeta@unimi.it from our analysis of epidemiological data in the early phase of this epidemic [ , ] . unavoidably, we will also here and there refer to the covid case. some observations deviating from the main line of discussionor which we want to pinpoint for easier reference to them -will be presented in the form of remarks. the symbol marks the end of remarks. in the sir model [ ] [ ] [ ] [ ] [ ] , a population of constant size (this means the analysis is valid over a relatively short time-span, or we should consider new births and also deaths not due to the epidemic) is subdivided in three classes: susceptibles, infected (and by this also infectives), and removed. the infected are supposed to be immediately infective (if this is not the case, one considers so called seir model to take into account the delay), and removed may be recovered, or dead, or isolated from contact with susceptibles. we stress that while in usual textbook discussions of the sir model [ ] [ ] [ ] [ ] the removed are either recovered or dead, in the framework of covid modeling the infectives are removed from the infective dynamics -i.e. do not contribute any more to the quadratic term in the eqs. ( ) below -through isolation. this means in practice hospitalization in cases where the symptoms are heavy and a serious health problem develops, and isolation at home (or in other places, e.g. in some countries or region specific hotels were used to this aim) in cases where it is estimated that there is no relevant risk for the health of the infective. in this sense, the reader should pay attention to the meaning of r in the present context. the nonlinear equations governing the sir dynamics are written as d s/d t = − α s i d i/d t = α s i − βi ( ) d r/d t = βi. these should be considered, in physicists' language, as mean field equations; they hold under the (surely not realistic) assumption that all individuals are equivalent, and that the numbers are sufficiently large to disregard fluctuations around mean quantities. note also that the last equation amounts to a simple integration, r (t) = r + β t t i(y ) dy ; thus we will mostly look at the first two equations in ( ) . we also stress, however, that epidemiological data can only collect time series for r ( t ): so this is the quantity to be compared to experimental data [ ] . in fact, as stressed in remark , in the case of a potentially dangerous illness (as covid), once the individuals are identified as infective, they are effectively removed from the epidemic dynamic through hospitalization or isolation. according to our eqs. ( ) , s ( t ) is always decreasing until there are infectives. the second equation in ( ) immediately shows that the number of infectives grows if s is above the epidemic threshold γ = β/α. ( ) thus to stop an epidemic once the numbers are too large to isolate all the infectives, we have three (non mutually exclusive) choices within the sir framework: (a) do nothing, i.e. wait until s ( t ) falls below the epidemic threshold; (b) raise the epidemic threshold above the present value of s ( t ) by decreasing α; (c) raise the epidemic threshold above the present value of s ( t ) by increasing β. in practice, any state will try to both raise β and lower α, and if this is not sufficient await that s falls below the attained value of γ . in order to understand how this is implemented, it is necessary to understand what α and β represent in concrete situations. the parameter β represents the removal rate of infectives; its inverse β − is the average time the infectives spend being able to spread the contagion. raising β means lowering the time from infection to isolation, hence from infection to detection of the infected state. the parameter α represents the infection rate , and as such it includes many thing. it depends both on the infection vector characteristics (how easily it spreads around, and how easily it infects a healthy individual who gets in contact with it), but is also depends on the occasions of contacts between individuals. so, roughly speaking, it is proportional to the number of close enough contacts an individual has with other ones per unit of time. it follows that -if properly implemented -social distancing results in reducing α. each of these two actions presents some problem. there is usually some time for the appearance of symptoms once an individual is infected, and the first symptoms can be quite weak. so early detection is possible only by fast tracing and laboratory checking of all the contacts of those who are known to be infected. this has a moderate cost (especially if compared to the cost of an intensive care hospital stay) but requires an extensive organization. on the other hand, social distancing is cheap in immediate terms, but produces a notable strain of the societal life, and in practice -as many of the contacts are actually work related -requires to stop as many production and economic activities as possible, i.e. has a formidable cost in the medium and long run. moreover, it cannot be pushed too far, as a number of activities and services (e.g. those carrying food to people, urgent medical care, etc.) can not be stopped. let us come back to ( ) ; using the first two equations, we can study i in terms of s , and find out that as we know that the maximum i * of i will be reached when s = γ , this allows immediately to determine the epidemic peak . in practice, i is negligible and for a new virus s corresponds to the whole population, s = n; thus note that only γ appears in this expression; that is, raising β or lowering α produces the same effect as long as we reach the same γ . on the other hand, this simple formula does not tell us when the epidemic peak is reached, but only that it is reached when s has the value γ . but if measures are taken, these should be effective for the whole duration of the epidemic, and it is not irrelevant -in particular if the social and economic life of a nation is stopped -to be able to evaluate how long this will be for. acting on α or on β to get the same γ will produce different timescales for the dynamics; see fig. , in which we have used values of the parameters resulting from our fit of early data for the northern italy covid- epidemic [ ] . this observation can be made more precise considering the scaling properties of ( ) . in fact, consider the scaling numerically integrated and i ( t ) plotted in arbitrary units for given initial conditions and α, β parameters (solid), the maximum i * being reached at t = t * . then they are integrated for the same initial condition but raising β by a factor ϑ = / (dashed) with maximum i β = r i * reached at time t β = σ β t * ; and lowering α by the same factor ϑ = / (dotted) with maximum i α = i β reached at time t α = σαt * . time unit is one day, α = ( / ) * − , β = / ; these parameters arise from our fitting of data from the early phase of covid epidemics in northern italy [ ] ; the population of the most affected area in the initial phase is about million, that of the whole italy is about million. the numerical simulation is ran with n = * ; it results it is clear that under this scaling γ remains unchanged, and also the equations are not affected; thus the dynamics is the same but with a different time-scale . the same property can be looked at in a slightly different way. first of all, we note that one can write α = β/γ ; moreover, α appears in ( ) only in connection with s , and it is more convenient to introduce the variable now, let us consider two sir systems with the same initial data but different sets of parameters, and let us for ease of notation just consider the first two equations of each. thus we have the two systems we can consider the change of variables ( λ > ) with this, ( ) becomes we can thus eliminate the factor λ in both equations. however, if we had chosen λ = β/β, we get ˆ β = β; if moreover γ = γ , the resulting equation is just but we had supposed the initial data for { s, i } and for { s , i } (and hence also for ϑ and ϑ ) to be the same. we can thus directly compare ( ) with ( ) . we observe that { ϑ , i } have thus exactly the same dynamics in terms of the rescaled time τ as { ϑ, i } in terms of the original time t . in particular, if the maximum of i is reached at time t * , the maximum of i is reached at τ * = t * , and hence at t * = λ τ * = λ t * . ( ) analytical results on the timescale change induced by a rescaling of the α and β parameters have recently been obtained by m. cadoni [ ] ; see also [ ] . we have supposed infected individuals to be immediately infective. if this is not the case an "exposed" class should be introduced. this is not qualitatively changing the outcome of our discussion, so we prefer to keep to the simplest setting. (moreover, for covid it is known that individuals become infective well before developing symptoms, so that our approximation is quite reasonable.) one of the striking aspects of the ongoing covid- epidemic is the presence of a large fraction of asymptomatic infectives [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; note that here we will always use "asymptomatic" as a shorthand for "asymptomatic or paucisymptomatic", as also people with very light symptoms will most likely escape to clinical detection of covid -and actually most frequently will not even think of consulting a physician. in order to take this aspect into account, we have recently formulated a variant of the sir model [ ] in which together with known infectives i ( t ), and hence known removed r ( t ), there are unregistered infectives j ( t ) and unregistered removed u ( t ). note that in this case removal amounts to healing; so while the removal time β − for known infected corresponds to the time from infection to isolation, thus in general slightly over the incubation time t i (this is t i . days for covid), the removal time η − for unrecognized infects will correspond to incubation time plus healing time. in the model, it is supposed that symptomatic and asymptomatic infectives are infective in the same way. this is not fully realistic, as one may expect that somebody having the first symptoms will however be more retired, or at east other people will be more careful in contacts; but this assumption simplifies the analysis,and is not completely unreasonable considering that for most of the infection-to-isolation time β − the symptoms do not show up. the equations for the a-sir model [ ] are note that here too we have a "master" system of three equations (the first three) while the last two equations amount to di- the parameter ξ ∈ [ , ] represents the probability that an infected individual is detected as such, i.e. falls in the class i . in the absence of epidemiological investigations to trace the contacts of known infectives, this corresponds to the probability of developing significant symptoms. in the first (arxiv) circulated version [ ] of our previous work [ ] , some confusion about the identification of the class j was present, as this was sometimes considered to be the class of asymptomatic infectives, and sometimes that of not registered ones . while this is not too much of a problem considering the "natural" situation, it becomes so when we think of action on this situation. actually, and unfortunately, this confusion has a consequence exactly on one of the points we want to discuss here, i.e. the effect of a campaign of chasing the infectives, e.g. among patients with light symptoms or within social contacts of known infectives; let us thus discuss briefly this point. if j is considered to be the set of asymptomatic virus carriers, then a rise in the fraction of these who are known to be infective, and thus isolated, means that the average time for which asymptomatic infectives are not isolated is decreasing. in other words, we are lowering η − and thus raising η. on the other hand, in this description ξ is the probability that a new infective is asymptomatic, and this depends only on the nature of the virus and its interactions with the immune system of the infected people; thus in this interpretation ξ should be considered as a constant of nature, and it cannot be changed. (this is the point of view taken in [ ] ; however some of the assumptions made in its first version [ ] were very reasonable only within the concurrent interpretation, described in a moment.) on the other hand, if j is the class of unknown infectives, things are slightly different. in fact, to be in this class it is needed ( a ) that the individual has no or very light symptoms; but also ( b ) that he/she is not traced and analyzed by some epidemiological campaign, e.g. due to contacts with known infected or because belonging to some special risk category (e.g. hospital workers). in this description, η is a constant of nature, depending on the nature of the virus and on the response of the "average" immune system of (asymptomatic) infected people, while effort s to trace asymptomatic infectives will act on raising the probability ξ . we want to discuss the effect of early detection of infectives, or tracing their contacts, within the second mentioned framework. note that a campaign of tracing contacts of infectives is useful not only to uncover infectives with no symptoms, but if accompanied by effective isolation of contacts with known infectives, and thus of those who are most likely to be infective, it will also reduce the removal time of "standard" (i.e. symptomatic) infectives, possibly to a time smaller than the incubation time itself. in this sense, we will look at an increase in ξ as early detection of infectives , and at an increase in both β and η (thus a reduction in the removal times β − and η − ) as tracing contacts of infectives . this should be kept in mind in our final discussion about the effect of different strategies. as mentioned above, one should also avoid any confusion between asymptomatic and pre-symptomatic infection. in our description, pre-symptomatic infectives -i.e. individuals which are infective and which do not yet display symptoms, but which will at a later stage display them -are counted in the class of "standard" infectives, i.e. those who will eventually display symptoms and hence be intercepted by the health system with no need for specific test or contact racing campaigns, exactly due to the appearance of symptoms. actually one expects that except for the early phase of the epidemics in the countries which were first hit in a given area (such as china for asia, or italy for europe), when symptoms could be attributed to a different illness, most infections by symptomatic people are actually pre-symptomatic , as with the appearance of symptoms people are either hospitalized or isolated at home; and even before any contact with the health system they will avoid contacts with other -and other people will surely do their best to avoid contacts with anybody displaying even light covid symptoms. in the case of asymptomatic infectives, instead, unless they are detected by means of a test or contact tracing campaign -see the forthcoming discussion -they remain infective until they recover, so that in this case removal is indeed equivalent to (spontaneous) recovery. this approach, indeed, was taken in one of the areas of early explosion of the contagion in northern italy, i.e. in vò euganeo; this had the advantage of being a small community (about residents), and all of them have been tested twice while embargo was in operation. in fact, this was the first systematic study showing that the number of asymptomatic carriers was very high, quite above the expectations [ ] . apart from its scientific interest, the approach proved very effective in practical terms, as new infectives were quickly traced and in that specific area the contagion was stopped in a short time. while testing everybody is not feasible in larger communities, the "follow the contacts" approach could be used on a larger scale, especially with the appearance of new very quick kits for ascertaining positivity to covid. the model will thus react to a raising of ξ by raising the fraction of i within the class of infectives, i.e. in k = i + j; but at the same time, as critical patients are always the same, i.e. represents always the same fraction of k , we should pay attention to the fact they will now represent a lower fraction of i . the chinese experience shows that critical patients are about % of hospitalized patients (i.e. of those with symptoms serious enough to require hospitalization); and hospitalized patients represented about half of known infected, the other being cured and isolated at home. similar percentages were observed in the early phase of the covid epidemic in italy; the fraction of infectives isolated at home has afterwards diminished, but it is believed that this was due to a different policy for lab exams, i.e. checking prioritarily patients with multiple symptoms suggesting the presence of covid rather than following the contacts. actually this policy was followed in most of italy, but in one region (veneto) the tracking of contacts and lab exams for them was pursued, and in there the percentages were much more similar to those known to hold for china. in our previous work [ ] we have considered data for the early phase of covid epidemics in italy, and found that β − best fits them while the estimate η − was considered as a working hypothesis. this same work found as value of the contact rate in the initial phase α . * − , and we will use this in our numerical simulations. it should be stressed that the extraction of the parameter α from epidemiological data is based on the number s n of susceptibles at the beginning of the epidemic, thus α and hence γ depend on the total population. the value given above was obtained considering n = * , i.e. the overall population of the three regions (lombardia, veneto and emilia-romagna) which were mostly affected in the initial phase. our forthcoming discussion, however, does not want to provide a forecast on the development of the covid epidemic in northern italy; we want instead to discuss -with realistic parameters and framework -what would be the differences if acting with different strategies in an epidemic with the general characteristics of the covid one. thus we will adopt the aforementioned parameters as "bare" ones (different strategies consisting indeed on acting on one or the other of these) but will apply these on a case study initial condition; this will be given by one important parameter is missing from this list, i.e. the detection probability ξ . following li et al. [ ] we assumed in previous work that ξ is between / and / . later works (and a general public interview by the head of the government agency handling the epidemic [ ] ) suggested that the lower bound is nearer to the truth; moreover a lower ξ will give us greater opportunity to improve things by acting on it (we will see this is not the best strategy, so it makes sense to consider the setting more favorable to it). we will thus run our simulation starting from a "bare" value as for the total population, we set n = * . with these choices we get a projection of what could have happened if no action was undertaken. a note by an oxford group [ ] , much discussed (also in general press [ ] ) upon its appearance, hinted that in italy and uk this fraction could be as low as ξ = / . we have ascertained that with this value of ξ , and assuming α was not changed by the restrictive measures adopted in the meanwhile, the a-sir model fits quite well the epidemiological data available to the end of april. however, despite this, we do not trust this hypothesisat least for italy -for various reasons, such as (in order of increasing relevance): ( i ) a viral infection showing effects only in % of affected individuals would be rather exceptional; ( ii ) albeit in our opinion the effect of social distancing measures adopted in italy is sometimes overestimated, we trust that there has been some effect; ( iii ) if only % of infected people was detected, in some parts of italy the infected population would be over %. on the other hand, the main point made by this report [ ] , i.e. that only a large scale serological study, checking if people have covid antibodies, will be able to tell how diffuse the infection is -and should be performed as soon as possible -is by all means true and correct. see also [ ] . a look at eqs. ( ) shows that i will grow provided where again γ = β/α, and we have introduced the ratio x ( t ) of known infectives over total infectives. in other words, now the epidemic threshold depends on the distribution of infectives in the classes i and j . note that if x = ξ (as one would expect to happen in early stages of the epidemic), then γ i = γ . needless to say, we have a similar result for j , i.e. j will grow as far as thus the epidemic threshold for unregistered infectives is it is important to note that x is evolving in time. more precisely, by the equations for i and j we get dx dt the behavior observed in fig. , which displays x ( t ) and related quantities on a numerical solution of eq. ( ) , can be easily understood intuitively. in the first phase of the epidemic, there is an exponential growth of both i and j ; due to the structure of the equations, they grow with the same rate, so their ratio remains constant; on the other hand, once the dynamics get near to the epidemic peak, the difference in the permanence time of the two (that is, the time individuals remain in the infect class) becomes relevant, and we see (plots (a ) and (b ) of fig. ) that not only the peak for j is higher than the one for i , but it occurs at a slightly later time. moreover, descending off the peak is also faster for i , as β − < η − , and thus x further decreases, until it reaches a new equilibrium while both classes i and j go exponentially to zero. if we look at ( ) we see that for fixed s the variable x would have two equilibria (one stable with < x < and one unstable with x > , stability following from β − η > ), easily determined solving d x/d t = . numerical simulations show that -apart from an initial transient -actually x ( t ) stays near, but in general does not really sticks to, the stable fixed point determined in this way. a relevant point should be noted here. if we consider the sum ( ) of all infectives, the a-sir model can be cast as a sir model in terms of s, k , and q = r + u as as x varies in time, this average removal rate is also changing. on the other hand, the basic reproduction number (brn) ρ (this is usually denoted as r , but we prefer to change this notation in order to avoid any confusion with initial data for the known removed r ( t )) for this model will be in other words, not taking the asymptomatic infectives into account leads to an underestimation of the brn. if the standard sir model predicts a brn of ρ , the a-sir model yields a brn ˆ ρ given this means that the epidemic will develop faster, and possibly much faster, than what one would expect on the basis of an estimate of ρ based only on registered cases, which in the initial phase are a subset of symptomatic cases as the symptoms may easily be leading to a wrong diagnosis (in the case of covid they lead to a diagnosis of standard flu). with our covid-related values β = / , η = / , and assuming that in the early phase there is thus a good reason for being surprised by the fast development of the epidemic: the actual brn is substantially higher than the one estimated by symptomatic infections [ ] . more generally, one would wonder what is the effect of the "hidden" infectives j ( t ) on the dynamics of the known infectives i ( t ) -which, we recall, include the relevant class of seriously affected infectives -and it appears that there are at least two, contrasting, effects: . on the one hand, the hidden infectives speed up the contagion spread and hence the rise of i ( t ); . on the other hand, they contribute to group immunity, so the larger this class the faster (and the lower the i level at which) the group immunity will be reached. the discussion above shows that the balance of these two factors leads to a much lower epidemic peak, and a shorter epidemic time, than those expected on the basis of the standard sir model (albeit in the case of covid with no intervention these are still awful numbers). on the other hand, we would like to understand if uncovering a larger number of cases (thus having prompt isolation of a larger fraction of the infectives) by early detection , i.e. raising ξ , would alter the time-span of the epidemic. it appears that this effect can be only marginal, as it appears only past the epidemic peak. we stress that this statement refers to "after incubation" analysis; if we were able to isolate cases before they test positive -i.e. to substantially reduce β − -the effect could be different. we will discuss this point, related to contact tracing , later on. an ongoing epidemic is not a laboratory experiment, and apart from not having controlled external conditions, i.e. constant parameters, the very collection of data is of course not the top priority of doctors fighting to save human lives. there has been considerable debate on what would be the most reliable indicator to overcome at least the second of these problems. one suggestion is to focus on the number of deaths; but this is itself not reliable, as in many cases covid is lethal on individuals which already had some medical problem, and registering these deaths as due to covid or to some other cause depends on the protocol adopted, and in some case also on political choices, e.g in order to reassure citizens (or on the other extreme, to stress great care must be taken to avoid contagion). another proposed indicator, possibly the most reliable in order to monitor the development of the epidemic, is that of patients in intensive care units. this appears to be sufficiently stable over different countries, and e.g. the italian data tend to reproduce in this respect -at least in regions where the sanitary system is not overstretched -the chinese ones. in this case, ic patients are about % of the total number of hospitalized cases; in china and for a long time also in italy (when protocols for choosing would-be cases to be subject to laboratory analysis have been stable), hospitalized cases have been about half of the known infection cases, the other having shown only minor symptoms and been cured (and isolated) in their home. the other, more widely used, indicator is simply the total number of known cases of infection. in view of the presence of a large class of asymptomatic infectives, this itself is strongly depending on the protocols for chasing infectives. on the other hand, this is the most available indicator: e.g., the w.h.o. situation reports [ ] provide these data. each of these indicators, thus, has advantages and disadvantages. we will just use the who data on known infected. in particular, in the case of covid we expect that with ξ the "bare" constant describing the probability that an infection is detected, out of the class i ( t ) we will have a % of infected with little or no symptoms ( i l ), a % of standard care hospitalized infected ( i h ), and a % of ic hospitalized infected ( i ic ). needless to say, this class is the most critical one, also in terms of strain on the health system. more generally, we say that with ξ the "bare" constant describing the probability that the infection under study is detected, there is a fraction χ (of the detected infections) belonging to the i ic class; that is, i ic (t) = χ i(t ) . we stress this depends on the protocol used to trigger laboratory tests; in our general theoretical discussion, this is any such protocol and we want to discuss the consequences of changing this in the sense of more extensive tests. we are now ready to discuss how modification of one or the other of the different parameters ( α, β, ξ ) on which we can act by various means will affect the a-sir dynamics. as it should be expected, this will give results similar to those holding for the sir model, but now we have one more parameter to be considered and thus a more rich set of possible actions. fig. . the effect of a change in ξ on the i ic class. we have used β = / , η = / , and α = . * − as in fig. , with a total population of n = * , and ran simulations with ξ = / (solid curve) and with ξ = / (dashed curve). the substantial increase in ξ produces a reduction in the epidemic peak and a general slowing down of the dynamics, but both these effects are rather small. a more extensive test campaign will raise ξ , say from ξ to ξ ; but of course this will not change the number of the most serious cases, as these are anyway getting to hospital and detected as being due to the infection in question. thus the new fraction χ of detected infections which need special care will be such that in order to describe the result of raising ξ , we should thus compare plots of this is what we do, indeed, in fig. . raising ξ corresponds to having more infective detected, and has some advantages from the point of view of the epidemic dynamics. in practical terms, this means extending tests to a larger class of subjects, and be able to isolate a larger fraction of asymptomatic infectives with the same speed and effectiveness as symptomatic ones. a different strategy for rapid action is also possible, and it consists of rapid isolations of subjects who had contacts with people known to have been infected, or who have themselves been in contact with known infectives (and so on). in other words, the strategy would be to isolate would-be infection carriers before any symptom could show up. this means that β − could be even smaller than the usual infection-to-isolation time (about seven days for covid) for symptomatic infectives, and even shorter than the incubation time (about five days for covid). it should be stressed that as each of these "possible infected" might have a small probability of being actually infected (depending on the kind of contacts chain leading to him/her from known infectives), here "isolation" does not necessarily mean top grade isolation, but might amount to a very conservative lifestyle, also -and actually, especially -within home, where a large part of registered chinese contagions took place. (the same large role of in-home contagion was observed in italy in the course of lockdown.) we have thus ran a simulation in which ξ is not changed, but β is raised from β = / to β = / ; the result of this is shown in fig. . in this case we have a marked diminution of the epidemic peak, and a very slight acceleration of the dynamics. . the effect of a change in β on the i ic class. we have used ξ = / , η = / , and α = . * − as in fig. , with a total population of n = * , and ran simulations with β = / (solid curve) and with β = / (dashed curve). the substantial increase in β produces a marked reduction in the epidemic peak and a very slightly faster pace in the dynamics. we have so far not discussed the most basic tool in epidemic containment, i.e. social distancing. this means acting on the parameter α by reducing it. direct measurement on the epidemiological data for northern italy show that this parameter can be reduced to about % of its initial value with relatively mild measures. in fact, albeit the media speak of a generalized lockdown in italy, the measures have closed schools and a number of commercial activities, but for the rest were actually more pointing at limiting leisure walk and sports and somewhat avoiding contacts in shops or in work environment than to a real lockdown as it was adopted in wuhan. this is a basic action to be undertaken, and in fact it is being taken by all nations. it is also the simplest one to be organized (albeit with high economic and social costs in the long run) and an action which can be taken together with other ones. no doubt this should be immediately taken when an epidemic is starting, and accompanied by other measures -such as those discussed above. but here we want to continue our study of what it means by itself in terms of modification of the epidemic dynamics. it is not clear what can be achieved in terms of reduction of social contacts. in fact, once the epidemic starts most of the dangerous contacts are the unavoidable ones, such as those arising from essential services and production activity (e.g. production and distribution of food or pharmaceutical goods), contacts at home, and above all contacts in hospitals. thus, after a first big leap downward corresponding to closing of schools and universities on the one side, and a number of unessential commercial activities on the other, and restrictions on travels, it is difficult to further reduce social contacts, not to say that this would have huge economic and social costs, and also a large impact on the general health in terms of sedentariness-related illness (and possibly mental health). a number of countries tried to further reduce social contacts by forbidding citizens to get out of their home; this makes good sense in densely populated areas, but is useless in many other areas. the fortunate slogan "stay home" risks to hide to the general public that the problem is not to seclude oneself in selfpunishment, but to avoid contacts . we point out that there is a further obstacle to reducing social contacts: as seen in the context of the simple sir model, reducing α will lower the epidemic peak, but it will also slow down the whole dynamic . while this allows to gain precious time to prepare fig. . the effect of a change in α on the i ic class. we have used β = / , ξ = / , η = / , with a total population of n = * , and ran simulations with α = . * − (solid curve) and with α = . * − (dashed curve). the reduction in α produces a marked reduction in the epidemic peak and also a marked slowing down in the dynamics. hospitals to stand the big wave, there is some temporal limit to an extended lockdown, and thus this tool cannot be used to too large an extent. we have thus ran a simulation in which β and ξ are not changed, while α is reduced by a factor . (smaller factors, i.e. smaller α, produce an untenable length of the critical phase); the result of this is shown in fig. . in this case we have a relevant diminution of the epidemic peak, and also a marked slowing down in the dynamics. an important remark is needed here. it may seem, looking at this plot, that social distancing is less effective than other way of coping with the epidemic. but these simulation concern a sir-type model; this means in particular that there is no spatial structure in our model [ ] . the travel ban is the most effective way of avoiding the spreading of contagion from one region to the others; while the "local" measures of social distancing can (and should) be triggered to find a balance with other needs, travel ban is the simplest and most effective way of protecting the communities which have not yet been touched by the epidemic. we can thus compare the different strategies we have been considering. this is done in fig. where we plot together i ic ( t ) for all our different simulations; and in table where we compare the height of the epidemic peak -again for i ic ( t ) -and the time at which it is reached. fig. . the effect of different strategies. we plot i ic ( t ) for n = * in the "bare" case, i.e. for α = . * − , β = / , ξ = / , η = / , and in cases where (only) one of the parameters is changed. in particular we have the bare case (solid line), the case where ξ is changed into ξ = / (dotted), the case where β is changed to β = / (dashed), and that where α is changed to α = . * − (solid, blue). we also plot a horizontal line representing a hypothetical maximal capacity of ic units. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) table epidemic peak (for i ic ) and time for reaching it (in days) as observed in our numerical simulations. all simulation were ran with n = * and η = in fig. we have also drawn a line representing the hypothetical maximal capacity of ic units. this stresses that not only the different actions lower the epidemic peak, but they also -and to an even larger extent -reduce the number of patients which can not be conveniently treated. in looking at this plot, one should remember that the model does not really discuss permanence in ic units, and that i ic are the infected which when detected will require ic treatment; this may go on for a long time -which is the reason why ic units are saturated in treating covid patients. so the plots are purely indicative, and a more detailed analysis (also with real parameters) would be needed to estimate the ic needs in the different scenarios. it should be stressed that the strategies of contacts tracing and early detection are usually played together; but as confusion could arise on this point, let us briefly discuss it. we have tried to stress that these two actions are not equivalent: one could conduct random testing, so uncovering a number of asymptomatic infectives, and just promptly isolate them without tracing their contacts;or on the other extreme one could just isolate everybody who had a (direct or indirect) contact with a known infective, without bothering to ascertain if they are themselves infective or not. this strategy would be as effective in containing the contagion (and less costly in terms of laboratory tests) than that of tracking contacts, test them (after a suitable time for the infection to develop and test give positive if this happens), and isolate only those who really turn infective. the difference is that if we isolate everybody this would involve a huge number of people (e.g. all those who have been in the same supermarket the same day as an infective; and their families and contacts etc etc); so in this context early detection actually should be intended as early detection of non-infectives , so that cautionary quarantine can be kept reasonably short in all the cases where it is not really needed. finally we recall that it is a triviality, and it was already mentioned in the introduction, that in real situations one has not to choose between acting on one or the other of the parameters, and all kind of actions should be pursued simultaneously. the numerical computations of the previous subsections suggest that increasing ξ -that is, detection of a larger fraction of asymptomatic -is not a very efficient strategy to counter the diffusion of an infection with a large number of asymptomatic infectives, while a prompt isolation of infectives is a more effective action. it should be recalled, however, that in our computations -and in particular on fig. , where their outcomes are compared -we are focusing on the number of patients needing ic support, i.e. the most critical parameter from the point of view of the health system. in order to substantiate our conclusions, it is worth considering also different ways to evaluate the effect of different strategies. we have thus considered also a different indicator, i.e. the total number of infectives we have run several simulations, with total population n = * and with parameters α = μ α α β = μ β β η = μ η η ξ = μ ξ ξ . the outcome of these simulations is displayed in fig. ; see its caption for the parameter (that is, the modulation factor) values in different runs. we see from fig. that action on α slows down substantially the epidemic dynamics and reduce the epidemic peak, while action on ξ or on β alone produce only a moderate effect. on the other hand, actions affecting the value of η (alone or together with the value of β) reduce substantially the epidemic peak and slightly slow down the dynamics. it may be noted that the shapes of the i ic ( t ) (see fig. ) and of the k ( t ) (see fig. ) are different; in particular, the decay of i ic ( t ) after attaining its peak is faster than the decay of k ( t ). this corresponds to what is observed in the epidemiological data for italy. we have considered epidemic dynamics as described by "mean field" models of the sir type; more specifically, we have first considered the classical kermack-mckendrick sir model [ ] [ ] [ ] [ ] [ ] and then a recently introduced modified version of it [ ] taking into account the presence of a large set of asymptomatic -and thus most frequently not detected -infectives. these models depend on several parameters, and different types of measures can to some extent change these parameters and thus the epidemic dynamics. in particular, this action can effect two basic characteristics of it, i.e. the height of the epidemic peak and the time-span of the epidemic. while it is clear that in facing a real lethal epidemics (such as the ongoing covid epidemic) all actions which can contrast it should be developed at the same time, in this paper we have considered the result -within these models -of different tools at our disposal, i.e. (generalized) social distancing, early detection (of asymptomatic infectives) and contacts tracing (of symptomatic and asymptomatic infectives). it turns out that -both in the classical sir model and in the modified a-sir one -social distancing is effective in reducing the epidemic peak, and moreover it slows down the epidemic dynamics. on the other hand, early detection of asymptomatic infectives seems to have only a moderate effect in the reduction of the epidemic peak for what concerns critical cases, and also a very little effect on the temporal development of the epidemic. in contrast, contact tracing has a strong impact on the epidemic peak -also in terms of critical cases -and does not substantially alter the temporal development of the epidemic, at least for what concerns the curve describing the most serious cases. remark . the conclusion that early detection of asymptomatic has only a moderate effect may appear to be paradoxical, and requires some further discussion. first of all we should remind that we are here actually talking about an increase of the parameter ξ (see remark ) , while in a real situation early detection of asymptomatic will most likely go together with early detection of symptomatic, and hence a reduction in β as well. the increase of ξ per se means that some fraction of asymptomatic will be recognized as infective and be isolated on the same timescale β − as the symptomatic infectives, while the other asymptomatic will escape recognition and still be infective on a timescale η − . on the other hand, a realistic contact tracing campaign will lead to prompt isolation of symptomatic and asymptomatic alike, and thus correspond to a reduction in β − and in η − , and we have seen that this action is indeed the most effective one in terms of contrasting the spread of the epidemic. in other words, our result suggests that the key to fight covid is not so much in detection , but in prompt isolation of infectives, and most notably of asymptomatic ones. this can be achieved only by contact tracing -as already suggested by experienced epidemiologists. slowing down the epidemic dynamic can be a positive or negative feature depending on the concrete situation and on the desired effects. it is surely positive in what concerns getting ready to face the epidemic peak, in particular in the presence of a faltering health system. on the other hand, it may be negative in that maintaining a generalized lockdown for a long time can have extremely serious economic and social consequences. balancing these two aspects is not a matter for the mathematician or the scientist, but for the decision maker; so we will not comment any further about this. it should also be recalled that our analysis was conducted in terms of very simple sir-type models, with all their limitations. in particular, we have considered no age or geographical or social structure, and only considered a population of "equivalent" indi-viduals. in particular, as we have noted above, in the early stage of an epidemic, which presumably develops in very populated areas, a generalized travel ban can simply stop the contagion to propagate to other (possibly less well equipped in medical terms) areas; moreover, social distancing measures can be implemented very simply -basically, by a government order (albeit if we look at the goal of these measures, i.e. reducing the occasion of exchanging the virus, a substantial role would be played by individual protection devices, such as facial masks; in many european countries, these were simply not available to the general public, and in some cases neither to medical operators, thus substantially reducing the impact of these measures) -and are thus the first action to be taken. in fact, in relation with the ongoing covid epidemics, one of the reproaches made to many governments is usually to have been too slow or too soft in stopping crowd gatherings, surely not the contrary. on the other hand, we hope that this study makes clear what are the consequences of different options. in particular, our study shows that contacts tracing , followed by prompt isolation of wouldbe infected people -is the only way to reduce the impact of the epidemic without having to live with it for an exceedingly long time. the veneto experience [ ] shows that this strategy can be effectively im plemented without hurting privacy or personal freedom. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. we are now going to briefly discuss these matters; we point out that this appendix was inserted in the revised version of this paper, so it can make use of knowledge not available at the time of writing the first submitted version, nd mentions papers appeared after the first submittal. compartment models, i.e. sir-type ones in this context, are based on several implicit and explicit assumptions, which are not realistic in many cases and surely when attempting to describe the covid epidemic, in particular in a full country. that is, among other aspects, sir-type models are (in physics' language) mean field (averaged) models and as such describe the dynamics and the underlying system as if: • all individuals are equivalent in medical sense, i.e. they all have equivalent pre-existent health status and equivalent immune system and react in the same way to contact with the pathogen; • in particular, as we know that covid is statistically more dangerous for older people, we are completely disregarding the age structure of the population, as well as the existence of other high risk classes related to pre-existent pathologies: all these contribute to an average over the whole population; • all individuals are equivalent in social sense, i.e. they all have an equivalent social activity and hence the same number and intensity of contacts with other members of the group, thus the same exposure to (possible) infectives; • in particular, this means we are completely disregarding any geographical structure in the population, and consider in the same way people living in large cities or in remote villages, just considering them in the same global average; • similarly, we do not consider that work can cause some people to be specially exposed through contact with a large number of people (e.g. shop cashiers) or even with a large number of infected people (e.g. medical doctors or nurses). thus one cannot hope to retain, through such models, effects like the faster spreading of the infection in more densely populated areas or the specially serious consequences of the covid infection among older people. we stress that these could be obtained by including geographical, age or social structures into the model, i.e. increasing the number of considered classes; in principles this should provide a finer and more realistic description of the epidemic dynamic, and in fact it is done in cases for which there is a large set of data, e.g. for influenza. such structured models would of course loose the main attractive of the sir model, i.e. its simplicity -which also allows to understand in qualitative terms the mechanisms at work. in particular, a relevant intermediate class of models is that of sir-type models on networks: these take into account geographical and social structures and make use of known information about contacts between different groups of individuals and about different health characteristics of different groups. the problem with these networked or however more structured models is that the network should be inferred from data. in this respect, it could be objected that the influenza monitoring over many years could give us the relevant data for reconstruction of such network; but it is everybody's experience, by now, that the social behavior of people are completely different if dealing with a well known and not so serious (except for certain categories) illness like influenza or with an unknown and potentially lethal one like covid; this not to say that the restrictive measures put into effect by many governments have completely changed the interaction patterns among people, so that previously accumulated data cannot be used in the present situation. when thinking of covid; it should be kept in mind that even if the countries which were first hit by the epidemic, we only have data over some months; e.g. for italy we have about days of data. if we were trying to give the model a geographical structure at the level of departments (which are themselves administrative units mostly with a very varied internal geographical structure), as there are departments in italy this would require in the simplest form to evaluate a × interaction matrix, and i cannot see any way to reliably build this out of such a scarce set of data. moreover, the epidemiological data are to some extent not reliable, especially around the epidemic peak, in that they are collected in an emergency situation, when other priorities are present in hospitals (e.g. in italy the data show a weekly modulation, which appears to be due simply to the procedure of data collection); so an even larger amount of data would be needed to filter out statistical noise and random fluctuations. in this sense, the weak point of sir-type models, i.e. their being based on an average over the whole population, turns out to be an advantage: they contain few parameters (two for the sir, four for the a-sir) and are thus statistically more robust in that fluctuations are averaged efficiently with less data than for more refined models with a large number of parameters. similar considerations hold when one compares sirtype models to a purely statistical description or to an "emerging behavior" approach. these approaches are extremely powerful, but are effective when one has a large database to build on and to which compare the outcome of the "experiment" (in this case the epidemic) under consideration. when we deal with a completely new pathogen,like for covid, we simply don't have a database, and we can only rely on the very general features of infective dynamics -which are well coded by sir and sir-like models. in other words, we are not claiming the sir approach to be superior to others, but only that it is appropriate when we have few data -as for covid. within the sir-type class, the a-sir model is specially simple; from the theoretical point of view its appeal lies in that it is the simplest possible model taking into account the presence of a large class of asymptomatic infectives; thus it focuses on the effect of this fact without the complications of a more detailed model. but, of course, it makes sense to rely on this model only if it is able to give a good, or at least a reasonable, agreement with observed data. of course each infective agent has its own characteristics, and using only the general sir model would completely overlook them, apart from the different values of the α and β parameters. thus we have to do something more than just evaluating the sir parameters. in our study we have identified the presence of a large class of asymptomatic infectives as one of the key problems in facing the covid epidemic, and we have considered a simple model which allows to focus precisely on this aspect. one should be aware that the a-sir model is focusing on this and not considering other features of covid, and indeed other more detailed sir-type models for covid have been formulated and studied (see also below). here we are taking an approach which is classical in mathematical physics and mathematical modeling, i.e. try to build and study the simplest model describing the phenomenon of interest. this will give results which are quantitatively worse than a more detailed models, but which are qualitatively good in that the model is simple enough to see more clearly what are the mechanisms at work and to understand the qualitative features if the dynamics and the qualitative outcome of any intervention able to modify the parameters of the model. having said that, it remains true that -as mentioned aboveit makes sense to rely on this model only if it is able to give a reasonable agreement with observed data. this is not the argument of this paper, and it was discussed in a previous paper [ ] ; the success of this model was the justification for this paper,i.e. for dis- however, the first version of this paper was submitted at mid-may, hence with two and half months of data available, while at the time of preparing this revised version we have four months of data; that represents a substantial increase in the available data, and it makes sense to wonder if the model is still describing the covid epidemic in italy. this is indeed the case, as shown in fig. ; they represent epidemiological data as communicated by the italian health ministry and by who (and widely available online through the standard covid databases) against a numerical integration of the a-sir eqs. ( ) . we refer to gaeta [ ] for a discussion of the parameters and their determination. note that the contact rate α is assumed to vary in response to the restrictive measures (and to the availability of individual protection devices); as these measures were taken in different steps, we also have different values of α in different time intervals. more precisely, the equations were integrated for a total population of n = * for the period february , through june , with initial data at day ( we stress that the parameter values are the same as in [ ] , even for the most recent time, not considered in that paper: the model continues to reasonably well describe the development of the epidemic in italy. we focused on a specific sir-type model, but several models of this type have been considered in the context of covid modeling. here we give a very short overview of these, with no attempt to completeness -which cannot even be imagined in such a rapidly evolving field. first of all, we note that other researchers have considered, motivated by the ongoing covid epidemic, the temporal aspects of the standard sir dynamics. we mention in particular cadoni [ ] (a related, but quite involved, approach had been considered by harko, lobo and mak [ ] ) and barlow and weinstein [ ] , who obtained an exact solution for the sir equations in terms of a divergent but asymptotic series [ ] ; see also [ , ] for a different approach to exact solution of sir and sir-type models. we also note that nonlinear modifications of the bilinear infection term of the standard sir model have been proposed -explicitly or implicitly -in the attempt to relate the standard sir model to covid dynamics [ , ] . we find [ ] of special interest, as this work introduces a model for the epidemic dynamics coupled to the immune system, and is thus able to take into account aspects related to the viral charge of infectives. extension of the sir model in the direction of allowing timedependence of the parameters -also to account for shifting public attitude -has also been considered [ ] . as mentioned above, see remark , considering the delay between infection and beginning of infectiveness would lead to consider seir-type models. the problem of temporal aspects of the dynamics for this class of models has been considered by becaer [ ] . the role of asymptomatic transmission in this class of models has also been considered [ , ] . the approach to sir by barlow and weinstein [ ] leading to exact solution has been extended to seir model [ ] . a generalization of the a-sir model, allowing for different infectiveness of symptomatic and asymptomatic infectives, has been considered by neves and guerrero [ ] . more elaborated compartment models with a larger number of compartments have been considered by a number of authors. we would like to mention in particular two papers which we consider specially significant, i.e. the work by the pavia group, in which mathematicians, statisticians and medical doctors collaborated [ ] , and the work by fokas, cuevas-maraver and kevrekidis [ ] , in which such a model -involving five compartments like the present paper, but chosen in a different way -is used to discuss (as in the present paper) exit strategies from the covid lockdown. as mentioned in the main text, one could -and should -consider epidemic dynamics on networks [ ] . attempts to analyze the covid epidemic in this way have of course been pursued, both on a small scale, with a network structure which can be determined by direct sociological study [ ] , and on a nationwide scale [ ] where the network structure has to be determined. this latter study [ ] also attempted to evaluate the effect of the containment measures; such a matter is of course very relevant and has been considered by many authors in many countries; even a cursory mention of these is impossible, and we will just mention one study applying to italy [ ] . we also stress that many of the papers mentioned above, see in particular [ , ] aim at using the models they study to evaluate the effect of interventions and containment measures. finally we would like to end on a positive note, and mention that while on the one hand it was found that the presence of asymptomatic makes that the basic reproduction number of covid is higher than initially estimated [ , , ] , the fact that the social contact rate is not uniform in the population makes that the herd immunity level should be lower than predicted on the basis of the standard sir-type models [ ] ; this is a specially nice result of the analysis on networks,as it only depends on general -and very reasonable -properties of the network and not on its detailed structure, thus overcoming the low statistics problem mentioned in remark above. contributions to the mathematical theory of epidemics mathematical biology. i: an introduction essential mathematical biology the mathematics of infectious diseases mathematical models in biology. siam arxiv: . ; data analysis for the covid- early dynamics in northern italy a simple sir model with a large set of asymptomatic infectives a simple sir model with a large set of asymptomatic infectives how to reduce epidemic peaks keeping under control the time-span of the epidemic accurate closed-form solution of the sir epidemic model asymptomatic transmission, the achilles heel of current strategies to control covid- (editorial) presumed asymptomatic carrier transmission of covid- pre-and asymptomatic individuals contribute up to % of covid- transmission evidence supporting transmission of severe acute respiratory syndrome coronavirus while presymptomatic or asymptomatic temporal dynamics in viral shedding and transmissibility of covid- the rate of underascertainment of novel coronavirus ( -ncov) infection: estimation using japanese passengers data on evacuation flights estimation of the asymptomatic ratio of novel coronavirus infections (covid- ) estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) covid- : four fifths of cases are asymptomatic, china figures indicate presymptomatic sars-cov- infections and transmission in a skilled nursing facility prevalence of asymptomatic sars-cov- infection suppression of a sars-cov- outbreak in the italian municipality of vÓ substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) interview to the newspaper la repubblica fundamental principles of epidemic spread highlight the immediate need for large-scale seologicalsurvey to assess the sage of the sarscov- epidemic asymptomatic infectives and r for exact analytical solutions of the susceptible-infected-recovered (sir) epidemic model and of the sir model with equal death and birth rates on the summation of divergent, truncated, and underspecified power series via asymptotic approximants path integral approach to uncertainties in sir-type systems should the rate term in the basic epidemiology models be second-order? immuno-epidemiological model of two-stage epidemic growth a time-dependent sir model for covid- with undetectable infected persons un modèle mathématique des débuts de lépidémie de coronavirus en france accounting for symptomatic and asymptomatic in a seir-type model of covid- covid- pandemic: a mobility-dependent seir model with undetected cases in italy analytic solution of the seir epidemic model via asymptotic approximant predicting the evolution of the covid- epidemic with the a-sir model: lombardy, italy and sao paulo state modelling the covid- epidemic and implementation of population-wide interventions in italy a quantitative framework for exploring exit strategies from the covid- lockdown spread of epidemic disease on networks heterogeneous contact networks in covid- spreading: the role of social deprivation spread and dynamics of the covid- epidemic in italy: effects of emergency containment measures the impact of a nation-wide lockdown on covid- transmissibility in italy the impact of undetected cases on tracking epidemics: the case of the disease-induced herd immunity level for covid- is substantially lower than the classical herd immunity level the work was carried out in lockdown at smri. i am also a member of gnfm-indam. our discussion was based on sir-type models, and in particular on the a-sir model. this raises several kind of questions, which we address in this appendix. key: cord- -fwmb rr authors: monzani, alice; ragazzoni, luca; della corte, francesco; rabbone, ivana; franc, jeffrey m. title: covid- pandemic: perspective from italian pediatric emergency physicians date: - - journal: disaster medicine and public health preparedness doi: . /dmp. . sha: doc_id: cord_uid: fwmb rr objectives: to document the lived experience of italian pediatric emergency physicians during the coronavirus disease (covid- ) pandemic. methods: we developed a structured interview to collect the lived experience of the staff of the pediatric emergency department (ped) of a tertiary referral university hospital in northern italy. the open-ended questions were draft according to the suggestions of canadian colleagues and administered by interviewer, who was part of the ped staff, at the end of march . all the ped staff was interviewed, on a voluntary basis, using purposive sampling. results: most respondents declared to be afraid of becoming infected and of infecting their families. the number of patients seen in the ped has decreased, and the cases tend to be more severe. a shift in the clinical approach to the ill child has occurred, the physical examination is problem-oriented, aiming to avoid un-necessary maneuvers and to minimize the number of practitioners involved. the most challenging aspects reported are: ( ) performing a physical examination in personal protective equipment (ppe), ( ) being updated with rapidly evolving guidelines, and ( ) staying focused on the possible covid- clinical presentation without failing in differential diagnosis. conclusions: during the covid- pandemic, it seems that pediatric emergency physicians are radically changing their clinical practice, aiming at prioritizing essential interventions and maneuvers and self-protection. t he coronavirus disease pandemic is now threatening several health systems around the world. in northern italy, being of the most affected areas so far, emergency departments are still struggling to deal with mass influxes of patients. even though severe covid- in children is rare and the number of confirmed cases in the pediatric population is significantly smaller compared with adults, pediatric emergency departments (peds) remain a crucial hospital service during such a public health crisis. , pediatricians are required to apply infection prevention and control (ipc) measures and operational public health skills, also encompassing direct patient care for suspected or confirmed patients with covid- and the proper application of personal protective equipment (ppe). unfortunately, these competencies are beyond the experience and knowledge base of many health-care workers who are not familiar with public health emergencies arising from infectious disease outbreaks. clinicians may feel uneasy about treating an infectious disease for which there is no vaccine, for which there is no specific therapy, and which is highly transmissible. in italy, the risk for covid- secondary infections among healthcare workers is extremely high, which highlights the probably inadequate ipc practices and protections in place in many hospitals and primary health-care services. in this scenario, numerous research studies and practical and clinical guidelines have been published by the world health organization and other academic institutions to increase the safety among practitioners and to enhance the quality of care. however, to the best of our knowledge, few scientists have studied the current pandemic from the perception of health-care workers providing pediatric care on the frontline. fear and challenges to respond to infectious emergencies, including pandemic influenza, are already well documented. nevertheless, little is known about the health-care workers' perspective on covid- pandemic, especially from pediatric emergency physicians. the objective of this study was to document the lived experience of pediatric health-care providers in italy during the initial phase of the covid- pandemic. the main areas of investigation were the variation in their hospital daily activities and the challenges encountered during direct patient care. we developed a structured interview to collect the lived experience of pediatric health-care providers of the maggiore della carità university hospital (novara, italy), the second largest third level referral hospital of the piedmont region, one of the most covid- affected regions in northwest italy. the open-ended questions, draft according to the suggestions and interests of canadian pediatric emergency physicians, were collected at the end of march , when , confirmed cases were reported in italy, with , deaths, whereas canada was not already critically involved in the covid- outbreak. canadian pediatric emergency physicians were interested in knowing the real-life experience of their italian counterparts. one of the authors (j.m.f.) collected by email the questions of his canadian colleagues. these open-ended questions represented the basis for the development of the interview. questions with similar content were merged to produce the final open-ended questions included in the interview. the ped staff of the maggiore della carità university hospital agreed to partake in the interview process. the questions were administered by interviewer (a.m.) who was part of the ped staff. all the staff of the ped were interviewed, either by face-to-face or phone interviews, on a voluntary basis. we used a purposive sampling, including the complete target population. the face-to-face interviews took place in the hospital, whereas the phone interview took place when the respondents were either in the hospital or at home. the day, time, and location of the interview were scheduled according to the respondents' availability and convenience, within a -wk frame, given the timeliness of the study. the comments and perceptions were collected and summarized to answer the open-ended questions. the written transcripts were read several times to obtain an overall feeling for them. relevant phrases or sentences that pertained directly to the lived experience were identified. integrated meanings were then formulated from the relevant statements and phrases, to provide an exhaustive summary of single perceptions, as detailed by creswell and poth. the participation in the interview was considered as the consent to participate in this report. confidentiality of information was ensured, and no financial incentive to participate in the study was offered. because no identifying personal data are reported and the responses to the interview are presented in aggregate form, the study was deemed exempt from the requirement for formal approval by the local ethics committee. all the physicians working in our ped (m:f = : ; aged - years) took part to the interviews, which lasted approximately min. theme . are you afraid of becoming infected or infecting your family? most of the respondents said that they are afraid of becoming infected, mainly because the use of ppe is reserved for patients with fever and/or respiratory symptoms, but it is known that also asymptomatic subjects may spread the virus. therefore, the only way to protect yourself would be if patients wore surgical masks, but it is not so feasible for younger children or, even more, for neonates. all the respondents are afraid of infecting their families so that most of them have changed their lifestyle. some pediatricians are still living at home with their families, but sleeping in a separate bed or bedroom, keeping distances from their family members, and, in some cases, wearing surgical masks also at home. a few other colleagues decided to leave their families and went to stay in hotels or empty houses to minimize the interactions with their cohabitants and the risk of infecting them. the number of patients seen in our ped has dramatically decreased (approximately - % reduction) during the covid- pandemic. all the respondents thought this could be due both to the restriction measures that came into force on march , and to the fear of the patients to be infected coming to the hospital. as a result, also the type of patients admitted has changed. the less severe codes (white and green codes) are decreased, alongside an increase in more severe ones was seen, suggesting that patients with minor and milder conditions tend to avoid seeking the ped. a considerable increase in traumatic injuries was noticed, perhaps linked to the motor hyperactivity often experienced by children under restrictive measures, forced to play in the limited space of their houses. moreover, surely, there are children referring to the ped for suspected covid- , presenting with fever, cough, sore throat, abdominal pain/diarrhea, skin lesions, and often with family members with covid- . nonetheless, as it is known that covid- in children may often be asymptomatic, it is important to keep a high level of suspicion with all the children admitted to the ed, also for conditions not directly linked to covid- (ie, injuries, burns, foreign body ingestions…). the respondents' attitude during physical examination has considerably changed during the covid- pandemic. certainly, wearing ppe represents a substantial change and makes it difficult to perform some maneuvers, for example, the face shield prevents an accurate otoscopy. more broadly, a radical shift in the approach to the ill child has occurred. pediatricians are used to meticulous head-to-toe physical examinations. in this unique situation, the focus has changed. physicians must protect themselves as a priority, so that the physical examination is problem-oriented and aimed at avoiding unnecessary maneuvers, also in a ppe-sparing perspective. for example, if a child is referred for a foot injury, a throat examination is not performed, so that the physician would not require to wear complete equipment, such as goggles or face shields. another substantial change is the attitude toward fever. if pediatricians used to consider as a noteworthy fever only a body temperature higher than - . °c, now even a rising of a few degrees in body temperature requires attention. it must be highlighted that in the lockdown period, when children are not attending schools, common community-acquired diseases are extremely uncommon so that the detection of fever should suggest the possibility of covid- . in this case, the patient has to be treated as potentially infected, following a specific intra-hospital patient flow, with early isolation, and should be tested for the presence of severe acute respiratory syndrome coronavirus (sars-cov- ) rna. all the respondents have changed their attitude and now only one parent is allowed in the examination room, aiming at limiting the contacts for health-care practitioners, even if it could be less reassuring for the youngest patients. it is a completely new situation. some of the respondents pointed out that children could be scared by such unusual stuff, but one can pretend to be an astronaut to try to add a little magic to a potentially frightening setting. there was wide agreement about the fact that most of the parents look comforted by the use of ppe and are cooperative in reassuring their kids. this seems to be one of the main concerns of the respondents. on the one hand, they are worried about the possibility that the focus on covid- may lead to fixation errors, detracting from other differential diagnoses that should be considered in the presence of fever and respiratory symptoms. on the other hand, there is also concern that seriously ill children could delay the admission to the ped for fear of becoming infected in the hospital. most of the respondents emphasized that the interactions with the colleagues are limited to the strict minimum necessary, keeping the right distances also when discussing patients, and minimizing the number of practitioners involved in the same procedure. from the interviews, multiple challenging aspects in the management of the covid- pandemic in a ped were collected. physicians have to face with the new dimension of performing a physical examination in ppe; they have to be trained and skilled in proper donning/doffing procedures; they have to keep constantly updated with rapidly evolving guidelines and protocols; they have to be focused on the possible sars-cov- infection, but, at the same time, they should not forget differential diagnoses. exploring for the first time the perception of pediatric emergency physicians in one of the most affected countries, this study represents a unique attempt to provide hints to those pediatricians not already critically involved in facing the covid- pandemic across the world. similar insights from chinese colleagues, initially involved in covid- outbreak management, would have been extremely useful at the very onset of the pandemic, to be at least in part prepared in advance to what would expect pediatricians around the world. one of the key points is that the clinical practice changed, and individual attitude was modified, aiming at prioritizing essential interventions and maneuvers; at the expense of routine but nonvital procedures. health-care practitioners should be able to keep updated with the rapidly evolving knowledge and guidelines, and to comply with a way of acting that would probably be new and unfamiliar for most of them, encompassing not only disease epidemiology, ipc protocols, and application of ppe, but also basic principles of disaster medicine, ie, surge capacity and scarce resource allocation, triage, and ethical dilemmas of rationing medical care. another paramount notion is the absolute priority of the safety of the health-care practitioner, not only in an individual perspective but also in the community interest. indeed, physicians not protecting themselves from the infection would turn out to be harmful for the global health-care system, both because they could not be able to work if they get sick and, probably even more, because they may spread the infection if they are asymptomatic and keep having contacts with patients and colleagues. the crucial role of proper ppe use is actually highlighted by ad-hoc covid- pediatric resuscitation algorithms, released by the american heart association, having the donning of ppe as the first point. in this perspective, pediatricians should consider each patient as a potentially covid- -positive patient but should be able not to fixate only on covid- disease, always bearing in mind more common differential diagnoses. underneath the finally, in countries facing with covid- pandemic, it is likely that a substantial reduction in the amount of access to the ped will be seen, similarly to our experience and to that reported in other italian centers, as a result of both the reticence on the part of the parents and caregivers to risk exposure to sars-cov- in a hospital setting, and the lower rates of acute infections in this period. this could result in a detrimental delayed provision of care for critically ill children. lazzerini et al. reported cases admitted to intensive care unit and deaths related to delayed access to care, in a small series of reports. on the other hand, the dramatic reduction in ped visits should provide food-for-thought on the actual appropriateness of a large part of the ordinary use of the ped to manage not urgent/emergent needs. it is advisable that it become a matter of debate for health-care policies. this work is not intended to recommend a code of conduct but just to apprise other pediatric health-care providers not already deeply involved in the management of covid- pandemic of what they would probably face in the next future. we think that the main strength of this work is sharing real-life experiences of front-line practitioners with their peers in countries not already deeply facing the pandemic, offering insights to be to some extent prepared in advance to a completely new and unknown situation. however, some limitations should be pointed out. first, the crisis pediatricians were facing in italy at that time prevented us from a strict methodological structure and sample size, thus, not allowing us to provide evidence-based data. therefore, it was just for documentation. second, the interviews were not audio-recorded, preventing us to report unedited narratives. finally, the fact that the interviewer was also a physician who worked with the target population in the same hospital may represent both an advantage and a disadvantage. on the one hand, it could be an advantage because it would allow a real empathetic listening of the colleagues' opinions during the interview and the possibility to easily keep in touch with them regardless of the complexity of the covid- period. on the other hand, it could represent a disadvantage possibly preventing an objective and unemotional interpretation of the responses, but this limitation was overcome by the other authors' contribution in reviewing and integrating the written transcripts of the interviews. in conclusion, pediatricians directly experiencing the effects of covid- pandemic in their ped have radically changed their clinical practice, aiming at prioritizing essential interventions and maneuvers and self-protection. their point of view could help their peers around the world to be prepared to face this unique situation, just trying to answer what they are wondering. translating covid- pandemic surge theory to practice in the emergency department: how to expand structure systematic review of covid- in children shows milder cases and a better prognosis than adults coronavirus disease and children: what pediatric health care clinicians need to know italian doctors call for protecting healthcare workers and boosting community surveillance during covid- outbreak assessment of medical reserve corps volunteers' emergency response willingness using a threat-and efficacy-based model qualitative inquiry and research design: choosing among five approaches coronavirus (covid- ): resources for cpr training & resuscitation -resources for healthcare providers delayed access or provision of care in italy resulting from fear of covid- we thank the staff of the pediatric emergency department of the maggiore della carità university hospital (novara, italy). the authors have no conflicts of interest to declare. key: cord- - ueqx v authors: mauri, eleonora; abati, elena; musumeci, olimpia; rodolico, carmelo; d’angelo, maria grazia; mirabella, massimiliano; lucchini, matteo; bello, luca; pegoraro, elena; maggi, lorenzo; manneschi, letizia; gemelli, chiara; grandis, marina; zuppa, angela; massucco, sara; benedetti, luana; caponnetto, claudia; schenone, angelo; prelle, alessandro; previtali, stefano c.; scarlato, marina; d’amico, adele; bertini, enrico; pennisi, elena m.; de giglio, laura; pane, marika; mercuri, eugenio; mongini, tiziana; ricci, federica; berardinelli, angela; astrea, guja; lenzi, sara; battini, roberta; ricci, giulia; torri, francesca; siciliano, gabriele; santorelli, filippo m.; ariatti, alessandra; filosto, massimiliano; passamano, luigia; politano, luisa; scutifero, marianna; tonin, paola; fossati, barbara; panicucci, chiara; bruno, claudio; ravaglia, sabrina; monforte, mauro; tasca, giorgio; ricci, enzo; petrucci, antonio; santoro, lucio; ruggiero, lucia; barp, andrea; albamonte, emilio; sansone, valeria; gagliardi, delia; costamagna, gianluca; govoni, alessandra; magri, francesca; brusa, roberta; velardo, daniele; meneri, megi; sciacco, monica; corti, stefania; bresolin, nereo; moroni, isabella; messina, sonia; di muzio, antonio; nigro, vincenzo; liguori, rocco; antonini, giovanni; toscano, antonio; minetti, carlo; comi, giacomo pietro title: estimating the impact of covid- pandemic on services provided by italian neuromuscular centers: an italian association of myology survey of the acute phase date: - - journal: acta myol doi: . / - - sha: doc_id: cord_uid: ueqx v introduction: since february , the outbreak of covid- in italy has forced the health care system to undergo profound rearrangements in its services and facilities, especially in the worst-hit areas in northern italy. in this setting, inpatient and outpatient services had to rethink and reorganize their activities to meet the needs of patients during the “lockdown”. the italian association of myology developed a survey to estimate the impact of these changes on patients affected by neuromuscular disorders and on specialized neuromuscular centers during the acute phase of covid- pandemic. methods: we developed an electronic survey that was sent to neuromuscular centers affiliated with the italian association of myology, assessing changes in pharmacological therapies provision, outpatient clinical and instrumental services, support services (physiotherapy, nursing care, psychological support) and clinical trials. results: % of surveyed neuromuscular centers reported a reduction in outpatient visit and examinations ( . % of centers in northern regions; % of centers in central regions; % of centers in southern regions). twenty-two% of centers postponed in-hospital administration of therapies for neuromuscular diseases ( . % in northern regions; . % in central regions; % in southern regions). diagnostic and support services (physiotherapy, nursing care, psychological support) were suspended in % of centers ( / / % in northern, central and southern centers respectively) overall, the most affected services were rehabilitative services and on-site outpatient visits, which were suspended in % of centers. strategies adopted by neuromuscular centers to overcome these changes included maintaining urgent on-site visits, addressing patients to available services and promoting remote contact and telemedicine. conclusions: overall, covid- pandemic resulted in a significant disruption of clinical and support services for patients with neuromuscular diseases. despite the efforts to provide telemedicine consults to patients, this option could be promoted and improved further. a close collaboration between the different neuromuscular centers and service providers as well as further implementation of telehealth platforms are necessary to ensure quality care to nmd patients in the near future and in case of recurrent pandemic waves. italy has been one of the first countries, after china, facing cases of local interhuman transmissions of severe acute respiratory syndrome-coronavirus- (sars-cov- infection) worldwide , . the first confirmed case of coronavirus disease- (covid- ) was reported in italy on the st of february . since then, more than . individuals across italy became infected . to assist covid- patients, territorial health care services were reorganized and some hospitals were converted into "covid hubs" (appointed by authorities), particularly in the most affected areas. moreover, general population was imposed strict preventative measures to limit unnecessary gatherings and movements. despite national laws and guidelines, regional differences in the management of the crisis exist [ ] [ ] [ ] [ ] [ ] . in this emergency setting, providing services to patients with chronic diseases and disability represented a major issue for the health care system . due to preventive home isolation, fragile patients and their caregivers had to face many problems in terms of diagnosis, therapy, rehabilitation and support. neuromuscular diseases (nmd) may represent a risk factor causing a more severe course and outcome of sars-cov- infection. indeed, several factors may contribute, namely respiratory and cardiac involvement, which are common nmd complications, muscle weakness, disability and, in some cases, dependency from caregivers [ ] [ ] [ ] . the discontinuation of care and treatments can worsen the underlying condition, exacerbate symptoms, and increase anxiety, leading to a vicious circle and increased management concerns [ ] [ ] [ ] [ ] [ ] . health services tried to address these needs by promoting remote patient contacts, referring patients to available nearby services and offering on-site visits only for urgent cases. across the country, specialized nmd centers devoted their efforts and shared their knowledge in order to ensure quality care for nmd patients during these dramatic and unusual months, under the guidance of the italian association of myology (aim). aim associates developed a survey to quantify the extent of changes experienced by nmd centers and patients during these months, and to outline the most effective solutions undertaken, with the ultimate goal of improving nmd patients' care. we designed a cross-sectional study based on a multicentric survey (appendix ). the period considered in the survey runs from february rd to april th , i.e. the most acute phase of the pandemic in italy. thirty-seven referral centers for neuromuscular diseases were enrolled. the questionnaire was sent via e-mail to members of the aim responsible for the enrolled centers. geographical areas were divided into three macro-areas: northern italy, comprising the regions valle d'aosta, piedmont, liguria, lombardy, veneto, trentino, friuli venezia giulia, emilia romagna; central italy, including tuscany, lazio, marche, umbria; southern italy accounting for abruzzi, molise, campania, apulia, calabria, basilicata, sicily and sardinia. the survey included questions addressing how the activity of the center and clinical practice had changed and which services reported the worst difficulties from february rd , to april th , . the questionnaire was divided into five sections: . center and neurologist identification information; . access of patients to standard home pharmacological therapy; . access of patients to hospital-administered pharmacological treatments, including nusinersen, intravenous immunoglobulins (ivig), intravenous corticosteroids, rituximab, enzyme replacement therapy (ert) for pompe disease, plasma exchange (plex), edaravone and others; . management of clinical trials; . management of care-related services such as physiotherapy, home nurse service, psychological support for the patient and the caregiver, prenatal diagnosis. thirty italian neuromuscular centers completed the survey: / centers from northern italy, (response rate -r.r. %); / centers from central italy (r.r. %); / centers from south italy, (r.r. %). four centers were exclusively dedicated to the pediatric population ( in northern italy and in central italy), while assisted both pediatric and adult patients. regional differences in the impact of covid- epidemic emerged, partly as a consequence of the different geographical viral spread. the impact on activities and services provided by neuromuscular center on the national territory is summarized in table i . from february th , , a marked reduction in both outpatient visits and exams was observed in all italian neuromuscular centers. only . % of centers all over the country continued to provide in-hospital visits and . % of them limited the visits to medical emergencies and urgencies (fig. ) . however, neuromuscular specialists were generally available on-call for neurological emergencies and a substantial part of the planned visits were replaced by remote telephone contact. in northern italy, the worst-hit area, . % of centers were not able to provide outpatient visits and instrumental procedures, while in central and southern italy outpatient visits were not performed in and % of centers respectively. cancelled visits were delayed or replaced with remote contact in the same proportion. the % of pediatric-dedicated centers guaranteed outpatients visits and exams. overall therapeutic pharmacological provisions (chronic pharmacological treatments, in-hospital administered therapies, experimental therapies) were guaranteed in % of interviewed centers (fig. ) . chronic outpatient treatments remained available for the majority of patients ( . % of italian centers and the totality of the exclusively-pediatric centers). neuromuscular patients in regular clinical practice often refer to specialized centers for day-hospital (dh) treatments, particularly for intravenous drug administration. in this regard, % of centers provided in-hospital therapeutic administration without changes and % of centers with minor changes in northern italy. in central and southern italy this service was mostly provided without major interruptions, but some difficulties were reported by and % of centers in central and southern regions respectively. we analyzed effectively performed dh treatments. pulse high dose intravenous corticosteroids were administered in % of scheduled patients, while intravenous immunoglobulins (ivig) administrations were provided in % of planned patients. conversely, the monoclonal antibody rituximab was administered only in % of expected cases. referral centers for nusinersen succeeded in % of scheduled administrations. in centers providing these treatments, edaravone was administered in % of programmed patients, while ert was maintained in % of patients with pompe disease. conversely, plasma exchange was performed in a higher number of patients than expected (fig. ) . the most relevant changes in treatment administration in this period were those for myasthenia gravis, followed by those affected with neuropathies, spinal muscular atrophy (sma), amyotrophic lateral sclerosis (als), inflammatory and metabolic myopathies. two centers referred their patients (with a diagnosis of myasthenia gravis, als, sma and pompe disease) to other institutions for the prosecution of treatment. the reasons why patients did not receive in-hospital therapy administration were mainly related to personal decisions, e.g. fear of the contagion. in most cases, pharmacological in-hospital treatments were postponed or rearranged, while, in few cases, drug administration was temporarily interrupted due to the high risk for the patient to develop serious covid- complications in case of infection. clinical trials continued with scheduled visits and exams in % of centers, with a minor percentage of inter- ruptions in the regions of central italy. reasons for interruption were logistic problems, safety issues and patients' fear of contagion. experimental trials involving als and pompe disease faced greater difficulties. as regards als patients, % of trial-related visits were delayed or cancelled for precautionary reasons. patients with pompe disease did not undergo visits and exams in % of cases. conversely, more than % of the centers participating in clinical trials for other neuromuscular diseases, including myasthenia gravis, inflammatory myopathies and neuropathies, dmd and sma, performed the scheduled visits and exams with only minor lags. particularly, visits and exams scheduled for clinical trials were conducted in the . % of pediatric-dedicated centers. services provided by neuromuscular centers, such as rehabilitation, home care nursing, psychological support and prenatal diagnosis, faced a reduction in % of centers, showing higher reduction rates in geographical areas with higher numbers of sars-cov- cases (aggregate services reduction in % of northern nmd centers, % of central centers and % of southern centers) (fig. ) . the most affected service was rehabilitation, since physical therapy sessions were suspended in % of centers, and postponed or delayed in a minor number of centers due to patient-related travel problems. indeed, only % of surveyed centers reported that physical therapy sessions continued without variations (all northern centers faced the interruption of sessions, while . % of central centers and % of southern centers maintained the service). despite some difficulties, psychological support to families was provided in . % of centers upon request. home nursing care service was not provided in % of centers, with a higher reduction rate in northern italy regions, where only % of expected patients obtained regular assistance. also, session duration was reduced. the suspension was mainly determined by a choice of the families to protect their relatives from external contacts. services associated to prenatal diagnosis remained accessible in . % of centers providing this service, in some cases with minor delays. fifty% of pediatric-dedicated centers reduced physiotherapy and home care nursing, while no center reported any defection in psychological support and prenatal diagnosis services. during the first wave of covid- pandemic, italian health care system had to rapidly and heavily reorganize inpatient and outpatient services, particularly in the worst-hit areas , , , . in this setting, specialized nmd centers were no exception. under the guidance of aim, they tried to address the needs of fragile nmd patients and to maintain essential services. however, despite these common intents, many nmd patients suffered from home isolation and temporary interruption of a part of outpatient visits and exams, thus struggling to receive standard therapies and rehabilitative sessions and facing the risk of worsening of their disease or exacerbation of symptoms , . with this survey, aim aimed to assess the impact of sars-cov- pandemic on nmd centers and nmd patients' care in italy. we found that changes related to the pandemic partly followed the regional burden of infections across italy. centers in northern italy experienced a heavier reduction of inpatient therapy administration and ancillary services if compared to nmd centers in central and southern italy. on the other hand, outpatient neurological visits and exams suffered from a marked reduction in southern regions. concerning these results, however, we acknowledge the possibility of a sampling bias, since centers from southern regions were less represented. from the survey, it emerged that most centers ensured in-hospital visits for urgent cases and tried to limit the worsening of nmd diseases through remote contact advice. provision of chronic pharmacological therapy was unaffected, whereas many centers faced difficulties in the administration of in-hospital therapies. particularly, rituximab and ivig were the most challenging to deliver appropriately, and patients who suffered the greatest change in in-hospital treatment delivery were myasthenia gravis patients. indeed, patients with auto-immune and inflammatory nmd receiving immunosuppressive treatment generally present a higher risk of complications from infectious diseases , . thus, treatments might have been cancelled in order to let these patients avoid unnecessary travels. furthermore, the pandemic partially affected the regular course of clinical trials as well. scheduled visits and exams were performed without major problems and with the necessary precautions only in % of centers. ancillary services such as rehabilitation, home care nursing and psychological support were markedly affected nation-wide. due to the additional risk of infection from close patient-therapist contact , rehabilitation services were generally suspended. remote physiotherapy was not provided, possibly because of the difficulties experienced by disabled patients and their caregivers in conducting physical sessions without support. home nursing care was provided in a higher number of cases as compared to physical therapy, despite the risks deriving from close contact with operators being similar. psychological support for patients and caregiver was generally available, since it could be provided through remote contact. finally, services related to prenatal diagnosis were accessible in most centers. exclusively-pediatric centers deserve a further comment, since their activity resulted less affected and services were generally maintained throughout the pandemic; however, rehabilitative activities requiring personal contact were reduced and extra-regional travels were avoided. this is partly due to an effort of the health care system to guarantee assistance to little fragile patients. moreover, we hypothesize that buildings and spaces dedicated to pediatric population was less involved in the conversion to assist covid- patients. this cross-sectional, multicentric study was the first to specifically address the issues experienced by nmd patients in our country and to provide a picture of the pandemic burden for nmd specialists in different regions. limitations of the study include the fact that we depicted the pandemic burden during a reduced time span, in a rapidly developing context in terms of preventative measures, organizational settings and law framework. a further limitation consists in the different restrictive measures applied regionally that may have led to a heterogeneous impact on the access to care for nmd patients. in addition to the abovementioned sampling bias, methodological limitations associated to the survey itself could be the adhesion bias, as centers volunteering for the study might not represent a uniform sample, and the referral bias, as centers which encountered problems might have been more prone to answer promptly. on the other hand, centers heavily involved in the emergency might have experienced difficulties in answering the survey due to lack of time. nonetheless, surveys are an effective tool to rapidly gather information without direct in-person contacts. in addition, our survey reached a consistent part of nmd centers across italy. overall, covid- pandemic caused a public health crisis with a potentially severe impact on the most fragile part of our society -including many nmd patients. in this context, neuromuscular centers played a pivotal role in ensuring an adequate support and care to these patients, and to reach this aim they had to rapidly reorganize their services. some of the strategies and innovations that were experimented, such as telemedicine, could prove useful in the nearby future, after the acute phase of the pandemic , and deserve diffusion and standardization for their use in clinical practice. surveys, at-distance meetings and virtual platforms will likely be valuable tools to help addressing the future concerns and challenges of nmd centers, aiming at maintaining the best standards of care for nmd patients in these difficult times. the survey has been performed in accordance with the ethical standards laid down in the declaration of helsinki and its later amendments. no targeted funding reported. survey on the impact of the covid- pandemic on the management of neuromuscular patients from / / - / / of abbreviations aim: italian association of myology als: amyotrophic lateral sclerosis covid- : corona virus disease dh: day hospital dmd: duchenne muscular dystrophy ert: enzyme replacement therapy ivig: intravenous immunoglobulins nmds: neuromuscular diseases plex: plasma exchange sars: severe acute respiratory syndrome sma: spinal muscular atrophy references who. coronavirus disease (covid- ) situation report clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study covid- bergamo hospital crisis unit. adaptations and lessons in the province of bergamo italian doctors call for protecting healthcare workers and boosting community surveillance during covid- outbreak balancing evidence and frontline experience in the early phases of the covid- pandemic: current position of the italian society of anti-infective therapy (sita) and the italian society of pulmonology (sip) sars-cov- invades the west. how to face a covid- epidemic in lombardy delayed access or provision of care in italy resulting from fear of covid- covid- pandemic and the crisis of health systems: the experience of the apulia cancer network and of the comprehensive cancer center istituto tumori "giovanni paolo ii" of bari covid- and neuromuscular disorders the care of patients with duchenne, becker, and other muscular dystrophies in the covid- pandemic spinal muscular atrophy care in the covid- pandemic era covid- and people with neuromuscular disorders: world muscle society position and advice. executive board of the wms th update www guidance for the management of myasthenia gravis (mg) and lambert-eaton myasthenic syndrome (lems) during the covid- pandemic factors associated with acute exacerbations of myasthenia gravis mental health in the coronavirus disease emergency -the italian response health risks and potential remedies during prolonged lockdowns for coronavirus disease (covid- ) the response of milan's emergency medical system to the covid- outbreak in italy at the epicenter of the covid- pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation. nejm catalyst innovations in care delivery a preliminary evaluation of the public risk perception related to the covid- health emergency in italy case-fatality rate and characteristics of patients dying in relation to covid- in italy impact of covid- outbreak on rehabilitation services and physical and rehabilitation medicine (prm) physicians' activities in italy. an official document of the italian prm society (simfer) coding in the world of covid- : non-face-to-face evaluation and management care of neuromuscular and neurodegenerative disorders, "bambino gesù" children's hospital, irccs, rome, italy; center for neuromuscular and rare neurological diseases, unit of neurology, "s. filippo neri" hospital, rome, italy; key: cord- -j kw a x authors: boccia, stefania; cascini, fidelia; mckee, martin; ricciardi, walter title: how the italian nhs is fighting against the covid- emergency date: - - journal: front public health doi: . /fpubh. . sha: doc_id: cord_uid: j kw a x nan italy, with more than , cases as of april nd ( ) has the second highest burden of coronavirus disease in europe after spain, and the third highest worldwide. the speed with which the epidemic grew took all concerned by surprise ( ) . within a week of the first case being identified in codogno, lombardy, the number had grown to , with deaths. this placed the local health services under exceptional pressure and, as in spain ( ), created tensions within the decentralized italian health system. italy comprises regions, with differing levels of autonomy. the italian prime minister threatened to take back powers from the regions and autonomous provinces as they were "in charge of implementing healthcare but not prepared to face a national emergency" ( ). the national response came in the form of a series of seven decrees from the presidency of the council of ministers (in effect the prime minister's office) progressively extending countermeasures. after the first declaration of emergency of january st, a decree (february rd) isolated cities with covid- clusters within the northern italian regions (lombardy and veneto) ( table ) . the following deecrees adopted further restrictions, closing schools and universities, prohibiting all public events, such as concerts and major sports competitions, and limiting business hours. the last three decrees imposed restrictions on mobility of the population. early on, several towns had introduced varying forms of quarantine, but further clusters continued to emerge. as a consequence, the new decrees extended restrictions from the region of lombardy to all of northern italy and, by march th, to the entire country (figure ) . detailed surveillance is being conducted by a task force in the department of infectious diseases of the instituto superiore di sanità ( ) . according to the latest available reports, threequarters ( . %) of cases were over years of age and only . % were aged years or younger, with . % between and years old. the majority ( . %) were men, the same as in early reports from china ( ) . healthcare workers represented . % of the reported cases, and among them lethality was . %. as of april nd, nearly half of all cases were diagnosed in lombardy ( , ), followed by emilia romagna ( , cases) and piemonte ( , cases) (supplementary table s ). with clinical data available for , cases, most ( . %) were classified as having mild pneumonia but . % were severe (dyspnoea, respiratory rate ≥ /min, blood oxygen saturation ≤ %), and . % were critical (respiratory failure, septic shock, and/or multiple organ dysfunction or failure), while % had few or no symptoms. obviously, international comparisons of case fatality must be interpreted with caution due to differences in the intensity of testing and, with deaths, the criteria for establishing the underlying cause. at present it appears that all deaths in someone who has tested positive for covid- are attributed to the virus and this may, and probably is not the case everywhere. by april, nd, where we had , deaths, giving a case fatality rate of . %. this is higher than has been reported in many other countries but is likely to be explained, at least in part, by the age distribution. the median age of cases in italy is years, compared to in china ( ) . however, the median age of those dying in italy is years. again, noting the need for caution because of issues with denominators, there was a clear association between age and outcome. there were no deaths among those aged under years old, but the case fatality rate was . % in those aged to , increasing to . % in those years and older. outcomes were also strongly associated underlying conditions: . % of deaths were among people with or more comorbidities, . % had two, . % had one, and only deaths ( . %) were of people who had apparently been healthy. suspension to the entire productive chain (unless "essential", e.g. food production and distribution) in the country figure | number of new cases, deaths and total cases due to covid- in italy, from st february to nd april . gray arrows represent the legislative decrees with a regional impact. black once the legislative decrees with national impact. the challenge to the national health service has been immense starting from the red zones in the northern italy. for instance, before the current crisis lombardy had approximately intensive care beds ( . % of all hospital beds in the region) ( ) . in the first days of the epidemic, of them were required to treat patients with covid- ( ) . in these circumstances the national health service has had to innovate. first, separate testing sites were established, and the ministry of health asked general practitioners to refer anyone meeting certain criteria based on their symptoms, to divert them from health facilities facing extreme pressure. second, the ministry of health put in place measures to recruit additional doctors and nurses to increase the capacity of intensive care units (icu). this included an extraordinary plan, launched on march th, to employ medical students and retired healthcare professionals. meanwhile, on march th, e million was allocated for additional medical devices and equipment ( ) . unfortunately, these measures have been implemented against a backdrop of the loss of many health care workers who have been quarantined or fallen ill with the infection, some of whom, tragically, have died. the approaches taken by the italian health system to the covid- emergency have varied among the most severely affected regions fall into three broad types ( ). type is a hospital based model, adopted in lombardy. type is a territorial based model, in veneto. type is a combined hospitalterritorial model, as in emilia-romagna and piedmont. the first type places the main emphasis on the role of hospitals, with a relatively low level of community testing. this has, as might be expected, been associated with substantial pressure on hospitals and, particularly, icu beds. an average of % of those diagnosed with covid- have been admitted to hospital in lombardy (vs. an average of % in other regions). although this seems a small difference, the duration of stay in icus means that, at any one time, he ratio of patients treated in icus to those treated at home is twice as high in lombardy than in veneto, emilia romagna and piedmont. this also means that daily occupancy of icu beds has been exceeding %, in contrast to emilia-romagna, the second most severely affected region, where occupancy is % ( ). the territorial management approach is characterized by a lower hospitalization rate and a higher incidence of testing. an extreme example is the town of vò, in veneto region, where all , inhabitants were tested ( ) . in veneto, only % of patients with a positive result are hospitalized (compared to the - % of the other italian regions) and nasopharyngeal swabs, which are also administered to asymptomatic individuals, reached . % of the regional population (vs. an average of . % of the other regions) ( ). the combined hospital-territorial management model, adopted in emilia-romagna and piedmont, is characterized by an intermediate level of hospitalization and an intermediate level of testing. in a situation such as the current pandemic, where the optimal course of action is uncertain, italy's decentralized structure has provided an important natural experiment. while there is still much to be learned, the emerging evidence points to the territorial management model being the best response to this emergency. sb, wr, and fc: substantial contributions to the conception and design of the work. mm: critical revision of the manuscript, interpretation of data for the work. available online at what other countries can learn from italy during the covid- pandemic the resilience of the spanish health system against the covid- pandemic covid- : preparedness, decentralisation, and the hunt for patient zero integrated surveillance of covid- in italy clinical characteristics of coronavirus disease in china critical care utilization for the covid- outbreak in lombardy, italy: early experience and forecast during an emergency response covid- , in gazzetta ufficiale il decreto per il potenziamento del ssn we showed mass testing could eradicate the coronavirus we thanks leonardo villani for his support in the elaboration of the figure the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fpubh. . /full#supplementary-material key: cord- -i gy dhj authors: loconsole, daniela; accogli, marisa; de robertis, anna lisa; capozzi, loredana; bianco, angelica; morea, anna; mallamaci, rosanna; quarto, michele; parisi, antonio; chironna, maria title: emerging high-risk st and st carbapenem-resistant klebsiella pneumoniae clones from bloodstream infections in southern italy date: - - journal: ann clin microbiol antimicrob doi: . /s - - -y sha: doc_id: cord_uid: i gy dhj background: carbapenem-resistant klebsiella pneumoniae (cr-kp) is an urgent public health issue in italy. this pattern of resistance is due mainly to dissemination of carbapenemase genes. molecular characterization of carbapenem-resistant klebsiella pneumoniae (cr-kp) strains was performed over a three-year period. in-depth analysis was performed on a subset of emerging cr-kp st and st clones. methods: a prospective study was performed on patients with cr-kp bloodstream infections hospitalized in hospitals located in three large provinces in southern italy. carbapenemase genes were identified via genotyping methods. multi-locus sequence typing (mlst) and whole genome sequencing (wgs) were carried out on st and st isolates. results: among the cr-kp isolates, bla(kpc) was found in . %, bla(vim) was found in . %, bla(ndm) was found in . % and bla(oxa- ) was found in . %. the bla(kpc- ) variant was identified in all characterized kpc-kp isolates. mlst of representative isolates revealed st in . %, st in . % and st in . % of the isolates. cgmlst of st and st isolates revealed presence of more than one beta-lactam resistance gene. amino acid substitution in the chromosomal colistin-resistance gene pmrb was found in two st isolates. conclusions: st is widespread in southern italy, but st and st are emerging since they were found in a significant proportion of cases. aggressive infection control measures and a continuous monitoring of these high-risk clones are necessary to avoid rapid spread of cr-kp, especially in hospital settings. the spread of antimicrobial resistance (amr) was recently identified by the world health organization (who) as a priority threat to human health. in particular, carbapenem-resistant enterobacteriaceae (cre) were included in the most critical group of antibiotic-resistant gram-negative bacteria [ ] , as infections with these organisms show high mortality rates, especially in cases of bloodstream infections (bsis) [ , ] . cre infections, mainly with carbapenem-resistant klebsiella pneumoniae (cr-kp), have become endemic in several countries [ ] . in italy, an abrupt and notable increase in the proportion of cr-kp invasive infections was reported in (from . % in to . % in ), making cr-kp the main antibiotic-resistance threat [ ] [ ] [ ] [ ] . a peak of prevalence was reached in ( . %), but a significant decreasing trend was reported in ( . %) [ ] . nationwide surveillance in the period - showed that . % of the bsis caused by carbapenemase-producing enterobacteriaceae (cpe) were due to kp strains carrying the klebsiella pneumoniae carbapenemase (kpc) enzyme [ ] . metallo-beta-lactamases, including ndm (new delhi metallo-beta-lactamase) and vim (verona integron-encoded metallo-beta-lactamase) enzymes, were identified in . % of the bsis, whereas oxa- (carbapenem-hydrolysing oxacillinase- ) was found in . % of the cases. kp strains coproducing several classes of carbapenemases were identified in . % of the bsis. among the kpc-kp isolates in italy, the bla kpc- gene was the most prevalent [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in our country, recent studies on the molecular epidemiology of kpc-kp from invasive infections showed an expansion of strains belonging to sequence types (sts) and of the hyperepidemic clonal complex (cc) [ , ] , which was detected for the first time in italy in [ ] . recent epidemiological reports showed that st and st are emerging in several countries; therefore, these sts are considered candidates for becoming the prevalent high-risk cr-kp in the near future [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . genetic features of these clones may contribute to their adaptation to hospital environments and to the human host, both of which result promote further spread of antibiotic resistance [ , ] . in italy, the emerging st and st have been rarely reported in patients with invasive infections to date [ , , , , ] . few data are available on the molecular characteristics of the cr-kp strains involved in bsis in the puglia region in southern italy. recently, calia et al. reported that out of strains ( . %) collected from patients with sepsis in puglia and basilicata harboured the bla kpc- gene and that st was the main sequence type detected in the cr-kp isolates from several clinical samples [ ] . st was reported in five cases, whereas none of the characterized strains belonged to st . in this study, we present the results of molecular characterization of cr-kp strains isolated from hospitalized patients with bsis detected during the -year period from to in three large provinces of the puglia region in southern italy. the observed increasing prevalence of st and st in the study period prompted us to conduct in-depth analyses of the lineage relatedness, resistance determinants, and core and accessory genome contents of these clones via whole-genome sequencing. a prospective study was designed at the university hospital policlinico in bari, italy, after that the italian ministry of health (moh) issued a directive to monitor and control cpe in (moh dgpre n° / / ). the study involved three provinces in the puglia region (bari, brindisi and barletta-andria-trani), which have a population of about million inhabitants, representing half of the overall regional population (source demographic data: istat, ; http://demo.istat .it/pop / index .html). nineteen hospitals were enrolled: eight were located in the province of bari, six in brindisi, and five in barletta-andria-trani. during the - period, the microbiology laboratories of the hospitals involved in the study collected the consecutive non-replicate clinical cr-kp isolates from the blood of patients with bsis hospitalized in intensive care units (icus) or medical or surgical wards. the collected strains included those with intermediate resistance or resistance to imipenem and/or meropenem based on the eucast breakpoints (http://www.eucas t.org/clini calbr eakpo ints/). all of these isolates were then sent to the laboratory of molecular epidemiology and public health of the hygiene unit of the university hospital policlinico of bari for genotypic confirmation of their carbapenem resistance and identification of their carbapenemase genes. further molecular characterization was performed on a representative subset of the isolates. for each hospitalized patient, demographic data (e.g., sex, age, etc.), the name and province of the hospital, the type of ward, the presumptive source of the bsi, and phenotypic resistance data were collected. all of the cr-kp isolates were tested using a commercial multiplex real-time pcr kit (genexpert platform, cepheid, sunnyvale, ca, usa) to identify the following carbapenemase-encoding genes: kpc (bla kpc ), vim (bla vim ), ndm (bla ndm ), imp (bla imp ) and oxa- (bla oxa- ). variant identification was performed on representative kpc-kp isolates from all the hospitals involved and the -years period of the study via pcr, as previously described [ ] , and subsequent sequencing. allelic variants were identified via the blast program on the national center for biotechnology information website (http://blast .ncbi.nlm.nih.gov). multi-locus sequence typing (mlst) of representative cr-kp isolates was performed by amplifying and sequencing internal fragments derived from seven specific housekeeping kp genes (rpob, gapa, mdh, pgi, phoe, infb, and tonb) [ ] . the allelic profiles were compared with those included in the electronic database of the pasteur institute to identify the st (bigsdb.web.pasteur. fr/klebsiella/klebsiella.html). seven cr-kp isolates, previously typed as st (n = ) and st (n = ) were available for wgs, which was performed at the istituto zooprofilattico di puglia e basilicata, foggia (italy) using an illumina miseq (illumina inc., san diego, ca, usa) with a paired-end run of by bp after nextera xt paired-end library preparation. sequencing reads were assembled using spades genome (version . ). the virulence-and amr-associated genes located on both the bacterial chromosome and on mobile genetic elements in the characterized cr-kp isolates were identified by uploading the assembled contigs into the online tool bigsdb-kp (http://bigsd b.paste ur.fr/klebs iella /klebs iella .html). the plasmid incompatibility types were identified via plasmidfinder [ ] . a % identity cut-off was used. for each isolate, a panel of genes, defined as the strict cgmlst set, was identified submitting the draft genomes to the bigsdb-kp (http://bigsd b.paste ur.fr/ klebs iella /klebs iella .html) [ ] . during the -year study period, a total of patients with bsis were included in the study. of these, . % (n = ) were in the icus, . % (n = ) were in the medical wards and . % (n = ) were in surgical wards. the remaining . % (n = ) of the bsis occurred in patients whose ward was not known. the mean age was . years (range: - years), and patients ( . %) were men. among the included cases, the origins of the bsis were distributed as follows: central/peripheral venous catheter infections, . % (n = ); ventilatorassociated pneumonia (vap), . % (n = ); urinary tract infections, . % (n = ); pneumonia, . % (n = ); skin and tissue infections, . % (n = ); abdominal infections, . % (n = ); surgical site infections, . % (n = ). in . % (n = ) of the cases, the bacteraemia was of unknown origin, while data regarding the infection site were missing for . % (n = ) of the cases. all the collected isolates were subjected to detection of carbapenemase genes (table ) . over these years, the following resistance genes were detected in cr-kp isolates at the given frequencies: bla kpc , . % (n = ); bla vim , . % (n = ); bla ndm , . % (n = ); bla oxa- , . % (n = ). the bla imp gene was not detected. carriage of multiple carbapenemase genes was reported in four isolates ( bla kpc + bla vim and bla ndm + bla oxa- ). of the three bla kpc + bla vim strains, two were isolated from male patients of and years, who were hospitalized in the icu of the same hospital (province of bari) in june and july . in both cases, the origin of infection was vap. the third bla kpc + bla vim strain was isolated in an -year-old female hospitalized in in a medical ward of another hospital in the province of bari, and her bacteraemia originated from a urinary tract infection. the bla ndm + bla oxa- isolate was identified in a -year-old male hospitalized in an icu in in the province of brindisi, and his bacteraemia was of unknown origin. the bla kpc variant was identified in kpc-kp isolates. in particular, / ( %) isolates collected in , / ( %) in and / ( %) in were subjected to variant detection. all of the strains carried the bla kpc- variant. mlst was performed on isolates ( table ). in the . % of the characterized isolates, st and st , which belong to cc , were identified. of the isolates, . % and . % belonged to st and st , respectively. significant proportions of st and st strains were observed over the study period. peaks for st and st were reported in ( . % and . %, respectively). other sts were reported in . % of the isolates. wgs was performed on five st and two st isolates. based on eucast guideline, all of these strains were multidrug-resistant. a phylogenetic tree using cgmlst is shown in fig. . the isolates of the present study were compared with other representative st and st present in the ncbi pathogen detection database (https ://www.ncbi.nlm.nih.gov/patho gens/). very high similarity was observed among the st strains, whereas, st strains identified in puglia region were phylogenetically more distant from the strains identified in italy and in other countries. among the isolates identified in puglia region, all the st carried the kpc- variant, whereas the five st showed the vim- variant in two cases and the kpc- variant in three cases. the classes of antimicrobial resistance genes and the plasmid replicons identified in the characterized isolates (i.e., a resistome analysis) are shown in table . all of the characterized isolates carried multiple beta-lactam resistance genes. a plasmid replicon analysis revealed the presence of several plasmid replicon-types among the characterized isolates. the highest number of plasmid replicon-types was found in the st isolates (n = ; inc types and col types). the most frequently identified plasmid replicon-types were incfii(k) (n = isolates), incfib(k) (n = isolates), incr (n = isolates) and the small colrnai plasmid (n = isolates). in addition, plasmid types incfib, incfii, incq , and col ii were exclusively detected in the st isolates, while plasmid types incfib(k), incfib(mar), incfib(pqil) and incn were detected only in st isolates. none of the isolates carried any plasmid-borne mcr genes that confer colistin resistance; however, amino acid substitution l m in the colistin resistance-conferring chromosomal pmrb gene was identified in the two st isolates ( br and br) (reference strain: cga_ . in genbank), which were also phenotypically resistant to colistin (minimal inhibitory concentrations (mics) > mg/l for both isolates). table provides the virulence and heavy metal resistance genes identified in the characterized isolates. all but one isolate ( br, belonging to st ) harboured the pcoabcders and silcers heavy metal resistance genes. isolate br carried the pbra heavy metal resistance gene, which was not present in any other isolates, while only isolate acq carried the tercz genes. sequencing of the capsular loci showed that all the st isolates carried the wzi allele, while the st isolates carried the wzi alleles. during the study period, almost all of the cr-kp strains isolated from the patients with bsis in the puglia region (southern italy) carried the bla kpc gene ( %), confirming that the production of the kpc-type carbapenemase was the most common carbapenem-resistance mechanism, as previously reported in italy [ , ] and southern italy [ ] . in our study, the kpc- variant was found in all of the analysed kpc-kp isolates, and this is consistent with the finding that this variant is the most prevalent in other kp surveillance studies in italy [ , , , , ] . we found a higher proportion of bla vim and bla ndm genes ( . %) in the bsis compared with the proportion reported by iacchini et al. ( . %) for italian isolates [ ] . calia et al. identified the bla vim gene in . % of cr-kp isolates. however, the data reported in the latter study, which was conducted in the puglia and basilicata regions (southern italy), included strains isolated from any infection site and not bsis specifically [ ] . moreover, we found one isolate carrying the bla ndm gene, whereas no ndm-kp was reported by calia et al. [ ] . carriage of multiple carbapenemase genes is rarely reported [ ] , and our study followed this trend ( . %). molecular characterization of such isolates could be of particular interest. unfortunately, the four isolates carrying multiple carbapenem-resistance genes included in our study were not characterized because they were not available for wgs. almost half of the cr-kp isolates included in the present study belonged to st . however, over the study period, we found an increasing proportion in bsis with st and st strains, which accounted for approximately % and % of all the characterized isolates, respectively. these sts have been rarely reported in italy, except for in the report of bonura et al. and in fasciana et al. in which prevalence of % ( / ) and of % ( / ) were reported for st , respectively [ , ] . nevertheless, these data included cr-kp strains isolated from any site of infection or colonization and not bsis specifically. furthermore, calia et al. found only five isolates belonging to st ( . %) [ ] , which is substantially lower than the prevalence of this st in the present study. in milan (lombardy), st was found in . % of isolates [ ] , whereas no strains belonging to this st were found in sassari (sardinia) [ ] . these data regarding the spread of st in italy seem to suggest that this st is replacing the predominant hyperendemic sts belonging to cc [ ] . st is an emerging nosocomial high-risk clone associated with infections with increased mortality compared with non-st infections [ ] . moreover, st is a high-risk for mediating further spread of carbapenem resistance [ , ] . in italy, this st has generally been reported as sporadic [ , , , , , ] , with higher prevalence when the studied strains were isolated from any infection site [ , , ] . we found that approximately % of the characterized strains belonged to st , while no isolates belonging to this st were identified in the report of calia et al. in southern italy [ ] . all of the st isolates identified : in the present study were kpc- -producing strains, whereas other italian studies reported st isolates that only produced the kpc- variant [ , , ] . our resistome analysis showed that all of the characterized isolates harboured genes conferring resistance to aminoglycosides, beta-lactams, and fluoroquinolones. in particular, the st isolates carried a significantly higher number of resistance genes compared to the numbers in the st isolates. however, no clear correlation between genetic background and phenotypic resistance in these strains could be established [ ] . in the present study, three out of five st isolates ( pol, pol, and acq) carried bla ctx-m- , bla shv- and bla kpc- . this pattern has been previously reported in italy [ , , ] . furthermore, the three isolates that carried bla ctx-m- , bla shv- and bla kpc- showed the same allelic profile for efflux pump genes, which differed from that identified in the st isolates. the analysis of virulence genes showed that the two st isolates seemed to be potentially extremely virulent strains, as they carried the siderophore gene (irp ), the ferric uptake system (kfuabc), the yersiniabactin siderophore cluster genes (ybtaepqstux), and the mannose-resistant klebsiella-like (type iii) fimbriae cluster (mrkabcdfhij). these virulence factors have been previously reported in only one kp-st isolate identified in northern italy in [ ] . all of the isolates characterized in our study harboured at least one heavy metal resistance gene, which, as previously suggested, can contribute to antibiotic-resistance gene dissemination and maintenance [ ] . non-antibiotic compound, such as metals, may contribute to the spread of antibiotic resistance through the co-selection process [ ] . in fact, genes conferring resistance to heavy metals and antibiotics may coexist on the same genetic element (e.g. plasmid), which could promote a further dissemination of the antibiotic-resistance mechanisms [ ] . moreover, a single resistance mechanism, such as an efflux pump, could confer resistance to both antibiotics and metals (crossresistance) [ ] . interestingly, we also identified many plasmids in the characterized isolates, especially incf-type and colrnai plasmids. this finding is consistent with studies reporting that these plasmids are frequently associated with successful dissemination of carbapenemase, ctx-mtype extended-spectrum beta-lactamase, and plasmidmediated quinolone resistance genes, as well as other virulence determinants in enterobacteriaceae [ , ] . when treating infections involving antimicrobial resistance (amr), colistin is the last-resort antibiotic for treatment of patients with enterobacteriaceae infections. colistin resistance associated with plasmid-borne mcr genes in kp has been reported in italy, although rarely [ ] . traditionally, colistin resistance in kp is associated with mutations in chromosomal genes [ ] . among the isolates characterized in our study, two carried the l m substitutions in the chromosomal pmrb gene, although, according to lomonaco et al. [ ] , these mutations seem not to be related to colistin resistance. however, these two isolates showed phenotypic resistance to colistin. finally, our findings regarding the k-antigens were consistent with previously reported results for st isolates carrying the wzi allele, but not for the st isolates, which carried the wzi allele instead of the wzi allele [ ] . this study has some limitations. first, our data did not cover the entire puglia region but only three provinces. nevertheless, the population of the provinces included in this study represents half of the total population of puglia. a future option would be to implement epidemiological and molecular surveillance of cr-kp infections in the entire region. second, wgs analysis was conducted on a limited number of isolates. unfortunately, we could not characterize either the cr-kp strains that carried more than one carbapenem-resistance gene nor all the identified st and st isolates. nevertheless, we believe that our data could provide important information about the hospital-based dissemination of two emerging highrisk clones, which have, to date, been rarely described in italy. these clones have characteristics that might lead to increased fitness, persistence in and adaptation to hospital environments and, consequently, an ability to supplant the most frequently reported cc clones. whole-genome sequencing is a promising surveillance tool that will be useful for early identification and characterization of both existing and emerging amr clones, especially in hospital settings where antibiotic-resistant pathogens pose a great challenge to clinicians due to limited available treatment options. in order to face the antimicrobial resistance, italy adopted its first national action plan on antimicrobial resistance (pncar - ) [ ] , which represents the tool for implementing actions aiming at reducing the amr at national, regional and local level. the objectives set by the plan included the monitoring of the antibiotic use and the implementation of prevention and infection control measures. according to the national plan, puglia region adopted measures of infection prevention and control, aimed at minimizing the transmission and the spread of cr-kp in hospitalized patients. among these measures, training courses and audit aimed at improving the awareness of health care professionals on amr were planned. moreover, a routine screening of patients admitted to hospitals, standardized procedures for management of colonized patients, and an active surveillance of bloodstream infections were implemented. the results of the present study underline the importance to further implement infection control measures in healthcare settings. in conclusion, st and st clones are emerging in southern italy since they were found in a significant proportion of cases of sepsis. whole-genome sequencing of isolates revealed the presence of more than one beta-lactam resistance gene. the analysis of virulence genes showed that these clones seemed to be potentially extremely virulent strains. to avoid rapid spread of cr-kp strains, especially in hospital settings, aggressive infection control measures and continuous monitoring for high-risk and potentially high-risk clones as well as effective antimicrobial stewardship strategies are necessary. world health organization predictors of mortality in bloodstream infections caused by klebsiella pneumoniae carbapenemase-producing k pneumoniae: importance of combination therapy attributable mortality rate for carbapenem-resistant klebsiella pneumoniae bacteremia annual report of the european antimicrobial resistance surveillance network (ears-net) european centre for disease prevention and control (ecdc) stockholm: ecdc european centre for disease prevention and control (ecdc) stockholm: ecdc epidemic diffusion of kpc carbapenemase-producing klebsiella pneumoniae in italy: results of the first countrywide survey carbapenem non-susceptible klebsiella 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colistin-resistant kpc carbapenemase-producing klebsiella pneumoniae strain of sequence type molecular mechanisms of polymyxin resistance: knowns and unknowns publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the we are greatly indebted to cinzia germinario, valerio giannico and jolanda prisco for invaluable help in collecting data on carbapenem-resistant klebsiella pneumoniae infections. this study was supported by the apulian regional observatory for epidemiology. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. this study was approved by the apulian regional observatory for epidemiology (aoo / ago /n. ) and was conducted according to the principles of the declaration of helsinki. written informed consent was obtained from all individuals who provided specimens or from their legal guardians when applicable. not applicable. the authors declare that they have no competing interests. key: cord- -ax n ss authors: semeraro, federico; scquizzato, tommaso; scapigliati, andrea; ristagno, giuseppe; gamberini, lorenzo; tartaglione, marco; dell’arciprete, oscar; mora, fabio; cordenons, fiorella; del giudice, donatella; picoco, cosimo; gordini, giovanni title: new early warning score: off-label approach for covid- outbreak patient deterioration in the community date: - - journal: resuscitation doi: . /j.resuscitation. . . sha: doc_id: cord_uid: ax n ss nan the spread of the severe acute respiratory syndrome coronavirus (sars-cov- ) emerged in china at the end of is challenging most health systems worldwide . in italy, the first "wave" hit lombardia on february , , with such a high proportion of patients to impact substantially both admission to emergency department and intensive care treatment, and arising new concerns . the italian ministry of health and the national health service strongly recommended to all citizen to reduce access to healthcare facilities (e.g. emergency departments, ambulatory and elective surgery) for non-urgent reasons. government also imposed several measures to contain the outbreak (e.g. closing of schools, limited access to public places, social isolation and preventive public health interventions). on april th , at about forty eight days from the beginning of outbreak in italy, the total number of cases have been . , with . positive tests, and . discharged in healthy condition . unfortunately, the amount of death cases ( . ) has claimed italy at the first place worldwide since the beginning of the outbreak. the emilia romagna is the second most affected italian region after lombardia. this condition leaded the local infectious disease task force to plan a change of paradigma basing prevention actions directly at home of the patients with infection. the aim is to assess patients at home to allow prescriptions of potential effective therapies (e.g. antiviral and chloroquine) when possible or intercepting those in borderline or warning conditions. accordingly with this new approach, a research project was started on voluntary bases by a multidisciplinary teams (e.g. healthcare professionals, software developers, engineers, social manager etc.) with the aim to develop a web app based system to monitor deterioration of patients directly at home. the idea is based on the national early warning score (news) applied to a home context: by the use of an experimental app for smartphones, the in-built features of the device can measure directly parameters included in news (e.g. accelerometer for respiratory rate and photo camera for heart rate measurements). this news "off-label" approach in community setting with "homemade" measurements could facilitate self-calculation of the score, thus helping pre evaluation by healthcare professionals and recognising deterioration of patients at risk especially in conditions of overwhelming requests as during a pandemic. the hypothesis of early interception of deterioration could be useful to prevent potential increase in mortality that unfortunately did happen last month in italy. we created a free open source platform called covidup ad hoc for citizens to increase awareness about potential deterioration in case of covid- infection. we invited all the international health system researchers to allocate time and resources on this innovative approach for potential "off label" application of news in covid- patients. global incidences of out-of-hospital cardiac arrest and survival rates: systematic review of prospective studies fast reshaping of intensive care unit facilities in a large metropolitan civil protection department covid- italy monitoring key: cord- -znmpheia authors: simione, luca; gnagnarella, camilla title: differences between health workers and general population in risk perception, behaviors, and psychological distress related to covid- spread in italy date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: znmpheia in this study, we investigated the perception of risk and the worries about covid- infection in both healthcare workers and the general population in italy. we studied the difference in risk perception in these two groups and how this related to demographic variables and psychological factors such as stress, anxiety, and death anxiety. to this aim, we administered an online questionnaire about covid- together with other questionnaires assessing the psychological condition of participants. first, we found that the exposition to infection risk, due to living area or job, increased the perceived stress and anxiety (i.e., medical staff in north italy was more stressed and anxious with respect to both medical- and non-medical participants from center and south italy). then, we conducted hierarchical logistic regression models on our data to assess the response odds ratio relatively to each regressor on each dependent variable. we found that health workers reported higher risk perception, level of worry, and knowledge as related to covid- infection compared to the general population. psychological state, sex, and living area were less related to these factors. instead, judgments about behaviors and containment rules were more linked to demographics, such as sex. we discussed these results in the light of risk factors for psychological distress and possible interventions to meet the psychological needs of healthcare workers. on december , , some cases of pneumonia of unknown etiology have emerged in the hubei region of china. then, on january , , the causative agent has been identified by means of oropharyngeal swabs, i.e., a virus belonging to the coronaviridae family called sars-cov- (severe acute respiratory syndrome coronavirus ). this new coronavirus was responsible of the respiratory syndrome called , ). most patients with positive swab test developed only minor symptoms, such as fever, dry cough, and pharyngitis, with a benign evolution and spontaneous resolution of the clinical picture. however, some patients developed severe complications, such as interstitial pneumoniae with acute respiratory distress syndrome, pulmonary edema, multiorgan failure, septic shock, and even death (sohrabi et al., ) . patients at risk were especially males, aged older than years, suffering from cardiovascular comorbidities (e.g., arterial hypertension, diabetes, and chronic coronary artery disease), and affected by chronic pneumopathies or cancer (world health organization [who] , ; zhou et al., ) . in the following month, the disease spread to other countries outside china, including italy, where the first positive cases were found on february , (spina et al., ) . italy experienced a significant increase in new cases, mostly in the month of march, in particular in the north regions, and this caused in turn a growing alarm throughout the italian medical-hospital sector due to the imbalance between the resources of the national health system (indicated as ssn, i.e., "sistema sanitario nazionale") and the expected need for treatment required by forecasts on the virus spread. this concern was publicly expressed in the guidelines published by the italian society of anesthesia, analgesia, and intensive care (named siaarti, i.e., "società italiana anestesia, analgesia, rianimazione e terapia intensiva") on march , , which reported that in case of huge imbalance between the population clinical needs and the effective availability of intensive resources, medical doctors should have selected patients for intensive therapies based on their actual hopes of survival (siaarti, ) . in fact, in italy, there were about , beds in total for intensive care units, and on march , , , of these beds had already been destined to patients suffering from covid- . according to the predicted number of new cases, the peak of contagions would have been reached by mid of april, when at least , beds in the intensive care units would have been needed in order to treat patients with covid- (remuzzi and remuzzi, ) , with significant consequences also for patients not affected by covid- , who would have given less assistance in the aforementioned units. however, on the one hand, doctors and other health workers multiplied their alarms relatively to this critical situation and to the related recommendations regarding behaviors to be followed and the hygienic conduct to be implemented; on the other hand, there were daily episodes of violation of such medical recommendations by the population, apparently only scarcely aware of the problem. for this reason, i.e., the failure of the unanimous spontaneous compliance of the population to the proposed hygienic rules and health practices, since february the italian government implemented increasingly restrictive dispositions to limit the spread of the disease throughout the country with various prime minister decrees (named dpcm, i.e., decreto del presidente del consiglio dei ministri; see dpcm on february , march , march , march , march , and march , ) . in fact, a significant portion of population continued to engage in risky behaviors, prompting increasingly stricter rules emanated by the authorities. therefore, a gap appeared to emerge between the indications and requests from the national health system staff and the reception of these same indications by the population, as well as a general difference in the perception and evaluation of the risks associated with the covid- infection between the two groups. such a difference seemed more evident especially in the areas of central and southern italy, where the covid- spread was lower than those of northern italy, as reported by the daily data provided by the national civil protection (see cereda et al., ) . the spread of the sars-cov virus in has shown how this type of epidemic disease has important psychopathological consequences, in the short and long term, in particular on health workers (sim and chua, ; lung et al., ; maunder, ) . thus, in the actual spread of the new sars-cov- virus attention to psychological health of doctors and others healthcare workers had already been expressed regarding the chinese situation relating to covid- (see for example xiang et al., ) , with proposals for intervention and support from the hospital structures . in fact, chinese health workers in wuhan faced a situation characterized by poor safety and protection, with excessive workloads, high infectious risk, absence of adequate personal protective equipment, and shortage of staff. this risky situation for one's own and loved ones' health could have clinical consequences, but also psychic ones. in fact, these health workers showed a symptomatology characterized by tiredness, worry, fear, frustration, isolation, depression, anxiety, stress, insomnia, anger, and negation (kang et al., ) . in particular, in this group of workers, women, workers with more than years of service, and operators who had a history of psychological suffering showed higher risk of stress, anxiety, and depression (zhu et al., ) . a further risk factor for psychological distress has been also a reduced social network support, a protective factor in the stress resilience (ozbay et al., ) . in the emergency situation caused by sars-cov- , healthcare workers are indeed at high risk of acute stress, and this risk could be even higher if they feel such a disjunction from the social community formed by the other citizens, as the situation in italy seemed to lead. in addition to the personal consequences on the psychophysical health of the health professionals, this could easily lead to a progressive decline in their health services, with a worsening of the quality of care provided. the experience with the disease caused by h n in japan showed how policies that take care of healthcare and give physicians confidence positively affected the overall care they provide to the population (maunder, ; imai, ) . for all these reasons, it is very important to study the trait and state psychological variables of healthcare workers as risk or protective factors with respect to the actual stressful situation. in this manner, it would be possible to evaluate the analogies and the differences with the chinese model at both intracultural and intercultural levels (mccrae, ) , for considering which intervention strategies could be suited for italian healthcare workers and thus importing the most adequate recently developed for the chinese healthcare system in response to the spread of covid- . in such an emergency situation, characterized by contrasts between the opinions and the worries of medical doctors on one side and the behaviors and the attitudes of the general population on the other, we designed and conducted this study. according to the evidences reported above, our objectives were (i) to probe the opinions and the worries relative to covid- spread in both the general population and healthcare workers; (ii) to study which demographic, geographic, and psychological variables were related to a higher perception of the health risks; and lastly (iii) to assess any difference in risk perception relatively to covid- between the general population and healthcare workers. thus, our aim was to understand the influence of psychological and training/working experience in shaping opinions, worries, and risk perception relatively to covid- . to this aim, we administered an online battery including a questionnaire about the direct experience, the opinions and the worries relative to the covid- , and some questionnaires evaluating the psychological distress state. to evaluate the participants distress level, we administered questionnaires measuring perceived stress, anxiety, and death anxiety as they usually increased in the general population (brooks et al., ) and in healthcare workers (brady, ; kang et al., ) during emergency situation. as the social-health situation in italy was evolving continuously in the beginning of march, we limited the data collection in the days - march . based on the evidence reviewed so far, we hypothesized that healthcare workers would show higher levels of distress in terms of stress, anxiety, and death anxiety, particularly in north italy, where the contagion was higher. in fact, as reported by lai et al. ( ) , direct exposition to virus outbreak affected the psychological health of healthcare workers, with those living in the region of wuhan reporting higher distress than colleagues living elsewhere. then, we hypothesized that healthcare workers would perceive higher levels of risk for themselves and for their relatives and that this effect would be true even when controlling for such psychological distress. in fact, we expected that this higher risk perception was not linked only to a worse psychological state, but also to a greater knowledge of the covid- disease and of its possible consequences. thus, we also expected that healthcare workers would report higher levels of knowledge of the new coronavirus. about containment and prevention measures, we expected that healthcare workers would report a higher engagement in preventing measures with respect to other people and request for more stringent containment measures, in order to prevent ssn collapse due to an increased number of accesses in hospital. following all the previous hypotheses, we expected that participants not in the healthcare workers group would provide more optimistic forecast about the progress of the spread of covid- . three hundred fifty-three italian adults participated in this study (mean age = . years, sd = . years; females = , males = ). we divided our sample by means of their job or training: in the first group, we included medical doctors, nurses, paramedics, and students in medicine/nursing/other medical disciplines ("med" group; n = ; mean age = . years, sd = . years; female = , males = ), whereas in the second group, we included all the other participants (no-med or "nom" group; n = ; mean age = . years, sd = . years; females = , males = ). we recruited our participants with a convenience sample method via email and social media. participants received a brief description of the study together with an informed consent module. after providing the informed consent, they completed an online battery of questionnaires, as described afterward. data were collected in anonymous format, and participants were invited at the end of the battery to leave their email in order to be contacted for possible follow-up measures. in this study, we collected data not reported here, as fully specified in the "materials and methods" section. in this study, we administered questionnaires to evaluate the psychological condition and personality traits of each participant. where possible, we opted for short or brief version of each questionnaire, in order to contain the total number of items ( total items). we included in our battery the following questionnaires: • the four-item perceived stress scale (pss; cohen et al., ) , a questionnaire evaluating the stress perceived by the participant in the last month, that is, the participant's perceived feeling to be in control over external events, relationships, and emotional life. we used the short fouritem version. each item was evaluated on a five-point likert scale ranging from (never) to (very often). in our sample, the four-item pss showed a good reliability score, cronbach's α = . , similar to what was reported in the original version, α = . . • the six-item version of state-trait anxiety inventory (stai; marteau and bekker, ) , which assessed the anxiety of the participants on six items including emotions or feelings. each item was evaluated on a four-point likert scale ranging from (almost never) to (almost always). in our sample, the six-item stai showed a similar reliability score, cronbach's α = . , to that reported in the original version, α = . . • the death anxiety scale of the existential concerns questionnaire (ecq; van bruggen et al., ) , which evaluated the anxiety of the participant relatively to his/her sense of finitude, to the fear of diseases and death. the total score was computed overall five items. each item was evaluated on a five-point likert scale ranging from (never) to (always). in our sample, the ecq death anxiety scale showed a good reliability score, cronbach's α = . (in the original version, only the internal consistency for the global score was reported, α = . ). • the marlowe and crowne social desirability scale (m&c; manganelli rattazzi et al., ) , which assessed the tendency of answering in a socially desirable manner. this version of the scale implied nine items evaluated over a six-point likert scale ranging from (absolutely false) to (absolutely true). in our sample, the m&c scale showed an acceptable reliability score, cronbach's α = . , slightly lower than that reported in the cited italian validation, α = . . we also included in the battery other questionnaires, which results were not reported in the present work: the -item big five questionnaire (guido et al., ) , the acceptance and action questionnaire ii (pennato et al., ) , and the emotion regulation questionnaire (balzarotti et al., ) . we further developed a questionnaire about sars-cov- and covid- -related experience and personal opinion. both authors (l.s. and c.g.) compiled a first list of items, and then this list was revised by five experts (medical doctors and psychotherapists) in order to remove, change, or add relevant items. we obtained a final list including items. a complete list of the items was reported in appendix a. to keep the questionnaire simple and easy to understand, we preferred to include mostly yes/no questions. the questionnaire we administered included the following: data analysis was conducted with statistical software r, version . . (r core team, ). as first step, we assessed differences in our sample between the med and nom groups for the demographic variables in order to control for unbalanced factors in our sample. we conducted these comparisons by means of t-tests for numerical data and of χ tests for frequencies. then, we described the experiences about covid- infection in our sample and compared med and nom groups again and areas (north vs. center vs. south italy). we also compared the psychological state of our participants by group and area to assess difference in levels of anxiety, stress, and death anxiety. for these comparisons, we implied mixed-effects analyses of variance (anovas) with one between factor (group, two levels: med vs. nom) and one within factor (area, three levels: north, center, south). we further decomposed significant main or interaction effects by means of least significant difference-corrected post hoc pairwise comparisons. as main analysis, we computed hierarchical logistic regression on the dichotomic responses and reported overall our participants about preoccupations, opinions, and behaviors relatively to their experience with the new coronavirus. this analysis allowed us to estimate the odds to obtain a positive response to a particular question given a set of parameters. for non-dichotomous variables (e.g., contagious spread in the next days could either increase, decrease, or stay stable), we created n dichotomous dummy variables, where n was the number of possible alternative responses to "equal" response (e.g., for contagious spread in the next days, we created a dummy variable for increased forecast and a dummy variable for decreased forecast). we used as reference the middle-point response, i.e., "equal" response, and evaluated the propension to respond "more" or "less" with respect to this point. moreover, we did not analyze the questions for which we obtained identical or almost identical responses by all our participants, i.e., question with > % of equal responses. in fact, for such questions, it was easy to find one of the outcome categories so underrepresented that it could lead to rare event outcome or be linearly separated by only one of the independent variables (ivs). we introduced the regressors in the model at different steps of computation. at the first step, we introduced the demographic variables such as sex, age, and living area (with the north italy as reference). at the second step, we added to these variables the psychological state factors of perceived stress (pss score), anxiety (stai score), and death anxiety (ecq score), in order to investigate the contribution of these regressors. as last step, we investigated the difference between med and nom groups in responding to the questionnaire. for this aim, at the third step, we introduced the group variable as regressor. when conducting logistic regression analysis, we should check for assumption violations. first, we considered the sample size issue. in the full model, i.e., model at step , we had a total of eight ivs including all the regressors and the covariates. considering our sample size of participants, this resulted in an event per variable (epv) of approximately , computed as the ratio between number of participants and number of ivs. this epv could be considered as fairly sufficient to make the interpretation of our global model meaningful (harrell, ; ogundimu et al., ) , even if the more stringent bujang et al.'s rule of thumb bujang et al. ( ) would suggest to include at least participants for such a number of variables. moreover, for each tested model, we checked for influential outliers and for multicollinearity. to test for influential outliers, we computed cook's distance for each data point and check for values larger than sd from the mean, as a large value of cook's distance indicates an influential observation (martín and pardo, ; zhang, ) . to test multicollinearity, we computed the variance inflation factor (vif) for each regressor and check for any value greater than . , considered as more strict threshold with respect to the usual value of or (midi et al., ) . for all our logistic regression models, we found no influential outliers or any vifs greater than the threshold value. the results of these tests, together with the reported epv greater than , testified that our logistic regression analyses could be considered sufficiently reliable. to further support our logistic regression model results, we conducted semipartial correlation analysis by means of the ppcor package for r (kim, ) . we assessed the degree of relationship between group (coded as nom = and med = ) and each dependent variable of the covid- questionnaire while controlling for sex, age, living area, anxiety, death anxiety, and stress. semipartial correlations were reported as pearson r for each computed correlation, with values ranging from − , very strong negative relationship, to , very strong positive relationship. even if we conducted a great number of statistical analyses on the same sample, we decided not to apply a general correction to significance level for multiple tests. because of the exploratory nature of this study, we preferred not to strictly control over false-positive rate (type i error) while avoiding to inflate falsenegative rate (type ii error); i.e., we decided to collect all the significant results emerging from our analysis so to guide further, confirmatory experiments and studies (see fiedler et al., , for an overview of the problem on multiple testing correction). table reports the descriptive statistics for the two groups and the relative tests for samples' comparison. as shown, participants in the med group were younger (mean = . vs. . ), studied more years (mean = . vs. . ), had less children (mean = . vs. . ), reported to sleep in average less time per night (mean = . vs. . ), and were more frequently vaccinated for annual flu in ( % vs. %). in this first results section, we reported the analysis of the data relatively to the experience with the covid- . we thus referred to the data in the first part of the questionnaire, in which we asked if participants had personal experiences or contacts with covid- infection. we reported data overall participants and divided by groups in table . frequencies were compared by means of χ test. for the overall sample, we found an effect of the living area on question , about the presence of symptoms related to covid- , χ ( ) = . , p < . ; question , about thinking that the symptoms relate to a covid- infection, χ ( ) = . , p < . ; question , about the quarantine status, χ ( ) = . , p < . ; question , about contact with people at risk of infection, χ ( ) = . , p < . ; and question , about the presence of positive case in the living area or city, χ ( ) = . , p < . . in answering to all these questions, participants from north italy reported a greater direct experience with covid- than participants from center or south italy, whereas participants from center italy reported more personal experiences than participants from the south. then, we compared the frequencies between the two groups, med versus nom. we found significant differences in question , about contact with people at risk of infection, χ ( ) = . , p < . , and in question , about contact with people positive for covid- test, χ ( ) = . , p < . , with participants in the med group reporting more frequent contacts with people at high risk of infection or already positive. we measured various indexes of psychological distress state, i.e., anxiety, death anxiety, and stress. here, we tested if any difference existed between groups in the psychological state and if this difference was modulated by the living area. to this aim, we conducted mixed-effects (anovas) with a between-variable of group (med vs. nom) and a within-variable of living area (north vs. center vs. south italy). we controlled for the effect of age and sex as covariates. we probed significant effects by means of post hoc corrected tests. for the death anxiety score (ecq; see figure , left panel), we found no significant main effects or significant interaction, all p's > . . for the perceived stress score (pss; see figure , middle panel), we found a significant main effect of the living area, f( , ) = . , p < . , with participants from north italy reporting higher stress levels than participants from both center, p < . , and south italy, p < . . the analysis also revealed a significant group × living area interaction, f( , ) = . , p < . , with med participants from north reporting higher stress score than other med participants from both center, p < . , and south italy, p < . , as well as higher stress score than the nom group participants from all living areas, all p's < . . for the anxiety score (stai; figure , right panel), we found a significant main effect of living area, f( , ) = . , p < . , with participants from north italy reporting higher anxiety levels than participants from center, and a significant group × living area interaction, f( , ) = . , p < . . the interaction was due to a significant difference in anxiety between med participants from north with respect to the med participants from center and south italy, p's < . , and with respect to nom participants from center italy, p < . . this analysis thus revealed that the med group participants from north italy reported higher levels of anxiety and stress than the general population and the medical and paramedical staff from other living areas. before conducting the regression analysis on the questionnaire data, we reported some descriptive information and statistics about the response frequency of participants. response frequencies for each item overall sample as well as divided by group are reported in table , left group of columns. here we reported also a χ test comparing the frequency of "yes" responses for the two groups. of note, % of participants were thought to be at risk of contagion, but only % were thought to be at risk when the first cases appeared in italy. they also thought that their loved ones would be at risk ( %). the med group reported higher frequency of thinking to be at risk ( %). many participants in this group ( %) were scared about health consequences or death if infected, but almost all ( %) were more worried for family or loved ones' consequences of infection. similarly, the % of them reported worries about the global sociopolitical implication of virus spread, and the % about the possible collapsing of the national health system. moreover, % of them thought that people's behavior could be ever scaring of the infection and % were worried by the increased aggression risk for health workers in the near future. most of our sample ( %) reported to adhere to hygiene measures and to avoid public events or places ( %), and only a few participants reported to have risky behaviors for themselves ( %) or their family ( %). however, only % of nom and % of med believed that people's behavior was adequate to the situation. about violation of the public health dispositions, most of participants thought that violation should be punished more severely ( %) or that the national army should be implied ( %), as they reported to be preoccupied or angry toward such violations ( %). few reported to have bought more canned food ( %), and very few participants reported that they would try to escape if the infection would spread in their living area ( %), even if a great part of them ( %) believed that the infection fear could be considered a valid reason to break the containment rules. about the possible problem of accessing healthcare services, most of participants ( %) believed in the national health system, whereas few thought that it was right to give priority to people with greater hope of survival in case of shortage of hospital beds ( %) and even less ( %) that they would accept an exclusion for them or their loved ones at all kindly. about their opinions on the containment measures disposed, % of the participants thought that these measures were necessary, but only % thought that these same measures were adequate (the med group was more skeptical than the nom group), and % proposed to strengthen them. in line with this, most participants thought that it was right to limit people's freedom for controlling the virus ( %), as well as one's own freedom ( %), as they already limited their behaviors ( %). about the information, they reported to be properly informed about the virus ( %) and the social situation related to it ( %), but also requested more information from experts ( %). about perception of risk in public opinion, % of participants reported to think that it was lesser than it should be and % that it was greater. interestingly, % reported to think that there was some hidden information about the virus, and % of these that such hidden information was related to a greater danger related to the infection. the med group, instead, reported to be less convinced of the existence of hidden information ( % vs. % of the nom group). lastly, about the spread of the virus, the med group was more pessimistic than the nom group. in fact, they reported less likely that the spread would slow down in some days ( % vs. %) or in some weeks ( % vs. %). in this subsection, we present logistic regression results on the covid- questionnaire. we used each question as a dependent variable in a three-step hierarchical logistic regression. at step , we used as regressors the demographic variables (see "data analysis" section) and the living area, considering north italy as the reference (the coefficients reported should be interpreted as the odds that a participant from center or south italy would answer "yes" to a question compared to a participant from north italy). at step , we added as regressors the psychological factors of perceived stress, anxiety, and death anxiety. finally, at step , we included the group effect. along with step results, we also provided semipartial correlation score for the relationship between each dependent variable and the group (coded as = nom and = med). for the sake of brevity, we reported only the questions for which we obtained significant regressors. at step (see table , step block of columns), we included in the model only demographic variables. of these, the most influential were sex and age. with respect to male sex, female sex was linked to higher odds to be concerned by the following risks: being infected ( . ), loved ones being infected ( . ), developing serious complication or dying ( . ), global crisis ( . ), people's behavior in response of virus outbreak ( . ), infecting family members or love ones ( . ), and people's violating the containment provisions ( . ). in fact, females had higher odds to report that the public opinion had less riskrelated perception about covid- than it should be ( . ), that risky behavior should be punished more severely ( . ), that containment provisions should be improved ( . ), and that it would be right to limit people's freedom in this situation ( . ). in line with these results, they reported more likely to have not continued to attend public places and events ( . ). about age factor, older age was related to lower odds of reporting worries about the risk of infection for the loved ones ( . ), or about people's behavior as more dangerous that virus infection ( . ), or about the perception of risk in public opinion as lower that is should be ( . ). older age people also reported table | hierarchical logistic regression odds for demographics (step ), psychological (step ), and group (step ) factors for the covid- questionnaire. frequency of "yes" responses step step step questions in italics showed imbalanced responses (almost all "yes" or "no"). sex was coded as = male, = female. group was coded as = nom and = med. ecq = existential concerns questionnaire (death anxiety scale); pss = perceived stress scale; stai = state-trait anxiety inventory. rightmost column (sp. cor.) reports pearson r for semipartial correlations between group and questionnaire responses (coded as = "no" and = "yes") controlling for all the other variables, i.e., sex, age, living area, anxiety, death anxiety, and stress. significance level marked as follows: + p < . , *p < . , **p < . . frontiers in psychology | www.frontiersin.org lower odds to be concerned about their behavior as risky for themselves ( . ) or loved ones ( . ), but higher odds to be concerned about their health status in case of covid- infection ( . ) and by people reaction to virus spreading ( . ). lastly, they reported lower odds to request for more information by experts on media ( . ). also, the living area had a relative impact on the outcome variables at this step. with respect to participants from north italy, those from both center and south italy showed greater odds to judge the actual containment measures as adequate (center = . , south = . ) and to think that some information about covid- was hidden from them (center = . , south = . ), whereas they reported less likely to be at infectious risk (center = . , south = . not significant) or to consider themselves at risk when the first cases were discovered in italy (center = . , south = . ). of note, participants in south area reported lower odds with respect to participants in north area to consider fear of infection as a valid reason to violate the containment measures ( . ). at step (see table , step block of columns) of hierarchical model, we added psychological factors of perceived stress (pss), anxiety (stai), and death anxiety (ecq). we found that these factors were related to few, but interesting outcomes. in particular, the pss score was related to a higher worry to be currently at infection risk ( . ) and a major need of information by experts ( . ), while their opinion on the virus spread was that it would show equal speed in the weeks following the compilation ( . for both accelerated or slowed-down spread). instead, the stai score was related to higher concerns of accelerated spread of virus in the weeks following the compilation of the questionnaire ( . ). lastly, the ecq score was related to a higher level of worrying about the covid- situation, in particular about possible severe outcome of the disease for themselves ( . ) or loved ones ( . ) and marginally related to higher level of worrying about possible catastrophic social global outcomes ( . ) or violation of containment measures ( . ). at step , we added to the logistic regression model the group factor to check for the predictive effect of being in the med or nom group while controlling for both demographic and psychological variables. results are reported in table , step column (see the rightmost column). participants in the med group reported higher odds of thinking to be at actual risk of infection ( . ) and also to be at risk from the beginning of the covid- spread in italy ( . ). they also reported more likely to think that their family or loved ones were at risk of infection ( . ). the med group showed higher odds to report that the fear of contagion would be a valid reason to violate the containment measures ( . ) and that the ssn would adequately cure them in case of infection ( . ) and to report a sufficient level of information about the characteristics of the disease ( . ) and about the social situation relative to . they also reported less likely that some information about the virus was hidden ( . ), but the ones who answered affirmatively to this question had more than two times the odds with respect to the nom group thinking that such hidden information was related to a greater virus-related danger ( . ). about the spreading of the virus, participants in the med group were less probably convinced that the virus spread would slow down in the following days after the compilation of the questionnaire ( . ). lastly, the med group participants more likely reported that perception of risk in public opinion was lower than it should be ( . ). semipartial correlations mostly confirmed this pattern of results. however, differently from the logistic regression, this analysis revealed that the med group was related to the opinion that people's behavior was not adequate to the situation, r = − . , and to agree to give care priority to people with greater hope of survival, r = . . also, semipartial correlations did not confirm the regression results for the questions about the fear of contagion as a valid reason to violate the containment measures, r = − . , and the adequacy of the ssn to take care of people in case of infection, r = . . in this article, we investigated the worries and the perception of risk toward the health and social situation in italy related to the outbreak of covid- . to this aim, we conducted a crosssectional study by means of online questionnaires administered to a convenience sample of volunteer participants including both health workers and the general population. we asked participants to report their worries and opinions about covid- in about different questions combined with psychological variables measuring stress, anxiety, and death anxiety. we obtained and analyzed data from italian adult, divided in participants in the med group (medical doctors, paramedics, health workers, and students) and participants in the nom group. we mainly compared the answers given to the questionnaires by these two groups. we also investigated the effect of the living area in italy, as the northern regions were more involved than the central and southern ones (cereda et al., ) . first, we assessed risky situations in which people were involved relatively to covid- . as expected, people from north italy reported more direct experiences with covid- , including more symptoms related to the infection, more prolonged quarantine status, more contacts with people at risk, and higher numbers of positive cases in their zone. the med group, instead, reported a higher number of contacts with people currently infected or at risk. thus, both living area and group predicted a major or minor probability to be involved in risky situations or contacts. following this, we found that participants from north italy reported higher levels of stress and anxiety and in particular that health workers in north area showed a higher level of both health workers from other areas and the general population from the same area. thus, both living area and job combined with the higher exposition to infection risk in order to increase the level of stress and anxiety in health workers from north italy. we would caution about the relatively small number of participants in each area divided by group: our results about living area should be considered strictly as preliminary. further studies are welcome in order to confirm or refute the results that we presented on this topic. however, we should note that our result was in line with the psychological response of health workers in china, where lai et al. ( ) found that psychological distress increased for workers closer to the outbreak of epidemic (i.e., who lived and worked in wuhan region) or assigned to patients affected by covid- . thus, the same rule applies here: the closer to the risk of infection, the higher the risk of acute psychological distress. similar results were found in previous researches on new disease outbreaks. for example, wong et al. ( ) reported higher levels of anxiety in university students during the sars epidemic, in particular among medicine students and students living in the area in which the infection spread more. also wheaton et al. ( ) reported higher levels of anxiety in students in response to pandemic spread of h n . more generally, anxiety emerged in response to various viral diseases, from the annual influenza virus to the h n pandemic (coughlin, ) . in the period of viruses spread, anxiety seems to increase in population along with mood disorders, and this increase was related to exposition and infection risk. in line with these results, participants of our study reported higher levels of perceived stress and of anxiety proportional to their risk of infection, i.e., health workers from north were more stressed and anxious than both their colleagues in center and south italy and the general population. while our result supports an acute increase of stress and anxiety, we should carefully monitor the psychological state evolution in order to assess also the effect of covid- over time. in fact, we expected that the virus spread and the quarantine state endurance in italy could have also mid-and long-term consequences. survivors from sars reported posttraumatic stress, anxiety, and depression symptoms month after discharge, suggesting that lifethreating condition could have important psychic sequelae (wu et al., ) . such sequelae could be even more significant in health workers, showing higher levels of psychological distress both during and after a quarantine period (brooks et al., ) . for this reason, supporting psychological intervention for healthcare workers could be crucial in the first phase of an outbreak (xiang et al., ) , in particular considering that a timely and effective intervention could greatly reduce the later onset of posttraumatic stress disorder symptoms following a catastrophic event (watson et al., ) . we analyzed the answers to our questionnaire on covid- by means of logistic regression. for each item, we computed the response odds related to each regressor in three successive steps, by adding sequentially demographic factors, psychological factors, and the group factor. here we discussed the implication of all these computational steps by dividing the questionnaire items by content. in this section, we discuss the variable that we found for the items relatively to risk perception and worries related to the covid- outbreak in italy. group was strongly related to risk perception: healthcare workers showed about . times the odds of other participants to perceive themselves at risk of infection, as well as about two times the odds to think they were at risk even at the very start of virus outbreak in italy. moreover, they worried about their family situation and about virus spread as they reported that it would not slow down. this supported the idea that medical doctors, nurses, and paramedics had greater risk perception about the covid- infection, probably due to also a greater exposition to danger and to suspect positive cases. also, living area predicted the perception of risk, as both participants from center and south italy reported . times less preoccupation about risk of infection with respect to participants from north italy. again, combination of work, i.e., health workers, and area, i.e., north italy, combined for the greater perception risk. about the demographics, the stronger regressor of such worries was female sex, which was related to higher perception of risk, both at personal and family levels, and of a number of worries about social situation and people's behaviors. in particular, female healthcare workers were reported to be at higher risks of stress, anxiety, and depression during the covid- outbreak in china (lai et al., ; zhu et al., ) . this increased distress level in female health workers could be related to an increased perception of risk for themselves and for their relatives as we found in our study, as also reported usually in researches about risk perception in female participants (gustafsod, ) . our results suggested carefully supporting female healthcare workers implied in covid- treatments, as they could be more exposed to risk-related stress compared to their male colleagues. another important demographic variable was age, as we found that aged people were more worried than younger people about severe consequences of covid- , as they already knew that the disease was more dangerous for older people, in particular when older than years (novel coronavirus pneumonia emergency response epidemiology team, ). lastly, also psychological factors influenced the odds of perceived risk of infection. in fact, stress was related to increase in perceived risk, while death anxiety was related to the concern about fatal or severe consequences of covid- . while the effect of both stress (traczyk et al., ; sobkow et al., ) and death anxiety (langford, ) on risk perception and risk taking was already reported in literature, it should be noted that higher levels of stress could also be due to actual exposure to contagion risk in the case of covid- , as shown by our results about comparisons on levels of perceived stress between healthcare workers from north, center, and south italy. taken together, all these results suggested a higher risk perception relative to covid- in healthcare workers living in outbreak areas, especially if females and with high levels of stress. for covid- , knowledge on medicine and on virus could thus increase risk perception, whereas in other fields such as nuclear radiation usually knowledge was associated to lower risk perception (e.g., sjöberg and drottz-sjöberg, ) . it should be noted that, in case of nuclear radiation, knowledge could be associated to an increase capacity of avoiding risky behavior or situations, whereas in case of covid- spread knowing, the health risks related to disease, but feeling powerless against its containment could exacerbate the danger perception. a reducing stress intervention by means of psychological support to medical workers could reduce the worries due to the perceived risk, so that they could avoid both risky behaviors and overwhelming, stressful concerns. we proceed here by discussing the variables related to risky behaviors, judgments about behaviors, and confinement actions. in this respect, female participants reported higher levels of worries about their own behavior, as well as other people's behaviors as risky. related to this, they also were four times more likely than men to report the thought that it would be right to limit people's freedom in order to block the virus spread and three times more likely than men to request more severe punishment for risky behaviors. capraro and sippel ( ) showed that females adopted stricter moral judgments than men in personal dilemmas, such as behaving appropriately in the actual covid- outbreak scenario. females seemed more prone to strict adherence to rules and even to imply stricter rules, probably also in relationship to their increased perception of risk (see section "risk perception and worries about covid- "). also, the living area showed a strong relationship with these dependent variables. participants from center and south italy were more likely to judge the containment measures as adequate compared to participants from north italy. on note, participants from south also reported less likely than north ones that concerns about covid- were a valid reason to violate the containment measures. this result could be related to the recent great "escape" of people from the north italy (when virus spread initially) toward the south, increasing worries in south population, politicians, and medical staff. again, please consider results on living area no more than preliminary because of the limited number of participants per area in our sample. lastly, we should mention that both the group variable and the psychological factors had none or little impact on these variables thus, our data suggest that opinions and judgments about behaviors and containment actions rely more on demographic variables than on psychological or work-related ones. conflict of interests or searching media visibility. the same criticisms, however, are often not applied, for the antiscientific community, to the studies supporting their theories (kata, ) . all these factors could have an effect also on the underestimation of medical advices and warning on covid- infection by the general population, resulting in the unappropriated behaviors expressed. thus, providing more information to population could be ineffective, if not supported by psychological evaluation of social dynamics underlying the antiscientific phenomenon, for example, the questioning of the legitimacy of traditional authorities (see kata, ) . understanding how to contrast such a phenomenon could be even more important in case a vaccine for covid- is provided, as already happened for the h n flu in , when many people refused to vaccinate despite the availability of a vaccine (see offit, ). further studies are needed in order to investigate these contrasting hypotheses for planning effective interventions relative to public health problems. this study is not free from limitations. first, it implied a crosssectional design; thus, a relationship between variables could be interpreted only with cautions. second, we implied a convenience sample method to recruit our volunteer participants, with a possibility for introducing biases that could undermine the possibility to generalize our results to the entire population. we also collected a small sample with respect to the optimal one, i.e., about participants (as suggested by bujang et al., ) , thus calling for caution while interpreting our results. for all these reasons, we should underline that our results could not be considered as conclusive and they should be confirmed with further experiments or studies. however, we should note that we conducted this study with two major difficulties. the first was a time-related issue: we had a very short time to collect data as the containment rules and the virus spread vary at a day-by-day rate. thus, we should collect our data in a concise and brief timeframe. the second issue was a logistic one: most people in italy, including the authors of this article, were quarantined at the time we collected and analyzed the data, so we were forced to opt for an online methodology of data collection. while methodologically limited, our results could open a number of possible future studies. first, this study could be considered as a time-zero data collection for a longitudinal study. in this regard, we would contact our previous participants in order to ask if they will participate to further data collection. thus, we could follow the change in risk perception and psychological situation in the general population and healthcare workers during the evolution of covid- infection spread. more experimental and cross-sectional studies are requested in order to better understand the relationship between healthcare workers' and the general population's information gap and risk perception in a pandemic disease scenario. this could help scientific community to find new strategies for conveying lifesaving information to population. reducing such information gap could also help in reducing the sense of separation between the healthcare workers and the rest of population and thus the sense of isolation with its negative psychological consequences on both groups. our study supports that a difference in risk perception between health workers and the general population exists and suggests a number of explanations for its causes as well as possible solutions to reduce it, with benefits in the psychological conditions of both groups of participants. more efforts need to be done in this direction, also because reducing psychological distress could advantage physical health state (prince et al., ) , in particular for medical staff facing such a difficult time, improving the quality of care they could provide (maunder, ; imai, ) . the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. the patients/participants provided their written informed consent to participate in this study. ls and cg designed the study and administered the questionnaire. ls conducted the data analysis. ls and cg wrote and revised the manuscript. both authors contributed to the article and approved the submitted version. an italian adaptation of the emotion regulation questionnaire death anxiety among emergency care workers the psychological impact of quarantine and how to reduce it: rapid review of the evidence sample size guidelines for logistic regression from observational studies with large population: emphasis on the accuracy between statistics and parameters based on real life clinical 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health workers we would like to thank all participants to our study, who voluntarily donated their time. in particular, we thank healthcare workers, whose time is even more precious in this difficult situation for all the country, who participated and distributed the questionnaire to their peers and colleagues. finally, we would like to thank our colleagues and friends martina formisano, alessio montemagno, antonello catinari, francesco romano, elisabeth prevete, carola de berardinis, and salvatore chiarella for their valuable help and sustain. the supplementary material for this article can be found online at:https://www.frontiersin.org/articles/ . /fpsyg. . /full#supplementary-material in this section, we discuss how demographic, psychological, and group variables impacted on the perceived level of knowledge relative to covid- and to its related sociopolitical situation. in this regard, the group was the strongest factor. in fact, healthcare workers reported higher odds than nonmedical participants of being properly informed about both covid- and its related social situation. they also were less likely convinced that some information about coronavirus was hidden from public opinion, but those who credited such secret information more likely believed that this information was about a greater virus threat. also, they reported the opinion that perceived risk in the population was not adequate. this result pattern suggests a large gap between the two groups about the perception of being properly informed.this information gap could explain the risk perception difference, because a greater knowledge could actually influence the personal risk awareness. it should be noted that, in general public opinion, the risk related to the new coronavirus was mistakenly considered as similar to that related to the common cold or annual influenza viruses, an error that could have been induced by the similarity in the spreading strategy and of some of the symptoms. this underrepresentation of fatal or serious outcomes of covid- led to a poor adherence to health recommendations in the very first phase of the coronavirus outbreak in italy, with important consequences afterward. these considerations seem to suggest that the reduction of such an information gap could eventually mitigate the disproportion in risk perception between groups and consequently increase the adherence to public health rules. also, our results seem to support this possibility because of the lack of information from experts lamented by more stressed participants, who also perceived a higher level of personal risk. to this aim, an information campaign about the novel coronavirus characteristics, its related disease symptoms and consequences, and public health problems linked to that could greatly support population in this moment, reducing the stress and also the risky behaviors.however, increasing the communication and the information could not be the most appropriated solution to the problem. in the last decades, especially because almost everyone has a large access to internet resources, we have witnessed not only a significant spreading of online information, but also misinformation; this is causing the diffusion of baseless rumors, difficult to erase from common people system of beliefs (kata, ; del vicario et al., ) . misinformation spreading combines with people's distrust in experts' authority, a more and more rising phenomenon despite the increase in the general education level. as a result, as proposed by gawande (gawande, , p. ): "to defend those beliefs, few dismiss the authority of science. they dismiss the authority of the scientific community. people do not argue back by claiming divine authority anymore. they argue back by claiming to have the truer scientific authority." this kind of problem is well known in the field of the unfounded, yet persisting, vaccine fear. in anti-vaccination movement, this mistrust phenomenon has been also exasperated by conspiracy theorists and other actors moving criticisms toward physicians and other experts, accused of having conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © simione and gnagnarella. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -boo au authors: borrelli, enrico; sacconi, riccardo; querques, lea; zucchiatti, ilaria; prascina, francesco; bandello, francesco; querques, giuseppe title: taking the right measures to control covid- in ophthalmology: the experience of a tertiary eye care referral center in italy date: - - journal: eye (lond) doi: . /s - - - sha: doc_id: cord_uid: boo au nan a rapidly increasing number of people has been infected with severe acute respiratory syndrome coronavirus (sars-cov- ), the virus causing coronavirus disease (covid- ) [ ] . to date, the italian covid- outbreak represents one of the largest outside china [ ] . in order to face it, the italian government has placed the whole of italy-a country with an estimated population of . million-under quarantine. in detail quarantine is defined as people are forbidden from assembling in public places and are recommended to keep at least -m distance from each other, and restaurants, pubs, and shopping centers-except for pharmacies and food markets-are obligated to remain close. these norms are even more rigorous in lombardy, a large region in northern italy, where most hot zones (i.e., where covid- cases are more concentrated) have been reported to date. a growing evidence suggests that human-to-human transmission of sars-cov- occurs among close contacts through droplets, contacts, and fomites [ ] . health workers may be contaminated during patient care. to date, more than health workers have been affected by covid- in italy, and among them more than medical doctors have died from this infection. although there is no information regarding the number of ophthalmologists affected, it is known that they are at high risk of infection assuming the close proximity between eye doctors and patients during examination and that a contamination may be present in ocular discharge and tears. in order to achieve continuity of care and to diminish the risk of contamination for both healthcare workers and patients, we adopted a series of measures. here we document the measures approved at our referral medical retinal practice at the san raffaele hospital in the metropolitan area of milan, italy. we adopted three levels of organizational hierarchy. first, patients were screened by telephone for: (i) complaints of fever or upper respiratory symptoms; (ii) domicile in one of the hot zones; (iii) contact with suspected or confirmed cases of covid- . patients who fulfilled any of these criteria and requiring urgent ophthalmology visit were redirected to a separate room. otherwise, patients were recontacted after days. second, patients not fulfilling any of the above criteria were asked for complains of reduced visual acuity or increase/appearance of other visual symptoms (e.g., metamorphopsia or photopsia). cases with stable symptoms were recontacted after days. otherwise, patients were asked to attend the visit with no more than one accompanying person in order to reduce the risk of exposure of uninfected people to potential subjects with covid- disease. third, healthcare personnel were appropriately trained and equipped. they were required to measure their body temperature and report any upper respiratory symptoms. ophthalmologists were equipped with visors or protective eyewear, gloves, and surgical masks. slit lamps were equipped with breath protector using acrylic sheets. moreover, rooms' equipment as slit lamps and imaging devices (e.g., optical coherence tomography) were disinfected before and after being utilized. in order to reduce gathering of people, patients in outpatient departments were asked to come into the visiting room without accompanying person. in addition, students and fellows were not allowed to attend the clinic. anti-vascular endothelial growth factor injection and laser treatments were immediately performed in patients that required them by following the same cautions. in conclusion, italy has just started to combat this tough battle. specific protocols are necessary to reduce healthcare * giuseppe querques giuseppe.querques@hotmail.it department of ophthalmology, university vita-salute, irccs ospedale san raffaele, milan, italy professionals' and patients' contamination and, at the same time, guarantee the continuity of care for ophthalmology patients. characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of , cases from the chinese center for disease control and prevention the response of milan's emergency medical system to the covid- outbreak in italy world health organization. coronavirus disease (covid- ): situation report, . world health organization publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -g sfh nf authors: mercadante, sebastiano; adile, claudio; ferrera, patrizia; giuliana, fausto; terruso, lidia; piccione, tania title: palliative care in the time of covid- date: - - journal: j pain symptom manage doi: . /j.jpainsymman. . . sha: doc_id: cord_uid: g sfh nf after covid- crisis in italy, serious restrictions have been introduced for relatives, with limitations or prohibitions on hospital visits. to partially overcome these issues “whatsapp” has been adopted to get family members to participate in clinical rounds. family members of patients admitted to the acute palliative care unit and hospice were screened for a period of weeks. four formal questions were posed: ) are you happy to virtually attend the clinical round? ) are you happy with the information gained in this occasion? ) do you think that your loved one was happy to see you during the clinical rounds? ) this technology may substitute your presence during the clinical rounds? the scores were = no, = a little bit, = much, = very much. relatives were free to comment about these points. sixteen of screened family members were interviewed. most family members had a good impression, providing scores of or for the first three items. however, the real presence bedside (forth question) was considered irreplaceable. they perceived that their loved one, when admitted to hospice, had to say good-bye before dying. limitations or prohibitions on hospital visits. to partially overcome these issues "whatsapp" has been adopted to get family members to participate in clinical rounds. family members of patients admitted to the acute palliative care unit and hospice were screened for a period of weeks. four formal questions were posed: ) are you happy to virtually attend the clinical round? ) are you happy with the information gained in this occasion? ) do you think that your loved one was happy to see you during the clinical rounds? ) this technology may substitute your presence during the clinical rounds? the scores were = no, = a little bit, = much, = very much. relatives were free to comment about these points. sixteen of screened family members were interviewed. most family members had a good impression, providing scores of or for the first three items. however, the real presence bedside (forth question) was considered irreplaceable. they perceived that their loved one, when admitted to hospice, had to say good-bye before dying. in the last months italy, the first country to face covid- in europe, has been overwhelmed by the spread of the contagion. after the coronavirus has reached northern italy, it spread throughout the entire country. despite the good palliative care network existing in italy, this crisis has highlighted some weaknesses of the health care system that could not be foreseen ( ). one of the most important was the lack of availability of equipment required for the protection of patients and health workers ( ) . face masks, for example, were unavailable for many weeks and have fueled a disreputable black market. moreover, there were no exact provisions for physicians who were following patients at home. in palermo, a metropolitan city of southern italy, in march the requests of home care assistance doubled compared to the same period of the previous year, putting stressed teams accustomed to following fewer patients. this was due to a reduced possibility of hospitalization and the shortage of beds for patients with cancer or noncancer chronic diseases. ironically, some patients refused the visits for fear of contagion ( ). on the other hand, hospital activity has changed dramatically, due to the barriers imposed for security reasons. the government issued increasingly stringent decrees to limit the conduct of daily life with the aim of preventing contagion. irrespective of the obligations for health personnel, serious restrictions have also been introduced for relatives, with limitations or prohibitions on visits. they too were forced to use personal protective equipment. all this caused a surreal atmosphere for palliative care in which the presence of a family member next to the patient is essential. patients remain alone all the day without support, except that provided by the health workers. it is well known that family support in a mediterranean country is of paramount importance. in response some patients can ask to be discharged earlier than necessary. sadly, some others die without any final contact with relatives or say good-bye before being admitted to hospice. before this crisis, in the acute supportive/palliative care unit, family members were allowed to stay "eight days a week" alongside their loved ones, as it occurs in the hospice, possibly even at night. in the first two weeks after the restrictions enacted by the health minister, we have patients and family members been discouraged to do this, drastically changing our way of operating according to the philosophy of palliative care. this also happened in the hospice, where a caregiver who wanted to stay in the room with his loved one, had to remain all day, without the possibility of going out ( ). this was a compromise obtained with our administration. other visits were prohibited. in such cases patients died alone, without any relatives' comfort. indeed, some hospices stopped their activities. to partially overcome these issues we have been adopted "whatsapp" to get family members to participate in clinical rounds ( ) . the family members were called live from the patient's telephone number during the doctors' visit to exchange information on the clinical progress and share the choices to be made. we interviewed the family members of a consecutive sample of patients who were admitted to our acute palliative care unit ( beds) and hospice ( beds) in a period of two weeks. the family members of patients who were unable to communicate were excluded. twenty-five patients-relatives couples were screened in the period of weeks. two relatives refused, six had their loved one unable to communicate. one patient did not agree with the relatives' interview. in such cases information was given by telephone. thus, sixteen family members were interviewed. seven patients did not have a smart telephone or an e-paid. in these cases, a smart phone was offered by the team. the characteristics of patients and family members who agreed with the interview and scores regarding the questionnaire are reported in table . most family members were happy to virtually attend the visit: "i was happy to see him and you while talking about the treatment"; "it was a pleasure to note the smart atmosphere surrounding my loved one you created "; "i was happy to be updated on the results of the investigations and treatment you proposed, explaining the reasons for such decisions"; "i liked to share your information about the treatments and the next follow-up, albeit at a distance". "i was happily surprised by this initiative". however, most of relatives also added: "i recognize the actual situation is regulated by law, but no technology can provide the same effects experienced by my presence" or "it could be the last time i see him/her". thus, some of the problems related to government restrictions, could be overcome with simple technology available to most population, proposing a temporary, hopefully, communication model of adaptation to this crisis. however, as expected, this modality of communication cannot substitute the real presence bedside and should considered as a temporary measure. while patients admitted to the acute palliative care unit in the majority of cases will be discharged home, hospice patients have a high mortality rate. in fact, in italy the mean admission time in hospice is less than three weeks, and % of patients will die in hospice ( ) . it is sad to say that patients who are admitted to hospice will say good-bye before dying. covid- and italy: what next? covid- in italy: momentous decisions and many uncertainties perché è inaccettabile la morte solitaria telemedicine in the time of coronavirus pattern and characteristics of advanced cancer patients admitted to hospices in italy key: cord- -gl y c authors: cesari, matteo; montero-odasso, manuel title: covid- and older adults. lessons learned from the italian epicenter date: - - journal: can geriatr j doi: . /cgj. . sha: doc_id: cord_uid: gl y c on march th, , the world health organization effectively established that europe is the new the covid- pandemic world epicenter, as cases in italy and other european nations soared. the numbers in italy have climbed with over , cases as of march th, and with over deaths, placing italy now as the country with the highest mortality rate. importantly, older adults are particularly vulnerable to get severe illness, complications, and to have a higher mortality rate than any other age group. the clinical presentation in older adults with severe illness, in the experience from geriatricians in lombardy, is described as quite sudden; patients can develop severe hypoxemia with the need of ventilation support in few hours. geriatric syndromes are not a common form of presentation for covid- in severe illness. it is suggested that stratification by frailty level may help to detect the most vulnerable, and decisions about healthcare resource prioritization should not be taken based only on age itself or previous diagnosis, such as having dementia. this quote from dr giampiero giron, an -year-old italian anesthetist and member of the team that performed italy's first heart transplant, encapsulates the commitment of doctors in difficult times and also the importance of older adults in our societies. on march th, , the world health organization (who) effectively established that europe is the new the covid- pandemic epicenter of the world, as cases in italy and other nations on the continent soared. as of march th , across the world, cases reached over , and deaths reached , . ( ) this dramatic and exponential increase in the prevalence and deaths related to covid- is spreading rapidly through europe, as it is now reporting more cases and deaths than the rest of the world combined. specifically, the numbers in italy have climbed, with over , cases and , deaths as of march th, , placing italy now as the country with the highest mortality rate ( ) importantly, older adults are particularly vulnerable to get severe illness and complications from this disease, and they also have a higher mortality rate than any other age group. the case fatality rate in italy jumped from the . % in those to years of age, to . % in those to years of age, and to . % in those years and older (table ) . dr matteo cesari, professor and chair of geriatric medicine of the university of milan, replied to our questions on march th, , which are transcribed below. mmo: thank you, matteo, for making this time for our questions. we know you are under extreme pressure and busy with unprecedented clinical demands, therefore we appreciate you providing us with direct information. how is the situation in lombardy and, specifically, milan, now (march , )? the situation in milan is still very complicated. we have not yet reached the peak of the number of new covid cases, which is expected to arrive in the very next few days. figure shows the distribution of cases among italian regions, and it can be seen how the north of italy took the first hit. the hospitals have been struggling since the beginning of the crisis because they needed to substantially redraw their matteo cesari, md, phd , and manuel montero-odasso, md, phd, agsf, fgsa, frcpc , , figure . number of covid- cases regionally in italy pathways in an attempt to isolate covid-positive patients from the others. at the same time, the system was not ready to sustain the massive number of patients with respiratory symptoms arriving all together at the emergency departments. as we can see in figure , % of our patients presented with severe disease and % are critical, all of them require hospitalization. this, combined with the shortage of icu beds and the gradual decrease in health-care professionals that were falling sick, has made things extremely difficult. how is the clinical presentation of the symptomatic disease in older adults? have you found any differences when compared with younger adults? do older patients with severe covid- illness present with severe dyspnea and hypoxemia, as do the younger adults? or do older patients present with geriatric syndromes, such as delirium? the clinical manifestation is usually characterized by fever and cough. it might appear as a normal pneumonia. in fact, very frequently, the covid- case manifests itself as a pneumonia resistant to antibiotics and standard care. the worsening of respiratory symptoms is today considered a clear sign suggestive of covid infection. since the very beginning, we have tried as much as possible to isolate patients with respiratory symptoms or flu-like manifestations from the others, given the high contagiousness of the coronavirus. this was also to allow the time to test for the possible sars-cov- infection. in this context, however, things are made difficult by the relatively high number of false-negative cases found from the nasopharyngeal swab. for this reason, people tend to be kept isolated even when tested negative, if the clinical suspicion remains high. overall, the istituto superiore di sanitá (iss) from italy reports that the most common complications are the following: acute respiratory distress syndrome ( . % of cases), acute renal failure ( . %), acute cardiac injury ( . %), and superinfection ( . %). nevertheless, the clinical manifestation of the covid in older persons can be associated with delirium. the main difference is the unexpectedly rapid course of the disease. many patients convert in - min from relatively stable conditions into severe respiratory distress. in other words, preliminary signs can be there, but their identification and management is often impossible given the quick evolution of the case. it is also noteworthy that hospitals have relatively few geriatricians, and they had to reorganize their workforces creating mixed teams. this was needed to face the shortage of personnel. i have to say that, in this emergency situation, it is very difficult for non-specialized clinicians to pay attention and detect geriatric syndromes that are largely overlooked and underestimated even in normal times. mmo: how are access to emergency room (er) care, inpatient beds, and critical care beds being managed? there have been reports in the media that older citizens should get a lower priority for access to medical services that are now limited, such as intensive care facilities and ventilation. this begs the question of whether this practice should go unchallenged as a form of "pragmatism", or whether we should think through how to engage with this in order to at least reduce the risk of the most egregious forms of discrimination. ers have been exponentially overwhelmed over the past weeks. the shortage of beds, especially in icus, has sometimes led to very difficult decisions. the relatively few number of ventilators, in fact, has generated discussions about the ethics of procedures applied to certain patients. the italian society of analgesia, anaesthesia, resuscitation, and intensive care (siaarti) released a document recommending a triage in a low-resource scenario based on age, comorbidities, and functional status. ( ) unfortunately, the ageistic approach diffused in our society and in traditional medicine, as well as the poor knowledge about how to operationally apply these recommendations, makes critical decisions still (too largely) governed by the age criterion. i do not/cannot justify these decisions, but we also need to consider that the rapid course of the disease leads to taking quite rapid decisions. related to the previous question, in the stratification or triage of sick patients to maximize health resources, do you use a functional scale, or frailty status scale, or dementia severity scale for clinical decisions? age of the patient itself has been the easiest parameter to obtain in an emergency to decide on allocation of care. it is wrong and we know it. unfortunately, our system does not yet consider more robust and reliable alternatives for measuring the resilience of the person. perhaps, we should have thought about nesting more geriatric principles (i.e., meaning of the disease construct at old age, importance of functions) in other specialties. the simple adoption of the clinical frailty scale measuring the pre-illness status of the patient might have already represented an important parameter in order to take better decisions. ( ) ( ) ( ) mmo: how does a covid unit look like in italy for geriatricians? a covid unit is very far from our usual geriatric world. the communication between health-care professionals and patients is extremely limited by the personal protective equipment (ppe), as well as by the patient's clinical conditions and respiratory devices. the unit environment is not age-friendly; its design is not oriented to individuals with sensory deficits or cognitive and/or mobility impairment. these units are busy, noisy, and only distinguish the "dirty" areas from the "clean" ones. family members are not allowed to enter the unit. therefore, patients have no contact with them, and family members are usually not receiving news of their relatives. for this reason, we have volunteers who are regularly keeping families updated about the status of the patients over the phone. we try to maintain such contacts, knowing that the worst thing happening with covid is the isolation of the person. mmo: how is the situation in long-term care facilities? do you have any recommendations to provide-particularly in this setting? figure . percentage of clinical presentation of patients who tested covid- -positive mc: long term facilities have initially tried to contain the diffusion of the virus within their facilities by reducing/ closing admissions from the hospitals. suspected cases have been isolated and/or sent to the emergency units in order to guarantee the safety of the rest of the residents. the work in long-term care is not easy these days, also because resources to test patients (and personnel) remain limited. there have also been limitations in some cases of protected personal equipment's (ppes). just in these last few days, the who has released recommendations for the management of covid in long term care. (https://apps.who.int/iris/bitstream/handle/ / /who- -ncov-ipc_long_term_care- . -eng.pdf) ( ) medication access for older adults mmo: as many older adults suffer from chronic conditions requiring medication, the question of how medication access is being managed is of paramount importance. how are you managing medication access to older patients with chronic conditions? mc: outpatient clinics were closed at the very beginning of the coronavirus outbreak. only visits that could not be postponed or that met urgency criteria were maintained. a lot of older persons have found themselves completely isolated from medications, but also from the simple access to groceries. in this scenario, consider that general practitioners have found themselves in huge difficulties (shortage of ppes, a lot of cases of covid, some even died). the consequent shortage of primary care physicians has further complicated things. frail older persons with chronic diseases will likely pay a huge toll for this coronavirus pandemic, even if not directly infected by it. this emergency situation has destroyed our already weak integration of care services, critical for the health of frail individuals. mmo: an additional fact is that the case mortality rate is higher in older adults, but particularly in those with cardiovascular disease, hypertension, and diabetes or having more than three comorbidities ( table ). it has been suggested that angiotensin-converting enzyme (ace- ) inhibitors may explain the higher risk and severity of the disease in older adults ( ) because they increase the up-regulation of ace- receptor expression-the receptor that the sars cov- virus uses to enter the human cells. ( ) are clinicians changing the ace- inhibitor medication in their patients for another medication such as calcium channel blockers? mc: there have been many news items around about the positive or negative effects of certain medications in face of the covid. to my knowledge, most of the clinicians have not changed the prescriptions of cardiovascular drugs of their patients, also given the recommendations from the american heart association, the heart failure society of america, and the american college of cardiology. another news item was related to a possible association of ibuprofen to negative outcomes in covid cases. this led to the suggestion of using paracetamol over ibuprofen. in this other case, the who explained that the news was not supported by evidence and discouraged the non-utilization of ibuprofen. is there something else that you want to add? mc: thank you, manuel, for reaching out and i hope our experience will help canadian geriatricians and health-care workers dealing with older adults during this overwhelming pandemic. we are in uncharted territory. . older adults present the highest mortality rate in this pandemic, in the italian epicenter and worldwide. . clinical presentation in older adults with severe illness is quite sudden; patients can develop severe hypoxemia and need ventilation support in very few hours. . geriatric syndromes are overlooked as a form of presentation of covid- severe illness, being this dominated by severe respiratory symptoms. . stratification by frailty level may help to early detect the most vulnerable. . decision concerning health-care utilization resources should not be taken based only on age itself or isolated diagnoses, such as the presence of dementia; rather, advanced directives or functional/frailty stratification may be considered. md: bloomberg school of public health clinical ethics recommendations for the allocation of intensive care treatments, in exceptional, resource-limited circumstances a global clinical measure of fitness and frailty in elderly people the frailty phenotype and the frailty index: different instruments for different purposes clinical frailty scale in an acute medicine unit: a simple tool that predicts length of stay infection prevention and control guidance for long-term care facilities in the context of covid- : interim guidance are patients with hypertension and diabetes mellitus at increased risk for covid- infection? the authors declare that no conflicts of interest exist. key: cord- -c np bar authors: vaira, luigi a.; salzano, giovanni; deiana, giovanna; de riu, giacomo title: anosmia and ageusia: common findings in covid‐ patients date: - - journal: laryngoscope doi: . /lary. sha: doc_id: cord_uid: c np bar in a not negligible number of patients affected by covid‐ (coronavirus disease ), especially if paucisymptomatic, anosmia and ageusia can represent the first or only symptomatology present. laryngoscope, europe and america currently represent the new front where the battle against severe acute respiratory syndrome coronavirus (sars-cov- ) is being fought. in the management of the health crisis, the identification of paucisymptomatic patients is emerging as a crucial factor in order to interrupt the transmission chain of the virus. in the centers that are facing this emergency, a significant number of patients presenting anosmia and ageusia associated with fever (> . c) as onset symptoms are being detected. this minor and nonspecific symptomatology can represent the only manifestation of the disease. mao et al. unfortunately, the actual emergency situation does not allow us to provide a precise incidence of the manifestation, but our estimates from history and physical examination of the first patients for whom we have data indicate that chemosensory dysfunction is present in . %. this incidence can be underestimated because the gustatory and olfactory function is not always investigated. anosmia has already been reported in the course of sars and other coronavirus , infections; however, it represents a rare occurrence. interestingly, in covid- patients ageusia and anosmia are not accompanied by nasal obstruction or other rhinitis symptoms. therefore, this is probably due to the direct damage of the virus on the olfactory and gustatory receptors. currently, it is not possible to determine whether there will be a full recovery of the olfactory and gustatory functions or how long it will take. however, on the basis of the experience that is being acquired in italy, we believe it is important to inform otolaryngologists colleagues to pay attention to these possible manifestations of sars-cov- infection. in a non-negligible number of patients, especially if paucisymptomatic, ageusia and anosmia can represent the first or the only symptomatology manifestation. it will be important, when feasible, to obtain olfactory and gustatory testing data on patients with confirmed covid- testing to provide quantitative data on the incidence and severity of these sensory losses. finally, understanding mechanisms of sensorineural olfactory loss with coronavirus infections might provide novel insights into aspects of viral pathogenesis. the authors would like to thank all italian health workers for the efforts and sacrifices they are making during this serious health crisis. neurological manifestations of hospitalized patients with covid- in wuhan, china: a retrospective case series study olfactory neuropathy in severe acute respiratory syndrome: report of a case long term serious olfactory loss in cold and/or flu identification of viruses in patients with postviral olfactory dysfunction olfactory mucosal findings and clinical course in patients with olfactory disorders following upper respiratory viral infection from the maxillofacial surgery unit (l.a.v., g.d.r.), university hospital of sassari, sassari, italy; department of medical, surgical and experimental sciences (g.d.), university of sassari, sassari, italy; and the maxillofacial surgery unit (g.s.), university hospital of naples "federico ii", naples, italy.editor's note: this manuscript was accepted for publication on march , .the authors have no funding, financial relationships, or conflicts of interest to disclose.send correspondence to luigi a. vaira, viale san pietro /b, sassari, italy. e-mail: luigi.vaira@gmail.com key: cord- -n c x q authors: ussai, silvia; armocida, benedetta; formenti, beatrice; palestra, francesca; calvi, marzia; missoni, eduardo title: hazard prevention, death and dignity during covid- pandemic in italy date: - - journal: front public health doi: . /fpubh. . sha: doc_id: cord_uid: n c x q on march , italy passed the prime minister's decree n. , establishing urgent measures to contain the transmission of covid- and prevent biological hazards, including very restrictive interventions on public holy masses and funerals. italy banned burial procedures based (i) on the recent acknowledgment about the virus environmental stability as well as (ii) its national civil contingency plan. hence, only the cremation process is admitted for covid- deaths. viewing of the body is permitted only for mourners, which are allowed to perform the prayer at the closing of the coffin and the prayer at the tomb (cf. rite of succession, first part n. and n. ). the dead cannot be buried in their personal clothes; however, priests have been authorized to put the family clothes on top of the corpse, as if they were dressed. burying personal items is also illegal. the dignity of the dead, their cultural and religious traditions, and their families should be always respected and protected. among all the threats, covid- epidemic in italy revealed the fragility of human beings under enforced isolation and, for the first time, the painful deprivation of families to accompany their loved ones to the last farewell. ethics poses new challenges in times of epidemics. on march , italy passed the prime minister's decree n. , establishing urgent measures to contain the transmission of covid- and prevent biological hazards, including very restrictive interventions on public holy masses and funerals. italy banned burial procedures based (i) on the recent acknowledgment about the virus environmental stability as well as (ii) its national civil contingency plan. hence, only the cremation process is admitted for covid- deaths. viewing of the body is permitted only for mourners, which are allowed to perform the prayer at the closing of the coffin and the prayer at the tomb (cf. rite of succession, first part n. and n. ). the dead cannot be buried in their personal clothes; however, priests have been authorized to put the family clothes on top of the corpse, as if they were dressed. burying personal items is also illegal. the dignity of the dead, their cultural and religious traditions, and their families should be always respected and protected. among all the threats, covid- epidemic in italy revealed the fragility of human beings under enforced isolation and, for the first time, the painful deprivation of families to accompany their loved ones to the last farewell. ethics poses new challenges in times of epidemics. on march , , the world health organization (who) released its guideline on "infection prevention and control for the safe management of a dead body in the context of covid- " ( ) . the document offers the most updated recommendations on the safe and dignified burial procedures of deceased persons with suspected or confirmed covid- . these include, among others: ( ) the lack of evidence-which does not mean that we may exclude the possibility of future evidence emerging-of human transmission to subjects having become infected from exposure to the bodies of persons who died with/for covid- . this, in contrast with ebola or marburg diseases, where dead bodies are known to be associated with contagion; ( ) the option for decedents with confirmed or suspected covid- to be buried or cremated; ( ) respect of customs, with family's chance to view the body after it has been prepared for burials, using standard precautions at all times including hand hygiene; ( ) body wrapping in cloth and deceased transfer as soon as possible to the mortuary area. who recommendations are released in the form of interim guidance, subjected to revision as new evidence becomes available. national healthcare authorities are fully empowered in leading local actions according to the context and customs. italian government adopted the highest level of precautions given its exceptional number of deaths ( . , june ) and the limited knowledge on this novel virus. on march , italy passed the prime minister's decree n. ( ) , establishing urgent measures to contain the transmission of covid- and prevent biological hazards, including very restrictive interventions on public holy masses and funerals. in coordination with the measures launched by the italian authorities, the italian conference of bishops (conferenza episcopale italiana) issued a statement describing actions taken by the vatican to limit the spread of covid- ( ) . severe measures that entail stringent restrictions on freedom of movement and association affecting the right to decent burials can be hugely distressing for families, exacerbating their grief. when balanced against public health interests, a basic rule is that governments should employ the least restrictive means necessary to protect public health. during the emergency phase, italy banned burial procedures based (i) on the recent acknowledgment about the virus environmental stability ( ) as well as (ii) its national civil contingency plan. hence, only the cremation process was admitted for covid- deaths. the visit to the body was equally forbidden by the health authority. therefore, in addition to the funeral ceremonies, any prayer at the closing of the coffin was suspended as well. viewing of the body was permitted only for mourners, which were allowed to perform the prayer at the closing of the coffin and the prayer at the tomb (cf. rite of succession, first part n. and n. ). the dead could not be buried in their personal clothes; however, priests have been authorized to put the family clothes on top of the corpse, as if they were dressed. burying personal items was also illegal. funeral gatherings were not permitted and family members of sars-cov- victims were either denied to participate at the burial as they themselves were, most of time, under quarantine. in order to minimize delays between time of death and cremation, deceased were taken straight to the cemetery where a brief rite of burial was celebrated. all the masses in suffrage of the deceased with the family have been postponed after the emergency. italy's mortuary industry has been overwhelmed as the number of dead kept rising. in bergamo, a city in lombardy region with the highest number of covid- cases in italy, the capacity to manage dead bodies exceeded. the time frame set by law from the death to the burial was up to h. however, due to the unprecedented amount of deaths, certain areas experienced a min turnover procedure because of the pressure created by the number of corpses, as caskets have been piling up in churches instead of the local cemeteries, which were full. the military stepped in to move about coffins to other provinces and regions for timely burial procedures ( ) . the cremation followed the standard procedure foreseen for biological hazard risk. for instance, in order to increase the capacity of each burial facility and in compliance with all hygiene requirements, safety and environmental regulations, alternative technical solutions have been allowed for each cremation to shorten the burial execution time, for example by accelerating the ignition of the coffin. the use of easily inflammable wooden coffins has been encouraged in cremation and only the use of a zinc inner bonnet was permitted. in case of massive transportation of crematorium coffins, these have been carried out with a closed truck, also military, to be disinfected properly after use, preferably internally covered with waterproof material easily washable. furthermore, in the cemetery register it was mandatory to indicate that the coffin was packed for the burial of the deceased with a contagious infectious disease by affixing the code "y" ( ) . currently, the recovery phase is easing restrictions on funeral and burial procedures. family members (up to people) can participate in the holy mass; any physical contact between the participants must be avoided. the funeral should preferably take place outdoors and it is mandatory the use of personal protective equipment (ppe) as well as the strict observance of the interpersonal safety distance of at least m ( ). on march , the italian government proclaimed a national day of mourning, inviting all public institutions to expose the italian flag at half-mast "as a sign of mourning for the victims of the coronavirus, of proximity to their families and of national participation in the condolences to the most affected communities." authors acknowledge that in the absence of any pharmaceutical intervention, the only strategy against covid- was to reduce mixing of susceptible and infectious people through early ascertainment of cases or reduction of contact. although biologic hazard prevention actions (including death management) have been praised by who, the possibility of imposing severe restrictions on death during covid- as adopted by the italian government raises important questions. the population requires and deserves assurance that the decision to enact these measures affecting vital cultural practices as faith-based services has been informed by the best attainable evidence. it is therefore relevant that policy makers maintain the public's trust through use of rigorous scientific assessment of risk and effectiveness. yet, burial restrictions in italy have been imposed without any individualized risk assessment. difficult questions will then arise, though. for example, was complete funeral ban necessary, or might the final goodbye be still said by families while practicing physical distancing? the dignity of the dead, their cultural and religious traditions, and their families should be always respected and protected. among all the threats, covid- epidemic in italy revealed the fragility of human beings under enforced isolation and, for the first time, the painful deprivation of families to accompany their loved ones to the last farewell. infection prevention and control for dead body management in the context of covid- : interim guidance ulteriori interventi urgenti di protezione civile in relazione all'emergenza relativa al rischio sanitario connesso all'insorgenza di patologie derivanti da agenti virali trasmissibili. (ordinanza n. ) -serie generale decreto "coronavirus": la posizione della cei persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents altro giorno di lutto per bergamo: bare sui camion dei militari oggetto: indicazioni emergenziali connesse ad epidemia covid- ministry of health publicly available datasets were analyzed in this study. this data can be found here: http://www.protezionecivile. gov.it. all persons listed as authors have contributed to preparing the manuscript and their authorship meets the international committee of medical journal editors (icmje) criteria. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © ussai, armocida, formenti, palestra, calvi and missoni. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - zu mna authors: borghese, a.; moioli, b. title: buffalo: mediterranean region date: - - journal: reference module in food science doi: . /b - - - - . - sha: doc_id: cord_uid: zu mna the buffaloes reared in the mediterranean region are the asian buffalo or water buffalo, that is, bubalus bubalis. this species includes two types: ( ) the river type, with chromosomes, with an adult male weighing between and kg and with an annual milk production of – kg; and ( ) the swamp type, with chromosomes, with an adult male weighing between and kg and with an annual milk production of up to kg. the river buffalo is reared mainly for milk, whereas the swamp buffalo is reared mainly for draught. only % of the world buffalo population is reared in the mediterranean region. significant numbers of buffaloes are at present found only in italy, romania, egypt, turkey, azerbaijan, iraq and iran. in all these countries, buffaloes represent only a very small portion of total livestock, except in egypt, where buffaloes are more numerous than cattle. because of the strong market demand for buffalo cheese, the number of buffaloes has increased in italy and there is a preference for buffalo dairy products compared to cows' milk products in a few countries. major morphological differences between the buffalo populations of different countries include ( ) the variable size, ranging between a minimum of and kg liveweight for adult females and males, respectively, in egypt to a maximum of and kg in iraq, the most frequent weights being and kg; ( ) the shape of the horns; and ( ) the coat color, from dark gray and dark brown to black, showing white spots in some cases. a dual-purpose line of buffaloes is maintained, and a maximum of % in italy, where males have no market. in countries where the majority of buffaloes are reared in very small herds there are also a few bigger private, cooperative or state herds (bulgaria, egypt and turkey) ( figure ). the number of calves produced per cow per year varies from . in egypt to . in azerbaijan and syria, with average of . . the age at first calving averages months. in italy a good proportion of buffaloes calve at months of age, whereas in egypt and syria a high number are months or more at first calving. in mediterranean countries, all herds have their own bull except in the areas with very small herds ( - breedable buffaloes) in romania, bulgaria, egypt and turkey, where there are groups of bulls for breeding at village level. official milk recording for the productivity of buffaloes is performed in egypt, italy, bulgaria, romania, the united kingdom, azerbaijan and iran. genetic evaluation of buffaloes is done in italy, bulgaria, romania, egypt, iran and azerbaijan. choice of breeding bulls/heifers is made on the basis of: where milk recording is not practiced, a better bull is judged on his appearance, size and strength. natural breeding stations exist in egypt and bulgaria. bulls in the breeding stations are provided by development programs of the government or other agencies. due to difficulties in estrus detection and lack of organization, artificial insemination (ai) is still practiced only to a very limited extent for buffaloes in the mediterranean region: in italy for . % of the buffaloes, in egypt and iran for . % and in romania for . %. in the large cooperative and state farms in bulgaria, ai is used on % of the buffaloes. in the other countries it is not used at all. the low use of ai has slowed down the implementation of national selection schemes for genetic improvement of milk productivity. research trials conducted in italy have indicated that artificial insemination is feasible and successful in buffaloes using the following technique to synchronize estrus and boost fertility. a progesterone-releasing intravaginal device (prid) of silicon coils is inserted and left for days; on the seventh day injections of iu of ecg (equine chorionic gonadotropin) and mg of luprostiol (prostaglandin f a ) are given; artificial insemination is performed at , and h after removal of the prid on day . the most common housing system is that referred to as 'traditional,' consisting of keeping buffaloes indoors at night and confined in fenced areas during the day (egypt, turkey, iraq, syria); in the favorable season they are allowed to graze during the day (romania, turkey and on some farms in italy). in the marshes in the southwest of iran, buffaloes are kept outdoors on pasture throughout the year, whereas in the northern areas, around the caspian sea, they are kept in barns in winter. lactating buffaloes are kept tied throughout the year in bulgaria, romania and azerbaijan. in italy, they are housed loose in paddocks all year, with the same modern systems used for dairy cows; one-third of italian buffaloes are also put on pasture in spring. one-third of iraqi buffaloes wallow in marshes all year, the water reaching a level halfway up their bodies. they swim far and wide to find food and when the water is high, they stand on platforms made of papyrus, reeds and mud; on these platforms the farmers sometimes build huts to house the buffaloes; these platforms can be pushed to different places in the marshes. the length of lactation varies from to days, the most frequent being . average lactation milk yield is kg. italian buffaloes give the highest average yield (almost kg) (figure ) , while in turkey, average yield is below kg. in the iraqi marshes buffaloes produce no more than kg in days. the fat content of the milk throughout the lactation is over % in italy, turkey, azerbaijan and iraq, around % in bulgaria, romania and egypt, and less than % in iran. the average daily milk yield of river buffaloes shows very wide variability, depending on the breed, the country and especially the management and feeding system. it can range from to kg day À for poorly fed animals (grazing and fed by products) to kg day À in intensive management systems. in the large commercial herds in italy and bulgaria, buffaloes are machine-milked, twice a day. in the smaller herds elsewhere in the mediterranean, buffaloes are usually hand-milked, and often the calf is allowed to stand with its dam to assist letdown which otherwise may be slow. extensive management systems, as practiced in europe and the near east, include grazing in the favorable seasons. in all cases, green forage 'cut-and-carry'composed of legumes varying from country to countryconcentrates and by-products are the basic foodstuff. green forage and hay are made mainly of lucerne (alfalfa) in italy, bulgaria, romania and turkey and trifolium alexandrinum in egypt. the most common by-products fed to buffaloes are brewers' grains in italy and bulgaria, sugarbeet pulp in italy, bulgaria and iran, cotton waste in egypt and azerbaijan, tomato peel in italy, apple-juice wastes in iran, sugar cane wastes in egypt and iran, maize stalk and cobs in iran, egypt and romania and straw everywhere. in the iraqi marshes, when the buffaloes return at night to the floating islands where they live, they are fed green forage cut by the farmer during the day; this forage is composed of reeds, papyrus, various water plants, and rice hulls when available. in italy, dairy buffaloes are managed in the same intensive way as dairy cows, maintained in loose housing paddocks throughout the year. maize silage and grass silage are the main feeds. average yearly milk production for buffaloes in italy is kg although % of recorded buffaloes yield more than kg. an example of feeding schedules for high-yielding buffaloes is given in table . similar high-energy diets ( . - . milk feed units (mfu) kg À dry matter) are used in italy for particular selected genotypes where highest milk yield is desired because of the high prices for buffalo milk and because such diets increase milk protein ( . - . %) and milk fat ( - %) resulting in higher cheese yields. buffalo pathologies are similar to those described for cattle. few diseases are peculiar to buffaloes. they tend to show more resistance to diseases than cattle, perhaps because of their adaptation to hot-humid climates. parasitic infections, particularly in developing countries, are very common in buffaloes. they include: gastrointestinal helminths (strongyloides, toxocara, moniezia, mammomonogamus) and coccidia (eimeria, giardia, cryptosporidium), liver parasites (fasciola), tick parasites (hyalomma, sarcoptes) blood parasites (theileria), all of which produce important economic losses in buffalo breeding. escherichia coli can cause gastroenteric pathologies in buffaloes, particularly in calves, associated with other bacteria (enterobacter, pseudomonas, klebsiella) or with coccidia or verminosis or virosis. respiratory diseases are caused by pasteurella, staphylococcus, streptococcus, e. coli, and can result in high mortality, if the animals are not treated with antibiotics. pasteurella multicida is responsible for hemorrhagic septicemia, the most serious disease in buffaloes because of the high mortality particularly in tropical asian countries; however, it can be controlled with antibiotics and vaccines. tuberculosis, produced by mycobacterium, is a serious zoonosis, which could be eradicated after tuberculin diagnosis. brucellosis, caused by brucella, is another zoonosis that needs to be eradicated after serological diagnosis. it can cause serious disease in the human population and reproductive disorders and infertility in buffaloes. vaccination can be applied only in developing countries. leptospirosis is another zoonosis, produced by infection with leptospira from water sources contaminated by rodents. listeriosis, caused by listeria, produces meningoencephalitis, abortion and septicemia: the source of the infection may be silage in the buffaloes' diet. chlamydia, rickettsia and johne's disease (caused by mycobacterium tuberculosis) are present even in developed countries also, and the evidence of mastitis incidence links them to dairy buffaloes. prophylaxis is very important to control some viral infections that could cause neonatal diarrhea (rotavirus, coronavirus), particularly in intensive systems. bovine rhinotracheitis (ibr), bovine diarrhea (bvd) and bovine herpesvirus (bhv) have all been diffused by animals in intensive buffalo farms. buffaloes are also susceptible to foot-and-mouth disease. buffaloes are affected by fungal infections, tumors and reproductive disorders including prolapse of the uterus. compared to that of cattle, buffalo milk is richer in fat ( - . %) and protein ( - %); it has a lower cholesterol content and higher tocopherol content. it is richer in calcium and phosphorus and has less sodium and potassium. peroxidase activity is - times higher than in cows' milk so that it can be preserved longer; b-carotene is more completely transformed to retinol and therefore the milk looks whiter than cows' milk. the milk can be consumed in liquid form or processed into a wide range of products, either alone or mixed with milk from other livestock: fermented milks, butter, ghee, condensed and powdered milk, and cheeses are produced. many farms produce their own cheese and cream which they sell directly. classifying the types of cheese according to water content, the following are typical: soft cheese (water content > %): karish, mish and domiati in egypt; madhfor in iraq; mozzarella in italy semi-hard cheese (water content - %): beyaz peyneri in turkey most of the cheeses produced in the mediterranean area, including mozzarella, belong to the acid-enzymatic category, meaning that acid coagulation prevails. the demand for high-quality in bulgaria, romania and albania from either buffaloes', cows' or sheep's milk. in egypt, rayat is produced from natural acidification of raw milk, after the removal of cream, with no addition of bacteria, and zabadi is produced industrially. raha is the fermented milk of iraq, produced either from whole or skim milk, and laban or khather are the fermented milks of syria, all of which are often produced from buffaloes' milk. creams creams also show variable techniques of production. in egypt, queshta mosakhana is the floating cream removed after boiling milk. gaymar in iraq is obtained both from spontaneous floating or from spinning, in which case it is then pasteurized. in italy, cream from buffalo milk is obtained after spinning the water buffalo conception rate in synchronized and artificially inseminated buffalo cows in two different seasons under field conditions improving reproductive efficiency in female buffaloes nutritional value and technological suitability of milk from various animal species used for dairy production proceedings of the th world buffalo congress buffalo production and research. fao, regional office for europe mediterranean milk and milk products reproductive cycles of buffalo proceedings of the international symposium on prospects for buffalo production in the mediterranean and middle east the husbandry and health of the domestic buffalo nutritional management for buffalo production proceedings of the th world buffalo congress key: cord- -mcru j g authors: indolfi, ciro; spaccarotella, carmen title: the outbreak of covid- in italy: fighting the pandemic date: - - journal: jacc case rep doi: . /j.jaccas. . . sha: doc_id: cord_uid: mcru j g nan the outbreak of covid- in italy has caused a never-seen-before disaster in terms of hospitalizations and deaths. on january , , the chinese center for disease control and prevention reported that a new coronavirus, severe acute respiratory syndrome coronavirus- (sars-cov- ), had been identified as the causative agent of coronavirus disease- , and the genomic sequence was made public. italy was the first european nation to be affected by covid- with , confirmed total cases and , deaths to date ( ) . the pandemic has mainly been located in northern italy (figure ), partially sparing, for the moment, the southern part of the country. italy was not prepared for covid- , currently a planetary health emergency with , , cases and , deaths worldwide ( ) . the italian crisis provoked by covid- is the most serious event in italian history after world war ii; it is a national human, health, and economic tragedy. covid- mortality in italy has been %, higher than that in china. the reasons for this high mortality are unclear. however, the infected fatality rate may actually be lower because the tests have not been widespread compared to other countries such as south korea. furthermore, the oldest population in italy may have increased mortality. in fact, the median age in italy of those who have died is w years (figure ). no patient < years of age has been hospitalized or has died. only % of the deaths have been detected in patients < years of age. on february , , the first italian patient with covid- was diagnosed, a -year-old man hospitalized at codogno hospital, lodi, in northern italy. also, in northern italy, on february , , another outbreak of viruses was discovered in vò euganeo (padua) and, in the veneto region, the first death was reported, a -year-old man in a hospital in padua. he was the first of a long series of deaths. the mortality rate in the lombardy region alone, with a total of , deaths, is greater than the number of deaths in china ( , total deaths). social containment, early and rapid throughout a nation, is the most effective measure for controlling the spread of covid- ; this social containment perhaps was delayed in italy ( figure ) . italy was the first nation in europe affected by covid- and was therefore caught unprepared. the rapid spread of covid- and the dangerousness of the disease, very different from the normal seasonal influenza, were perhaps initially underestimated. today, the entire nation is on lockdown, and cities and towns have become isolated, as seen in figure ( ). healthcare workers in italy paid a very high price with more than , confirmed cases, clinicians killed by covid- and many with burnout syndrome. the epidemic in italy has also found territorial medicine to be unprepared, which has not been able it has been previously reported that the quality of the universal italian health system and healthy behaviors have contributed in the past to the country's favorable overall health ( ). in a race against time in an unprecedented health national health emergency, with an organizational effort never seen before in italy, hundreds of doctors and nurses (many then sars-cov- positive) are fighting this horrible disease. the advantage of the italian health system is that all citizens may have access to medical therapies. however, in recent years, health policy has profoundly changed in italy. the number of beds has been reduced, and regional autonomy has accentuated inequalities in the quality of services on the national territory. there has been recent concern in the cardiology community about the possible negative effect of covid- in italy - : ---italy, as it has done in the past, will improve its health and economic systems after this tragedy. probably nothing will be like before, and this catastrophe will be a great opportunity to further improve an efficient and effective national universal health system. critical case utilization for the covid- outbreak in at the epicenter of the covid- pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation covid- and italy: what next? italy collaborators. italy's health performance, - : findings from the global burden of disease study key: cord- -w aa elj authors: tonetti, tommaso; grasselli, giacomo; zanella, alberto; pizzilli, giacinto; fumagalli, roberto; piva, simone; lorini, luca; iotti, giorgio; foti, giuseppe; colombo, sergio; vivona, luigi; rossi, sandra; girardis, massimo; agnoletti, vanni; campagna, anselmo; gordini, giovanni; navalesi, paolo; boscolo, annalisa; graziano, alessandro; valeri, ilaria; vianello, andrea; cereda, danilo; filippini, claudia; cecconi, maurizio; locatelli, franco; bartoletti, michele; giannella, maddalena; viale, pierluigi; antonelli, massimo; nava, stefano; pesenti, antonio; ranieri, v. marco title: use of critical care resources during the first weeks (february –march , ) of the covid- outbreak in italy date: - - journal: ann intensive care doi: . /s - - -z sha: doc_id: cord_uid: w aa elj background: a covid- outbreak developed in lombardy, veneto and emilia-romagna (italy) at the end of february . fear of an imminent saturation of available icu beds generated the notion that rationing of intensive care resources could have been necessary. results: in order to evaluate the impact of covid- on the icu capacity to manage critically ill patients, we performed a retrospective analysis of the first weeks of the outbreak (february –march ). data were collected from regional registries and from a case report form sent to participating sites. icu beds increased from to ( . %), and patients receiving respiratory support outside the icu increased from ( . %) to ( . %). patients receiving respiratory support outside the icu were significantly older [ vs. years], had more cerebrovascular ( . vs. . %) and renal ( . vs. . %) comorbidities and less obesity ( . vs. . %) than patients admitted to the icu. pao( )/fio( ) ratio, respiratory rate and arterial ph were higher [ vs. ; vs. breath/min; . vs. . ] and paco( ) and base excess were lower [ vs. mmhg; . vs. . ] in patients receiving respiratory support outside the icu than in patients admitted to the icu, respectively. conclusions: increase in icu beds and use of out-of-icu respiratory support allowed effective management of the first days of the covid- outbreak, avoiding resource rationing. data regarding the impact of covid- outbreak on the capacity of the health-care system to accomplish the need for icu care are limited. the estimated need for intensive care unit (icu) admission is variable, ranging between . [ ] , . [ ] and . % [ ] . reported icu mortality ranges between [ ] , [ ] [ ] , and % [ ] . this extreme variability has been attributed to differences in terms of beds availability, staff and organization of intensive care units [ ] . on thursday, th february , the first cases of positivity for sars-cov- were recorded in lombardy region, northern italy. since then, the number of patients with corona virus and acute hypoxemic respiratory failure in three regions of northern italy (lombardy, veneto and emilia-romagna) dramatically increased, subsequently leading to the call of a national emergency status [ ] . a mathematical model of the occupation of intensive care resources in italy predicted the saturation of the theoretical availability of beds on the national territory by mid-april [ ] . in order to respond to such predicted growing need for icu resources, on march st the italian government published a notice, ordering to increase the number of icu beds (https ://www.salut e.gov.it/porta le/homem obile .jsp) and approved a law decree that allocated million euros to the public health service to bring the number of icu beds for invasive mechanical ventilation to the % of the total hospital beds (https :// www.gazze ttauf ficia le.it/eli/id/ / / / g / sg). since the spread of the sars-cov- virus is growing and critical care resources of public health systems are dramatically challenged [ ] , we reasoned that a better understanding of clinical management and icu requirements for patients with severe covid- at the very beginning of the outbreak may support resources planning and may help to set effective organizational and clinical interventions for the most seriously affected patients. the objective of the study was therefore to ( ) describe the process of expansion of the icu capacity in response to the covid- outbreak during the first weeks of the pandemics; ( ) describe settings and modalities of care of acutely ill covid- patients; ( ) compare outcomes between critically ill patients with covid- receiving care in or outside the icu. we retrospectively studied consecutive critically ill patients with confirmed covid- who were referred to the hospitals of the lombardy, veneto and emilia-romagna regions during the first weeks of the italian outbreak (february - march , ) . a confirmed case of covid- was defined as a patient with a positive result on high-throughput sequencing or real-time reverse transcriptase-polymerase chain reaction assay of nasal and pharyngeal swab specimens [ ] . in total, hospitals ( in lombardy, in emilia-romagna and in veneto) participated in the study. institutional review boards reviewed the protocol and authorized data collection. data on icu beds expansion and on total hospital and icu admissions were gathered from registries of the regional icus coordinators of lombardy (ap), veneto (pn) and emilia-romagna (vmr) [ ] . moreover, a data collection form was circulated among participating icus and de-identified data on patients admitted in the icu and receiving respiratory support outside the icu were recorded h after admission. in particular, demographics, comorbidities and basic physiological data were collected. in the initial days of the epidemics in northern italy, icu beds and personnel were made available by closing elective surgical admissions and centralizing to a limited number of single non-covid- hub hospitals all neuro-and cardiac-surgical admissions. moreover, ordinary availability of icu beds in the three regions was increased from to ( . %); in particular, icu capacity increased by . % (from to ), . % (from to ) and . % (from to ) in lombardy, emilia-romagna and veneto, respectively. this was achieved by converting operating rooms, coronary units, step-down units and recovery rooms to fully equipped covid- icus. furthermore, the use of outof-icu respiratory support in the form of cpap or niv [ ] [ ] [ ] was extended to many different wards, although initial reports suggested caution in the use of non-invasive respiratory support in covid- patients due to the risk of transmission of infection [ ] . all patients included in the study underwent evaluation by a senior intensivist, who decided according to her/ his clinical judgment and to local protocols whether to treat the patient in a ward under supervision of the icu team or to admit the patient to the icu. the criteria for icu admission were: (a) failure of noninvasive respiratory support, defined as persistent hypoxemia, tachypnea and respiratory distress or development of hypercapnia despite the application of cpap/niv; (b) expected imminent need for invasive mechanical ventilation; (c) absence of a do-not-intubate order, as discussed collegially by the intensivist and the ward staff physicians caring for the patient. at all sites out-of-icu respiratory support was provided by care teams that included at least (i) a senior clinical staff with certified experience in intensive care medicine available around the clock; (ii) nurse support provided with a nurse/patients ratio ranging from : to : ; (iii) continuous monitoring of electrocardiogram trace, non-invasive blood pressure, oxygen saturation, and respiratory rate. conventional oxygen therapy was referred as applied through venturi or no-rebreathing masks. helmets were the interface systematically used to deliver cpap. niv was equally delivered through mask and helmets. highflow oxygen therapy was adopted in some units as an alternative to cpap. classification into oxygen therapy and non-invasive respiratory support followed the rule of the highest degree of support; accordingly, a patient receiving oxygen therapy at first and then escalating to non-invasive support was classified as receiving non-invasive support. continuous variables were expressed as medians and interquartile ranges (iqr). categorical variables were summarized as counts and percentages. no imputation was made for missing data. statistical analyses were descriptive. comparisons between groups were made using wilcoxon rank-sum and pearson's chi-square. all tests were -tailed and were considered significant if p < . . twenty-eight-day mortality of patients admitted in the icu through the period february -march , and of patients receiving respiratory support outside the icu through the same period was evaluated using the method of kaplan-meier. cumulative incidence of patients extubated and disconnected from mechanical ventilation was calculated and death was considered a competing event. patients were followed up until april th. all the analyses were performed with the use of sas software, version . (sas institute inc., cary, nc). in the period february th-march th, registries of the coordinating centers of lombardy, emilia-romagna and veneto showed that a total of patients were hospitalized for covid- and a total of were admitted and treated in the icu ( . %). data collection forms collected from the participating centers provided information on patients treated in the icu and on patients who received respiratory support outside the icu ( patients in total). notably, the number of patients receiving respiratory support outside the icu increased from ( . %) on february to ( . %) on march (fig. , top) , and the proportion of patients admitted to the icu declined from the . % of hospitalized covid- patients on february to the . % of hospitalized covid- patients on march (fig. , bottom) . compared to patients admitted to the icu, patients receiving respiratory support outside the icu were significantly older, had more cerebrovascular and renal comorbidities and fewer of them were obese. the attending intensivists deemed patients ( . % of the patients treated outside the icu) as non-eligible for further escalation of respiratory support (i.e., for invasive mechanical ventilation). in patients treated outside the icu, conventional o therapy was applied in the . % of the cases and non-invasive respiratory support (including niv, cpap and high-flow o therapy) in the . %, while . % of icu patients were intubated. pao /fio ratio, respiratory rate and arterial ph were higher and paco and base excess were lower in patients receiving respiratory support outside the icu than in patients admitted to the icu. (table ). the infectious disease and the pneumology wards were the most more common locations where out-of-icu respiratory support was delivered ( . % and . %, respectively) ( table ). patients receiving conventional o therapy outside the icu had less cerebrovascular comorbidities and obesity and had significantly higher values of pao /fio and arterial ph than patients receiving noninvasive ventilatory support outside the icu (including niv, cpap and high-flow o therapy). mortality did not differ between patients receiving conventional o therapy and non-invasive respiratory support ( . % vs. . %, respectively; table ). analysis of -day mortality showed a proportion of deaths of . % ( out of ) in patients treated in the icu and of . % ( out of ), in patients receiving respiratory support outside the icu (p = . ). nonsurvivors treated in the icu died within ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) days while in non-survivors receiving respiratory support outside the icu death occurred within ( - ) days. fortyfour patients in the icu group ( . %) and patients ( . %) in the out-of-icu group were still hospitalized through april th (last day of follow-up). the present study describes how the italian health-care system of three northern italian regions responded to the increasing need for clinical resources for critically ill patients during the first days of the covid- outbreak through the . % increase in icu beds and the increasing use of non-invasive respiratory support outside the icu. data to evaluate the impact of covid- outbreak on the capacity of the health-care system to accomplish the need for icu resources are limited. xie and coworkers reported that in wuhan as of feb , , there were about patients requiring ventilatory support with new patients every day. however, since only icu beds were available, three general hospitals were rapidly converted to critical care hospitals with a total of about beds dedicated to covid- critically ill patients [ ] . griffin and coworkers described the process to implement an icu surge capacity at the greater new york presbyterian system. in their experience, new covid- icus had to be rapidly assembled after the first weeks from the admission of the first critically ill covid- patients [ ] . concomitantly to the increase in icu bed capacity, there was a progressive increase in the number of patients who received respiratory support outside the icu (from . to . %) under the daily supervision of an intensivist. this allowed to reduce the percentage of patients admitted to the icu from . % on february th to . % on march th. the response between the italian and the greater new york presbyterian systems was similar, despite the different icu capacity ( . % of the total hospital beds in the usa [ ] vs. . % in italy (https ://www.salut e.gov.it/imgs/c_ _pubbl icazi oni_ _alleg ato.pdf ). this might be explained by the extensive use of out-of-icu respiratory support we adopted in italy [ ] [ ] [ ] . our data show that, compared to patients admitted to the icu, patients receiving respiratory support outside the icu were significantly older, had more comorbidities and had a higher pao /fio ratio and a lower paco . among patients treated outside the icu, proportions of patients treated with conventional o therapy and noninvasive respiratory support were comparable ( . vs. . %, respectively). the median age of our icu population [ years ( - )] is consistent with the one reported at national level in pre-pandemic times [ ] and, although it is difficult to draw conclusion from these data, it is probable that the same age criteria were adopted during the first weeks of the covid- epidemics in northern italy. patients receiving conventional o therapy outside the icu showed a pao /fio ratio higher than those receiving non-invasive support outside the icu, without differences in age and mortality. although a crude comparison of mortality is not very informative because of the baseline differences between the icu and outof-icu populations, we show here that the difference in survival at days in patients treated in the icu and those receiving respiratory support outside the icu was small ( . vs. . %, respectively). altogether these data seem to suggest that treatment outside the icu has been offered as a therapeutic setting proportional to patient's conditions and not as a 'limited' standard of care, always remaining within the ethical perimeter of standard clinical practice [ , ] . nevertheless, is unlikely that all eligible patients were transferred to an icu, and we cannot exclude that at least some patients who matched criteria for icu admission did not survive long enough to be transferred to icu or comorbid disease or goals of care precluded escalation to icu level care. non-invasive ventilation was suggested to be avoided in covid- patients due to the risk of transmission of infection [ ] . in our hospitals, the risk might have been reduced for the following reasons: (a) helmets equipped with high-efficiency particulate air filters at the peep port were the interface of choice for delivering non-invasive respiratory support in almost / of patients treated outside the icu; this interface might have avoided the dispersion of the multiphase turbulent gas cloud from coughing and sneezing on part of the patients, possibly reducing the transmission of covid- [ ] ; (b) about % of the patients receiving respiratory support outside the icu were treated in infectious disease wards that are commonly equipped with negative pressure rooms [ ] . moreover, there is growing evidence that niv can be safely performed outside the icu in covid- patients, and even advanced maneuvers such as prone positioning have been successfully tested in these patients [ ] . these data have may important implications for the reorganization required by health-care systems necessary to manage the covid- outbreak. the italian society of anesthesia, analgesia, resuscitation, and intensive care (siaarti) recommended an approach for resource allocation based on "clinical appropriateness" and "distributive justice" in case of significant mismatch between the number of patients requiring icu admission and the available resources and acknowledged that: "it is not about making choices on value, but to reserve possibly scarce resources first to who has higher probability of survival and second to who can have higher saved years of life, with the purpose of maximizing benefits for the highest possible number of people" [ ] . our data show that increasing the icu capacity by . % obtained through the reorganization of available facilities (conversion of operating rooms, coronary units, closure of all scheduled surgical activity) and use of out-of-icu respiratory support [ ] [ ] [ ] , the healthcare system was able to accomplish the clinical needs for respiratory support in covid- patients and may suggest that end-of-life practices might have remained within the ethical perimeter of standard clinical practice [ , ] . the retrospective nature represents the major weakness of this study. although data have been collected by personnel with experience in clinical research and strongly motivated to share their experience, the enormous clinical load and the risk of contagion have certainly influenced the quality of the data and limited the number of information that has been possible to collect. moreover, further analysis is needed to provide information regarding use of resources, allocation of beds, staffing choices, timing of opening up of new beds, and what resources were most stretched in the first weeks. moreover, the expected heterogeneity in hospital capacity and care practices between study hospitals may limit the practical utility of the description for clinicians facing an imminent surge of patients with covid- disease. despite these limitations, this study represents the first and most detailed description of the clinical reality of the first western country overwhelmed by the covid- epidemic. in conclusion, although our analysis confirms the grave concerns regarding the capacity of health-care systems to effectively respond to the covid- outbreak, these data show that the rapid increase in beds obtained through the reversal of already available resources into intensive care facilities and the use of out-of-icu respiratory support allowed to manage the first terrible days of the covid- outbreak. the present analysis shows that only rapid acquisition of new intensive care facilities with appropriate equipment and personnel and use of out-of-icu respiratory support [ ] [ ] [ ] may avoid the rationing of health-care resources that may be acceptable for "battlefield medicine", but should be incompatible with health-care systems founded on the principles of universality, solidarity and distributive justice (article of the constitution of the italian republic and law number december rd, ). manerbio (italy), benvenuto.antonini@asst-garda.it; nicolangela belgiorno, istituto clinico san rocco ), massimo_borelli@asst-bgovest.it; luca cabrini, ospedale di circolo e fondazione macchi, varese (italy), luca.cabrini@uninsubria.it; livio carnevale busto arsizio (italy), daniel.covello@asst-valleolona.it; gianluca de filippi, asst rhodense-presidio ospedaliero g milan (italy), deipolimd@gmail.com; paolo dughi, asst franciacorta -presidio ospedaliero di iseo vimercate (italy), giorgio.gallioli@asst-vimercate.it; paolo gnesin saronno (italy), stefano.greco@asst-valleolona.it; luca guatteri, ospedale "sacra famiglia" fatebenefratelli seriate (italy), roberto.keim@asst-bergamoest.it; giovanni landoni melegnano (italy), giovanni.marino@asst-melegnanomartesana.it; guido merli, asst crema-ospedale maggiore di crema, crema (italy), guido.merli@asst-crema.it; dario merlo fondazione poliambulanza istituto ospedaliero, brescia (italy), giuseppe.natalini@gmail. com; nicola petrucci, asst garda-ospedale di desenzano d/g, desenzano del garda (italy), nicola.petrucci@asst-garda legnano (italy), danilo.radrizzani@asst-ovestmi.it; maurizio raimondi, asst pavia-ospedale civile di voghera ), enrico.storti@asst-lodi.it; mario tavola zingonia (italy), giovanni.vitale@ grupposandonato.it mirano (italy), mauroantonio.calo@aulss .veneto.it; vinicio danzi, u.o.c anestesia e rianimazione ospedale cà foncello, treviso (italy), antonio.farnia@aulss .veneto.it; francesco lazzari, u.o.c. anestesia e rianimazione, ospedale dell azienda zero del veneto, padova (italy), mario.saia@azero.veneto.it; nicolò sella, u.o.c. istituto di anestesia e rianimazione, azienda ospedale università padova, nico.sella@hotmail.it; eugenio serra, u.o.c. istituto di anestesia e rianimazione, azienda ospedale università di padova, padova, eugenio.serra@ aopd.veneto.it; ivo tiberio, u.o.c. anestesia e rianimazione, azienda ospedale università padova, ivo.tiberio@aulss .veneto.it (italy), martina.bordini @studio.unibo.it; fabio caramelli, policlinico sant'orsola malpighi (italy), guido.frascaroli@aosp.bo.it; maurizio fusari, ospedale "santa maria delle croci (italy), costanza.martino@auslromagna.it; raffaele merola, policlinico sant'orsola-malpighi (italy), mrofrc@ unife.it; giuseppe nardi, ospedale "infermi rimini (italy), antonella.potalivo@auslromagna.it; francesca repetti, ausl piacenza, piacenza (italy), francesca.repetti@hotmail.it; pierpaolo salsi, azienda ospedaliera santa maria nuova, reggio emilia (italy), salsi.pierpaolo@ausl.re.it; marina terzitta modena (italy), tosimartina@gmail.com; sergio venturi, emergency commissioner for emilia-romagna region, sergio arcispedale sant' anna clinical characteristics of coronavirus disease , in china clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a singlecentered, retrospective, observational study clinical characteristics of hospitalized patients with, novel coronavirus-infected pneumonia in wuhan china baseline characteristics and outcomes of 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sars-cov- hospital preparedness for covid- : a practical guide from a critical care perspective critical care bed growth in the united states: a comparison of regional and national trends the influence of gender on the epidemiology of and outcome from severe sepsis changes in end-of-life practices in european intensive care units from to withholding or withdrawing of life-sustaining therapy in older adults (>/= years) admitted to the intensive care unit turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid- protecting healthcare workers from sars-cov- infection: practical indications respiratory parameters in patients with covid- after using noninvasive ventilation in the prone position outside the intensive care unit it/sitea ssets /news/covid % -% d ocume nti% s iaart i/siaar ti% -% c ovid- % -% c linic al% e thics % r eccom endat ions members of the covid- northern italian icu network: lombardy: giovanni albano, humanitas gavazzeni, bergamo (italy), giovanni.albano@gavazzeni.it; armando alborghetti, policlinico san pietro-ponte san pietro (italy), armando.alborghetti@grupposandonato.it; giorgio aldegheri, irccs tt, gg, az, pn, mc, fl, pv, ma, sn were responsible for study design, data acquisition, analysis, interpretation, and preparing the first draft of the manuscript. gp, rf, sp, ll, gi, gf, sc, lv, sr, mg, va, ac, gg, ab, ag, av, iv, dc, cf, mb mg, were responsible for data acquisition and data interpretation. ap and vmr were responsible for study design, data acquisition, analysis, interpretation, finalize the manuscript and study data integrity. all authors had an opportunity to review the manuscript and approved its final submitted version. no funding was provided. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the study was approved by the coordinating center's irb (comitato etico avec, bologna, italy) with approval number / /oss/aoubo; participant centers obtained approval from their respective irbs; consent to participate was waived for unresponsive, uncommunicative or deceased patients, in accordance to rule / of the italian privacy authority. key: cord- -kl aq ut authors: de leo, diego; trabucchi, marco title: the fight against covid- : a report from the italian trenches date: - - journal: international psychogeriatrics doi: . /s sha: doc_id: cord_uid: kl aq ut nan our senior citizens are the most exposed to the consequences of the covid- . frailty caused by comorbidities makes the advanced age of many people particularly vulnerable to the infection of this new coronavirus, the characteristics of which are still largely unknown to fellow scholars. what is clear is that older adults die more frequently than younger age groups, everywhere in the world. in the veneto region, . % of individuals who died were + years old, with the mean age of those who lost their lives to coronavirus being years (regione del veneto, ). in the last days, italian citizens were hugely impressed by watching on television a long series of military trucks transporting the coffins of dead people away from their home places because there is no more room in the close cemeteries or opportunities for cremation. the mental health of our seniors is particularly challenged: they feel scared by the news and aware that, if infected, they would not receive the same attention (e.g. intubation, a bed in resuscitation unit, etc.) of younger individuals. older adults with mental health conditions feel more frail and vulnerable than before: contacts with carers are now reduced to the minimum, with loneliness and abandonment becoming an excruciating reality. checking on regular assumption of drug therapies may become problematic; eating properly and keeping with personal hygiene at a sufficient level can also be quite difficult. this may increase the sense of demoralization and despair in people. a few cases of suicide have been signaled by media (gazzettino, ) , but in the present chaotic situation, it would not be surprising if similar fatalities would remain mostly undetected. demented people are particularly exposed to the impact of covid- ; there are anecdotal reports from domiciliary care nurses and staff in nursing homes that cases of delirium are on sharp increase. this could be justified by the positioning of the virus in the central nervous system (li et al., ) . on the other hand, in the present situation, caregivers of people with dementia are also exposed to extra stress: limited opportunities to offer the usual level of care; food and cleaning management more problematic; worries and concerns for the possibility of contaminating an older adult that would not survive the disease; and, in a situation like the italian one, the many "badanti" (carers from eastern europe) without a regular contract (rugolotto et al., ) , now impeded to reach the home of the older adults they take care of because they are intercepted by the police at check points. while we are writing this commentary from italy (april th), it appears that the number of people tested positive to the virus in the united states has almost quadrupled the number of people found positive in our country; however, italy counts a disproportionately high number of deaths. chinathe country where everything reportedly originatedhas now smaller figures than those of the us, italy, spain, and germany. these countries, together, make more than one million people detected as infected (worldometers, ) . not much can be said about the reliability of these figures or their comparability. it is good to say that the numbers are based on subjects who tested positive to the swab. unfortunately, italy has lost the opportunity to keep reliable records of those subjects. so, it is not known what is the percentage of those who repeated the test several times but are represented as different individuals; equally unknown, because not standardized, are the criteria for which the tests were performed, if to patients with initial symptoms (fever? fever and coughing? fever, coughing, and dyspnea? what?); with "full-blown" clinical syndromes (severe symptomatology); to individuals potentially exposed by contiguity or direct contact (living in the same environment does not necessarily imply proximity or contact), etc. in essence, it is not known in which of these situations the swabs have been made. in this waywe believea big opportunity to study contagion modalities has been lost. each region of italy has run autonomous strategies to protect its citizens and counteract the disease. this has applied also to testrelated policies. thus, aggregating data from regions looks like a problematic, though unreliable, process. we were expecting tests to be performed to all health personnel of common health settings: from general medicine surgeries to protected residences (including those for older adults), to emergency departments, and to general hospitals. unfortunately, this elementary strategy to guarantee that health professionals were sufficiently protected toward the virus was not applied with due care, and up to date the number of doctors who died from the infection during their professional activity is unbearable ( victims, at the time of writing). the number of other health workers who have lost their life is also tragically unbearable: . it has been reported that the costs involved in performing the tests in appropriate numbers (at least to all health professionals and people presenting with light symptoms) are too heavy, and the availability of skilled technicians and reagents for carrying out the tests is scarce. in italy, and probably elsewhere, nothing is and will be like before the pandemic. for the moment, we must overcome the crisis, but there could be a lot to say, on a clinical level, on that of the organization of services, and on how to protect the life of our older adults. the latter have been too affected by imprudent and superficial colleagues to feel accepted by the community again; in a few days, in deciding if the death was "by coronavirus" or "with coronavirus," we have undone decades of geriatric education, but above all, we have destroyed a fundamental relationship of trust. how will we approach patients if they are now convinced that today we ask for their age to make critical decisions (intubation) and maybe tomorrow the administration of expensive drugs? a discrepancy between the gift of a long life and public acceptance of older adults is increasingly noticeable and clearly expressed by the deprecated document on "damages caused by longevity," which christine lagarde (now the president of the european central bank) spoke about a few years ago ( ): "old people live too long and this is a risk for the global economy. we must do something, urgently." the scenario we are witnessing today is characterized by different situations in which people affected by covid- are forced to live. the main ones are schematically summarized below, even if there are significant differences, related to the different organization of services in the regions: a. older adults who remain at home even when the first symptoms appear are entrusted in most cases to themselves and to the care of their loved ones. family doctors are afraid; they tend to block visits, and families do not call emergency services because they fear seeing the family member get on the ambulance and not being able to greet him/her before they die. so they keep the sickespecially if a seniorin their homes, assist them with fear and affection until the negative or positive resolution of the clinical picture. there is no mention of swabs or blood chemistry analyses, let alone ct or other scans. our fellow citizens risk ending their lives without even being told what is happening to them. many seniors live alone; many have limited or no familiarity with the internet and are poorly connected with other family or community members (newman and zainal, ) . some die in complete isolation and their corpses are discovered many days after death (gazzettino, ) . b. in the last couple of months, the people who live in nursing homes have been completely forgotten by administrative powers. frail older adults are certainly the subjects who have suffered the most from the difficulties of the italian health system. in cities of lombardy such as bergamo and brescia, the deaths have followed at an impressive rate giving rise to sad images in the televisions of the italians of long lines of military trucks carrying coffins to incinerators, often very far from the place of origin of the deceased. as reported elsewhere (trabucchi and de leo, in press) , in just days, in the nursing homes of the province of bergamo (lombardy), which have a total accommodation capacity of , beds, there were more than deaths, which is a hecatomb. the real dimensions of the phenomenon will only be known after some time; it is probable that the number of infected people is far greater than that officially reported. it is also likely that the number of deaths is far greater than that officially attributed to covid- . no attention was paid to those who manage the residences, no support in terms of means of protection, the possibility of making swabs, or even economic support in the face of damage caused by the release of beds caused by the death of guests. the access of new guests has been blocked, but the residences are increasingly pressed to admit elderly people discharged from hospitals. the staff is exhausted, as a consequence of heroic commitment. in small communities, the warmth that stirs around the residences compensates for the solitude of guests and operators, but this is not the case in large centers, where the bigger turnover of personnel makes the atmosphere more impersonal. in any case, since doctors and other operators get sick in rapid progressionand despite the extreme commitment of those on dutyguests feel progressively more fearful and abandoned. c. in italy, hospitals are at the center of the covid- cyclone: the attention of the media and the whole nation is focused on them. schematically, the hospital system moves according to this model: (a) the emergency department, where patients arrive after days spent at home, sometimes already exhausted by the disease. it is rarely possible to send them back home: in most cases, they are kept under observation, waiting for a bed; (b) resuscitation units, where patients are intubated and followed mainly by doctors specialized in anesthesia and resuscitation; and (c) departments where patients are treated with oxygen therapy (with different levels of intensity) and checked for overall health conditions, including psychological problems. at this point in time, all remaining wards for the management of the diseases that "normally" refer to hospitals are in progressive reduction. to cope with the emergency, resuscitation units were built in record time, and entire hospitals were reconverted into the care of covid- . unfortunately, the pandemic has found the country largely unprepared and unable to provide the necessary protection in time even to its health workers, resulting in serious shortcomings regarding the supply of eyeglasses, masks, gloves, and gowns. as said, this has resulted in the loss of far too many health professionals' lives. the very serious difficulties in assisting critical situations, combined with the scarcity of places suitable for the reception of patients in severe conditions and the lack of a sufficient number of ventilators, have given rise to very painful ethical choices for health professionals on whom to privilege in the care with available equipment (rosenbaum, ) . d. from hospitals, patients are discharged when they achieve clinical recovery; they can return home, or to low-intensity "hotels," where they can manage themselves in isolation. the problem of older adults remains unsolved; often, after reaching clinical recovery, they show such a degree of disability that returning home is not possible, and it is difficult to resort to rehabilitation or long-term care facilities. the described picture seems to apply to many northern territories of italy. any criticism must be postponed; however, we seriously doubt that transferring people discharged from hospitals to rest homes may represent an appropriate choice. and certainly we should find a way to respond to those families that cannot accompany their loved ones to intensive care units and are left without any information and prognostic indication. this often means that the patients were kept too long at home, without effective tools to catch the signs of aggravation in time, which is frequently sudden and characterized by the discrepancy between symptoms and objectivity (e.g. oxygen saturation or temperature). the south of italy is generally less equipped than the north of the country, and grave concerns rest on a possible dramatic evolution of the next few days over there. social distancing and isolation remain at the moment the only strategies available to citizens. however, the latter need to feel supported by governments; mental health should soon regain a very high status on the agenda of all nations, especially if confinement at home expects to be very long, and financial, family, and relationship problems risk to aggravate a future now seen with serious concerns and deep anxieties. there are too many unanswered questions regarding the disease, and even the duration and quality of immunity post-infection remain a big question mark. while waiting for a vaccine to be ready, several protocols and drugs are being proposed by clinicians. noneat the momentappears as particularly promising. however, we remain confident that a serious and open collaboration among researchers, institutions, and countries across the globe will offer concrete hope for our future. for the time being, the mental health of people needs to be supported in any possible way. social distancing, loneliness, forced isolation, and fear of contracting the illness are all big challenges for the general population facing the expansion of the epidemic, but the risk of psychological consequences can be greater for the frail senior (armitage and nellums, ) . we need to activate all possible opportunities to offer help, at least in the form of tele-assistance, to our patients (krysinska and de leo, ) . psychological support should be made available to all via ngos and public services, with contact with psychiatrists and other physicians actively established. as much as possible, health professionals should contact their patients and make the continuity of care a reality as soon as possible. active outreach seems to be imperative, especially for older adults (de leo et al., ) , in order to counteract feelings of abandonment and disempowerment that covid- is imposing on all community members, especially to the most fragile ones. covid- and the consequences of isolating the elderly suicide among the elderly: the long-term impact of a telephone support and assessment intervention in northern italy e' contaminato" [coronavirus positive: -year-old businessman kills himself in the house. those tickets in front of the house telecommunication and suicide prevention: hopes and challenges for the new century presentation of the global financial stability report the neuroinvasive potential of sars-cov may play a role in the respiratory failure of covid- patients the value of maintaining social connections for mental health in older people daily reports on covid- facing covid- in italy -ethics, logistics, and therapeutics on the epidemic's front line how migrants keep italian families: badanti and the private care of older people nursing homes or besieged castles: covid- in northern italy coronavirus update live key: cord- - h rmwk authors: michelozzi, paola; de’donato, francesca; scortichini, matteo; de sario, manuela; noccioli, fiammetta; rossi, pasqualino; davoli, marina title: mortality impacts of the coronavirus disease (covid- ) outbreak by sex and age: rapid mortality surveillance system, italy, february to april date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: h rmwk data from the rapid mortality surveillance system in major italian cities were used to carry out a timely assessment of the health impact of the covid- epidemic. by april, a + % excess in mortality was observed, with a higher impact in the north of the country (+ %). the excess was greatest among men, with an increasing trend by age. surveillance data can be used to evaluate the lockdown and re-opening phases. italy has been one of the countries worst hit by coronavirus disease , with over , cases and around % registered in north of the country [ ] . numbers in the initial phase of the outbreak in italy seem to suggest a greater severity of the disease, with a higher case fatality rate (cfr) than previously observed in china ( . % vs . %) [ ] . the aim of the study was to estimate the excess in total mortality by age and sex during the epidemic in italian cities. since , italy has had a rapid mortality surveillance system (sismg) for real-time monitoring of daily deaths in major italian cities and allows routine evaluation of the health impact of extreme events and influenza epidemics [ , ] . this surveillance system was a valuable tool for an early evaluation of the direct or indirect impact of covid- on health. it is a standardised surveillance system capable of detecting variations in total mortality in the entire population rather than only on the infected cases, and it does not depend on a specific case definition (i.e. covid- -related deaths). briefly, sismg is based on an ad hoc daily flow of mortality data (resident population by age and sex) from local municipal registry offices to the department of epidemiology, lazio regional health authority -asl roma (deplazio) which manages the system on behalf of the ministry of health [ , ] . the standardised methodology to evaluate excess mortality typically used in the italian national surveillance system was considered when estimating the excess related to covid- [ , [ ] [ ] [ ] . specifically, the excess was defined as the difference between observed and baseline daily mortality (mean daily value by week and day of the week in the past years). in this report, we show results for a subgroup of cities, representative of almost all italian regions, with timely updates of data, corresponding to million residents ( % of the italian population). figure shows the weekly trend in observed and expected (baseline) mortality in northern vs central and southern cities of italy included in sismg. shaded areas represent the confidence bands calculated as the estimated baseline +/− . × standard deviation of the series of observed deaths contributing to the baseline. from the first week of march, a steep rise in mortality was observed in cities in northern italy ( figure ). from the start of the covid- epidemic until april, an overall , (+ %) excess deaths were observed in italian cities, with a significantly higher excess in cities in the north (+ %, + , deaths) compared with the centre and south (+ %, + deaths). city-specific mortality data showed the highest excess in the cities in the north, especially brescia (+ %) and milan (+ %), lombardy, genoa, liguria (+ %), turin, piedmont (+ %), verona, veneto (+ %) and bologna and emilia-romagna (+ %). in contrast, the south and central cities recorded a more contained (+ % in rome, + % in messina) or no excess. figure shows excess mortality by sex and age groups among cities in the north and in the centre and south of italy caused by the covid- outbreak up until april . overall, the excess in mortality was higher among men than among women in cities in the north vs the centre and south of italy (men:+ % and + % and women: + % and + %, respectively), with an increasing trend by age. the greatest excess in the north was among elderly men (+ % in - yearolds, + % in - year-olds and + % in those years and older). in central and southern italy, the excess in mortality among men was lower, with a statistically significant excess only among elderly men: + % and + %, respectively, in the - years and ≥ years age group. our results confirm that the epidemic had the strongest impact in the most affected areas of the north. national covid- data show that the lombardy region alone accounted for % of cases in italy, followed by the neighbouring regions of emilia-romagna ( %), piedmont ( %) and veneto ( %) [ ] . it should be considered that these four northern regions accounted for ca % of national trade and economic activity [ ] , which could have accelerated and amplified the spread of infection to and within this area of italy. the regions of central and southern italy registered nearly a week's delay in the outbreak and cases are to date much lower, with ca % of total cases [ ] . centralsouthern cities were comparatively less affected, possibly because containment measures were put in place before the emergency stage was reached. when considering the impact of covid- mortality, we should take into account the low mortality of the / winter, below the baseline from october to the end of february, which was attributable to the mild intensity of the influenza season and a warmer than average temperatures as shown in the latest sismg report [ ] . this phenomenon of lower mortality may have led to a greater number of vulnerable individuals exposed to the covid- epidemic, thus amplifying the impact on mortality in our country. prevalence of chronic diseases is an important risk factor to take into account when analysing excess mortality related to covid- . previous studies have shown that both disease severity and death risk are higher in patients with co-existing illnesses [ , ] . recent data from italy show that ca % of covid- deaths occurred in people with three or more comorbidities, mainly hypertension ( %), type- diabetes ( %), chronic renal failure ( %) and ischaemic heart disease ( %) [ ] . in italian cities, the higher mortality impact in men confirmed findings from a previous study in italy [ ] . furthermore, experimental studies suggest that men are more susceptible to respiratory viral infections because of hormonal and epigenetic mechanisms involving innate immunity [ ] . this has also been shown in animal studies for severe acute respiratory syndrome (sars) coronavirus [ ] . another potential risk factor reported in the literature is smoking. firstly, smoking is detrimental to the immune system and its responsiveness to infections and, specifically, it is able to upregulate the ace- receptors in the airways, the key for sars-cov- virus entrance into the epithelial cells [ ] . a recent review showed that smoking is most data source: sismg surveillance system, ministry of health italy [ , ] . probably associated with a negative progression and adverse outcomes of covid- [ , ] . the higher prevalence of male smokers for all ages in italy, especially among the elderly [ ] , may explain their higher predisposition to covid- . furthermore, the potential role of air pollution has been as a possible factor influencing covid- transmission and an effect modifier of the number of cases [ , ] . populations with a chronic exposure to high levels of air pollution, such as in the north of italy [ ] , may have a greater predisposition to develop respiratory symptoms, which may make them more susceptible to covid- . however, evidence on the direct and indirect role of air pollution is to date limited and the causal pathways and the differential risk attributable to this in different geographical areas need to be further investigated. considering our findings in the european context, the latest euromomo bulletin shows a steep increase in excess all-cause mortality in several european countries, mostly in the age groups ≥ and - years [ ] . a comparison of official covid- deaths in europe and the united states suggests that adults younger than years had a lower death risk than elderly people, with high geographical variability, and that more than % of deaths occurred among people with previous illnesses [ ] . the different risk factors associated with covid- predisposition and severity need to be taken into account when assessing the geographical heterogeneity in the risk of covid- -related deaths in the different population subgroups. since the beginning of the italian epidemic, efforts have been made to estimate the impact, which could possibly be greater than indicated by official data [ ] because of a delay in death ascertainment and lack of specificity of covid- -related deaths. despite these methodological challenges, rapid surveillance systems can help provide timely updates of the overall impact at population level and reference data for evaluation studies on lockdown measures during the epidemic. they can also help guide the re-opening phase in different areas. rome: department for civil protection case-fatality rate and characteristics of patients dying in relation to covid- in italy the impact of the february cold spell on health in italy using surveillance data excess all-cause and influenzaattributable mortality in europe surveillance of summer mortality and preparedness to reduce the health impact of heat waves in italy sistema di sorveglianza della mortalità giornaliera -rapporto settimanale. settimana - aprile department of epidemiology regional health service lazio (deplazio) on behalf of the ministry of health. mortalità giornaliera (sismg) ed analisi della mortalità cumulative nelle città italiane in relazione all'epidemia di covid- . sesto rapporto febbraio - aprile statistiche import export. graduatoria delle regioni italiane per valore delle esportazioni/importazioni in base ai dati clinical characteristics of coronavirus disease in china sex difference and smoking predisposition in patients with covid- sex hormones regulate innate immune cells and promote sex differences in respiratory virus infection do men have a higher case fatality rate of severe acute respiratory syndrome than women do? covid- and smoking: a systematic review of the evidence la sorveglianza passi d'argento. i dati per l'italia: abitudine al fumo the short-term effects of air pollutants on influenza-like illness in jinan, china exposure to air pollution and covid- mortality in the united states estimation of daily pm and pm . concentrations in italy, - , using a spatiotemporal land-use random-forest model the european mortality monitoring bulletin for week populationlevel covid- mortality risk for non-elderly individuals overall and for nonelderly individuals without underlying diseases in pandemic epicenters the authors would like to acknowledge the ministry of health -ccm program for funding the sismg. none declared. pm planned the study and revised manuscript; fdd conceived the analysis plan and wrote manuscript and coordinate sismg; ms conceived and did the analysis and manage sismg; pr was in charge for overall direction and planning; mds helped in writing manuscript; fn helped in sismg data management; md contributed to the interpretation of the results and drafting the manuscript. all authors provided critical feedback and helped shape the research, analysis and manuscript. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- -i t v authors: cesari, matteo; proietti, m. title: geriatric medicine in italy in the time of covid- date: - - journal: j nutr health aging doi: . /s - - -z sha: doc_id: cord_uid: i t v nan on march , , the world health organization declared the sars-cov- outbreak a pandemic ( ) . to date, the number of coronavirus cases has passed , worldwide, and more than , persons have died. after china, italy is the country that has been most violently hit by the covid- tsunami. with , cases and , deaths (updated on march th, ), the case fatality rate due to covid- in italy is one of the highest in the world (about . %), well above what has been recorded in china ( ) . in particular, the lombardy region (with its cities of bergamo, brescia, lodi, and milan) is paying the heaviest toll to the coronavirus in terms of fatalities ( ) . the vast majority of deaths is occurring in older persons, a fact that can only be partially explained by the older age of the italian population. in fact, although current data indicate that persons aged years and older contribute to about the % of the death events in italy, it cannot be overlooked the fact that japan has substantially smaller figures despite being the oldest country in the world. it is likely that the strategies adopted for testing the population as well as the robustness of the healthcare system may play a much more relevant role than age in the explanation of the scenario. unfortunately, numbers can only provide a partial description of what is happening, and probably underestimate the ongoing tragedy ( ) . right in these days, the international media have broadcasted worldwide the dramatic column of military trucks leaving the city of bergamo laden with the coffins of many patients died of covid- . people cannot even celebrate the funeral of their relatives who died isolated in covid- facilities. schools, universities, activities…all of them have been closed; the entire country is locked down since march st. the worldwide web is full of videos showing how healthcare professionals are today working around the clock, often with limited resources. the limitation of resources has indeed been a major issue, especially during the very first days of the crisis when there still was a certain dose of unpreparedness and surprise in front of the magnitude of what was happening. low number of ventilators, saturation of beds in intensive care units, shortage of personal protective equipment, overcrowding emergency departments, inability to adequately isolate suspected cases... have been the most urgent and critical problems faced by clinicians, especially at the beginning. it is thus not surprising why about % of the persons infected by the sars-cov- is today constituted by physicians and nurses. working in such an emergency to counteract the enormity of what was arriving unavoidably led to healthcare professionals falling sick (and sometimes even die) under the coronavirus. the consequent reduction of the healthcare professionals due to covid- has been threatening the sustainability of the system. to overcome this critical moment, many healthcare professionals from everywhere in the world have answered to the call for help, and converged to italy to put their expertise at disposal of our community. to keep the healthcare machine running and support the colleagues overwhelmed in the management of covid patients, there have been pediatricians working with older patients, or surgeons taking care of internal medicine issues... everyone is giving what he/she can. how is the life of a geriatrician in such an apocalyptic scenario? geriatricians are sadly used to the fact that our hedonistic society often finds no place for older people ( ). in normal times, geriatricians regularly battle for guaranteeing the proper care to persons who are otherwise excluded from interventions just because they are "old". geriatric medicine has produced substantial evidence showing that frail older persons require adaptations in the clinical approach, and that the environment plays a critical role for the wellbeing of the aging individual ( , ). well…everything we know has simply been wiped away by this period of crisis we are living. it can be understandable that the focus is today exclusively on the sars-cov- , and the hospitals are primarily acting for defeating it. nothing else seems important in front of the enormity of the crisis. the differences across specialties have been cancelled because sars-cov- is everywhere, probably more diffused than what is generally thought or objectively measured by tests. the only priority in the clinical setting is to isolate covid patients (or suspect cases) as soon as possible, and offer prompt respiratory support when symptoms appear and start to dramatically worsen. the coronavirus has deleted everything that can be felt superfluous, redundant, and/or unnecessary. unfortunately, geriatric medicine did not start the coronavirus crisis with solid roots. it has traditionally been considered a "poor specialty", always struggling for recognition. it was indeed not in the best position compared to other disciplines to protect the results it has been accomplishing over the past decades for the care of frail older persons. furthermore, it has never been a winning ticket for geriatrics to specifically serve those frail older persons, often at the margin of the society. under this scenario, it has not taken a lot before everything related to geriatrics was forgotten in the name of the battle against the coronavirus. ubi major minor cessat… is this right? is this the way things should go? of course, not! the fact that the emergency has forced drastic (and sometimes cynical) decisions does not justify the loss of attention to the person's values. listening a physician stating "luckily, in our hospital no one younger than years has died" provides a clear taste of the well-known ageism of our society. this kind of attitude was before (more or less) hidden behind a façade of politically correct, but is receiving now a sort of legitimation in front of the dramatic emergency. when media have been underlining that older persons with comorbidities were the most likely to die, a relief breath was made by many (as they had no older relatives, or as they will not be old as well one day!). nevertheless, geriatricians are also to blame for the failure we experience today. we have lost ourselves and wasted time running after futile controversies, missing opportunities for contaminating other disciplines with the correct principles of geriatric medicine. we probably remained closed inside our ivory towers, and left others design their (inadequate) solutions for our patients ( ) . the costs of the coronavirus for older persons are and will likely remain underestimated. we can count deaths events occurring in individuals resulting positive to the test. however, these figures do not consider that: ) tests have not been routinely available, especially for older persons. for example, long term care facilities and primary care physicians cannot yet easily perform the test for the identification of sars-cov- . ) the test is limited by a relatively high number of false negative results. clinicians are quite confident that many patients died of covid despite not officially being diagnosed for it. although these patients could still be managed with all the precautions as they were covid, they have still been excluded from the statistics. there has been a patient in our hospital who resulted three times negative at the nasopharyngeal swab, before finally being found positive at the broncho-alveolar lavage. ) the already fragile integration of care services completely collapsed under the coronavirus diffusion. hospitals closed within themselves for limiting the diffusion of the virus, while post-acute/long-term care facilities reduced admissions being afraid of a possible contamination arriving from outside. primary care physicians have been paying a huge tool (even with their lives), finding themselves isolated in the community with no means and no support from the hospitals. many older persons (with their chronic conditions and care needs) remained isolated after the sars-cov- outbreak. the consequences of this are not part of the standard covid narrative... ) in a situation of constant emergency, it is almost impossible to apply many interventions against geriatric conditions across clinical settings. physical therapists for mobilization of the older patient, avoidance of stressors for prevention of delirium, social contacts for re-orientation of the person, teamwork for the design of person-tailored interventions… all this does not exist anymore. there is no time, no personnel, no resources for supporting the good practice of geriatric medicine. the same human interaction between the patient and his/her physician is lost behind the burdening personal protective equipment in covid- facilities. are we redundant? we prefer to think that we have not yet been able to communicate how certain care standards solicited by geriatricians are necessary for practicing a modern medicine. what will remain after the sars-cov- will be defeated? we will count our deaths. we will probably take some time for thinking at our acts and decisions. we will surely see many errors, but this will give us a unique opportunity for improvement. after all, albert einstein said "the crisis is the greatest blessing for people and nations, because the crisis brings progress". hopefully, we will learn from our mistakes and contribute for a better society… a society able to give more value to persons, independently of their age. disclosures: no conflict of interest to declare. who director-general's opening remarks at the media briefing on covid- - case-fatality rate and characteristics of patients dying in relation to covid- in italy at the epicenter of the covid- pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation the courage to be a geriatrician the end of the disease era forging a frailty-ready healthcare system to meet population ageing the geriatric management of frailty as paradigm of "the end of the disease era key: cord- -w sk m authors: caduff, carlo title: what went wrong: corona and the world after the full stop date: - - journal: med anthropol q doi: . /maq. sha: doc_id: cord_uid: w sk m this article examines the global response to the covid‐ pandemic. it argues that we urgently need to look beyond the virus if we want to understand the real seriousness of what is happening today. how did we end up in a space of thinking, acting, and feeling that has normalized extremes and is based on the assumption that biological life is an absolute value separate from politics? the author suggests that today's fear is fueled by mathematical disease modeling, neoliberal health policies, nervous media reporting, and authoritarian longings. it is as though mankind had divided itself between those who believe in human omnipotence (who think that everything is possible if one knows how to organize masses for it) and those for whom powerlessness has become the major experience of their lives. ---hannah arendt the measures that governments across the world have taken to contain the spread of coronavirus disease are massive and unprecedented. as a result of these measures, life has come to an almost complete standstill, with many countries under lockdown. never in the history of humanity have such drastic interventions into the lives of populations occurred in the name of health on such a scale and in such a short period of time. as a result of the world's largest and perhaps most stringent lockdown, millions of daily wage laborers have lost their source of income in india. health care workers have been attacked and evicted from their homes because they are seen as potential spreaders of contagious disease (kalra and ghoshal ) . neighborhoods have been scared into panic when an ambulance appears on the street. due to the sudden ban on any form of transportation, migrant workers have been stranded between the cities where they used to work and the villages where their families are living (daniyal et al. ) . cancer patients have been unable to receive essential medical care because they cannot reach the hospital. it is the poor, the marginalized, and the vulnerable who are most affected by drastic measures, exacerbating already existing inequalities. in kenya, the police enforced a coronavirus curfew using teargas and excessive force against presumable violators of lockdown law (namwaya ) . in bangladesh, the government created a special unit to monitor social media and arrest people for spreading "misinformation" about the virus (hrw ). in hungary, parliament passed a law allowing prime minister orbán to limit freedom of speech, defer elections, and suspend rules and regulations by decree (gebrekidan ) . in india, state governments released companies from the purview of labor laws, including occupational health laws, to stimulate the economy (sharma ) . in lebanon, the currency collapsed, leaving % of the population in need of food aid (chulov ) . in the united states, over million people have filed for unemployment benefits (rushe and aratani ) . unfortunately, as of the writing of this article, many things remain completely unknown in this pandemic despite intensive investigation. for example, we don't know what helped contain the outbreak in china, and particularly whether government interventions reduced the spread of the virus or if the virus burned out there before moving on to other susceptible populations. the fact is: we simply don't know. nevertheless, many actors and institutions have proceeded as if they did know, imposing extreme measures that have affected billions of people and that have pushed societies to the edge of collapse by creating poverty, hunger, misery, debt, and unemployment. today, many wonder how we ended up where we are. how was it possible for a virus to trigger such a massive response that continues to threaten society and the economy, with so little discussion about the costs and consequences of extreme measures? why is there widespread agreement that aggressive interventions to "flatten the curve" were necessary and justified? it seems that this unprecedented public health experiment occurred without sufficient consideration of the social, political, and economic consequences. the failure to consider the impact of extreme measures that have become the norm in many places in the covid- pandemic has been stunning. the destruction of lives and livelihoods in the name of survival will haunt us for decades. the coronavirus disease outbreak seems to have started in the chinese city of wuhan in december . in january, the chinese government put wuhan and other major cities in the province under lockdown. a lockdown of million people "is unprecedented in public health history, so it is certainly not a recommendation the who has made," dr. gauden galea, the world health organization's (who) representative in beijing, emphasized at the time (reuters ). in other provinces, the chinese government implemented tailored measures, including factory shutdowns and school closures, but not a lockdown or restriction of movement to limit the spread of disease. major media outlets in the united states called china's locked-city strategy deployed in and around wuhan "harsh," "extreme," "severe," and "controversial," emphasizing that it offered "no guarantee of success" (qin et al. ) . a new york times article noted that "china is trying to halt a coronavirus outbreak using a tactic … with a long and complicated history fraught with social, political and ethical concerns" (levenson ) . experts quoted in the article called the lockdown of cities "an unbelievable undertaking" that would be "patently unconstitutional in the united states." "that type of thing," said james hodge, a professor of law, "is obviously an excessive response." another expert cited in the article, historian howard markel, pointed to the "darker side of quarantine-its use as a social tool rather than its scientific use as a medical tool." in the united kingdom, newspaper articles suggested that the chinese government would not be able to keep the city of wuhan "closed for business indefinitely" (graham-harrison ). in february, the virus continued to circulate and soon appeared in other countries. in march, the who declared the covid- outbreak a global pandemic. despite the criticism of china's approach, a crude and extreme version of lockdown became the international norm promoted by experts, officials, and the media across the world. concerns with the dark side of quarantine faded rapidly. a few countries like south korea veered from this norm and chose instead a classic infectious disease intervention: test-trace-isolate, with a highly centralized approach to public health intelligence gathering. emphasizing mass testing and meticulous contact tracing to interrupt the chain of transmission, south korean health officials closed schools and managed the crisis successfully without any lockdowns or roadblocks, and few restrictions of movement. significantly, south korea learned from earlier outbreaks of infectious disease (sars in particular), and imposed central control, used digital technologies, and enforced quarantines, and it witnessed one of the lowest covid- mortality rates. by the end of april , around , cases of infection had been detected there, but only people had died. germany developed its own testing protocol, which was published on january by the who (beaumont ) . when the first case was detected on january , germany launched mass testing, systematic contact tracing, and early hospitalization, keeping the mortality rate low and hospitals functional even when cases of infection increased (mohr and datan-grajewski ) . health officials relied on an extensive network of laboratories and were able to conduct over , sars-cov- (covid- ) tests per week (buck ) . along with south korea, germany put testing and contact tracing at the heart of the response. despite the who's emphasis on testing and south korea's and germany's early success in reducing the spread of the virus, most countries considered testing at scale as a low priority and relied on an extreme version of the chinese approach of lockdown. however, in china, the approach was tailored and regionalized; as a who report noted, "specific containment measures were adjusted to the provincial, county and even community context, the capacity of the setting and the nature of novel coronavirus transmission there" (who ). the lockdown focused on the major cities in the most affected province, constraining the life of million people in a country of . billion. in other words, it was a limited lockdown affecting % of china's total population. in contrast to the tailored and regionally differentiated intervention that sought to minimize the socio-economic impact of the response, many other governments across the world imposed nationwide lockdowns that went far beyond china's locked-city approach. in practice, these lockdowns amounted to curfews (often legalized after the fact by emergency laws). italy was the world's first country with a nationwide lockdown/curfew. many countries followed suit, partly motivated by shocking images of overwhelmed hospitals in italy's north and partly driven by a disease model report released in the united kingdom a few days after italy's surprising national lockdown announcement . this moment of shock and surprise triggered a chain reaction in the pandemic response. the horizon shifted, the inconceivable became possible, and life suddenly felt surreal. the u.k.'s disease model garnered a lot of attention, creating a sense of urgency that amplified the political pressure because the numbers were alarming . published by a group of experts without peer review on an institutional website, the report compared covid- with the great pandemic of , which killed over million people worldwide and suggested, without any evidence, that sars-cov- was "a virus with comparable lethality to h n influenza in ." most frightening in all this was not so much the lethality of the sars-cov- virus but the license to rush forward with predictions, abandon basic standards of science, and make dramatic claims to scare people. for covid- , the report predicted , deaths in the united kingdom and . million deaths in the united states. it presented possible strategies to reduce the impact of the pandemic, but the focus was exclusively on "non-pharmaceutical interventions." there was no discussion of testing and contact tracing. a proven public health strategy known to be effective was systematically sidelined in one of the most influential reports to emerge in the covid- pandemic. significantly, the report claimed to focus on "feasibility" of measures and promoted the idea that systematic suppression of transmission would work best-in other words, lockdowns. however, it excluded from any consideration the social, political, and economic implications of lockdowns, noting that "no public health intervention with such disruptive effects on society has been previously attempted for such a long duration of time." in addition to ignoring testing and contact tracing as a possible strategy, disregarding the social, political, and economic implications of lockdowns, and conceding that there is no "easy policy decision to be made," the authors of the report felt confident enough to claim that "suppression" was the "preferred policy option" and the "only viable strategy" that countries across the world needed to implement "imminently." the lockdowns that were required for suppression would need to be maintained "until a vaccine becomes available" (which they predicted to be "potentially months or more"). a crude, extreme, and ultimately unsustainable version of the chinese approach became the international norm. shutting down society and the economy until a preventive medical treatment becomes available was advanced as an appropriate response and the only possible way of dealing with the crisis, despite the costs and consequences. italy figured as an important but fundamentally ambivalent model, shifting the locked-city into a locked-country approach. the italian scenario was sobering and frightening but also inspiring and motivating. to avoid italy's disaster, governments appropriated the italian approach of mass confinement and rigid restriction of movement as a one-size-fits-all intervention. in many places throughout the world, including italy and france, the locked-country approach took a militarized form with massive deployment of the police to enforce lockdown restrictions. the locked-country approach seemed to obviate the necessity of justifying a differentiated strategy that might have looked unequal and unfair and that might have intensified social and political conflicts along multiple internal fractures and fault lines. to avoid the political fallout of a differentiated strategy, which would have required systematic testing, government officials in europe and elsewhere invoked the politically expedient image of a total threat and suggested that "we are all in the same boat" and that "we are all in this together." the idea that regional lockdowns would not be possible and that it was best to treat the virus as a global threat that would uniformly impact all people involved conjuring an image of a united nation confronting a total threat that required everyone's sacrifice. this image relied on a false assumption of equality. solidarity came to mean not mutual support in a situation of uneven risk, but rather generalization of a sense of danger across a national population perceived as a homogeneous body under attack. the ideology of national pandemic solidarity-putting everybody under confinement and treating everyone the same-obscured the reality that lockdowns mean different things to different people, and that not everyone is equally exposed or equally vulnerable. both the virus and the lockdown disproportionately affected those who were already vulnerable along lines of age, class, and race. dramatic references to the magnitude of the threat served as justification for nationwide lockdown policies. this extreme and unprecedented blanket approach systematically imposed on entire populations was driven by a number of factors that variously prevailed in different countries across the world: a growing sense of panic, constant media sensationalism, deep authoritarian longings, increasing political pressure to contain the spread of the virus, disturbing accounts of overwhelmed hospitals unable to cope with the surge of patients, misleading mortality calculations, and, most importantly, a trust in the power of mathematical disease modeling. throughout the covid- pandemic, there has been an abiding assumption among observers and the public that it is clear what is happening; that everyone knows what is going on because everyone can see it on television. however, what an endless stream of media reports from around the world have continued to obscure is the fact that it is impossible to know what is happening in a population when there is no systematic testing. the lack of testing created a void that was filled by the flexible evidence of disease modeling. in the absence of robust data, disease modeling emerged as the presumably best and only available science to inform policy. media hyperbole focused on absolute numbers independent of context and made covid- deaths politically visible. flexible disease modeling (often based on data derived from viruses such as influenza) took the place of accurate epidemiological surveillance. scientific papers published online without peer review made scary projections and painted a grim picture. widely reported simulation models created shock effects that shaped government policies. a narrow focus on numbers played an important role in understandings of the magnitude of the threat, fueling fear and panic in the absence of actual evidence. a distinct imaginary took hold, "the imaginary of an unprecedented event," which seemed to require an unprecedented response (kelly ). there was a widespread sense, among experts and the media, that the sars-cov- virus was much more lethal than influenza. that this pandemic was different from influenza and thus necessitated a different approach was typically claimed on the basis of the case-fatality rate, the number of deaths as a subset of those infected with sars-cov- . the casefatality rate played a crucial role in the justification of the public health experiment now unfolding before our eyes. estimates of the case-fatality rate initially varied hugely from . % to %. in an article published in the lancet, scientists claimed the case-fatality rate could even be as high as % (baud et al. ). in early march , the who directorgeneral stated that the case-fatality rate for sars-cov- was . %. he added: "by comparison, seasonal flu generally kills far fewer than % among those infected." whatever the estimates, the fact remains that it is impossible to calculate the case-fatality rate in the absence of systematic testing. given the lack of evidence, the only scientifically valid statement at the time would have been to say that we simply don't know how lethal the virus is. early on in this pandemic, it became clear that over % of infected people were experiencing no symptoms at all at the time of testing (gudbjartsson et al. ) . this means that a surveillance regime where only people with symptoms were tested will automatically exclude a large number of infections. additionally, patients with symptoms are much more likely to die than asymptomatic people. the result is an exaggerated case-fatality rate. testing strategies differed across countries and changed within countries over time. for example, on february , , the italian ministry of health published a revised policy for testing, prioritizing patients with severe clinical symptoms (and thus higher chances of dying). this change in policy resulted in an apparent increase in the case-fatality rate of . % on february to . % on march (onder et al. ) . suddenly, the virus seemed to have become much more deadly. however, this increase was a numerical illusion-a statistical artifact. there was no change in the lethality of the virus. changes in testing policy occurred in many countries and even across regions where different tactics for counting deaths were used. in china, test-positive asymptomatic patients were excluded from being counted as cases of infection (wu et al. ) . in belgium, deaths were counted independent of any testing (schultz ) . of % of all deaths, only . % turned out to be confirmed by laboratory test as covid- positive. almost half of all victims were merely suspected to be linked with the virus but had never actually been tested. there was and remains no agreement among experts and officials on what counts as a death caused by the virus. in italy, covid- -related deaths were defined as those occurring in test-positive patients, "independently from preexisting diseases that may have caused death" (onder et al. ) . this is particularly concerning in terms of data quality because the vast majority of deaths occur in patients who are older than with one or more comorbidity. test-positive patients who die because of heart disease or terminal cancer are not necessarily dying because of sars-cov- infection. yet they appear in the statistics of some countries. this confusion between patients who die with the virus and those who die from it has had an impact on the data and their quality, making comparisons between countries impossible. further, almost all tests that are done use rna tests, which can detect an infection only as long as the virus is present in the body. these tests, however, cannot tell whether a person had the virus in the past. only serological tests for antibodies against the virus can provide an accurate picture of how many people have been infected in a given population. and yet, such systematic serological studies were and are missing. given the lack of testing and taking into account the role of selection bias, the large number of asymptomatic cases, the confusions in case definitions, the changes in testing policies, and the difficulty of knowing who is dying with versus dying from the disease, the denominator for calculating actual death rates cannot be reliably determined. without a denominator, it is mathematically impossible to calculate the case-fatality rate. nevertheless, despite the lack of data, experts, officials, and the media have remained transfixed by the assumption of clarity and reliability of numbers, and they continued to circulate wild estimates, unleashing a pandemic of scary charts with exponential curves. over the last weeks of march, more and more testing was done globally, and more testing continues as of the time of this publication. not surprisingly, estimates of the case-fatality rate have come down significantly, because the denominator has gone up due to the increase of testing. in iceland, % of the population has been tested using rt-pcr-based tests independent of symptoms, suggesting a case-fatality rate of . %. this figure is six times lower than who's official estimate for covid- . the center for evidence-based medicine at the university of oxford noted that if one assumes that % of iceland's population is infected, then the corresponding infection-fatality rate would be . %. a study using both rt-pcr-based and serological tests conducted in one of germany's most affected regions indicated a case-fatality rate of . % and an infection-fatality rate of . % (streeck et al. ) . we know from epidemics and pandemics of the past that the case-fatality rate is often massively overestimated at the beginning of an outbreak because case detection is limited, largely based on hospital patients and typically biased toward the severest cases of disease. when the h n swine flu pandemic occurred in , the estimated case-fatality rate varied between . % to . % in the first weeks of the outbreak. in , a decade after the pandemic, the who reported that the swine flu pandemic turned out to have a case-fatality rate of . %. this means that the actual casefatality rate was five times lower than the lowest estimate. social science scholarship has shown how numbers can deceive. numbers have the ability to reveal as well as conceal. therein lies their magic. they appear as seemingly neutral bearers of truth. they offer a sense of mathematical precision, making things seem more certain than they actually are and displacing attention away from the conditions under which they were produced. abstracting from limitations on the conditions of their production and treating numbers as absolute is dangerous because it makes things comparable that are not comparable, because it suggests scientific knowledge where there is lack of evidence, and because it creates the sense of a major threat obscuring the differential nature of risk. what using numbers this way fails to account for is the fact that not everyone is at risk in the same way. among the more interesting figures of the sars-cov- pandemic is the number of deaths per million inhabitants per country. this number is probably more reliable than the case-fatality rate because deaths are less likely to be missed (ignoring for now the case of belgium and the difficulty of defining deaths caused by sars-cov- ) and because the denominator, a country's population, is known. here are the current numbers of deaths per million inhabitants for five countries as of may , : spain: italy: france: germany: south korea: the staggering differences between countries cannot solely be explained by demography or rates of infection (some countries seem to have more infected people per million inhabitants than others and so might be overwhelmed, though this is also a question of time-how many cases per week per region). what the differences might reveal (and it is important to note that they may well change) is that some health care systems are able to deal with the crisis in a better way than others. the structural fragilities of an underfunded, understaffed, overstretched, and increasingly privatized and fractured health care system contribute to higher mortality rates (adams ) . in a sense, each society has the mortality it deserves (canguilhem : ) . where medical care is easily accessible, with sufficient and well-trained staff, and with capacity flexibility, patients are more likely to receive better care and survive. in this sense, it matters that spain turns out to have beds per , inhabitants, italy . , france , germany , and south korea . . although beds per inhabitants is a crude indicator, it is noteworthy that germany can rely on over , staffed intensive care beds, out of which only , were occupied in early april (see mohr and datan-grajewski ) . this at a time when there were more cases in germany than in france and the united kingdom and slightly less than in spain and italy. germany's was clearly not a health care system overwhelmed by a sudden surge of patients. ironically, organizations such as the oecd frequently scolded germany's health care system in the past for "oversupply" of hospital beds and its "inability" to "rationalize hospital capacity" (kumar and schoenstein ) . this means that the case-fatality rate is not just dependent on the biological nature of the virus and the age and health profile of the population (people most at risk of death are older than with one or more comorbidity). the case-fatality rate also depends on systematic testing, meticulous contact tracing, well-trained health care workers, nursing homes with adequate resources, and the ability of the health care system to cope with the crisis (excess as well as surge capacity) and provide high-quality medical care, particularly keeping medical workers safe and healthy. in this sense, the pandemic has and will continue to brutally expose policy failures and structural health care system deficits. the situation in many hospitals in italy, spain, and france is troubling, especially in densely populated areas. but it is important to understand why some of these highly visible institutions of care were overwhelmed. lombardy, italy's most affected region, has long been an experimental site for health care privatization: community-centered care "has been all but wiped out" (bagnato ) . the lack of general practitioners and the defunding and low emphasis on community care have increased the pressure on hospitals in urban centers. these hospitals have neither excess nor surge capacity to cope with a sudden rise in demand. over the past five years, hospitals across europe held numerous strike actions "with doctors and health workers complaining of funding cuts, a government reduction in the number of beds and a serious lack of medical staff leading to dire working conditions for emergency room staff" (chrisafis ). hospital systems in italy, spain, and france were on the brink of collapse even before the virus arrived. the most telling demonstration of the structural contradictions of pandemic preparedness under neoliberalism occurred, not surprisingly, in the united states. as american newspaper articles reported, hospitals across the country deferred regular medical services to free up space, equipment, and staff for the pandemic response. when patients started to avoid hospitals due to fear of infection, a main source of income was drastically cut off, "causing huge losses that have forced some hospitals to let go of health care workers as they struggle to treat infected patients" (harris and schneider ). facing a "financial nightmare," hospitals filled their intensive care units with patients who did not really need intensive care so that they could charge more and make up for the financial loss. additionally, administrators cut salaries, laid off hundreds of staff, and sent others on unpaid leave, weakening the health care system further in the midst of the pandemic response. when a new virus appears, things start to fall apart. once everyone gets scared, extreme measures are implemented, in a more or less improvised manner, and trillions of dollars, euros, and pounds are pumped into the economy to make up for the loss. once the worst is over, however, the normal crisis continues, and the structural fragilities remain (caduff ) . this pandemic will haunt us all for decades in ways that we can barely imagine at this point. the nature and sheer scale of the interventions that we have witnessed are staggering, and the consequences-social, political, and economic-remain unforeseeable. there are no systematic accounts of the implications and repercussions seen so far, nor do we have any idea about the number of indirect deaths due to the lockdowns/curfews, the social distancing and the self-isolation. we have yet to see a realistic plan that would outline how we might learn to live with a virus that is unlikely to disappear any time soon (sullivan and chalkidou ) . in the meantime, i suggest that we reframe the corona conversation to cut through the confusion and dimness that is pervading this pandemic in the following ways: the emergence of new viruses in human populations is normal. it has happened before; it will happen again. coronaviruses are common and circulate widely in humans. they have infected people and killed thousands year after year, especially in winter. worldwide, between , to , people die from influenza viruses every year. the sars-cov- virus has killed , people so far. there is no doubt, sars-cov- is causing a serious infectious disease, but so far it is still in the range of what we observe in terms of mortality during a severe influenza season. the main difference is the speed of infection, the clinical picture of the disease, and the impact on demographically older populations causing massive compression of morbidity and mortality that is overwhelming weak health care systems with no excess and little surge capacity. the influenza pandemic killed between and million people worldwide, and the influenza pandemic killed between and million people. as of the writing of this article, covid- has killed , people, according to the official numbers. clearly, the world has witnessed worse pandemics, including . million deaths due to tb each year, , deaths due to hiv infections each year, and , deaths due to malaria, all preventable and treatable conditions. this observation does not mean that influenza and covid- are clinically similar or that nothing should be done to contain the spread of sars-cov- and mitigate the consequences. however, it raises the question of why fear and panic are spreading like wildfire, provoking such extreme measures, and why experts and government officials are willing to mount an unprecedented effort for sars-cov- but have never considered similar interventions for the , - , people who die every year due to influenza. influenza is a relatively well-known virus. to say that sars-cov- is an unknown virus doesn't automatically justify the most extreme measures that the world has ever seen. what makes this pandemic unprecedented is not the virus but the response to it. extreme measures to contain the spread of the virus have resulted in extreme fallouts. it is difficult to overestimate what we are witnessing today. the pandemic response has pushed the world into a space of fragility and uncertainty. there hovers a "perhaps" over everything now (caduff ) . blinded by the urgency of the immediate moment, the response has created an opening for actors and institutions to push agendas and reorder the world. we will grapple for years to come with the changes that are happening today. the response to the disease is driven by a fantasy of control that overestimates and overreacts. this fantasy has caused and is causing enormous harm. it is unrealistic, misleading, and bound to fail. a pandemic like this cannot be controlled; it can only be managed. if we keep using words such as control, we are only setting ourselves up for disappointment. this pandemic is far from having found a language that is adequate to the problems it is posing. we urgently need new concepts but seem to have little imagination. the urgency of the crisis has displaced reliance on basic standards for quality and control of quality of scientific research. papers are published without peer review. claims are made without evidence. perhaps not surprisingly, given the fragile health care infrastructures in some countries, speed appears to be more important than quality, rigor, and integrity. underscrutinized science, lack of data, speculative evidence, strong opinions, deliberate misinformation, exaggerated mortality rates, the / news media attention, and the rapid spread of dramatic stories on social media have led to poor political choices and major public anxiety. we are afraid of covid- . we are not afraid of influenza. we see one thing as a public health emergency and another as a fact of life. today, we are learning an old insight the hard way: not every life and not every death are equal. some deaths are more important than others, drawing more attention, triggering a bigger response and mobilizing more resources. in the covid- pandemic, the belief seems to have taken root that health is an absolute value and that every life needs to be saved by all means. meanwhile, millions of people are dying of influenza, tb, hiv, malaria, and diarrhea, not to mention chronic diseases and accidents. there seems to be less political urgency for these preventable deaths. some health care systems were overwhelmed in this pandemic. others were not. for decades, governments have underfunded, understaffed, and privatized health care systems across the world, and these trends have exacerbated the impact of the pandemic. the response to sars-cov- took a particular shape, converging in extreme measures that have become the norm in many countries. questions that remain include: was it the only possible way of managing the crisis? why has a crude version of china's approach become the dominant model? at the heart of this pandemic was and is the widespread assumption that there were and are no alternatives to extreme measures implemented on entire populations with little consideration of cost and consequences. this is not true. as some countries have shown, adequate testing and less drastic policies of social distancing work well to manage the pandemic. it seems that some officials saw covid- as a disease that could be contained. as the who director-general suggested in early march , "we don't even talk about containment for seasonal flu-it's just not possible. but it is possible for covid- ." this perception may have contributed to the radically different approach seen across many countries. the idea of "flattening the curve" is often seen as the optimal solution, but there is no guarantee that the effort to do this will actually impact the total number of deaths over the long run of the disease's presence in any community. it may ultimately simply spread the same number of deaths over a longer period of time and thus perhaps reduce the pressure on hospitals but not overall mortality. nationwide lockdowns are not a solution. they prevent infection as long as they are in place, but they also keep people susceptible. this is particularly concerning in a pandemic where the virus has become endemic. once lockdowns are lifted, the number of infected people may well rise again later. this is why it has been so hard for countries who adopted this strategy to return to normal life-the strategy is not sustainable over the long run. as andrea bagnato noted about the stay-at-home strategy: "it is not in the harshness of its lockdown, but in the effectivity of separating the infected from the non-infected, that china's response has excelled: a centralized system of dedicated structures (called fangcang) was built in no time, where all patients and their contacts were treated and divided in four groups according to severity. instead, lombardy simply closed everything down. and it becomes clearer by the day that the main landscapes of infection were not public spaces, but hospitals, retirement homes, workplaces, and indeed private homes" (bagnato ). in germany, % of the people who died due to covid- are years or older (mohr and datan-grajewski ) . a majority of the patients who died have one or more underlying health condition such as hypertension, diabetes, cardiovascular disease, chronic respiratory disease, or cancer. this means that the pandemic is killing predominantly people with an already reduced life expectancy. the key question then becomes excess deaths-the difference between the statistically expected number of deaths and actually occurring deaths over a period of time. there is no doubt that there will be excess deaths due to covid- , but it is unclear how large that number will be. the pandemic response has produced a substantial rise in the number of people who now live with untreated illness. prohibition of public transport has made it difficult for patients and staff to reach hospitals. patients with conditions other than covid- avoid doctors because they are afraid of getting infected. emergency room attendance dropped substantively the world over. cancer referrals decreased and cancer screening services stopped entirely. rural health services in countries such as india crashed. essential public health programs have been paused; many resources have been reallocated. this means that patients are neglected, receiving no or less medical care, leading to untreated illness and a rise in mortality. a virus causes disease, not hunger. it is not the pandemic, but the response to it that threatens the livelihood of millions of people. in many countries, both rich and poor, the trends are shocking. in india, children die of starvation. farmers commit suicide because they are unable to harvest crops. stranded daily wage laborers drop dead after walking hundreds of miles. the poor, marginalized, and vulnerable bear the brunt of the pandemic response. the lockdown is a political mechanism not simply for the prevention but for the redistribution of negative effects. lockdowns shift negative effects away from hotspots of public attention to places where they are less visible and presumably less serious. in this way, they are part and parcel of a politics of inequality. this pandemic is not just about health, it is about fear, and the objects that are singled out and then made the ground and motivation of systematic thought and action. to be afraid has become an obligation, a responsibility, a duty. people are afraid not just because of what they experience but because they are told to be afraid and encouraged to inhabit the world with fear of "foreign bodies" and "invisible enemies." public discourse is highly moralized. looking for someone to blame, individuals are exposed as "super-spreaders" responsible for the rising number of cases. on social media, "lockdown warriors" accuse citizens of lack of patriotism and failure to "do their duty" in the face of danger. in this highly moralized public discourse, life is considered an absolute value that can justify almost every form of disciplinary intervention in the name of health. public health needs to be front and center in any infectious disease intervention. investing in strong public health infrastructures should happen even when there is no pandemic. mathematical disease modeling cannot replace systematic epidemiological surveillance on the ground. the most effective way to manage an infectious disease outbreak is to test, trace, and isolate. interventions need to be phased over time; they need to be dynamic, regionally targeted and risk based. all interventions must take into account the social, political, and economic impact, as well as the indirect impact on other health conditions. interventions that do this will create management strategies that work to minimize collateral damage. absolute numbers cannot be used for policy, they only fuel fear and panic. national lockdowns are not a solution. they protect people temporarily, but they also leave them susceptible. once restrictions are lifted, cases of infection are likely to increase again. there is no exit from the pandemic; there is only an exit from the response to it. we are still at an early stage of understanding how best to clinically manage covid- both as a disease and as a risk factor to potentially vulnerable populations. it is vital to find better ways of sharing quality data and effective practice to ensure health systems learn and adapt quickly. what this pandemic shows is a lack of preparedness. this will come as a surprise, given the billions of dollars, euros, and pounds that were spent over the last years on pandemic preparedness, including experience with past epidemics and pandemics such as ebola and swine flu. how can it be that hospitals ran out of n masks in week one? where did all the billions spent on preparedness go? outsourced production capacity and insufficient stockpiles of personal protective equipment put nursing home residents, community health care workers, and hospital staff at risk, weakening health care systems further. key preparedness concepts need to be at the heart of the response. fifteen years of pandemic preparedness seem to have evaporated into thin air in this pandemic. instead of activating existing plans and drawing on concepts such as the pandemic severity assessment framework, countries imposed a massive, untested, and unproven generic lockdown with unforeseeable social, political, and economic repercussions. sars-cov- is less lethal than every single scenario exercise that has been conducted for preparedness planning by governments and non-governmental organizations in europe and america. it will be important to understand why key preparedness concepts were sidelined in this pandemic, despite the attention that preparedness received and the substantial resources it consumed for over a decade. the fear of death is powerful in societies eager to repress the inescapable reality of death. in such a context, it is important to flatten the curve of extreme speaking, feeling, and acting. what was and will always be urgently needed is moderation and perspective. to continue to engage in today's competition for ever more extreme predictions is dangerous. it will only support those who ignored the virus initially and who are more than willing to blame it now for the mess. equally dangerous is a public health populism of clapping hands that leaves out any consideration of the social, political, and economic costs and consequences of sweeping interventions. attempts to obscure political failures are growing rapidly. those who contribute to extreme predictions and apocalyptic readings of the current situation are only contributing to the obfuscation of the policy failures and underlying structural issues that are responsible for many of today's problems. there are already attempts in countries such as the united kingdom and the united states to rewrite failure as success. not surprisingly, governments are calling on citizens to participate in public performances, demonstrate national unity in the face of danger, and celebrate collective strength and resolve. fighter jets soaring through the sky and helicopters showering rose petals on "frontline warriors" are militarized state spectacles. but health care workers deserve more than patriotic feelings and symbolic gestures; they deserve better health care policies. to challenge and critique now is essential. the story of how the chinese approach became a model for generic lockdowns in the global north and then exported to countries in the global south is important to note, particularly considering the dramatic consequences for millions of people struggling to survive without any source of income. ironically, these extremely restrictive lockdowns were sometimes demanded by people eager to criticize the authoritarianism of the chinese state. across the world, the pandemic unleashed authoritarian longings in democratic societies, allowing governments to seize the opportunity, create states of exception and push political agendas. commentators have presented the pandemic as a chance for the west to learn authoritarianism from the east. this pandemic risks teaching people to love power and call for its meticulous application. pandemic time is an auspicious time for all kinds of political projects. as a result of the unforeseeable social, political, and economic consequences of today's sweeping measures, governments across the world have launched record stimulus bills costing trillions of dollars, pounds, pesos, rand, and rupees. earmarked predominantly for individuals and businesses, these historic emergency relief bills are pumping staggering amounts of money into the economy, but, ironically, they are not intended to strengthen the public health infrastructure or improve medical care. the trillions that governments are spending now as stimulus packages surpass even those of the financial crisis and will need to be paid for somehow. today, there is a massive global recession in the making. if austerity policies of the past are at the root of the current crisis with overwhelmed health care systems in some countries, the rapidly rising public debt is creating the perfect conditions for more austerity in the future. the pandemic response will have major implications for the public funding of education, welfare, social security, environment, and health in the future. if you think something good will come out of this crisis, you should think again. today we are just driving faster and with a much bigger car, but it is the same road with the same destination. wolf bukowski notes that the political discussion in italy is now dominated by an "uncritical 'responsibility'" that cannot find a place outside the imperative to contain the virus. "the right intuition that 'we should not question the reality of the epidemic' shifts all too easily into 'we should not question the government's response to the epidemic'" (bukowski ). in such a context, any control intervention imposed by the state is perceived as lawful, and no democratic discussion and debate appears necessary ("let the experts speak!"). in other places, critique has become difficult for other reasons. the tragedy of today's political moment in the united states, the united kingdom, and brazil is that right-wing politicians pushed many into embracing measures that one thought were only possible in authoritarian regimes. here, an engagement in critical analysis has become almost impossible because it is seen as playing into the hands of trump, johnson, and bolsonaro, political figures who seem unconcerned with public health and the staggering inequalities that afflict our world and whose public statements have reached an unmatched level of ignorance and incompetence. however, it is important to understand that the strategic combination of confusion, contradiction, and the play of extreme opposites is foundational for authoritarian rule. everything that instills a sense of disorder and that intensifies the crisis magnifies the desire for decisive action. in this article, i have tried to carve a path through the morass of fear, panic, and desire for control to see how one can sustain a critical analysis of the pandemic response. as scholars and citizens, we have the obligation to think beyond the crisis, create openings in the world, and consider, critically and democratically, how we want to govern ourselves. as veena das underscores, it is important that we do not let our "love for the subtle and nuanced understanding of issues disappear on the grounds of needs for the rough and the ready in an emergency" (das ) . the pandemic and the response to it will require us to reimagine lives, rebuild conditions of existence, and find better ways of doing science and politics. like every engagement in a serious pedagogical project, it will entail a reconsideration of the objects we desire. today's fear is fueled by four main forces: mathematical disease modeling-a flexible and highly adaptable tool for prediction, mixing calculations with speculations, often based on codes that are kept secret and assumptions that are difficult to scrutinize from the outside. neoliberal policies-systematic disinvestments in public health and medical care that have created fragile systems unable to cope with the crisis. nervous media reporting-an endless stream of information, obsessed with absolute numbers, exploiting the lack of trust in the health care infrastructure and magnifying the fear of collapsing systems. authoritarian longings-a deep desire for sovereign rule, which derives pleasure from destruction and tries to push the world to the edge of collapse so that it can be rebuilt from scratch. this set of forces inspires thought, action, and passion in powerful ways. energized by the thrilling experience of witnessing "history in the making," actors and institutions have seized the opportunity to reorder the world, push political agendas in the name of survival, and shape life for years to come. the pandemic has become an auspicious moment to change the rules of engagement and expand the scope of scientific, medical, and political authority over bodies and populations. it is an occasion to publish papers and make dramatic statements, to feel relevant and important to the world, and enjoy the moment in the limelight. in the midst of death and destruction, the pandemic creates opportunities for innovation, domination, and profit-making. this unexpected opening connects elites in science, politics, and the media, releasing shocks of information, instruction, and command that are pushing hard against our confined, anxious, restless bodies. mathematical disease modeling, neoliberal policies, nervous media, and authoritarian longings fuel a fatal spiral centered around the fear of collapse. this fear is now literally in the air; it moves in and out of us with every breath; it operates as animating medium of our intense isolation and immobility. pandemic fear is unnerving and mentally exhausting. yet for those who embrace the feeling, it has the power of sustaining a state of excitement-excitement derived from the secret pleasure of spoiling a precious thing, wasting enormous resources, and engaging in an all-consuming project with total dedication. what we might call the provocation of the crisis-its intensification, expansion, and totalization beyond any notion of utility-seems so excessive and extreme that it borders on sheer madness. what could be more dangerous, more daring, more exciting than a walk on the wild side, an excursion to the other side of reason? melodramatic phrases such as "beating the virus," "winning the war," and "defeating the darkness" are rhetorically powerful and contagious. equally popular notions like "corona heroes" and "lockdown warriors" are symptoms of overidentification in a hegemonic discourse of power. all these terms reveal how this pandemic is "fabulously textual, through and through," and, at the same time, is lacking a source of symbolization strong, creative, and disturbing enough to move our engagement with the world beyond the most conventional of tropes (derrida ) . the language that we are asked to adopt today, in the midst of this outbreak, is contaminated with words that are stiff, stale, and corrupt like putrid air. given that so much of today's response is based on and driven by mathematical disease modeling and that millions of lives and livelihoods are being destroyed before our eyes, it is not an option anymore to exclude the "externalities" of a pandemic response that lacks imagination and that has resorted to the crudest interventions of all: the full stop. for those with permanent jobs, a comfortable couch, and no daycare duties, this unforeseen interruption may feel like a gift, a welcome relief from the non-stop world of global capitalism. but for millions of people living in less privileged parts of the planet, the pause button spells unemployment and hunger, not breaktime and downtime. without income, food, and access to basic health care, people are not making the most of the confinement outside in the garden; they are desperate and dying. we urgently need to look beyond the virus if we want to understand the real seriousness of what is happening today. how did we end up in this strange space of thinking, acting, and feeling that has normalized extremes and that is based on the assumption that biological life is an absolute value separate from politics? never has it been more important to insist that another politics of life is possible. the latest imperial college disease model report summarizes the staggering blindness that has prevailed in this pandemic: "we do not consider the wider social and economic costs of suppression, which will be high" (walker et al. ) . the time to suppress the costs of suppression and cast the consequences of interventions as an externality to model-based policy is over. these claims are utterly misleading, ignoring the influenza pandemics of and , including the hiv/aids pandemic, ebola, and many other infectious disease outbreaks that have killed millions of people worldwide with no vaccine available. last but not least, the toll of covid- is not even close to the toll of the influenza pandemic. . the report distinguishes between two strategies these days i sometimes catch myself wishing to get the virus-in this way, at least the debilitating uncertainty would be over… a clear sign of how my anxiety is growing is how i relate to sleep. till around a week ago i was eagerly awaiting the evening: finally, i can escape into sleep and forget about the fears of my daily life… now it's almost the opposite: i am afraid to fall asleep since nightmares haunt me in my dreams and awaken me in panic-nightmares about the reality that awaits me disasters and capitalism … and covid- . somatosphere website staying at home. e-flux real estimates of mortality following covid- infection. the lancet coronavirus testing: how some countries got ahead of the rest. the guardian website germany's coronavirus anomaly controllo e autocontrollo sociale ai tempi del covid- the pandemic perhaps: dramatic events in a public culture of danger the normal and the pathological french medics warn health service is on the brink of collapse. the guardian website anger and poverty grip lebanese city. the guardian as covid- pandemic hits india's daily-wage earners hard, some leave city for their home towns. scroll.in website facing covid- : my land of neither hope nor despair no apocalypse, not now impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and health care demand. imperial college website for autocrats and others corona virus is a chance to grab even more power. the new york times website wartime conditions" as global coronavirus deaths reach . the guardian website spread of sars-cov- in the icelandic population cash-starved hospitals and doctor groups cut staff amid pandemic. the washington post bangladesh: end wave of covid- "rumor" arrests. human rights watch website indian doctors evicted over coronavirus transmission fears, says medical body. the wire website, march ebola vaccines, evidentiary charisma and the rise of global health emergency research managing hospital volumes germany and experiences from oecd countries scale of china's wuhan shutdown is believed to be without precedent. the new york times so stark ist die krankenhaus-auslastung mit corona-patienten. mdr website kenya police abuses could undermine coronavirus fight. human rights watch website . case-fatality rate and characteristics of patients dying in relation to covid- in italy china tightens wuhan lockdown in "wartime" battle with coronavirus. the new york times website shows commitment to contain virus: who representation in china. reuters website us unemployment rises another m, bringing total to m since pandemic began. the guardian website india moves big labour law changes to limit coronavirus impact others make these changes. financial express website why belgium's death rate is so high: it counts lots of suspected covid- cases. npr website vorläufiges ergebnis und schlussfolgerungen der covid- case-cluster-study urgent call for an exit plan: the economic and social consequences of response to covid- pandemic. centre for global development website the global impact of covid- and strategies for mitigation and suppression who. . report of the who-china joint mission on coronavirus disease (covid- estimating clinical severity of covid- from the transmission dynamics in wuhan, china is barbarism with a human face our fate? critical inquiry blog stavrianakis, nancy tamini, and laurence tessier. i am particularly grateful for conversations with richard sullivan. maria josé de abreu insisted i start writing and put thoughts on paper. for suggestions, i would like to thank the editor of medical anthropology quarterly, vincanne adams, and the two anonymous reviewers. none of these colleagues and friends are responsible for the arguments in this article. key: cord- - w wn u authors: pedersini, paolo; corbellini, camilo; villafañe, jorge hugo title: italian physical therapists’ response to the novel covid- emergency date: - - journal: phys ther doi: . /ptj/pzaa sha: doc_id: cord_uid: w wn u nan this epidemiological transition in italy is continuously updated with characteristics of patients with covid- who have died, including: average age ( years old), sex ( % men), pathologies before hospitalization (patients with pre-existing pathologies %; patients with pre-existing pathology %; patients with pre-existing pathologies %; patients with or more pre-existing conditions %). lombardy has registered % of the covid- deaths in italy . on february , , in lombardy, a man was admitted with an atypical pneumonia that later proved to be covid- . when it was identified that community transmission was occurring, including several cases without any connections to previous cases or travel histories to china, lombardy raised its pandemic preparedness alert level establishing red zones in the affected areas. a series of gradually increased countermeasures has been implemented involving all professional groups, including physical therapists. in conjunction with this, an important document was drafted by the ) to transform all non-essential treatments into a tele-rehabilitation modality and to manage clinical cases through telephone or webcam counselling in order to skills. in fact, in the acute phases of the pathology, the respiratory physical therapists support the work of intensive care physicians, pulmonologists and nurses by: providing qualified care in the different modalities of non-invasive mechanical ventilation (niv); monitoring and addressing respiratory fatigue; preventing the complications of prolonged immobility; prone positioning to improve gas exchange in severily ill subjects; participating in the weaning from invasive mechanical ventilation protocols; and assisting in the recovery of the patient's autonomy in activities of daily life, which is often compromised by long periods of sedation and prolonged hospitalization. in a quick response to the increasing demand of patients in intensive care unit, the "associazione dei riabilitatori dell'insufficienza respiratoria" (arir) had written an outstanding position paper that covers everything regarding physical therapy practice in intensive care units. in italy, at the beginning of the spread of the epidemic, there were approximately , beds in intensive care units. the situation in the epicenter area has been described as terrifying with overcrowded hospitals and lack of medications, ventilators, oxygen, and personal protective equipment. so, it is also very important to reduce the length of hospitalization and therefore to free up places more quickly and allow other patients to be accommodated, given the serious shortage of beds compared to the growth of the contagion. many colleagues from milan, lombardy, and surrounding cities are organizing themselves into a sort of "physical therapy task force", to help each other to quickly improve their skills and knowledge but also to take action where necessary. in addition, covid- emergency preparedness frequently does not take into account issues specific to rehabilitation patients, when patient and staff safety have to be at the forefront of providing rehabilitation services to our community. besides being physical therapists, they are first and foremost health professionals and, before that, citizens who want to make their contribution to support the whole population and all the components of the health system responding efforts to this emergency. covid- -situazione in italia coronavirus disease (covid- ) in italy emerbenza covid- : indicazioni per fisioterapisti e pazienti rapid response to covid- : health informatics support for outbreak management in an academic health system covid- : fighting panic with information. the lancet clinical research as foundation for the advancement of respiratory physiotherapy. monaldi arch chest dis covid- and italy: what next? lancet at the epicenter of the covid- pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation. nejm catal mental health strategies to combat the psychological impact of covid- beyond paranoia and panic coronavirus: more countries will adopt italy's measures, says austrian leader. support the guardian web site wind of change: brexit and european rehabilitation the authors completed the icmje form for disclosure of potential conflicts of interest and reported no conflicts of interest.the authors certify that they have no affiliations with or financial involvement in any organization or entity with a direct financial interest in the subject matter or materials discussed in the article. key: cord- - bwtg xz authors: novara, giacomo; bartoletti, riccardo; crestani, alessandro; de nunzio, cosimo; durante, jacopo; gregori, andrea; liguori, giovanni; pavan, nicola; trombetta, carlo; simonato, alchiede; tubaro, andrea; ficarra, vincenzo; porpiglia, francesco title: impact of the covid‐ pandemic on urological practice in emergency departments in italy date: - - journal: bju int doi: . /bju. sha: doc_id: cord_uid: bwtg xz the severe acute respiratory syndrome coronavirus (sars-cov- ) and the disease it causes, coronavirus disease (covid- ), are causing a rapid and tragic health emergency worldwide [ , ]. italy was the first european country to experience a virus outbreak, starting on feb, st . it resulted in national quarantine, and the official lockdown of the country's non-essential businesses and services began on mar, th . although several reports are available in the literature on recommendations for reorganization of the clinical and surgical activities [ - ], to our knowledge, no data is available on the effective impact of covid- pandemic on the outcomes of other medical conditions. s evere acute respiratory syndrome coronavirus (sars-cov- ) and the disease it causes, coronavirus disease (covid- ), are causing a rapid and tragic health emergency worldwide [ , ] . italy was the first european country to experience a virus outbreak, starting on february . it resulted in a national quarantine, and the official lockdown of the country's non-essential businesses and services began on march . although several reports are available in the literature providing recommendations for the reorganization of clinical and surgical activities [ ] [ ] [ ] [ ] [ ] , to our knowledge, no data are available on the effects of the covid- pandemic on the outcomes of other medical conditions. for this reason, we aimed to evaluate the urological component of emergency department activities during the covid- pandemic, assessing the requests for urgent urological consultations in a network of academic and nonacademic hospitals in italy. to assess the impact of the covid- pandemic on urgent outpatient urological practice, we evaluated the urological consultations performed in emergency departments in a specific week, after the national lockdown of the country starting on march . accordingly, we considered those patients assessed between and march ( th week of the year). data on the patients observed in the same week of (between and march ) were also collected to provide a control group. a total of eight academic and non-academic urological centres ( continuous variables are reported as median and interquartile ranges. differences in variables with a continuous distribution across dichotomous categories were assessed using the mann-whitney u-test. pearson's chi-squared test was used to evaluate the association between categorical variables. all reported p values are two-sided, and statistical significance was set at p < . . all statistical analyses were performed with ibm spss statistics for macos, version (ibm corp., armonk, ny, usa). we identified urological consultations in emergency departments in and in the corresponding week of . that corresponds to a % year-on-year decrease in the overall number of consultations. table summarizes the clinical characteristics of the observed patients. on the whole, we identified significant geographical differences, with the decrease being higher in the centres from northern italy (À %), the region of the country more severely hit by the covid- pandemic, and, surprisingly, in the only centre in southern italy (À %). patients observed in were significantly older than those in consultations (p = . ). they had received a previous emergency department consultation evaluation for the same clinical complaints in a lower percentage of cases ( % vs %; p < . ). the most common clinical diagnoses were colicky flank pain, gross haematuria, and acute urinary retention. notably, a significant difference in the distribution of the diagnosis was observed (p = . ). following the emergency department consultation, a similar percentage of patients was hospitalized for urological conditions ( % and % in the and weeks, respectively). a higher percentage of patients with gross haematuria received early endoscopic management in ( % vs %; p = . ). similarly, the proportion of patients with colicky flank pain who received immediate jj placement or endoscopic lithotripsy was higher in ( % vs %); however, the difference between the two groups did not meet conventional levels of statistical significance (p = . ). to our knowledge, the present report is the first analysis of data on the modification of clinical activity secondary to the virus outbreak in a branch of medicine unrelated to the covid- pandemic. there were several interesting findings. firstly, a substantial decrease in the number of urgent urological consultations was observed. secondly, that decrease was heterogeneously distributed across the country, with hospitals in the northern part of italy that was more severely hit by the covid- pandemic reporting a more considerable reduction. conversely, the number of consultations was stable in the institutions in central italy. thirdly, the patients who arrived at hospital were significantly older and had a slightly different spectrum of urological conditions, including more cases of gross haematuria and acute urinary retention, conditions commonly considered as undeferrable. likewise, life-threatening conditions such as trauma were mostly unchanged, whereas the prevalence of less severe clinical diseases, such as uncomplicated urinary infections, decreased. lastly, we identified a pattern of more aggressive treatment in two specific conditions; specifically, the number of early haemostatic transurethral resections for gross haematuria was higher in the patients. that was mainly attributable to the need to reduce the need for blood transfusions as much as possible, considering the shortage of blood products resulting from decreased donation [ , ] , and to shorten the length of stay. similarly, the number of jj stenting and ureteroscopy procedures for urinary stones was numerically higher in the current year as compared to . that was mainly attributable to the need to resolve the patient's clinical problem (i.e. recurrent pain and/or urosepsis), thus preventing them from repeated presentation to hospital, which could have ultimately increased the risk of contagion. in this context, the choice of the most appropriate treatments (stenting alone vs endoscopic lithotripsy) was mainly related to patients' condition, size and position of the stones, in agreement with the currently available guidelines [ ] , as well as the local availability of urologists, anaesthesiologists and other health workers in this challenging period [ ] . the present report is noteworthy for several reasons. all urology departments in italy and worldwide are facing reorganization and restriction of their activity to differing extents. recommendations have been recently implemented on the way to perform such a reorganization [ , ] , and on which surgical procedures to prioritize [ , , ] . however, in addition to this, the present report suggests that the urological departments of the different institutions should be prepared and equipped to manage approximately % of their usual urological activities in their emergency departments, including specialized services for urgent management of lower and upper urinary tract diseases. however, from the emergency department perspective, it should be taken into consideration that the amount of activity related to the urgent urological conditions has been approximately halved but not reduced to zero. although data from other medical and surgical specialties are lacking, our findings imply that physicians in emergency departments must be present to handle non-covid patients in a proper measure according to the level of diffusion of the virus outbreak in the different countries. finally, all clinicians suspect and fear that a significant number of patients with major medical conditions unrelated to covid- are not arriving at hospital in the present period because of movement restrictions and fear of the contagion. however, our data seem to suggest that, at the moment, this is not happening on a massive scale, at least not for urgent urological diseases. however, only in the coming months will we be able to understand how many and eventually which urological conditions experienced a diagnostic delay as a result of the pandemic. the present report has some limitations, first and foremost, those related to its retrospective design. second, the involved institutions collected information on the experience of institutions. finally, the present assessment is only a snapshot of the situation in a specific week. consequently, the number of cases was not extremely large. a more prolonged evaluation would be needed to report the evolution of the problem, along with the diffusion of covid- across the country. in the present report evaluating urological consultations in emergency departments during one week of the covid- pandemic, we found a substantial decrease in the number of urgent consultations. the reduction was heterogeneously distributed across the country. the patients observed in the group had a slightly different spectrum of urological conditions compared with those in the control group. the most common diagnoses were colicky flank pain, gross haematuria and acute urinary retention. the prevalence of life-threatening conditions, such as trauma, was stable. as a consequence of the current situation, we observed an increase in the adoption of some endoscopic urological procedures to treat mainly bladder cancer. clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china covid- and italy: what next? considerations in the triage of urologic surgeries during the covid- pandemic urology practice during covid- pandemic advice for medical oncology care of urological cancer patients during the covid- pandemic guidelines office rapid reaction group: an organisation-wide collaborative effort to adapt the eau guidelines recommendations to the covid- era pathways for urology patients during the covid- pandemic eau guidelines eau guidelines on urolithiasis members of the research urology network (run) key: cord- -wtdzad authors: tuite, ashleigh; ng, victoria; rees, erin; fisman, david title: estimation of covid- outbreak size in italy based on international case exportations date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: wtdzad italy is currently experiencing an epidemic of covid- which emerged in the lombardy region . during the interval between february - , , we identified cases of covid- reported in countries in europe, africa, north america, and south america which were either in individuals with recent travel from italy, or who had presumed infection by a traveler from italy . in six cases, in four of the affected countries (switzerland, france, austria, croatia), land travel was a likely route of introduction, or was documented to have been the route of introduction. we used air travel volume between italian cities and cities in other countries as an index of connectedness, using data available from the international air transport association (iata) for february ( . million total departing international air passengers from italy). we used the methods of fraser et al. to estimate the size of the underlying epidemic in italy necessary in order for these cases to be observed with a reasonable probability. to estimate the time at risk of covid- exposure for travelers departing italy, we obtained data from the united nations world tourism organization (unwto) for the proportion of international travelers that are non-residents of italy ( %) and the average length of stay of tourists to italy ( . days), and assumed the italian epidemic began one month prior to february , . we also performed sensitivity analyses in which we included outbound travel to all countries regardless of reported case importations, inflated travel volumes by %, to account for the relative increase in flight numbers from - , and excluded cases in bordering countries and which were documented to have been introduced by overland travel. when all cases were considered we estimated a true outbreak size of cases ( % ci - ), as compared to a reported case count of on february , , suggesting non-identification of % ( - %) of cases. in sensitivity analyses, outbreak sizes varied from to cases (implying non-identification of - % of cases). we recently used similar methods to estimate a much larger epidemic size in iran, with a far greater degree of under-reporting, based on many fewer exported cases. the reason for this difference relates to the relatively high volume of travel from italy, relative to iran. in summary, we suggest that the numerous covid- case exportations from italy in recent days suggest an epidemic that is larger than official case counts suggest, and which is approximately on a par with that currently occurring in south korea, which reports cases (and fewer deaths) as of february , . were either in individuals with recent travel from italy, or who had presumed infection by a traveler from italy . in six cases, in four of the affected countries (switzerland, france, austria, croatia), land travel was a likely route of introduction, or was documented to have been the route of introduction . . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint we used air travel volume between italian cities and cities in other countries as an index of connectedness, using data available from the international air transport association (iata) for february ( . million total departing international air passengers from italy). we used the methods of fraser et al to estimate the size of the underlying epidemic in italy necessary in order for these cases to be observed with a reasonable probability. to estimate the time at risk of covid- exposure for travelers departing italy, we obtained data from the united nations world tourism organization (unwto) for the proportion of international travelers that are non-residents of italy ( %) and the average length of stay of tourists to italy ( . days) , and assumed the italian epidemic began one month prior to february , . we also performed sensitivity analyses in which we included outbound travel to all countries regardless of reported case importations, inflated travel volumes by %, to account for the relative increase in flight numbers from - , and excluded cases in bordering countries and which were documented to have been introduced by overland travel. when all cases were considered we estimated a true outbreak size of cases ( % ci - ), as compared to a reported case count of on february , , suggesting nonidentification of % ( - %) of cases. in sensitivity analyses, outbreak sizes varied from to cases (implying non-identification of - % of cases) (table) . we recently used similar methods to estimate a much larger epidemic size in iran, with a far greater degree of under-reporting, based on many fewer exported cases. the reason for this difference relates to the relatively high volume of travel from italy, relative to iran . in summary, we suggest that the numerous covid- case exportations from italy in recent days suggest an epidemic that is larger than official case counts suggest, and which is approximately on a par . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint with that currently occurring in south korea, which reports cases (and fewer deaths) as of february , . . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint covid- : italy confirms deaths as cases spread from north tracking coronavirus: map, data and timeline. available via the pandemic potential of a strain of influenza a (h n ): early findings tourism statistics: italy: country-specific: basic indicators (compendium) united nations, encyclopedia of the nations: average length of stay of visitors -tourism indicators -unctad handbook of statistics -country comparison first report of covid- in south america estimation of covid- burden and potential for international dissemination of infection from iran key: cord- -ab o xol authors: giuliani, diego; dickson, maria michela; espa, giuseppe; santi, flavio title: modelling and predicting the spatio-temporal spread of coronavirus disease (covid- ) in italy date: - - journal: nan doi: nan sha: doc_id: cord_uid: ab o xol official freely available data about the number of infected at the finest possible level of spatial areal aggregation (italian provinces) are used to model the spatio-temporal distribution of covid- infections at local level. data time horizon ranges from february , which is the date when the first case not directly connected with china has been discovered in northern italy, to march . an endemic-epidemic multivariate time-series mixed-effects generalized linear model for areal disease counts has been implemented to understand and predict spatio-temporal diffusion of the phenomenon. previous literature has shown that these class of models provide reliable predictions of infectious diseases in time and space. three subcomponents characterize the estimated model. the first is related to the evolution of the disease over time; the second is characterized by transmission of the illness among inhabitants of the same province; the third remarks the effects of spatial neighbourhood and try to capture the contagion effects of nearby areas. focusing on the aggregated time-series of the daily counts in italy, the contribution of any of the three subcomponents do not dominate on the others and our predictions are excellent for the whole country, with an error of per thousand compared to the late available data. at local level, instead, interesting distinct patterns emerge. in particular, the provinces first concerned by containment measures are those that are not affected by the effects of spatial neighbours. on the other hand, for the provinces the are currently strongly affected by contagions, the component accounting for the spatial interaction with surrounding areas is prevalent. moreover, the proposed model provides good forecasts of the number of infections at local level while controlling for delayed reporting. many scholars are trying to give a contribution to the problem, both in open online venues and on scientific publications, debating about reproductive number, mortality distributed for age and gender and, more in general, epidemic features, without neglecting to avail of mathematical and statistical models. the main contributions use deterministic epidemiological models (see kucharski et al., ; liu et al., a; sun et al., , among others) , all focussing on the time evolution of the phenomena, especially with predictive purposes. the prediction of new infected, deceased and healed is certainly essential for health policy in order to estimate the capacity of a health system to cope with the stress caused by a pandemic. nevertheless, in the recent literature the relevance of space in the diffusion of the covid- is treated only marginally (chinazzi et al., ; li et al., b) , although the importance of spatial and spatio-temporal autocorrelation in epidemiology was already highlighted in the seminal book by cliff and ord ( ) . the authors show the effectiveness of statistical methods in analyzing the incidence of some epidemics, such as e.g. measles in cornwall, cornwall, - in london in and tuberculosis and bronchitis in wales, wales, - in the last years spatial epidemiology has been evolving rapidly and it has found a great response in medical applied research (for a recent review on methods and applications in the field, see kirby et al., ) . the importance of the space in the study of disease transmission, like all natural phenomena, answers to the first law of geography (tobler, ) , for which everything is related to everything else, but near things are more related than distant things". reformulating this concept, it could be said that the phenomenon external to an area of interest affects what goes on inside. in light of this, we found imperative to include a component that can account for spatial dependence among areal units in the study of covid- diffusion, in order to explain the strength and direction of the spreading on the territory as well as in time. this is particularly relevant in countries such as italy or germany in which the local health systems interested by the disease are strongly regionalized. the territorial specificity, which can result in more or less drastic containment measures, cannot be neglected in a model construction that has the ambition to explain how the contagion moves in space and time and to allow reliable spatio-temporal predictions. the purpose of the present paper is to model and predict the number of covid- infections, drawing out the effects of its spatial diffusion. forecasts about where and when the disease will occur may be of great usefulness for public decision-makers, as they give them the time to intervene on the local public health systems. the italian department of civil protection according with the italian ministry of health, has started to release a daily bulletin about covid- infections in italy since february . by march data were also released at nuts- level (provinces), having been previously released only at the highly aggregated nuts- level. data are available from the websites of the main italian newspapers, such as, il sole ore. therefore, immediately after the release, we were able to collect the infectious disease count time series at provincial level for the period nuts (nomenclature of territorial units for statistics) is a european union geocode standard for referencing the subdivisions of countries for statistical purposes, established by eurostat in agreement with each member state. the level of nuts are four, nested into each other: nuts- are the sovereign countries; nuts- are the major socio-economic regions; nuts- are basic regions for the application of regional policies (italian regions); nuts- are small regions for specific diagnoses (italian provinces). https://lab .ilsole ore.com/coronavirus. from february to march using two sources. since spatial dependence may result stronger than it actually is at a coarse spatial resolution (arbia et al., ) , we have wanted to use data at the provincial level of aggregation, which is the freely available finest possible level of aggregation. for the first six days, data were provided only to media outlets and published by newspapers in the form of interactive maps. for this period, we have used web scraping techniques to track raw data upon which maps have been constructed. for the remaining days, we simply downloaded data about provinces released by the department of civil protection. clearly, at this stage, data suffer from several problems of quality. the number of contagions is continuously updated and positive swabs need to be confirmed by the national institute of health, not to mention several cases of delayed reporting by the local authorities. therefore, the results presented in the paper may benefit from availability of more accurate data. the overall temporal distribution of daily counts of covid- infections is depicted in figure , while the spatial distribution is showed in the map in figure . the two plots indicate that while the growth of cases over time in italy has been globally exponential, it is characterized by strong local heterogeneity and also by a relevant spatial pattern in the form of a hot-spot in the northern part of the country (the plot of the time series disaggregated for all italian provinces is reported in the appendix). the evolution of the number of daily infections is studied by means of a statistical model which has been adapted from that proposed in held et al. ( ) and paul and held ( ) , where both temporal dynamics of the infections and its geographical mechanism of spread are considered and estimated (see adegboye and adegboye, ; cheng et al., , for an implementation of the model in epidemiology). the expected number of infections occurred in a province in the course of a day may be decomposed according to three additive components representing three different channels through which the contagions may increase. the first component of contagion is represented by the spread mechanism of the covid- within each province, which depends on the cumulative incidence of the disease and determines the speed of diffusion of the virus in the future. this component determines the temporal dynamics of the contagion within each province and for this reason is referred to as epidemicwithin component (see appendix). another channel is attributable to contagions originated from other provinces, and in particular from provinces which are geographically close to each other. the origin of such spatial dimension of contagion can be found in the high mobility of people across provinces, and substantially contributes to explain the observed diffusion of the virus from a limited number of focuses to a wider area which, at the time we are writing, heavily affects the most part of northern italy. in the following, this source of contagion is referred to as epidemic-between component, as it concerns the inter-province spread of covid- (see appendix). the last portion of cases should be attributed to province-specific conditions which determined the first centres of infections and the initial exposure to the risk of contagion. such a local component of the overall daily province infections is referred to as endemic component, according to terminology introduced in paul and held ( ) , and which, in this context, does not implies any epidemiological qualification of the covid- in the population of italian provinces. we point out that in this paper both terms epidemic and endemic have been inherited from paul and held ( ), whereas terms within and between are introduced in order to distinguish between the temporal and spatial terms which paul and held ( ) jointly refers to as epidemic component. the statistical model is structured in three parts which contribute to the number of daily infections per province according to the three described components. each part of the model considers also the presence of heterogeneity both in the temporal and in the geographical mechanisms of contagion, so that the different dynamics existing inside the provinces, both in terms of temporal and geographical mechanisms spread of covid- are modelled. all analyses presented in this paper were performed using the r software (r core team, ). the estimated model provides useful insights about the evolution and spread of covid- occurrences across the territory of italy. the most striking evidence is that the epidemic potential across areas is, on average, very strong; however, it is also highly heterogeneous among provinces. to better assess the degree of spatial heterogeneity, figure shows three maps depicting the decomposition of the estimated expected number of infections into its three components -namely the within-epidemic, between-epidemic and endemic contributions -by provinces. for each province, the three fitted components are expressed as proportions of their sum. we can clearly see that only few provinces, the most affected by the disease until now, are mainly influenced by the local endogenous transmission of the contagion (map on the left). for a relatively higher number of provinces, mostly located in the north and center parts of the country, a relevant number of cases is instead explained by the transmission from neighboring provinces (map on the centre). for the majority of provinces, located mainly in the south, the contagions follow essentially the endemic trend (map on the right). the comparison among provinces in terms of the three investigated components is robust to spuriousness because the italian population is entirely and homogeneously susceptible, that is immunologically "naive", to this new virus. moreover, being vaccines still missing, there are no differences among provinces deriving from vaccination coverage. to gain further insights about the relative importance of the three components we take into consideration some paradigmatic provinces located in the part of the country with the highest number of cases, that is the northern areas (see figure ). in particular, we focus the attention on the northern part of italy because after the illness started to occur in the country, it took several days to spread throughout the territory, thus leading to an unbalanced spatial distribution of infections. figure depicts the mean number of cases estimated by the model along with the observed number of cases for the paradigmatic provinces. first of all, the province of lodi (lombardy region), which is the first area that reported cases, sees a predominance of the component related to the internal diffusion of the disease. this evidence agrees with the quarantine to which some of its municipalities underwent since the beginning of the outbreak and with the hypothesis that the epidemic in italy started right there. the province of lodi shares the eastern border with the province of cremona (lombardy region), that in turn shares the northern border with the province of bergamo (lombardy region) as depicted in figure . the latter two are so far some among the most severely affected provinces in italy. their proximity with the province of lodi makes them widely susceptible to the contagion effect between neighboring regions. this suggests that the contagion of infections among inhabitants occurred before containment measures were carried out and that the disease remained undetected before patient was discovered. a similar situation arose for the province of parma (emilia-romagna region), which share the norther border with the province of cremona. even here the between-epidemic component is relevant and it may be explained with the strong social and economic ties existing among the areas. the second in time discovered outbreak of covid- in italy was detected in a small town in the province of padova (veneto region). even in this case, strong control measures to reduce transmission outside have been carried out, as evidenced by the almost total influence of the within-epidemic component. this shows that there have been no "return infections" from the surrounding areas but it does not mean that, before the quarantine, the infection could not have circulated in the neighboring provinces. in fact, the province of venice (veneto region), which is geographically, culturally and economically near to that of padova demonstrates to be affected by these form of interactions. the cases of the provinces near the national borders are also of particular interest, such as the provinces of turin (piedmont region) and trieste (friuli venezia giulia region), adjoining with france and slovenia, respectively. these provinces may be affected by the so-called boundary problem for which the analysis of a phenomenon may be biased by the presence of arbitrary administrative geographic borders. thus, on one side we might lose information regarding what happens beyond the country border, due to different ways of collect information and monitor the diffusion of the disease. one the other side, we might not have a clear overview about cross-border phenomena as job trajectories. in addition, the two considered provinces seem to be not connected with the aforementioned outbreaks originated in lombardy and veneto regions. due to these reasons, the provinces of turin and trieste show a limited effect of epidemic components and a prevalence of the endemic component, which is due only to the evolution of the disease over time. looking at the national aggregated counts, the exponential evolution of covid- contagions is clear. on the country as a whole, the main component seems to be the temporal trend but also the epidemic components have a not negligible role. the observed decomposition is probably mainly due to the central and southern provinces, which are characterized almost exclusively by the spread over time component. moreover, to check the ability of the proposed model in explaining the spatio-temporal distribution of the covid- occurrences in italy, we examine how it is good at predicting the future daily counts of infections. therefore, we re-estimate the model using the data between february - march and we make prediction for march . the observed total number of cases at march , obtained by aggregating the province data, is ; the model provides a prediction of cases. therefore, the model underestimates by only units. naturally, the advantage of the proposed model is that it provides predictions for each individual province (point predictions can be found in tables and of the appendix) but it is clear that the use of predictions at local level can have positive effects on the prediction of total number at country level. we report (in figure of the appendix) the % prediction intervals of the provincial counts of infections at march . given the brevity of the observed time series, to obtain a reasonable degree of precision we cannot require higher confidence levels. despite the fact that the intervals are quite wide, the results are promising if compared with the known observed counts. we expect that the level of precision will improve as the data are updated and the observed time series become longer. in this article, we analyzed, by modelling, the trend of covid- epidemics along time and space. the use of spatio-temporal models can greatly improve the estimation of number of infected and can help the public decision-makers to better plan health policy interventions. italy has viewed a massive spread of the disease with peculiar patterns on the territory. started from some provinces in the northern area, this serious illness descended down the boot and is nowadays present in all italian provinces. several discrepancies among provinces, especially between north and center-south of the country, persist but coming weeks seem to be decisive to understand the behavior of the contagion trend. containment measures in italy have followed an application in three steps: first, some municipalities in lombardy and veneto regions underwent to quarantine; second, the entire lombardy and some provinces in other northern regions (veneto, piedmont, emilia-romagna and marche) were isolated from the rest of the country; third, all italian territories were subjected to a complete lockdown. such stepwise approach in imposing hard control measures to the entire national territory might have conducted to a temporal shifting of the contagion dynamics. emblematic is the case of the province of lodi, the first area submitted to the quarantine. after an average increment of cases when the illness began to be systematically discovered, in the last days of the time series, which correspond to three weeks after the lockdown, this province seems to have settled the average number of newly discovered contagions equal to cases (so the contagions have been halved). this fact suggests that policies of contagion containment seem to exert a mild success so far in the areas in which there was an effective enforcement of the control measures. instead, in metropolitan areas where the population density and the more active social behavior make more difficult to comply properly to the recommendations, the whished effects are not yet taking place. this is the case of the province of milan, which is experiencing an increasing number of cases in last days (from an average of new cases detected of the beginning to an average of new cases of last days). and then there is the case of center and southern provinces, which have viewed a delayed start of the epidemics but also the arrival, in the last two weeks, of flows of people escaped from northern regions undergone to lockdown. as an example, the province of florence went from an average increment of new cases of the first period to an average increment of new cases of last days, and, similarly, the province of naples, has gone from an average increment of new cases to an average increment of new cases, confirming the hypothesis of downhill race of the disease along the peninsula. all this has led to the failure in applying homogeneous measures at national level, also due to regional autonomy in force in italy and to important local differences, which may lead to delays or failures in containing the contagions from a global point of view. this evidence suggests that consider the peculiarities of local territories may be effective in planning specific strategies for enforcing the containment measures established at national level. these differences in the dynamics of the epidemics in italy demonstrate the crucial importance of a strong national coordination level for the homogeneous enforcement of control measures, but also reveal how essential predictions at local level are. since the epidemics started, a frequent question of the public decision-maker is about when the peak of contagions will manifest. probably a more appropriate request should concern the emergence of different local peaks in different moments, which italy should expect in next weeks. the dramatic events in northern provinces can serve as a test bench for the health system, offering an overview about what southern provinces might be confronted. analyses and predictions both in space and time offer a decisive perspective about which areas may be more affected and when, given the time to the decision-makers to intervene on the local policies. dg and fs were responsible for statistical modeling, data analysis and writing of the manuscript. mmd and ge were responsible for result presentation, discussion and writing of the manuscript. all authors reviewed, revised and approved the final manuscript. • none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. • no competing interests are at stake and there is no conflict of interest with other people or organizations that could inappropriately influence or bias the content of the paper. the data about the spatio-temporal distribution of coronavirus disease infections at province level consist in multivariate count time series whose spatial references are in the form of irregular spatial lattices. therefore, the proper regression modelling framework for this empirical circumstance is the class of the so-called areal generalized linear models (glms). by extending the seminal model originally introduced by held et al. ( ) , paul and held ( ) proposed an endemic-epidemic multivariate time-series mixed-effects glm for areal disease counts, which proved to provide good predictions of infectious diseases (see adegboye and adegboye, ; cheng et al., , among the others). the main equation of the model describes the expected number of infections µ r,t in a region (province) r at time (day) t as follows: where y r,t is the number of infections reported in the region r at time t, which follows a negative binomial distribution with region-level overdispersion parameter ψ r . if ψ r > the conditional variance of y r,t− is µ r,t ( + ψ r µ r,t ), while if ψ r = the negative binomial distribution reduces to a poisson distribution with parameter µ r,t . the three terms on the right-hand side of equation ( ) correspond to the three components of the model: the epidemic-within, the epidemic-between, and the endemic. the first component models the contribution of temporal dynamics of contagions to the expected number of infections within region r. the term includes the number of infections observed in the previous day (time t − ), which affect µ r,t depending on the value of the coefficient λ r > . as the notation suggests, λ r changes amongst the provinces because of a random effect which allows for heterogeneous behaviour in the dynamics of contagions. the epidemic-between component models the contagion between neighbouring provinces by including the average incidence of the infections q r ,t− of provinces r which are neighbours of province r. in particular, the coefficients w r ,r in the summation r =r w r ,r q r ,t− are positive if either province r and r share a border or province r and r share a border with the same province, whereas w r ,r is zero otherwise. the coefficient φ r determines the magnitude of the effect of inter-province spread of contagion, and changes amongst provinces according to the population as well as to unobserved heterogeneity in the diffusion process. the last component determine the province-specific contribution to the number of infections, once the temporal and spatial autoregressive effect are accounted for. the term e r is the population of province r, whereas term ν r,t consists of a national time trend component, and a province-specific effect depending on the share of population over , and on a random effect which catches the heterogeneity due to unobserved factors. paul and held ( ) suggested that the endemic and epidemic subcomponents can be modelled themselves through log-linear specifications: log(ν r,t ) = α (ν) where the α (·) r parameters are region-specific intercepts and z (·) r,t represent observed covariates that can affect both the endemic and epidemic occurrences of infections. the varying intercepts allow to control for unobserved heterogeneity in the disease incidence levels across regions due, for example, to under-reporting of actual infections (paul and held, ) . given the regionally decentralized health system in italy, non-negligible differences in case reporting of covid- infections among italian regions are very likely, which make the opportunity to have regionspecific intercepts very important. following paul and held ( ) region-specific intercepts can be obtained through the inclusion of random effects. in particular, we assume here that . the paul and held ( ) model with normally distributed random effects can be estimated through penalized likelihood approaches that have been implemented in the r package surveillance (meyer et al., ) . see paul and held ( ) for futher details. given the brevity of the observed time series, the epidemic-within autoregressive parameter is assumed to be constant over time and, in absence of useful exogenous covariates, the model of equation ( ) takes the form log(λ r ) = α (λ) r , that is, the "internal" infectiousness depends only on a spatially varying intercept. following meyer and held ( ) , the subcomponent model for the epidemic within autoregressive parameter, equation ( ), is here specified as log e r , which accounts for the fact that the regions may have different propensities to be affected by the other neighbouring regions, and this may depend by their resident population share. the rationale is that the more populated regions tend to be more susceptible to transmission across regions. since some first recent empirical evidences suggest that the number of covid- infections seems to grow exponentially over time (liu et al., b; maier and brockmann, ) , the endemic component model assessing the temporal dynamic of disease incidence, equation ( ), is specified as a second-order polynomial log-linear regression: log(a r ), where t = , , . . . is the time in days and a r is the proportion of inhabitants over years old. in the global model of equation ( ) the endemic predictor ν r,t is multiplied by the offset e r , which in our case is the regional share of resident population. we do not include higher-order terms to avoid the risk of introducing spurious endemic patterns and overfitting. spatially correlated time series and ecological niche analysis of cutaneous leishmaniasis in afghanistan effects of the maup on image classification analysis of heterogeneous dengue transmission in guangdong in with multivariate time series model the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak spatial processes: models and applications clinical characteristics of coronavirus disease in china a statistical framework for the analysis of multivariate infectious disease surveillance counts advances in spatial epidemiology and geographic information systems early dynamics of transmission and control of covid- : a mathematical modelling study. the lancet infectious diseases early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (covid- ) evolutionary history, potential intermediate animal host, and crossspecies analyses of sarscov the reproductive number of covid- is higher compared to sars coronavirus the reproductive number of covid- is higher compared to sars coronavirus effective containment explains sub-exponential growth in confirmed cases of recent covid- outbreak in mainland china power-law models for infectious disease spread spatio-temporal analysis of epidemic phenomena using the r package surveillance authors thank umberto agrimi of the istituto superiore della sanit (italian national institute of health, rome), who read a preliminary version of this article and provided several valuable comments and suggestions. key: cord- -dmgd d authors: berardi, giammauro; colasanti, marco; levi sandri, giovanni battista; del basso, celeste; ferretti, stefano; laurenzi, andrea; guglielmo, nicola; meniconi, roberto luca; antonini, mario; d’offizi, gianpiero; ettorre, giuseppe maria title: continuing our work: transplant surgery and surgical oncology in a tertiary referral covid- center date: - - journal: updates surg doi: . /s - - - sha: doc_id: cord_uid: dmgd d covid- is rapidly spreading worldwide. healthcare systems are struggling to properly allocate resources while ensuring cure for diseases outside of the infection. the aim of this study was to demonstrate how surgical activity was affected by the virus outbreak and show the changes in practice in a tertiary referral covid- center. the official bulletins of the italian national institute for the infectious diseases “l. spallanzani” were reviewed to retrieve the number of daily covid- patients. records of consecutive oncological and transplant procedures performed during the outbreak were reviewed. patients with a high probability of postoperative intensive care unit (icu) admission were considered as high risk and defined by an asa score ≥ iii and/or a charlson comorbidity index (cci) ≥ and/or a revised cardiac risk index for preoperative risk (rcri) ≥ . patients were operated, including ( . %) liver and kidney transplantations. patients had few comorbidities ( . %), low asa score ( . ± . ), cci ( . ± . ), and rcri ( . ± . ) and had overall a low risk of postoperative icu admission. few patients had liver cirrhosis ( . %) or received preoperative systemic therapy ( . %). ( %) high-risk surgical procedures were performed, including major hepatectomies, pancreaticoduodenectomies, total gastrectomies, multivisceral resections, and transplantations. despite this, only patients ( . %) were admitted to the icu. only oncologic cases and transplantations were performed during the covid- outbreak. careful selection of patients allowed to perform major cancer surgeries and transplantations without further stressing hospital resources, meanwhile minimizing collateral damage to patients. since december , the world is struggling against coronavirus disease and in the past months, the life of more than one-third of the planet's population has radically changed [ ] . severe acute respiratory syndrome coronavirus (sars-cov- ) has shown to be a very contagious virus potentially causing complicated atypical pneumonia and associated with significant mortality [ ] . despite initially confined to hubei province, china and asia, covid- promptly spread to western countries, and in march , , the world health organization (who) declared a public health emergency of international concern [ ] . by early april, we are counting over , , cases and , deaths worldwide, with countries involved over continents [ ] . italy was one of the first western countries diagnosing covid- patients in late january and was certainly the one suffering the most by the sudden outbreak of the disease. indeed, sars-cov- rapidly spread throughout the country by the end of february, challenging the healthcare system toward its collapse, increasing the mortality rate and raising the question concerning the need for special measures to contain the crisis. as a matter of fact, by mid-march the italian cases of covid- were already , with almost patients requiring mechanical ventilation in the intensive care unit (icu) [ ] . consequently, the italian government announced extraordinary measures to contain the spread of the pandemic, by means of small confinements in northern italy, reaching a nationwide lockdown that is still limiting the population [ ] . covid- outbreak seriously stressed the healthcare system worldwide, with the urgent need of extra icu beds, dedicated hospital paths, personnel training, and infection control measures [ , ] . care of diseases outside the covid- has rapidly changed, and healthcare providers are currently adapting to maximize and properly allocate resources [ ] . surgical activity has shifted to pursue only emergency and elective cancer cases as described in different specialties [ ] [ ] [ ] [ ] . furthermore, the big picture and the full impact is still unknown as its effects on economy, hospital infrastructure, healthcare strategy, and prognosis of oncologic patients [ ] . indeed, people with cancer might suffer the most by this worldwide outbreak as the access to surgery is limited by the significant reduction in available resources, eventually influencing the decision making and the therapeutic allocation of patients [ ] . the italian experience anticipated what the world is currently facing, and the measures for containment, the healthcare adaptations, and the changes in hospital practice have been already tested and are somehow established in our country where the peak of the infection seems to be reached and overcome [ ] . as the italian national institute for the infectious diseases, we have hospitalized the first italian covid- patients and since then, our general surgery department had to face this reality [ ] . the aim of this study was to demonstrate how our surgical activity was affected by the covid- outbreak, what adaptations were made and how our practice changed, eventually sharing the lessons learned as anticipators in a health issue of international concern. on january , two covid- -positive individuals were hospitalized at the italian national institute for the infectious diseases "l. spallanzani" of rome. in this hospital, four departments and five daily outpatient clinics are dedicated to the diagnosis and treatment of various infectious diseases. health emergencies, such as sars in and ebola in , have been nationally referred and managed at our hospital previously [ , ] . in the same institution, our general surgery department operates in two daily operative rooms (or) more than cases per year, including liver, kidney, and pancreas transplantations. the icu of the hospital admits both medical and surgical patients in a -bed department. the records and official bulletins of our hospital were reviewed to retrieve the number of daily covid- patients hospitalized, those requiring icu admission and those discharged home. furthermore, all the records of consecutive surgical procedures performed during the covid- outbreak were reviewed, and the following data were extracted: age, gender, disease, comorbidities, neoadjuvant therapy, cirrhosis, previous surgery, type of surgical procedure, type of approach (open vs. laparoscopic), conversion, operative time, blood loss, and admission to the icu. the search was limited between the nationwide lockdown (march , ) and april , . informed consent was obtained from each patient, and every oncologic case was discussed in a multidisciplinary meeting involving surgeons, medical oncologists, gastroenterologists, radiologists, and pathologists. comorbidities were graded using the american society of anesthesiology (asa) score, the charlson comorbidity index (cci), and the revised cardiac risk index for preoperative risk (rcri). patients with a high probability of postoperative icu admission were considered as high risk and defined by an asa score ≥ iii and/or a cci ≥ and/or a rcri ≥ as previously validated [ ] [ ] [ ] . major hepatectomies were defined as the resections of three liver segments or more [ ] . pancreatic resections, total gastrectomies, major hepatectomies, and multivisceral resections as well as liver and kidney transplantations were considered as the high-risk surgical procedures because of the increased likelihood of postoperative icu admission. distribution of variables was assessed using kolmogorov-smirnov and shapiro-wilk tests. data are expressed as the mean ± standard deviation for parametric continuous data and as median and interquartile ranges for non-parametric distribution. categorical data were expressed as number and percentages. categorical data are expressed as number and percentages. chi-squared or fisher exact test with yates correction when appropriate was used to compare differences in categorical variables. unpaired student's t-test was used to compare differences in continuous parametric variables and the mann-whitney test for continuous non-parametric variables. statistical analysis was performed with spss software (version . . armonk, ny, ibm corp) for macosx. a p value of < . was considered statistically significant. on january , (day ), the first two covid- -positive patients in italy were admitted to the department of infectious diseases of our hospital with mild fever and atypical pneumonia requiring no invasive treatment. on february (day ), due to the worsening clinical conditions, mechanical ventilation was needed, and the two patients were admitted to the icu. three days later (day ), a third patient with positive nasopharyngeal swab was admitted and monitored at the department of infectious diseases. from day to day (march , ), diagnostic nasopharyngeal swabs for symptomatic patients were implemented at our institution as part of a regional referral policy; however, no further patients tested positive to sars-cov- , picturing a stable phase of the epidemic. on march (day ), two individuals had a positive swab and were hospitalized; since then, an exponential growth of covid- cases admitted to our hospital was registered as part of a national trend. sixty-five hospitalized cases were reached on day , nine of which required mechanical ventilation, therefore saturating the capacity of the icu. on the evening of march , the italian government announced the national lockdown, and the following day (day ), the first institutional guidelines on extraordinary covid- measures were released by our hospital (fig. ) : ( ) all healthcare workers were required to enhance hygienic measures and wear surgical masks and gloves within the hospital building. ( ) construction works in the icu started with the aim of increasing the number of beds from to . ( ) one of our daily general surgery operating rooms was closed to reallocate anesthesiologists and nurses to the management of covid- cases. ( ) the or personnel was trained on how to wear and un-wear special equipment and how to deal with contagious patients. ( ) our surgical department was asked to re-consider and re-work the or planning to perform only oncological cases and transplantations; outpatient clinics were significantly reduced. ( ) questionnaires on possible infection or contacts with covid- cases were administered to all patients admitted and scheduled for surgery; nasopharyngeal swabs were performed in suspicious cases. ( ) all gatherings, including our multidisciplinary meetings, were forbidden and reworked on a webinar platform. on march (day ), further institutional guidelines were released: ( ) relatives were forbidden to visit patients before and after surgery. ( ) nasopharyngeal swabs were administered to all patients admitted and scheduled for surgery and isolation was maintained until test's response. as a result, the first covid- patient of our surgical ward was identified on day : the man had a klatskin tumor and was scheduled to receive a right hepatectomy with hepaticojejunostomy. unfortunately, he was shifted to percutaneous transhepatic biliary drainage and transferred to the infectious disease department. seventy-two patients ( males and females) with a median age of ( - ) were operated at our surgical department in the study period (table ) . most patients had colorectal cancer ( . %) and colorectal liver metastases ( . %), followed by cholangiocarcinoma ( . %) and pancreatic ductal adenocarcinoma ( . %). twelve patients ( . %) had liver or kidney disease requiring transplantation and no patients died while on the waiting list. compared to the same period of time in , fewer procedures were performed in ( in vs. in ); furthermore, a statistical significant difference in terms of type of operations was found between years ( . % benign, . % malignant, and . % transplantations in vs. % benign, . % malignant, and . % transplantations in ; p < . ). overall, patients operated during the covid- outbreak had few comorbidities ( . %), low asa score ( . ± . ), cci ( . ± . ), and rcri ( . ± . ), being overall at lower risk of postoperative icu admission (table ) . furthermore, few patients had liver cirrhosis ( . %) or received preoperative systemic therapy ( . %). type of surgical procedures and operative details are depicted in table . we have performed ( . %) high-risk operations in the study period of which ( . %) were liver and ( . %) were kidney transplantations. despite this, few patients were admitted to the icu ( . %). one patient ( . %) died during the study period (one hyperacute allograft dysfunction following liver transplantation for autoimmune hepatitis). morbidity rate was . % and five patients ( . %) developed a major grade complication according to clavien-dindo. as mentioned above, during the first period (january -march , ), covid- hospitalizations were stable at our hospital, with two cases admitted to the icu and one monitored at the department of infectious diseases. in the second period (march -april , ), an exponential growth in number of admissions was registered, with a median of ( - ) positive cases per day (fig. ) . at the same time, a median of ( - ) cases per day were operated by our surgical team in the first period, while fewer cases were operated in the second (median of ( - ) per day; p = . ). thirty-nine ( . %) benign conditions, ( . %) oncological patients, and ( . %) transplantations were performed in the first period, while only malignant cases and transplantations (n = , %) were performed in the second. considering only the transplantations and the operations performed for cancer, patients in the second period had fewer comorbidities, lower asa score, cci, and rcri, being overall at lower risk of postoperative icu admission (table ) . furthermore, fewer patients had previous surgery ( . % vs. . %; p < . ) and/or received preoperative systemic therapy ( . % vs. . %; p < . ). the number of high-risk operations was higher in the second phase of the pandemic ( . % vs. . %; p = . ). despite this, fewer patients were admitted to the icu in period and this was statistically significant ( . % in period vs. . % in period ; p = . ). in this study, we have shown how the recent covid- outbreak affected our department of surgery and transplantation. fewer surgeries were performed and these were only for cancer and for urgent liver or kidney transplantations. notwithstanding, we have maintained our standard practice by enhancing selection of patients, meanwhile allowing for the re-allocation of the hospital resources. sars-cov- virus infection has dramatically changed the world recently. more than two hundred countries all over the world had to undertake extraordinary measures to contain the epidemic and reduce possible side effects on healthcare and economy [ ] . indeed, many eastern and western governments followed the example of china and are currently in lockdown, with few exceptions. a deep impact was expected on the healthcare systems all over the world: re-allocation of resources was urgently required in an effort to contain the devastating consequences of the pandemic. as a domino effect, patients with other medical conditions, such as cardiovascular diseases, cancer, and chronic conditions, suffered and are still suffering as a collateral damage [ ] . in response to the rapid increase of covid- cases, institutional guidelines called for the re-arrangement of the anesthesiologists and nurses were re-allocated, and dedicated teams for both covid- patients and surgical practice were created. our department suffered the sudden closure of one daily or and the significant reduction in the possibility to use icu beds after surgery, as these were day by day used for the management of covid- patients. as surgeons, we had to completely re-consider our roles and our surgical activity, reckoning with the limited resources. we had to face a healthcare emergency with uncertain future course and adapt accordingly. surgeries for benign conditions were stopped and the waiting list for cancer was handled and reworked entirely. the most important limitation was the hampered access to the icu as many patients require intensive monitoring after surgery for cancer, especially when major procedures are needed. furthermore, we had to deal with the fact that transplantations normally require intensive care management in the postoperative. we were advised to limit the number of surgeries requiring icu admission as much as possible, to avoid the saturation of resources and failure of the system. as an institutional policy, our transplant center remained opened and we decided to continue with our standard surgical oncology activity, improving selection of patients to limit the need for postoperative intensive care management. waiting list for liver and kidney transplantations was maintained respecting the same inclusion and exclusion criteria and using the same prioritization. transplantations were still admitted to the icu for monitoring through a dedicated pathway with dedicated personnel, despite limiting their length of stay and discharging them to the surgical ward as soon as possible. notably, we have performed standard numbers of the high-risk surgical procedures, selecting more surgically fit individuals. furthermore, patients were less commonly having preoperative neoadjuvant chemotherapy and/or previous surgery, significantly lowering intraoperative risks. all the transplantations performed were admitted to the icu, while only three oncologic patients required intensive care management after surgery: these latter were patients with significant comorbidities that were scheduled for the high-risk surgeries (two pancreaticoduodenectomies for adenocarcinoma and one right hepatectomy for hepatocellular carcinoma). our policy was pursued in an effort to prioritize patients by cancer prognosis. indeed, malignancies requiring high-risk surgical interventions (i.e., cholangiocarcinoma, liver metastasis, pancreatic adenocarcinoma, and sarcoma) are generally those with the worst prognosis and more likely to suffer by a delay in treatment [ ] . a different approach would have been to rework the waiting list for cancer prioritizing less-invasive procedures (i.e., colorectal resections) with no strict selection of candidates in order to cope with the limited resources [ ] . the main issue here is that both strategies will generate a gap in cure that has to be considered in the near future when the covid- crisis will be over. surgical delay should not affect oncological prognosis, in order to minimize future repercussions of this international health crisis [ ] . the alternatives to surgery and their efficacy should be reviewed and discussed for each type of cancer and case by case, in an effort to minimize the waiting list and the loss of patients due to cancer progression [ , ] . in some cases, systemic treatment could be prolonged or adapted, while in others the window of cure might be missed; sometimes surgery can be postponed, while some other patients need surgical treatment to stay on their pathway [ , ] . in this setting, multidisciplinary meetings should be reworked to consider patients with cancer in the covid- era, in which resources and future are uncertain. balancing the prognosis of cancer with the potential alternatives and the characteristics of patients might eventually lead to improve the treatment allocation exploiting resources and maintaining oncologic principles [ ] . finally, an "hub and spoke" system based on a regional policy should be encouraged in the near future to allow for immediate referral and faster access to treatments, eventually improving quality of care. patients with cancer are paying the price at different levels, not only because of the limited access to cure but also because of the delay in diagnosis [ ] . indeed, the significant reduction of outpatient clinics and the limitation in imaging modalities and/or endoscopies might hamper a rapid diagnosis and a prompt referral. as a further issue, it has already been shown that cancer patients are more susceptible to covid- infections: % of covid- patients in china had a history of cancer as compared to . % among normal chinese population [ ] . these patients were also having more severe infections and most commonly required intubation and invasive ventilation. one lesson that we have learned in our hospital is that the diagnostic modalities for covid- are important not only to contain the infection and avoid the spread of the disease but also to ensure safe treatment of patients. questionnaires are useless in our opinion, as we are at the point that this pandemic cannot be detected by recalling contacts or possible exposure to the infection [ ] . indeed, once we have shifted to nasopharyngeal swabs for all patients admitted to our department, we had our first sars-cov- -positive surgical candidate, this patient earlier tested negative to the questionnaire. on one hand, this highlights the importance of swabs for all surgical candidates to contain the infection among patients and healthcare workers; on the other hand, it sheds the light on the need for further evidence concerning the management of positive covid- patients with cancer. indeed, our patient with cholangiocarcinoma was shifted to non-surgical treatment: our team was unprepared at that time as no recommendations were available and issues regarding potential complications and transmission were raised. recently, lei et al. have demonstrated that positive asymptomatic covid- patients undergoing surgery had a % mortality rate, and all developed atypical pneumonias. furthermore, it is very important to stress that our decision unfortunately altered the patient's prognosis, and this highlights the impact of this pandemic on our healthcare system. finally, special considerations should also be made concerning covid- transplanted patients [ ] [ ] [ ] : indeed, immunosuppressive therapy might lead to an atypical clinical manifestation, such as unspecific viral disease or gastrointestinal symptoms [ ] . this should be taken into account in order to minimize unexpected clinical complications and unpredictable outcomes. in our institution, we have recently standardized the use of ppe to limit the possible transmission of disease during surgery. it has been speculated that laparoscopy might increase transmission due to the aerosolized biological fluids [ , ] . in line with this, we are now using extra protection (double gloves, shield, and filter mask) and filtered gas evacuation systems to avoid any possible transmission during minimally invasive procedures. however, as possible aerosolized fluids happen also as a result of cauterization during open surgery, the surgical community needs more evidence and guidelines concerning the proper use of equipment and resources to be used during surgery [ ] . this study has some limitations mainly being the retrospective analysis that might have introduced selection bias. in this setting, anesthesiologists could have decided subjectively whether the patient required icu admission or not. furthermore, as mentioned above, our hospital is a national referral for the infectious diseases and we have already faced previous emergencies [ , ] ; therefore, the rapid adaptation that we have shown in this manuscript could be the result of experience and competences gained over years. since the recent worldwide spread of the disease, many editorials, letters, and expert opinions have been published on covid- crisis and many have speculated that surgical activity has suffered the spread of this pandemic [ , , , [ ] [ ] [ ] [ ] [ ] . to the best of our knowledge, this is the first manuscript presenting numbers and statistical analysis highlighting the changes in surgical practice during covid- outbreak. as we write, italy is already in its fourth week of lockdown, and the emergency seems to have reached the peak. we will be probably moving toward more relaxed containment measures despite a total opening is unlikely. meanwhile, countries worldwide are facing the ascending phase of this outbreak with a delay of days or even weeks. we believe it is important to share our anticipatory experience on how we have changed and adapted our surgical practice to face this crisis and optimized resources. future evolution of this infection is uncertain: evidence, policies, and recommendations may change on daily bases hopefully improving the quality of care and minimizing the side effects on more patients. we must be prepared to face the consequent need for care in the coming months. by then, a progressive inverse trend should be encouraged, reallocating resources to assist those who suffered the most by the outbreak, both re-enforcing diagnostic steps, and treatment possibilities to counteract the delay we have so far accumulated. besides the emergency of patients infected by sars-cov- infection, our healthcare system is facing the side effects of covid- pandemic. adjustments were necessary in many environments as well as in the surgical field. a shift toward the exclusive management of oncologic diseases and urgent cases has been recently recommended worldwide. rearrangements of resources require adaptations in clinical practice. multidisciplinary meetings should be reworked in the context of covid- reality and selection of patients should be enhanced to ensure cure and minimize loss of patients. we foresee a difficult phase for the time being in which all the non-covid health issues will re-emerge. international cooperation is required, and resources should be allocated accordingly to better 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protective equipment (ppe) during the covid- pandemic covid- outbreak and surgical practice: unexpected fatality in perioperative period covid- outbreak in northern italy: viewpoint of the milan area surgical community impact of the covid- pandemic on surgical services: early experiences at a nominated covid- centre response of a european surgical department to the covid- crisis neurosurgery during the covid- pandemic: update from lombardy, northern italy key: cord- -f mbdfr authors: cartenì, armando; di francesco, luigi; martino, maria title: how mobility habits influenced the spread of the covid- pandemic: results from the italian case study date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: f mbdfr abstract starting from december the world has faced an unprecedented health crisis caused by the new coronavirus (covid- ) due to the sars-cov- pathogen. within this topic, the aim of the paper was to quantify the effect of mobility habits in the spread of the coronavirus in italy through a multiple linear regression model. estimation results showed that mobility habits represent one of the variables that explains the number of covid- infections jointly with the number of tests/day and some environmental variables (i.e. pm pollution and temperature). nevertheless, a proximity variable to the first outbreak was also significant, meaning that the areas close to the outbreak had a higher risk of contagion, especially in the initial stage of infection (time-decay phenomena). furthermore, the number of daily new cases was related to the trips performed three weeks before. this threshold of days could be considered as a sort of positivity detection time, meaning that the mobility restrictions quarantine commonly set at days, defined only according to incubation-based epidemiological considerations, is underestimated (possible delays between contagion and detection) as a containment policy and may not always contribute to effectively slowing down the spread of virus worldwide. this result is original and, if confirmed in other studies, will lay the groundwork for more effective containment of covid- in countries that are still in the health emergency, as well as for possible future returns of the virus. in december , the city of wuhan (hubei, china) experienced a cluster of pneumonia cases that were monitored by the chinese health authorities. this was caused by the new coronavirus sars-cov- pathogen, also known as covid- (e.g. chinazzi et al., ) . the global spread was so rapid that the world health organization (who) on january officially declared that the covid- epidemic was a public health emergency of international concern and later, on march , a global pandemic . in june , who ( ) counted a total of more than million confirmed cases globally and about thousand deaths, reaching all countries worldwide. countries that were initially heavily impacted by this pandemic (e.g. china and south korea) through a massive testing regime as well as strict mobility and travel restrictions were successful in limiting the number of new locally transmitted cases (e.g. kucharski et al., ) . in these few months, scientists have made advances in characterising the novel coronavirus and have worked extensively on therapies and vaccines to combat it. furthermore, the medico-scientific community has investigated incubation times for the virus. for example, lauer et al. ( ) estimate that the average incubation time is . days, while in most cases ( . %) the symptoms occur within . days of infection. the above results were used to derive the commonly applied quarantine period of days (e.g. backer et al., ) applied in many countries (e.g. china, the usa and major european countries like france, germany, italy, spain and the uk). with respect to the detection of positive cases, previous studies have revealed the presence of a significant fraction of asymptomatic patients infected with the virus surveillances, ) , in addition to an unknown percentage of false positive and/or negative of each diagnostic tool among patients with covid- , which may have slowed down the detection time of new infections. while the scientific community has focused in recent months mainly on health issues to defeat this virus, other key topics addressed in the literature seek to correlate the cases (deaths) of covid- to both meteorological (e.g. temperature and relative humidity - qi et al., ; shi et al., ; j o u r n a l p r e -p r o o f tosepu et al., ; zhu and xie, ) and air quality (e.g. pm pollution - conticini et al., ; pluchino et al., ; variables. by contrast the incidence of human mobility on the spread of the covid- was not still deeply investigated. indeed, the lockdown of cities and regions together with specific mobility restrictions (e.g. restricted hours and/or areas, specific restrictions for citizen categories) have been common practices performed worldwide to contain and delay the spread of the covid- epidemic. for example, according to fang et al. ( ) without the wuhan lockdown, the number of covid- cases would have been . % higher in the chinese cities outside hubei province, and . % higher in the other cities within hubei (fang et al., ) . many countries are using expedients such as strict mobility, travel restrictions, minimum distance, and quarantine (e.g. muller et al., ; wells et al., ) the main european countries (such as germany, spain, france and lastly the uk) as well as china and the us, have implemented a -day quarantine period based exclusively on medical considerations related to incubation time, that is the time that elapses from initial infection to manifestation of the symptoms (or no symptoms for the asymptomatic patients). this common practice is based on the consideration that, as the incubation of infected suspects takes place within days, the national health system/world health organization will be able to detect a new case in this time interval. by contrast, the hypothesis discussed in this research is that the time period (days) in which a new positive case of coronavirus is identified and certified, which could be called a sort of a positivity detection time, is longer than the incubation time because of possible delays between contagion and detection caused, for example, by the significant percentage of tests that prove false negative to covid- , or by the fraction of people who, although infected, are asymptomatic and/or initially show only mild symptoms, and therefore do not resort to health care. furthermore, this positivity detection time, as well as the spread of covid- , is correlated with j o u r n a l p r e -p r o o f mobility habits, in the sense that the number of certified cases of coronavirus in one day is directly related to the number of people who made trips several days before. to the authors' knowledge, this issue has not been investigated elsewhere, and the appropriate definition and estimation of positivity detection time and its correlation with mobility habits (e.g. daily origin-destination trips) could avoid a slowdown in detecting the infection and hence a slowdown in taking restrictive/mitigative measures. starting from these considerations, the aim of the paper was twofold: i) to discuss the spread of coronavirus in italy; ii) to investigate, for the first time in the literature, the incidence of citizen mobility within the spread of the coronavirus (covid- ) pandemic, also quantifying the positivity detection time for the italian case study. to do this, we referred to the mobility habits of the - year-old population defined in italy as the "active population" (source: istat, ), that is the fraction of citizens who are individually able, unless temporary impediments, to carry out activities (e.g. work, leisure, shopping) and that therefore have autonomous mobility habits. the proposed case study is very suitable for the purposes of this research because italy was the first european country to experience mass contagion starting from the first outbreak. furthermore, by may the spread had almost stopped. it is therefore possible to analyse the huge quantities of detailed contagion data (on a daily basis) and citizen mobility observed at a national scale and for a long time (before, during and after the lockdown), in addition to the effects of specific injunctions adopted by the italian government. to do this, quantitative estimation was also performed from the transportation perspective: the hypothesis according to which the number of certified cases of coronavirus in one day is directly related to the mobility habits made several days before, in addition to other context factors, was investigated. after all, citizen mobility could increase the probability of contagion both directly, for example via trips made by public transport where social distancing cannot be guaranteed, or indirectly (e.g. car trips) because such trips are a measure of the number of activities that a population undertakes in a certain area (trips are made for a purpose), j o u r n a l p r e -p r o o f activities that are generally based on human interactions (e.g. work, leisure, shop, sports, events, cinema), which favor the spread of the virus. estimates were made through a multiple linear regression model linking the number of certified daily cases (day-to-day) to socio-economic indices (e.g. number of residents; population density), environmental variables (e.g. temperature, pm pollution), health care indicators (e.g. number of swabs taken daily) and mobility habits (e.g. number people who performed trips several days before). the paper is organized as follows. section reports methods and materials discussing data collection and model formulation; section describes and discusses the main results. finally, conclusions are reported in section . as stated above, one of the aims of the paper was to investigate the incidence of citizen mobility within the spread of the coronavirus (covid- ) pandemic. estimates were made through a regional (zonal) aggregation level following the classification of territorial units for statistics (nuts) of ec ( ) , although some regional-scale variables were estimates starting from a subzonal (provincial) analysis as described below (see the traffic zones considered in figure  the average daily temperature measured by ilmeteo ( ) from january to may ;  the italian mobility rates estimated by isfort ( ) . precisely, the mobility rates considered are those estimated by the official national monitoring observatory "audimob" of isfort ( ) , which periodically carries out continuous sample surveys on the mobility of italians through telephone and computer interviews. through this observatory it was possible to analyse the mobility habits before and during the national lockdown in italy. in all, , interviews were conducted by isfort ( ) between january and february (before the covid- epidemic) and , interviews immediately after the lockdown ( march ) on a representative population sample between and years old. about % of interviews were conducted by the computer assisted telephone interview (cati) system and about % by the computer assisted web interviewing (cawi) system. the model estimation was performed through a multiple linear regression model linking the number of certified daily cases (day-to-day) to socio-economic, environmental, health care and mobility traffic count sections j o u r n a l p r e -p r o o f habits variables. this tool is among those most commonly used in econometric analysis (e.g. greene, ) . for example, within the transport and/or economic sectors, such methods are commonly used to explain the economic development of an area driven by a transportation infrastructure considered as an input, in addition to other social, economic and territorial variables. with this type of model it is possible to separate the effects of one group of variables with respect to the others (which is the aim of the research), or, in other words, to estimate the effects of a specific variable (e.g. mobility habits), other things being equal. the daily regional new positive cases of covid- (delta new certified infections per day) provided by the italian ministry of health ( ), were considered as dependent variables, while different independent variables were tested at regional scale:  health care variables (e.g. number of covid- tests per dayrelative to the year );  mobility habits variables (e.g. number of citizens who make at least one trip per day; transport accessibility; distance from the main italian clustersrelative to the year ). although several model specifications and independent variables are significant the best model formulation obtained with respect to the validation tests (adj. r-squared and t-value) was: where: mob t,i-x is the average number of - year-old people have done at least one trip (here defined as "mobility habits") "x" days before the i-th day with respect to the t-th region [ , *people/day]; this variable measures the circumstance investigated in this research that the number of certified cases of coronavirus in one day is directly related to the mobility habits made "x" days before; temp t,i-x is the average daily temperature observed "x" days before the i-th day with respect to the t-th region [°c] (source: ilmeteo, ); constant variable was also estimated, accounting for all the attributes not otherwise included (explained) in the model. as mentioned in the previous section, the first real covid- outbreak in italy was in codogno (lombardy). starting from this, the epidemic spread first to neighbouring regions and then increasingly greater distances to the whole of italy. to take into account this proximity effect, a specific variable, ttd t,i , measuring the proximity to the outbreak of codogno was considered in the model. to take this effect into account, we considered that this proximity variable could follow a time-decay principle. the most common non-linear functions proposed for quantifying the time-j o u r n a l p r e -p r o o f decay effect (in the literature well known as the distance-decay principle) are the inverse power and the negative exponential functions (e.g. cheng and bertolini, ; martínez and viegas, ; hooper, , kwan, . starting from such considerations, different time-decay specifications were tested for the proposed ttd t,i variable, resulting that the following inverse power function was the best variable formulation for the case study in question: where: tt t,i is the average weighted (on the population) travel time from the t-th region toward the codogno cluster on the i-th day; ctt t,i,p is the average minimum car travel time from the p-th province toward codogno on the i-th day, estimated on the current national transport network; pop p is the population of the p-th province (source: istat, ); day i is the i-th day. to the authors' knowledge, an issue which has not yet been investigated in the literature is the proper definition and estimation of positivity detection time and its correlation with mobility habits (e.g. trips/day). this time period is the average number of days in which the national health system identifies and certifies a new positive case of coronavirus, and which does not coincide (this is the hypothesis supported in this research) with the incubation time that epidemiological studies quantify as being within days (with an average value of days). for this purpose, through the model formulation ( ), the number of new covid- cases in one day was correlated with the number of people making at least one trip "x" days before, which is to say that the number of today's certified new cases are a function of the mobility habits (and hence of people's interactions) performed some days before. to do this, reference was made to the average number of - yearold people making at least one trip a day (mobility habits) estimated through the equation: where: mob t,i is the i-th day average number of - year-old people have done at least one trip in the t-th region; pop t is the - year-old population (that is, as said, the socio-economic category considered in this study) living in the t-th regional area (source: istat, ); admr t is the average daily mobility rate relative to the t-th regional area before the diffusion of covid- (source: isfort, ); the daily mobility rate was defined as the percentage of residents in the t-th region who during a day make at least one trip, for whatever purpose, with the exception of pedestrian trips shorter than five minutes; %var t,i is the i-th average daily percentage variation of admr t with respect to the pre-covid- condition and relative to the t-th regional area. on basis of the italian transport ministry ( ) data, regional weighted ( where: %var t,i,p is the i-th day average percentage variation of the p-th provincial daily mobility rate within the t-th region, with respect to the pre-covid- condition; pop p is the population living in the p-th province (source: istat, ); %var t,i,p,j is the i-th day average percentage variation of the j-th car traffic trips within the p-th province and the t-th region, with respect to the pre-covid- condition (source: italian transport ministry, ); f p,j,i is the i-th day average car trips relative to the j-th traffic count section within the p-th province and the t-th region (source: italian transport ministry, ). the decision to estimate the trend (day by day) of the daily average percentage variation (%var t,i ) starting from the trend in car trips observed (car traffic counts), instead of considering, for example, the trend in public transport passengers, was made for two reasons: i) public transport (transit) trips decreased over time faster than those observed for private cars due both to the reluctance of users to use such transport services during the pandemic (which do not guarantee adequate social distancing), and to the reduction in the supply of transport services (e.g. reduction in departures/day); whereas public transport trips decreased more rapidly over time, the overall mobility rate, i.e. the average number of people making at least one trip/day (e.g. trips to buy food, pharmaceutical products or other basic necessities), followed a more gradual trend comparable with that observed for car mobility (a consideration also confirmed in terms of model estimation results as described in section ); ii) moreover, for this transport mode, there were much more widespread data available at a national scale, which was therefore better suited to the purposes of the research. as said, one of the aims of the research was to discuss the spread of coronavirus in italy to investigate the incidence of citizen mobility within the spread of the coronavirus (covid- ) pandemic, the mobility habits trend was preliminary estimated applying the equation ( ). as said, through the official national monitoring observatory "audimob" of isfort ( ), were estimated the mobility rates of the italian "active population" ( - year-old population) before and during the national lockdown (as imposed by dpcm of march ). before the covid- , within the - year-old italian population, million residents ( % of the total) made at least one trip per day, while after the lockdown ordinance (dpcm, march ) a significant reduction in mobility habits was observed and in few days a reduction to about million people trips/day was observed (- %). the estimates for the period before covid- were used to evaluate the admr t rate, while those made in the first lockdown period ( march ) were used to validate the %var t,i estimates. results in term of daily mobility rates performed by isfort ( ) j o u r n a l p r e -p r o o f significant in regions in central italy (- %), and less marked in the southern regions and the islands (- %), while the northern regions fell just below the national average (- %). as regards the age of the interviewees, starting from the restrictions the collapse in mobility was clear, especially among the over- s where it fell by three-quarters: fewer than % of citizens made at least one trip by private or public transport during the lockdown. the reduction in the mobility rate was also striking among the young and very young, where the majority are schoolchildren or university students. looking at the employment status of the interviewees, the mobility rate during the lockdown was still around % among workers (a little higher among employees than the self-employed), who recorded a % decrease in trips, just - points below the general average. on the other hand, the trips of retirees were almost eliminated: their mobility rate fell from % pre-covid- to % under lockdown. there was also a very marked reduction in j o u r n a l p r e -p r o o f student mobility (from % to %), which was of course massively affected by school closures (dpcm of march ). for each of the italian regions, the - year-old population mobility habits and its day by day evolution before, during and after (beginning of business recovery "phase ") the covid- pandemic in italy was therefore estimated through the equation ( ) found.) show that the estimated mobility habits trend is consistent with those of the available databases, in addition to those of the isfort ( ) investigation. as stated above, the main aim of the paper was to investigate the incidence of mobility habits ( ), linking the number of certified daily cases (day-to-day) to socio-economic, environmental, health care and mobility zone-specific variables at regional scale. the length of time considered spans the period from the first new cases observed on february resulting from the outbreak in codogno near milan in lombardy, to april ( consecutive days) when the daily infection curve reached its lowest point (figure ) . furthermore, other time periods were also tested and not reported for brevity so as not to produce significant differences in estimation results. although several model specifications and independent variables are significant, in table with respect to socio-economic variables, italy is characterized by uneven population density within the area of the country and for this reason, although the average regional population density, the model that gave the best results includes a population density variable (popdensity t ) referring to the provincial capital of the region, that is the area where most of the population live. in the italian regions, the average population density is about inhabitants/km , while the corresponding provincial capital population density is about inhabitants/km (+ %). the territorial area where this difference is more evident is the campania region where the province of naples (the highest population density area of the country) with , inhabitants/km is % denser than its region average value ( inhabitants/km ). this circumstance means that areas with higher population densities have a higher probability of contagion, being (on average) less able to guarantee social distancing (increase in social activities with overcrowding). moreover, more than million inhabitants live in these provincial capitals of the region. (table ) , explains the circumstance that, all else being equal, the more tests are conducted, the greater is the probability of finding positive cases (especially with respect to the asymptomatic population). as mentioned, the first real covid- outbreak in italy was in codogno (lombardy). starting from this, the epidemic spread first to neighbouring regions and then increasingly greater distances to the whole of italy. to take into account that the areas close to codogno have greater daily exchange trips with the outbreak area, and therefore a greater probability of contagion, a specific variable (ttd t,i ) measuring the proximity to the outbreak of codogno was considered in the model. moreover, with the passing of the days this proximity effect from the initial outbreak (trips from codogno and lombardy) decreased, resulting in new contagion produced by the local mobility of residents in the region. to take this effect into account, we considered that this proximity variable could follow the time-decay principle described by the equation ( ). through this variable j o u r n a l p r e -p r o o f formulation the proximity effect was greatest within the first days and then "decayed" in its incidence with the passing of the time. mobility habits were the variable (mob t,i-x ) that best explained the number of covid- infections (in term of "weight" with respect to the standardized coefficients estimated and reported in table ). this variable measures the circumstance investigated in this research that the number of certified cases of coronavirus in one day is directly related to the mobility habits made "x" days before. to estimate the most representative number of "days before" that influence the new cases in a day, many thresholds were tested in terms of model validation tests, obtaining that trips days before was the best variable to reproduce the data observed. this result is also qualitatively observable from figure through which it may be seen that the daily mobility habits shifted days ahead (left axis of figure ) closely reproduce the observed trend of new daily cases of covid- (right axis of figure ). furthermore, among the environmental measures, a particulate matter pollutant variable (pm t ) was significant, measuring the number of days in in which the national pm daily limit set at µg/m was exceeded. this measure on a regional scale was obtained as a weighted (on the population) average of the corresponding variables referring to provinces within each region, meaning that areas with higher population and with lower air quality have have a higher probability of contagion. overall, the data analysis shows how the areas of the country with the highest pm pollution are those of northern italy (e.g. province of milan in lombardy and turin in piemonte), where are mainly located the industrial areas and/or most of the population live, according to with the circumstance that pms are mainly generated by industries, heating (e.g. home, office) and transport sector (e.g. mobility habits). the opposite occurs for areas in the south, characterized by an economy mainly based on tourism and agriculture. this variable explains, as observed in other case studies, the (positive) correlation between the number of cases per day and the average pollution in the area. with respect to air quality impacts upon the spread of covid- , some recent research has shown that people living with long-term exposure to air pollution are more likely to j o u r n a l p r e -p r o o f become infected by coronavirus. for example, conticini et al. ( ) conclude that a prolonged exposure to air pollution may partly explain a higher presence of viral agents such as sars-cov- . finally, temperature (temp t,i-x ) was also significant and represents the third variable in "weight" with respect to the standardized coefficients estimated ( j o u r n a l p r e -p r o o f relative humidity positively influence the spread of covid- (e.g. qi et al., ; shi et al., ; tosepu et al., ; zhu and xie, ) . for example, in hubei (china) qi et al. ( ) observed that every °c increase in the average temperature with relative humidity in the range from % to % led to a % to % reduction in confirmed covid- cases. in addition, the authors also concluded that every % increase in relative humidity led to an % to % reduction in daily confirmed cases with average temperatures in the range from . °c to . °c. the research discussed in this paper concerns the topics of both the "atmosphere" (air quality and temperature impacts) and the "anthroposphere", in the sense of the earth's research area dealing with the part of the environment that is made or modified to satisfy human activities and habits, where transportation system and the corresponding people mobility trips cover a central role. precisely, the aim of the paper was to investigate the incidence of citizens' mobility habits within the spread of the coronavirus (covid- ) pandemic for the italian case study. the conjecture that the number of new certified cases of coronavirus in one day is directly related to the number of trips made several days before, in addition to environmental and other context factors, was investigated. another issue discussed in this paper and unexplored in the literature is the appropriate definition and estimation of the positivity detection time and its correlation with mobility habits. the thesis was that this time period generally exceeds the incubation time due to many external factors such as false negative test results to covid- or people who, albeit infected, are asymptomatic and/or initially show only mild symptoms, and therefore do not resort to health care. to pursue the research aims, quantitative estimation was made through a multiple linear regression model. estimation results showed that mobility habits represent the variable that mainly covid- mobility trends (driving data) regionale per la protezione ambientale incubation period of novel coronavirus ( -ncov) infections among travellers from wuhan, china presumed 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environment the research was carried out as part of the activities of the transportation system laboratory of the university of campania "luigi vanvitelli," italy. the research was also carried out within the activities of funding program valere: vanvitelli per la ricerca; send research project, university of campania "luigi vanvitelli", italy. key: cord- -wfiqbu m authors: upadhyay, ranjit kumar; chatterjee, sourin; saha, satvik; azad, rajeev k. title: age-group-targeted testing for covid- as a new prevention strategy date: - - journal: nonlinear dyn doi: . /s - - -x sha: doc_id: cord_uid: wfiqbu m robust testing and tracing are key to fighting the menace of coronavirus disease (covid- ). this outbreak has progressed with tremendous impact on human life, society and economy. in this paper, we propose an age-structured siqr model to track the progression of the pandemic in india, italy and usa, taking into account the different age structures of these countries. we have made predictions about the disease dynamics, identified the most infected age groups and analysed the effectiveness of social distancing measures taken in the early stages of infection. the basic reproductive ratio [formula: see text] has been numerically calculated for each country. we propose a strategy of age-targeted testing, with increased testing in the most proportionally infected age groups. we observe a marked flattening of the infection curve upon simulating increased testing in the – year age groups in india. thus, we conclude that social distancing and widespread testing are effective methods of control, with emphasis on testing and identifying the hot spots of highly infected populations. it has also been suggested that a complete lockdown, followed by lockdowns in selected regions, is more effective than the reverse. covid- , a respiratory disease caused by a new strain of coronavirus (sars-cov- ), has spread to almost every part of the world, since first reported in december [ ] . who declared this outbreak a 'public health emergency of international concern' on january . as of april , it has caused , , deaths with , , confirmed cases of infection. till date, usa has the largest number of fatalities, followed by italy. usa reports , , cases, with cases per million people, though the statistics have still been evolving due to the large variability in testing performed by different countries as of now [ ] . in the absence of any vaccine to prevent and contain the spread of novel coronavirus disease, covid- , as well as the lack of an established treatment regimen to cure this disease (beyond mitigation of symptoms), effective non-pharmaceutical interventions are needed to contain the epidemic and minimize morbidity and mortality associated with this respiratory disease. as the covid- pandemic has swept across the globe, now affecting almost all countries, measures for mitigation have been put in place that include strict lockdown to less restrictive people movement but all aiming to achieve social distancing of different degrees to balance the socio-economic impacts of the lockdown and the disease. robust testing and tracing are key to containing the pandemic and effectively 'flattening' the infection curve, both by distributing cases over a longer period of time and by reducing the total number of cases, and thus lowering the epidemic peak. governments and health agencies have banked on mathematical models to guide towards the goal of optimizing available resources to attain maximal benefits of mitigation measures. mathematical models are often based on certain assumptions; however, these are continually improved upon through adjustments guided by emerging data, and eventually, these models become more reliable in helping navigate through such situations. here, we leverage the emerging information from covid- in different countries, mainly usa, italy and india, to develop a covid- specific model that can inform on effective interventions for coronavirus containment. as people of different age groups have responded differently to coronavirus, we utilized the age-stratified data of covid- to develop a system that can inform on more effective prevention strategies. we particularly focused on india where covid- seems to have not peaked yet despite the most restrictive lockdown imposed for over a month now. our model recommends that testing and tracing be ramped up in the - -year age-group population in india in order to flatten the infection curve in shortest time possible in the current situation. we further demonstrate this by computing the basic reproductive ratio r at different times and following an age-grouptargeted intervention. the novel coronavirus is thought to have originated in bats and eventually infected humans, due to the similarity in the genome sequence of sars-cov- to that of a bat coronavirus [ ] . human-to-human transmission has also been established. so far, we observe different transmission and fatality rates in different countries. one of the main reasons for this is differing age groups and social contact structures. in order to study this effect, we use social contact matrices, which show contact patterns of an age group with others and are used to parametrize mathematical methods to understand the transmission patterns. schenzle [ ] used an age-structured seir model with age groups to study the spread of measles, a disease which mostly affects children. this method has previously been applied to respiratory diseases like influenza [ ] , pertussis [ ] and varicella [ ] . given that covid- transmission patterns are very similar to those of respiratory diseases caused by other viruses, we can get valuable information by studying covid- disease dynamics through an age-structured model. it has been observed that social distancing, isolating infected populations and quarantine are effective ways of containing the epidemic. after receiving the best available medical intervention to date, a quarter of critically ill patients still die, signifying that the host response to the virus is an important factor [ ] . thus, the government and hospitals need to procure supportive care equipment in sufficient amounts. initiating the process of flattening the curve by these abovementioned methods provides the time to prepare for supportive care. with social distancing becoming a preventive strategy, many countries have announced partial or complete lockdowns. numerous companies are also advising their employees to 'work from home'. due to this, patterns of mixing between people change, the effects of which are hard to represent with classical compartmental disease models. however, it is essential to understand these changing contact patterns in order to more accurately model the disease dynamics. considering the differential impacts of an infectious disease on people in different age groups, perhaps due to a number of reasons including physiology, immunity, mobility, and social contact and behaviour, an infectious disease model must consider differential age-group disease dynamics. recently, some new approaches about age structure population models have been proposed in [ , ] . age-structured models offer better approximations of reality and also give health organizations better tools to develop age-group-targeted control strategies. here, we have simulated the spread of novel coronavirus using such an age-structured siqr model. we have fitted our model to the current situations in italy and usa and have estimated age-wise mortality rates. side by side, we compared this with the scenario in india. we have also analysed the success of lockdown measures adopted by these countries qualitatively and have projected the effects of further lockdowns. we examine whether starting off with a complete lock-down which is then gradually lifted in specific areas is more effective than the reverse, i.e. declaration of lockdown regionally followed by a nationwide lockdown. finally, we have proposed a novel method of age-group-targeted testing to tackle the situation and have also showed how it can help in flattening the curve effectively. this manuscript is organized as follows. in sect. , we formulate the mathematical framework of an siqr epidemic model. in sect. , we analyse the predictions made by a numerical simulation of our model. in sect. , we propose control strategies and their intended impacts. finally, in sect. , we discuss the conclusions. the basic reproductive ratio r is calculated in "appendix a", and various parameters used in our numerical simulation are tabulated in "appendix b". the mathematical framework of an age-structured siqr epidemiological model is formulated. in order to construct the model, our assumptions are stated as follows: . the entire population, n , is divided into four compartments: susceptible population, s (which are under risk of contracting the infection), infected population, i (which consists of infectious, both symptomatic and asymptomatic, or untested individuals), quarantined population, q (which are removed from all contacts within the entire population and are hence not infectious) and recovered class, r (which are recovered from infection). therefore, we have n = s + i + q + r. in order to keep track of disease-induced death, we assign an additional compartment of fatalities, f. . each population of siqr and f is further subdivided into m age classes. individuals within the same compartment interact with other individuals proportional to a coefficient of interaction c i j , which specifies the average contact between age classes i and j, with < i, j ≤ m. . there is no recruitment of the susceptible population and no natural death in any compartment. there is also no aging of individuals. . the disease is transmitted from the infected to the susceptible population with age class i, i.e. compartment s i , at a rate βλ i . here, β is the transmission probability, and λ i is the weighted coefficient fig. schematic diagram of the interacting population within a certain age class, for the model ( ) of contact of age class i with the entire infected population. individuals from compartment s i move into compartment i i and become infective immediately. . individuals from compartment i i move into compartment q i at a rate δ i and are no longer infectious. . the infected and quarantined populations, i i and q i , recover and move into the recovered population, r i , at rates and γ , respectively. . the populations i i and q i suffer disease-induced death, at a common rate of μ i . a schematic diagram of the interacting population is presented in fig. . thus, the transmission process is formulated by the following system of differential equations: the initial conditions are s i ( ) > , all the parameters in the system are positive quantities. we break down the age-structured social contact matrix, c = [c i j ] m×m , into the contributions from households, workplaces, schools (all educational institutions) and other areas (market places, restaurants, cinema halls, shopping malls, etc.), represented by c h , c w , c s and c o , respectively. each of these is weighted with coefficients α h , α w , α s and α o , which we change over time to reflect the effect of lockdown on social contact. for example, during the time period when all educational institutes are closed, we set α s = . we note that when italy and usa announced partial lockdowns, the coefficients α have been fitted to the existing data. it must also be noted that even during a complete lockdown, the contributions to the contact matrix from work and other areas are never zero, as people involved in essential services continue work and marketplaces must operate to some degree. lockdowns also induce an increase in household contact, as people are staying at home more [ ] . for numerical simulation, we have collected data on the times and nature of lockdown imposed, starting from the closure of schools and universities to complete lockdowns [ ] [ ] [ ] . a complete lockdown in india was declared on march . usa declared a national emergency on march , followed by various statewise guidelines and orders to 'stay at home'. italy had also proceeded towards lockdown step by step, with a lockdown of the northern provinces on march and a nationwide lockdown from march onwards. the parameters δ i , γ and have been assumed, using known rates of infection, recovery and the first appearance of symptoms. we assume the onset of symptoms, detection and isolation in . days on average [ ] for infected individuals. we also assume that an individual stays quarantined for days, which is the average recovery period for a symptomatic individual [ ] . in addition, we assume that unidentified infected individuals, either asymptomatic or untested, can proceed straight to recovery after days of infection on average. the parameters β and μ i have been fitted to existing case and fatality data. the values of β for india, usa and italy are . , . and . , respectively. the values of the remaining parameters are listed in "appendix b" (tables , ). it must be noted that we choose to interpret currently reported cases to belong in the quarantined compartment, q, which is a subset of the total infected population. we have also chosen our initial conditions such that i i ( ) = q i ( ), on the assumption that cases are underreported where exactly half of all infected individuals are identified (quarantined). for india and italy, we use m = age classes, equally dividing the range of ages - years. due to unavailability of data, we use m = age classes for the usa, over a range - years. for each country, we have set up m differential equations, which we have integrated using the python module 'numpy'. we have collected covid- case and mortality data up to april for each country from worldometers [ ]. we have also collected age-structure data from popu-lationpyramid [ ] and social contact data from prem et al. [ ] . the age classes and initial susceptible population data are presented in table . the infection curves predicted by our model are shown in fig. . we note that our model predicts a fairly symmetric infection curve, whose peak trails behind the peak of the quarantined population. usa and india continue an upward growth, while italy's infection curve has begun to drop. if current conditions continue without any new measures being taken, active cases are predicted to reach peak within months for usa and around months for india. we also predict that everything will be normal in months for italy, without considering the effect of herd immunity. on the other hand, it may take a year at worst for india and usa to fully recover. by 'normal', we mean that the number of infected individuals has dropped below one thousand, which represents a sufficiently small number of cases relative to the populations of the countries in consideration to be fully identified and isolated. we observe a common trend in infected age groups across all three countries, in that the young-and middleaged groups (between and years of age) have the largest numbers of infected people, relative to the initial susceptible population size of that age group. we estimate this by measuring the drop in s i from its initial value across all age groups i over time, as shown in fig. . this may be explained by larger contact coefficients α among themselves and other age groups. the population within this age group is also highly mobile, in each country. on the other hand, data suggest that infection is less common in children [ ] . dr. calum semple, professor at liverpool university, stated that 'we know that lung development doesn't finish until teenage years. ace is highly regulated in lung development. because of that the "lock" might be expressed differ-ently in kids' lungs' [ ] . hence, we have identified the - -year-old age group to be the most infected age group across all three countries (fig. ) . in our proposed control strategy, we thus place less emphasis on individuals younger than years. considering different mortality rates across different age groups has very little effect on the total infection and mortality curves, both qualitatively and quantitatively. we note that italy's mortality curve in fig. f has begun to flatten, far quicker than our model would sug- gest. we may explain this by noting that the reported mortality rates, which we used to fit our model, are likely inaccurate due to factors such as sampling bias and the changing capability of health care system. with time, as italy continues to improve medical facilities and mobilize doctors and nurses, patients receive better care and facilities are no longer swamped as they were in the early stages of the pandemic. we also note that with time, the mechanisms by which covid- causes death might be better understood by scientists and practitioners, so the observed lower mortality rate may also be explained by better, more effective treatment and the identification of drugs which improve survival chances. presently, the overall mortality rate is the highest in italy, followed by india and the usa (fig. ) . interpreting these mortality rates is complicated by the fact that pre-existing medical conditions play a major role and are somewhat correlated with age. fatality rates may also be inflated by limited testing and the resulting selection bias in which asymptomatic individuals are not accounted for. in addition, the fact that our model omits - -year age group in the usa, despite a high expected mortality rate in that group, may explain why our model predicts a lower mortality rate in usa, compared to india and italy. the basic reproductive ratio r can be interpreted as the expected number of cases directly caused by a single infected individual in a completely susceptible population. when r > , the infection spreads in the population, and it does so more rapidly with higher r . when r < , the infection eventually dies out, and the system proceeds towards the disease-free equilibrium. we have calculated r for the three countries, as presented in table . a lower r value for india initially indicates a comparatively slower spread of disease than in usa and italy. figure illustrates the decrease in r with the implementation of social distancing measures. the drop in r is significant in all cases, although only italy shows r < , which is enough for the disease to die out. concerningly, our model shows very little impact of the lockdown in india, compared to the projections without one. after fitting our model to case numbers before the implementation of social distancing measures, our model predicts that complete stop of contact between people outside their home, or even a % reduction in such contact, is not consistent with subsequent reported cases. the three different countries show different degrees of reduction in contact, in terms of different control coefficients α. this effective reduction in contact in india seems to be far less than in usa, which in turn is less than in italy. in the case of italy, data look promising, suggesting that the implementation of lockdown was more successful. the effectiveness of control strategies can be measured by the basic reproductive ratio r . the parameters which can practically reduce r are the contact coefficients, α and the rate of quarantine of infected individuals, δ i . social distancing works well at reducing interpersonal contact, but we can see that implementation issues can severely damage its effectiveness. by sufficiently increasing the number of tests carried out, we can identify and quarantine infected individuals more quickly, thus indirectly increasing δ i . this means that infected individuals would have a lower probability of infecting a susceptible individual. on the other hand, testing rates are limited by the medical resources of each country. while south korea has managed to test a large fraction of their population, in countries with very large populations such as india, randomized testing for the entire population is not feasible. hence, we suggest an age-group-targeted testing initiative, where the age groups with the largest number of infected individuals are targeted. in addition, economic activities have come to a halt during lockdown in india. barclays has estimated a loss of up to . billion usd in india [ ] . practically, it may not be possible to continue a complete lockdown indefinitely. only a multipronged approach can successfully combat an outbreak of infectious disease. though we have not provided any medical insights, they should go hand in hand with the strategies we propose here. we discuss two strategies below. . keeping in mind that individuals in the age group - years are most likely to catch infection, we emphasize testing more people from this age group rather than randomized testing. this will help in isolating infected people and restrict their disease transmission. the impact of such a strategy is evident from the curves in fig. . the corresponding change in r is illustrated in fig. b . we see that although r has not dropped below , our strategy offers a significant improvement. for this simulation, we have increased the value of δ i for the targeted age group - years, which corresponds to i ∈ { , , , , } ( table ). the initial population sizes of these age groups are shown in table . we have assumed that infected members of these groups can proceed to quarantine in . days, on average. for the remaining groups, we increase the detection period to . days. concentrating testing on groups most likely to have infected members helps bring them out of contact with the susceptible population and hence flatten the infection curves. this in turn lowers the peak number of critical cases, thus distributing the workload of medical facilities over a longer period of time. the peak is also observed far later than with normal rates of testing, although normalcy is restored not much later. . we suggest that a complete lockdown, followed by lockdowns in selected regions, is more effective than the reverse. as symptoms take time to manifest, infection can spread very rapidly to areas not under sufficient lockdown. however, if the majority of infected individuals can by identified and isolated by testing during the lockdown period, subsequent lockdowns can target those areas with larger infected populations. this would effectively reduce disease transmission across a country. we suggest that introducing a lockdown in slow phases, as was done in italy and usa, may not have been as effective as a complete lockdown introduced in the early stages of the pandemic. however, we do acknowledge differences in the socio-economic structures and dynamics of different countries, which demand differential strategies tailored individually to their underlying structures. our model is ill equipped to model lockdowns in selected regions, but we have approximated this effect with reduced contact coefficients α. our model of covid- dynamics allows us to make some useful predictions and modify pre-existing strategies to obtain better results. this model has been tuned with available data of social contact matrices and reported deaths and infected individuals available till april . methods of testing and social distancing are known to tackle this kind of situation. in the context of this novel disease, we have re-examined these methods. it must be noted that in our model, we have interpreted the number of reported cases as the number of symptomatic or tested cases, and we assume that they are transferred to quarantine as soon as they are confirmed positive. the actual number of covid- cases is much higher than reported, which is consistent with the nature of the reported infection curve. we must note that as of april , india has conducted , , tests. this is a major step up from the initial , tests before the declaration of lockdown. at this milestone of tests, india has recorded around , positive cases, as opposed to usa's , , and italy's , cases [ ] . india has also observed a comparatively lower mortality rate. this can be explained by her disproportionately young population, together with the low rate of infection and transmission in the younger age groups. the mean age of india's population is . , compared to usa's . and italy's . [ ] . we acknowledge that there are uncertainties in determining the model parameters due to unavailability of proper data and that this may lead to incorrect predictions. we note that the use of mortality data is partially motivated by the fact that such figures are more likely to be reliable, as opposed to infection numbers which suffer from under-reporting. the choice of a single transmission coefficient β across all age groups is because of the lack of age-group-specific estimates on transmission probabilities, as well as the complexity of fitting such coefficients for each of the age groups even if available. similarly, disease-induced mortality rates across the infected (both symptomatic and asymptomatic) and quarantined populations have been assumed to be the same, μ. while these rates may indeed differ among these groups in reality since severe cases are frequently quarantined, we justify this assumption with our simulation which closely mimics the trends in available data without introducing additional parameters. furthermore, we emphasize, again because of these reasons, that factors such as agedependent immunity have not been incorporated into our model, and the coefficients λ i merely reflect normalized amounts of contact between age classes. our initial conditions i i ( ) = q i ( ), which reflect the assumption that half of the cases are reported, may be adjusted with better estimates of the fraction of reported cases. the assumption that births, natural deaths and aging are absent is valid only over relatively short periods of time. we have also focused on the population below years of age, as the rest of the population is significantly small. with these assumptions, we can clearly say that our model can make short-term predictions, but cannot reliably make long-term forecasts. in this instance, we have run our simulation for a maximum time period of months. we also acknowledge that partial lockdowns of infection hot spots are not well modelled by our method, which considers a given region as a whole. with the availability of reliable data, we may be able to apply our model on smaller populations and make region-wise predictions. we also post-pone the application of targeted testing to other countries, such as usa and italy, for further study. through this study, we have offered a more efficient, country-specific covid- model for informing on strategies to contain the sars-cov- pandemic. our contribution of a new age-stratified model will aid government and health agencies and will spur further research in covd- modelling. the implications of our proposed work are timely as this is still an emerging situation in many countries and perhaps broad as well, as viral pandemics are predicted to keep re-emerging in the near future, perhaps in different shades or shapes. keeping in mind that all models are merely approximations of reality, we hope that this model can aid in developing policy, with economic and medical perspectives. here, we find the basic reproduction number using the next-generation method [ ] . we first linearize the system of equations ( ) at disease-free equilibrium, where hence, when the infected and quarantined populations are small, their dynamics are described by the following system. we collect the infected and quarantined compartments in the vector v = (i · · · i m q · · · q m ) t . we can thus rewrite the system ( ) in the form d we further break l = t − v , where the transmission matrix, t , represents the influx of newly infected individuals, and the transition matrix, v , represents the movement between the infected compartments. they are calculated numerically as follows: the basic reproductive number r = ρ(t v − ) is simply calculated as the spectral radius of the matrix t v − (table , fig. ). here, we list the parameters used in our numerical simulation (tables , and ). all population numbers are in millions covid- ) situation reports- a pneumonia outbreak associated with a new coronavirus of probable bat origin an age-structured model of pre-and postvaccination measles transmission the contribution of social behaviour to the transmission of influenza a in a human population clustering of contacts relevant to the spread of infectious disease contact network structure explains the changing epidemiology of pertussis surviving covid- nonlinear physiologicallystructured population models with two internal variables on first-order hyperbolic partial differential equations with two internal variables modeling population dynamics of two physiological structures modeling infectious diseases in humans and animals the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application who: report of the who-china joint mission on coronavirus disease projecting social contact matrices in countries using contact surveys and demographic data characteristics of pediatric sars-cov- infection and potential evidence for persistent fecal viral shedding scientists seek reason why coronavirus has less impact on children reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission key: cord- -t ejmq authors: ugolini, francesca; massetti, luciano; calaza-martínez, pedro; cariñanos, paloma; dobbs, cynnamon; ostoic, silvija krajter; marin, ana marija; pearlmutter, david; saaroni, hadas; Šaulienė, ingrida; simoneti, maja; verlič, andrej; vuletić, dijana; sanesi, giovanni title: effects of the covid- pandemic on the use and perceptions of urban green space: an international exploratory study date: - - journal: urban for urban green doi: . /j.ufug. . sha: doc_id: cord_uid: t ejmq urban green space (ugs) is an essential element in the urban environment, providing multiple ecosystem services as well as beneficial effects on physical and mental health. in a time of societal crisis these effects may be amplified, but ensuring that they are maintained requires effective planning and management – which is a complex challenge given the rapid changes in modern society and the need for continual adaptation. this study aims to identify the drivers that normally attract visitors to ugs, and to assess the effects of social isolation on the usage and perception of ugs during the covid- pandemic. we conducted an online survey during the period in which restrictive measures were imposed in response to the pandemic (march-may ), in croatia, israel, italy, lithuania, slovenia and spain. results showed that urban residents normally have a need for accessible ugs, mainly for physical exercise, relaxing and observing nature. the reduction in ugs visitation during the containment period was related to distinct changes in the motivations of those who did visit, with a relative increase in "necessary activities" such as taking the dog out, and a reduction in activities that could be considered non-essential or high-risk such as meeting people or observing nature. behavioral changes related to proximity were also observed, with an increase in people walking to small urban gardens nearby (e.g. in italy) or tree-lined streets (e.g. in spain, israel), and people traveling by car to green areas outside the city (e.g. in lithuania). what the respondents missed the most about ugs during the pandemic was "spending time outdoors" and "meeting other people" – highlighting that during the covid- isolation, ugs was important for providing places of solace and respite, and for allowing exercise and relaxation. respondents expressed the need for urban greenery even when legally mandated access was limited – and many proposed concrete suggestions for improved urban planning that integrates green spaces of different sizes within the fabric of cities and neighborhoods, so that all residents have access to ugs. in a world of increasing urbanization and with more than half of the population living in cities, urban green space (ugs) is a crucial element for urban transformation and the sustainability of cities (andersson et al., ) . prominent tendencies and movements in urbanism, such as landscape urbanism (waldheim, ) , ecological urbanism (mostafavi and doherty, ) , ecological landscape urbanism (steiner, ) and ecosystem urbanism (rueda, ) emphasize that prioritizing nature and ecological considerations can improve the quality of life in citiesand that ugs is the key. ugs directly and indirectly promotes a better quality of life (sanesi et al., ) , as it can provide refuge from an increasingly stressful everyday lifestyle j o u r n a l p r e -p r o o f (van der berg et al., ) , encourage social cohesion (zijlema et al., ) , stimulate physical activity (hunter et al., ) , improve health (van den bosch and sang, ) and even enhance a person's wellbeing and mental state (nath et al., ; tsai et al., ) . ugs includes a diversity of biophysical structures and their ecological processes, which combine to support the city's "green infrastructure" and provide multiple "ecosystem services": namely provisioning services, regulating services and cultural services (haines-young and potschin, ) . the european commission (ec, ) has highlighted the importance of integrating these divergent approaches to service provision in cities, and transforming the traditional concept of isolated ugs (parks and gardens) into a comprehensive vision of green infrastructure. this vision is also reinforced in the eu biodiversity strategy (ec, ) and new green deal (ngd) (ec, ) . the intended optimization of es that this implies is seen as a necessary response to the rapid changes in lifestyle that modern urban society is experiencing, as the role and functions of ugs are shifting to adapt to new demands and human needs (derkzen et al., ) . ugs represents an urban design resource which is not only aesthetic, but fully functionaland one that must be utilized and nurtured in accordance with the holistic and comprehensive concept of green infrastructure, integrating the perceptions and preferences of citizens in planning proposals and management of this resource in the future (ryan, ) . human perceptions and preferences towards nature are believed to be either innate (e.g. evolutionary theories such as "biophilia" by wilson ( ) , or attention restoration theory by kaplan & kaplan ( ) ), or learned (according to cultural theories)in the latter case being shaped by social, cultural and personal characteristics (tveit et al., ) . in many studies carried out on the attitudes that people have toward ugs, significant differences have been found between various localities (de la barrera et al., ; riechers et al., ) and countries (schipperijin et al., ; bertram and rehdanz. ) indicating that cultural background as well as environmental factors can condition the expectations and behavior of a particular population. there is much more to be learned about how these perceptions and usage patterns may change in response to external events. research on how a global event of great magnitude may be perceived differently in different j o u r n a l p r e -p r o o f geographical contexts is not frequent. given the worldwide health emergency caused by the pandemic, it is of great interest to explore whether and how citizens' behavior and perceptions of ugs in different places may have changed, above and beyond the ways in which the particular areas were affected by the pandemic and the social restrictions that were imposed. while several major epidemics have occurred throughout the world in recent decades (e.g. sars, mers), the covid- pandemic has already had a global impact which is unprecedented in its scope and scale. as governments around the world have responded to the public health threat by imposing social distancing protocols, economic shutdowns and various forms of home quarantine, it has been hypothesized that these measures may have fundamentally changed the relation between humans and public spaces, in terms of use and perception. the purpose of this study was to explore, from an international perspective, the ways in which human behavior, perceptions and attitudes toward ugs may have changed due to restrictions imposed by the containment of the covid- outbreak in different countries, in comparison to the period prior to the restrictions. in addition, the study aimed to evaluate citizens' satisfaction with ugs in their locality and elicit constructive recommendations for improvement. the study was conducted in croatia, israel, italy, lithuania, slovenia, and spain. these countries constitute a diverse range of societies and cultures, each with norms and customs that might influence the use and perception of ugseven, and perhaps especially, during the emergency situation triggered by the covid- pandemic. the measures implemented to contain the covid- outbreak in the six countries studied began on different days, and with different levels of restrictions (supplementary materials (sm) - table ). italy was the first country to adopt restrictive measures at a national level. initially (between the th and th of march ), sports activities and walking outdoors were allowed, even though public gatherings were prohibited. but the rapid increase in positive cases led the ministry of health to ban any kind of physical exercise or walking j o u r n a l p r e -p r o o f farther than m from home, and going outdoors was allowed only for essential reasons. similar restrictions were imposed on march in israel, only allowing movement m away from home. a few days after the outbreak in italy, spain began recording a dramatic increase in positive cases and on the basis of the italian experience, imposed on march the strictest level of limitationsaccording to which going outside was allowed only for essential activities, and thus the frequentation of ugs was banned. in slovenia, croatia and lithuania, outdoor activities were allowed throughout the period but with strong warnings to keep distance from other people (other than family or close relatives) and to avoid gatherings even between a few people. these restrictions lasted until april in slovenia (where some outdoor activities were allowed), until april in lithuania, and until the beginning of may in the other countries. in italy, although with regional distinctions, vegetable gardening was generally allowed from the th of april. (see further details in sm - table ). at the beginning of the isolation period in italy (march ), an online questionnaire (using google forms) was developed for distribution to the general public. respondents were asked for informed consent prior to their participation in the survey. a link was provided with information on the researchers conducting the survey, what data would be collected, how the data would be stored, analyzed and reported, and respondents' rights regarding the provided data. participation was voluntary, and participants had the right to leave the questionnaire at any point. the questionnaire was divided into nine sections and contained between and questions, depending on whether respondents self-identified as those who usually visit ugs ("ugs visitors") or those who do not ("non-ugs visitors") and on whether they visited or not ugs during the covid- pandemic. the results presented below refer to sections - (see the structure of the questionnaire in sm - table ) and consider responses to an open question (which was included in all countries except slovenia) on the relationship between urbanization, people and nature. the questionnaire was originally developed as an initial exploration in italy, where it was pre-tested by people of different ages and education levels in order to ensure the clarity of the questions and overall j o u r n a l p r e -p r o o f structure. the preliminary version was corrected based on the suggestions and feedback, and then translated from italian into english and into other languages. data collection was concurrent in most countries, starting on the th of april and ending on the th of may (sm -table ) when a relaxation of restrictive measures to contain the covid- outbreak was declared in most countries. restrictions on ugs visitation during the pandemic were largely similar in most countries, despite stricter limitations in italy, spain and israel. in each country, non-probability samples were obtained through an unrestricted self-selected survey (fricker, ) . distribution of the online questionnaire initially started through the authors' networks of professional and personal contacts, by email and through social media (facebook, whatsapp, etc.) or posts on websites. participants were kindly asked to fill in the questionnaire and distribute further to their contacts. thus, the distribution proceeded according to a snowball effect, and did not allow for personal identification of individual respondents. the datasets were analyzed by performing descriptive statistics, while logistic regression analysis was used to compare differences among countries. we transformed each item in the questionnaire into a binary variable according to the indicated preference by the respondent (y/n) and calculated the odds ratio (or) and % confidence interval (ci) of the association of each item and nationality of the respondents, considering the countries with the highest score for that item as the reference group. the level of significance was set to p< . . differences within each country were also investigated by the chi-square test between pairwise options. the same test was also used for comparisons between two datasets (e.g. attitudes of ugs visitors before and during covid- restrictions). statistical analyses were performed using r software (https://www.r-project.org/) and particularly the odds ratio (or) package. it should be noted that due to the sample type (purposive sampling) and mode of administration (internet survey), conclusions could be made only for the sampled population. textual responses were analyzed separately. if text provided under the option "other" expressed a concept that was analogous to one of the predefined choices, the answer was coded with that choice, and if a concept that was different from the predefined options was indicated by several respondents, we assigned a common code to those responses to allow further comparative analysis. for the open-ended question, respondents were invited to share "thoughts about urbanization and the relationship between people and nature in the city" which were analyzed using text mining. text mining is a computer-based technique that extracts information from large data sets (feldman and sanger, ; fuller et al. ) . all responses to open-ended questions were translated into english in order to unify them in a single dataset. the analysis was performed using statistica (release , statsoft, inc. - . the preliminary phase consisted of filtering the text in order to reduce the presence of insignificant words, and words with the same meaning were combined. "indexing" produced a draft list of words that could be further "cleaned" by combining further synonyms and deleting more words. the program computed the raw frequencies of word occurrences, enumerating the number of times that a given word occurs in each text unit. the list of frequencies was used to create a matrix of all the words, which indicates the concurrent presence of words in the same text units. exploratory analysis of the word matrix was done to find the most relevant words and connections between them from the perspective of the respondents. two approaches were applied: correlation analysis (with mapping of p-values to visualize the results), and principal component analysis (pca) . in order to derive the content of the text units with much greater detail, we also performed content analysis (krippendorff, ) and thematic analysis (braun and clarke, ) . collected data (n= ) were checked for consistency, and records that were missing essential information were excluded (n= ). the final sample thus consisted of responses distributed over the six countries, ranging from respondents in israel to in spain. the majority of the respondents in all countries were female (see sm - table ) , with especially high percentages in lithuania ( %), croatia ( %) and slovenia ( %) (α< . ). respondents were mostly adults in the age range of - years old, with some differences between countries: the dominant age groups j o u r n a l p r e -p r o o f in spain were - and - and in slovenia - and - , while in other countries the proportions of respondents were more evenly distributed between and . in israel, younger respondents ( - ) were also well represented (sm - figure ). the majority of respondents in most of the national samples had completed some form of higher education, most notably in lithuania, israel, slovenia, and spain (sm - figure a ). the italian and croatian samples also included some respondents (on average %) whose educational level was listed as a high school diploma (α< . ). the large majority were employed in a public or private company (α< . ) (sm - figure b ), while israel counted the highest percentage of students ( %). most respondents (table ) reported living in large towns or cities with more than , inhabitants. the percentage was highest in croatia ( %), and exceeded % in every country except italy ( %). if we consider all the respondents living in urban areas, the largest proportions were found in croatia and lithuania ( % and %, respectively), followed by italy, israel, spain and slovenia. respondents were firstly asked whether they generally (pre-pandemic) tended to visit or spend significant periods of time in ugs in the place where they live. most respondents ( - % depending on country) stated that they do visit ugs frequently (table ) . despite croatia and slovenia showing different percentages of respondents living in urbanized contexts, both recorded the highest rates of visitation, especially among those living in urban areas, with a score significantly different from italy, lithuania, or spain. in italy and lithuania, respondents from large towns and cities were also much more frequent visitors of ugs than those living in small towns and villages or in rural areasthough this was not the case in israel, croatia and slovenia. j o u r n a l p r e -p r o o f table . percentage of ugs visitation in the place of residence and percentage of visitation within each category of place of residence. statistical differences between countries are identified by the odds-ratio test, taking as reference the country with the highest percentage. different lowercase letters in italics indicate that or were significantly different (p< . ), ns means no difference. significant differences between town size within the same country were identified by the chi-square test and differences are indicated by capital letters at α< . . regarding the habits of ugs visitors (sm - table ). most respondents declared that they reach the ugs by foot, despite some differences among countries (table b ). for instance in slovenia, the percentage was significantly higher than in italy, croatia, and lithuania. private cars were selected as a means of transportation to ugs by lithuanians, significantly more than italians, slovenians, spaniards and israelis. croatians and slovenians tend to use bikes to visit ugs more than spaniards, israelis, and lithuanians, while some italians go by motorcycle. on the other hand, the option of public transportation was only selected by a small minority, though higher in italy than in croatia, slovenia or israel. regarding the type of ugs visited (table c ), in all countries except slovenia most respondents indicated visiting an urban park. the largest proportion of visitors to urban parks was in italy ( %), slightly higher than in croatia ( %) and significantly higher than in lithuania, spain, israel and slovenia. urban gardens were most frequented by israelis ( %), with percentages relatively lower among italians and spaniards and near-zero among lithuanians, croatians and slovenians. in slovenia, over half of the respondents ( %) selected green area outside the town; which included forest, as slovenians listed this term separately. riverbanks were frequented by % of spanish respondents and j o u r n a l p r e -p r o o f slightly less by respondents from other countries (except israel, with only %)while the option of treelined was largely selected by lithuanians ( %). in all countries, most respondents declared that they visit a green space more than once a week (table d) , with the largest majorities in slovenia and spain. italy also recorded the highest percentage for the option several times a month as compared to other countries, whereas slovenia recorded the highest percentages of respondents visiting a ugs once a month. few respondents, ranging from % in slovenia to % in lithuania, declared going to ugs less than once a month. and significantly more than israel, italy or croatiawith an even greater difference from lithuania. however, the option of relaxing was also frequently selected, especially in slovenia, italy and croatia, and this was the main reason mentioned for visiting a green space in lithuania ( %). observing nature, going out with children and walking the dog were selected as secondary options. in particular, observing nature was selected mainly by italians, significantly more than croatians and slovenians. respondents from the latter countries more often selected walking the dog than those from lithuania, italy, spain or israeland lithuanians selected taking the kids outdoors more than spaniards, slovenians, italians or israelis. meeting people was scarcely selected, though registering more as a priority for croatians, israelis and slovenians than for italians, lithuanians or spaniards. respondents were asked whether or not they had visited any ugs during the period of covid- containment measures. those who used to visit some type of ugs before the pandemic (ugs visitors) generally continued to do so (table ) significant differences between the two categories (ugs visitors vs. non-ugs visitors) were identified by the chi-square test. differences are indicated by * at α< . , ** at < . and *** at α< . . regarding the pre-pandemic ugs visitors, who represent the large majority in all countries, significant differences were observed during the containment period in the distance traveled to ugs with respect to the pre-covid situation (fig. ). in general, there was a tendency to visit green spaces at closer distances (< m) in israel, italy and spain, although the differences were only significant in spain (α< . ). however, in croatia, italy, lithuania and spain the percentage of respondents traveling farther was slightly higher than the percentage of those going shorter distances (α< . ) (sm - j o u r n a l p r e -p r o o f fig. percentage of variation in the distance traveled to ugs during the containment period relative to the pre-pandemic period, among ugs visitors. significant differences between the two situations (before vs. during covid- ) were identified by the chi-square test. differences are indicated by * at α< . , ** at α < . and *** at α< . . the or cross-analysis comparing travel distance with place of residence (large city, small town, rural etc.), showed significant correlations in israel, italy, lithuania and slovenia (sm -table ). people living in a village or a rural area in israel were more likely to travel more than meters to visit a green area than those living in a large town or city. italy and lithuania showed opposite results, with people living in big towns or cities more likely than people living in small towns (or in villages/rural areas, in italy) to travel over meters. the latter trend was also found in slovenia, particularly among people living in villages or rural areas, who preferred travelling shorter distances than urbanites. the most commonly reported way of reaching ugs in all countries during the containment period was on foot (α< . ). israel showed the highest percentage, while croatia and lithuania had significantly lower percentages (sm -table b ). bicycle travel was mostly selected by slovenians, to a considerably greater extent than israelis and spaniards, while the car was mainly a choice of lithuanians. very few differences within countries were observed between visitor behavior before and during the covid- containment in terms of the preferred means of transportation ( figure ). the only statistically significant difference was a decrease in car use in israel (- %, α< . ), which was similar to an increase of israelis reaching the green j o u r n a l p r e -p r o o f area on foot when compared to the pre-pandemic behavior. an even larger increase (of %, though not statistically significant) in going on foot was seen in italy. fig. . percentage of variation in the means of transportation used by ugs visitors during the containment period relative to the pre-pandemic period. significant differences between the two situations (before vs. during covid- ) were identified by the chi-square test. differences are indicated by * at α< . , ** at α < . and *** at α< . . regarding the type of green space visited during the pandemic containment period, the majority of respondents in most countries chose urban parks, though to a lesser extent than before (sm - table c ). the main exceptions were in slovenia (α< . ) where green areas outside the town were preferred by a large margin, and in italy where the margin was narrower. in fact a relative increase in travel to green areas outside the respondents' place of residence was seen in several other countries as well, reaching % (α< . ) in croatia-where there was a % reduction in visitation to urban parks (α< . )and smaller percentages in israel, lithuania and spain (figure ). in italy there was a similar reduction of % (α< . ) in frequentation of urban parks, and in spain there was a significant % increase in visitors of tree-lined streets (α< . )a preference also noticed in israel and croatia. urban gardens were also increasingly appreciated, especially by italiansas compared with croatians, lithuanians, and slovenians. figure . percentage of variation in the type of ugs reached by ugs visitors during the containment period relative to the pre-pandemic period. significant differences between the two situations (before vs. during covid- ) were identified by the chi-square test. differences are indicated by * at α< . , ** at α < . and *** at α< . . excluding the option "i have not gone", which was mainly selected option by respondents in italy and spain ( % in both countries), the main motivation to visit a green area in many countries was to do physical exercise (indicated as walking or running) followed by relaxing, which was particularly common in lithuania (sm - table d ). the motivations showing a large decline during the containment (fig. ) were those that could increase the risk of contagion, such as meeting people, and those we might consider "non-essential", such as observing naturewhich was less selected in every country (reduction by - %), but was still one of the top motivations for lithuanians (sm -table d ). physical exercise was the most frequently mentioned motivation during the pandemic in every country except spain and lithuania, most notably in italy, israel and croatia. finally, taking the dog out increased in relative importance in every single country, most significantly in spain (where it rose by over %). taking the kids outdoors was most cited by croatians. j o u r n a l p r e -p r o o f figure . percentage of variation in the reasons given for visiting ugs during the containment period (among those who did so) relative to the pre-pandemic period, by ugs visitors. significant differences between the two situations (before vs. during were identified by the chi-square test. differences are indicated by * at α< . , ** at α < . and *** at α< . . surprisingly, respondents who visited ugs during the covid- pandemic declared that they did it quite often (sm -table e) , with the option of more than once a week selected by a majority in nearly every country (reaching % in slovenia). only in spain once a week was selected more frequently ( % vs. %), and italy was the country with the highest proportion of respondents ( %) declaring that they only went out oncemoderately higher than croatia ( %), lithuania ( %) and israel, and much higher than slovenia and spain. when respondents were asked if the ugs visited during the period of containment was the same as the one they visited pre-pandemic, the majority in all countries (except croatia) answered that it indeed was (sm - table ). the percentage of those who declared that their visitation changed to a different ugs ranged from % in croatia to % in slovenia. in an open-ended question (sm -table ), the majority of these respondents in israel, italy and in spain wrote that their main reason was to stay closer to home (presumably following the rules set by local legislation)while in lithuania and slovenia (and by a small margin also croatia) the main rationale given was to avoid people. another consideration that was linked to the local j o u r n a l p r e -p r o o f restrictions was the non-accessibility of ugs in italy and in spain, for the simple reason that they were closed ( % in both countries). respondents were also asked about their perceptions regarding ugs during the period of home quarantine, specifically: a) the possibility of seeing some outdoor "greenery" from the window of their residence, b) the extent to which they "miss" accessing green areas directly, and c) what particular activities related to ugs they miss the most (sm -table ). in terms of a "green view" from the window (table a) , the great majority of respondents reported being able to see some form of ugswith highest percentages lacking any such view found in lithuania ( %), italy ( %) and spain ( %). overall the most commonly reported views were of private gardens (ranging from % in israel to % in spain), with the notable exception of slovenia, where respondents instead referred to natural landscape ( %) or other ( %), represented mostly by a generic green space near buildings, vegetable gardens and playgrounds ( %, and % respectively within other). also, the possibility of seeing tree-lined streets was prominently mentioned in spain ( %), israel ( %) and croatia ( %). in response to the question "during the period of closure, how much do you miss going to a green area?", over half of the lithuanians (and nearly half of the slovenians) declared that they did not miss ugs at all, or if so just a little. these cases were exceptional, however, as a clear majority reported missing ugs a lot in italy ( %), spain ( %) and israel ( %)and this was also the top response in croatia ( %) and even in slovenia ( %), where the distribution of responses was especially wide (table b) . regarding the aspects of ugs that respondents missed the most (table c) , there was considerable variation among countries but several significant patterns do emerge. first of all, respondents from most countries prioritized spending time outdoors, which was the top response in italy ( %) and israel ( %) and nearly so in lithuania ( %) and croatia ( %). in slovenia, on the other hand, this sentiment was not expressed at all; instead meeting other people was missed the most ( %), which was also the top response in croatia ( %) and among the top responses in israel ( %). for spaniards, exercising outdoors was the activity most missed ( %), followed by observing nature ( %)both of which were also missed in israel j o u r n a l p r e -p r o o f ( % and % respectively) and the latter in italy ( %). finally, it may be noted that lithuanian respondents missed, above all else, breathing fresh air ( %). the extent to which citizens missed ugs depended on their frequency of visitation. those who never went out during the pandemic missed the ugs the most (sm -table ) , and this relationship also occurred with the group who did visit ugs during the isolationas visitors who went to ugs more frequently (e.g. more than once a week) missed ugs less than the other groups (sm -table ). respondents missed visiting ugs, although those who could see natural landscape missed it only to a minor extent (sm -table ). finally, the questionnaire respondents were asked for an open-ended reflection on the relationship between urbanization, people and nature. a total of valid responses were collected, in which the automated text mining identified a total of significant words as the most commonly used by the respondents (sm - table ). the five most used terms were "green area" ( counts), "city" ( counts), "nature" ( counts), "trees" ( counts) and "human" ( counts). the correlation analysis of the word matrix (sm - fig. ) showed how "green areas" were conceptually linked to other key terms such as "awareness", "city", "greenery", as well as "lack" and "urbanization". moreover, the word "city" was frequently used together with the words "nature" and "trees," and the word "man" was often connected to "health," "nature" and "trees". another perspective was offered by the pca analysis, conducted on the same word matrix: thirteen components were selected explaining % of the word matrix variability (sm - table ). the most relevant association (p ) was between "green areas", "trees" and "citizens" ( % variability explained). associated to such words were several terms from p : "nature", "man" and "to create" ( % variability explained). the concepts of "urban" and "life" appeared from p , and "health", "quality" and "urbanization" from p . thematic analysis of the entire body of text, identifying text units by their content within the respondent's answer for a total of valid text units, allowed us to identify individual concepts which were grouped into nine categories, on the basis of the objective and conceptual meaning derived from the original text (sm -table ). these categories were further grouped into three macro-categories, in which % of the individual concepts were assigned to the macro-category governance, % to awareness and % to benefits and services. the concepts associated with the macro-category of governance were the most mentioned in all countriesmost prominently in croatia (accounting there for % of the total), followed by lithuania j o u r n a l p r e -p r o o f ( %) and by italy, spain and israel with  % each (see table in sm for a comparison of the five countries; the slovenian survey did not include this open question). within governance, the greatest number of concepts were related to the category of planning ( %)including tangible recommendations such as the need to "increase the quantity of green surfaces," "adopt approaches to urban planning which integrate green space and tree plantations into new neighborhoods," and "reduce paving and building." an additional % of all the individual concepts were related to the category of management, such as "maintenance of green areas" and "quality of management." respondents showed a certain level of awareness of the importance of green areas, emphasizing environmental consciousness, and to a somewhat lesser extent also expressed the need to educate adults and children to care for the environment. indeed, under awareness, the majority of texts referred to consciousness ( %), a term that identifies the text units expressing respondents' own personal awareness in relation to the importance of urban greenery, including have a greater contact with nature and respect for nature. minor categories included education ( %) and valuation ( %), which included concepts that express the importance of recognizing the added value of green areas. texts indicated respondents' awareness either in general terms or with special emphasis on specific benefits ( %)mostly related to health and well-being and psychological state. this study was motivated by the social isolation that was imposed during the covid- pandemic in . findings from our online survey in six european countries highlighted some of the changes in behavior and attitude related to the visitation of urban green space that resulted from this altered reality. the samples in croatia, israel, italy, slovenia, lithuania, and spain were all overwhelmingly urban, with most respondents living in large cities (table ). this is highly reflective of the european population overall, and it indicates that most people cannot take ugs for granted. the results demonstrate how adults not only share this need, but they are willing, in general, to travel long distances within or beyond the city to access ugs. in order for ugs to be accessible to the population, they need to be created and maintained within the urban fabric, which is often densely built and characterized by competing land uses. in terms of the reasons that urban dwellers have this need for ugs, it was clearly seen from pre-pandemic results that the most sought-after activities in ugs were physical exercise and relaxing (sm -table) . this was the case for all countries, though italians and lithuanians in particular also expressed an appreciation for nature observation. the use of ugs for running, walking and outdoor sports has been widely studied and found to depend on numerous interconnected variables and aspects of the urban fabricincluding built density and green surface area, pedestrian access, safety and protection from traffic and crime (sreetheran and van den bosch, ) . taking children outdoors and walking the dog were also commonly cited activities, the latter also associated with physical exercise (christian et al., ) . a key objective of the survey was to discern changes in ugs visitation after the covid- restrictions were enacted. in italy and spain, the two largest countries in the survey and the two hardest hit by the pandemic, nearly two-thirds of those who previously visited ugs on a regular basis responded that they simply stopped going (sm - table ) . while this can easily be attributed to the government restrictions on personal mobility during the period of containment, % of respondents in each of these two countries did continue making their way to some ugs. this indicates that the need for greenery and open air certainly did not disappear with the legal restrictions to access to such places. the reduced visitation was in fact not evenly distributed among different population groups; for example a larger reduction was seen among female respondents (sm -table )many of whom may have been put off by other duties or the insecurity of parks that were relatively deserted (sreetheran and van den bosch, ) . while the restrictions may not have changed people's basic need for ugs, they did lead to an increase in the diversity of ugs that were accessedwith some traveling out of town, and others relying more on tree-lined streets and urban gardens (fig. , sm - table ). this finding points to a possible dichotomy in which limitations on regular access to parks could result in trends toward either more localized or more longdistance travel (sm -table ), both of which have environmental implications. the evidence for this dichotomy is also suggested by changes in the modes of transportation used for reaching ugs. while in most countries such changes were not pronounced, a significant reduction in the use of private cars was registered in israel, accompanied by an increase among those arriving on footand in an especially striking result, and one that could have tangible implications for ugs planning, pertains to the stated motivations for changing one's behavior. a strong reduction was seen among people going to ugs for reasons that could be seen as non-essential, such as observing nature or which could pose a risk for possible contagion, such as meeting people (fig. ) . this is significant because it shows that parks and other ugs have essential functions that are fundamentally different from other types of public places like shops or restaurants. while ugs can indeed serve as centers of public gathering, they can also be vital for the opposite reasonto serve as places where people can find respite and isolation within the crowded city. all of these findings have implications for the design, management, and indeed the appreciation of ugs. when respondents were asked how much they missed ugs, the response was clear, with the large majority missing ugs to a large extent ugs (sm -table ). however this feeling depended on their frequency of visitation, with deeper feelings expressed by those who never went to ugs or went just once during the pandemic (sm - table ). the view of greenery from peoples' windows played only a minor role, although those who could see natural landscapes missed ugs only to a minor extent, likely because such views induced a perception of freedom or sense of exploration (sm -table ). regarding what they missed the most during the period of home quarantine, respondents in most countries prioritized the simple idea of spending time outdoorsand while this might seem like a fairly straightforward reaction to the circumstances of being confined to one's home for an extended period, it should be stressed that the question referred specifically to green space, and not to outdoor areas in general. this could be an indicator of the special biophilic importance that greenery has for city dwellers, above and beyond the need for open space such as may be found in a public square that is relatively devoid of vegetation. in this sense, parks and tree-lined streets may once again be seen as having added value not just as public open spaces for gathering, but alsoas indicated during the time of the pandemicplaces of personal refuge within the city (fig. ) . this in no way diminishes the tremendous value of large public parks, and in fact urban parks were the most selected type of ugs during the pandemic. what many respondents missed most during the pandemic was park-related activity like exercising outdoors and meeting other people (sm -table ); in other words, parks are seen as vital places. in some cases (e.g. israel and italy), observing nature and breathing fresh air j o u r n a l p r e -p r o o f were also highly missed. these findings express the wide range of needs fulfilled by ugsfrom the physical to the cognitive and emotional, and from the tangible to the ethereal. when it came to expressing their general feelings about the relationship between people and nature in the context of urbanization, most respondents focused on tangible issues related to the planning and management of ugs. many focused on design and planning decisions made by local actors, such as increasing the spatial extent of green areas and improving residents' access to them. this reflects a forward-looking, action-oriented attitude on the part of many respondents within the survey sample. it also reflects the large percentage of respondents with higher education (see supplementary material) and presumably with a high level of knowledge or interest in the topic, as demonstrated by the familiarity with specific definitions and laws related to the management of urban green (as expressed in individual textual responses). a concurrent theme, appearing through the selected options of ugs and the responses to the open-ended questions, is related to the importance for urban green infrastructure to be well articulated and inclusive of different types of greenery. in order to create resilient cities, urban planning and design should consider a diverse mix including large parks which can offer spacious open-air green settings, which are useful for visitation (even without large gatherings) of ugs during times of pandemic, together with smaller pocket parks and gardenswhich can guarantee, as much as possible, that all residents will have access to ugs within walking distance from their home. from this perspective, it becomes important to foster the multiple services that different types of ugs provide in the city and to re-think urban policy and planning to respond to new behaviors and needs that have arisen from the covid- pandemic (honey-rosés et al. ) . for instance, the rise of pedestrian movement and green mobility observed in some countries should be accompanied by the provision of streets, sidewalks and biking trails that are shaded with healthy, appropriately selected and well nurtured trees, connecting green spaces throughout the city through vital green corridors. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. j o u r n a l p r e -p r o o f conceptualization, methodology, formal analysis, writing-original draft preparation formal analysis, writing-original draft preparation; pedro calaza-martínez: investigation, data curation, writing-original draft preparation; paloma cariñanos: investigation, data curation, writing-original draft preparation silvija krajter ostoic: investigation, data curation, writing-original draft preparation ana marija marin: investigation, data curation; david pearlmutter: investigation, data curation, writing -review & editing writing-original draft preparation, ingrida Šaulienė: investigation, data curation, writing-original draft preparation writing-original draft preparation; andrej verlič: investigation, data curation, writing-original draft preparation; dijana vuletić: investigation, data curation, writing-original draft preparation cultural ecosystem services as a gateway for improving urban sustainability preferences for cultural urban ecosystem services: comparing attitudes, perception and use using thematic analysis in psychology encouraging dog walking for health promotion and disease 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the impact of interventions to promote physical activity in urban green space: a systematic review and recommendations for future research the experience of nature: a psychological perspective content analysis: an introduction to its methodology ecological urbanism urban green space and well-being in kuala lumpur cultural ecosystem services provided by urban green change along an urban-periurban gradient el urbanismo ecosistémico the social landscape of planning: integrating social and perceptual research with spatial planning information urban forests and their ecosystem services in relation to human health factors influencing the use of green space: results from a danish national representative survey association between indoor-outdoor green features and psychological health during the covid- lockdown in italy: a cross-sectional nationwide study a socio-ecological exploration of fear of crime in urban green spaces-a systematic review statistica electronic manual landscape ecological urbanism: origins and trajectories relationships between characteristics of urban green land cover and mental health in u.s. metropolitan areas scenic beauty: visual landscape assessment and human landscape perception green space as a buffer between stressful life events and health urban natural environments as nature-based solutions for improved public health -a systematic review of reviews landscape as urbanism: a general theory biophilia. cambridge (ma) the relationship between natural outdoor environments and cognitive functioning and its mediators we would like to thank all the people and organizations who promoted and distributed the survey in the six countries, as well as all survey respondents.in croatia, this work has been supported in part by the croatian science foundation under the project uip- - - . cd was supported by fondecyt . in spain a special thank to the spanish association of parks and public gardens. key: cord- -h rnyww authors: gherghina, Ștefan cristian; armeanu, daniel Ștefan; joldeș, camelia cătălina title: stock market reactions to covid- pandemic outbreak: quantitative evidence from ardl bounds tests and granger causality analysis date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: h rnyww this paper examines the linkages in financial markets during coronavirus disease (covid- ) pandemic outbreak. for this purpose, daily stock market returns were used over the period of december , –april , for the following economies: usa, spain, italy, france, germany, uk, china, and romania. the study applied the autoregressive distributed lag (ardl) model to explore whether the romanian stock market is impacted by the crisis generated by novel coronavirus. granger causality was employed to investigate the causalities among covid- and stock market returns, as well as between pandemic measures and several commodities. the outcomes of the ardl approach failed to find evidence towards the impact of chinese covid- records on the romanian financial market, neither in the short-term, nor in the long-term. on the other hand, our quantitative approach reveals a negative effect of the new deaths’ cases from italy on the -year romanian bond yield both in the short-run and long-run. the econometric research provide evidence that romanian -year government bond is more sensitive to the news related to covid- than the index of the bucharest stock exchange. granger causality analysis reveals causal associations between selected stock market returns and philadelphia gold/silver index. with globalization, urban sprawl, and ecological transformations, contagious disease outbursts turned out to be worldwide risks demanding a joint reply [ ] . according to the international monetary fund (imf), coronavirus disease (covid- ) generated an economic crisis different from the others [ ] for the reason that it is much more multifaceted (interconnections between the economy and the health system), uncertain (the related treatment is established gradually, alongside the measures concerning how to streamline isolation and the means to start over the economy), and has a worldwide character. both supply and demand reductions occur since individuals work and consume lower, whereas companies diminish their productivity and investment [ ] . hence, erokhin and gao [ ] explored developing states and established that food security status of individuals and the strength of food supply chains are impacted by consequently, governments have taken unprecedented actions, respectively fiscal measures figuring to around $ trillion, whereas central banks injected liquidity getting up to over $ trillion [ ] . the imf has implemented exceptional measures by doubling its emergency loaning volume to $ billion and deferring debt outflows for poor nations [ ] . preparing for the economic recovery raised a number of issues such as the way to maintain fiscal stimulus and unconventional monetary policy, managing high unemployment, low interest rates, and preserving financial stability [ ] . hence, market inefficiencies to acquire abnormal returns [ ] . on the contrary, yan, et al. [ ] recommended the tourism industry, technology sector, leisure industry, and gold as suitable investments. li, et al. [ ] endorsed health sector in line with chong, et al. [ ] which suggested over sars to buy medical stocks and sell tourism stocks. in terms of cryptocurrencies, chen, et al. [ ] argued that augmented concerns of the coronavirus caused negative bitcoin returns and large trading volume, whereas conlon and mcgee [ ] advised that it does not perform as a hedge. with reference to the influence of the pandemic on the enterprise's activities, mazur, et al. [ ] contended that companies reply in various means to the covid- revenue shock because many sectors were locked throughout the quarantine stage. hence, xiong, wu, hou and zhang [ ] evidenced that companies belonging to sectors that are exposed to the pandemic have significantly lower cumulative abnormal returns, but enterprises with good financial conditions endure less opposing effect of the disease. nguyen [ ] established that energy segment experienced the utmost abnormal negative returns amid all sectors. fallahgoul [ ] established that the financial segment is the most doubtful, whereas health is the most hopeful over the covid- pandemic. he, sun, zhang and li [ ] claimed that manufacturing, information technology, education and health-care chinese sectors remained stable to covid- . gu, et al. [ ] found that chinese manufacturing sector was hardly hit by corona crisis, but construction, information transfer, computer services and software, and health care and social work were positively influenced by covid- . financial markets worldwide confronted with the flight-to-safety phenomenon which engendered a severe deterioration in asset appraisals and amplified volatility around the world [ ] . baker, bloom, davis, kost, sammon and viratyosin [ ] stressed that there was no prior illness that determined such daily stock market jumps. albulescu [ ] emphasized that the fatality rate has a positive and very significant influence on financial volatility, whereas albuquerque, koskinen, yang and zhang [ ] found that green stocks are highly valued and register lower volatility and larger trading volumes than the rest of stocks. markets are a function of government, hence responding reliant on authority reply [ ] . alfaro, chari, greenland and schott [ ] confirmed that a doubling of projected contaminations is linked with a to percent deterioration of aggregate market value. alber [ ] showed that stock market return is influenced by covid- cases more than deaths, as well as by aggregate measures more than new ones. however, attributable to local features, the influence of novel coronavirus may diverge across equity markets [ ] . onali [ ] revealed that variations in the amount of cases and deaths in the usa and other highly impacted nations by the coronavirus do not influence stock market returns out of usa, except the number of cases for china. the spread of covid- globally driven an upsurge of yields on sovereign securities more than proportionally in developing and emerging states [ ] . nozawa and qiu [ ] noticed that corporate bonds supplied by companies showing a strong link with china respond more to the quarantine of wuhan at early . hence, m.al-awadhi, alsaifi, al-awadhi and alhammadi [ ] concluded that the covid- disease negatively influence stock market returns of the companies covered in the hang seng index and shanghai stock exchange composite index. adenomon, maijamaa and john [ ] strengthened that the coronavirus disease negatively influences the stock returns in nigeria. on the contrary, there was proved that everyday cases of new contagions have a low adverse effect on the crude oil quotations in the long-term [ ] . albulescu [ ] explored whether the covid- and crude oil influence the economic policy uncertainty of the united states and observed no impact when considering the global coronavirus data, but a positive effect when assessing the condition outside china. sharif, et al. [ ] established a unique responsiveness of stock market of usa, related economic policy uncertainty, and geopolitical risk to the joint shocks of the coronavirus and oil instability. for the case of colombia, cardona-arenas and serna-gómez [ ] argued that the depreciation of national in addition, we have included a wide range of variables that allow us to achieve our goal, such as covid- measures, commodities, currencies, and -year government bond spreads. in order to gain insights towards the linkages in stock markets during covid- pandemic outbreak, we will use the autoregressive distributed lag (ardl) model similar albulescu [ , ] , erokhin and gao [ ] , as well as granger causality test alike mamaysky [ ] . checking for unit root in ardl approach is not fundamental in as much as it can examine for the occurrence of cointegration among a set of variables of order i( ) or i( ) or a mixture of them. hence, the leading benefit of ardl model consist in its versatility. however, the ardl methodology impose that no variable should be integrated of second order or i ( ) . therefore, in line with prior research [ , , ] , the augmented dickey-fuller (adf) test will be applied for unit root testing. the null hypothesis of the adf test claims the presence of unit root in the time series. the adf test involves estimating the following equation: where t denotes the time trend, t signifies the length of the sample, while k is the length of the lag in the dependent variable. further, ardl model examines the long and short-term cointegration, being specified as a sole equation framed with adaptable choice of lag extents. the general form of an ardl (p, q) model is as follows: the lag orders p and q are established by means of the akaike information criteria and may differ over the explanatory variables covered in our quantitative framework. the granger causality test can be applied to analyze the causality between variables, as in mamaysky [ ] . the null hypothesis is that w does not granger-cause z and that z does not granger-cause w. the following bivariate regressions will be estimated: w t = α + α w t− + · · · + α p w t−p + β z t− + · · · + β p z −p + u t ( ) the descriptive statistics of the variables are provided in table . the distributions of all stock market returns, as well as most of included commodities are negatively skewed. thus, negative returns are more prevalent than positive returns, supporting a greater likelihood for very high losses. kurtosis shows the thickness of the tail and highlights a high level of risk for selected stock markets, especially spain and italy. in addition, except eur/cny and natural gas futures contract , the jarque-bera test provides evidence that selected series are not normally distributed. figure shows the evolution of the number of new cases due to covid- , whereas figure reveals the progress of the number of new death due to covid- . there is noticed that usa registers the highest figures in this regard. source: authors' own calculations. notes: for the definition of variables, please see table . figure shows the evolution of the number of new cases due to covid- , whereas figure reveals the progress of the number of new death due to covid- . there is noticed that usa registers the highest figures in this regard. figure shows the evolution of stock market returns amongst the explored period. there is reinforced the significant volatility, especially for ftse mib on march , and march , , as well as for dow jones industrial average on march , . in the first two months of , dax declined by . percent, cac dropped by . percent, whereas ftse plunged . %. in the same vein, dow jones throw down by percent and s&p by . percent. the bucharest stock exchange also encountered instabilities and registered a decay of . percent [ ] . capelle-blancard and desroziers [ ] contended that prior to february , stock markets disregarded the pandemic, but over february -march , the reaction to the rising number of diseased people was strong. as such, mazur, dang and vega [ ] emphasized that the failure of stock quotes in march marked one of the major financial market collapses in history. baiardi, et al. [ ] developed a three-regime switching model and concluded that in the most common state for the dow jones industrial average was turbulent. figure shows the evolution of stock market returns amongst the explored period. there is reinforced the significant volatility, especially for ftse mib on march , and march , , as well as for dow jones industrial average on march , . in the first two months of , dax declined by . percent, cac dropped by . percent, whereas ftse plunged . %. in the same vein, dow jones throw down by percent and s&p by . percent. the bucharest stock but over february -march , the reaction to the rising number of diseased people was strong. as such, mazur, dang and vega [ ] emphasized that the failure of stock quotes in march marked one of the major financial market collapses in history. baiardi, et al. [ ] developed a three-regime switching model and concluded that in the most common state for the dow jones industrial average was turbulent. table . figure reveals the evolution of oil futures. there is noticed the sharp decline registered on april . figure shows the progress of philadelphia gold/silver index returns. therewith, high volatility is prevailing. table . figure reveals the evolution of oil futures. there is noticed the sharp decline registered on april . figure shows the progress of philadelphia gold/silver index returns. therewith, high volatility is prevailing. table . table . for the definition of variables, please see table . table reveals the correlations among selected variables. there are acknowledged high negative correlations (below − . ) between the number of new cases and new deaths due to covid- in italy and crude oil, wti, as well as nymex light sweet crude oil. in case of the number of new cases and table . table reveals the correlations among selected variables. there are acknowledged high negative correlations (below − . ) between the number of new cases and new deaths due to covid- in italy and crude oil, wti, as well as nymex light sweet crude oil. in case of the number of new cases and new deaths due to covid- in china, there are not recorded high correlations with the included measures. therewith, high positive correlations (over . ) are registered amongst the stock market returns, except sse (china). non-stationary variables lead to inadequate results, which means insignificant results. the verification of the stationarity of the selected data is performed through adf stationarity test. this test is most commonly used to confirm the stationarity of a data series. table shows the results of the adf test at the level and in the first difference, as well as the level of integration of the stock indices. the outcomes of adf test provide support that all covered stock indices are stationary at the first difference, showing an integration order of i( ), except the stock market index from the shanghai stock exchange. we also notice that the indicators related to the evolution of covid- for the most affected regions, china and italy, show a mixed integration order (i( )and i( )). after studying the stationary of the data series and due to the mixed results, we conclude that the ardl model is the most appropriate for exploring the linkages between variables. further, the purpose is to assess whether new cases and new deaths due to covid- in china and italy, along with chinese and italian stock market returns, several commodities, and currencies are related to the romanian stock market as measured by bet index return and romania -year bond yield. the ardl (autoregressive distributed lag) model is used especially when the variables i( ) and i( ) are integrated. for the accurate choice of the ardl model that would allow us to research the relationships that are established between variables, it is imperative to choose the correct number of lags. therefore, we will analyze the akaike information criteria (aic) to select the optimal lags for the variables included in the ardl model. we will apply the criteria graph, which will indicate the suitable lags for the ardl model and the lowest value is preferred. figure shows the results of criteria graph for the ardl model that takes into account the number of new cases and new deaths in china, both for the bet stock index return and for the romanian government bond ( y). according to the results, in total, , , ardl model specifications were considered for each of the four cases given the information related to covid- in china. the top results are presented in the criteria graph. further, table summarizes the selected lags for the model romania and covid- (china) according to criteria graph out of figure . table . according to the results, in total, , , ardl model specifications were considered for each of the four cases given the information related to covid- in china. the top results are presented in the criteria graph. further, table summarizes the selected lags for the model romania and covid- (china) according to criteria graph out of figure . figure shows the results of criteria graph for the ardl model that takes into account the number of new cases and new deaths in italy, both for the bet stock index return and for the romanian government bond ( y). likewise, in case of italy, in total, , , ardl model specifications were considered for each of the four cases. table . table exhibits the selected lags for the model romania and covid- (italy) in line with criteria graph out of figure . the results reported in tables and provides the ardl bound test for cointegration. if the f-statistic is greater than the upper bound, then the variables comprised in the model are cointegrated and a long-run relationship befall. with reference to new cases in china models (see table ), the f-statistic for bet_r ( . ) and ro_bond ( . ) models is greater than the upper bound of bounds value at %, which is suggesting that long-run relationship occur between the variables. the same result is achieved in the case of new deaths in china models, where the value of the f-statistic is greater than the upper bound critical value. hence, the null hypothesis is rejected, meaning that the variables in the model are cointegrated. figure . the results reported in tables and provides the ardl bound test for cointegration. if the fstatistic is greater than the upper bound, then the variables comprised in the model are cointegrated and a long-run relationship befall. with reference to new cases in china models (see table ), the fstatistic for bet_r ( . ) and ro_bond ( . ) models is greater than the upper bound of bounds value at %, which is suggesting that long-run relationship occur between the variables. the same result is achieved in the case of new deaths in china models, where the value of the f-statistic is greater than the upper bound critical value. hence, the null hypothesis is rejected, meaning that the variables in the model are cointegrated. table . regarding italy, in all four estimated ardl models the existence of cointegration is confirmed (see table ) since the f-statistic is significantly higher than the critical values in i( ) and i( ). consequently, the examined variables are cointegrated and will move together in long-run. further, we will analyze the results of the long-term linkages between selected measures. table shows the outcomes regarding the long-run causal connections among variables for the model romania and covid- (china)-new cases. the short-run estimates of ardl approach are presented in table s . in the first model, the number of new infection cases from china have no effect on the bet index return. however, a decrease of crude oil price leads to a higher uncertainty, consistent with salisu, ebuh and usman [ ] , suggesting the necessity for policymakers to diminish fears in financial markets. in addition, the exchange rate negatively influences stock market return in the long-run. the philadelphia gold/silver index coefficient is positive and significant at the % level of significance. hence, the coefficient of xau_r indicates that an increase of one unit in philadelphia gold/silver index leads to over . units increase in bet index return in the long-run. the error correction term or adjustment speed provides evidence regarding the rate of convergence to equilibrium, being highly statistically significant. the adjustment speed of − . shows that deviations from the long-term equilibrium in bet index return are corrected the following day by approximately . percent. regarding the second model from table , similar to the first model, the new infection cases from china does not influence romania -year bond yield in the long-run. unlike the previous model, the ro_bond is negatively affected by xau_r and indicates that an increase of one unit in philadelphia gold/silver index leads to over . units decrease in ro_bond return in the long-term. besides, in the long-run, the return of stock market index sse negatively influences romania -year bond yield. the coefficient of the error correction term is highly statistically significant. hence, the romanian -year bond will reach equilibrium with a speed of . percent in next day. as well, the short-run results strengthen the lack of impact regarding new infection cases of covid- from china on ro_bond. table reveals the outcomes of the long-term connection amongst variables for the model romania and covid- (china)-new deaths. the short-run results are shown in table s . the empirical findings reveal that the impact is stronger in this case as compared to the model that depends on the number of new cases in china due to covid- (see table ). however, both models shows that the number of new deaths in china due to covid- has no influence on the bet index return, respectively, on the romania -year bond yield, neither in the short-term, nor in the long-term. therefore, both research hypotheses are rejected for chinese covid- figures, similar topcu and gulal [ ] which established that emerging european countries experienced the lowest influence of the outbreak. tables and reveals the results of serial correlation and heteroscedasticity tests for the models romania and covid- (china)-new cases and romania and covid- (china)-new deaths. the results support that the models are free from autocorrelation and heteroscedasticity. in the case of models that take into account the effects of new cases and new deaths in italy, unique relationships are identified between the selected variables, as opposed to the models that explored the impact of coronavirus from china. table exhibits the outcomes of the long-term causal associations between variables for the model romania and covid- (italy)-new cases. the short-run outcomes are exhibited in table s . in the long-run, the results of the first model show the lack of any effect from the number of new cases of covid- in italy on bet index return. in contrast, the return of milan stock market index ftse mib has a positive long-term impact on the bet index return. as well, the short-run results reveal no impact of new infection cases of covid- from italy on the bet index return. in contrast to covid- figures from china, in case of italian new cases of coronavirus, the first hypothesis is still rejected, but the second hypothesis is confirmed. moreover, in the second model, several statistically significant relationships are identified. there is found a positive impact of the number of new cases in italy on the romania -year bond yield in the long-term. in addition, a natural gas futures contract has a positive effect on ro_bond, while the wti oil and philadelphia gold/silver index has a negative impact in the long-run. another outstanding outcome is that new infection cases of covid- from italy negatively influence ro_bond in the short-run, consistent with sène, mbengue and allaya [ ] . therefore, the related uncertainty triggered by the health emergency may determine investors to get rid of their securities. table exposes the findings towards long-run linkages between variables for models related to romania and covid- (italy)-new deaths. the results of short-run estimates are presented in table s . the first model out of table exhibits that the number of new deaths from italy have no effect on the bet index return in the long-run. the philadelphia gold/silver index coefficient is positive and significant at the % level of significance. hence, the coefficient value of xau_r indicates that an increase of one unit in philadelphia gold/silver index leads to over . units increase in bet index return in the long-term. however, the short-run results show a negative impact of new deaths cases of covid- from italy on the bet index return, in line with okorie and lin [ ] which underlined a transitory contagion effect in the stock markets due to novel coronavirus. in addition, erdem [ ] claimed that the index returns decline and volatilities rise due to corona crisis. hence, the first hypothesis is confirmed. the second model shows a negative effect of the new deaths' cases from italy on the romania -year bond yield in the long-run. in addition, the philadelphia gold/silver index and the ok crude oil future contract negatively influence ro_bond in the long-term. besides, in the long-run, the returns of the stock market index ftse mib has no impact on the -year romanian bond. nevertheless, in the short-run, results show a negative impact of new deaths cases of covid- from italy on the ro_bond. therefore, the second hypothesis is established. tables and exhibit the outcomes of breusch-godfrey serial correlation lm test and breusch-pagan-godfrey heteroscedasticity test for the models romania and covid- (italy)-new cases and romania and covid- (italy)-new deaths. hence, the models are not threatened by autocorrelation and heteroscedasticity. with the purpose of exploring the causality between included variables, the granger causality test is employed. in order to be able to apply the granger causality test, the data series must be stationary and therefore they were turned it into stationary series. table displays the results of granger causality test for the stock market returns and covid- measures. there were identified some bidirectional causal relations between bet_r and ftmib_r ( st lag), as well as among bet_r and ibex _r ( st lag). besides, some unidirectional causal relations arise from ftse_r ( st lag), djia_r ( st lag and rd lag), sse _r ( st lag, nd lag, and rd lag), and xau_r ( st lag, nd lag, and rd lag) to bet_r. nevertheless, no relationship was found between bet_r and the covid- variables. table shows the outcomes of causalities for the variables concerning commodities, currencies, governmental bonds, and covid- . the causalities for the whole world stock indexes, commodities, currencies, and covid- variables are reported in table s . some bidirectional relationships were found merely for the st lag between the -year romanian bond and few stock market indices returns, namely cac , dax, and ibex . besides, unidirectional relationships for st lag, nd lag, and rd lag occurred from returns of djia, s&p , ftse , ftse mib, sse , and the number of new cases in italy due to covid- to the -year romanian bond. one of the most severe stock market crashes was registered in march [ ] due to the occurrence of the novel coronavirus covid- pandemic [ ] . the research contributions are twofold. first, we investigated whether the romanian stock market is affected by the covid- pandemic outbreak. second, our paper explored the causalities among covid- and major stock market returns, as well as between pandemic measures and several commodities. in this regard, we used daily stock market returns over the period december , -april , for the following economies: usa, spain, italy, france, germany, uk, china, and romania. we have selected a wide range of variables that allow us to achieve our goal, such as stock market indices, new number of cases of illness, new number of deaths in china and italy, exchange rate, commodity indices, romanian bonds. as far as we know, this is the first study addressing the impact of the covid- from both china and italy crisis on the romanian capital market and the -year romanian bond. after examining the stationarity of the selected data series and due to the mixed results, we conclude that the ardl model is the most appropriate to explore the short-term and long-term causal associations among romanian stock market and novel coronavirus. in the case of the model that includes the number of new deaths in china due to covid- , it is found that the impact of the coefficients is stronger compared to the model that depends on the number of new cases in china due to covid- . at the level of these two models, no effect was identified from the number of new deaths in china due to covid- on the bet index return, respectively on the romania -year bond yield, neither in the short-term, nor in the long-term. with reference to the model that cover the new cases of coronavirus from italy, short-run results provide support for a negative impact of new italian covid- cases on the romania -year bond yield. taking into account the number of new deaths in italy we found that it has no effect on the bet index in the long-term, but the short-run results exposes a negative effect. besides, the ardl models showed a negative effect of the new deaths' cases from italy on the romania -year bond yield both in the long-run and short-run. granger causality test exhibits bidirectional causal relations between returns of bet and ftse mib, ibex, as well as a unidirectional causal relation from ftse , djia, sse , and philadelphia gold/silver index to bet index return. however, no relationship was found between the bet index return and the covid- variables. some bidirectional relationships were found between the -year romanian bond and a few stock market indices (cac , dax, and ibex ). unidirectional relationships occurred from returns of djia, s&p , ftse , ftse mib, sse , and the number of new cases in italy due to covid- to the -year romanian bond. therefore, the empirical findings from ardl model and granger causality test confirmed both the presence of a long-term and short-term relationship between romanian capital market and covid- variables. the findings show that the chinese covid- numbers have no impact on the romanian financial market. in addition, it was found that the -year romanian bond is more sensitive to the news related to covid- than the index of the bucharest stock exchange, similar to pavlyshenko [ ] , mamaysky [ ] . the paper may have some policy implications. as long as the bet index is not influenced by covid- variables, this may suggest evidence of an inefficient market, in line with beck, flynn and homanen [ ] , mensi, sensoy, vo and kang [ ] . there are required policies to increase market efficiency though longstanding and sustainable growth rather than administering short-term interest rates [ ] . the investors should seek long-term horizons of investing since the monetary and fiscal policies set by governments will alleviate the harmful effects of covid- . the policymakers should be aware that corona crisis may be an occasion to improve the discrepancy among romania and developed nations of european union. in this regard, a substantial share of the budget should be expended to alleviate this pandemic [ ] . a suitable clinical stream is vital so as to ensure a reliable supervision of patients [ ] . rearrangement of public expenditure to enlarge the absorptive volume of healthcare organizations is essential [ ] . therefore, public health expenditures should be increased, along with offering direct income funding to exposed populations via cash transfers, 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the authors declare no conflict of interest. key: cord- -bzfrd xj authors: abenavoli, fabio massimo title: plastic surgery in the age of coronavirus date: - - journal: plast reconstr surg glob open doi: . /gox. sha: doc_id: cord_uid: bzfrd xj nan t he history of the appearance of the coronavirus disease , which is caused by the virus later renamed severe acute respiratory distress syndrome coronavirus (sars-cov- ), is well known by now. it first appeared in the city of wuhan in china's hubei province and then quickly spread to the rest of the world. within days, the virus had thus spread from a single city to the entire country and beyond. articles published in international journals, first by chinese authors, then by others, up to the last week of march, help us to understand the evolution and the causes of this rapid spread. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] what has now become apparent is that the novelty of the virus and the possibility of its rapid spread were underestimated. [ ] [ ] [ ] even the first epidemiological studies identified potential scenarios that were later disproved by the data that emerged. , [ ] [ ] [ ] within a very short time, the world health organisation (who) put out guidelines that quickly had to be revised following the rapid evolution of the spread of the virus. on january , , the who declared the covid- outbreak a public health emergency of international concern. the chances of contracting the disease through close proximity to people carrying the virus, even those with mild symptoms, appear to have been grossly underestimated. [ ] [ ] [ ] the ability of the chinese authorities to build hospital facilities for infected patients within a very short time appeared to be a test of "strength." however, little attention was paid to the conclusions that should have been drawn about how to assist new patients during the emergency situation. the political authorities in many countries viewed the lockdown and the complete suspension of all public activities in china as a dictatorial measure that was not based on any real need. on january , , the us centers for disease control and prevention and the department of homeland security's customs and border protection began health screenings at us airports for passengers returning from wuhan city. it took until the end of january for more stringent measures to be implemented, before flights from china were suspended altogether. italy responded in much the same way, by canceling all flights to and from china beginning on february , , but without ensuring the necessary precautions for people traveling from china on flights with stopovers in other countries. the option of testing potentially infected patients for the disease appeared to be a far-off measure that was not urgently considered. however, the illusions outlined above were drastically shattered by the nature of the virus itself, which spread very rapidly and turned out to be highly contagious. for italy, which found itself with the largest number of infections in europe within a relatively short time, the outbreak soon turned into a crisis of unprecedented proportions. [ ] [ ] [ ] despite being aware of what might happen, our political and health authorities, like those, it would seem, in much of the world, did not respond quickly enough to what was happening in china and was already publicly known. the italian authorities did not realize quickly enough that the virus would require immediate attention and would soon dictate drastic measures in response to a situation that was quickly deteriorating. for instance, as late as february , , thousands of people from the city of bergamo traveled to milan to watch a champions league football game. nevertheless, the italian government only placed the country under quarantine and ordered serious containment measures after march. this time, the historical division of our country into a northern part, characterized by greater economic development, and a southern one-which has seen large migrations of its population to the north and abroad due to the lack of local jobs and economic opportunities-was oddly reversed. lombardy, in particular, one of the richest and most densely populated regions in europe, has been the most severely affected. on march , , the who declared covid- a global pandemic, with > , clinical cases and over deaths in affected countries. to date, as of april , , there have been , cases and , deaths in italy, most of them in lombardy. today, precisely through the development of new systems for tracking the spread of the virus, it is possible to monitor the rapid global rise in infections. this provides useful data for attempts to counter the evolution of the virus by means of containment measures. most hospitals in northern italy are overcrowded and approaching the absolute limits of their capacity, and at the same time, there is a lack of drugs, mechanical respirators, oxygen, and personal protective equipment (ppe). the healthcare system is struggling to provide normal services. military field hospitals have been established in several areas, for example, in bergamo, an italian city with a rich history and traditions that is also one of the worst affected. within a few days, even the pavilions of the milan world exposition were partially transformed into a large resuscitation facility for thousands of new patients. this was all made possible, thanks to the generosity of italian companies and citizens who are donating millions of euros to "rebuild" the italian healthcare system, a healthcare system which, precisely because it is accessible to everyone without distinction and at no cost, has always been a point of pride for italy, and which in recent years has been tragically weakened and impoverished due to completely inadequate policy choices and a lack of investment. furthermore, commitment to patients is of particular importance, given that the virus primarily targets the lungs. severe acute respiratory distress syndrome coronavirus (sars-cov- ), which results in a high percentage of infected patients, has been enormously difficult to manage. the incidence of mortality appears to be high in elderly people and in those who suffer from comorbidities, such as hypertension, obesity, diabetes, or other pathologies, but there is a great deal of uncertainty on this topic. moreover, the italian society of environmental medicine has published an interesting position paper highlighting the fact that atmospheric particulate matter, in addition to being a carrier, acts as a substrate that permits the live virus to remain in the air for some time, namely for hours or even days. in the context of lombardy, which is a highly industrialized area, the greater degree of contamination may also be a significant reason for the higher incidence of infection among the local population. to complicate the situation, much of the information that is transmitted through the various social networks is quickly proved to be false, creating a situation of additional confusion and uncertainty. furthermore, many experts, continually solicited by the mass media, have issued contradictory messages, which have further increased confusion about the containment measures that urgently needed to be taken. in response to the emergency, the healthcare system is currently holding up as best as it can, thanks to the generosity of the healthcare operators and the many gestures of solidarity and collective mobilization. however, due to a lack of or inadequate ppe, the rate of infections of medical and nursing staff has reached unacceptably high levels. despite the who recommendations that health personnel should be equipped with ppes such as n or ffp masks, eye protection, gowns, and gloves to protect themselves against infection, there is a great shortage of this material. the deputy minister of health, dr. sileri, himself a surgeon, has tested positive for the disease and is currently under quarantine. the mortality of infected health workers is also very high. more than doctors have died as a result of the infection, and almost % of infected italians work in the health sector. doctors and nurses are arriving from cuba and russia to support their italian colleagues, who are often working > hours a day. the number of intensive-care hospital beds in italy is one of the lowest in europe, partly due to misguided health policy planning that has left the country unprepared to face the high number of patients in need of intensive care. there are similarities as well as differences in the progression of covid- in various european countries. the statistics on the rates of infection have shown that the lockdown measures in italy have had a positive effect (but only after various attempts to contain the spread of contagion failed, by quarantining first parts of the north and then the whole country). in italy, the disease has now been stopped from spreading freely, and the same is foreseeable for germany and france. in spain, on the other hand, the rate of infections still continues to grow substantially. the next few days will show if this growth will be exponential and continue on a trajectory that is even worse than the italian one. plastic surgeons also have to face this new type of challenge. although not directly involved in assisting patients affected by this pathology, the situation has disrupted all normal surgical and diagnostic activities. as plastic surgeons, we find ourselves experiencing a very stressful situation. on the one hand, we are dealing with healthy patients who require surgery to remediate an aesthetic problem and who, by not suffering any urgent medical need, will again be able to resort to cosmetic surgery at some time in the distant future. on the other hand, we are also dealing with patients who need a reconstructive surgery, which is necessary but not urgent and therefore also has to be postponed until the situation has "normalized". however, the date when this will happen is impossible to forecast or predict. consequently, we are faced with a situation of absolute uncertainty. as doctors, we would like to contribute to the emergency response by supporting our hospitals. however, without specific experience in the field, there is little we can do. for those of us who run a private practice that is not associated with any hospital facility, active participation is even more unlikely. we can, however, carry on our specialist activities through online medical practice or telemedicine tools. whatsapp, for example, enables us to receive images from postoperative patients or to analyze clinical examinations and thereby ensure a quick response. on a daily basis, i receive photographs from patients who have recently undergone surgery but who are afraid to go out and come in for a checkup, and who thus remove their dressings themselves and have their partners or relatives photograph them. in this manner, we can evaluate the wounds and the healing process and reassure our patients about the progression of their procedures. obviously, this implies assuming greater responsibility, also from a medical-legal point of view. nevertheless, this situation has unexpectedly led to a reestablishment of trust and amicable relationships between doctors and patients. we are all keenly listening to the needs of patients, and patients are listening to doctors. patients are asking us doctors to be "present," to be supportive, and to abide by our hippocratic oath. as plastic surgeons, we can be part of a wider project to put doctor-patient relationships on a new footing and to promote a renewed alliance, not just between doctors and patients, but also between people. as such, this time of crisis represents a chance for a renewal of interpersonal relationships, an opportunity that must not be missed. it will then be up to us to figure out how to manage and maintain this new spirit in the future. another important issue involves upholding the financial commitments that we must normally fulfil. the lack of paid surgical activities, as well as the impossibility of generating any revenue through procedures such as fillers, botox, or laser treatments, is a great cause for concern. the fact that we cannot predict when we will return to "normal" operations is an absolutely new and unexpected experience that no one can remedy. as the president of an international ngo, www.emergenzasorrisi.it, that performs reconstructive surgeries in developing countries, i have had to suspend surgical missions in benin, afghanistan, and iraq without knowing when we will be able to return to work in these countries. not to mention the fact that if the containment measures adopted prove to be sufficient, these countries will soon also have to face this terrible virus. a vaccine would certainly make for a triumphant end to the sars-cov- saga; however, it is difficult to create effective vaccines against rna viruses such as this one. to sum up, we are all stuck at home in line with the italian government decree. all businesses, factories, and shopping centers are closed, and it is uncertain whether they will be able to reopen quickly. the forecast is that even after the resumption of normal activities, the ensuing economic crisis will bring italy to a standstill for several months or even years; all of this obviously without any prospective solutions or renewed opportunities. the european union, which could and should act as an umbrella for all member countries, has so far proved unable to launch a joint response, so that all member states have had to pursue autonomous emergency measures without any central coordination or synergies. in short, the pandemic that has hit the world, and italy especially, should make us realize how suddenly our lives can change and enable us to reflect on the true values that must guide us in the future. for many of us, reaffirming "in god we trust" leads to a path of faith and hope. on the other hand, for many others, faith in man's ability and resourcefulness may serve as a beacon of light at the end of the tunnel-but once again, we must reiterate the principle that together, we will find a way out of this horrible nightmare. and thence we came forth to see again the stars e quindi uscimmo a riveder le stele -dante alighieri, inferno, xxxiv, verse via salaria roma italy e-mail: f.abenavoli@mclink.it the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- clinical features of patients infected with novel coronavirus in wuhan discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus (sars-cov- ): facts and myths genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding features, evaluation and treatment coronavirus 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cases from the chinese center for disease control and prevention available at https:// www.who.int/news-room/detail/ - - -statement-on-thesecond-meeting-of-the-international-health-regulations-( )-emergency-committee-regarding-the-outbreak-of-novel-coronavirus-( -ncov) epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study novel coronavirus ( -ncov) early-stage importation risk to europe from sars to covid- : a previously unknown sars-related coronavirus (sars-cov- ) of pandemic potential infecting humans-call for a one health approach feasibility of controlling covid- outbreaks by isolation of cases and contacts secretary azar declares public health emergency for united states for novel coronavirus transmission of -ncov infection from an asymptomatic contact in germany -ncov (wuhan virus), a novel coronavirus: human-to-human transmission, travel-related cases, and vaccine readiness first known person-to-person transmission of severe acute respiratory syndrome coronavirus (sars-cov- ) in the usa isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus ( -ncov) outbreak public health screening to begin at u.s. airports for novel coronavirus us department of health and human services, cdc; laboratory readiness and response for novel coronavirus ( -ncov) in expert laboratories in eu/eea countries the response of milan's emergency medical system to the covid- outbreak in italy critical case utilization for the covid- outbreak in lombardy differential diagnosis of illness in patients under investigation for the novel coronavirus (sars-cov- ) covid- and italy: what next? geographical tracking and mapping of coronavirus disease covid- /severe acute respiratory syndrome coronavirus (sars-cov- ) epidemic and associated events around the world: how st century gis technologies are supporting the global fight against outbreaks and epidemics at the epicenter of the covid- pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation position paper: relazione circa l'effetto dell'inquinamento da particolato atmosferico e la diffusione di virus nella popolazione world health organization. coronavirus disease (covid- ) advice for the public: myth busters european centre for disease prevention and control. geographical distribution of -ncov cases globally. european centre for disease prevention and control innovations in the plastic surgery care pathway: using telemedicine for clinical efficiency and patient satisfaction key: cord- -k zq zc authors: micheli, v.; rimoldi, s. g.; romeri, f.; comandatore, f.; mancon, a.; gigantiello, a.; brilli, m.; mileto, d.; pagani, c.; lombardi, a.; gismondo, m. r.; laboratory of clinical microbiology, virology and diagnostic of bioemergencies group title: geographic reconstruction of the sars-cov- outbreak in lombardy (italy) during the early phase date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: k zq zc the circulation of sars-cov- in italy has been dominated by two large clusters of outbreaks in northern part of the peninsula, source of alarming and prolonged infections in lombardy region, in codogno and bergamo areas especially. the aim of the study was to expand understanding on the circulation of sars-cov- in the affected lombardy areas. to this purpose, twenty full length genomes were collected from patients addressing to several lombard hospitals from february th to april th, . the obtained genome assemblies, available on the gisad database and performed at the referral center for covid- diagnosis, identified main monophyletic clades, containing and isolates, respectively. the molecular clock analysis estimated a clusters divergence approximately one month before the first patient identification, supporting the hypothesis that different sars-cov- strains spread all over the world at different time, but their presence became evident only in late february along with italian epidemic emergence. therefore, the epidemiological reconstruction carried out by this work highlights multiple inputs of the virus into its initial circulation in lombardy region. however, a phylogenetic reconstruction robustness will be improved when other genomic sequences will be available, in order to guarantee a complete epidemiological surveillance. the positive-strand rna virus family coronaviridae includes subfamilies for a total of genera and more than species, which infect a broad spectrum of vertebrates; in particular, seven . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . coronaviruses are related to human diseases, among which two represented a public health concern in the past years: in november , the severe acute respiratory syndrome related coronavirus (sars-cov) emerged in guangdong, china, resulting in more than , confirmed cases and deaths in countries; similarly, starting from in saudi arabia, the middle east respiratory syndrome related coronavirus (mers-cov) infected , individuals and caused fatalities. , the covid- related virus was classified as a β -betacoronavirus and, considering its close correlation to sars-cov, it was renamed sars-cov- . in europe, italy is one of the most affected areas, accounting for more than , cases on june th , . northern italy has reported the highest prevalence in the country, especially in milano, brescia and bergamo provinces, which registered more than , , , and , cases , the aim of the present study was to assess the potential presence of different viral clusters belonging to the six main provinces involved in lombardy covid- cases in order to highlight peculiar province-dependent viral characteristics. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the study included sars-cov is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . for each sample, the genome assembly was obtained using a mapping-based approach, as follows. low quality reads bases were trimmed out using trimmomatic software, is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the basis of the identified snps and the reference sequence. reference bases were called in conserved positions with coverage above five, otherwise n were introduced. the procedure produced thirteen genome assemblies per sample, corresponding to the thirteen different trimming parameter sets. for each sample, the genome assembly with the lowest number of undetermined bases (n) as the final genome assembly was selected. a dataset of genome assemblies of sars-cov- strains isolated in italy between february and march were retrieved from gisaid database , (see supplementary table for details). a global dataset including these gisaid (table ) genome assemblies and the genome assemblies produced in this study was produced and aligned using mafft. the low quality alignment regions at the extremities of the alignment were removed using gblocks with default parameters. the obtained alignment was subjected to maximum likelihood phylogenetic analysis using raxml. the obtained tree was then analysed using tempest then the phylogenetic analysis was repeated with the selected coalescent priori with million states and sampling every , steps. the convergence of mcmc chain was checked using tracer v. . . . the maximum clade credibility (mcc) trees were obtained from the tree posterior distribution using treeannotator (http://beast.community/index.html). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . a total of samples from different subjects were successfully sequenced and included in phylogenetic analysis, attributing the progressive id hsacco-n (from hsacco- to hsacco- ). all patients were resident in lombardy region, distributed in different provinces, as reported in table x: in particular, the category 'milano' contains also patients hospitalized for non-covid- disease, for whom sars-cov- hospital acquisition was supposed; in addition, hsacco- , in 'other' category, was a nurse living in bergamo and working at lodi hospital. the obtained sars-cov- genome assemblies are available on the gisad database (see table for details). the comparison of the marginal likelihoods of constant and exponential coalescent models under a log-normal relaxed clock showed that the best fitting model was the exponential coalescent prior (ps bf exponential growth vs. constant = , ; ss bf exponential growth vs. constant = - , ). the obtained phylogenetic tree is reported in figure . the tree shows the existence of two major monophyletic clades, containing and isolates respectively (coloured in green and blue, respectively, in figure ). the smaller clade is characterized by a non-synonymous mutation in position three of the gene m (mutation d g) and its time of the most recent common ancestor (trmca) resulted th february (hpd % interval from th january to th february); the bergamo and hsacco- sequences mapped inside . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . this group. the larger clade contains two monophyletic sub-clades, characterized by nonsynonymous mutations in the n gene ( figure ). the first cluster includes isolates, mainly from central italy, in which r k and g r variants were found; the other one, instead, accounted for three sequences from friuli venezia giulia region, presenting the v i substitution. the tmrca calculated for the two sub-clades were nd march (hpd % interval from february th to march th ) and th february (hpd % interval from february th to march nd ), respectively ( table ) . the lombardy region has the highest prevalence of covid- in italy, thus being the likely epicentre of country outbreak. however, it is still unclear how sars-cov- circulation started: no epidemiological link was found for the first identified autochthonous patient. moreover, despite the diagnosis was made on february , emerging evidences suggest a multiple virus introduction at least in january . , , the present work tried to expand understanding on the circulation of sars-cov- during the early epidemic period in lombardy, especially in most affected areas. a phylogenetic analysis was conducted on full length genomes collected from patients addressing to several lombard hospitals from february to april , , creating a dataset including italian viral genome assemblies available on gisaid database as of th march, . in spite of restricted geographical origin, these sequences were not included in the same cluster, but two main monophyletic clades containing isolates collected across different regions were found; in addition, the main clade accounting for / assemblies was divided in two subclades. interestingly, the molecular clock analysis estimated a clusters divergence approximately one month before the first patient identification. such evidences are in accordance with other studies, further supporting the . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint hypothesis that different sars-cov- strains spread all over the world at different time, but their presence became evident only in late february along with italian epidemic emergence. , , noteworthy, bergamo (hsacco- , hsacco- , hsacco- ) and hsacco- sequences had the m gene d g mutation of the first clade, in contrast with all the other ones located in the most represented clade. two main observations consequently arose: firstly, the virus found in bergamo area seemed to had a more restricted circulation, probably for a delayed introduction. in addition, the nurse working in lodi and living in bergamo likely acquired the infection in a personal contest rather than hospital environment, since lodi patients mapped within the other cluster. another interesting outcome was the presence of the n gene mutations r k and g r subclade. these two aminoacid changes appear to coexist, since they were always found together into the dataset, as well as they both are present almost exclusively in the gisaid b clade. these mutations have an impact on structure and function of the mutated n protein. the phylogenetic tree also clearly showed that the subclade is predominantly made up of isolates from abruzzo region, suggesting a segregation of this specific virus in central italy area. given that the role of these mutations is still unclear, an indian group investigated their possible influence on virus replication interference mediated by mirna: they found out how some mirna, present in different pathological conditions, are likely to bind to native n gene and repress its expression, thus helping in disease progression limitation; on the contrary, mutated variants could increase their chances of interference escape. another small subclade was found in the main one, characterized by the presence of n gene mutation v i in three sequences, all from friuli venezia giulia region. besides the unique geographical origin, it is noticeable that in gisaid map 'geography' the v i mutation is actually present only in italy, as well as the v a one only in israel. their rarity could have two probable explanations: on the one hand, available data are still limited, making difficult to have a reliable distribution; on the other hand, these variations could have a negative influence on viral fitness, diminishing efficacy in replication and consequently virus transmission. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint world health organization a pneumonia outbreak associated with a new coronavirus of probable bat origin world health organization; who director-general's remarks at the media briefing on -ncov on world health organization; who director-general's opening remarks at the media briefing on covid- - international committee on taxonomy of viruses; virus taxonomy genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding sars and mers: recent insights into emerging coronaviruses coronaviridae study group of the international committee on taxonomy of viruses; the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- dipartimento della protezione civile; covid- italia -monitoraggio della situazione trimmomatic: a flexible trimmer for illumina sequence data a framework for variation discovery and genotyping using next-generation dna sequencing data disease and diplomacy: gisaid's innovative contribution to global health - ) parallelization of mafft for large-scale multiple sequence alignments selection of conserved blocks from multiple alignments for their use in phylogenetic analysis raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies exploring the temporal structure of heterochronous sequences using tempest jmodeltest : more models, new heuristics and parallel computing bayesian phylogenetic and phylodynamic data integration using beast . virus evolution posterior summarisation in bayesian phylogenetics using tracer . . systematic biology. syy a doubt of multiple introduction of sars-cov- in italy: a preliminary overview genomic characterization and phylogenetic analysis of sars-cov- in italy emergence of genomic diversity and recurrent mutations in sars-cov- mutations in sars-cov- viral rna identified in eastern india: possible implications for the ongoing outbreak in india and impact on viral structure and host susceptibility decoding sars-cov- transmission and evolution and ramifications for covid- diagnosis insights on early mutational events in sarscov- virus reveal founder effects across geographical regions other mutations found in the present work had negligible influence on phylogenetic analysis, even a biological significance can not be excluded: viral genome and proteins are key factors in patients management and any variation can extremely burden the efficacy of drugs, vaccines and diagnostic tools or be related to a more severe clinical presentation. , in conclusion, this study gave insights on early dynamics of sars-cov- circulation in italy, underling peculiar strains localization and supporting multiple virus introductions at least in january . key: cord- - p ez i authors: boriani, giuseppe; palmisano, pietro; guerra, federico; bertini, matteo; zanotto, gabriele; lavalle, carlo; notarstefano, pasquale; accogli, michele; bisignani, giovanni; forleo, giovanni battista; landolina, maurizio; d’onofrio, antonio; ricci, renato; de ponti, roberto title: impact of covid- pandemic on the clinical activities related to arrhythmias and electrophysiology in italy: results of a survey promoted by aiac (italian association of arrhythmology and cardiac pacing) date: - - journal: intern emerg med doi: . /s - - -w sha: doc_id: cord_uid: p ez i covid- outbreak had a major impact on the organization of care in italy, and a survey to evaluate provision of for arrhythmia during covid- outbreak (march–april ) was launched. a total of physicians from italian arrhythmia centres took part in the survey. the vast majority of participating centres ( . %) reported a significant reduction in the number of elective pacemaker implantations during the outbreak period compared to the corresponding two months of year ( . % of centres reported a reduction of > %). similarly, . % of participating centres reported a significant reduction in the number of implantable cardioverter-defibrillator (icd) implantations for primary prevention, and . % a significant reduction of icd implantations for secondary prevention (> % in . and . % of the centres, respectively). the majority of participating centres ( . %) reported a significant reduction in the number of elective ablations (> % in . % of the centres). also the interventional procedures performed in an emergency setting, as well as acute management of atrial fibrillation had a marked reduction, thus leading to the conclusion that the impact of covid- was disrupting the entire organization of health care, with a massive impact on the activities and procedures related to arrhythmia management in italy. electronic supplementary material: the online version of this article ( . /s - - -w) contains supplementary material, which is available to authorized users. the world health organization declared covid- a pandemic on march , ( ) and italy was the first european country that had to take urgent decisions for limiting the transmission in the population of sars-cov- [ ] . on march th, italy became the second most affected country in the world after china, and specific rules for restricting social contacts in the whole country were applied by the italian government in march [ ] . at the end of june the total number of subjects found affected by in italy was reported to be around with more than , deaths [ ] . the covid- outbreak had a devastating and massive impact on the organization of social activities, as well as a disruptive impact on the organization of care in italy, with a dramatic reduction in traditional contacts for ensuring care to non covid- diseases [ ] . as a matter of fact, hospital admissions for acute myocardial infarction were significantly reduced during the early phase of covid- pandemic across italy, with a parallel increase in fatality and complication rates [ ] . moreover, a % increase in the occurrence of out of hospital cardiac arrests was documented in some provinces from lombardy in the first months of the the list of investigators participating to aiac ricerca network is reported in the acknowledgements section. the online version of this article (https ://doi.org/ . /s - - -w) contains supplementary material, which is available to authorized users. pandemic, and this increase was associated with worse in hospital outcomes [ ] . within this complex scenario, corresponding to a profound re-arrangement of health care system organization in italy, no data are available on the different aspects of care in the field of arrhythmia and electrophysiology, either with regard to the period of lockdown or with regard to the so called "phase " (post-covid- recovery phase) that started on may , targeted to a re-organization of all the activities, including health care, after the period of massive emergency. the italian association of arrhythmology and cardiac pacing (aiac) launched a survey among its members in order to report the situation of cardiac care for arrhythmia in these particular phases. from april to may , a survey endorsed by the aiac was published on the official aiac website (https :// aiac.it/). the survey was open to physicians operating in all italian centres involved in arrhythmia care. participation in the survey was voluntary. the questionnaire could be completed by more than one physician from the same centre. the questionnaire consisted of questions: five of them focused on the characteristics of the participating centre (i.e. involvement of the centres and of the physicians in the management of suspected and confirmed patients with covid- , volume of annual cieds implantations and ablation procedures); seven of them focused on the impact of covid- pandemic on the number of cied implantations and ablation procedures performed in both elective and emergency settings, and on the number of cases of acute pharmacological and non-pharmacological treatment of atrial fibrillation (af) in emergency setting; two of them focused on the impact of covid- pandemic on the management of remote monitoring (rm) of cieds; the remaining four were focused on the possible organizational strategies for post-covid- recovery phase. seventeen of the questions were multiple-choice questions (see online supplementary material for details). descriptive statistics were reported as means for normally distributed continuous variables. continuous variables with skewed distribution were reported as medians with - th percentiles. categorical data were expressed as percentages, reported in contingency tables, and compared by means of χ test or fisher's exact test, as appropriate. p values < . were considered statistically significant. a total of physicians from italian arrhythmia centres took part in the survey. for centres, more than one physician responded to the survey (mean; ; range - ). a complete list of participating centres is reported in appendix. the centres which participated in the survey accounted for . % of all arrhythmia centres operating in italy in [ ] . the participating centres displayed a wide geographical distribution (fig. b) : a mean of three centres per region (range: - ; interquartile range: - ) responded. in six regions there were five or more participating centres. the response rate was similar in northern, central and southern italy ( . , . , and . % of all operating centres, respectively, p = . ). after dividing the italian regions into four groups, according to incidence of covid- cases (confirmed cases < . , from . to . , from . to . , and > . per populations, fig. a ) ( ), the response rate was similar in the regions with higher incidence of covid- cases (confirmed cases > per population, n = ) compared to other regions (n = ) ( . vs. . %; p = . ). many participating centres ( . %) had three operators, . % had only one operator, and . % > operators (fig. c) . fifty-nine of participating centres ( . %) were located in hospitals designated to treat patients with covid- . of these, ( . %) reported that during covid- pandemic at least one operator (median: ; range: - ) was directly involved in the management of patients with covid- . in these centres a mean of . % of operators was involved in assistance to patients with covid- and in centres ( % of those involved in the care of patients with covid- ), all the operators of the electrophysiology team were involved in assistance to patients with covid- . the majority of participating centres ( . %) had implanted from to cieds during ; . % had implanted from to cieds, and the remaining . % < or > (fig. d) . in . % of centres, < ablation procedures had been performed during ; . % had been performed from to ablation procedures; . % had been performed > ablation procedures (fig. e ). procedures performed in elective setting the vast majority of participating centres ( . %) reported a significant reduction in the number of elective pacemaker (pm) implantations procedures during the two months march-april compared to the corresponding two months (march-april) of year . specifically, . % of centres reported a reduction of > %. only . % of centres reported no significant variations (fig. a) . similarly, . % of participating centres reported a significant reduction in the number of implantable cardioverter-defibrillator (icd) implantations for primary prevention in the same period. the majority of these ( . %) reported a reduction > %. only . % of centres reported no significant variations (fig. b) . covid- pandemic seemed to have an impact also on the number of icd implantations for secondary prevention; in fact, . % of centres reported a significant reduction (of > % in . % of centres), while . % reported no significant variations (p < . compared to icd implantations for primary prevention, fig. b ). no significant difference was found in the answers between the centres located in regions with higher incidence of covid- cases and the other ones ( figure s , panel a-c). the majority of participating centres ( . %) reported a significant reduction in the number of elective ablations performed during the two months march-april compared to the months march-april (reduction of > % in . % of the centres); . % reported no significant variations (fig. c) . the impact of the pandemic on the number of elective ablations performed was greater in the regions with higher incidence of covid- cases where there was a significantly higher rate of the centres that reported a reduction in the number of procedures of > % ( . vs. . %; p = . ), and a significantly lower rate of the centres that reported no significant variations ( . vs. . %; p = . ) compared to other centres ( figure s , panel d). ablation procedures volume of participating centres (e). *data from italian civil protection department [ ] . cied cardiac implantable electronic device during covid- pandemic, the participating centres globally reported a mean reduction in the number of elective pm implantations, icd implantations for primary prevention, icd implantations for secondary preventions, and elective ablations of . , . , . , and . %, respectively. procedures performed in emergency setting the majority of participating centres ( . %) reported a significant reduction in the number of cied implantation procedures performed in emergency setting (including temporary and definitive pm implantations for severe, life-threatening bradyarrhythmias, and icd implantations for secondary prevention) during covid- pandemic compared to the same period of the previous year; . % of centres reported no significant variations; . % reported a significant increase (of - % in most cases, fig. d ). about half of the participating centres ( . %) reported a significant reduction in the number of ablation procedures performed in emergency setting (including urgent ablation of electrical storm, or of refractory ventricular or supraventricular tachycardias) during covid- pandemic compared to the same period of the previous year (of > % in . % of the centres); . % reported no significant variations; only . % reported a significant increase (fig. e) . no significant difference was found in the answers between the centres located in regions with higher incidence of covid- cases and the other ones ( figure s , panel a and b). the majority of participating centres ( . %) reported a significant reduction of cases of acute pharmacological and non-pharmacological treatment of af in emergency setting (including pharmacological rate or rhythm control, and urgent electrical cardioversion); . % reported no significant variations; only . % reported a significant increase (fig. f) . in the regions with higher incidence of covid- cases a significantly higher rate of centres reported a reduction of > % in the number of cases of acute af treatment in emergency setting compared to other regions ( . vs. . %; p < . ; figure s , panel c). during covid- pandemic, the participating centres globally reported a mean reduction in the number of urgent cieds implantations, urgent ablations, and in the number of cases requiring acute treatment of af in emergency setting of . , . , and . %, respectively. based on the reported procedure volumes, we estimated that, during the two months march-april in the centres that participated in the survey, globally about fewer cieds had been implanted and about fewer ablations had been performed (in both elective and emergency settings) compared to the same period of the previous year. remote monitoring of cieds eighty-one of participating centres ( . %) used remote monitoring (rm) for the follow-up of patients with cieds. almost half of these centres ( . %) reported no significant variations in the number of patients followed by rm during the two months that we analysed (march-april ), while . % reported a significant increase; . % declared to offer rm to all available cied patients (fig. a) . about half of the centres ( . %) indicated that during covid- pandemic performed inoffice evaluation of cied patients followed by rm only in case of alerts triggered by device/lead malfunction or by clinical events; . % performed in-office evaluation only in case of alerts related to device/lead malfunction; finally, . % declared that during the pandemic no in-office evaluation was performed (fig. b the following results refer to the whole group of physicians who responded to the questionnaire. the majority of the interviewed physicians ( . %) considered, as main strategy for the post-covid- recovery phase, the adoption of new organizational structures for patient admission in order to minimize the risk of infection. besides, . % of respondents considered as main strategy the implementation of short-stay hospitalization for patients undergoing elective procedures (i.e. day-case admission or ordinary admission with a single night stay). finally, . % of respondents considered as the main challenge for post-covid- phase to overcome the distrust of patients to go to the hospital. for the majority of the interviewed physicians ( . %) the procedures that could be performed under day-case admission were cieds replacements, followed by supraventricular tachycardias (svts) ablations ( . %) and by elective pm implantations ( . %, fig. a) . instead, the procedures, that could be performed under ordinary admission with a single night stay, were elective pm implantations for . % of respondents, elective icd implantations for . %, svts ablations for . %, and cieds replacements for . % (fig. b) . concerning the time needed to return to pre-covid procedure volumes, about a third of respondents ( . %) thought that it will take at least months; . % that it . cied cardiac implantable electronic device, icd implantable cardioverter-defibrillator, pm pacemaker, svt supraventricular tachycardia will take from to years; . % thought that the pre-covid- procedure volumes will be achieved within months. the present survey highlights that the outbreak of covid- pandemic had a disrupting impact on health care organization that profoundly affected the organization of care in the hospitals and the cardiology divisions of many areas, specifically in northern italy, with an important impact on the activities of the teams involved in management of arrhythmias and electrophysiology. indeed, around % of centres that participated to this survey were located in hospitals directly involved in treatment of patients with covid- and around % reported that during the pandemic at least one physician of the arrhythmia team was directly involved in the management of patients with covid- . the extraordinary consequences of the pandemic are even more evident by considering that in % of the centres involved in management of covid- all the operators of the electrophysiology team were involved in tackling the emergency situation. the elective procedures related to device implants had a very important reduction in march-april as compared to the same months of the previous year, with the majority, or even the large majority of centres reporting a greater than % reduction in the number of elective pms or of icds implanted in primary prevention of sudden cardiac death. the indication to limit hospital admissions to emergencies or, anyway, to non-deferrable procedures, combined with the fear of patients to be infected in the hospital are all factors that can explain this phenomenon whose impact on future events is unpredictable. the reduction in icd implants for secondary prevention was less impressive, but these data should be interpreted in a larger perspective, taking into account the increase in out of hospital cardiac arrests observed during the covid- outbreak [ ] . it is noteworthy that no significant difference was found in the analysis on device implants between the centres located in regions with higher incidence of covid- cases and the other ones, suggesting that the impact of the pandemic on patient behaviours and organization of care was, in general, independent on the peaks of covid- epidemiological pressure. it is unknown whether the reduction in elective implants for prophylactic icds will imply in the future a relative increase in malignant ventricular tachyarrhythmias or cardiac arrests, leading to a rebound increase in icds implanted for secondary prevention. ablation of af is currently one of the main activities of italian electrophysiology centres, and is performed with different approaches and techniques both in patients with no underlying heart disease and in selected patients with heart failure [ ] [ ] [ ] [ ] [ ] . in the present survey around % of the centres reported a significant reduction in the number of elective ablations performed in march-april compared to the previous years, but with some differences related to areas with higher incidence of covid- cases. the elective nature of af ablation procedures and the re-organization of care related to covid- , that obliged many centres to cancel elective procedures may explain the heterogeneity of this finding. also the electrophysiological procedures and the interventions performed in an emergency setting were markedly reduced during the observation period. in interpreting these findings, it should be considered that the covid- outbreak markedly changed the pattern of emergency departments (eds) referral in italy, with figures up to a % reduction accesses to hospitals and eds unrelated to covid- [ , ] . a reduction up to % in urgent pacemaker implants for severe bradyarrhythmias was previously reported, in agreement with our national survey, by analysis performed on a single hospital basis or on a regional basis [ , ] . the reduction in urgent pacemaker implants may imply a lack of prevention for the potential harmful consequences of bradyarrhythmias and, indeed, a relative increase in the proportion of patients presenting with syncope due to bradyarrhythmias was already observed [ ] . it is possible that this trend will increase also in the post-lockdown phase and it will be interesting to analyse if it will lead to a rebound in pacemaker implants. in recent years rm of implanted devices has been implemented in clinical practice in a substantial proportion of italian centres, despite the problems linked to lack of reimbursement or lack of official general plans for large-scale implementation [ ] [ ] [ ] [ ] . as compared to patient monitoring with external devices, the use of remote monitoring with implanted devices offers the advantage of an easy implementation, simply requiring patient and caregiver's education coupled with availability of dedicated transmitters. therefore, covid- offered a great chance to enhance implementation of rm among patients with implanted devices [ ] , although with a variable extent from centre to centre. as a matter of fact, more than % of the centres participating to this survey reported some increase in the use of rm for the follow-up of their patients. however, the extent of rm implementation as a consequence of the limitations of covid- lockdown actually ranged from an increase in the range of - % of assisted patients to more than % or even (in around % of centres) to a complete shift to a strategy based on offering rm to all available cied patients. although it is clear that the pressure of the limitations due to covid- lockdown offered a great opportunity for a larger implementation of rm, overcoming a series of bureaucratic and administrative barriers, it emerges a substantial heterogeneity in the extent of implementation of rm, that should be object of future re-assessments. the very drastic limitations linked to the period of massive pressure of civid- are highlighted by the relatively important proportion of centres (one in five) reporting that during the pandemic no in-office evaluations were performed. currently remote programming of implanted devices is not allowed, in view of safety concerns, so it remains crucial the adoption of specific recommendations for device programming according to patient profile, thus minimizing troubleshooting during follow up [ , ] . in the specific context of covid- lockdown the potential advantages of rm should not be limited to device checks. as known, rm can be used with the purpose of remote device check or for monitoring patients' status (heart rhythm, fluid overload, right ventricular pressure, oximetry, etc.), thus with a shift from strictly device-centred follow-up to perspectives centred on the patient (and patient-device interactions) [ ] [ ] [ ] [ ] [ ] [ ] . the organization of disease management of heart failure, though rm in patients with implanted devices is complex, requires an interplay between the competence on devices and heart failure management and, therefore, should be object of promotion for the post-covid- recovery phase. the assessment of quality of care delivered through rm [ ] , with appropriate involvement of the patients and the caregivers [ ] , will become of primary importance for outcome improvement. anyway, as stressed in official documents of the major international associations in the field of arrhythmia management, the crisis precipitated by the pandemic has surely catalysed the adoption of rm across many specialties and heart rhythm professionals are in the front line for full adoption of this technological and clinical advancement even beyond the emergency of covid- pandemic, making rm as the true standard of care in this field [ ] . af is a very common arrhythmia and its acute management carries a high burden of workload to eds and cardiology clinics [ ] . in view of its epidemiological profile, af affects subjects in the range of age at highest risk of adverse outcomes if infected by sars-cov- [ ] and the caution in avoiding admissions to hospital may explain the important reduction in acute pharmacological and non-pharmacological treatments applied for af in emergency setting reported during the study period, as reported in this survey. since appropriate prescription of oral anticoagulants in patients at risk of stroke is a major determinant of outcome at long term [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it will be necessary in the near future to establish even stricter connections between hospital and out of hospital care, for a re-assessment of patients who presented af and these months with regard to clinical evaluation and appropriateness of treatment for ensuring continuity of care. it will also be interesting to assess to what extent untreated or undiscovered af occurred during the lockdown will result in major consequences, such as syncope, heart failure, stroke/systemic embolism [ ] [ ] [ ] . it is surprising that the reduction in activities performed by arrhythmia services during march-april also involved ablations performed in emergency setting (including urgent ablation of electrical storm, or of refractory vt or svt), that require high competence and usually cannot be deferred [ ] . the patients' tendency to avoid hospitalization that characterized the peak phase of covid- pandemic could have resulted in an increased amount of cardiovascular deaths occurred at home, but this of difficult to assess now. the implications of the gap of care that the reduction in emergency ablations and electrophysiological interventions implied will require further assessments in the future and should suggest a reorganization of care, with networks able to guarantee these procedures, following an appropriate referral, even in case of national emergencies. one of the key questions after the outbreak of covid- is how to re-organize care in the post-covid- recovery phase and our survey indicated that according to italian physicians in the field. according to our survey there is absolute need of adopting new organizational models for patient admission in order to minimize the risk of infection. a short-stay hospitalization for patients undergoing elective procedures (i.e. day-case admission or ordinary admission with a single night stay) appears to be a suitable strategy, although up to now it was adopted with substantial heterogeneity, according to administrative reasons and reimbursement policies [ ] . according to the majority of respondents, not only device replacements but also ablations for svts and elective pm implants could be performed with a short hospital stay, with the advantage to improve efficiency of the system. this perspective will require an increased compliance with prospective registries on electrophysiological procedures [ , [ ] [ ] [ ] [ ] , with our scientific association providing specific reports on complication rates and outcomes associated with the different procedures programmed in the field of interventional electrophysiology. this will also be the basis for working with policymakers and regulators for planning audits targeted to verify the quality of care in a virtuous circle where daily practice provides a continuous feedback on health care system performance [ ] . this will be the appropriate response to the challenging battle against covid- and will allow to improve the performance of our health care system, with the premise for achieving full confidence of the citizens on the overall appropriateness and safety of our care processes. our survey has some limitations since it was not based on a precise computation of activities and procedure in every specific centre; however, this is a method that allows a rapid feedback and was chosen for having a general view of covid- pandemic in italy at a short time from its onset. only out of arrhythmia centres operating in italy took part in the survey ( . % of the italian centres). for this reason, our findings should be interpreted with caution, as they may not accurately reflect the impact of covid- pandemic on the activities of all italian arrhythmia centres. seventeen of the questions of the questionnaire were multiple-choice questions. this type of questionnaire is time-efficient, and responses are easy to code and interpret. on the other hand, the surveys based on multiple-choice questions have some limitations. respondents are required to choose a response that does not exactly reflect their answer. in addition, the arbitrary design of questionnaires and multiple-choice questions with pre-conceived categories represents a biased and overly simple view of reality. the impact of covid- was disrupting on the entire organization of health care, particularly for hospital care, and had a massive impact on the activities related to arrhythmia management and electrophysiology occurred in italy in march-april . our survey focused on real-life activities in this field showed that in hospitals with wards specifically dedicated to care of patients with covid- physicians usually involved in the field of arrhythmias and electrophysiology were frequently moved to take care of patients infected by sars-cov- . in this period a reduction of > % in the number of implants of cardiac electronic devices was reported, and involved pacemakers and icds, with an important reduction not only on icd implants for primary prevention of sudden death, but also on icd implants for secondary prevention. the number of ablation procedures was markedly reduced and the reduction also affected emergency procedures, especially for centres directly involved in the care of covid- . in this context, a wider use of rm among patients with implanted devices was achieved, although with a variable extent from centre to centre. it is clear that for the post-covid- recovery phase there is absolute need for adopting new organizational models for patient admission in order to minimize the risk of infection, and a short-stay hospitalization for patients undergoing elective procedures (i.e. day-case admission or ordinary admission with a single night stay) appears to be a suitable strategy. an increased compliance with prospective registries on electrophysiological procedures will allow a continuous monitoring of the type and number of interventions needed in this new phase, with potential differences with regard to historical series, and will also allow a check of centres' performances in specific procedures, with an enormous potential for quality improvement. conceptualization, gb and rpr.; methodology, gb and pp; software, pp; validation, gb, rpr and gb; formal analysis, pp; investigation, gb; resources, gb; data curation, gb and pp; writing-original draft preparation, gb, pp; writingreview and editing and visualization, fg, mb, gz, cl, pn, ma, gb, gbf, ml, ad, rpr and rdp. altamura (bari); sardegna: nissardi v, clinica cardiologica-utic -policlinico "d. casula università degli studi di firenze e aou careggi, firenze; giaccardi m, uos di cardiologia ed elettrofisiologia firenze azienda sanitaria firenze approaches to daily monitoring 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management of atrial fibrillation in the emergency room and in the cardiology ward: the blitz af study the epidemiological burden of atrial fibrillation: a challenge for clinicians and health care systems direct oral anticoagulants vs non-vitamin k antagonist in atrial fibrillation: a prospective, propensity score adjusted cohort study changes to oral anticoagulant therapy and risk of death over a -year followup of a contemporary cohort of european patients with atrial fibrillation final report of the eurobservational research programme on atrial fibrillation (eorp-af) pilot general registry increased burden of comorbidities and risk of cardiovascular death in atrial fibrillation patients in europe over ten years: a comparison between eorp-af pilot and ehs-af registries atrial fibrillation and prediction of mortality by conventional clinical score systems according to the setting of care the euro heart survey and eurobservational research programme (eorp) in atrial fibrillation registries: contribution to epidemiology, clinical management and therapy of atrial fibrillation patients over the last years long-term relationship between atrial fibrillation, multimorbidity and oral anticoagulant drug use association between antithrombotic treatment and outcomes at -year follow-up in patients with atrial fibrillation: the eorp-af general long-term registry syncope in the german nationwide inpatient sample-syncope in atrial fibrillation/flutter is related to pulmonary embolism and is accompanied by higher in-hospital mortality effects of remote monitoring of cardiac implantable electronic devices after stroke or transient ischemic attack occurrence of atrial fibrillation in pacemaker patients and its association with sleep apnea and heart rate variability nationwide survey on the current practice of ventricular tachycardia ablation clinical and organizational management of cardiac implantable electronic device replacements: an italian survey promoted by aiac (italian association of arrhythmology and cardiac pacing) the pacemaker and implantable cardioverter-defibrillator registry of the italian association of arrhythmology and cardiac pacing-annual favorable trend of implantable cardioverter-defibrillator service life in a large single-nation population: insights from -year analysis of the italian implantable cardioverter-defibrillator registry radiotherapy-induced malfunctions of cardiac implantable electronic devices in cancer patients influence of frailty on anticoagulant prescription and clinical outcomes after -year follow-up in hospitalised older patients with atrial fibrillation health technology assessment in interventional electrophysiology and device therapy: a position paper of the european heart rhythm association acknowledgements no funding was received for this work.we acknowledge the aiac ricerca investigators who participated in this survey: abruzzo: luise r, ospedale san salvatore, l'aquila; basilicata: grieco p, osp. san carlo, potenza; calabria: pangallo a, grande ospedale metropolitano di reggio calabria, reggio calabria; conflict of interest gb: small speaker fee from medtronic, boston, biotronik, boehringer ingelheim and bayer. pp: small speaker fee from bayer, boehringer ingelheim, bristol-myers squibb, medtronic. novartis and personal fees from proctorship agreement with abbott, medtronic, biotronik, and boston scientific. mb: small speaker fee from boston scientific, abbott, boehringer ingelheim and bayer. ml: modest speaker fee from bayer, boehringer ingelheim, boston scientific, livanova, medtronic, and pfizer. rpr: small speaker fees by boston e biotronik; advisory board modest fees by dompé. rdp: lecture fees from biosense webster and biotronik. no conflict of interest reported by the other authors. the study procedures were in accordance with the ethical standards of the latest helsinki declaration.informed consent all the participants provided written consent for their participation in this survey at the time of online data collection. due to the anonymous and voluntary nature of the study, no identifying information about participants was mandatory for inclusion. key: cord- - bwlxlfo authors: ciufolini, ignazio; paolozzi, antonio title: mathematical prediction of the time evolution of the covid- pandemic in italy by a gauss error function and monte carlo simulations date: - - journal: eur phys j plus doi: . /epjp/s - - -y sha: doc_id: cord_uid: bwlxlfo in this paper are presented mathematical predictions on the evolution in time of the number of positive cases in italy of the covid- pandemic based on official data and on the use of a function of the type of a gauss error function, with four parameters, as a cumulative distribution function. we have analyzed the available data for china and italy. the evolution in time of the number of cumulative diagnosed positive cases of covid- in china very well approximates a distribution of the type of the error function, that is, the integral of a normal, gaussian distribution. we have then used such a function to study the potential evolution in time of the number of positive cases in italy by performing a number of fits of the official data so far available. we then found a statistical prediction for the day in which the peak of the number of daily positive cases in italy occurs, corresponding to the flex of the fit, that is, to the change in sign of its second derivative (i.e., the change from acceleration to deceleration), as well as of the day in which a substantial attenuation of such number of daily cases is reached. we have also analyzed the predictions of the cumulative number of fatalities in both china and italy, obtaining consistent results. we have then performed monte carlo simulations to have a more robust prediction of the day of the above-mentioned peak and of the day of the substantial decrease in the number of daily positive cases and fatalities. although official data have been used, those predictions are obtained with a heuristic approach since they are based on a statistical approach and do not take into account either a number of relevant issues (such as number of daily nasopharyngeal swabs, medical, social distancing, virological and epidemiological) or models of contamination diffusion. by considering the cumulative diagnosed positive cases of covid- infections and fatalities available in the web site of the italian "ministero della salute" [ ], worldometer [ ] and world health organization [ ], we found that they can be well approximated by a cumulative distribution function (cdf) with four parameters of the type of the gauss error function, that is, the integral of a normal, gaussian distribution (see sect. ). by positive cases, we mean the positive cases actually diagnosed plus, for future days, the positive cases that we expect a e-mail: ignazio.ciufolini@gmail.com (corresponding author) to be diagnosed. indeed, it is well known among the virologists that the actual number of positive cases is much higher than the diagnosed ones [ ] . however, it is assumed that the diagnosed cases are a good statistical representation of the entire population of the positive cases. in fig. , we report the result of our fit of the cumulative diagnosed positive cases in china. we have then applied such a cdf to study the evolution in time of the number of positive cases in italy, in the attempt to possibly, statistically, predict the peak in the number of daily positive cases and the possible date of a substantial decrease in the number of daily positive cases. furthermore, we have also applied our method to the cdf of the number of fatalities in both china and italy, confirming our predictions obtained with the cumulative positive cases. finally, in sect. , we have performed a number of monte carlo simulations to get a more robust prediction of both the date of the peak in the number of positive cases, diagnosed each day, and in the date after which the number of new positive cases will be below a certain threshold [ ] . based on the number of diagnosed positive cases and fatalities of covid- in china, we have fitted the cumulative numbers with a function of the type of the gauss error function containing the four parameters a, b, c, d, that we have fitted using the available official data. a similar distribution is also observed in other studies of seasonal influenza and pandemic [ , ] . the result of the fit is reported in fig. , which shows the good level of the fit using function ( ) with those four parameters. in fig. , we report the fit of the cumulative number of fatalities due to covid- in china, which also shows the good level of the fit using function ( ). in sect. , the same procedure was applied to the italian data. in this section, we report the results of the fit of the cumulative diagnosed positive cases of covid- in italy using a function of the type of the gauss error function, given in sect. . we obtained very similar results using a logistic function with four parameters, which are not reported here for the sake of brevity. figure shows the fit of the data from february , , to march , . according to this fit, the flex, i.e., the point where the second derivative of the fit is becoming negative, that is, the difference between two successive daily cases becomes negative, or in other words the point where there is a deceleration in the number of positive cases is reached at march , , plus or minus days ( -sigma), this uncertainty is derived in sect. . according to this fit, the date of a substantial reduction in the number of cumulative positive cases in italy (about cases), is april , , plus or minus . days ( -sigma) (see sect. ). to check whether we obtain the same predictions using the cumulative number of fatalities of covid- in italy, instead of using the number of positive cases of covid- in italy, we repeated the previous analysis using the cumulative fatalities enumerated in the web sites of the italian "ministero della salute" (fig. ) , thus confirming the consistency of our results from february , , to march , , and so on, until march , (included), thus getting evaluations of the date of the flex. we then evaluated the standard deviation of these points, and we obtained a -sigma standard deviation for the flex of days. the -sigma ( . % probability) and -sigma ( . % probability) uncertainties in the day of the flex are then days and days, respectively. in regard to the date of a substantial reduction in the number of cumulative positive cases in italy (about cases), the standard deviation is days ( . % probability), days ( . % probability) and days ( . % probability). both the date of the flex and the date of a substantial reduction in the number of cases have a quasi oscillating behavior whose amplitude is decreasing by increasing the final date of the analysis, i.e., increasing the number of points used in the fit. indeed, using the last seven evaluations out of the ones, the previous standard deviations reduce by a factor of about two. however, using a second more robust method, by a monte carlo analysis [ ] [ ] [ ] , we evaluated narrower uncertainties for the day of the flex and for the day of a substantial reduction in the number of diagnosed positive cases, as described in sect. . the monte carlo simulations [ ] [ ] [ ] have been designed to possibly take into account the measurement error in each daily number of the cumulative positive cases of covid- in italy. this error should describe the uncertainty in the process of measuring the daily number of positive cases due to fluctuations in the measurement procedures (such as a different number of performed daily nasopharyngeal swabs of one day with respect to another day); of course, this error does not describe the difference between the actual total positive cases and the diagnosed ones which can be very large [ ] . however, the diagnosed cases are hypothesized to be a representative sample of the actual population (i.e., of the total number of positive cases, which is unknown). to get an estimate of the uncertainty in each daily number, we applied the following heuristic approach. we have assumed a measurement uncertainty in the total number of positive cases equal to % of each daily number (gaussian distributed). the second step was to generate a random matrix (m × n), where n (columns) is the number of observed days and m (rows) is the number of random outcomes, which we have chosen to be . each number in the matrix is part of a gaussian distribution with mean equal to and sigma equal to . (i.e., % of ), both row-wise and column-wise. in such a way, each day will be characterized by simulated outcomes that allow to apply a statistical approach. the outcomes represent a reasonably large number of simulated deviations from the official data. we then multiplied the nominal value of the diagnosed positive cases of the jth day for the numbers of the jth column of the random matrix mentioned above. in such a way, each day will be associated with a series of numbers (gaussian distributed with a % standard deviation), which simulate the statistical nature of a single datum. the index j will run from february , , to march , . finally, we integrated the daily data to obtain series of the cumulative diagnosed positive cases that allowed to perform the statistical analysis. in summary, starting from the n nominal values of the daily data, we generated n gaussian distributions with outcomes, with mean equal to the n nominal values and with % standard deviation. then, for each of the simulations, these n values (corresponding to the cumulative positive cases of n days) were fitted with a four-parameter function of the type of the gauss error function, and we then determined the date of the flex with such fitted function for each simulation. using the fitted function, we also determined the date at which the number of daily positive cases will be at a certain threshold that, for example, we have chosen to be . finally, we calculated the standard deviation of these simulations. in fig. and fig. , we report the values (red dots) and the mean (horizontal solid line) of the monte carlo simulations, respectively, for the date of the flex and for the date of a substantial reduction in the number of daily positive cases. using n = days (i.e., the number of daily diagnosed positive cases up to march , ), the mean of the monte carlo simulations gives the expected dates of march , , and april , , for the flex and the day of a substantial reduction in the number of daily cases (about ), respectively. we then obtained a standard deviation ( -sigma) of day for the date of the flex and of . days for the date in which a substantial reduction in the number of daily cases would be about . this result corresponds to a probability of . % that the date of the flex will be at a certain date plus or minus day and that the date of a substantial reduction in the number of cases will be at a certain date plus or minus . days. a -sigma standard deviation will give a more robust probability of . % of the day of the flex and of the day of a substantial reduction in the number of cases. the -sigma values correspond to plus or minus days for the day of the flex and plus or minus . days for the day of a substantial reduction in the number of cases. a similar uncertainty was obtained for the day of the flex and the day of a substantial reduction in the number of fatalities. in this paper, we considered the first days for fitting the cumulative diagnosed positive cases of covid- in italy (i.e., from february , , to march , ) and the first days for the monte carlo simulations. the function used for the fitting is of the type of the gauss error function with four parameters (a distribution function which well fits the corresponding cumulative diagnosed positive cases in china). we obtained that the day of the flex (i.e., the day of deceleration in the number of daily positive cases) is in italy, with a . % probability, between march , , and march , ; the -sigma uncertainty of +/− days was obtained with monte carlo simulations. in regard to the day of a substantial reduction in the number of daily positive cases (which, for example, we took to be ), this day will be in italy, with a . % probability, between april , , and april , ; the -sigma uncertainty of +/− . days was also obtained with the monte carlo simulations. in regard to the day of the flex and the day of a substantial reduction in the number of fatalities, we obtained a four-day forward shift with respect to the corresponding dates of the positive cases. the predictions discussed in this paper are statistical in nature and do not explicitly take into account the relevant factors of the daily number of nasopharyngeal swabs, social distancing, and epidemiological and virology studies, which are outside the analysis of the present paper. it is important to stress that the mathematical predictions reported in this paper provide an indication of the approximate period when a substantial reduction of positive cases and fatalities is expected and not the end of the extremely important mitigation measures. substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) prediction of the time evolution of the covid- pandemic in italy by a gauss error function and monte carlo simulations incubation periods of acute respiratory viral infections: a systematic review forecasting covid- impact on hospital bed-days, icu-days, ventilator-days and deaths by us state in the next months data reduction and error analysis for the physical sciences monte carlo simulations of the lares space experiment to test general relativity and fundamental physics acknowledgements we gratefully thank richard matzner of the university of texas at austin for helpful suggestions, alessandro paolozzi, and claudio paris of centro fermi.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. key: cord- -zc huo j authors: capone, alessandro title: simultaneous circulation of covid- and flu in italy: potential combined effects on the risk of death? date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: zc huo j based on data updated to may , , in italy the total recorded number of patients who died due to covid- -related reasons is , . demographic and clinical characteristics of died patients (including the number of comorbidities) are extremely relevant, especially to define those with a higher risk of mortality. health authority recommends flu vaccination in a number of categories at risk of serious medical complications: subjects over , patients with diabetes, cardiovascular diseases, copd, renal failure, cancer, immunodeficiencies, chronic hepatopathies and chronic inflammatory bowel diseases. the peak of the seasonal flu certainly preceded the pandemic one; however, it would seem clear that for a while the two viruses have been circulating simultaneously in italy. hence, after its peak, influenza-like illness-related (ili) deaths started to grow again. while some of the excess mortality reported in the ili group may be attributable to covid- , a question arises: do we have to consider this observation as a result of a random sequence of events or a potential relationship between the two viruses play a role? a cooperation mechanism intended at establishing an absolute advantage over the host could also be assumed. this system often takes place to boost their reproductive probabilities. a characterization of patients died due to virus-related reasons can be done by cross-linking data stored in different warehouses of the same geographical area and developing electronic health records. it would be of great relevance to identify patients at a very high risk of mortality as a result of an overlapping or combination of risk factors reported separately in patients died from covid- or influenza. the description of the subgroup of patients at higher risk of mortality will be crucial for the prioritization and implementation of future public health prevention and treatment programmes. social distancing and other forms of precautionary public health measures have led to an effective control of beta coronavirus circulation in china, in italy, in other european countries, and in the usa as well. the blood toll paid in italy has been early and particularly high. at present, a "ceasefire" has been successfully achieved, but the war against covid- may not yet be over. indeed, a resurgence of infections due to beta coronavirus could be observed at the time of possible simultaneous circulation of covid- and influenza. such an event could be expected between late autumn and early winter this year. based on data updated to may , , in italy the total recorded number of patients who came into contact with covid- and died corresponds to , [ ] . preliminary case-fatality rate (cfr) among patients with covid- was estimated by onder et al [ ] . the authors calculated the overall crude cfr corresponded to , %, a remarkably higher value than the average reported in china ( , %) [ ] . demographic and clinical characteristics of died patients (including the number of comorbidities) are extremely relevant, especially to define those with a higher risk of mortality. age was recognised as one of the main risk factors for death associated with covid- . the median age of deceased patients corresponded to years (interquartile range: - ). this value is actually years higher compared with the median age of infected patients (died patients years, and infected patients years, respectively) [ ] . indeed, italy is one of the countries with the highest mean age in j o u r n a l p r e -p r o o f the world, along with japan and germany. pre-existing chronic diseases (diagnosed before the covid- infection) is an additional and substantial risk factor. the most frequent comorbidities reported include high blood pressure ( , %), type diabetes ( , %), ischemic heart disease ( , %) and renal failure ( , %) [ ] . unfortunately, the comorbidity assessment was derived from a carefully analysis of medical records of only , out of , died patients (about % of the overall supposed number) [ ] . the average number of diseases observed in this population was , ± , . on the other hand, a study lately performed by the italian national institute of health (iss), has shown that the excess of mortality associated with influenza-like illness (ili) was between , and , per , , reaching a total of over , deaths in a -year time period [ ] . in other words, patients with diabetes or other metabolic diseases (including those with a bmi > ), cardiovascular diseases, copd, renal failure, cancer, immunodeficiencies, chronic hepatopathies and chronic inflammatory bowel diseases [ ] . the comparison between individuals at risk of mortality associated with both diseases (i.e. covid- and influenza) is of the utmost importance. indeed, a sub-stratification of patients at a very high risk of mortality might be predictable, in particular when the circulation of both viruses is supposed to occur in the same period. although attempts are being made to develop unique data collection platforms in italy, the information needed for accurate mortality risk estimation are commonly located in different repositories. this is true for both flu and covid- . the national sentinel influenza surveillance system (known as influnet) is a network of general practitioners and pediatricians working as sensors and providing health care to at least % of the whole italian population [ ] . as a consequence, estimating the role of flu in the overall mortality calculation is quite complex, since the diagnosis is not always confirmed by laboratory tests and most death cases are due to complications. for these reasons, although the death case is flu-related, flu may not be officially reported in all death certificates or registries. an indirect measure of its impact on mortality is given by the attributable excess of mortality, which can be calculated as the differential between the number of deaths observed during the flu season and the expected baseline value (in the absence of flu). several statistical regression models have been adopted to estimate the excess mortality attributable to influenza, considering the potential confounding effect of variables such as temperature, viral genotypes and age distribution patterns of the population [ , [ ] [ ] [ ] . as far as covid- related deaths are concerned, the national institute of health (iss) has set up a specific web platform (similar to influnet) for epidemiological and immunological data collection, including recommended procedures for molecular diagnostics. however, as not all covid- related death are assumed to have been hospitalized (i.e. some deaths occurred at home or often in health care homes), mortality data are supposed to be underestimated. nonetheless, it is very important to point out that the cause of death may not be surely or purely virus-dependent. this circumstance might be more frequently detected in patients with multiple comorbidities (more than % of died patients were affected by ≥ comorbidities) [ ] . moreover, data pertaining to drugs previously prescribed in patients with chronic clinical conditions (such as high blood pressure, diabetes, ischemic heart disease or copd) may not be detailed enough to identify patients at higher risk of death or those having factors increasing the risk itself. when we look at the shape and trend of curves over time, something immediately jumps out: the two death curves show an absolutely overlapping profile, although the magnitude of numbers associated with covid- is remarkably greater. that's not surprising. in any case, ili-related deaths have also started growing again. the peak of the seasonal flu has certainly preceded the pandemic one, however, it would seem clear that for a while the two viruses have been circulating simultaneously in italy. while some of the excess mortality reported in the ili group (in over ) may be attributable to covid- infection actually, a question arises: do we have to consider this observation as a result of a random sequence of events or a potential relationship between the two viruses play a role? although they show a different binding affinity for their own specific redundant receptors (i.e. sialic acids of glycoproteins or glycolipids and ace for influenza and covid- , respectively) [ , ] , a cellular access pattern with different effectiveness, and consequently a diverse pathophysiology, both have an elective tropism for the respiratory tract. taking viral signaling into consideration, a j o u r n a l p r e -p r o o f cooperation mechanism intended at establishing an absolute advantage over the host could also be envisaged [ ] . this system often takes place between different viral strains sharing the same interest in boosting their own reproductive probabilities. an example of effective viral cooperation was found in several oncogenic genotypes of high-risk and low-risk human papillomavirus [ ] . moreover, the virulence also has usually turned out to be based on a cooperative effect between different genes which encoding for specific viral capabilities such as transmissibility and fatality [ ] . hence, it's become increasingly clear that many viruses actively work together, teaming up to co-infect hosts and neutralise antiviral immune procedures. although at this stage it is merely a hypothesis that needs further investigation, the simultaneous or sequential infection of both viruses (covid- and influenza) leading to strengthen the effectiveness of the single infection cannot be excluded. at present, several data needed to estimate the real impact of influenza and coronavirus on overall mortality are still missing. in addition, assuming the potential simultaneous circulation of the two viruses (during the flu season), it is suggested that health authorities undertake a study (or perform a full assessment) specifically aimed at establishing the predominant risk factors of virus-related death. in particular, it would be of great relevance to identify patients at a very high risk of mortality as a result of an overlapping or combination of risk factors reported separately in patients died from covid- or influenza. subjects aged - years and over, suffering from hypertension or diabetes and especially those with metabolic syndrome or with - comorbidities (mentioned above in the two groups separately) are likely to be the elective target of prevention programmes. this is the reason why a similar investigation is mandatory. a detailed characterization of patients died due to virus-related reasons can be done by cross-linking data stored in different archives or warehouses of the same geographical area (classically a region). this guarantees a high intrinsic quality of the analysis and at the same time reduces the need to use j o u r n a l p r e -p r o o f alternative proxies or sources that could introduce a systematic bias. administrative and demographic databases can be queried retrospectively using a unique code referring to a single individual in order to develop a longitudinal electronic health record with a predetermined time interval. the description of the subgroup of patients at higher risk of mortality will be crucial for the prioritization and implementation of future public health prevention and treatment programmes such as a mass flu vaccination campaign. italy was not as ready to manage a so-rapid and dramatic pandemic, as many other countries were, actually. the gathering of data and assessment of consequences associated with covid- infections in italy took some time and appropriate responses (i.e. quarantine and social distancing measures) could have been delayed a bit. this event may also have contributed to smoothing the viral transmission and worsening some patients' clinical conditions. now, probably nothing will happen in the late fall, but an integrated pandemic plan for italy we must be prepared as soon as possible. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: characteristics of sars-cov- patients dying in italy case-fatality rate and characteristics of patients dying in relation to covid- in italy characteristics of and important lessons from the coronavirus disease national institute of health (istituto superiore di sanità) investigating the impact of influenza on excess mortality in all ages in italy during recent seasons ( / - / seasons) prevenzione e controllo dell'influenza: raccomandazioni per la stagione multinational impact of the hong kong influenza pandemic: evidence for a smoldering pandemic mortality due to influenza in the united states -an annualized regression approach using multiple-cause mortality data trends for influenza-related deaths during pandemic and epidemic seasons impatto dell'epidemia covid- sulla mortalità totale della popolazione residente. primo quadrimestre . full text available for download from the istat website avian influenza a viruses differ from human viruses by recognition of sialyloligosaccharides and ganglosides and by a higher conservation of the ha receptor-binding site coronavirus infections -more than just the common cold communication between viruses guides lysislysogeny decisions koilocytosis: a cooperative interaction between the human papillomavirus e and e oncoproteins fenner and white's medical virology key: cord- -okvvajms authors: lazzarini, luca; barzon, luisa; foglia, felice; manfrin, vinicio; pacenti, monia; pavan, giacomina; rassu, mario; capelli, gioia; montarsi, fabrizio; martini, simone; zanella, francesca; padovan, maria teresa; russo, francesca; gobbi, federico title: first autochthonous dengue outbreak in italy, august date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: okvvajms in august , during the coronavirus disease (covid- ) pandemic, five locally acquired cases of dengue virus type were detected in a family cluster in vicenza province, north-east italy where aedes albopictus mosquitoes are endemic. the primary case was an importation from west sumatra, indonesia. this is the first outbreak of autochthonous dengue reported in italy. during the covid- pandemic, screening of febrile travelers from endemic countries is crucial in areas where competent vectors are present. in august , during the coronavirus disease (covid- ) pandemic, five locally acquired cases of dengue virus type were detected in a family cluster in vicenza province, north-east italy where aedes albopictus mosquitoes are endemic. the primary case was an importation from west sumatra, indonesia. this is the first outbreak of autochthonous dengue reported in italy. during the covid- pandemic, screening of febrile travelers from endemic countries is crucial in areas where competent vectors are present. in europe, dengue is almost always imported from endemic countries, and only very few autochthonous cases and limited outbreaks have been reported. we present the first autochthonous outbreak of dengue in italy, due to an imported case from indonesia. a woman in her thirties from vicenza province, veneto region, italy, (case ) stayed in pulau weh, a tropical island in west sumatra, indonesia, for months and returned to her home town in vicenza province on july . she had a four-day stopover in djakarta before flying back to italy. after her return, she stayed at home for days to complete the coronavirus disease (covid- ) quarantine, mandated by the italian government for travellers outside european union (eu) countries at the time. since july, she experienced fever ( ° c), malaise, back pain and upper limb itching. on july, she was tested with oral and nasal swab for severe acute respiratory syndrome coronavirus (sars-cov- ) rna and found negative. clinical symptoms resolved within days. between and august, five of her seven household contacts started having symptoms of fever (> ° c), malaise, headache, and upper limb itching. the contacts included a female and male in their fifties (case and case , respectively), two males in their twenties, and a preschool child (cases , and , respectively). clinical symptoms resolved within days in all of them. case had stayed in in pulau weh in january for days. she was asymptomatic during and after travel. cases , , and never travelled abroad. case presented to the infectious diseases unit of our hospital on august, when clinical symptoms had already disappeared. she was investigated for west nile virus, usutu virus, dengue virus (denv), chikungunya virus (chikv) and zika virus (zikv) by molecular and serology testing, because she reported that a family member (case ) had had similar symptoms after a recent travel in indonesia. she was pcr tested for sars-cov- and found negative. the results were available on august, and she was consecutively diagnosed with denv type (denv- ) infection based on positive realtime reverse transcriptase (rt)-pcr on plasma, urine, and saliva, and positive denv ns antigen in plasma, while serology testing was negative. following the dengue diagnosis of case , cases , , , and were invited to present to the same unit, where they were tested on august and also found positive for denv- (table) . all serological tests for chikv, zikv, west nile and usutu vires were negative. all patients recovered fully without complications, and none may be classified as severe dengue. following the notification to the public health authority of veneto region on august, public health technicians inspected the house where the affected family lives, which is in a rural, underpopulated area. on the same day, and for three consecutive days, mosquito control was conducted in the area comprised in a radius of m around the house, and extended to sensible places nearby such as a hospital and a touristic castle, as indicated by the national plan against arbovirus infections [ ] . disinfestations included larvicides, adulticides and removal of the breeding sites. on august, an entomological monitoring was set up using bg-sentinel traps, ovitraps and manual aspiration in order to control the effectiveness of the disinfestations, to define the mosquito density outside the area and to search for the virus. a retrospective laboratory investigation was performed in all the patients from vicenza province who were referred for suspected arbovirus infection during the previous month, which excluded denv- infection in all of them. general practitioners in the district where the outbreak occurred were alerted to refer cases of unexplained fever for investigation to the local infectious diseases department. during and soon after disinfestation activities, local health professionals, entomologists and representatives of the local police went from door-todoor in the radius of m to interview the inhabitants on symptoms, and raise awareness for the need to avoid mosquito bites and the need to remove possible larval breeding sites. the population of the city where the family resides is about , inhabitants, and was provided with informative brochures about dengue. in addition, on august, the national transplant centre activated denv nat screening in organ, tissue and haematopoietic stem cell donors, as well as in blood donors, who reside in vicenza province or had stayed for at least one night in vicenza province in the days before donation. blood collections from donors in the town where the outbreak occurred have been suspended. this analysis was conducted as part of public health usual practice, and was not conducted for research. patients gave informed consent to anonymously publish their clinical and laboratory data for the purpose of this paper. [ ] . real-time rt-pcr assays were carried out using the one-step, real-time kit (thermo fisher scientific, waltham, massachusetts, us) and run on abi ht sequence detection systems (thermo fisher scientific). b denv ns antigen was detected in plasma by using a rapid immuno-chromatographic assay (dengue ns ag strips, bio-rad, hercules, california, us). c denv igm and igg antibodies were detected by a chemiluminescence immunoassay (virclia, vircell, granada, spain) in a thunderbolt instrument (vircell). between and , autochthonous cases of dengue fever in europe were reported in from france [ ] and croatia [ ] , in from madeira, portugal [ ] , from france in , , , , [ ] [ ] [ ] and from spain in and [ , ] . these cases were caused by denv- , as in cases described here, and denv- [ ] . previously, in italy no autochthonous cases of dengue had been reported, while two outbreaks of chikv disease were detected in emilia-romagna region and in central-southern italy in and in , respectively [ , ] . the veneto region started an integrated surveillance for imported fevers in [ ] . the surveillance, active between june and november, aims to increase the detection rate of denv and chikv (since , also zikv) infection in travellers from endemic areas and to promptly identify potential autochthonous cases. patients with fever > ° c in the last days and recent (less than days) return from endemic countries, after ruling out malaria, are referred to an infectious diseases department of the region to perform a rapid test for denv and to collect samples for the regional reference laboratory in padua, for second line testing. entomologic investigations are also performed in the areas surrounding ( m) the residence of human cases of denv, chikv and zikv infection, if notified as viraemic [ ] . in ten years of surveillance ( - ), ca , patients were tested and were positive for denv, for chikv and for zikv. each year, a variable proportion of febrile cases returning from endemic areas, ranging from . % in to . % in , are diagnosed with an arbovirus infection. all these cases were imported. a mathematical model was applied to epidemiological and entomological data to assess the risk of a denv outbreak in northern italy [ ] . the risk for denv outbreaks was estimated lower than for chikv, because of the lower competence for transmitting denv of ae. albopictus, the dominant vector in our area. we cannot exclude that unnoticed autochthonous cases of denv may have occurred in italy, especially if asymptomatic. however, we believe that any cluster of unexplained summer fever should be detected by existing clinical surveillance plans. the index patient in the outbreak described here was not promptly referred to an infectious disease department, but the mandatory covid- restriction measures also limited the risk of denv spread from the index case during the viraemic phase. at the same time, the ongoing covid- pandemic caused a diagnostic delay, as the first clinical suspicion was covid- . however, the high density of mosquitoes in the area where the source case (case ) was living, as well as her permanent presence at home due to quarantine, led to a high attack rate among household contacts. active clinical surveillance in the control area, aimed at diagnosing other possible secondary cases is still ongoing, as well as entomological surveillance, including installation of ovitraps, larval search and manual aspiration of adult mosquitos within and outside the control area. we suggest that risk of further local spread may be possible, depending on the number and movement of potential viraemic people outside the control area before clinical diagnosis and on the local mosquito density. clinical surveillance of summer fever and surveillance of febrile travellers returning from endemic areas should not be limited to rule out sars-cov- infection, and should include arbovirus screening in all countries where competent vectors are present. west nile virus and usutu, the surveillance and response plan. rome: ministry of health first two autochthonous dengue virus infections in metropolitan france autochthonous dengue fever in croatia clinical presentation and laboratory findings for the first autochthonous cases of dengue fever in madeira island autochthonous dengue outbreak in nîmes european centre for disease prevention and control (ecdc) european centre for disease prevention and control (ecdc) infection with chikungunya virus in italy: an outbreak in a temperate region detection of a chikungunya outbreak in central italy surveillance for west nile, dengue, and chikungunya virus infections human and entomological surveillance of west nile fever, dengue and chikungunya in veneto region potential risk of dengue and chikungunya outbreaks in northern italy based on a population model of aedes albopictus (diptera: culicidae) analytical and clinical performance of the cdc real time rt-pcr assay for detection and typing of dengue virus none declared. luca lazzarini and vinicio manfrin managed the patients. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- - j vuqer authors: kim, a. s. title: transformed time series analysis of first-wave covid- : universal similarities found in the group of twenty (g ) countries date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: j vuqer as of april , , the number of cumulative confirmed coronavirus disease (covid- ) cases exceeded million worldwide and million in the us with an estimated fatality rate of more than percent. because the patterns of the occurrence of new confirmed cases and deaths over time are complex and seemingly country-specific, estimating the long-term pandemic spread is challenging. i developed a simple transformation algorithm to investigate the characteristics of the case and death time series per nation, and described the universal similarities observed in the transformed time series of nations in the group of twenty (g ). to investigate the universal similarities among the cumulative profiles of confirmed cases and deaths of individual nations in the g , a transformation algorithm of the time series data sets was developed with open-source software programs. the algorithm was used to extract and analyze statistical information from daily updated covid- pandemic data sets from the european centre for disease prevention and control (ecdc). two new parameters for each nation were suggested as factors for time-shifting and time-scaling to define reduced time, which was used to quantify the degree of universal similarities among nations. after the cumulative confirmed case and death profiles of a nation were transformed by using reduced time, most of the nations, with few exceptions, had transformed profiles that closely converged to those of italy after the onset of cases and deaths. the initial profiles of the cumulative confirmed cases per nation universally showed - week latency periods, during which the total number of cases remained at approximately ten. the latency period of the cumulative number of deaths was approximately half the latency number of cumulative cases, and subsequent uncontrollable increases in human deaths seemed unavoidable because the coronavirus had already widely spread. immediate governmental actions, including responsive public-health policy-making and enforcement, are observed to be critical to minimize (and possibly stop) further infections and subsequent deaths. in the pandemic spread of infectious viral diseases, such as covid- studied in this work, different nations show dissimilar and seemingly uncorrelated time series profiles of infected cases and deaths. after these statistical phenomena were viewed as identical events occurring at a distinct rate in each country, the reported algorithm of the data transformation using the reduced time revealed a nation-independent, universal profile (especially initial periods of the pandemic spread) from which a nation-specific, predictive estimation could be made and used to assist in immediate public-health policy-making. a brief history of the first deaths on december , , chinese health authorities treated a patient cluster of pneumonia caused by the newly recognized coronavirus, i.e., severe acute respiratory syndrome-coronavirus (sars-cov- ): they had closely monitored the cluster from the beginning of december, . the first domestic death in china was reported on january , , and afterward the city of wuhan, with a population of · million, was locked down. outside china, the first death was reported by the philippines on february , , followed by france on february , italy on february , and the u.s. on february . this rapid transmissibility of the coronavirus was estimated by using the instantaneous reproduction number (r t ) and confirmed case-fatality risk for four megacities and multiple provinces reporting the highest number of confirmed cases in china. the aggressive non-pharmaceutical interventions reduced only the first wave of covid- outside of hubei, and this effort that might have been more successful if foreign importation had been limited to prevent viral reintroduction. effects of various non-pharmaceutical intervention attempts were reported to be effective in reducing the transmission of covid- (as well as influenza) in hong kong, such as border restriction, quarantine and isolation, and social distancing. further nation-specific situations of g nations can be found elsewhere: argentina, australia, , brazil, canada, china, , , german, france, indonesia, india, italy, , , japan, korea, mexico, , russia, saudi arabia, south africa, , turkey, uk, us, and multiple european nations. because the failure of non-pharmaceutical intervention was ascribed to overseas travel, a global metapopulation disease transmission model was used to project how travel limitations contributed to the mitigation of the global covid- spread. within weeks after the first death in china on january , the coronavirus appeared to have already been transmitted to other major cities within mainland china. the reported data suggest that nonpharmaceutical interventions were effective only within cities within china but did not significantly affect the trans- port of covid- overseas. as of april , , the global number of confirmed cases and deaths exceeded · million and thousand, respectively. nevertheless, the initial routes of covid- transmission from china to other countries are not well identified, and the correlations among patterns of the occurrence of confirmed cases and deaths in each nation are still ambiguous. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint the objective of this study, therefore, was set to analyze the ccc and ccd of a number of selected countries and investigate any possible universal similarities in the patterns of increases in ccc and ccd over time from december , until april , . the observed universal similarities were used to predict a few cases in mid may, . for this purpose, a simple mathematical data transformation model was conceptually developed and implemented by using open-sourced software packages and utilities. all data sets used for this work were downloaded from the ecdc website as a comma-separated value (csv) format file, named "download" without a file extension. the data file contained the daily numbers of confirmed cases and deaths for locations, according to country or territory codes. open-sourced utilities such as bash, sed, and awk were used as needed to extract the daily ccc and ccd data for individual countries or territories. a total of files were generated, with file names identical to specific countries or territories. each extracted csvformat file had rows of six selected integer items (i.e., columns): date, day, month, year, cases, and deaths, where the date format was dd/mm/yyyy. in octave (open-sourced software, an alternative clone to matlab), the ccc and ccd were calculated and plotted against the number of days elapsed after december , . for each country or territory, eight graphs -new (daily) vs. cumulative, cases vs. death, and linear vs. logarithmic (with base ) profiles -were plotted against the number of days. the script-generated graphs were automatically saved as image files for visual investigation after the data were plotted. on a desktop computer (linux os, ubuntu · · lst (bionic beaver), intel(r) xeon(r) cpu e - v @ · ghz, gb ram), this task required only - minutes for all countries and territories. close visual inspection of these graphs per nation provided an initial understanding of each nation's ccc and ccd time series in , where january was set as day . although the basic data extraction and mining were completed for all nations by using the daily updated data file, analysis of all countries or territories was challenging. to potentially include nations representing all continents (excluding antarctica), individual nations were selected in g for the current analysis. the european union (eu) was excluded because it contains multiple member countries, and ecdc reports covid- data only for individual countries and territories. functions embedded in calc. the date-information in "dd/mm/yyyy" format was converted into "yyyy-mm-dd" format to calculate the number of days between two specific dates by using the "days" function, embedded in calc software. this time conversion from lexical date format to number of days (as a countable integer) was an important basic step for further data investigation and analysis. in this work, the cumulative data of cases and deaths (i.e., ccc and ccd) were primarily used instead of data of new daily occurrences with respect to the number of days after december , . in principle, using the cumulative information in statistical analysis is equivalent to using a cumulative density function as an integral of a probability density function with respect to control variables. the fundamental advantages of using the cumulative data are as follows. first, the daily data often fluctuate too much to capture specific variations and trends in target variables, so that statistically meaningful characteristics not only are subject to the data observer's viewpoint but also are often challenging to extract. second, the cumulative profiles never decrease, so that either rapid/gradual or local/global variations can be systematically captured by using semi-log plots, i.e., common-logarithmic ccc and ccd on the y−axis vs. the linear day number on the x−axis. third, the ever-increasing trend in logarithmic cumulative data is often considerably smoother than the original daily time series. therefore, variation trends can be captured without significant statistical noise. in addition, with a fixed time interval, i.e., day in the current case, the original time series data can be easily retrieved by calculating the difference of the cumulative data between two consecutive days. for a consistent analysis, we define new variables, such as c (n) and d (n) of day n, indicating the ccc and ccd, respectively, where n increases from n = of january , to the day for which the latest data are available. the time series analysis in this study primarily used the cumulative data from january to april , , and the developed algorithm was tested using the data from may to , . because the time series of cases and deaths are updated daily, a unit time interval is set as day, i.e., δn = . then, ccc and ccd are represented as functions of day number n: respectively, where δc (n) and δd (n) are the increased numbers of cases and deaths, respectively, on day n. some nations do not have complete data sets: the missing days either are in early january, when no pandemic effects were found, or are intermittent - days after the pandemic report was started. because cumulative data are processed and analyzed in this study, the missing days are treated as days with no new occurrences, which is to avoid arbitrarily altering the statistical results by interpolation processes. data similarity was found unexpectedly between the infection and death time series available online and the phase transition patterns of matter in thermodynamics. in statistical physics, the clausius-clapeyron equation describes a discontinuous transition between two phases of single-constituent matter. various materials have their unique material properties, often represented by using material constants, for example, evaporation enthalpy called latent heat at a specific temperature. although many organic or inorganic solutions have various trends in their phase-diagrams, e.g., pressure vs. temperature (p − t ) curves, the overall pattern of how p increases with temperature t is universal in physics. along the liquid-gas equilibrium, p and t are related as where ∆s = l h (t ) /t is the molar entropy change from liquid to gas phases, l h [joule/mol] is the molar latent heat as a function of absolute temperature t [k], and ∆v [liter/mol] is the molar volume difference per molecule between . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the two phases, which is is often approximated as the gas-phase molar volume. recent analysis of the clausius-clapeyron equation for water has been described elsewhere in detail. theoretical ideas for the current work stem from fundamental thermodynamics and statistical physics that the clausius-clapeyron equation provides a universal functional form that covers a number of materials undergoing transitions between two or more phases. among the nations belonging to g , the cases and deaths reported in italy were selected as principal data. furthermore, we hypothesized that the ccc and ccd profiles of other nations would have certain degrees of similarity to those of italy. because minimizing deaths is a more immediate task than reducing the number of confirmed cases, we investigated the ccd data first. to directly compare the ccd time series of italy (it) and that of nation x, a characteristic day is defined as the first day when italy's ccd exceeded a certain threshold number selected here as , denoted ν d = . the day number of the date when italy's total deaths exceeded the threshold is denoted m it d = (indicating february , ). then, the x−axis of italy's ccd vs. time graph moves from n to n − m it d , which is equivalent to moving italy's profile as many as m it d = days to the left on the time axis. in general, reduced time τ d (for the total deaths), specifically for italy, is defined asτ by definition because italy's ccd data form an international baseline of ccd and therefore do not need to be scaled. a general definition of the reduced time ccd is for nation x, which will be replaced by the two-character abbreviations of nations investigated. in this case, n − m it d indicates the number of days after the sudden increase in italy's ccd. the universal value ν d = is determined, as it is frequently done for order-parameter estimations in statistical physics, because italy's ccd drastically increases after it exceeds . a negative value of n − m it d indicates the number of days before the explosive ccd onset. for nation x, the reduced time τ x d can be obtained by identifying the nation's day number of ccd onset m x d , interpreted as a time-shifting parameter, and calculating β x d , defined as a time-scaling parameter. the physical meaning of β x d is explained as if β x d = · ; then the ccd rate of nation x is · times slower than that of italy. in this study, it was found that most nations (except a few outliers) have β values greater than · . after , is plotted on the same graph with respect to the nation's reduced time τ x d = n − m x d /β x d by using an initial guess of β x d = . italy's variation in ccd has the stiffest slope among all countries after its first covid- -related death was reported on february , following france's first reported death on february . in this regard, the ccd profiles of other countries, especially the five european nations of germany (de), france (fr), russia (ru), turkey (tr), and the united kingdom (uk), are on the right-hand side in the time axis of n to italy's ccd with lower ccd profiles. while m x d moves the ccd profile of nation x to the left to match its onset to that of italy, the β x d value proportionally shortens (or lengthens) the ccd profile of nation x on the shifted axis of n − m x d . the ccd profiles of italy and nation x are numerically integrated with n and τ x d , respectively, from to min max n − m it values of the integrals are the areas under the ccc curves for italy and nation x, whose absolute difference is minimized by iterative adjustment of β x d in calc software. that is, the optimal value of β x d maximizes the overlapping degree of italy's linear ccd (without use of the time-scaling parameter β it d ) and nation x's transformed ccd profiles. because β x d is determined by comparing two finite integrals, β x d is independent of the pre-selected values of m x d . moreover, ccc profiles are analyzed with the same method for ccd profiles, i.e., for nation x, identification of the ccc onset day, calculation of time-shifting parameter m x c , and determination of the time-scaling parameter β x c . the reduced time for nation x's ccc is similarly defined as where m c and β c are different for each nation. the threshold of ccc is preset as ν c = using the same criteria to preset ν d . specific values of τ and β for ccd and ccc are listed in table for all g nations excluding the eu (denoted g ) nations. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . this work was motivated by interesting similarities among the ccd data for six nations in the continent of europe included in g : de, fr, it, ru, tr, and the uk. these six nations are denoted e throughout the manuscript, and e denotes all those nations except italy, i.e., the nation with the highest number of ccc and ccd at most times to date. figure shows the pandemic time series of the e nations: ccc profiles on (a) linear and (b) logarithmic scales and ccd series on (c) linear and (d) logarithmic scales. notably, the y−axis maxima of (a) and (b) for ccc are much higher (approximately a tenfold or more) than those of (c) and (d) for ccd. the current fatality rate of covid- is estimated to be on the order of o − , i.e., a few percent. herein, ccc and ccd data are plotted on the y−axes of the linear and logarithmic (base ) scales along (linear) x−axes of time, i.e., the number of days after december , , which are denoted in linear and logarithmic plots, respectively. in general, two data lines in a linear plot can be compared by observing the apparent dominance of one line over the other in terms of magnitude. the logarithmic plots allow for comparison of two data lines at various orders of magnitude. for example, the onset-time and latency-period of each nation are better visualized in the logarithmic plots, whereas their one-to-one comparison is more straightforward in the linear plots. several unique aspects identified by simple visual investigation in figure became the major motivation for this study. first, the linear ccc profiles, shown in figure (a) appear to follow an ordered previously unknown pattern. except for the ccc case of the uk, no intersections between two nations are seen. this trend is exceptionless in the ccd profiles shown in figure (c), thus strongly implying that, if the ordered pattern continues for all e nations, none of the e nations will have more severe situations than italy, which has the highest ccc and ccd numbers. this argument was found to be reasonably valid until mid-april, (i.e., day or later). individual nations' immediate enforcement of public health policies may be able to alter increasing ccc rates, but non-pharmaceutical interventions are known to be inefficient after covid- spread become prevalent. second, figure (b) shows a sudden onset followed by a latency period of approximately month. during this latency period, the ccc remains more or less , specifically, between and . one exception is turkey, showing only a few days of ccc latency: turkey's ccc also intersects with that of russia before it exceeds the threshold around . determination of the onset time and latency more importantly, the latency periods of ccd profiles are often too short to recognize, and the subsequent death bursts are very abrupt, showing stiff slopes. the onset profiles of the e 's ccd suggest that after a nation identifies the first death related to and/or caused by the coronavirus, the death burst will inevitably occur within no more than a week. updated plots of e 's ccc and ccd profiles until may , , are included as figure a. in appendix. the pandemic profiles of the e nations are investigated by calculation of each nation's m and β values for ccc and ccd, which are compared with those of italy as baseline values. figures (a) and (b) show the apparent similarities between the e 's transformed profiles and italy's linear profiles of ccc and ccd, respectively. this universality of the pandemic time series data is present in both confirmed cases and deaths, because an infection is a necessary condition for death unless complications of the covid- pandemic develop unexpectedly. since the key process in the current study is transforming the cumulative profiles of nation x onto those of the reference country (italy), the threshold values for ccc and ccd must be preset to calculate m and β values. these threshold values are subject to intuitive data observation, allowing qualitative human perceptions to be input into numerical calculations for more meaningful data interpretations. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint ccc of the e figure (a) shows that not all e nations have a similar onset trend after a certain latency period, but most appear to follow italy's profile after their ccc numbers exceed approximately no more than , denoted here as the default ccc threshold ν c = . during the transformation, the actual threshold value of a nation's ccc is flexibly and manually adjusted to identify the best profile-matching values of m c and β c per nation. in general, the best matching is obtained by concurrently searching ν c , m c and β c by using the "solver" function in openoffice calc. the target matching zone of the reduced time is where italy's ccc varies from to , . germany, france, and the uk clearly have longer latency periods of approximately - weeks, and three other nations have latency periods of less than week. italy's latency period appears as almost four weeks with a small ccc number of . much in-depth research is required to understand the heterogeneous onset trends in neighboring nations, but the similarity of their transformed ccc profiles after τ c > is, to the best of my knowledge, a unique finding in this work. all e nations' table . this onset time difference is graphically represented as the distance between the highest ccc values of the uk and italy. in addition, β uk c = · indicates that the propagation speed of the confirmed cases in the uk is · times (or · percent) slower than that of italy, so that the uk and italy have the same rate of increase in ccc over time. the difference in the reduced time between the latest ccc numbers between italy and the uk is - · = · days in figure (a) , and if these values are multiplied by β uk c = · , then · days is obtained, which is the time distance in real days for the uk to reach the highest level seen in italy. that is, the uk's ccc will increase from thousand (as of april ) and reach that of current italy ( , as of april ) by may or (i.e., or and conceptually visualized by a double arrow at the end of italy's ccc profile in (a). in addition, germany and france have ccd onsets days after that of italy, and france's ccd is approximately . % slower than that of italy, but germany's ccc propagation is as fast as that of italy because of β de c = · · . turkey and russia both started their ccc onset days after italy's: in terms of ccc propagation rates, turkey (β tr c . ) has an equal pace to that of italy, and russia (β ru c = · ) is the slowest among the e nations and is close to the us (β us c = · ). these nation-specific, complex trends of ccc onset and burst are explained using the simple transformation method that can predict the time distance to the future situations using the reduced time concept. ccd analysis methods are the same as those of ccc, described above. higher β d values than β c : β fr d = · , thus indicating that the death rate of france is /β fr d = · times (or · percent) faster than that of italy. because france is the only nation with β d < β c , its slower ccc and faster ccd rates than those of italy may be ascribed to the first death in france occurring days earlier than italy's first death on february . similarly to t c (uk/it), the ccd time distance of the uk, denoted t d (uk/it) is calculated as where τ it is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint any future scenario. after april , , the uk's ccc and ccd numbers exceeded those of italy, as shown in figure a. (a) and (b), including may data on to figure (a) and (b), respectively. russia appears to start the secondary ccc burst after april ; however, its ccd record is still one order of magnitude lower than that of italy because russia's first death is days after that of italy. more one-to-one comparisons between nations and italy could be used to provide specific information for decision-making but are beyond the scope of this work's focus on developing and implementing the transformation algorithm with the newly defined time-shifting and time-scaling parameters of m and β, respectively. nevertheless, the transformation methods used above for e nations are extended to additional nations. we expand the list of nations to test whether the ccc and ccd similarities among e nations are also present in more nations on six different continents: africa, north and south america, asia, australia/oceania, and europe. in this regard, we select g as described above. because the eu is not an individual nation but a group member of european countries, it is not included in the pandemic analysis in this study. data used for the g analysis implicitly include those of the e used above. figure shows the g nations' linear profiles of ccc and ccd, denoted c (n) and d (n), respectively, and their scaled profiles of c (τ ) and d (τ ), respectively. the overall trend in c (n) profiles in figure (a) is similar to that of d (n) in figure (c) slightly moved to the left in the time axis, because a nation's ccc profile precedes its ccd profile in time. as previously discussed for the e cases, the threshold cut and latency periods of ccc onsets are generally higher and longer than those of ccd onsets, respectively. most g nations show a two-step ccc burst: a first burst from o ( ) to o − , followed by a certain latency period before a second burst from o − to at least o or higher. as graphically shown in figures (b) and (d), the universality of the cumulative profiles is not limited to e nations in the european figure (b) , i.e., in the most recent days, shows a much stiffer slope than those of china, south korea, and even italy. it is concerned that c us τ us c will increase more rapidly than those for any other nations in g . except for a few abnormal or outlying ccc profiles, figure (b) shows a strong universality among g nations in converging to the baseline ccc of italy, especially during the initial reduced time τ d of approximately days, despite the large population differences and the different continents. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint of deaths is manageable. the universal linearity of all g nations shown in (d) in the reduced time τ from to mathematically implies a simple power law for a nation i such as log d (i) = ατ where α is the universal slope and γ (i) is an y−intercept. on the basis of italy's ccd data from the first days after onset, α = · and γ it = · are calculated. in figure (a) and (c), as of april , c us (n = ) = , k is approximately · times c it (n) = k, and this ratio is in good agreement with the population ratio (in ) of the us ( · m) and italy ( · m), i.e., · ÷ · = · . however, the ratio of d us (n) = · k and d it (n) = · k, i.e., · , is below half the population ratio as of april . the other, more rigorous way to estimate the death rate is as follows. the which is closer to c us (n = ) /c it (n = ) = · and the population ratio of · . although the ratios calculated above are not a common academic standard but instead are empirical, they provide a quantitative method to estimate the future ccc and ccd of the us, as derived from the baseline values for italy. the coincidence of the total case and population ratios is limited to the us and further in-depth research is crucial for more fundamental evaluations. for the us, β us c = · and β us d = . indicate that both the case and death rates are · percent and · percent slower than those of italy, respectively. the ccc and ccd onsets of the us are only and days after those of italy. however, after the us's ccd onset, only days (from march to april ) are required to exceed italy's linear ccd profile, d it (n), in figure (c) . the same trend in the us is found in figure (d) , such that ∆τ us figure (b) should be further analyzed in depth, because the us currently has the largest number of ccd in the world, and, more severely, the greatest potential for further deaths. closely monitoring the uk, russia, and brazil, in addition to the us, indicates that italy's role as the reference country may end for this first wave of onset and burst of global cases and deaths. the second wave is not yet predictable using the current knowledge, obtainable from the data released by ecdc. figure shows a scatter plot of β c vs. β d for all g nations. italy is, in principle, positioned at ( , ) on the diagonal line. a position above the diagonal line indicates that the nation's death rate is slower than the infection case rate, i.e., β d > β c . most nations are located between the diagonal and upper lines, with the exceptions of australia and south korea. in australia, even if the number of infection cases were to increase as rapidly as those of most nations, such as india, south africa, and canada, the death rate is approximately three times slower than the infection rate. south korea is located at ( . , . ) far above the italy position ( , ), but below that of australia. although south korea initially had a faster increase in the infection number (β kr c < ) than that of italy, their death rate significantly decelerates after day in figure (c) . three nations, france, mexico, and the us, are below the diagonal line. although the positions of these three nations are still near the diagonal line, their death rates are of concern because of β d < β c . for more meaningful analyses, various domestic conditions in the nations should be considered systematically in addition to the actual number of ccc and ccd; however, such analysis is beyond the scope of this research. the present study used a mathematical transformation to identify universalities among many nations (on multiple continents) lacking apparent similarities in population, land size, and socioeconomic conditions. two time-related parameters were newly introduced in this study for the ccc ccd: the time-shifting parameter m and the time-scaling parameter β. these parameters move a nation's cumulative profile to a new time-origin and allow the nation's profile to be matched to italy's baseline by stretching or shrinking the profile (anchored at the new origin) along the time coordinate (i.e., x−axis). the m and β values were obtained individually for the ccc and ccd of each nation and used to define the reduced time τ . by transforming a nation's data relative to italy's baseline ccc and ccd, the short-term estimation of cumulative profiles becomes possible, and the results can be used for broad types of decisionmaking. because the large number of individual nations and territories where the covid- pandemic caused severe public health problems, the current study is restricted to the time series of the ccc and ccd of independent nations within g (excluding the eu). the primary research idea originated from the sequentially ordered patterns of ccd time series found in six nations on the european continent during the early stage of the pandemic spread, i.e., within days. with the transformation methods of reduced time, both the ccc and ccd profiles of the five european nations converged to those of italy, which were used as baselines for the rest of the present study. exceptions observed were china, south korea, and the us, owing to their noticeable deviations from the ccc and ccd profiles from those of italy. when the transformation of the cumulative data was extended to all g nations, the universality of the profile convergence was found to be valid for as many as nations within g , excluding the three exceptions above and the reference country, italy. the common plateau profiles of china's ccc and ccd, reached in the middle of february , showed early deviation from italy's baseline profile. south korea's ccc profile appeared to be a down-sized version of china's, representing only a small number of new recent cases per day. if the ccc profile is assumed to be a good precursor of incoming ccd, south korea's ccd profile already have appeared to deviate from that of italy (see figure ) . on a linear time-scale, the ccc and ccd profiles of the us already exceeded those of italy in late march and early april, respectively, but the us profiles in the reduced time-scale became the world largest values much earlier than those observed on the linear time-scale. visual investigation of the transformed ccc and ccd profiles implied that, unlike those of other nations, the us profiles intrinsically did not follow italy's profiles but increased much faster over the most recent ccd and ccc values of italy. a rough but conservative prediction of the us's ccc is approximately five fold higher than that of italy, a result similar to the us-to-italy population density ratio. more importantly, for future responses to a similar pandemic spread, i emphasize new fundamental insights obtained and findings from the transformation model developed herein. the ccd and ccc profiles of different nations show subtle but distinct characteristics in their onset behaviors. the six nations in europe had drastic increases in ccd immediately after their onsets. only france and russia showed graphically recognizable ccd latency periods. however, five nations in europe (except turkey) showed a longer latency period of ccc, close to four weeks. for the six nations in europe, the threshold values of ccc and ccd were on the order of o ( ) and o − , respectively, which correlated with the fatality rate of covid- , close to . % as of april and may, . similar characteristics of the latency periods and threshold values of ccc and ccd were found for all g nations, implicitly including e nations. first, in general, the ccc profile of a nation usually has a longer latency period and a higher threshold value than those of ccd of the nation, respectively. second, there exists a baseline ccc and ccd profiles of a nation whose status are more severe than that of any other nation in the initial pandemic period of at least days; this nation was italy in the current study. the three outlier nations whose ccd/ccc profiles did not converge to those of italy were the us,china, andsouth korea. on the basis of visual investigation of transformed ccc/ccd plots of these nations, the ccc/ccd of the us, which already exceed those of italy, is expected to increase much faster than those of any other nations, to an unprecedented level. the ccc and especially the ccd of south korea have almost certainly already stabilized to seemingly constant values, thus indicating that the number of new confirmed cases per day should decrease down to one-or two-digit numbers. profiles of china has the longest time series with an abrupt daily jump of , in ccd but no similar variations in its ccc. in recent engineering disciplines, big data research and applications have become an essential component of future development. in this regard, two representative approaches are data-driven and data-oriented: in the former, progress is compelled by data, excluding human inputs, whereas the latter is originally intended to optimize software programs against the object-oriented programming of a poor data locality. in decision-making processes during the current global health crisis, a novel paradigm should include the principal advantages of the robust data structures of datadriven approaches, relaxing the excessive dependence on data and ensuring efficient data-utilization in data-oriented approaches for prompt, accurate decision-making. in addition, data-informed approaches can be also considered to create proper balances in decision-making by fully utilizing demonstrated knowledge, experience, skills, and predictive ability. in conclusion, the current study of the mathematical transformation (i) combined three data-utilization approaches by using the pandemic data from ecdc on human infection and death caused by covid- without any mining processes, (ii) developed program scripts by using open-sourced software, and (iii) intuitively applied basic principles of statistical physics to pandemic time series analyses. new outcomes of the current work are the developed transformation model of the reduced time τ with two parameters of m and β, tabulated for the ccc and ccd time series of a total of nations. specifically, this study suggests that the universality found by using the transformation model can be effectively and efficiently applied in public health policy-making for any nations whose ccc and ccd profiles follow those of the leading nation, i.e., italy. because neighboring nations have similar trends in ccc and ccd propagation over time, close international collaborations involving sharing of human and medical resources would effectively decelerate the pandemic spread and risk to human lives. ask designed the study, collected the data, developed scripts of open-sourced software, analyzed and interpreted the data using the scripts, and wrote the manuscript. the author declares no competing interests. this paper was reviewed by thomas c. hardy, a professional medical editor, for accuracy and correct language use. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint table for details.) the double arrow toward the end-point of italy's profile in (a) indicates the estimated time distance for the u.k. to reach the same ccc level as that of italy. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint and ccd have gradually exceeded those of italy so that the current model with italy as the reference country might not be as predictable as the present analysis. a population-based pandemic dynamics model is of great necessity. table for details.) the double arrow toward the end-point of italy's profile in (a) indicates the estimated time distance for the u.k. to reach the same ccc level as that of italy. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint novel coronavirus -china, disease outbreak news first-wave covid- transmissibility and severity in china outside hubei after control measures, and second-wave scenario planning: a modelling impact assessment. the lancet impact assessment of non-pharmaceutical interventions against coronavirus disease and influenza in hong kong: an observational study. the lancet public health a simulation of a covid- epidemic based on a deterministic seir model. arxiv modelling transmission and control of the covid- pandemic in australia. arxiv covid- national incident room surveillance team. covid- so what?". the lancet diagnosis and management of first case of covid- in canada: lessons applied from sars critical review of the present situation of corona virus in china epidemiology of corona virus in the world and its effects on the china economy sustaining containment of covid- in china. the lancet covid- : why germany's case fatality rate seems so low cluster of covid- in northern france: a retrospective closed cohort study review and analysis of current responses to covid- in indonesia: period of india under covid- lockdown. the lancet similarity in case fatality rates (cfr) of covid- /sars-cov- in italy and china estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship transmission potential and severity of covid- in south korea predicting covid- distribution in mexico through a discrete and time-dependent markov chain and an sir-like model. arxiv a data driven analysis and forecast of an seiard epidemic model for covid- in mexico. arxiv covid- as a tool of information confrontation: russia's approach. ssrn electronic journal covid- : preparing for superspreader potential among umrah pilgrims to saudi arabia. the lancet preparedness and vulnerability of african countries against importations of covid- : a modelling study. the lancet looming threat of covid- infection in africa: act collectively, and fast. the lancet impact of weather on covid- pandemic in turkey. science of the total environment universal weekly testing as the uk covid- lockdown exit strategy. the lancet covid- in the usa: a question of time. the lancet clinical and virological data of the first cases of covid- in europe: a case series. the lancet infectious diseases the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak risk for transportation of coronavirus disease from wuhan to other cities in china. emerging infectious diseases rapidly increasing cumulative incidence of coronavirus disease (covid- ) in the european union/european economic area and the united kingdom estimating number of cases and spread of coronavirus disease (covid- ) using critical care admissions predicting the cumulative number of cases for the covid- epidemic in china from early data. arxiv strong correlations between power-law growth of covid- in four continents and the inefficiency of soft quarantine strategies. chaos: an inter- disciplinary journal of nonlinear science effects of chinese strategies for controlling the diffusion and deterioration of novel coronavirus-infected pneumonia in china. medrxiv free software foundation. bash (born again shell) ( . . ) gnu octave version . . manual: a high-level interactive lan- guage for numerical computations the document foundation, libreoffice [free office suite a two-interface transport model with pore-size distribution for predicting the performance of direct contact membrane distillation (dcmd) key: cord- - e rj authors: barone-adesi, francesco; ragazzoni, luca; schmid, maurizio title: investigating the determinants of high case-fatality rate for coronavirus disease in italy date: - - journal: disaster medicine and public health preparedness doi: . /dmp. . sha: doc_id: cord_uid: e rj case-fatality rate (cfr) for covid- in italy is apparently much higher than in other countries. using data from italy and other countries we evaluated the role of different determinants of this phenomenon. we found that the italian testing strategy could explain an important part of the observed difference in cfr. in particular, the majority of patients that are currently tested in italy have severe clinical symptoms that usually require hospitalization and this translates to a large cfr. we are confident that, once modifications in the testing strategy leading to higher population coverage are consistently adopted in italy, cfr will realign with the values reported worldwide. t he epidemiologic features of the recent pandemic of coronavirus disease (covid- ) have been shown to relevantly vary among countries, with substantial differences in terms of incidence, mortality, and case-fatality rate (cfr), a parameter whose crude estimation is obtained by dividing the number of deaths by the total number of cases. in particular, a substantially higher cfr for covid- was reported for italy compared with other countries, and several authors put forward different hypotheses to explain it. [ ] [ ] [ ] using data from china and italy, paper suggested that this phenomenon could be due to mainly different factors : ( ) a diverse age distribution; ( ) a different definition of covid- -related deaths; and ( ) a countryspecific strategy in testing. expanding on this, we tried to single out the effect coming from each of the suggested causes. we used direct standardization and applied chinese age-specific risk displayed in the onder et al. study to the italian age structure to accommodate the possible effect of age. as expected, the corresponding standardized cfr was higher than that shown in the original article ( . % as compared with . %). however, it was still far from the currently reported in italy ( . %), thus suggesting that the different age distribution between the samples accounts for only a part of the difference in the cfr. moreover, this would hardly explain why the italian cfr is also substantially higher than that of countries, such as greece, portugal, and germany, which share a very similar age structure with the italian population. regarding the definition of covid- -related deaths, the criteria used by each country are often not clearly defined, making comparisons difficult. however, this might not apply to the comparison between italian and chinese data used in the onder et al. study, because all confirmed cases of patients who eventually died were considered as covid- -related deaths in china, a definition very similar to that used in italy. on the other hand, we think that the italian testing strategy could explain an important part of the observed difference in cfrs. the majority of patients who are currently tested in italy have severe clinical symptoms that usually require hospitalization. indeed, the proportion of positive patient-cases that are admitted to the hospital in italy is about % (and used to be much higher in the past weeks), whereas it was about - % in china. as the positive cases resulting from this testing strategy are so skewed toward more serious conditions, it is not surprising that such a high cfr is observed. this phenomenon could also explain the substantial heterogeneity of cfrs observed among italian regions, which can independently determine their own testing strategy. figure shows a clear association between the proportion of hospitalized cases and cfrs. we are confident that, after modifications in the testing strategy leading to higher population coverage will be consistently adopted in italy, cfrs will realign with the values reported worldwide. cross-country comparison of case fatality rates of covid- /sars-cov- . osong public health res perspect case-fatality rate and characteristics of patients dying in relation to covid- in italy as covid- cases, deaths and fatality rates surge in italy, underlying causes require investigation monitoring transmissibility and mortality of covid- in europe italian civil protection. daily report of cases in italy estimates of the severity of covid- disease the authors have no conflicts of interest to declare. fba, lr, and ms conceived the study, processed data, and drafted the final version of the manuscript. the study was based on publicly available aggregate data. no ethics committee approval was necessary. key: cord- -scg h b authors: atzori, l.; mugheddu, c.; addis, g.; sanna, s.; satta, r.; ferreli, c.; atzori, m.g.; montesu, m.a.; rongioletti, f. title: psoriasis health care in the time of the coronavirus pandemic: insights from dedicated centers in sardinia (italy) date: - - journal: j eur acad dermatol venereol doi: . /jdv. sha: doc_id: cord_uid: scg h b psoriasis is a major chronic inflammatory skin disease, affecting about % of the population in italy, whose management require experienced specialists in order to guarantee high‐quality standards of care. the pandemic coronavirus ( ‐ncov; covid‐ ) has changed the approach to all patients requiring close contact during a visit, including dermatologic consultations. in italy, true outbreak begun in lombardy, by february , with exponential contagion, surpassing china in the number of deaths. this article is protected by copyright. all rights reserved copyright: the authors certify that the manuscript is original, never submitted to other journal for publication before. all authors contributed equally to the manuscript and had the opportunity to revise and approve the final text. dr. l atzori has nothing to disclose dr. mugheddu has nothing to disclose. dr. addis has nothing to disclose. dr. sanna has nothing to disclose. dr. satta has nothing to disclose. dr. ferreli has nothing to disclose. dr. mg atzori has nothing to disclose. dr. montesu has nothing to disclose. dr. rongioletti has nothing to disclose. dear editor, psoriasis is a major chronic inflammatory skin disease, affecting about % of the population in italy, whose management require experienced specialists in order to guarantee high-quality standards of care. the pandemic coronavirus ( -ncov; covid- ) has changed the approach to all patients requiring close contact during a visit, including dermatologic consultations. in italy, true outbreak begun in lombardy, by february , with exponential contagion, surpassing china in the number of deaths. the health care system was overwhelmed, best hospitals in northern italy unable to cope with the huge number of desperately ill patients. concern about the impact on the south was great due to the chronic shortage of facilities and limited intensive care equipment. in sardinia, with a population of inhabitants, the institutional recognition found only intensive care beds available to welcome covid- cases. a strategic plan will increase the number to beds, but full implementation would require time (resolution of the regional council / of the / / ). as isolation is the main weapon to control the spread, and on march , a decree of the president of the council of ministers ordered the suspension of all outpatients' services, including clinics for psoriasis patients. unfortunately, thousands of patients were suddenly deprived of dermatologic care. the dedicated psoriasis clinics in the major hospital of cagliari, nuoro, and sassari account approximately patients. about % of them afflicted with severe psoriasis and psoriatic arthritis, requiring systemic treatments or phototherapy (table ) . this article is protected by copyright. all rights reserved we began calling all patients with scheduled visits and programmable procedures to remain at home, providing telephone consultation and counselling. patients were completely unprepared, upset, and disoriented by the lock down. some patients could not understand the crisis and demanded full attention, quite aggressively. other patients were more understanding. only patients requiring dose adjustment and/or blood chemistry controls were admitted, as well as patients requiring infusions, such as infliximab. the access to the phototherapy service was also maintained for selected cases. on march , the italian medicines agency (aifa) allowed the automatic renewal of all expiring therapeutic plan, required for biotechnological drugs, preventing patients from referral only to have their prescription renewed. as awareness of the severity of the covid- increased some patients were concerned about continuing their biologics. we followed the sidemast recommendations, and advised patients not to dismiss their drugs without consultation, especially biologics. clinical trials were continued for patients already enrolled; but new screening was discontinued, and prudently, not new treatment was started. patients were admitted only after a strict triage: body temperature was registered, and was required the compilation of a questionnaire, on recent symptoms (fever, cough, breathing difficulties) or travels outside sardinia. a quarantine of two weeks had become mandatory from march , . thankfully, sardinia covid- cases are small in magnitude, but we hold an unpleasant record. on march , the number of infections among health personnel was higher ( %) than the national average of - %. . in this uncomfortable setting, a covid- patient indicated the skin consultation at the dermatology clinic of sassari as the possible source of contagion. all the health personnel resulted negative for the infection, but we clearly felt the risk of being considered "spreaders" for the community, instead of essential care providers. we were more exposed to involuntary contagion from asymptomatic patients, and not provided with the necessary personal protective equipment. as dermatologists are specialists not directly involved in the management of critical patients, the supply of specific protective equipment against bio-hazards, as well as of environmental sanitizers, was very limited, and the massive consumption from critical care wards depleted all stocks. thus, all not essential sanitary personnel were placed on vacation, but with the possibility of being called back into service to support other critical specialties. educational programs for medical students, graduate and resident have been interrupted. effects of pandemic coronavirus infection are without precedents. restrictive measures were mandatory, and dermatologists had to adapt: more counselling to support patients, detect unmet needs, and find ways to reassure patients about their disease, to keep them safely home. italian guidelines on the systemic treatments of moderate-to-severe plaque psoriasis covid- in italy: momentous decisions and many uncertainties emergency management for preventing and controlling nosocomial infection of novel coronavirus: implications for the dermatology department istituto superiore sanità), epidemia covid- this article is protected by copyright. all rights reserved key: cord- -xbji g x authors: uccelli, matteo; cesana, giovanni carlo; de carli, stefano maria; ciccarese, francesca; oldani, alberto; zanoni, adelinda angela giulia; giorgi, riccardo; villa, roberta; ismail, ayman; targa, simone; d’alessio, andrea; cesana, giancarlo; mantovani, lorenzo; olmi, stefano title: covid- and obesity: is bariatric surgery protective? retrospective analysis on patients undergone bariatric-metabolic surgery from high volume center in italy (lombardy) date: - - journal: obes surg doi: . /s - - -z sha: doc_id: cord_uid: xbji g x introduction: on february , , a severe case of pneumonia due to sars-cov- was diagnosed in northern italy (lombardy). some studies have identified obesity as a risk factor for severe disease in patients with covid- . the purpose of this study was to investigate the incidence of sars-cov- infection and its severity in patients who have undergone bariatric surgery. material and methods: during the lockdown period (until may ), we contacted operated patients by phone and social networks (e.g., facebook) to maintain constant contact with them; in addition, we gave the patients a dedicated phone number at which to call us for emergencies. we produced telemedicine and educational videos for obese and bariatric patients, and we submitted a questionnaire to patients who had undergone bariatric surgery in the past. results: a total of patients ( male; female) replied to the questionnaire. mean presurgical bmi: . ± . kg/m( ). mean age: . ± . year. mean bmi after surgery: . ± . kg/m( ) (p < . ). from february to may , . % of patients reported that they suffered from at least one symptom among those identified as related to sars-cov- infection. thirteen patients ( . %) tested positive for covid- . six patients ( . %) were admitted to the covid department, and patients ( . %) were admitted to the icu. conclusions: although the reported rates of symptoms and fever were high, only . % of patients tested positive for covid- . among more than patients who underwent bariatric surgery analyzed in this study, only . % needed icu admission. supplementary information: the online version contains supplementary material available at . /s - - -z. the spread of coronavirus disease (covid- ) has reached-from the beginning of march-the epidemiological criteria to be declared a pandemic (affecting more than , people in countries) [ ] . only cases of covid- were identified in italy in the first half of february , and all involved people who had recently traveled to china. on february , , a severe case of pneumonia due to sars-cov- (severe acute respiratory syndrome-coronavirus ) was diagnosed in northern italy's lombardy region in a man in his thirties who had no history of possible exposure abroad. within days, many other cases of covid- in the surrounding area were diagnosed, including a substantial number of critically ill patients [ ] . italy, as reported in the data of the istituto superiore di sanità, had , confirmed cases and , deaths as of june [ ] . the mean age of patients dying from sars-cov- infection was years. the most common comorbidities diagnosed before sars-cov- infection were hypertension ( . %); type diabetes ( . %); ischemic heart disease ( . %); atrial fibrillation ( . %); chronic renal failure ( . %); chronic obstructive pulmonary disease ( . %); supplementary information the online version contains supplementary material available at https://doi.org/ . /s - - -z. heart failure ( . %); and obesity ( . %). a total of . % of the deceased patients had or more comorbidities simultaneously [ ] . based on the number of cases and advanced stage of the disease, it has been hypothesized that covid- has been circulating within the population since january [ ] . although the effects of covid- in patients with obesity have not yet been well described, it has been suggested [ ] that there is a need for increased vigilance, prioritization of detection and testing, and aggressive therapy for patients with obesity and covid- . some studies have identified a higher body mass index (bmi) as a risk factor for severe disease in patients with covid- [ , ] . patients with obesity have reduced chest wall elastance and lower total respiratory system compliance with a decreased expiratory reserve volume. difficult airway management, as well as altered physiology in combination with positional gas trapping, is routinely encountered in such patients [ ] . in clinical practice, the need for a ventilatory support/icu bed is one of the main problems in the management of the covid- pandemic [ , ] . there are also emerging data indicating that obesity is an independent predictor of intensive care unit (icu) admission, mechanical ventilation, and death [ , , ] , and in a recent report from a large cohort of covid- patients in new york, obesity was found to be one of the most common associated comorbidities in hospitalized patients [ , ] . during the outbreak of the epidemic, our hospital found itself in the italian epicenter: lombardy, and in particular the area near the city of bergamo. we surgeons have been involved, like all physicians, in the management of covid patients in our hospital, which was transformed into a covid hospital within a few days. during this period, we treated covid- patients who had undergone bariatric surgery. in particular, we provided treatment for covid- ex-bariatric patients operated on at our center and another ex-bariatric patient operated on at another center. we noted in these patients that the response to therapy was good, despite the severity of the infection. we therefore analyzed a significant number of patients to evaluate the spread and the effects of the sars-cov- infection in a population of patients who had undergone bariatric surgery. study design this study was a cross-sectional retrospective review of patients undergoing bariatric surgery (both primary and redo-surgery) for morbid obesity at the same bariatric surgery centre from december to march . starting from march to june in italy, there were limitations due to the covid epidemic regarding traveling from city to city and from region to region; in addition, there were limitations in accessing the hospitals and medical practices themselves. to promote continuity in the follow-up of our bariatric patients, we had to shift from in-person to remotely delivered bariatric care by telemedicine, according to the recent recommendations for metabolic and bariatric surgery during the covid- pandemic from ifso [ ] . we implemented several methods of remote communication with our patients: patients were provided a phone number to call in case of obesity-related questions. a surgeon was available at that number every day from p.m. to p.m. all patients were usually given the email address of our service at the time of discharge and were encouraged to send emails in case of any concerns. the e-mails were read by surgeons, and an answer was soon sent to each patient, whether the request was for medical advice or for general information. obesity-related and bariatric-related groups have been in contact on social networks (e.g., facebook, "chirurgiapoliclinicosanmarco"), through both our official social page and through self-help groups of obese patients and their associations (e.g., "apslarinascita", "la rinascita onlus", "sleeve la rinascita"); we also provided patients with the phone numbers of dieticians and psychologists. educational videos were edited and posted by surgeons to these groups, sharing updated information about covid- at our hospital, providing general information about eating behavior, and focusing on postoperative signs and symptoms that patients must take into account for contacting doctors. a survey consisting of questions (supplementary information -attachment ) was provided to patients electronically, giving the opportunity to respond in compliance with the rules in force in italy in april. the italian regulation indicated by the government prevented nonurgent outpatient visits from being carried out and made it impossible to travel from one's own town of residence, except for absolutely necessary and deferred reasons. the questionnaire was administered via social networks and through the mentioned self-help associations. to ensure the correct compilation of the questionnaire, dedicated videos were produced by the promoters of the survey. all patients had morbid obesity before surgery. data concerning patient demographic variables, age, gender, body mass index (bmi kg/m ), italian region of residence, comorbid conditions, and type of surgery, were collected. the preoperative characteristics of the population are summarized in table . we have considered the most common clinical presentations of covid- [ ] [ ] [ ] [ ] [ ] ; to avoid possible compilation errors by patients, we preferred to omit the symptoms related to loss of taste and smell, as bariatric patients often present these symptoms immediately and long after bariatric surgery [ , ] . we published the questionnaire and informed the patients on april at noon. the questionnaire was available for days until april at midnight. each operated patient could complete the questionnaire only once, answering all the questions anonymously. continuous demographic variables are expressed as the mean ± standard deviation and range. categorical variables and complications are reported as numbers and percentages. data were processed by xlstat to perform statistical analyses. statistical significance was predetermined as p < . . preoperative data patient characteristics and preoperative assessments are summarized in table comorbidities and follow-up are summarized in table . with an average follow-up of . ± . months (range: . - . ), we found an average bmi of . ± . kg/m (p < . ). reductions in the number of comorbidities were almost all statistically significant (graph ). a total of ( . %) patients were prescribed home isolation following the development of symptoms or contact with covid- patients (fiduciary home quarantine). a total of . % ( ) of the patients reported having had a fever above . °c. a total of patients ( . %) reported coughing, respiratory symptoms, or dyspnea. in total, patients ( . %) reported having had at least one symptom among those identified as related to sars-cov- infection. nevertheless, testing for covid- was performed only in cases ( . %). thirteen patients ( . %) tested positive for covid- (table ) , nine in the northern section of the country, where the epidemic has been more intense, and four in the south-central region. these covid- -positive patients were initially treated at home in cases ( . %). the average duration of home treatment was . ± . days. six patients ( . %) were admitted to the covid department. the average length of hospitalization at covid hospital was . ± . days. in two cases ( . %), patients were admitted to intensive care. both of these patients were intubated with invasive mechanical ventilation. the average duration of inva- coronavirus disease- (covid- ) is an infectious disease caused by the recently discovered coronavirus sars-cov- [ ] . the first case of covid- was reported to the world health organization (who) by chinese authorities on december , , as a result of a patient experiencing pneumonia in wuhan city, hubei province. following a rapid spread in china, new outbreaks occurred first in northern italy and then in several european countries. the spread in italy has been intense since january [ ] , and individuals with obesity, particularly its complications, such as diabetes and hypertension, might be more liable to develop serious illness, requiring hospital admission and invasive ventilation [ ] . graph follow-up: comorbidities, *p < . . gerd, gastroesophageal reflux disease from a strictly bariatric point of view, our patients reported weight loss and remission of comorbidities that fall within the normalcy [ ] . a total of . % of patients reported at least one covid- -related symptom. however, almost all patients ( . %) did not undergo any covid- testing. although this report highlights a possible underestimation of the spread of the infection in italy, few patients required home or hospital treatment, and even fewer ( . %) developed severe forms requiring intensive care. these data confirm what we noticed during the period of assistance to postbariatric covid- patients [ ] , with good response and resolution of pneumonia. hajifathalian et al. [ ] reported that obesity was associated with increased risk of icu admission or death whereas being underweight was not. the comparison, given the small size of our sample of patients who tested positive for the covid- test, is partial. however, in our patients, there were positive results for the infection ( patients), with less than half hospitalized. the icu admission rate in our case series (only two patients) was also lower than that in the literature data ( . - . %) [ , ] . stratifying by bmi, the literature data report icu admission rates greater than % for patients with bmi > [ ] . therefore, our data are encouraging, considering that these patients were obese: bariatric surgery and the consequent weight loss seem to significantly lower the risk of serious consequences due to covid infection. bariatric surgery, therefore, can be considered a protective factor with respect to the onset of severe respiratory disease resulting from sars-cov- infection and probably other respiratory diseases. furthermore, considering the changes in quality of life in the patient undergoing bariatric surgery [ ] , the weight-loss patient becomes a socially active individual who is more exposed to contacts with covid- -positive individuals. especially in the first phase of the pandemic in italy, when no social restriction measures were adopted, covid- circulated freely [ ] . our large population of potentially exposed postbariatric patients did not show a higher incidence than the general population, especially in forms of more serious infections [ ] [ ] [ ] . a strength of this study is the population size (over patients). furthermore, another strong point is the geographical distribution of the patients, who-although operated on and followed by a single bariatric center-came from areas all over italy with different diffusions of covid- in the january-may period. as already pointed out, the analyzed population is socially active, as often happens to ex-obese patients currently losing weight. the main limitations of the study were ( ) the unavailability of a comparable control group of obese patients not undergoing bariatric surgery and ( ) the use of a survey performed with a questionnaire without the possibility of a direct clinical and diagnostic evaluation. although the reported rate of symptoms was high, only . % of patients tested positive for covid- . out of more than patients analyzed in this study who underwent bariatric surgery, only patients ( . %) needed icu admission. bariatric surgery, therefore, can be considered a protective factor with respect to the onset of severe respiratory disease resulting from infection with sars-cov- . conflict of interest the authors declare that they have no conflict of interest. ethical approval study did not require approval from the ethics committee and for this type of study formal consent is not required. human and animal rights and informed consent this article does not contain any studies with human 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outbreak size in italy publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -e pwgnx authors: martelloni, gabriele; martelloni, gianluca title: modelling the downhill of the sars-cov- in italy and a universal forecast of the epidemic in the world date: - - journal: chaos solitons fractals doi: . /j.chaos. . sha: doc_id: cord_uid: e pwgnx in a previous article [ ] we have described the temporal evolution of the sars-cov- in italy in the time window february -april . as we can see in [ ] a generalized logistic equation captures both the peaks of the total infected and the deaths. in this article our goal is to study the missing peak, i.e. the currently infected one (or total currently positive). after the april the large increase in the number of swabs meant that the logistical behavior of the infected curve no longer worked. so we decided to generalize the model, introducing new parameters. moreover, we adopt a similar approach used in [ ] (for the estimation of deaths) in order to evaluate the recoveries. in this way, introducing a simple conservation law, we define a model with populations: total infected, currently positives, recoveries and deaths. therefore, we propose an alternative method to a classical sird model for the evaluation of the sars-cov- epidemic. however, the method is general and thus applicable to other diseases. finally we study the behavior of the ratio infected over swabs for italy, germany and usa, and we show as studying this parameter we recover the generalized logistic model used in [ ] for these three countries. we think that this trend could be useful for a future epidemic of this coronavirus. we briefly review the historical evolution of the sars-cov- in the earth. in early december the sars-cov- appeared in wuhan, china. the disease caused by the new coronavirus has a name: "covid- " (where "co" stands for corona, "vi" for virus, "d" for disease and " " indicates the year in which it occurred). the oms director-general tedros adhanom ghebreyesus announced it on february , , during the extraordinary press conference dedicated to the virus. the appearance of new pathogenic viruses for humans, previously circulating only in the animal world, is a widely known phenomenon (called spill over) and it is thought that it may also be at the basis of the origin of the new coronavirus (sars-cov- ). the scientific community is currently trying to identify the source of the infection. on december , , the municipal health commission of wuhan (china) reported to the oms a cluster of cases of pneumonia of unknown etiology in the city of wuhan, in the chinese province of hubei. on january , , the chinese center for disease prevention and control (cdc) reported that a new coronavirus (initially called -ncov and now called sars-cov- ) has been identified as the causative agent and has been rendered publishes the genomic sequence. oms on march , declared that covid- can be defined as a pandemic. after notification of the epidemic by china, italy immediately recommended postponing unnecessary flights to wuhan and, subsequently, with the spread of the epidemic, to all of china. consequently, the latter has canceled all flights from wuhan. this disease does not save italy that has become a protected area with the dpcm signed on the evening of march by the prime minister, giuseppe conte, who has extended the restrictive measures already applied for lombardy and the northern provinces most affected by the coronavirus infection to the whole national territory. the new action comes into force on march and will take effect until april . among the main innovations: it limits the movement of people, blocks sporting events, suspends teaching activities in schools and universities throughout the country until april . with the new ordinance of march issued by the minister of health and the minister of the interior, from march people are prohibited from moving with public or private trasportation in a municipality other than that in which they are located, except for proven work needs, absolute urgency or for health reasons. [ ] many growth models have been very recently applied to study the evolution of the covid- infection [ , , , , , , , ] . in [ ] we tried to analyze the time evolution of the sars-cov- in italy, using a logistic model [ ] at the beginning of the study and after with a generalization of that model. the logistic behaviour assumes that growth stops when maximum sustainable population density is reached through the carrying capacity k that depends on the environmental conditions. for example the ordinances of the prime minister g.conte, the people's hygiene habits are encoded in the carrying capacity k. we observe as the generalized model of [ ] works very well until the april . after this date the large increase in the number of swabs meant that the logistical behavior of the infected curve no longer worked. at first in italy, pharyngeal swabs were initially made only on seriously ill people. this choice gave us the possibility to have a sample of the infected that we can describe with a single population model, after april it becomes impossible. so we decided to use a different model to describe the new trend of the data and try to give different scenarios of the descent phase of the virus in italy, in the time window february -may . in [ ] we described two different peaks, the peak of the infected and the deaths one. in this paper we analyze the peak of the currently infected and the downhill of the propagation of the sars-cov- . to do this we define a new model similar to a sird (see for example [ ] ), but without the population of supsceptibles, because there are no criteria on defining the susceptible ones. we consider three couple differential equations for infected i(t), deaths d(t) and recovery r(t) with the following conservation law where p (t) represents the currently infected (or positive). in the last part of this article we observe as the following ratio (infected i(t i ) over swabs s(t i )) is the most important parameter to describe the evolution of the sars-cov- . indeed, we can describe the trend of this quantity only with a generalized logistic model with parameters even with data after april . this behavior suggest us to use this model for a future epidemic of this virus. if we will able to perform a greater and constant number of swabs everyday, using this model, we may have better control over the contagion curve, and consequently over the number of deaths. our idea is to use a model that adapts to the data of the problem. we explain better. let's consider the following data: some comments about these data: the points ) and ) describe perfectly that the sample of infected is not clean; at the beginning of the contagion the swabs are performed only on the severe infected, after month the number of swabs are increased of a factor and consequently also the midly infected are detected. point ) tells us that there is probably an incredible number of asymptomatics as a source of severe infected, we have no control about it. points ), ) indicate that while the death data is under control, the healed data are very oscillating in time. finally the points ) tells us that contribution of asymptomatics, portrayed in [ ] , changes in time, indeed from april - ( - days after the second ld, i.e. an incubation time ) the generalized logistic description fails. after these considerations we have decided to couple the following equations: with a conservation law where p (t) represents the currently infected (or positive). the parameters r represents the rates of growth of epidemic, k is the carrying capacity for the classical logistic model, α is a constant in order to have a power low initial growth before ld, β is the exponent of the second term of equation that represents the influence of asymptomatic; δ,a correction of the quadratic term of logistic, and γ are the constant parameters considering the influence of the government measures , k f is a proportionality constant between deaths and total number of infected, while t d and t r are the delays of deaths and recoveries respect to infected respectively; the constant a represents the contribution of asymptomatic people as introduced in [ ] and finally t is the time of ld start. a brief consideration about the function f (t): the great variability of t r suggest us that only the parameter t r is not sufficient to describe correctly the function r(t), so we decided to introduce a coefficient time dependent. we present two different scenarios, in fig. we consider a linear approximation f (t) = a + bt, while in fig. we consider a quadratic approximation f (t) = a + bt + ct . this choice is not random. indeed, considering the behaviour of the recovery time series in which a single recovery can heal with some delay in a window variable from few days to two months, the correct modeling could be a regressive linear function of type r(t) = n i= a i * i(t − t i ) (eventually introducing also no-linear term in the series), but in this way we introduce many degree of freedom how many are the coefficient a i of the regressive function. therefore, we consider an approximation using the two functions f (t) considered above. we desumed the following values for the principal parameters by means of stochastic simulation using direct method gillespie algorithm adapted to nonautonomous differential equations: t r = ± for quadratic approximation. some comments about these values: with respect to [ ] we observe that the total number of infected, positive, recovery and dead figure : the scenario with a quadratic growth for the recoveries: the black curve represents the deaths, the red one for the infected, the green one for the recovery and the pink one for the currently infected. peak of the severe infected is correctly estimated, i.e. t = − march and also the peak of the deaths, i.e. t = − march; also the time delay t d remained the same; the same t r approaches the experimental lower limit in quadratic approximation. with respect to the logistic model r is increased while the coefficient δ drops from the value to the value . , i.e. we are considering different models. in fig. - we observe as the peak of currently infected is close to april and finally we give us our prevision for a linear growth for the recoveries close to july ; for a quadratic growth we have close to june . the estimated numbers i(end) and d(end) are very close, but it is not surprising: the eqs. ( ) and ( ) for total infected head the model, while f (t) is present in eq. ( ) that is only a proportionality equation. obviously a linear approximation for f(t) leads to a slower recovery curve and therefore a small increase of infected. now we consider the following parameter: that represents the number of infected normalized with the number of swabs s(t i ). we study this quantity with generalized logistic equation used in [ ] : where α, r , k and a have the same meaning used in the previous section. compared to the previous section we observe as studying the parameter i norm (t i ) we can describe the contagion with a simple logistic equation and without the phenomenological terms introduced in eqs.( )- ( ) . in order to calibrate this model in the best way possible we use two algorithms, the first one based on simulated annealing [ ] and the second one on optimized simplex [ ] . we evaluate the function error defined as where x i is the real data at day i, y i (p) is the correspondent output of the model depending of vector parameter p and w i is a generic weight that we can use or can be equal to one. for our purpose we adopt as weight the derivative of data or the data at time (day) i: the use of derivative allows to calibrate better on average the curve, while the use of the data as weight permit to calibrate better the data of the last part of the curve. in fig. - cumulative rate real data model error + % error - % figure : the scenario of italy minus lombardia with the derivative weight. for the parameters of fig. r = . ± . , for the parameters of fig. r = . ± . , and finally for the parameters of fig. r = . ± . , we observe as the quantity i norm (t) is probably the most important quantity studying the evolution of the virus! we explain better: the contribution of asymptomatic people is essentially the same in lombardia and in the rest of italy, while the coefficient r is larger if we consider italy compared to the scenario of italy minus lombardy; this consideration is extremely coherent with the data: the infected of lombardia region represent the % of all the italian infected. moreover the ratio infected over swabs is a very reliable parameter, we can describe correctly the italian situation only with parameter and with a wellknown model. we stress that in the future if a nation is ready to carry out a large and constant number of swabs every day, using this model, we can have a reliable forecast of the epidemic! we consider also the scenario represented by eq. ( ) for germany in fig. and for usa in fig. . for germany we study the time evolution of the sars-cov- in the time window march -may and we obtain the following parameters for usa we study the contagion in the time window march -may and we have these values for the parameters speed. let's try to justify this idea: a different speed may depend on population density, work habits and the number of swabs at the beginning of the epidemic. about the last consideration we imagine to immediately carry out a large number of swabs: knowing as soon as possible the largest possible number of infected means limiting the contagion and therefore the propagation speed of virus. we described the evolution of the sars-cov- in italy in the time window february -may . to do this we have built a phenomenological growth model adapted on the data of civil protection. with respect to a classical sir(d) model we did not consider the supsceptible population, because there are not medical evidences on which sample of the population can be ill. so we have considered three couple differential equations for infected i(t), deaths d(t) and recovery r(t) with the a conservation law including the currently positive population p(t). as the time delay between the onset of symptoms and healing t r days is a very oscillating parameter we introduced a sort of regressive function f (t) to modelling better this delay. so we described two scenarios of the end of epidemic: • i(end) = , d(end) = , close to july , for f (t) linearly approximated, • i(end) = , d(end) = , close to june , for f (t) in a quadratic approximation. obviously a linear approximation for f(t) leads to a slower recovery curve and therefore a small increase of infected. in the second part of this manuscript we described the time evolution of the normalized data that represents the number of infected normalized with the number of swabs s(t i ). we have studied this parameter on four different scenarios: • italy, • data of italy minus data of lombardia ( about % of the italian infected belong to the lombardia region ), • usa, • germany. so we have found that all the evolutions are governed by the same generalized logistic equation [ ] , suggesting an universal feature of the propagation of sars-cov- virus. in particular the value of the parameter r is in descending order compatible with the respective apparent cfr ( acfr ) • for italy r = . and acf r = %, • for italy-lombardia r = . and acf r = %, • for usa r = . and acf r = %, • germany r = . and acf r = , %. finally we suggest that the data i norm (t i ) is the most important parameter to control the propagation of the virus for a new inauspicious propagation of this virus in the world, because, knowing its universal feature, we can forward know the number of infected preparing a relevant number of swabs. analysis of the evolution of the sars-cov- in italy, the role of the asymptomatics and the success of logistic model early phylogenetic estimate of the effective reproduction number of sars-cov- emerging coronaviruses: genome structure, replication, and pathogenesis data analysis on coronavirus spreading by macroscopic growth laws covid : an automatic, semiparametric estimation method for the population infected in italy analysis and forecast of covid- spreading in china a poisson autoregressive model to understand covid- contagion dynamics, ssrn -abstract-id= cdc covid- response team, severe outcomes among patients with coronavirus disease (covid- ) -united states how macroscopic laws describe complex dynamics: asymptomatic population and covid- spreading notice sur la loi que la population poursuit dans son accroissement on the nature of the function expressive of the law of human mortality and a new mode of determining life contingencies the simplex-simulated annealing approach to continuos non-linear optimization libelli parameter estimation of ecological models we thank many colleagues for interesting discussions, in particular andrea marzolla and domenico seminara. we also thank pierluigi blanc, s.o.c. infectious diseases santa maria annunziata hospital, for stimulating discussions on technical subjects on which we had no knowledge.the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- - s znn c authors: gallo, gaetano; sturiale, alessandro; de simone, veronica; mancini, stefano; di tanna, gian luca; milito, giovanni; bianco, francesco; perinotti, roberto; giani, iacopo; grossi, ugo; aiello, domenico; bianco, francesco; bondurri, andrea; gallo, gaetano; la torre, marco; milito, giovanni; perinotti, roberto; pietroletti, renato; serventi, alberto; fiorino, marina; de simone, veronica; grossi, ugo; manigrasso, michele; sturiale, alessandro; zaffaroni, gloria; boffi, ferruccio; bellato, vittoria; cantarella, francesco; deidda, simona; marino, fabio; martellucci, jacopo; milone, marco; picciariello, arcangelo; bravo, ana minaya; vigorita, vincenzo; cunha, miguel fernandes; leventoglu, sezai; garmanova, tatiana; tsarkov, petr; el-hussuna, alaa; frontali, alice; ioannidis, argyrios; bislenghi, gabriele; shalaby, mostafa; porzio, felipe celedon; wu, jiong; zimmerman, david; elbetti, claudio; mayol, julio; naldini, gabriele; trompetto, mario; sammarco, giuseppe; santoro, giulio aniello title: deadlock of proctologic practice in italy during covid- pandemic: a national report from proctolock date: - - journal: updates surg doi: . /s - - - sha: doc_id: cord_uid: s znn c proctology is one of the surgical specialties that suffered the most during covid- pandemic. using data from a cross-sectional worldwide web survey, we aimed to snapshot the current status of proctologic practice in italy with differences between three macro areas (north, centre, south). specialists affiliated to renowned scientific societies with an interest in coloproctology were invited to join a -item survey. predictive power of respondents’ and hospitals’ demographics on the change of status of surgical activities was calculated. the study was registered at clinicaltrials.gov (nct ). of respondents from italy, ( %) practiced in the north, ( %) in the centrer and ( %) in the south and islands. the majority were men ( %), at consultant level ( %), with a mean age of . years, practicing in academic hospitals ( %), where a dedicated proctologist was readily available ( %). southern respondents were more at risk of infection compared to those from the center (or, . ; %ci . ; . , p = . ), as were males (or, . ; %ci . ; . , p = . ) and those who routinely tested patients prior to surgery (or, . ; %ci . ; . , p = . ). the likelihood of ongoing surgical practice was higher in the south (or . , %ci . ; . , p = . ) and in centers that were not fully dedicated to covid- care (or . , %ci . ; . , p < . ). the results of this survey highlight important factors contributing to the deadlock of proctologic practice in italy and may inform the development of future management strategies. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. covid- pandemic has critically impacted the surgical world [ ] . more than million procedures would be cancelled or postponed during the -week peak according to a recent global expert-response study [ ] . the vast majority ( %) of operations would be treating benign diseases, with an estimated overall -week cancellation rate of %. this scenario has strongly challenged proctologic practice, which includes a large spectrum of conditions with a significant psycho-socio-economic burden [ ] . detection of the novel coronavirus (sars-cov- ) rna in patients' stool samples and gastrointestinal epithelium has led to enhance infection control precautions [ ] . consequently, several guidelines have been developed to optimize treatment strategies while ensuring healthcare workers' safety by means of adequate personal protective equipment (ppe) [ ] [ ] [ ] . however, the ever-changing situation observed in most countries has often hampered the attempts to put these guidelines into practice [ ] . proctolock is a survey aimed to assess the current status of proctologic practice worldwide. in our previous global report [ ] , the proportion of unaltered, reduced or fully stopped practice has been snapshotted in countries. the purpose of this study was to explore the impact of covid- on proctologic practice in italy, looking for differences between north, south and central regions. experts in the field who joined a previous qualitative study [ ] (n = ) were invited to complete a web survey. the survey link was sent to national scientific societies of interest to coloproctologists and disseminated to their members. all collaborators committed to further recruitment of participants by direct invitation. a -item survey (namely, 'proctolock ′; appendix ) was designed and developed by the authors using an online platform ('online surveys' [formerly bos-bristol online survey], developed by the university of bristol) in accordance with the checklist for reporting results of internet e-surveys (the cherries statement) [ ] . the finalized online survey was made available online from april th to th . the survey aimed to capture the current status of proctologic practice worldwide, first exploring the overall changes in terms of resource allocation, and second assessing in more detail the various fields of application for both proctologic surgery (i.e. elective [oncological and non oncological] and urgent) and outpatient practice, with a focus on sexually transmitted disease and pelvic floor clinics. the availability of anorectal physiology testing was also assessed. the study was registered at clinicaltrials. gov (nct ). logistic models for binary or ordinal variables were performed to assess the association between respondents' preferences and their characteristics (adjusted odds ratio [or]). multivariable models were fitted using a predefined set of covariates which included respondents' and hospitals' demographics (i.e. geographical area, age, gender, type of hospital, hospital rearrangement, external facilities for proctologic surgery, use of ppe, pre-operative testing policies for covid - ) . brant test to check the proportional odds assumption was performed for the ordinal logistic model. no formal correction for multiple testing has been made although we have critically assessed all p values < . . all analyses were performed using stata (statacorp llc, college station, tx, usa). from a total of , respondents worldwide, ( %) were europeans (appendix ), with ( %) from italy. among these, ( %) practiced in the north, ( %) in the centre and ( %) in the south and islands ( table ). the majority were men ( %), at consultant level ( %), with a mean age of . years, working in academic hospitals ( %), where a dedicated proctologist was readily available ( %). overall, %, % and % reported the presence of dedicated pathways for sexually transmitted disease, pelvic floor disorders and anorectal physiology testing, respectively. a multivariable logistic model showed that respondents from the south were more at risk of infection compared to those from the centre (or, . ; %ci . ; . , p = . ), as were males (or, . ; %ci . ; . , p = . ) and those who routinely tested patients prior to surgery (or, . ; ci . ; . , p = . ) compared to their counterparts. the majority of italian respondents worked in centres that were partially rearranged (n = [ %]) to guarantee the assistance to covid- patients, with a similar distribution between regions (table ) . conversely, hospitals fully converted to covid- centers (n = [ %]) and those not directly involved in covid- care (n = [ %]) were more prevalent in the north and south, respectively. more than a half of respondents had modified the surgical informed consent for both covid- positive (n = [ %]) and negative patients (n = [ %]), by mentioning the higher risk of infection and sars-cov- -related complications. ppe ready availability and routine pre-operative testing for covid- were more likely reported by respondents from the north (n = [ %] and n = [ %], respectively) compared to other regions (p = . and p = . , respectively). one-third of respondents (n = [ %]) faced patients refusing surgery, with the fear of sars-cov- infection as the main reason. forty percent (n = ) of respondents had yet to reschedule patients waiting for surgery or outpatient visit. compared to the rest of europe, elective proctologic surgery in italy was considerably reduced (with a test of heterogeneity at p = . ) (fig. ) . the likelihood of ongoing proctologic practice was higher in southern regions (or vs central regions . , %ci . ; . , p = . ) and in centers that were not fully dedicated to covid- care (or . , %ci . ; . , p < . ) ( table ) . among the ( %) respondents who found flaws or delay in the management of oncological patients, the majority was from the north (n = [ %]). more than % (n = ) of participants declared that elective non oncological surgery was fully stopped, with the main reasons being hospital directions and/or reduced referrals. emergency surgery was fully stopped according to onefourth (n = ) of respondents, while half (n = ) experienced a reduced activity. among those still performing emergency surgery (n = [ %]), the majority (n = [ %]) stated that patients were routinely tested for covid- pre-operatively, with similar interregional distribution. following national or local hospital directions, outpatient activity was fully stopped or reduced in italy according to % (n = ) or % (n = ) of respondents, respectively. the majority ( [ %)]) reported regular use of ppe during the visits. possible diagnostic delays resulting from a decreased outpatient activity concerned [ . %] respondents. in italy (and europe), the hunt for patient zero has proven unsuccessful and only served to fuel the confusion on the origin of the outbreak [ ] . sars-cov- spread across europe following multiple paths and heterogeneously impacted on countries and between different geographical areas within the same nation. the higher prevalence of male over female subjects was consistent with the results of our worldwide survey [ ] . despite proctology has been recognized worldwide as subspecialty, only two-thirds of italian respondents reported the availability of dedicated proctologist in their center. the alarmingly high prevalence of covid- positivity among italian respondents (twice that of all healthcare workers) [ ] might suggest that proctologists carry a higher risk of contagion compared to other specialists. several case studies have reported gastrointestinal symptoms and/or evidence that some patients with covid- have viral rna viable in stool or gastrointestinal epithelium, suggesting fecal-oral pathway as a further possible route of transmission [ , , ] . compared to those from the center, prevalence of covid- positivity among southern respondents was three times higher. in this geographic area, ppe were less frequently deemed readily available and the likelihood of continuing the surgical activity was % higher. such a worrying proportion of covid- positive respondents in the south suggests that the different timing of the epidemic and the prompt lockdown measures put in place by the italian government likely may have rescued this area from a potential catastrophe. as proof of resilience during troubled times, a significant number of respondents were redeployed to other activities [ ] . more than % of respondents reported to have amended the surgical consent form, reflecting a great awareness of growing evidence from the literature about the increased operative risks in covid- patients [ , ] . compared to the rest of europe, the reduction in elective surgical activity has been more pronounced in italy. to further confirm the deadlock of proctologic practice in this country, almost % of respondents had yet to reschedule patients' outpatient visits or operations. while being unable to access healthcare services, many patients refrained from attending the emergency department due to the fear of being infected [ ] . the suspension of oncological activity was reported by % of italian respondents, with a peak of % in the north (the worst-hit area), where all activities were more likely to be put on hold and hospitals forced to shift all resources towards covid- care. most respondents were concerned about the negative effects of delaying care, with potentially irreversible consequences, especially for cancer patients [ ] . as recently suggested [ ] , the outpatient/office surgical activity could have helped to diminish commitment to hospitals and optimize resource allocation in terms of operating spaces, staffing and beds. but this was not the case for at least two reasons, namely the full closure of all non-covid-related activities (as per national directions) and the currently very limited experience with delivering this type of proctologic surgery. undoubtedly, this should prompt health authorities and specialists to redesign and optimize the whole proctologic pathway across the national territory [ , ] . this study has some limitations that are commonly observed in survey-based studies (e.g. recall and selection bias). nevertheless, the high percentage of consultants among respondents vouches for a satisfactory level of experience and supports the reliability of collected data. the results of proctolock survey highlighted key critical issues that have emerged during covid- pandemic worldwide and in particular in italy, thus building foundations for future development of organizational solutions and nation-level initiatives. colorectal surgery in italy during the covid outbreak: a survey from the icral study group elective surgery cancellations due to the covid- pandemic: global predictive modelling to inform surgical recovery plans proctology in the covid- era: handle with care evidence for gastrointestinal infection of sars-cov- italian society of colorectal surgery recommendations for good clinical practice in colorectal surgery during the novel coronavirus pandemic global guidance for surgical care during the covid- pandemic coronavirus pandemic and colorectal surgery: practical advice based on the italian experience impact of covid- outbreak on healthcare workers in italy: results from a national e-survey ?term=proct olock &draw= &rank= . accessed contemporary surgical practice in the management of anal fistula: results from an international survey improving the quality of web surveys: the checklist for reporting results of internet e-surveys (cher-ries) covid- : preparedness, decentralisation, and the hunt for patient zero covid- : faecal-oral transmission? preventing transmission among operating room staff during covid- pandemic: the role of the aerosol box and other personal protective equipment being a doctor will never be the same after the covid- pandemic surgical ethics during a pandemic: moving into the unknown? unknown unknowns: surgical consent during the covid- pandemic covid- and the fears of italian senior citizens how covid- outbreak is impacting colorectal cancer patients in italy: a long shadow beyond infection tailored anal block (tab): a new anesthesia procedure for surgical treatment of hemorrhoids in an outpatient setting the impact of covid- on proctologic practice in italy acknowledgements proctolock working group: collabora-affiliations gaetano gallo · alessandro sturiale · veronica de simone · stefano mancini · gian luca di tanna · giovanni milito · francesco bianco · roberto perinotti · iacopo giani · ugo grossi on behalf of proctolock working group · domenico aiello · francesco bianco · andrea bondurri · gaetano gallo · marco la torre · giovanni milito · roberto perinotti · renato pietroletti · alberto serventi · marina fiorino · veronica de simone · ugo grossi · michele manigrasso · alessandro sturiale · gloria zaffaroni · ferruccio boffi · vittoria bellato · francesco cantarella · simona deidda · fabio marino · jacopo martellucci · marco milone · arcangelo picciariello · ana minaya bravo · vincenzo vigorita · miguel fernandes cunha · sezai leventoglu · tatiana garmanova · petr tsarkov · alaa el-hussuna · alice frontali · argyrios ioannidis · gabriele bislenghi · mostafa shalaby · felipe celedon porzio · jiong wu · david zimmerman · claudio elbetti · julio mayol · gabriele naldini · mario trompetto · giuseppe sammarco · giulio aniello santoro key: cord- - h tjs r authors: kuchenbuch, mathieu; d’onofrio, gianluca; wirrell, elaine; jiang, yuwu; dupont, sophie; grinspan, zachary m.; auvin, stephane; wilmshurst, jo m.; arzimanoglou, alexis; cross, j. helen; specchio, nicola; nabbout, rima title: an accelerated shift in the use of remote systems in epilepsy due to the covid- pandemic date: - - journal: epilepsy behav doi: . /j.yebeh. . sha: doc_id: cord_uid: h tjs r purpose: the purpose of the study was to describe epileptologists' opinion on the increased use of remote systems implemented during the covid- pandemic across clinics, education, and scientific meetings activities. methods: between april and may , we conducted a cross-sectional, electronic survey on remote systems use before and during the covid- pandemic through the european reference center for rare and complex epilepsies (epicare) network, the international and the french leagues against epilepsy, and the international and the french child neurology associations. after descriptive statistical analysis, we compared the results of france, china, and italy. results: one hundred and seventy-two respondents from countries completed the survey. prior to the covid- pandemic, . % had experienced remote systems for clinical care. during the pandemic, the use of remote clinics, either institutional or personal, significantly increased (p < (− )). eighty-three percent used remote systems with video, either institutional ( %) or personal ( %). during the pandemic, . % of respondents involved in academic activities transformed their courses to online teaching. from february to july , few scientific meetings relevant to epileptologists and routinely attended was adapted to virtual meeting (median: [ th– th percentile: – ]). responders were quite satisfied with remote systems in all three activity domains. interestingly, before the covid- pandemic, remote systems were significantly more frequently used in china for clinical activity compared with france or italy. this difference became less marked during the pandemic. conclusion: the covid- pandemic has dramatically altered how academic epileptologists carry out their core missions of clinical care, medical education, and scientific discovery and dissemination. close attention to the impact of these changes is merited. pandemics can lead to government regulations that limit social contact, decreased access to healthcare resources, and increased anxiety and fearall can disrupt the care path of patients with chronic diseases and decrease of face-to-face visits. in , a study on the outbreak of severe acute respiratory syndrome (sars) in china showed that the loss of contact with medical care providers led to an increase in the withdrawal of antiseizure medications resulting in an increase in seizure frequency [ ] . the current covid- pandemic is an important challenge for the management of patients with epilepsy worldwide. remote patient management systems [ ] (in use since the s and now integral to several national digital health strategies [ ] [ ] [ ] [ ] ) are a valuable tool during a pandemic to continue medical follow-up. they include different types of communications such as phone calls, one-way video links, and on live interactive communication. in addition, the epilepsy medical community is involved in educational activity and promoting knowledge dissemination through courses and scientific congresses. these activities also rely on face-to-face interactions and are also likely affected by the covid- pandemic. the aim of this study was to assess the impact of the covid- pandemic on the acute use of remote systems in clinics, education, and scientific meetings in the field of epilepsy and to explore the users' satisfaction and the perspectives of future use. we conducted a cross-sectional, electronic survey of epileptologists, neurologists, and pediatric neurologists mainly involved in the epilepsy field to determine the use of remote work during the covid- pandemic (supplementary data). to reach a wider public, this survey was addressed to adult and child neurologists specialized in epilepsy care through the european reference network for rare and complex epilepsies (epicare), international league against epilepsy (ilae), international child neurology association (icna), the french league against epilepsy (lfce), and the french society of child neurology (sfnp). the survey was comprised of questions divided into four sections: demographic and general information followed by remote work for clinical practice, education, and scientific meetings and symposia (for details, see supplementary data). items assessed practice before and during the covid- pandemic. the first two sections were mandatory (demographics and clinical practice). we used different types of questions: closed (n = ), semiopen (n = ) , and open (n = ). some questions used semiquantitative scales such as the likert scale. descriptive statistics included mean ± standard deviation for normal data, and median [ th- th percentile] for non-normal data. in the event of missing data, percentages were calculated per number of responses obtained, item by item. frequency of use of remote system was scored as follows: never = , used it once = . , few = , monthly = , weekly = , and daily = . wilcoxon signed-rank test were used to compare the frequency of the institutional and personal remote system use before and during the covid- pandemic and the frequency of use of these two systems during the same period. open-ended questions on free text allowed us to obtain qualitative data to illustrate respondents' feelings about their satisfaction with remote systems. we constructed a coding frame to analyze free-text data about satisfaction of remote clinic, online teaching, and virtual meeting. we subdivided into level categories to evaluate positive and negative aspects with some subcategory: cost, time, interaction, and target public. two authors (mk and rn) discussed the coding and interpretation of results. finally, we compared findings among the three countries with the highest number of respondents (france, china, and italy). quantitative or semiquantitative data were compared using kruskal-wallis h test followed, in case of significance (p b . ), by a dwass-steel-critchlow-fligner procedure. for qualitative data, we used chi tests. a p-value b . was considered as statistically significant, and a p-value b . as a tendency. the statistical analyses were performed using r software [ ] . this study was approved by the ethics committee of our institution necker hospital, aphp. participants were entirely free to participate and their consent was implicit. between april and may , , respondents in countries from continents completed the survey from all over the word (table , fig. ). responders were involved in caring for children with epilepsy (n = , . %), adults (n = , . %), or both (n = , . %). one hundred and fifty ( . %) worked in a public hospital. all had a clinical practice, were involved in clinical research ( . %), and in basic research activities ( . %). most of the participants were from europe (n = , . %). a containment policy due to the covid- pandemic was decreed in the countries of participants ( . %). indeed, participants ( %) belonged to the most impacted countries of the world in this period [ ] . the sections concerning remote work for education and scientific meetings were completed by participants ( % of all respondents). the questionnaire completion rate was % ( / , ). prior to the covid- pandemic, responders ( . %) had already experienced using a personal ( / , . %) or an institutional ( / , . %) remote system: / for patient direct care ( . %), for education of trainees ( . %), for clinical case discussions within other institutions ( . %), for research ( . %), and for clinical case discussions within their own institutions ( . %). for on the responders ( . %), this experience was at least monthly using institutional (n = / , . %) or personal (n = / , . %) remote systems ( fig. a) . there was no statistical difference between the frequency of use of institutional versus personal remote system (p = . ). the three main personal systems used were skype® (n = of the using personal remote system, . %), zoom® (n = , . %), and webex® (n = , . %). the means frequently used to contact remote respondents in an emergency were telephone calls (n = , %) and e-mails from families ( , %). other means (letters from families ( , . %) and letters ( , . %), telephone calls ( , %), and e-mails ( , . %) from the attending physician) were less frequently used. during the covid- pandemic, the use of remote systems increased, both institutional systems ( to ) and personal systems fig. b ). however, contrary to the pre-pandemic period, the use of institutional remote systems was significantly higher than that of personal systems (p = . ). only one respondent from china did not have to reschedule any face-to-face clinics compared with who rescheduled most or all their clinics ( . %). one hundred and sixty-two respondents ( . %) replaced faceto-face visits by various ways of remote connections with the families or the patients. this involved all clinics for ( . %), most of them for ( . %) and only a few for / respondents ( . %). sixty-eight ( %) used phone calls without any remote specific connection with or without video, and a remote system with video connection ( . %). this system was institutional for ( . %) either regularly available ( / , . %) or developed for this pandemic ( , . %). a personal remote system was used by ( . %). the duration of remote clinics was considered as identical as face-to-face ones for respondents ( . %), shorter for ( . %, including much shorter for ), and longer for ( . %, including much longer for ). regarding antiseizure medication changes, . % of respondents tended to make fewer amendments (n = ), . % same (n = ), and . % more (n = ). electroencephalogram (eeg) were less frequently requested for . % (n = ), without changing the frequency requested for . % (n = ), and more frequently requested for . % (n = ). blood test were less frequently requested for . % (n = ), without changing the frequency requested for . % (n = ), and more frequently requested for . % (n = ). respondents reported an increase in email and phone contacts by patients and their families (for , . % and , . % of respondents, respectively) but also by primary care physicians (for , . % and , . %, respectively for email and phone). one hundred and thirty-four respondents ( / , . %) were involved in educational activities. before the covid- pandemic, ( . %) had face-to-face lectures or small group teaching courses, and ( . %) had been involved in online teaching. educational activities were impacted by the covid- pandemic for respondents ( . %). eighty-two percent had at least a part of their activities canceled (n = / , %), postponed (n = , . %), or transformed to online teaching ( , . %). respondents' courses were either interactive ( / , . %), video recorded ( / , . %), or both ( / , . %). for / respondents, all courses were transformed to online teaching ( . %). before the covid- pandemic, . % ( / ) of the respondents had participated in remote scientific meetings, . %, ( / ) in workshops, . % ( / ) in clinical studies meetings, and . % ( / ) in research symposia. few had such experience with national . % ( / ) or international . % ( / ) congresses. during the period from february to july , responders had planned [ ] [ ] [ ] [ ] [ ] meetings. only a few were transformed to remote (median: [ - . ]) giving the opportunity to eighty-nine responders ( . %) who participated in at least one meeting transformed to remote. the rest of the meetings were canceled or postponed. sixty-one percent of respondents were satisfied by their remote clinics ( / , including very satisfied), . % by their online teaching ( / , including very satisfied), and . % by remote meetings ( / ) (fig. ) . feelings regarding family and patient satisfaction with the remote clinic were positive for . % of the respondents ( / , of which were very positive) and . % regarding students and online teaching ( / , of which were very positive). almost onequarter of responders reported dissatisfaction with remote work, mostly for remote education ( . %, n = / ), remote meetings ( %, / ), and remote clinics ( / , . %). respondents indicated they would likely continue greater use of remote work for remote clinics, education, and meetings after the covid- pandemic, in . % ( / ), . % ( / ), and . % ( / ), respectively (fig. ) . free text allowed us to have more qualitative data on the reasons to maintain remote working in their different activity axis. indeed, in their opinion, remote clinics had the advantage of decreasing time and cost for families and patients travel and consequently of work absenteeism. this was highlighted for follow-up visits but not for new patients having their first evaluation. for first visits, respondents declared a clear need for a face-to-face visit. saving time, adapting to the availability of students, and increasing the target audience due to the absence of the need to travel to attend the course seemed to be the positive factors identified by respondents regarding remote education. however, they identified several negative factors including a decrease in interactions, especially the immediate students' feedback reactions. workshops with a small number of participants was reported as particularly adapted to remote systems allowing a gain in term regarding travel, time, and cost. however, respondents agreed that national and international meetings are more adapted to in-person meetings as their major goal in addition to disseminate knowledge is to favor personal interactions and consolidate personal friendships and contacts to enhance collaboration and exchange of ideas. the pandemic began in december in china, late february in italy, and early march in france. these countries were all placed under quarantine (from january to april in china, from march to may in italy and from march to may in france). the peak of pandemic-related deaths occurred between february , [ ] . comparison of data from france (n = ), italy (n = ), and china (n = ) showed no significant differences in terms of age, gender, and practice (pediatric, adult, or both, public or private, epilepsy center or not). belonging to a healthcare network was statistically different between countries (p b − ). indeed, only four chinese respondents ( . %) belonged to a patient care network, whereas there were % in france ( responders) and . % in italy ( ) . before the covid- pandemic, the rate of respondents who had experienced remote working systems was higher in china than in the two other countries ( . % versus . % for france and % for italy, p = − ). in the same way, the number of respondents with an institutional remote work system was higher in china ( . % versus % for france and . % for italy, p b − ). however, the rate of respondents who had a personal remote work system was not statistically different (china: . %, france: % and italy: . %, p = ns). the frequency (scored from : never to : daily) of use of institutional remote systems was significantly different between the three countries the proportion of respondents using official remote systems or phone calls without video for remote clinics was not statistically different between france, italy, and china (official remote system: china: . %, france: . %, italy: . %, p = ns and phone call without video: china: . %, france: %, and italy: %, p = ns). however, in china, remote personal systems were more often used to manage patients than in other countries ( . %, italy: %, france: . %, p b − ). concerning educational activities, the proportion of respondents involved was not statistically different (china: . %, france: . %, and italy: . %, p = ns). before the covid- pandemic, the proportion of chinese respondents who had already experience online teaching was significantly higher ( . % versus . % for france and . % for italy, p = . ). the respondents who had their teaching activities impacted by covid- pandemic were % for china, . % for france, and . % for italy (p = ns). during the pandemic, all respondents in china replaced at least part of their course with online teaching ( / ) compared with % in france and % in italy (p = . ) in particular using interactive online teaching (china: . %, france: %, and italy: %, p = . ). concerning remote meetings, a large majority of respondents had already used this system without any statistical difference between countries (china: %, france: . %, and italy: . %, p = ns). for satisfaction scores (from very unsatisfied: − to very satisfied: + with a neutral position: ), only the impression on families' and patients' satisfaction for remote clinics had a tendency to be higher in france compared with china ( for france and [ - ] for china, p = . , italy: [ . ). all other satisfaction scores showed no significant difference. the covid- pandemic blockage has significantly strengthened the use of remote access technologies in medicine. our study showed that pandemic has increased the shift from classical to remote communication for epilepsy practitioners in all the fields of their activity, namely clinical activity, teaching, and scientific meetings. the satisfaction was acceptable, and almost all responders agreed on a possible future use of remote systems for some of the scientific and educational meetings or for occasional remote clinics excluding first visit. our study demonstrated that during the covid- pandemic, there has been a reduction of face-to-face visits with a replacement for most by remote clinics. in similar situations, remote systems had already been identified as a possible alternative to face-to-face visits, for example, during ebola or sars epidemics [ ] . in the same way, our study showed an increase of remote clinic frequency use during the pandemic compared with the pre-pandemic period. prior work on remote systems in epilepsy has shown notable benefits. a pilot study in canada compared remote systems to face-to-face clinics showing a decrease of costs of . % ($ . versus $ ) with a satisfaction for patients of % and only % preferring a face-to-face next visit in both groups [ ] . the main barriers to remote clinics are the need for clinical examination, technical support, and reimbursement [ ] . in our survey, respondents identified the same advantages and barriers, the first visit being the most challenging. in another study comparing the impression of new patients on remote visits with face-to-face visits, patients' perceptions of the neurologist's understanding, their ability to say what they wanted, their confidence in the neurologist, and the usefulness of the visit were similar [ ] . however, they stated more difficulties in describing their symptoms and concerns about confidentiality. in our study, respondents used personal remote system for remote clinics. this raises concerns about privacy and protection. of note is that the explosion of remote working systems due to covid- attracted hackers [ ] . one attack, called "zoom bombing", consists of an unwanted intrusion, causing disruption and possibly disclosure of medical confidentiality. in order to regulate the security of health data during remote clinics, countries have established strict rules such as the health insurance portability and accountability act (hipaa) in the usa [ ] and general data protection regulation in eu [ ] . most of the free-access personal remote systems in our study are not hipaa compliant. this point should be better addressed by health authorities in future development of remote clinics. until , attendance in medical classes was correlated with passing the examination [ ] [ ] [ ] . since , however, some studies have found no clear correlation [ , ] . for example, th year medical school students have more absences than nd year students due to conferences, meetings, and residency interviews, but unlike personal absences, this type of absence is not significantly associated with lower academic test scores [ ] . this is likely due to the improvement of means of communication that have enabled the students to fill in the gaps. in a recent study using a combined approach between online teaching and face-to-face interactive medical course, online teaching attendance was higher than face-to-face, and the exam score was correlated to online teaching attendance. ninety-eight percent were satisfied with this teaching, and % wished to extend it to the entire second cycle [ ] . this is a good illustration of the change of perspective that is taking place in undergraduate and postgraduate university education. factors associated with a good adherence to online teaching are mainly the quality of the technical system, support system, learner and instructor, and the perceived usefulness [ , ] . the advantages and disadvantages identified by the providers in our study were in line with the literature, i.e., on the one hand, a greater flexibility, an increase of the dissemination of knowledge, a decrease of travel cost and time, and better accessibility, on the other hand, less peer-to-peer exchange and feedback difficulties, including nonverbal communication [ ] . in the symposia and meetings, the same advantages and disadvantages as with teaching were identified, but the proportion of respondents who recommended this method for the future were lower than for clinics and teaching. the use of remote systems seemed to be more adapted for research networks and workshops than congresses. but during the covid- pandemic, the european academy of neurology replaced its congress by a virtual meeting free-of-charge. with more than , participants, they claimed this to be "the biggest neurology meeting ever" [ ] . a virtual congress allows for lower prices, time savings, and a greater dissemination of knowledge both to and from all over the world. however, the respondents interviewed stressed the importance of face-to-face for the development of collaborative projects. our questionnaire highlighted, before the covid- epidemic, a stronger experience of remote systems in china compared with france and italy. this may be due to previous epidemic crisis in china, a larger geographic area of china compared with france and italy, and a lower density of neurologists and child neurologists ( . and . per , persons for neurologists and child neurologists, respectively in south-east asia region versus . and . per , persons in europe [ ] ). indeed, prior to the covid- pandemic, some studies and reviews identified remote clinics in the field of epilepsy as an opportunity in rural regions and in resource-poor setting where the access to a specialist is an important barrier to epilepsy diagnosis and treatment [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the covid- pandemic seemed to have accelerated the shift towards the implementation of remote clinics and had enabled france and italy to fill the gap with a strong development of remote patient management tools. the sample of this study was small, but respondents completed the survey just after the covid- in china and during the covid- pandemic and lock down in other countries, giving to this survey a value of "almost" real-time responses. responses were from many countries around the world thanks to the involvement of international societies. we cannot rule out the presence of a selection bias since this questionnaire was sent online. however, we believe that this study can present a picture about practitioners' opinion on remote work in epilepsy and help to develop future perspectives. in addition, a significant proportion of respondents in our sample focus on pediatric care. the use of remote clinics in this population is probably easier than in adults. indeed, parents may be able to successfully complete a visit on their child's behalf but adults with cognitive impairment or other limitations may have more difficulty negotiating the technical requirements of such a visit. finally, we did not request a detailed description of the applied methods of online teaching. the survey aimed to have answers on the three activity fields of the respondents without adding much details relatively long survey. the covid- pandemic has increased the shift from classical to remote communication for epilepsy practitioners in all the fields of their activity, namely clinical activity, teaching, and scientific meetings. the advances of these methods of communication have allowed a rapid adaptation to confinement policies using their flexibility and their accessibility. this allowed a maintained link between practitioners and patients, professors and students, and between groups and colleagues. the satisfaction was acceptable, and almost all responders agreed on a possible future for remote work, for some of the scientific and educational meetings or for occasional teleconsultations. in addition, the positive ecological impact of such approaches might be interesting in addition to the economic impact on health and academic costs. it is likely that in "the world after covid", the shift process to the implementation of these new modes of communication is moving forward, although the balance between face-to-face and remote work has yet to be determined in our different fields of activities, and the long-term benefit of such shift to virtual interaction should be evaluated. m. kuchenbuch, g d'onofrio, y. jiang, zm grinspan, j wilmshurst and r nabbout have any conflict of interest to disclose. s dupont has received honoria from eisai, ucb, gw, novartis, advicenne and shire. e wirrell has acted as an investigator for gw pharma and zogenix and has received consulting fees from biocodex and biomarin. s auvin has served as consultant or received honoraria for lectures from arvelle therapeutics, biocodex, eisai, gw pharma, novartis, nutricia, ucb pharma, zogenyx. he has been investigator for clinical trials for advicenne pharma, eisai, ucb pharma and zogenyx. a arzimanoglou has served as consultant, received honoraria for lectures from arvelle therapeutics, eisai, gw pharma, ucb pharma and zogenix. on behalf of his instiitution he has been investigator for clinical trials sponsored by eisai, gw, ucb pharma and zogenix. jh cross has acted as an investigator for studies with gw pharma, zogenix, vitaflo and marinius. she has been a speaker and on advisory boards for gw pharma, zogenix, and nutricia; all remuneration has been paid to her department. her research is supported by the national institute of health research (nihr) biomedical research centre at great ormond street hospital, nihr, epsrc, gosh charity, eruk, the waterloo foundation. n specchio has acted as an investigator for studies with zogenix, marinus, biomarin, and livanova, and has received consulting fees from zogenix, biomarin, arvelle, livanova. the impact of sars on epilepsy: the experience of drug withdrawal in epileptic patients advantages and limitations of teleneurology scotland ' s digital health & care strategy n.d danish ministry of health south african national department of health. national digital health strategy for south africa rdct. a language and environment for statistical computing. r found stat comput world health organization. coronavirus disease (covid- ) situation report- coronavirus covid- global cases by johns hopkins coronavirus resource center telemedicine: potential applications in epidemic situations feasibility of epilepsy follow-up care through telemedicine: a pilot study on the patient's perspective telemedicine and epilepsy care-a canada-wide survey randomised controlled trial of telemedicine for new neurological outpatient referrals hackers' new target during pandemic: video conference calls n health insurance portability and accountability act of regulation on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, and repealing directive / /ec (data protection directive) class attendance in college: a meta-analytic review of the relationship of class attendance with grades and student characteristics student attendance and academic performance in undergraduate obstetrics/gynecology clinical rotations absenteeism among medical and health science undergraduate students at hawassa university, ethiopia relationship between classroom attendance and examination performance in a second-year medical pathophysiology class does class attendance matter? results from a second-year medical school dermatology cohort study the impact of student absences on grade outcomes in a neurology clerkship setting assessment of a newly-implemented blended teaching of intensive care and emergency medicine at paris-diderot university evaluating e-learning systems success: an empirical study e-learning success determinants: brazilian empirical study emerging themes in e-learning: a review from the stakeholders' perspective ean congress highlights challenges -predict, prevent, repair atlas -country resources for neurological disorders tele-neurology in sub-saharan africa: a systematic review of the literature can technology help reduce risk of harm in patients with epilepsy? pediatric teleneurology: a model of epilepsy care for rural populations epilepsy field workers, a smartphone application and telephone telemedicine: safe and effective epilepsy care in rural nepal telemedicine for epilepsy: a useful contribution telemedicine for epilepsy support in resource-poor settings. front public heal managing epilepsy by telemedicine in resource-poor settings telemedicine in epilepsy: how can we improve care, teaching, and awareness? epilepsy behav the authors wish to thank the practitioners who participated in this study and the networks without whom this study could not have been carried out, namely the international league against epilepsy, the international child neurology association, the french league against epilepsy, the french society of pediatric neurology, and the ern epicare network. rima nabbout was supported by state funding from the agence nationale de la recherche under "investissements d'avenir" program (anr- -iahu- ) and the "fondation bettencourt schueller". this research was supported by the agence nationale de la recherche under "investissements d'avenir" program (anr- -iahu- ) and the "fondation bettencourt schueller", paris, france. supplementary data to this article can be found online at https://doi. org/ . /j.yebeh. . . key: cord- -vz rsy authors: wodarz, dominik; komarova, natalia l. title: patterns of the covid epidemic spread around the world: exponential vs power laws date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: vz rsy we have analyzed the covid epidemic data of more than countries (excluding china) in the period between january and march , . we found that some countries (such as the us, the uk, and canada) follow an exponential epidemic growth, while others (like italy and several other european countries) show a power law like growth. at the same time, regardless of the best fitting law, most countries can be shown to follow a trajectory similar to that of italy, but with varying degrees of delay. we found that countries with ``younger" epidemics tend to exhibit more exponential like behavior, while countries that are closer behind italy tend to follow a power law growth. we hypothesize that there is a universal growth pattern of this infection that starts off as exponential and subsequently becomes more power law like. although it cannot be excluded that this growth pattern is a consequence of social distancing measures, an alternative explanation is that it is an intrinsic epidemic growth law, dictated by a spatially distributed community structure, where the growth in individual highly mixed communities is exponential but the longer term, local geographical spread (in the absence of global mixing) results in a power-law. this is supported by computer simulations of a metapopulation model that gives rise to predictions about the growth dynamics that are consistent with correlations found in the epidemiological data. therefore, seeing a deviation from straight exponential growth may not be a consequence of working non-pharmaceutical interventions (except for, perhaps, restricting the air travel). instead, this is a normal course of raging infection spread. on the practical side, this cautions us against overly optimistic interpretations of the countries epidemic development and emphasizes the need to continue improving the compliance with social distancing behavior recommendations. to some extent in younger segments of the population. the overall mortality rate further depends on the prevalence of the infection in the population: once the number of infected individuals has reached sufficiently large sizes, health care systems become overwhelmed and lose the capacity to treat all patients in need [ ] . in particular, the number of respirator units poses an important limitation, which has resulted in difficult triaging policies in some countries, most notably italy and spain. non-pharmaceutical intervention methods, such as social distancing, have become an important tool in the fight against coronavirus spread [ ] . the idea is to slow down the rate at which the virus spreads through human populations, and thus to maintain the number of infected individuals that require treatment below a threshold level, thus avoiding a situation in which hospitals become overwhelmed. hence, wide-spread closures of schools and businesses, and stay-home orders, have been implemented across asia, america, europe, and other affected regions. a number of studies have been performed that investigated the kinetics of coronavirus spread through human populations. the pattern of virus spread was found to be approximately exponential, at least during the early stages of virus spread [ , ] , and importantly, the basic reproductive ratio of the virus has been estimated [ , , , ] . the implementation of social distancing measures are expected to have a significant impact on the virus spread kinetics [ ] , which has been observed in some asian countries, such as china [ ] . while the effect of containment efforts have been very pronounced in these countries, they might be more subtle in other countries across the globe, especially during the initial phase following their implementation. to better assess observed virus spread trajectories following the implementation of social distancing measures, a more detailed understanding of the basic kinetics of virus spread in different countries is required. while the virus spread pattern is often quoted to be exponential [ , ] , power law patterns have been reported in data from china [ ] . in this study, we compare the per capita virus spread kinetics observed for many countries around the globe, in order to obtain a better understanding of similarities and differences. interestingly, we find that as per capita infections grow towards larger numbers, the growth pattern deviates from exponential and is better described by a power law. this can be hypothesized to be due to social distancing measures [ ] , or potentially to the build-up of immunity in the population [ ] . we show, however, that the longer term per capita infection levels over time across a wide array of countries can be remarkably similar, and follow the same power law trajectory as seen in countries that are most strongly affected, such as italy. this indicates that the long-term dynamics of covid spread might be intrinsically governed by a power law, even in the absence of non-pharmaceutical interventions. we interpret these findings with computer simulations of a metapopulation model, which can account for an initial exponential spread phase, followed by a longer-term power law behavior, by assuming that the infection spreads in a well-mixed manner inside local demes, while also spreading spatially across different demes. we relate model predictions to epidemiological correlations found in the data. these insights into the spread kinetics of covid might be useful for assessing the impact of non-pharmaceutical intervention methods in different countries. the data of confirmed covid cases over time have been obtained from the data repository maintained by johns hopkins university center for systems science and engineering (csse) (https://datahub.io/core/covid- #data-cli). as of march , countries were represented in the database, as well as the cases on "diamond princess" (which were not used in the analysis). we only included the total counts for each country, even though information on the different provinces was available for several countries. the number of confirmed cases has been recorded since january , , and has been updated daily. to compare the time course of covid cases across different locations, the per capita incidence was calculated, normalizing the numbers by the total population size of the country. the information on the population size and the area of different countries was taken from wolfram mathematica's database, "countrydata". here, we present the comparison of the kinetics according to which cumulative covid cases grow over time in different countries around the world. figure (a) presents the raw data showing total case counts for a select number of key countries. figure (b) shows the corresponding per million case counts. a complication for comparing the growth dynamics is that the timing of the onset of community spread varies across locations. the growth curve of confirmed cases was therefore shifted in time to make them comparable, according to the following procedure. the cumulative confirmed covid case counts in italy were chosen to be the example against which the growth curves in all other countries were compared, due to italy being a current epicenter of the outbreak. the (normalized, cases per million) infection growth curves of the other locations were shifted in time such that the difference between all data points of the country under consideration and italy was minimized. the shift that minimized this euclidean distance between the curves was assumed to indicate the number of days that the country lags behind italy. some examples of such results are presented in figure . we note that this assumes that all the countries test for covid at comparable levels, which is an over-simplification. if a country tests less than italy, it will lag behind italy to a lesser extent. conversely, if a country tests more than italy, . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . it is predicted to be further behind italy. reliable data on the total number of tests in different countries are currently not available to our knowledge. in this way, we obtained a time course of confirmed covid cases that are temporally synchronized with italy, which allows for a more straightforward comparison of the kinetics. heterogeneity in growth laws across the different countries are observed. while some countries appear to show exponential growth of confirmed covid cases, other countries appear to exhibit growth laws that deviate from exponential. previous work [ ] has suggested that a power law might be a better description of the cumulative covid cases over time in china during the earlier stages of the epidemic (before it was controlled). therefore, we hypothesized that for a subset of the countries that were analyzed, a power law is an appropriate description. to test this hypothesis, we fit both exponential and power law curves to the data for each country and determined the goodness of fit. this data fitting was performed as follows: only the data points were considered where the number of covid cases had risen above a threshold, which we set at case per million people (see appendix for variations of this threshold). we fit both a power function and an exponential function to the data to determine which model fits the data better. for the power law function, a complication arose because fitting requires knowledge of the "zero" point, that is, the moment of time when the growth (according to the power law) began. the fits to the data change if the time scale is changed. hence, we started by assuming the first data point to be the day when the infection frequency first exceeded case per million, and fitted the power law, a x b , for some constants a and figure : the same data as in figure (bottom), presented by shifting individuals lines to match the italy curve. the table shows the lag, that is, by how many days each country is behind italy. b . then we shifted the time series incrementally by one day, and for each shift the power law was fitted. for each fitting frame, a different value of the power law exponent, b , was obtained. subsequently, we fit an exponential function to the same data (a e b x ). the estimated exponent does not depend on the time shift, so fitting the exponential function was straightforward and yields a unique value b for all the fitting frames. for both the exponential and the power law fits, we determined the sum squared error between observed and expected, and compared them. for more details of the fitting procedure, see appendix a. figure shows the fitting errors calculated for countries; we included a country if the number of cases reached per million, and excluded china and south korea, since their epidemics clearly deviate from an exponential or a power law. the yellow horizontal lines represent the exponential fitting and the blue lines the power law fitting. we observe that there are several different configurations that are repeatedly encountered. • for some countries (like the us, see also figure (a)), the power fitting error is always above the exponential fitting error. such countries are clearly showing an exponential epidemic growth. • there is another group of countries (such as italy, see also figure (b)), where the power law fitting error is always below the exponential error; here we clearly have a power law growth. • there are some other countries that we can classify as power law-like and exponential- . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . like. suppose a power law error curve crosses the exponential error line (see greece, figure (c)), at a given frame shift. in this case, we will classify the growth as power law-like if the value of the exponent b that corresponds to this frame shift satisfies b < . otherwise, we will classify the growth law as exponential-like. for the examples mentioned here, figure shows the best fits obtained by this method. for (b) and (c) it is clear that the power law provides more satisfactory fits. more details are provided in appendix. since in smaller countries (such as for example luxembourg) the laws may be harder to determine and the data are subject to a higher degree of noise, for classification purposes we restricted the pool of countries to those with over a million inhabitants. then the following country's epidemics were classified as exponential and as a power law, see also figure : in the lists above, the countries that are classified as power law-like and exponentiallike are printed in gray. there are countries in the exponential law class, with of them being truly exponential and the rest exponential-like. there are countries in the power law class, with of them being truly power law and the rest power law like. it is interesting that the countries showing a power law (or power law-like behavior) are different with respect to some characteristics compared to the countries showing exponential or exponential-like behavior. figure (a) shows a numerical probability distribution for the day when the infection in each country reached the level of case per million. blue represents the power law set and yellow the exponential set (grey means an overlap of the two colors). the average date of reaching case per million (counting from jan ) is about days for the power law and days for the exponential set (p = . by t test). in other words, the countries that are demonstrating a power law infection spread have had the relative infection level slightly longer than the exponentially developing countries. this points us towards a hypothesis that perhaps it is typical to observe a transition between an early, exponential stage of growth, and a later, power-like stage of growth. figure (b) shows the difference between the two classes of countries in terms of . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint their area. we find that the exponentially growing infection class is associated with larger countries (mean area of about . × km ) compared to the power law class (mean area about . × km , p = . by t test). similarly, exponential epidemic spread tends to correlate with lower density countries (figure (c)). it is possible that it takes longer for a larger country of a lower density to transition to a power law growth. we provide a possible explanation of this correlation in the context of metapopulation modeling. it is important to understand the dynamics with which the cumulative case counts increase over time, such that we have a better ability to judge whether non-pharmaceutical . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . interventions (e.g. social distancing) have an impact on the course of the epidemic. the covid epidemic is often thought to grow exponentially. if this is the case, a deviation from exponential growth following the introduction of non-pharmaceutical interventions can indicate success of those interventions. in contrast, if the infection grows according to a power law while growth is incorrectly assumed to be exponential, a slow down of the cumulative covid cases over time on a log scale can result in the false conclusion that the non-pharmaceutical intervention methods are working. if the cumulative cases grow like a power law, successful intervention would result in the growth deviating from the power law, and not from exponential growth. one interpretation of our analysis could be that in the absence of non-pharmaceutical interventions, the disease burden grows exponentially, but that over time, the nonpharmaceutical interventions slow down the spread. hence, overall, the disease dynamics are described better by a power law than by an exponential function, because the power law is characterized by a slow-down of the growth rate over time. italy, where one of the most pronounced power laws was observed, implemented some degrees of social distancing relatively early, although the more strict measures were implemented only recently. . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . while this cannot be excluded, the comparative plots in figure might argue against this hypothesis. these graphs shows the spread dynamics for a few countries, whose epidemics are relatively advanced, and they have all been shifted to be temporally synchronized with italy. as the infection spreads to higher levels, the curves for the different countries converge towards a common trajectory of the per capita cases of covid . in other words, as the infection spreads, the per capita number of cases converge and follow the same power law. if this deviation from exponential growth was due to nonpharmaceutical interventions, then it is surprising that those countries follow the same trajectory because they implemented those interventions at different times and to different degrees. the percentage of countries with a given delay that belong to the power law group and to the exponential law group. the trend that the percentage of exponential growth increases with the time lag (that is, decreases with the epidemic "age") is significant (p < − by linear fitting). alternatively, it is possible that the local epidemics characterized by power laws demonstrate an intrinsic power law spread that is independent of interventions. computational models suggest that infection spread across networks or in spatially structured populations can lead to dynamics that follow a power law rather than an exponential trajectory, see below. if some countries truly show infection spread that is governed by a power law, the question arises why other countries show clear exponential spread, and why yet other countries are more difficult to classify. we hypothesize that different countries are at different stages of epidemic development, but they all roughly follow the same trajectory, where an initial exponential growth is gradually replaced by a more power like behavior. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint tries until, for each country, the best match with the italian curve was obtained. as we can see in figure (a) , there are only a few countries that are just behind italy, and as the number of lag days increases, the number of countries grows. this corresponds to the world wide spread of the pandemic: more and more countries become affected as time goes by. figure (b) calculates, for each lag time, the percentage of countries that were classified as following power law or exponential dynamics. we can see that for the countries that are just a few days behind italy, % of them belong to the power law group. as the lag time increases, indicating an earlier stage of the epidemic, more and more countries exhibit exponential growth (p < − ). the same trend is also corroborated by figure (a) that shows that the epidemics in the exponential group are "younger". how can we explain the existence, to different extents, of power law like behavior in different countries? one possible reason could be non-pharmaceutical interventions that are only partially effective, see a schematic in figure . it is possible that in early stages of the infection, exponential virus spread occurs because people have not yet altered their behavior and continue to travel and socially mix with each other. mass-action dynamics are expected to yield exponential growth. as a result of non-pharmaceutical interventions that are only partially effective, people might stop traveling and thus slow down large scale mixing. however, they would still be interacting locally within their social network, which would lead to a transition to power law dynamics. it is possible that a number of countries are difficult to classify because they exhibit early mass action dynamics (exponential), followed by network interactions that lead to a transition to a power law. this would indicate that stronger non-pharmaceutical intervention methods need to be implemented. more generally, the results can be interpreted in the context of a minimally parameterized metapopulation model, see figure . assume that within a local deme (such as a local community), people interact with each other, resulting in mass action dynamics. for the infection to spread further, however, people have to enter other demes, and seed the infection there. we have performed computer simulations of such a model to explore outcomes. the model is a two-dimensional metapopulation consisting of n × n patches. in each patch, i, the infection dynamics are given by a set of ordinary differential equations (odes) that take into account the population of susceptible (s i ), infected (i i ), recovered (r i ), and dead (d i ) individuals: . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . here, infection is described by a frequency-dependent infection term [ ] , characterized but the rate constant β and a saturation constant . infected individuals die with a rate ga and recover with a rate g( − a). the migration terms include the outward migration to n neighbors and an inward migration from all the n neighboring demes that belong to neighborhood b n i of deme i. the migration rate is denoted by f and we assume that each patch has eight direct neighbors, i.e. n = . the boundary demes are characterize by fewer inward/outward migrations (that is, they have smaller neighborhood sets). using this model, we track the predicted dynamics for i + r + d over time, which represents the cumulative infection case counts. in a first scenario, we start the computer simulations with a small amount of infected individuals in a single patch, located in the center of the grid. all other patches contain only susceptible individuals. the resulting dynamics are shown in figure . we observe an initial exponential phase of infection spread, followed by a transition to a power-law spread. the spread is initially exponential, because within a single patch (the first patch), the dynamics are governed by well mixed populations. as the infection spreads to other patches by migration, the overall infection spread starts to be governed by spatial dynamics, which explains the . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint next we assumed that instead of starting with infecteds only being present in a single patch, a small amount of infected individuals are initially present in more than one patch around the same time. this could correspond to larger countries, in which the infection is simultaneously seeded in multiple areas (e.g. due to travel from other places). now, we observe overall growth dynamics that are are more exponential-like. the length of the predicted exponential phase becomes longer the more patches are initially seeded. the reason is that with more initial seeding events, the importance of spatial spread is de-emphasized. the metapopulation model can therefore predict an array of growth patterns where an exponential phase of varying length is followed by a transition to power law, depending on the initial conditions of the simulation. in this paper, we analyzed data that document the cumulative covid case counts over time in a large number of countries around the world, and examined the laws according to which the infection spreads. this suggests that although the initial phase of the spread may be exponential, the longer term dynamics tend to be governed by a power law. the analysis indicates that the countries that display clear evidence for exponential growth are currently in a relatively early phase of the epidemic. the data further suggest that countries that are further along in the epidemic converge to a common power law . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . behavior, and cumulative per capita case counts appear to converge over time. these observations were interpreted by computer simulations of a metapopulation model that takes into account both local spread and spread across geographical space. this model predicts an initial exponential phase (due to local transmission events driving the dynamics), followed by a transition to a power law (once spatial dynamics significantly drive spread). the duration of the exponential phase is determined by the number of patches that are initially seeded with the infection. if the infection originates in a single location (patch), the exponential phase is likely not very pronounced, and most of the growth curve is predicted to follow a power law. if the infection is seeded simultaneously or nearly simultaneously in multiple locations, the duration of the exponential phase becomes longer. the larger the number of initially seeded locations, the more pronounced the exponential phase of the infection spread. these predictions are further in agreement with the correlations that we found between the growth law of the infection and country size. exponential growth was associated with countries that are characterized by a larger area. more pronounced power laws tend to occur in counties with a smaller area. if a country has a larger area, it is more likely that multiple locations are seeded with the infection around a similar time, for example due to travelers returning from a country with a larger disease incidence. for this scenario, the metapopulation model predicts more pronounced exponential growth. if the country has a smaller area, it is more likely that the infection is seeded within one geographical area and spreads outward from there. in this case, the metapopulation predicts an infection spread pattern that mostly follows a power law. beyond scientific interest, a better understanding of the laws according to which covid spreads through populations is also of practical importance. currently, emphasis is placed on non-pharmaceutical intervention measures to slow down the spread of the infection such that the ability of the health care system to cope with the number of incoming patients is preserved. the success of these intervention measures should be reflected in slowed infection spread. an understanding of the infection spread laws is crucial to interpret such data. if we assume that infection growth is exponential and if we plot the cumulative number of covid cases on a log scale, a deviation and slow-down from exponential growth would lead to the conclusions that the non-pharmaceutical intervention measures are successful [ ] . if, however, the true spread dynamics are characterized by a power law, we expect deviation and slow-down compared to exponential growth over time, even if the infection continues to spread at full force. in this case, a deviation from exponential spread cannot be interpreted to mean that non-pharmaceutical intervention measures are sufficient. to come to that conclusion, we would need to observe a deviation and slow-down of the infection spread compared to a power law null model. as with any data and modeling studies, it is important to note that results can depend on assumptions and methodologies. these are clearly spelt our here. one of the larger challenges we faced in the data analysis is the lack of knowledge at what time the infection was initiated in the individual countries. this information is not available. the time frame in turn influences the fit of the power law to the data, which we have addressed with our time shifting methodology. if further information becomes available about the time when infections are estimated to have originated in the individual countries, the methodology can be updated. genetic studies could provide valuable data in this respect. another limitation of the data interpretation is the degree, to which different countries test for the coronavirus. if some countries test less than others, they will appear to be further behind italy, while in reality the lag could be shorter. this type of uncertainty however does not change the central finding that the long term dynamics of covid cases in different countries follow a power law, after an initial stage of exponential growth. here we present the details of the fitting procedure used to determine the growth laws for different countries. it is illustrated with the example of italy in figure . the full data for the number of cases per million in italy are presented in figure (b) , orange curve. in figure , the subset of same data starting from case per million, is plotted on the log-log scale (panel (a)) and on a linear scale (panel (b)), with varying horizontal shift, which corresponds to changing the position of time zero. this is what we refer to as a "fitting frame". the fitting frame number one is when the first data point in the selected subset corresponds to day . the ith fitting frame shifts this point to day i. for each fitting frame, we obtained the best fit with function a x b , by using a built-in mathematica routine "findfit". note that the natural logarithm of the data was fitted for the figures presented here. clearly, some fits are better than others, see figure (a,b) . the fitting error for each fitting frame, i, was calculated as the distance between the data and the fit, normalized by the number of points: where y exponential fits of the same data from italy are shown in figure (a,b) . in panel (a) we used a log scale, such that the exponential fits look like straight lines. it is clear that, first of all, these fits are all parallel lines and thus the error is exactly the same (thus the exponential fitting errors as functions of the fitting frame are horizontal lines, see figures and ). second, we note that these fits are not very good for italy, that's why the power fit errors is always below the exponential error, see figure (b). for comparison, panels (c) and (d) of figure show the exponential fits for the us data. we can see that the quality of the fits is better, see also figure (b). in the main text and in this appendix so far, we describe a fitting procedure where the "confirmed cases" data for each country were used only if the numbers exceeded case per million. here we demonstrate how this changes if a different choice is made and a minimum of cases per million is required for each data point to be included. in figure we demonstrate the difference for countries. the choice of countries for this graphics was somewhat arbitrary: we included the countries out of the subset used in figure that had the largest infection (cases per million). . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . figure : examples of exponential fits for italy (a,b) and the us (c,d), where the log scale is used in (a,c) and the linear scale in (b,d). the yellow lines show exponential fits for different frames from to , as in figure (a,b) . here we present plots of the best fits for different classes of countries. figure shows the countries that were classified as a power law countries. the plots are presented on a log-log scale, such that the power law fits are straight lines. we can see that the best power law fit (blue) is a visibly better match than the exponential fit (yellow). note that for all of these countries the power law fitting error for any frame shift is smaller than that obtained by the exponential fitting. the rest of the power law countries (those that were classified as power law like) are shown in figure . this list contains countries. for convenience, we present both a log log plot (such that the power law fits, blue, appear as straight lines) and a log plot (such that the exponential fits, yellow, appear as straight lines). figure shows the countries that are characterized by a straight exponential growth. for these data, we used a log scale, such that the exponential fits are straight lines. for all of these countries the power law fitting error for any frame shift is larger than that obtained by the exponential fitting. note however that power fits that are almost as good as exponential fits can always be found, if we we shift the frame far enough. these fits correspond to very large values of the power coefficient b in the power law, see for example figure (a) which presents the example of the us. as the fitting frame index increases, the power law fitting error (top, blue line) approaches the exponential fitting error (horizontal yellow line). this, however, is meaningless, and does not indicate the presence of a power law. figure presents the rest of the countries from the exponential class, that is, those that were classified as exponential-like. . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint figure : comparison of two different choices of the fitting procedure, for countries. each panel represents a country, with the hrizontal axes being the fitting frame, and the vertical the fitting error. red symbols correspond to the case per million threshold, and blue circles to the cases per million threshold. circles represent the error of the power law fitting; they form non-constant functions. squares represent the error of the exponential fitting and form horizontal lines, because these errors do not depend on the fitting frame. . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint figure : the countries that were classified as those following a power law. for each country, two panels are presented. one is the full data (cases per million) plotted on a log log scale. the other is the subset of data (with or more cases per mission) plotted on a log-log scale (black circles) together with the best power law (blue line) and exponential (yellow line) fits. . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint figure : the countries that were classified as power law like. for each country, three panels are presented: ( ) is the full data (cases per million) plotted on a log log scale. ( ) is the subset of data (with or more cases per mission) plotted on a log-log scale (black circles) together with the best power law (blue line) and exponential (yellow line) fits. ( ) is the same as ( ) except on a log scale. . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint figure : the countries that were classified as those following an exponential law. for each country, two panels are presented. one is the full data (cases per million) plotted on a log log scale. the other is the subset of data (with or more cases per mission) plotted on a log scale (black circles) together with the exponential fit (yellow line). note that for these countries, the power law fits correspond to very high values of the exponent and are therefore not significantly different from the exponential fits. figure : the countries that were classified as exponential-like. panels are as in figure . . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint the covid- epidemic the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study. the lancet fair allocation of scarce medical resources in the time of covid- impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and healthcare demand covid- and italy: what next? the lancet covid- epidemic outside china: founders and exponential growth. medrxiv estimation of country-level basic reproductive ratios for novel coronavirus (covid- ) using synthetic contact matrices. medrxiv the reproductive number of covid- is higher compared to sars coronavirus estimating the reproductive number and the outbreak size of novel coronavirus disease (covid- ) using mathematical model in republic of korea the reproductive number r of covid- based on estimate of a statistical time delay dynamical system. medrxiv covid- containment: china provides important lessons for global response scaling features in the spreading of covid- coronavirus may have infected half of uk population how should pathogen transmission be modelled? trends in ecology & evolution effective containment explains sub-exponential growth in confirmed cases of recent covid- outbreak in mainland china acknowledgements. support of grant nsf dms / is gratefully acknowledged. key: cord- -pezm tnw authors: riccardo, flavia; ajelli, marco; andrianou, xanthi; bella, antonino; del manso, martina; fabiani, massimo; bellino, stefania; boros, stefano; mateo urdiales, alberto; marziano, valentina; rota, maria cristina; filia, antonietta; d'ancona, fortunato (paolo); siddu, andrea; punzo, ornella; trentini, filippo; guzzetta, giorgio; poletti, piero; stefanelli, paola; castrucci, maria rita; ciervo, alessandra; di benedetto, corrado; tallon, marco; piccioli, andrea; brusaferro, silvio; rezza, giovanni; merler, stefano; pezzotti, patrizio title: epidemiological characteristics of covid- cases in italy and estimates of the reproductive numbers one month into the epidemic date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: pezm tnw background in february , a locally-acquired covid- case was detected in lombardia, italy. this was the first signal of ongoing transmission of sars-cov- in the country. the outbreak rapidly escalated to a national level epidemic, amid the who declaration of a pandemic. methods we analysed data from the national case-based integrated surveillance system of all rt-pcr confirmed covid- infections as of march th , collected from all italian regions and autonomous provinces. here we provide a descriptive epidemiological summary on the first , covid- cases in italy as well as estimates of the basic and net reproductive numbers by region. findings of the , cases of covid- analysed, . % were reported from three regions (lombardia, veneto and emilia-romagna). all cases reported after february th were locally acquired. estimates of r varied between . ( %ci: . - . ) in toscana and ( %ci: . - . ) in lazio, with epidemic doubling time of . days ( %ci: . - . ) and . days ( %ci: . - . ), respectively. the net reproduction number showed a decreasing trend starting around february - , in northern regions. notably, , cases were reported among health care workers. of the , reported covid- associated deaths, % occurred in people aged years or above with an overall crude cfr of . %. male sex and age were independent risk factors for covid- death. interpretation the covid- infection in italy emerged with a clustering onset similar to the one described in wuhan, china and likewise showed worse outcomes in older males with comorbidities. initial r at . in lombardia, explains the high case-load and rapid geographical spread observed. overall rt in italian regions is currently decreasing albeit with large diversities across the country, supporting the importance of combined non-pharmacological control measures. introduction sars-cov- infection in humans causing clusters of severe pneumonia ( ) ( ) ( ) was first detected in the city of wuhan, china, in december . this pathogen was confirmed as a new virus by the coronavirus study group (csg) of the international committee on taxonomy of viruses. based on phylogeny, taxonomy and established practice, they designated it as severe acute respiratory syndrome coronavirus (sars-cov- ). ( ) although of probable zoonotic origin, human-to-human transmission is rapidly fuelling the spread of sars-cov- infections globally, with the main known routes of transmission being droplet and fomites. the infection spread within china and rapidly across countries, with cases in europe initially limited to small clusters in germany, france ( , ) and the uk ( ). on the th of february , the first case of locally acquired sars-cov- infection was diagnosed in northern italy in a critically ill, hospitalized young man with no travel history to known areas of viral circulation or link to a probable or confirmed covid- case. prior to this date, only three cases of covid- had been reported in central italy, all with a travel history to wuhan. following this unexpected finding, extensive contact tracing and testing of close contacts unveiled ongoing transmission in several municipalities of the lombardia region ( , ) . in subsequent days and weeks, case counts, and death tolls increased rapidly, at first in northern italy and then in the rest of the country. the italian government imposed increasingly strict physical distancing measures starting with the closure of municipalities in the lodi province (lombardia) and one in the padua province (veneto) on the rd of february . this culminated with a national lock-down on the th of march . ( , ) in this paper, we summarize key epidemiological findings from data on the first , confirmed covid- cases in italy, including , associated deaths, and initial findings on sars-cov- transmissibility across different regions. with the aim to increase the understanding of the disease dynamics in italy and support the planning of public health actions, a case-based surveillance system was established on the th of february , building on a previously existing surveillance system focused only on suspected and confirmed covid- severe respiratory infections ( ) . the system contains data on all laboratory confirmed cases of covid- as per the case definition published and regularly updated online by the european centre for disease prevention and control (ecdc) ( ) . laboratory confirmation by rt-pcr on nasopharyngeal swabs is performed at regional level, as previously described ( , ) . from the beginning of the outbreak until the st of march , all initially confirmed cases were sent for final confirmation at the national reference laboratory in the istituto superiore di sanità (iss) and re-confirmed according to the world health organization guidelines using rt-pcr protocols described by corman et al. ( ) and by the us-cdc ( ). due to the high concordance ( %) among confirmation results with the engaged laboratories, the policy was then changed allowing selected regions with demonstrated confirmation capacity to directly confirm covid- cases ( ) . data is collected daily using a secure online platform or, alternatively, received as individual datasets from the regions and two autonomous provinces (ap) of the italian territory, according to an increasingly harmonized track-record. data collected includes information on: demographics, clinical severity, comorbidities, date of symptom onset, date of diagnosis, outcome, region of diagnosis and province of residence. clinical severity was defined as follows: (i) asymptomatic -a person found positive for sars-cov- with no apparent signs or symptoms of disease, (ii) paucisymptomatic -a person found positive for sars-cov- with general mild symptoms (e.g. with general malaise, low grade fever, tiredness etc.) but no clear signs of disease, (iii) mild -a person found positive for sars-cov- with clear signs and symptoms of disease (e.g., respiratory disease) but not severe enough to require hospitalisation, (iv) severe -a person found positive for sars-cov- with clear signs and symptoms of disease (e.g. respiratory disease) and severe enough to require hospitalisation, and (v) critical -a person found positive for sars-cov- with clear signs and symptoms of disease (e.g., respiratory disease) and severe enough to require admission to an intensive care unit (icu). the surveillance system captures whether the reported subject is a health care worker (hcw). we have defined a hcw broadly as a person who has ever worked in the health care sector regardless of role, profession or current working status. the system also records whether the affected person has one of the following comorbidities: cardiovascular diseases, respiratory diseases, diabetes, immune-deficiencies, metabolic diseases, oncologic diseases, obesity, kidney diseases or other chronic diseases. we have defined covid- associated deaths as any person who has died and was confirmed to be infected with sars-cov- . data is harmonized in a single dataset, cleaned and analysed daily to produce an infographic with main surveillance outputs. a more detailed bulletin is published biweekly. these outputs are publicly available on the web portal of epidemiology of the iss ( ) . in total, , cases were included in the analysis (data extracted on march , ) . the data was summarized by age group and sex. epidemic curves were made by date of diagnosis and of symptom onset. cases were aggregated by region/ap of diagnosis and by province of residence for cases residing in the same region/pa of diagnosis. attack rates per , population by region/pa were calculated using population estimates for , available from the italian national institute of statistics (istituto nazionale di statistica; istat) and adjusted using the age distribution of the italian population as a reference. we classified the attack rates in each region as high, intermediate and low based on the inter quartile range (iqr) of the adjusted attack rates as follows: (i) high: . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org / . / . / attack rates higher than the upper limit of the iqr; (ii) intermediatewithin the iqr; (iii) lowlower than the lower limit of the iqr. case fatality rates (cfr), not accounting for delays, were calculated by age and sex and smoothed with the locally weighted regression method. cfrs by age were also calculated by calendar period of diagnosis (i.e., before th , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a multilevel (clustered by region/ap) multivariable logistic model was applied to evaluate characteristics associated with death, including age group (i.e., ≤ , - , - , - , - , - , + years) , sex, hcw status, and week of diagnosis. adjusted odds ratios were estimated. the analyses were performed using stata (version ) and r (version . . ). the list of the r packages used for the analysis is available in the supplementary materials. as described previously ( ) , we define the basic reproduction number r as the average number of secondary cases that are generated in a fully susceptible population by a primary infector. this is an expression of the potential for transmission without any containment measure. however, once interventions are introduced or the susceptibility in the population decreases, the transmission potential at a given time t is measured as the net reproduction number rt. in this paper, we estimated both r and rt for italian regions in different epidemiological situations (high, intermediate and low age-adjusted attack rates), selected among those with highest data robustness. we used a previously described bayesian approach ( ) ( ) ( ) , informed by estimates of the serial interval from contact tracing data in lombardia ( ) . we defined the serial interval as the distribution of the time from symptoms onset in a primary infector and the symptoms onset in secondary cases and estimated it on average to be · days. details are reported in the supplementary materials. as of the th of march , , confirmed cases of covid- had been reported including , related deaths, and the number of reported cases was increasing ( figure ). locally acquired cases diagnosed at the end of february reported onset of symptoms from the th of january onwards, indicating undetected local transmission for at least three weeks before detection. the average delay between symptom onset and diagnosis in the first month of the outbreak was · days. by march , , all of italy's regions and aps had reported at least one locally acquired case of covid- . the country had high incidence areas with sustained local transmission (mainly in the north), low incidence areas with limited but growing numbers of locally acquired cases of infection and regions with intermediate incidence ( figure ). overall, % of , covid- cases diagnosed in lombardia, were among people residing in this region. among the remaining % for which the residence of the case was known, most ( cases) resided in the neighbouring region of emilia romagna. the index case of the outbreak was not found, and no clear chains of transmission were identified linking initial cases in newly affected regions/ap. age -adjusted attack rates per , were classified as high in lombardia (crude attack rate · and age-adjusted attack rate · per , ), valle d'aosta, trento, the region of emilia-romagna bordering lombardia and in the marche region in central italy. age -adjusted attack rates were classified as intermediate in bolzano/bozen, veneto, piemonte, liguria, friuli-venezia giulia, abruzzo, toscana, umbria, molise and puglia. in lazio, campania, sardegna, calabria, sicilia and basilicata age -adjusted attack rates were classified as low ( figure and table , supplementary material). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint most affected cases were male ( %) and the median age of cases was years. notably, , cases were reported among health care workers (median age years, · % male). among cases with known age, clinical severity was reported for , cases. as shown in figure , the proportion of severe cases in under years of age was below %. this proportion decreased to . % in the - year age group, then increased again gradually to · % in the age group - years. critical severity was reported in cases aged years and above, reaching % in the - year age segment ( table , supplementary material). among all cases, , ( · %) were reported to have been managed at home and , ( · %) were hospitalised. for , ( · %) cases this information was not available. as shown in figure (a), the proportion of hospitalised covid- cases decreased from . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org / . / . / - to - years of age and increased progressively from - to - years of age when it appeared to stabilize. figure (b) shows, among all reported hospitalised covid- cases with known unit of admission and age (n= , ) , the proportion of patients admitted in icu vs those admitted to any other hospital unit. overall, the icu admission rate was · % considering only cases for which admission hospital unit was reported. among all cases reported to have been hospitalised, the icu admission rate was · %. icu admissions were reported from the - age group onwards, increasing in proportion until ages - years. the proportion decreases in older age groups. of the , reported covid- associated deaths, % occurred in cases aged years or above with an overall crude case fatality rate (cfr) of · % (table , supplementary material). overall, deaths were reported in hcw. until march , , no deaths were reported among cases aged less than years. overall, % of the people who died were reported to have at least one co-morbidity. figure shows the cfr reported as of the th of march by single age of diagnosed cases stratified by sex smoothed using locally weighted regression curves. there was a strong association of the cfr with age. women had a lower cfr at each age point. when stratifying by age group and calendar period of diagnosis we confirmed the age effect on the cfr but also showed that cfr increases with time since diagnosis, with an overall cfr of % for people diagnosed with covid- before the th of march (table , supplementary material). after adjusting for age, sex, hcw profession, and calendar period of diagnosis, we estimated a higher adjusted odds ratios (aor) of death with increasing age decade and a higher aor for males compared to females. health care workers (hcw) diagnosed with covid- had lower aor of death compared to the non hcw (table , supplementary material). we performed additional models evaluating a possible interaction between sex and age group and between being a hcw and both age and sex. however, these did not provide a better fit to the data (p= · , p= · , p= . log-likelihood ratio test, respectively). similar results were found when restricting the analysis to cases aged - years old. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org / . / . . . doi: medrxiv preprint we estimated the transmission patterns for six italian regions (lombardia, veneto, emilia-romagna, toscana, lazio, and puglia) with adjusted ar classification ranging from low to high. these six italian regions are characterized by highly different epidemic trajectories. the variability is also clearly visible in terms of epidemic doubling time, which varies between · days ( %ci: · - · ) in emilia romagna and · days ( %ci: · - · ) in veneto (table ) , and basic reproductive numbers, which lie in the range · - · . in lombardia, we estimated the net reproduction number (rt) to be above the epidemic threshold since late january ( figure ). in february, the rt started to oscillate reaching maximum values around over the week from february to . starting from february , with the enforcement of a quarantined area around the most affected . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org / . / . / municipalities of the region, rt was estimated to follow a constantly decreasing trend. the second and third most affected regions in february (veneto and emilia romagna) show an increasing trend of rt until about february ( figure ). at that time a few tens of cases had been detected in those regions. the transmission patterns of toscana, lazio, and puglia are markedly different. in central and south italy, where tuscany, lazio, and apulia are located, the epidemic spread was largely undetected until early march. after an initial increase, rt remained nearly constant at values around · - until march - , when blanket physical distancing measures began being implemented at national level ( figure ). covid- emerged in italy with a similar clustering onset to the one described in wuhan, china ( ) with three major clusters around the cities of codogno, bergamo and cremona in the lombardia region in northern italy ( ) . subsequently, cases spread across the country with more sustained transmission in neighbouring regions in the north and in the central region of marche. we estimated r in the range of · - · in different italian regions with doubling time estimated between · and · days. this, alongside the short serial interval of covid- in italy ( · days on average) and the fact that the outbreak was detected amid ongoing transmission that had started at least three weeks earlier, explains the rapid case increase and geographical spread. it also explains the inability to identify the index case and clearly trace the initial spread of infection across the country. notably, in northern regions (lombardia, veneto and emilia-romagna), the net reproduction number rt shows a marked decreasing trend since the identification of the first cases in late february. the observed decrease of rt in northern regions is possibly due to increased population awareness and to the early effect of interventions. in other parts of the country, such as in central (toscana, lazio) and southern regions (puglia), transmission was largely undetected until the first days of march, with rt decreasing after the initial implementation of national blanket physical distancing measures. cases were for the most part detected in the same region/ap in which they reside. most initial cases that did not reside in lombardia resided in neighbouring emilia-romagna. this could be related to the fact that initial clusters occurred in industrial hubs with strong cross-border connections with neighbouring provinces in other regions, possibly favouring the rapid geographical spread of the infection in the north of the country. reported clinical severity was confirmed to be lowest among children, and in particular in the - years age group. the proportion of severe and critical cases increases with age until age or above. the slight decrease in the proportion of critical and severe cases, and of deaths, in the higher age groups might be due to the demographic structure of the population with a higher female to male ratio among older people ( ) . the proportion of hospitalized covid- cases appears to follow a similar trend with age. hospitalisation rates decreased from infancy to childhood and adolescence to increase again progressively with age among adults stabilizing from the - years age group. overall, the icu admission rate based on reported data ranged between . % and . % depending on the denominator used. both rates are much higher than the reported % in european union countries disclosed by the european centre for disease prevention and control (ecdc) ( ) . we are unable at this stage to speculate whether the reasons behind this difference are related with hospitalisation policies and practices or whether there are other factors at play. however, this observation confirms recent studies in lombardia that highlight the potentially catastrophic effects of an uncontrolled covid- epidemic on the healthcare system ( , ) . consistently with reported clinical severity, icu admissions were notified starting from the - years age segment, suggesting the potential for critical disease also among young adults. the proportion of covid- related icu admissions, increases until the - years age segment to decrease in older ages apparently more rapidly than the reported stabilization of more severe disease in the same age groups. this pattern, however, might simply reflect lack of completeness of the surveillance data available at this stage. further analysis on a larger number of records is required to confirm this finding. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org / . / . / as shown previously in china ( ), we also demonstrated worse outcomes in older males with comorbidities. male sex and older age are independent risk factors for covid- related deaths. the cfr of . % documented overall in italy, is higher than that observed in other countries. as recently described, this could be in part explained by the demographic structure of the italian population ( ) . however, other aspects such as testing policies and the surveillance system characteristics, including the choice in italy to count only laboratory confirmed cases and to define associated deaths in a very inclusive manner, may also play a role in making initial case fatality data poorly comparable across countries. it is however noteworthy to stress that the early phase of the covid- epidemic in italy is characterized by a large number of cases with a short follow-up time since diagnosis. this implies that the overall cfr is currently largely underestimated. this was confirmed when performing an analysis by period of diagnosis. we found that those diagnosed before march th, had an overall cfr of around %. future studies with longer follow-up will better clarify this aspect including studies evaluating overall population excess mortality, which will also be more comparable across countries. surveillance data shows clearly a very high number of covid- cases among hcw, underlining the fact that sars-cov- can be easily spread in health care contexts and the importance of strong infection prevention and control practices. in italy, as described in other countries ( ) , nursing homes and long term care facilities have emerged as particularly fragile environments in which infection can spread very rapidly with potentially more severe outcomes due to the vulnerability of the hosted populations ( ) . affected hcw, compared with the affected general population, are on average younger and more frequently female. considering the predominance of female professionals in working age in the italian health sector, this is the distribution that would be expected in professionally exposed groups. age and sex only explains in part the lower cfr, as a lower aor of death was observed among hcw also after adjusting for those variables and might be related to earlier detection and management. the data collected from the italian integrated covid- surveillance system during the initial phase of the emergency presents a number of limitations mainly due to completeness challenges. for this reason, some stratifications and analysis were not shown. the lack of completeness on the presence and type of comorbidities, did not allow us to include this in the multivariable analysis of deaths in order to assess, and/or adjust for, this factor. data on hospitalisation and icu admissions as well as cfrs are not adjusted for the expected time for disease evolution and might therefore be underestimated in the more recent period. finally, the estimation of r , rt and the doubling time were performed in regions selected on the basis of the robustness of data considering epidemiologically diverse settings. even in the presence of the mentioned limitations, our analysis suggests that the sars-cov- transmission potential in italy is decreasing, albeit with large diversities across the country. further, we observe that as of march , the rt it is still above the epidemic threshold. the progressively harsh physical distancing measures enacted since then may have enhanced the decreasing trend in transmissibility as happened in china [ , ] . the surveillance system will be key to monitor the effect of the implemented policies and guide the public health response as italy will enter the second phase of its outbreak. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org / . / . / evidence before the widespread sustained local transmission of covid- in italy was largely based on the experience shared by china ( , ) and specific other countries in asia, while evidence in europe was initially limited to small local clusters in germany, france ( , ) and the uk ( ). while some commentaries have been issued regarding the evolving situation in italy ( ) and initial studies are available on the epidemic in the first affected region lombardia ( , ) or based on different modelling approaches, there has been yet no communication to the scientific community of the italian epidemiological situation at national level based on surveillance data. in this paper, we summarize key epidemiological findings from data on the first , confirmed covid- cases in italy, including , associated deaths, and initial findings on sars-cov- transmissibility across different regions. the added value of this study is of providing an in depth review of the first month of the italian outbreak through descriptive and analytic epidemiology and an estimation of the r and rt taking into account the diversity of transmission across the country. we believe that the evidence collected in italy, with a demographic structure and health system organization that differs from those of other countries that had reported sustained disease transmission, can be of support to countries currently experiencing initial or escalating covid- transmission. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org / . / . / the basic reproduction number r represents the average number of secondary cases generated by a primary infector in a fully susceptible population. in general terms, when r is larger than the infection may spread in the population and the larger r the larger effort required to control the epidemic. once the number of susceptible individuals declines, the transmission potential of the disease at a given time t is measured in terms of the net reproduction number r(t). the net reproduction number is useful to track the effectiveness of performed control measures and other factors affecting the spread of the epidemic (e.g., the behavioral response of the population) over time. as soon as r(t) falls below , the epidemic starts to decline. to estimate r(t), we use the same methodology presented in reference [ ] [ ] [ ] . we assumed that the daily number of new cases (date of symptom onset) with locally acquired infection l(t) can be approximated by a poisson distribution according to the equation where  c(t), with t from to t, is daily number of new cases (date of symptom onset);  r(t) is the net reproduction number at time t;  ( ) is the distribution of the generation time (corresponding to the distribution of the serial interval) calculated at time s. from the analysis of observations of individual serial intervals in clusters, the distribution of the serial interval was estimated to follow a gamma distribution with mean . days (percentiles . th and . th of the distribution: . - . ) [ ] . the likelihood ℒ of the observed time series of cases from day to day t conditional on c( ) is thus given by where p(k; λ) is the probability mass function of a poisson distribution (i.e., the probability of observing k events if these events occur with rate λ). we then used mcmc metropolis-hastings sampling to estimate the posterior distribution of r(t). to estimate r , we assumed that during the period where the epidemic showed exponential growth r(t)=r and used the above described procedure. clinical features of patients infected with novel coronavirus in wuhan, china. the lancet epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study. the lancet a familial cluster of pneumonia associated with the novel coronavirus indicating person-toperson transmission: a study of a family cluster. the lancet severe acute respiratory syndrome-related coronavirus: the species and its viruses -a statement of the coronavirus study group. biorxiv first cases of coronavirus disease (covid- ) in france: surveillance, investigations and control measures transmission of -ncov infection from an asymptomatic contact in germany lessons for managing highconsequence infections from first covid- cases in the uk. the lancet potential short-term outcome of an uncontrolled covid- epidemic in the early phase of the covid- outbreak in disposizioni attuative del decreto-legge febbraio , n. , recante misure urgenti in materia di contenimento e gestione dell'emergenza epidemiologica da covid- ulteriori disposizioni attuative del decreto-legge febbraio , n. , recante misure urgenti in materia di contenimento e gestione dell'emergenza epidemiologica da covid- , applicabili sull first cases of coronavirus disease (covid- ) in the who european region case definition and european surveillance for covid- european centre for disease prevention and control whole genome and phylogenetic analysis of two sars-cov- strains isolated in italy in ministerial decree n. -covid- . aggiornamento della definizione di caso integrated surveillance of covid- : main national data ebola virus disease in west africa -the first months of the epidemic and forward projections measurability of the epidemic reproduction number in data-driven contact networks evolving epidemiology of novel coronavirus diseases and possible interruption of local transmission outside hubei province in china: a descriptive and modeling study. medrxiv demographic statistics -italian national institute of statistics -istituto nazionale di statistica istat rapid risk assessment: coronavirus disease (covid- ) pandemic: increased transmission in the eu/eea and the uk -seventh update european centre for disease prevention and control characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention case-fatality rate and characteristics of patients dying in relation to covid- in italy nursing homes are ground zero for covid- pandemic national survey on covid- spread in nursing homes and long term care facilities, first report world health organization ebola response team, ebola virus disease in west africa--the first months of the epidemic and forward projections measurability of the epidemic reproduction number in data-driven contact networks evolving epidemiology of novel coronavirus diseases and possible interruption of local transmission outside hubei province in china: a descriptive and modeling study the early phase of the covid- outbreak in lombardy, italy extended model formulas in r: multiple parts and multiple responses inspectdf: inspection, comparison and visualisation of data frames officer: manipulation of microsoft word and powerpoint documents rvg: r graphics devices for vector graphics output broom: convert statistical analysis objects into tidy tibbles with contributions from charles dupont and many others dates and times made easy with lubridate ggplot : elegant graphics for data analysis the split-apply-combine strategy for data analysis stringr: simple, consistent wrappers for common string operations forcats: tools for working with categorical variables (factors) scales: scale functions for visualization haven: import and export 'spss', 'stata' and 'sas' files readxl: read excel files tidyr: tidy messy data readr: read rectangular text data dplyr: a grammar of data manipulation eeptools: convenience functions for education data glue: interpreted string literals epidemic curves made easy using the r package incidence tibble: simple data frames purrr: functional programming tools the report package for r: ensuring the use of best practices for results reporting cartocolors' palettes simple features for r: standardized support for spatial vector data janitor: simple tools for examining and cleaning dirty data lattice: multivariate data visualization with r viridislite: default color maps from 'matplotlib' (lite version) _a package for survival analysis in s_. version . patchwork: the composer of plots epitools: epidemiology tools welcome to the tidyverse knitr: a general-purpose package for dynamic report generation in r table -distribution of diagnosed ) key: cord- -buzdfgyq authors: beccuti, g.; ghizzoni, l.; cambria, v.; codullo, v.; sacchi, p.; lovati, e.; mongodi, s.; iotti, g. a.; mojoli, f. title: a covid- pneumonia case report of autoimmune polyendocrine syndrome type in lombardy, italy: letter to the editor date: - - journal: j endocrinol invest doi: . /s - - - sha: doc_id: cord_uid: buzdfgyq nan we thank the european society of endocrinology (ese) for providing the global endocrinology community with the statement on coronavirus disease and endocrine disorders [ ] . regarding addison's disease, the ese statement affirms that there is no evidence that patients with adrenal insufficiency are at increased risk of contracting covid- , and there are no reported data on the outcomes of covid- infection in adrenal-insufficient individuals. herein, we present the case of a -year-old woman with autoimmune polyglandular syndrome type who developed covid- caused by severe acute respiratory syndrome coronavirus (sars-cov- ); she lived closed to the first lombardy cluster spreading from codogno, italy. her clinical, immunological, and genetic patterns have been previously described (as patient no. ) in a case series of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced), also known as autoimmune polyendocrine syndrome type (aps- ) [ ] . in summary, she carries a homozygous r x mutation in exon five of the autoimmune regulator (aire) gene, resulting in primary adrenal insufficiency (pai), hypoparathyroidism, hypogonadism, ectodermal dystrophy, candidiasis, pernicious anemia, and gastrointestinal dysfunction. because her chronic hypoparathyroidism was inadequately controlled by standard treatment alone, the patient had been on hormonal replacement therapy with recombinant human parathyroid hormone (rhpth) ( - ) since january . after starting rhpth ( - ) µg once daily as a subcutaneous injection, she stopped calcitriol and calcium supplementation, having achieved optimal and stable serum calcium levels. her treatment was specifically approved by the italian medicines agency (aifa) before the marketing authorization in italy, and the patient underwent regular follow-up at the university of turin (piedmont, italy). on february , the patient presented to the emergency department of the cremona hospital (lombardy, italy) with fever, cough, and dyspnea. high-resolution computed tomography showed multiple and bilateral ground-glass opacities of the lungs. bronchoalveolar lavage fluid was positive for sars-cov- . after an unsuccessful trial of non-invasive ventilation, her progressive respiratory failure required endotracheal intubation and mechanical ventilation. therefore, she was transferred to the intensive care unit (icu) of san matteo hospital in pavia (lombardy, italy) on february . pharmacological treatment included empirical antiviral regimens with lopinavir/ritonavir and ribavirin, and prophylaxis with hydroxychloroquine, azithromycin, piperacillin/tazobactam, and trimethoprim-sulfamethoxazole. hemodynamic support required norepinephrine and dobutamine infusion; intravenous hydrocortisone was subsequently introduced at a dose of mg divided in bolus injections over h. after days, the patient was extubated and started helmet continuous positive airway pressure. during hospitalization, the intravenous glucocorticoid dose was gradually tapered and eventually switched back to the pre-admission oral regimen. unfortunately, the patient's rhpth treatment was interrupted after admission. although the summary of product characteristics (smpc) does not include diseases requiring intensive care among reasons for discontinuation, rhpth has not been studied in this specific clinical setting. therefore, calcitriol and calcium supplementation was needed again, leading to suboptimal serum calcium concentrations. a few days before discharge, the patient resumed treatment with rhpth ( - ), with sufficient -hydroxy vitamin d stores. following the instructions in the smpc, oral calcium supplementation was progressively reduced, and calcitriol supplementation was stopped within days, as the patient achieved adequate serum calcium concentrations. the patient was discharged on march , after days of hospitalization, with complete resolution of symptoms and two negative tests for sars-cov- at an interval of h. her last chest x-ray showed bronchiolitis obliterans organizing pneumonia-like features that will need radiological and clinical follow-up. as of march , among , confirmed covid- patients in italy, . % required intensive care (data from the italian civil protection department website https ://www. prote zione civil e.it-english version available; last access on may ). baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus in lombardy have been recently published; median length of stay in icu was days, and almost all the patients required respiratory support, mainly invasive mechanical ventilation ( %) [ ] . as soon as information is also available on the characteristics of non-icu hospitalized covid- patients in italy, it would be interesting to explore whether pai may worsen covid- outcomes, such as length of hospital stay, incidence of acute respiratory distress syndrome, and the need for cpap therapy and mechanical ventilation. the ese statement notes the occurrence of impaired natural immunity function in pai, with defective action of neutrophils and natural killer cells, independent of the underlying etiology [ ] , suggesting that exogenous glucocorticoids play a role in modulating the immune system. our case might not be considered a paradigmatic example of pai; at variance with other forms of autoimmune addison disease, patients with aps- have primary immunodeficiency, which explains the t-cell deficiency-related chronic mucocutaneous candidiasis. this feature has relevant clinical consequences; in fact, a recent prospective study showed that the finnish aps- cohort had an increased mortality from infections (standardized mortality ratio ; % ci . - ) in comparison to the general population [ ] . similarly, an increased risk of infections has been observed also in other forms of addison's disease, with autoimmune or genetic basis. however, in these forms, there is no clear demonstration of primary immunodeficiency, and the infection risk in addison's disease could be linked to the types or doses of glucocorticoid replacement. a population-based, retrospective, open cohort study in the united kingdom from to showed that the addison's disease cohort, compared with matched controls, had a higher risk of infections of the lower respiratory [adjusted incidence rate ratio (airr) . ; % ci . - . ], urinary (airr . ; % ci . - . ), and gastrointestinal (airr . ; % ci . - . ) tracts, leading to increased use of antimicrobial agents in the primary care setting [ ] . interestingly, the same study showed no increased risk of infection in patients with untreated congenital adrenal hyperplasia (cah), but an increased infection risk in patients treated for cah, suggesting that nonphysiological delivery of glucocorticoid replacement may represent a risk factor for the development of infections [ ] . all of these findings are in line with the increased infection-related mortality described in the literature. a population-based, retrospective study on the swedish population from to found that the mortality rate resulting from infections in addison's disease was five times higher than expected (risk ratio . ; % ci . - . in women; risk ratio . ; % ci . - . in men) [ ] . another swedish population-based, retrospective study from to reported increased mortality from infections in patients with autoimmune pai (standardized mortality ratio . ; % ci . - . ) [ ] . in norway, a population-based, retrospective study from to reported an increase in mortality from infections associated with addison's disease [ % among causes of death ( % ci . - . ) vs. . % in the general population] [ ] . the first prospective study investigating causes of death in pai and secondary adrenal insufficiency (sai) was based on real-world data from patients ( pai, , sai) in the european adrenal insufficiency registry (eu-air; clinicaltrials.gov identifier nct ). the primary objective of eu-air is to monitor the safety of long-term treatment with once-daily, modified-release hydrocortisone and other glucocorticoid replacement therapies in adrenal insufficiency. of the deaths registered from to , only % occurred to subjects with addison's disease. infections accounted for % of the deaths. with the limitations of a small percentage of autoimmune pai among infection-related deaths and the eu-air inclusion of european adrenal insufficiency patients treated in highly specialized centers, the results of this observational, open-ended study appear to be consistent with the previous evidence from retrospective studies [ ] . in conclusion, it is not clear whether the disease per se or the hormonal replacement without physiological glucocorticoid rhythm plays a pre-eminent role in the impaired immune function of pai. our case report, along with the aforementioned studies, calls attention to the increased risk of infections in addison's disease, with or without associated primary immunodeficiency. further research worldwide covid- and endocrine diseases a statement from the autoimmune-polyendocrinopathycandidiasis-ectodermal-dystrophy in calabria: clinical, immunological and genetic patterns lombardy icu network baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region primary adrenal insufficiency is associated with impaired natural killer cell function: a potential link to increased mortality patients with apeced have increased early mortality due to endocrine causes, malignancies and infections increased infection risk in addison's disease and congenital adrenal hyperplasia premature mortality in patients with addison's disease: a population-based study increased death risk and altered cancer incidence pattern in patients with isolated or combined autoimmune primary adrenocortical insufficiency normal overall mortality rate in addison's disease, but young patients are at risk of premature death eu-air investigators mortality data from the european adrenal insufficiency registry-patient characterization and associations key: cord- -fc tmx authors: ciminelli, gabriele; garcia-mandicó, sílvia title: covid- in italy: an analysis of death registry data date: - - journal: j public health (oxf) doi: . /pubmed/fdaa sha: doc_id: cord_uid: fc tmx background: there are still many unknowns about covid- . we do not know its exact mortality rate nor the speed through which it spreads across communities. this lack of evidence complicates the design of appropriate response policies. methods: we source daily death registry data for municipalities in italy’s north and match them to census data. we augment the dataset with municipality-level data on a host of co-factors of covid- mortality, which we exploit in a differences-in-differences regression model to analyze covid- -induced mortality. results: we find that covid- killed more than . % of the local population during the first wave of the epidemic. we also show that official statistics vastly underreport this death toll, by about %. next, we uncover the dramatic effects of the epidemic on nursing home residents in the outbreak epicenter: in municipalities with a high share of the elderly living in nursing homes, covid- mortality was about twice as high as in those with no nursing home intown. conclusions: a pro-active approach in managing the epidemic is key to reduce covid- mortality. authorities should ramp-up testing capacity and increase contact-tracing abilities. adequate protective equipment should be provided to nursing home residents and staff. there are still many unknowns about covid- . we do not know its true mortality rate nor the speed through which it spreads across communities. this lack of evidence complicates the design of appropriate response policies. the case of the uk is illustrative. the government first opted for bare minimum mitigation. it then drastically reversed course after microsimulations by the imperial college covid- response team showed that that strategy could have resulted in hundreds of thousands of deaths. this paper provides first-hand evidence on some of these questions. we focus on italy's north, for long the world's worst affected area by covid- . using daily death registry data from the italian statistical agency, we first quantify the death toll from and by covid- and show that many deaths went unrecorded in official statistics. covid- was responsible for the death of about people-more than . % of the local population-during the first wave of the epidemic (from mid-february to mid-may ). its mortality is vastly undercounted in official statistics. a plausible estimate suggests that true covid- deaths might have been % higher than what is officially reported. at the peak of the epidemic, one additional death went undetected for each officially recorded covid- fatality. we then uncover large regional differences in mortality, which can be partly explained through the different approaches taken to deal with the epidemic. in the veneto region, which embraced mass testing, contact tracing, and at-home care provision, covid- -induced mortality was, respectively, three and six times smaller than in neighboring emilia-romagna and lombardy. we close the paper by discussing the management of nursing homes in lombardy, italy's covid- outbreak epicenter. nursing homes have been in the spotlight across europe and beyond for being possible hotspots of contagion and deaths. our results suggest that living in a nursing home may have significantly increased the probability of dying during the covid- epidemic. we find that in municipalities with a percentage point higher share of people living in a nursing home, mortality among the elderly was about twice as high as in those with no nursing home in town. this result helps to rationalize the serious undercounting of covid- fatalities in official statistics, which do not include deaths in nursing homes. we source daily death registry data from istat, the italian statistical agency. (accessed th may ). the data provide information on daily deaths by age and gender for the vast majority of italian municipalities, for the period january to may, for the years - . our focus is on the eight regions of italy's north (emilia-romagna, friuli-venezia giulia, liguria, lombardy, piedmont, trentino-south tyrol, valle d'aosta and veneto), which together account for roughly half of italy's population and about % of all covid- fatalities (as of may ). our data cover almost % of all municipalities in these eight regions and about % of their population. table a in the appendix reports descriptive statistics on the coverage of the dataset. figure below takes a first look at the data by comparing deaths in (red line) against those in each of the five preceding years. soon after the detection of the first covid- community case (vertical line), deaths increased rapidly. they jumped from about at the end of february to over at the end of march. from the beginning of april, as the government social distancing policies began to bear an effect, deaths slowly started to decrease and returned to normal levels around mid-may. our sample thus spans the entire first wave of the covid- epidemic in italy. we then source census population data from istat and match them to death registry data to analyze covid- induced mortality. census data provide information on the resident population by age and gender, as of january, for the years - . (accessed th april ). we impute values by using growth rates. we also source official covid- fatality data from protezione civile, to compare officially reported mortality to the mortality calculated from death registry data. lastly, we source municipality-level data on a host of cofactors of covid- mortality, such as population density, commuting, digital employment, air pollution and several demographic characteristics. we also source data on health care management, in particular nursing homes, for the lombardy region. deaths before the covid- outbreak were following fairly closely deaths in . assuming that this trend would have continued in the absence of covid- , we calculate a measure of excess deaths by subtracting deaths in to deaths in . to empirically evaluate the impact of covid- on mortality, we embed the concept of excess deaths in a differences-in-differences regression model, which allows us to control for a host of potentially confounding factors. more detail on the methodology is provided in the online appendix. using the concept of excess deaths, we calculate that the virus might have caused the death of about people-more than . % of the local population-during the first wave of the epidemic. next, we compare excess deaths in to official covid- fatality data. figure below plots the official daily number of covid- fatalities (dashed blue line) and excess deaths in relative , as recorded in municipal death registry data (solid red line). strikingly, excess deaths were higher than official fatalities throughout the end of april, suggesting that covid- deaths might have been vastly underreported in official statistics. what is the scale of underreporting? excess deaths include both indirect and direct covid- deaths, while official data only cover direct deaths. to gauge the importance of indirect deaths, we consider the regions of italy, which are not included in our sample because they were relatively unaffected by covid- . these display ratios of excess deaths to official covid- fatalities that are similar to the regions in our sample. assuming that these regions correctly detected all covid- deaths, we obtain a measure of indirect deaths as a share of the population, which we use to infer the number of indirect deaths in the eight regions in our sample. using this approach, we estimate that indirect deaths might account for - % of all deaths attributable to covid- . discounting indirect deaths, we calculate that covid- deaths may have been % higher than what was officially reported. at the peak of the epidemic (around end-march), deaths may have been underreported by about a factor of two. that is, for each officially recorded death, an additional one went undetected. what can explain underreporting? in italy, guidelines for the classification of covid- fatalities vary by region, but in most cases, deaths outside hospitals are not counted in official statistics. anecdotal evidence suggests that, as the health system struggled with a surge in demand, many old patients died of covid- at home or in elderly care facilities, without being tested. , this hypothesis seems plausible since the extent of underreporting was higher at the peak of the epidemic. limited testing capacity may be a complementary reason. while excess deaths from registry data decreased sharply after peaking at the end of march, official covid- fatalities peaked later. they also did not fall as sharply, suggesting that the authorities may have progressively ramped up testing capability. in may, the number of official fatalities was even higher than that of excess deaths. this may be due to lags in the official reporting of fatalities relative to the fatality date. data presented so far were aggregate for the entire sample of the municipalities covered in the dataset. in fig. below, we uncover variation in the extent of the epidemic both across different municipalities and over time. we assign different colors to municipalities, based on the mortality rate. municipalities with a mortality rate within pre-epidemic 'normal' levels ( - daily deaths per inhabitants) are denoted with blue. those with a mortality rate between and daily deaths per inhabitants are marked by different shades of orange, while red colors indicate those with very high rates (above per inhabitants). at the start of the epidemic, the mortality rate was generally within normal levels, except for a few municipalities. by march, two large outbreaks in the lombardy region had become apparent: one in the south, around the town of codogno (the epicenter), and another in the north, around alzano lombardo (a town close to bergamo). while deaths increased across most municipalities during the unfolding of the outbreak, the increase around these two clusters was much higher. along the border between lombardy and neighboring regions, death rates were high in both emilia-romagna and piedmont, while they stayed low in veneto. next, we use a differences-in-differences method to confirm the patterns observed in fig. and quantify the effects of covid- on the mortality rate by region. we estimate that the pandemic resulted in almost three deaths per inhabitants per day in lombardy, by far the region in which covid- had the highest effect on mortality. across the four regions bordering lombardy, we estimate the lowest impact in veneto, at around . daily death for every inhabitants. that is about three times less than trentino-south tyrol and piedmont and four times less than emilia-romagna-all sharing a long border with lombardy-and suggests that the bold approach adopted by the veneto region in managing the epidemic may have sensibly reduced covid- mortality there. details on the estimation and additional results are reported in the online appendix. the veneto region has been widely praised for its timely and proactive response to the pandemic. much has been written about the 'veneto model', , , and we only summarize its main characteristics: (i) mass testing, including testing of asymptomatic cases; (ii) at-home testing and care provision and (iii) tracing and quarantining contacts. both ramping up the testing capacity and developing a comprehensive tracking system have been recognized as essential elements to coping with the epidemic. , testing and providing care at home works towards preventing the spread of the virus. in a testament to the success of its strategy, on may, the veneto region registered zero new infections, while lombardy had more than . the veneto model provides useful lessons for policy makers around the world on how to manage new outbreaks of covid- in the future. living in a nursing home may have increased the chances of dying for the elderly next, we explore gender and age differences in covid- mortality. details on the estimation are provided in the online appendix, while here we only summarize the main findings. we first show that mortality increases exponentially with age, but at much higher levels for men, confirming that gender differences play a crucial role in understanding the distribution of risk from the epidemic. at the same time, underreporting greatly increases with age and is particularly high among older women. why do we observe so many undetected deaths among the elderly, and particularly so among women? all available anecdotal evidence points to nursing homes. , with a large number of residents sharing common spaces and having close contacts with multiple staff members, nursing homes may have acted as hotbeds of contagion. moreover, as in italy nursing homes do not qualify as medical centers, they were heavily understaffed and unprepared to deal with the crisis, lacking protective equipment for staff and emergency care equipment for infected patients. in lombardy, these inherent characteristics may have been particularly aggravating, as the regional authority decided to relocate covid- positive patients with mild symptoms from hospitals to nursing homes. since italy does not include fatalities in nursing homes in its covid- statistics, they likely account for a big chunk of the undetected deaths. we zoom in on lombardy, the worst affected region, to test whether covid- had an additional effect on mortality in municipalities with a higher share of people living in nursing homes (see the online appendix for details on the estimation). in fig. below, we compare the mortality effect of covid- in municipalities where % of the elderly population lives in nursing homes with no nursing home in town. the results suggest that living in a nursing home may have significantly increased the chance of dying during the covid- epidemic for both men and women. strikingly, we find that covid- mortality was twice as high in municipalities with a high proportion ( %) of the elderly living in a nursing home as in those municipalities with no nursing home in town. this paper provides first-hand evidence on the true death toll from and by covid- using italy's north-for long the world's worst affected area by covid- -as a case study. we show that covid- caused the death of more than . % of the local population during the first wave of the epidemic. we also show that many deaths go unrecorded in official statistics. a plausible estimate suggests that true deaths were about % higher than what was officially reported during the first wave of the covid- epidemic in italy. this undercounting is much more severe for the elderly, and in particular, for women. since italy does not include fatalities in nursing homes in its covid- statistics, they likely account for a big chunk of the undetected deaths. our analysis quantifies the catastrophic effects of covid- in nursing homes. in municipalities with a percentage point higher share of the elderly population living in a nursing home, mortality was about twice as high as those with no nursing home in town. our analysis shows that many of them could have been prevented through better preparedness. providing adequate protective equipment is key to protecting nursing homes' residents and staff. even more essential is the need to identify and isolate positive cases, and prevent staff from going to work if they are affected. the concept of excess deaths has been used by several statistical offices around the globe to quantify covid- mortality. a notable example is the uk statistical office (ons), which has produced relevant work comparing excess mortality across european countries. some media outlets have already relied on the concept of excess deaths to suggest that covid- fatalities are undercounted in official statistics. , others have provided some anecdotal evidence suggesting that nursing homes around the globe may have been hotbeds for contagion and mortality. for instance, the economist has analyzed data from european countries that count deaths in nursing homes in official statistics and found that these make up for percent of all official fatalities, on average. this ratio increases to percent in belgium. we use highly granular daily death registry data for thousands of municipalities in italy's north to conduct a precise estimation of the true effect of covid- on the mortality rate and compare the real death toll with what is reported in official statistics. in the next step, we extend the analysis by using detailed health care data as well as data on a host of sociodemographic, labor market and territorial characteristics to credibly estimate the 'nursing homes' effect on covid- mortality. this study relies on the definition of excess deaths to uncover the effect of covid- and draw some policy lessons. this approach does not allow us to calculate the case fatality rate of covid- , which is essential to understand how prevalent the virus is in the population and thus inform social distancing policies. moreover, our definition of excess deaths attributable to covid- includes both direct deaths-people dying of covid- -and indirect deathspeople dying for causes related to covid- , such as overcrowded hospitals. supplementary data are available at the journal of public health online. impact of non-pharmaceutical interventions (npis) to reduce covid mortality and healthcare demand dipartimento di protezione civile death at home: the unseen toll of italy's coronavirus crisis. united states: reuters world news the economist. many covid deaths in care homes are unrecorded , the economist group scovare i positivi casa per casa: così abbiamo sconfitto il virus a vo'. il virologo crisanti racconta il modello veneto managing the pandemic: lessons from italy's veneto region why mass testing is crucial: the u.s. should study the veneto model to fight covid- covid- testing for testing times: fostering economic recovery and preparing for the second wave mitigating the covid economic crisis: act fast and do whatever it takes covid- : the gendered impacts of the outbreak the economist. the impact of covid- on care homes, . london: the economist group liste di attesa nelle rsa. sistema socio sanitario regione lombardi fontana su anziani morti nelle rsa lombarde: 'l'utilizzo di quelle case di riposo per i malati covid è responsabilità dell'ats comparisons of all-cause mortality between european countries and regions global coronavirus death toll could be % higher than reported , london: the financial times group the economist. tracking covid- excess deaths across countries data gaps and the policy response to the novel coronavirus deaths from cardiac arrest have surged in new york city. london: the economist graphic detail we are deeply grateful to travers barclay child, hans genberg, coen van der kraats and eli remolona for extensive discussions in the early stages of this project. we also thank richard baldwin, xu bin, emanuele ciani, jacopo cimadomo, owen o'donnell, viktar fedaseyeu, pilar garcia-gómez, massimo giuliodori, sergi jiménez-martín, luca marcolin, magdalena rola-janicka, jonathan stöterau, stefan thewissen and seminar participants at ceibs and the oecd for helpful feedback. the views expressed in this paper are those of the authors and do not represent those of the oecd or its member countries. notes: 'municipalities' report the number of municipalities covered in the daily death registry data. '% of all municipalities' reports the share of municipalities covered over the region's total. 'population (in s)' indicates the number of residents in the municipalities covered by the death registry data, imputed from census population data, as of january . '% of all population' reports the share of the population covered over the region's total 'full sample' report statistics for the full sample of municipalities.sources: istat. , key: cord- - s v y authors: mira, francesco; purpari, giuseppa; di bella, santina; colaianni, maria loredana; schirò, giorgia; chiaramonte, gabriele; gucciardi, francesca; pisano, patrizia; lastra, antonio; decaro, nicola; guercio, annalisa title: spreading of canine parvovirus type c mutants of asian origin in southern italy date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: s v y canine parvovirus type (cpv‐ ) emerged as dog pathogen in the late s, causing severe and often fatal epizootics of gastroenteritis in the canine population worldwide. although to date cpv‐ is circulating in all continents, most of the current studies have analysed the amino acid changes accounted in the vp gene sequence, with limited information on virus introductions from other countries. the aim of this study was to analyse the genetic features of cpv‐ c strains currently spreading in italy. swabs and tissue samples were collected from dogs suspected of cpv infection. the nearly complete genome sequence from the cpv‐positive samples was obtained. the co‐circulation of two different but related cpv‐ c strains, with amino acid changes characteristic of cpv strains of asian origin (ns : v, f, f, p – ns : v, n, v ‐ vp : a/g, y, a, i, r), were observed. the phylogenetic analyses inferred from the ns and vp gene sequences confirmed the relationship with asian cpv‐ c strains. this study reports the spread of novel cpv‐ c mutants in italy and supports further studies to evaluate the coexistence of genetically divergent cpv strains in the same geographical environment. . after its emergence, the original type cpv- was replaced by three antigenic variants termed cpv- a, and cpv- c (buonavoglia et al., ; parrish et al., ; parrish, o'connell, evermann, & carmichael, ) . during the years, several amino acid (aa) changes were accounted in the vp gene sequence (battilani et al., ; geng et al., ; jeoung, ahn, & kim, ; nakamura et al., ; truyen, ) and, only recently, the analysis of the ns gene sequence was included in the cpv phylogenies (canuti, rodrigues, whitney, & lang, ; grecco et al., ; han et al., ; li et al., ; mira et al., ; pérez et al., ; zhuang et al., ) . previous studies provided information on the cpv strains spreading in italy (decaro, desario, et al., ; decaro et al., decaro et al., , dei giudici et al., ; mira, dowgier, et al., ; purpari et al., ; tucciarone et al., ) , suggesting the need of a continuous epidemiological survey to evaluate the cpv circulation and evolution, whereas limited data are available on the spread of novel strains imported from other continents (mira, purpari, lorusso, et al., ) . the aim of this study was the detection and molecular analysis of cpv strains displaying genetic features of asian viruses spreading in southern italy. during an epidemiological survey, rectal swabs (n = ) and tissue samples (n = ) from seven dogs suspected of cpv infection (table ) , collected in southern italy (sicily) from august to march , were analysed at the istituto zooprofilattico sperimentale della sicilia "a. mirri" (palermo, italy) for diagnostic purposes. dna and rna were extracted from swab/organ homogenates, obtained as previously described presence of cpv dna was evaluated by a diagnostic pcr using a primer pair targeting the vp gene (touihri et al., ) , as previously described (mira, purpari, lorusso, et al., ) , and each amplicon was analysed by electrophoresis on a % agarose gel supplemented with ethidium bromide. sequencing encompassing both orfs (ns and vp genes) was carried out using primer pairs developed by pérez et al. ( ) , as previously described . sequences were assembled according to an overlapping strategy and analysed using bioedit ver . . . software (hall, ) . assembled nucleotide sequences were submitted to nblast program (zhang, schwartz, wagner, & miller, ) to search related sequences in public domain databases. these sequence data have been submitted to the ddbj/embl/ genbank databases under accession numbers mk - . the obtained sequences were aligned with reference sequences retrieved from the genbank database, which included the sequence (accession number mf ) of a cpv- c strain collected from the same region and previously analysed (mira, purpari, lorusso, et al., to elucidate the genetic relationships of the analysed cpv strains, two phylogenetic trees, based on the full-length vp and ns gene sequences, were constructed with the mega x software (kumar, stecher, li, knyaz, & tamura, ) , using the maximum-likelihood f i g u r e maximum-likelihood tree based on full-length vp gene sequences of canine parvovirus type strains (bootstrap , replicates; bootstrap values greater than are shown). black dots markings (•) indicate cpv strains analysed in this study. each sequence is indicated with virus type (fplv: feline panleukopenia virus-cpv: canine parvovirus) or variant (cpv- , cpv- a, cpv- b, cpv- c), country and year of collection, and accession number method according to the tamura -parameter (t ) and hasegawa-kishino-yano (hky) models, with discrete gamma distribution (five rate categories) (g) and invariant sites (i) (bootstrap analyses with , replicates). the models selection was performed using the best-fit model of nucleotide substitution with mega x software (vp gene: t +g+i; ns gene: hky+g). extracted dna/rna were also amplified using a set of pcr assays for the detection of canine distemper virus (cdv) , canine adenovirus (cadv) type and type (dowgier et al., ) , canine herpesvirus (cahv- ) (decaro et al., ) , canine coronavirus (ccov) (decaro et al., ) and canine rotavirus (crov) (freeman, kerin, hull, mccaustland, & gentsch, ) . sequence analysis revealed amino acid changes previously described in asian cpv- c strains (ns : v, f, f, p-ns : v, n, v-vp : a/g, y, a, i, r) (table s ). cpv strain izssi_pa / evidenced an additional change at residue of ns protein (table s ). only one mutation (a/g) was observed among the analysed strains at residue of the vp protein, which suggests the circulation of two different but related cpv- c strains in southern italy. amino acid change i v in ns also lies at the same residue in the ns -encoding sequence, while change at codon of ns sequences did not result in any changes in the encoded ns protein. additional two amino acid changes in the ns -encoding sequences were observed among the analysed strains: d n and m v (table s ). these changes resulted in silent mutations in the corresponding encoded ns protein. phylogenetic analysis inferred from vp sequences indicated that analysed strains are more related to asian than to european cpv strains, clustering in a separate clade (figure ). phylogenetic tree inferred from ns gene sequences shows that strains clustered within the phylogeny according to the geographical origin and the year of collection rather than to the cpv variant ( figure ). the present molecular analysis of cpv strains detected in southern italy provides new data about the viral spread and dynamics of cpv mutants circulating in italy. in the last decades, several studies analysed the spread of cpv strains in italy, and in , the emergence of the cpv- c variant was firstly reported (buonavoglia et al., ) . in the following years, all three cpv variants were described in italy, with a slightly higher prevalence of the cpv- a and cpv- c variants (decaro, desario, et al., ; decaro et al., decaro et al., , tucciarone et al., ) . more recently, a cpv- c strain displaying genetic signatures typical of asian viruses was detected in southern italy (mira, purpari, lorusso, et al., ) , thus suggesting the introduction of the virus from other countries, as reported for other canine viruses (decaro, campolo, et al., ; martella et al., ; . therefore, a continuous molecular survey was assessed to point out eventual introduction and spread of cpv strains originated from other geographic areas in the italian canine population. since (hoelzer & parrish, ) . according to this study, the spread of asian cpv strains in a separate geographical area different from asian countries could be suggested, as previously described in south america (grecco et al., ; maya et al., ) . whereas cpv- a and cpv- b are the prevalent variants circulating in asia (yi, tong, cheng, song, & cheng, ) , and more recently, cpv- c has been described in the same continent (chiang, wu, chiou, chang, & lin, ; geng et al., ; nakamura et al., ; wang et al., ; zhao et al., ; zhou, zeng, zhang, & li, ; zhuang et al., ) , showing molecular signatures different from those of other continents. indeed, the asian cpv- c variant shows specific amino acids in ns ( v, f, v, p) and vp ( a/g, y, a, i, r) gene sequences. most of these amino acids have been described in the vp of cpv- a/ b/ c strains collected in china, vietnam, india, taiwan, south korea, thailand and japan (chiang et al., ; geng et al., ; han et al., ; jeoung et al., ; lin et al., ; mukhopadhyay et al., ; nakamura et al., ; phromnoi, sirinarumitr, & sirinarumitr, ; soma, taharaguchi, ohinata, ishii, & hara, ; xu et al., ; yi et al., ; zhang et al., ) . in particular, cpv- c strains displaying the amino acid glycine (g) instead of the highly conserved alanine (a) at residue of the vp have been previously detected in china (wang et al., ) and italy (mira, purpari, lorusso, et al., ) . more recently, molecular analyses including the ns gene sequence showed the presence of molecular signatures of the asian cpv strains even in this region (han et al., ; mira, purpari, lorusso, et al., ; zhuang et al., ) . indeed, it results critical to extend the analysis to other genomic regions to properly infer the spread of the genetic cpv variants (grecco et al., ) . (ns : v, f, f, p -ns : v, n, v) could contribute to further elucidate its evolution ). the classification system based on single amino acids ( and ) of the vp protein does not clearly reflect the phylogenetic relationships of the strains, better supported to proposed "clade" or "lineage/sub-lineage" new classification criteria (grecco et al., ; zhuang et al., ; mira et al., ) . as observed also in this study, phylogeny lacks any clustering based on the single vp aa residue (cpv- a/ b/ c), as well as on the geographic origin and period of sample collection. therefore, a wider evolutionary analysis further supports the thesis to consider the cpv antigenic variants as variants of cpv- a rather than distinct subtypes (organtini, allison, lukk, parrish, & hafenstein, ) and could be considered as a more reliable tool in outbreak tracing. this study reported the early evidence and spread of cpv- c strains of asian origin in the italian canine population. as observed in south america (grecco 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the vp gene of canine parvoviruses circulating in china a greedy algorithm for aligning dna sequences typing of canine parvovirus strains circulating in north-east china the genetic evolution of canine parvovirus -a new perspective genome sequence characterization of canine parvoviruses prevalent in the sichuan province of china additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article key: cord- -cmx q sc authors: amoroso, maria grazia; russo, danilo; lanave, gianvito; cistrone, luca; pratelli, annamaria; martella, vito; galiero, giorgio; decaro, nicola; fusco, giovanna title: detection and phylogenetic characterization of astroviruses in insectivorous bats from central‐southern italy date: - - journal: zoonoses public health doi: . /zph. sha: doc_id: cord_uid: cmx q sc in recent years, bats have been found to harbour many viruses, raising several questions about their role as reservoirs and potential disseminators of zoonotic viruses. we investigated the presence of six virus families in bats in three regions of central‐southern italy. astroviruses were identified in seven of bat species. sequence analysis revealed marked genetic heterogeneity among the astroviruses identified, with nucleotide identity ranging between . % and . %. astrovirus diversity was not associated with the bat species, the geographic areas or the bat colony, suggesting the circulation of several astrovirus strains in italian ecosystems. genetic diversification and interspecies transmission appear common in bat astroviruses and could provide, potentially, the bases for transmission to humans and other mammals. yet overemphasizing this risk might have detrimental consequences for bat conservation and preservation of the important ecosystem services bats provide. risk is crucial in western countries. several bat species avoid human settlements and have little chance to enter into contact with humans (russo & ancillotto, ) , and other species are at risk of extinction (e.g. conenna, rocha, russo, & cabeza, ; o'shea, cryan, hayman, plowright, & streicker, ) , making the risks negligible. however, increasing anthropization of the environment is altering the ecosystems, disrupting usual habitats and/or creating novel ecological niches that may overlap with human activities. it should be considered, however, that bats provide important ecosystem services (aizpurua et al., ; ancillotto et al., ; boyles, cryan, mccracken, & kunz, ; kunz, braun de torrez, bauer, lobova, & fleming, ) . overemphasizing the potential risks posed by bats to humans may generate unjustified alarmism, a fact that has raised considerable concern in conservationists (lópez-baucells, rocha, & fernández-llamazares, ) , as also highlighted by the eurobats agreement on the conservation of bat populations in europe (working group on "communication, bat conservation and public health"). under this perspective, surveillance may still be important to avoid or mitigate potential conflicts and eventually improve bat conservation policies. in italy, a few studies have been carried out to assess the presence of viruses in bats lelli, papetti, et al., ) but only in the northern regions. these studies detected orthoreoviruses and coronaviruses in various bat species. however, there are no data for the central-southern italian regions, which are characterized by a warmer, drier climate and a different biogeography. in this study, we screened bat species living in these geographic areas. the bat species were selected ad hoc to represent a range of environmental and behavioural differences. our data set covered species that form large colonies in caves and show high fidelity to their roosting sites (e.g. miniopterus schreibersii); species that roost in trees in small groups and switch roosts frequently (e.g. barbastella barbastellus); and synanthropic species that are more likely to enter into contact with humans (e.g. pipistrellus kuhlii). the bats were screened for a large panel of viruses, including rabies viruses, coronaviruses, reoviruses, caliciviruses, astroviruses and enteroviruses. we overall sampled individuals from three regions of central-southern italy. most bats we screened were caught on emergence from their roosts with harp traps or at drinking sites with mist nets. in such cases, bats were promptly removed from the trap or the net and their forearm length and body mass were measured, respectively, with a caliper to the nearest mm and a digital scale to the nearest . g. sex was assessed by inspecting genitalia (racey, ) , and wings were trans-illuminated to distinguish juveniles from adults (anthony, ) . in females, we ascertained pregnancy by palpation (racey, ) , and lactation by the presence of enlarged nipples surrounded by a hairless skin area and by extruding milk with a gentle finger pressure on the nipple base. bats were immediately released after processing. a few samples came from animals hosted at rehabilitation centres. saliva was sampled from each bat with a dry sterile swab, which was placed in viral transport medium (chu, poon, guan, & peiris, ) , and transported in a cool box to the laboratory for the analysis. bat droppings expelled during manipulation of specimens were collected, stored in sterile vials and preserved in cool boxes during transportation. nucleic acids were extracted with the magmax automated system the presence of rabies virus and mers-coronavirus (mers-cov) was investigated by real-time rt-pcr using two commercial kits, that is rabies virus real time rt-pcr kit (shanghai zj bio-tech co., ltd) and mers-cov real time rt-pcr kit (shanghai zj bio-tech co., • identification of astroviruses in four bat species never found positive before. • identification of astrovirus in bats living in italy. • strains of astrovirus identified revealed a high degree of genetic diversity, not related to bat species. • mechanisms other than host specificity may drive virus genetic diversification. ltd). the reaction was carried out following the manufacturer's instructions. all the tests were performed with a fast real time pcr system (applied biosystems, monza, italy). positive and negative controls were included in the kits. the primers employed for detection of the different viruses are indicated in table . all the assays were carried out with the flexid mastercycler nexsusx (eppendorf) using the superscript™one- step rt-pcr kit (life technologies italia). coronaviruses (covs) were searched as described by drosten et al. ( ) (table ) . for reovirus detection, the nucleic acids were preventively denatured ( μl viral extract with . μl of dmso at °c for min) and then reversetranscribed and amplified using a nested protocol, as described previously (leary et al., ; all the pcr products were analysed by tape station , an automated platform for electrophoresis, (agilent technologies), using the d screentape system. amplicons of the pcr-positive samples were sequenced as previously described (amoroso et al., ) . the nucleotide sequence similarity searches were performed using the blast server (http:// www.ncbi.nlm.nih.gov/genbank/index.html). nucleotide sequences of astvs were aligned using the program clustal w (larkin et al., ) with reference sequences of mamastrovirus (mastv), using an avian astrovirus (avastv) jf as outgroup (supporting information table s ). phylogenetic analyses were carried out by mr bayes (huelsenbeck & ronquist, ; ronquist & huelsenbeck, ) program implemented in the software package geneious v. . . (biomatters, new zealand). bayesian inference was performed using four "chains" run over one million generations (with the first trees discarded as "burn-in") and supplying statistical support with subsampling over replicates. jmodeltest (posada, ) all the screened bats were negative to reoviruses, caliciviruses, enteroviruses, rabies viruses and mers-cov. when analysed for the bat species b. barbastellus ( . %) and in an additional four bat species (table ) . geographic location of sampling sites from which positive samples were taken is indicated in figure . upon interrogation (november ) of genbank sequence database ( (table ) . ta b l e interrogation by blast search of ncbi nucleotide database (december ) based on the orf b (rdrp) sequences generated in this study. the strain with the highest % nucleotide identity (% pi) and e value (e-v) found in the database (bm, best match) is shown for each bat sequence generated in this study (table ) . upon sequence comparison, a marked genetic heterogeneity was information table s ) and supported by bootstrap values ≥ . in this study, we investigated the presence of various human viral pathogens in different species of bats captured in central and southern italy. none of the samples was positive to rabies virus that is the most serious concern for the potential human transmission. from an epidemiologic point of view, bat-associated rabies cases are rare, with the incidence rates in canada and the unites states being as low as . - . human cases per billion persons/year over a -year period (velasco-villa et al., ) . however, there is still a potential risk that needs attention, especially for bat specialists and rehabilitators, who handle bats and are often exposed to bites. our study, in spite of the relatively small number of sampled animals, confirms that this risk is negligible in italian territories. consensus diagnostic molecular assays are useful to detect novel viral species or genetically different viral strains, but they are usually not highly sensitive. using broadly reactive consensus primers, cov rna was detected in / ( . %) samples. these results could not be confirmed with sequence analysis. however, the samples also tested negative by a quantitative assay specific for mers-cov, thus ruling out the presence of this virus in the sampled population. using consensus primers universal for the astroviridae family, in europe and asia (drexler et al., ; fischer et al., fischer et al., , kemenesi et al., ; zhu et al., ) . the rt-pcr prevalence ( . %) observed in our study was higher than that observed in hungary ( . %) (kemenesi et al., ) and similar to that found in germany ( . %; fischer et al., ) . astv in % of the tested bats (chu et al., ; xiao et al., ) . the prevalence was also found to greatly vary by bat species (fischer et al., ) . as an example, a study carried out in china revealed the presence of astrovirus in . % of the samples of myotis pylosus analysed, whilst the prevalence was found much lower for other species (chu et al., ) . to detect and characterize the astv strains, we amplified and sequenced a fragment of bp of the rdrp gene, which represents the most conserved region of the astv genome. upon sequence analysis, we observed a remarkable genetic diversity among the various bat astv strains detected in italy. such a high degree of variation was observed also within species and in the same geographic area or colony. these findings do not confirm the bat species specificity of astv, proposed by other authors (fischer et al., astrovirus infection is associated with gastro-enteritis in most animal species, and humans astvs are regarded as a common cause of viral diarrhoea in children (mendez, aguirre-crespo, zavala, & arias, ; xiao et al., ) . avian astvs have also been associated with extra-intestinal diseases, such as nephritis in chicken (imada et al., ) and hepatitis in ducks (todd et al., ) . recently, astvs have been also detected in the nervous tissues of minks with shaking disease (blomstrom, widen, hammer, belak, & berg, ) and of bovines with neurological disorders (bouzalas et al., ; li et al., ) . neurological disease in immunocompromised human patients has been associated with astv infection (brown et al., ; fremond et al., ; lum et al., ; quan et al., ) . in our research, all the animals positive to astv appeared healthy, as also reported in previous studies in bats (fischer et al., ) . astroviruses could therefore simply be nonpathogenic members of the bat virome. however, more information is needed on bat-borne immune response to state whether these viruses are really nonpathogenic for bats. bats could play an important role in transmitting such viruses to humans, as astv transmission usually follows an oral-faecal route. contamination of food or drinking water could for example occur by bat droppings. on this regard, it is however important to underline that the probability that human ingest food and water contaminated by astv coming from human faeces looks much higher-see, for instance, the high percentage ( . %) of human astvs recently found in mussels harvested in the gulf of naples, italy (fusco et al., ) . interspecies transmission of astv has been documented on more occasions (de battisti et al., ; de benedictis, schultz-cherry, burnham, & cattoli, ; mihalov-kovacs et al., ; nagai et al., ) . also, novel human astvs (mlb , mlb , va , hmo-c, hmo-b, hmo-a, va- ) have been identified that are genetically unrelated to "classical" human astvs (banyai, meleg, moschidou, & martella, ; finkbeiner, le, holtz, storch, & wang, ; finkbeiner, li, et al., ; kapoor et al., ) and closer to animal astvs. the origin of animal-like human astvs has not been deciphered yet. the potential zoonotic risks associated with bats have attracted the attention of researchers, mostly after the discovery of sars-like and mers-like covs (two coronaviruses highly pathogenic for humans) in european bat species, 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source of emerging viruses capable of infecting humans identification of chicken enteroviruslike viruses, duck hepatitis virus type and duck hepatitis virus type as astroviruses the roles of taxonomy and systematics in bat conservation bats in the anthropocene: conservation of bats in a changing world the history of rabies in the western hemisphere isolation and phylogenetic characterization of bat astroviruses in southern china identification and characterization of multiple porcine astrovirus genotypes in hunan province, china comparative analysis of bat genomes provides insight into the evolution of flight and immunity detection of diverse astroviruses from bats in china prevalence and genetic characterization of caliciviruses among children hospitalized for acute gastroenteritis in the united states additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article key: cord- -xi zy ow authors: allam, zaheer title: the third days: a detailed chronological timeline and extensive review of literature documenting the covid- pandemic from day to day date: - - journal: surveying the covid- pandemic and its implications doi: . /b - - - - . - sha: doc_id: cord_uid: xi zy ow this chapter surveys the global unfolding of events during the third days of the covid- pandemic that originated from china. the third days of the unfolding of the events showcased how city-wide lockdowns were started to be considered globally, the moving of the epicenter from china to europe, and major industries being impacted worldwide. to document this, an extensive review of the literature provides a daily overview of the situation covering health, economic, political, and social perspectives and outlines key events during the unfolding of the pandemic. this chapter surveys, and establishes a chronological timeline of the outbreak from day to day , covering issues appertaining to health policy and dwells into socioeconomic measures and impacts during the unfolding of the pandemic. the past days of the covid- pandemic had been eventful, as the reality of the disease was clear globally, where no single continent had been spared. the next days (day to day ) were marked with major milestones, challenging and most trying in different spheres of life as is demonstrated in succeeding sections. to start with, during this period, the number of confirmed cases increased from slightly above , to a high of million and counting (spotlight, ) , with the number of casualties increasing from deaths to over , deaths across the globe by the end of the days. in addition, the number of affected countries increased from countries to countries and territories (worldometer, ) . another change that was witnessed within the third days is the shifting of the covid- pandemic epicenter from europe to the united states, where the numbers of confirmed cases in the country, beingdby that timedmore than a quarter of the confirmed cases in the rest of the world (kirby and stewart, ). as the impacts of the coronavirus continued to be felt, there was evidence of an economic downturn in different countries, including in developed economies, where the number of people filing for unemployment claims increased. due to the economic hardships, it became apparent that many countries were trying to ease the lockdown restrictions to allow for reopening of economies, but in a gradual and cautious manner to avoid the reemergence of cases. it is within these days that governments were seen to propose economic stimulus packages to bail out their citizens and economies, including companies that were already struggling due to reduced activities. at the same time, due to the economic and social strives, there was evidence of political tension between countries as they trade blame on responsibilities toward containing the coronavirus before it spread, and become global pandemic (business davidson, a; smith, ) . during these days, it also became clear that the world was a long way before a vaccine could be developed and thus, the demand for personal protective equipment (ppes) would continue, and we see countries and regions formulating policies to control the exportation of ppes and medical supplies to other countries. with the increasing scarcity of ppes and other basic, medical essentials, these were seen to rely on the world health organization (who) and well-wishers for the supplies. within these days, there is also evidence of there was no safe-haven against coronavirus, as even those in cruise ships and aircraft carriers were infected and with cases in such places spreading faster (cna, c; gajanan and mansoor, ; kaneko and kim, ; willsher and sabbagh, ) . it also dawned that even those in positions of power and authority are not immune to the virus, where some even succumbed to their injuries. within these days, it also became apparent that every single sector is highly dependent on the health sector, as those like sports and entertainment, religious sectors and others remained "grounded" with some high profile events such as professional football leagues, olympics, and wrestling being canceled, postponed, or suspended indefinitely (bbc sport, ; cacciola and deb, ; schad, ) . during this period also, it becomes apparent that it is possible for technology companies to set aside their competition and come together for the common goal of humanity (apple, ) . therefore, even as events of these third days had been devastating and heartbreaking, there is much that humanity can learn, and have learned, and going forward, even after the covid- is finally phased out, as people, governments, regions, and economies embark on rebuilding, some of the positives that have been learned will need to be kept alive. the following sections document the unfolding of the pandemic. during this month, every effort counted in the fight against the spread of covid- , and this was emphasized by the events of march . on this day, finally, the who conceded that without a doubt, coronavirus amounted to a global pandemic (who, ad) . the build-up to this global pandemic announcement saw a national wide lockdown declared in italy on th following an uncontrollable and astronomic increase in the number of new cases and deaths in the country (bbc, c) . following this, the country was beginning to experience unprecedented abandonment by its neighbors and country members of the eu who had continued to issue a travel advisory to their citizens against traveling into or from italy (gov.uk, a) . unsurprisingly, by th, all eu member states had experienced the outbreak of the covid- disease, and most of the first cases were related to travelers fleeing italy after the situation therein started to worsen (who, ad) . in north america, the situation in the united states was also getting out of hand, with over states including arizona, washington d.c., michigan, colorado, vermont and rhode island, and others declaring states of emergency (razek, ) . here, on march , the united nations headquarters in new york closed its doors to the public for fear of spreading the virus (krisel, ). as the situation escalated, with over confirmed cases in the country, and over deaths from the virus, the national basketball association (nba), one of the most popular and fancied sporting activity in north america abruptly suspended its season, as of march , when a player of the utah jazz tested positive for coronavirus, just before their game with oklahoma city began (cacciola and deb, ) . these unfolding attracted a host of intervention measures in the country to ensure people would observe the health guidelines, especially by staying at home and keeping social distancing. first, on march , vice president pence announced that medical insurance companies had agreed to waive all copayments on covid- testing and also extended their coverage for the treatment of the disease (office of the president, b). similarly, the country, through homeland security, announced a level travel advisory and subsequently temporarily restricting entry to all foreign travelers from china, iran, and certain countries of europe. the restrictions also demanded that all american citizens and legal permanent residents and their immediate families returning from countries already affected by the virus must undergo a self-quarantine for a minimum of days upon arrival (homeland security, ) . this announcement was affirmed by the oval office address by president trump stating that travel advisory applied to all the countries in the schengen area (collinson, ) . the decision by trump was disapproved by the eu leaders (gaouette et al., ) . elsewhere, to control and reduce the spread of the virus, the schooling system in different countries was seen to be disrupted. as of march , a un report indicated that about % of students across the globe were out of school and this included countries like italy, czech republic, part of spain (madrid region), greece, and austria among many others that took the decision of closing the school to protect students and their families, as a mean to comply with who's health guidelines (who, p) . surprisingly, as countries hastened to close down schoolsdhighlighting the worsening of this situation, reports from china indicated that normalcy was returning to a point that some schools were reopening especially in qinghai province (cgtn, ) . in regard to financing efforts against this disease and supporting technological advancement in the development of test kits and vaccine development, on march , the bill & melinda gates foundation, mastercard, and wellcome together committed approximately $ million (bill and melinda gates foundation, ). on the same date, the coalition for epidemic preparedness innovations on its part committed an extra $ . million to render the organization's total investment in vaccine development to $ . million, with the funds expected to help both the company novavax and the university of oxford to research and develop a vaccine for this disease (kff, ). furthermore, on march , the united kingdom announced more funding ($ . million) , this time from its national budget to aid vulnerable countries in their efforts against covid- (gov.uk, b) . two days after declaring the coronavirus a global pandemic, circumstances in the western part of the globe have led the who to make another declaration; this time, the new epicenter of the outbreak of coronavirus was europe (who, ae). this announcement was not surprising as of then, italy was already overwhelmed ( , cases and deaths already) (snuggs, ) , and spain had in the day declared a state of emergency (cnbc, ) . things in other member countries of the union were also worsening, with the region recording over deaths in a span of h from the disease, and over new cases in the same period (who, c) . and surprisingly, from the situation report by the who (who, c), all the cases in each of these countries were of local transmission; thus, warranting the decision different countries were making to restrict movements, and where possible, instituting total lockdowns like the case of italy. on the western side of the globe, more states in the united states were declaring a state of emergencies, restricting the number of people in gatherings, closing all learning institutions, and restricting movements among other things. these happenings were more pronounced on march , and when the worse came to worst, present donald trump, under the robert t. stafford disaster relief and emergency assistance act (stanford act), declared a state of national emergency (office of the president, a). by then, the number of cases in the united states had increased to and those who died from the disease reached deaths, with transmission happening locally (who, j) . in total, the global number of confirmed cases had reached a high of , cases, with of those reported in the past h. of the sum, , had been confirmed from countries, of which were reporting their first cases. the remaining cases were reported in china, where situations were coming back to normal, with only new cases reported in the populous country, and deaths were reported in the -h period. wuhan, the former epicenter only recorded five confirmed cases (reuters, c) . outside china, the deaths increased by , raising the total tally to cases (who, c). following these unprecedented unfolding globally, economies were facing numerous challenges, as most economic activities had stalled or were nonexistent. for instance, in the united states, the stock exchange recorded the lowest point, only reminiscence with situations of (mccabe and ostroff, ). for this reason, different agencies introduced financial packages to offer some support. for instance, the adb announced a $ million package that could be accessed by companies to supply critical essentials for combating covid- (adb, ) . in europe, the european bank for reconstruction and development is reported to have approved a $ . billion financial package to help companies in the region to remain afloat during this period the region was experiencing the most trying moment in the recent history (williams, ) . amid the challenges of covid- and its impacts, some countries like taiwan found some solace in the use of technology to track and instill the mandatory quarantine, especially for those coming from certain areas. with technology, enforcement officers were able to know those who were flaunting rules and their whereabouts, and this helped reduce the spread of the virus, despite it being just kilometers from china where things were worse some days ago (yun, ) . in europe, there was some use of technology, especially mobile apps, that helped in mapping and tracking cases, and to bolster this even further, the european commission (ec) was offering funding to a tune of v m ($ . ) for start-ups or small medium enterprises (smes) developing technologies with capacities to treat, test, monitor, or offer other aspects that could help in the fight against covid- (euroean commission, a). in the past month since the first case was confirmed in africa, the number of countries affected in the continent increased to by march , with seven reporting their first case in a span of h. also, a week prior, the number of cases in the continent was only , but they increased to with six deaths reported to have been imported, except for south africa, algeria, senegal, and cameroon (who, k) . in europe, most countries were receiving unprecedented numbers of new cases, especially in italy, spain, france, and germany, which recorded , , , , and new cases, respectively, and almost all cases in most of these eu countries were locally transmitted (who, k). the number of deaths in the region was also rising with italy losing people in a single day (snuggs, ) , while spain lost , france , and the united kingdom lost lives. in germany, which was reported to have embarked on mass testing as early as the situation warranted so, the number of deaths was relatively low (only two reported by th) (who, k) . following the dire state in the region, the european commission published guidelines (euroean commission, b) on the exportation of ppes out of the region. among such guidelines were the restriction of exportation of the said equipment unless with express green light from the eu member states. however, the eu was categorical that the measures taken were only within a specified period, especially during that period when those ppes were greatly needed locally; and thus, it was not a total ban on export, which would contravene the region's international obligations in matters of trade (euroean commission, b) . in the middle east, the severity of the disease was still present in iran, which until th had a total of , confirmed cases and deaths, with a high of deaths having been reported in a period of h. the situation in other countries within the region was controlled with only deaths reported ( in iraq, in lebanon, and in bahrain). in the american region, only a few confirmed cases were reported, and only deaths were reported in ecuador. however, the government there continued to institute proactive response measures to ensure the spread would be contained. for instance, the cdc announced that no gathering would be allowed in the united states that have more than people, thus putting into disarray functions such as weddings, concerts, and sporting events among others that are known to attract a large crowd (the new york times, a). more states declared a state of emergencies, while more schools in different states (more than states), including new york city with over . students (shapiro, ) calling for the closure of schools. one major news in the united states on that day was the negative test results for the country's president, who a few days ago had some contact with a brazilian official who turns positive after the testdprompting president trump to undergo testing (education week, ) . in other places, country borders were being closed. for instance, after confirming their first covid- cases, kazakhstan and uzbekistan closed their borders (reuters, j) . colombia also closed its border with venezuela, while turkey, with one of the leading airline services globally, suspended flights to nine european countries (liptak, ) . lebanon reported to have called for a -day lockdown in the entire country to curb the spread of the virus, which for the past week had claimed the lives of three people (france , b). the other goods news of the day came from south korea, which had shown a great resolve in bringing down the number of new cases, and this was bearing fruits after it implemented a number of strategies that involved the use of advanced technologies. first, it adopted the use of drive-thru clinics where people could be tested within min and receive their results the following day. this allowed them to test over people per hour, as there were such clinics countrywide, where six people were being tested in an hour per clinic (choon, ) . the strategy also reduced local transmissions as contacts were greatly reduced. the country also implemented the use of mobile apps to enforce quarantines and track the spread of the virus (park, ) . the success of the drive-thru clinics and apps were later adopted by the united states and germany (yamey, on th, the health landscape in different regions changed even further, with the western pacific region having the highest number of confirmed cases, mostly because of china. the european region was on its knees, health-wise, with , confirmed cases, with of these being reported in a single day. the number of deaths in this region had also increased by to take the region's tally to deaths. both north and south american regions had cases, and a total of deaths ( deaths reported in a day). the african and the south-east asia regions were relatively saved until then, with only and confirmed cases, respectively, and total deaths for both regions being (who, l). following the dire need in the european region, the eu closed its borders except for essential travels. russia also closed its borders, only allowing its citizens and legal permanent residents to travel back. spain was also reported to have instituted border restrictions for all noncitizens and residents. in france, besides instituting border restrictions like a majority of its eu counterparts, it also announced a countrywide lockdown, which meant that no gathering of any size would be allowed, with people expected to remain at home with some exceptions (onishi and méheut, ) . similar events of widespread lockdowns and border restrictions were also observed in latin america, with countries such as venezuela and peru leading the cue in countrywide lockdowns. colombia and costa rica also instituted border restrictions and control. however, brazil, which by then (march ) had the most confirmed cases in the region, overlooked all the measures that the rest of the world were implementing, and had a section of its population (supporters of their president; jair bolsonaro) demonstrate against his opponents (harris and schipani, ) . in iran, the government was forced to release over , prisoners as a way of curbing the spread of the virus in the country, which was already at alarming levels (hawkins, ) . as the global social fabric was being dismantled by the disease, its impacts were being felt in the economic sphere. for instance, on th, the airline industry in the united states was seeking government financial assistance of up to $ billion to help them remain afloat (sider and mann, ) . the dow jones industry also recorded its historical low after dropping . points; the worst day crash since the "black monday" crash (millhiser, ) . following those uncertainties and the threat covid- posed to the global fabric, a number of well-wishers and philanthropists were seen to be in the frontline of helping countries win this war. on th, the jack ma foundation donated assorted ppes and testing kits to the united states , which greatly needed these following the increasing demand, and the disruption of supply chain following the slow activities in china. on th, the world bank group committed another $ billion to help in the fight against the diseases (the world bank, b), while on the same day, bloomberg philanthropies gave a financial package of $ million, especially to help low-x and middleincome economies (bloomberg philanthropies, ). as the dark crowd of coronavirus continued to spread in italy, its impacts were becoming evident, as in a month, the number of deaths ( ) in the country surpassed those in china ( ) since the onset of the outbreak to march (quinn, b) . its health sector was completely overwhelmed with images of dejected nurses trending on social media, and while that was happening, china reported no new confirmed cases attributable to local infection, as the new cases that it confirmed were suspected to have been imported from other countries (siobhán o'grady et al., b) , as it had started to ease its border restrictions. the number of new cases in italy also increased by cases, bringing their country total to , , becoming the second most affected country after china. as for the european regions, italy recorded a total of over , cases on both th . the number of deaths for both days totaled , far much more than the rest of the regions combined. in the americas, new cases on th were : almost double of what was recorded the previous day ( new cases on th). the eastern mediterranean region recorded new cases to push their region total to , , and the number of deaths in the region reached after ( died on th), with most of these coming from iran. in total, the global confirmed cases had increased to , cases on th after , more cases were reported. the number of total deaths globally increased by a total of to reach a global tally of cases (who, m). on local scenes, the disease affected some prominent people, celebrities, and sports personalities. for instance, in the us nba teams, denver nuggets (wimbish, ), los angele lakers (whitcomb, b) , philadelphia ers (zagoria, ) , and boston celtics (ward-henninger, ) reported that each had some of their players confirmed positive, but they decline to give the names of those players. on the same land, congress representatives, ben mcadams (d-utah) and mario diaz balart of florida, confirmed that they had also contracted the disease (helsel, ) . in washington, two employees of the world bank group tested positive on the th with fears that more could have been affected. on the th, there were reports that prince albert of monaco also tested positive for covid- (romo, ) . another person is the executive director of the world food program, david beasley, who also tested positive after returning from canada (world food program, ) . to respond to the numerous cases and scenarios prompted by the covid- case, different regions, individual countries, and agencies adopted different and diverse measures. for instance, in india, the government was reported to have banned any export of ppes, ventilators, and certain medications and supplements as such were not enough locally (suneja, ) . in addition to this, to reduce further spread, especially from imported cases, it closed its borders for incoming flights (business today, b). in the united states, the number of interventions was observed, for instance, the two june scheduled party primaries in connecticut were postponed (pramuk and dzhanova, ) . president trump also signed into law the family first coronavirus response act that would see most americans receive a family relief of $ . the act also gave $ billion to be accessed by smes (erica werner et al., ) . the university of hong kong received $ , from the coalition for epidemic preparedness innovations to continue with vaccine testing for covid- (galford, ) . on the th, the rockefeller foundation committed a $ million to assist in response to covid- , especially in cities such as nairobi, new york, washington, bangkok, and bellagio (rockefeller foundation, ). since the onset of the coronavirus, there had been a theory that it was mostly affecting older people, but the who debunked this, by indicating that data on those affected in different countries and regions have shown that a significant proportion of those in hospitals are aged below years; hence, calling the younger generation to be also extra cautious. the organization also launched a health alert messaging services through whatsapp and facebook, demystifying the importance of technology in the fight against covid- , especially in sharing data and information (who, d). the organization also reported that it had reached an agreement with different producers in china who were ready to supply the organization with ppes, so that it could continue supporting countries in need of such across the world (schnirring, ) . while that was going on, they delivered an extra . million laboratory test kits for covid- to different parts of the world (watts and simon, ) . there was also news that first vaccine trials had begun, and the who together with its partners was to organize an international study dubbed solidarity trial in different countries aimed at trying different treatments that could be adopted to win over covid- (who, d) . regarding the reports on the spread and impacts of the coronavirus on this day (march ), the situation reported by world health organization indicated that the world was still in great danger of recording even more cases. for instance, italy reported the single largest number of deaths, where people died, and those confirmed increasing to , cases. in the united states, the cdc indicated that it had confirmed a total of , cases (cdc, ). as that was happening, president trump invoked the defense production act, to force general motors (gm) to produce ventilators to fill the gap after the situation in the hospital become dire (haynes, ) . on this, earlier on the day, gm rejected the move, prompting the president to apply his executive authority on them, and the new york governor equated the ventilators to missiles during world war ii (klein and raju, ) . the controversies in the united states were not over as it was reported that canada, through its prime minister, justin trudeau, would return all asylum seekers to the united states (austen, ). elsewhere, cuba invoked border restrictions for all noncitizens and nonresidents. it had delayed this decision to "keep its key tourism industry alive," but the threat of the coronavirus forced the president to take that crucial and bold decision (france , a). as time passes, the spread of the coronavirus virus took hold across the globe. on march , the who declared the virus outbreak as a global pandemic, and days later (on th), the director-general declared that the "pandemic is accelerating" (chappell, ) . and true to his word, in the past days, the number of new confirmed cases each day were averaging , new cases, to push the global total to , confirmed cases. the number of new deaths in those days was also increasing at an average of almost each day, and by rd, the total number of deaths had reached , globally (who, n). of great concern on those numbers is that most of them were coming from the european region, which by rd had reached a high of , confirmed cases and total deaths. the only regions that had shown a lot of resilience were the african region with only total confirmed cases and total reported deaths. the south-east asia region was also relatively spared having confirmed only cases and total deaths. the region of americas was showing worrisome trends, having reported a high of , new cases in a single day (march ) to push its total tally of confirmed cases to , cases and a total tally of deaths ( deaths) reported in a single day (who, n). while a majority of countries, especially in europe writhed in desperation from the impacts of covid- , china reported a third consecutive day with no local new case of coronavirus (the straits times, c). this meant that they could slowly transition back to their previous economic routine, and it also gave hope to those whose situation was worsening. in the other regions, including africa where confirmed cases were low, they were responding to the spread by escalating lockdowns and border restrictions (aljazeera, b) . other countries followed this trend. for instance, on march e , bolivia (reuters, b) , greece (stamouli, ), cuba (oppmann, ) , and the united kingdom (sparrow et al., ) imposed total lockdown in their countries (aljazeera, b) . egypt on the other hand called off all religious activities in mosques and churches for days (mourad, ) . during this period still, other countries including pakistan (reuters, n), vietnam, singapore (benner, ), uae, panama (aljazeera, b) , india, nigeria (reuters, m), and zimbabwe (the citizen, ) closed their borders to all foreign travels and nonresidents and banned international flights to or from their soils for a minimum of days. germany, on the other hand, banned gatherings of more than two persons, as cases in the country started to soar, while on the same day, spain extended the state of emergency for more days, as the situation internally was getting out of hand, with thousands hospitalized, and cases soaring each day (picheta, ) . the decision taken by each individual country was meant for the good of the citizens, but such also had a far-reaching impact on the economy. for instance, the banning of the international flights in these days, and others that had been instituted earlier mean that sectors such as tourism, hospitality industry, and others are grounded, with millions of workers employed in those sectors uncertain of what the future holds. in such circumstances, governments were forced to rely on external organizationsdlike the jack ma foundation, which on nd delivered a consignment of assorted ppes, and test kits to african countries to fight the covid- (meseret and meldrum, ). on rd, the world bank group's president was also pleading with bilateral creditors to extend debt relief to low-income economies to help them build some capacity that could allow them to fight the disease (bank, ) . while this was going on, african finance ministers were pleading for a $ billion economic stimulus in addition to the suspension of external debt to allow their individual countries to fight the coronavirus pandemic (uneca, ). china's progression toward containing the virus received a boost after the province of hubei was freed from the lockdown after almost months since the lockdown was instituted (associated press, ). it was a relief to the residents who, for such a long period, lived in fear and uncertainty of when they would return to their previous routines. the good news was that wuhan, the first epicenter of coronavirus was to open a month later, as the number of new cases had reduced significantly. in the last h, the entire country of china reported on new cases, of which, cases were reported to have been imported (cna, a). the number of deaths had also increased, with seven reported in the city of wuhan (the star, ) . the other good news came from germany where the chancellor's, angela merkel, the first test came back negative for coronavirus, but would undergo the same process a few days later to confidently confirm this result (mischke, ) . the fear that she would have contracted the disease came after a doctor who had attended her turned positive, forcing the chancellor into a safety procedure of self-isolation (mischke, ) . the day however did not present the good news to everyone, especially to governments, health workers, and security forces implementing lockdowns and other measures in different countries and to the general global population. on this, in the past h alone, the number of newly laboratory-confirmed cases neared , and the deaths on a single day accumulated to globally (cna, a). the most unfortunate report on this is that half of the new cases and deaths reported came from european countries ( , cases and deaths), with the american regions also experiencing a high of , new cases and deaths (who, e). regarding the accumulation of confirmed cases, it took at least months for cases to climb to , confirmed, and only days to reach , cases. from here, it only took days for the global total to surpass , confirmed cases, and even more startling, only days for the total to reach almost , confirmed cases. such trends prompted the international olympics committee and japan's olympic authority, led by prime minister shinzo abe, to postpone the olympics scheduled for summer to as more countries had expressed their fears of the virus (ramsay, ) , while others cited lack of preparation and other technicalities (aarons, ) . they also prompted the prime minister of india, narendra modi, to lock down the country for days, only a day after the later declared an immediate grounding of all flights in or out of india (shroff, ) . new zealand also went into lockdown (bbc, e), as australia announced a ban on all overseas travel (whiteman and sharma, ) . while in the united states, a troop of , us national guards was mobilized in states to help in response to the disease (gresik and altman, ), as it had already infected more than , people countrywide. while those were being mobilized, three of their colleagues in the navy (sailors) contracted the virus, when an aircraft carrier they were boardingdtogether with approximately other peopledset sail from vietnam. by now, the situation of covid- globally reached fever-pitch where the u.n. secretary-general antónio guterres argued that the disease was ravaging the whole of humanity, with approximately one-third of the population experiencing one or more covid- -related restriction (unicef, ). following this, there was shortages in all sectors, with the who warning that already, the world was facing a "significant shortage" of assorted medical supplies. on the social sphere, there was a shortage in the health sector prompting the un to launch a $ billion financial package on march targeting global humanitarian responses, especially to be advanced to vulnerable countries (un secretary-general, ). with the funds, those countries would manage to bolster their laboratory equipment, build and increase available sanitation (handwashing) stations, and increase medical supplies among other things. besides the financial package, the who was calling on developed economies to assist african countries with health machines such as ventilators and respirators as the continent cannot satisfy the demand for such, especially if cases of covid- were to increase (ighobor, ). those calls and interventions come at a time when most countries, especially in europe and america, were experiencing one of the darkest moments of their history. for instance, in spain, as of th, the number of reported deaths ( deaths) surpassed those reported in china ( ). but still, italy was leading in the number of people who had died with a total of , with having died in a span of only h. france had also started to lose a significant number of people as a result of the covid- , with already having succumbed in the past h. the islamic state of iran and the united states were also affected with (total deaths ) and (total deaths ) deaths having died in the past h. in regard to the number of confirmed cases, which were averaging , cases per day in the past days, the global total had increased to , by th according to the who data. africa was still showing some resilience despite reports of weak health system, with only deaths reported coming from confirmed cases across the globe (who, o). the economic situation globally was worsening, but even more in the united states where it was reported that by march , over . million american had filed for their employment benefit (casselman et al., ) ; a figure that is thought to be the highest in the history of the country. this could be attributed to the increasing number of people who were contracting the coronavirus especially in new york, which had become the epicenter of the outbreak in the northern american region, with the united states having more cases than any other country (kirby and stewart, ; the new york times, b). but, fortunately, the trump administration's stimulus plandworth $ trilliondwas unanimously passed the senate (carney, ) , thus allowing the government to offer some financial support, especially to those who continued to lose their livelihoods. elsewhere, more countries were responding to the pandemic by instituting lockdowns or other strict measures that would somehow suppress the local transmission. on this, on march , panama was reported to have suspended any form of domestic flights , days after, it had suspended international flights (aljazeera, b) . in thailand, the government declared a state of emergency forcing more areas within the country to shut down (techakitteranun, ) , while iraq and lebanon extended their curfews by days in each country. in russia, moscow shut down all forms of businesses and activities for a week (march to april ), except for essential businesses such as pharmacies and grocery stores (astapkovich, ) . the highlight of the day was the testing positive for coronavirus of uk prime minister boris johnson, which he announced to the world via a video on twitter (bbc, g). he reported that, henceforth, he would be in self-isolation and will continue working from. but, while this was breaking news, the greatest and most unfortunate news came from italy and spain. in spain, it had earlier been reported that it had recorded the highest number of deaths in a single day ( ) (rtve, ), but later only italy released its official report of the day where it indicated that approximately (who later reported (who, f)) people had died within a span of only h. italy had also confirmed new cases while spain reported new cases bringing the total confirmed cases to , and , cases, respectively. however, the united states reported the highest number of new confirmed cases ( , ), taking its total tally to , according to the data by who (who, f) but according to the us cdc data (cdc, ), the total number of cases reported in the country by th had reached , , thus leading globally. in the middle east, iran was reporting the highest numbers of new cases, with cases reported within h and new deaths during the same period (who, f). other countries in the region were relatively calm with no other reporting more than a cases a day. in africa, algeria reported the highest number of deaths ( ) (who, f) while south africa reported the first death from the virus. this death and the increasing number of confirmed cases in the country prompted the government to announce a -week nationwide lockdown (neuman, b) . elsewhere, china reported new cases and new deaths, in what seemed like a signal to a second wave of infection. following this, the government announced a ban on all foreigners, suspecting that they were the cause of this new trend in rise of cases, as out of those new cases, none were from local transmission (bbc, a; mai, ) . on the same date, the african development bank (afdb) provided aid to the continent with a $ billion social bond targeted to economic and social sectors facing stiff challenges from the impacts of covid- (afdb, ). at the same time, the who announced that the vaccine trial dubbed solidarity trialdthat it had announced about on march dwas to begin shortly, with the first trials administered to patients drawn from norway and spain, but overall, the trials would be extended to more than countries that had agreed to be part of the program (who, af). in the united states, the food and drug administration authorized the use of a -min diagnostic kit for coronavirus intended to speed-up the testing process, but a shortage of necessary equipment for collecting specimens was feared to derail the use of this kit (azad, ) . on the same land, donald trump, president of america, signed the $ trillion stimulus that was passed the previous day by the senate (foran et al., ) . even as the month of march came to an end, the incidences related to covid- continued almost unthwarted. for instance, the number of new confirmed cases for the past days has been increasing at an average of above , each day, and the number of deaths occurring each day likewise increased at an average of deaths. within the days, the total number of confirmed cases globally increased from , das captured by the who on march (who, f)dto , by march . another astonishing occurrencedespecially on march dwas the number of people that died in a single day in both spain and italy, which recorded and new deaths, respectively (who, p) . this happened while the number of patients being admitted to different hospitals in both countries exceeded their bed capacity and human resources allocation (cheng, ) . the united states was also going through thick and thin, as the number of new cases in the country continued to soar at alarming rates. even in africad which had shown some levels of resilience against the spread of this pandemicdthe numbers for the past days seemed to increase at an average of almost daily, with egypt, algeria, and south africa being the most affected (who, q) . in fact, the who's director-general highlighted the plight of the global health system and argued that there was a chronic global shortage of medical supplies such as ppes, ventilators, and other basic amenities required to saving lives (who, af). these unfolding events were happening amidst numerous responses and interventions both at local, regional, and international levels by government, international organizations like the who, the world bank, and others. among the new interventions that countries were implementing include the extension of the social distancing guidelines in the united states by president donald trump until the end of the following month (april ), and perhaps with a possible extension to june (shear, ) . the country had also accelerated the testing of individuals, and by th, the president announced that over one million people had been tested despite earlier hiccups in regard to faulty testing kits (tirrell et al., ) . in africa, on th, nigeria was reported to have directed the cessation of movement in two of its most populous cities, lagos and abuja, to reduce the chances of importation of the coronavirus to rural areas (akwagyiram, a) . in mexico, in a bid to reduce the soaring confirmed cases and growing number of deaths as a result of covid- , the government declared a health emergency (reuters, l) . in portugal, the government had resulted in treating everyone including foreigners with pending applications as permanent residents of the country so they could access public health facilities without encountering hitches (reuters, o) . but despite all those negatives, there was good news from the city of wuhan, which after undergoing a "dark moment" in the past months, saw authorities relaxing some quarantine measures, including rail services in and out of the city, meaning that people could eventually access the rest of the country (beaumont, ) . authorities in the province of hubei also announced that they would allow domestic flights to resume in all airports, except tianhe international airport in wuhan (xinhua, c) . the other goods news is that on , the bill & melinda gates foundation, wellcome, and mastercard together granted three institutions (the university of washington, university of oxford, and la jolla institute for immunology) financial support worth $ million to facilitate clinical trials for immunotherapies they were developing (mastercard, ) . in the wake of the new months, the reality of devastation of the covid- in different countries, regions, and globally was becoming clear. for instance, in spain, by april , the number of those affected reached , . but, by then, italy had more than these cases with , confirmed cases, while the united states, which had become the new epicenter, had , confirmed cases, as per the cdc data (cdc, ), while the who reported , (who, r). regionally, cases in europe reached more than half a million people ( , ), where , of those had succumbed to the virus. the region of the americas was the second most affected with a total of , confirmed cases and deaths. the western pacific region was the third hardest hit, with , infected and deaths. the eastern mediterranean regions had , confirmed cases and recorded deaths, while the african region had confirmed cases and reported deaths from the virus. overall, the global total number of confirmed cases had reached , , and an addition of , from the previous reported numbers. the number of deaths had also reached , , with a high of dying in the past h (who, r). by april , the global total exceeded the , , mark as per the john hopkins data, ncov .live, and other websites, but who reported the total numbers at , confirmed cases (who, g). the reality was reflected in the economic sector with the world bank reporting that the impacts of covid- pandemic would force more than million people into poverty (the world bank, a) . the reality of this statement was affirmed in the happenings in the united states, where it was reported that in a period of just week, . million more people filed for unemployment benefits, taking the number of those who had filed for the benefits to over million people (long and dam, a) . the same trends of unemployment were also being experienced in austria where the unemployment levels jumped to % in the country within the months since the onset of the pandemic (reuters, a) . the said reality prompted several unprecedented actions in different countries and organizations. for instance, on april , the un announced the postponement of the climate conference (cop ) scheduled for november due to covid- (un, ) . in the sporting world, the all england club announced that the wimbledon tennis tournament was canceled due to covid- , and this was the first time since world war ii that the championship was called off. on the same day, the health minister in italy announced that the countrywide lockdown would continue to be in force until the th of the month as the number of confirmed cases, and deaths continued to increase (reuters, i). the announcement was also confirmed by the country's prime minister giuseppe conte said that the situation in the country forced him to sign the decree to extend the measures (orihuela et al., ) . the same measures were taken by germany. on april , more actions continued, with saudi arabia extended its curfew to h on its most visited and the holiest cities in islam: mecca and medina (aljazeera, c) . a similar action was taken in thailand involving the entire country, with exception of medical personnel, and those transporting essential products, and for people moving to quarantine or health facilities (the nation, ). peru and panama establish a different strategy for reducing the number of people outside by imposing a gender divide, where certain days were set aside for only men and the others for men. this way, it would be impossible for those living together leaving their homes together (aquino and moreno, ). in the united states, the white house was encouraging people going out to wear masks (sun and dawsey, ) , and this came while most of the citizens were trying to come to terms with stay-at-home orders that had been declared in almost states in the country (nottingham, ) . the highlight of the day is the worsening health status of the uk prime minister boris johnson, prompting him to be moved into intensive care. he was taken to hospital in london the previous day (april ), after the covid- symptoms persisted, days after he tested positive (bbc, g). on the same day, in the same country, new deaths were reported, taking the country's total death toll to while the total number of confirmed cases increased to , after new cases were reported in a period of h. within the european region, the total number of confirmed cases had increased to , after , more cases were reported in a span of h. the number of deaths also had increased by a total of deaths to take the total tally for the region to , , and these were far much more than the total deaths of all other regions combined. on the same day, the united states reported , new cases to take their country's total to , (who, s) and also reported a high of deaths increasing the total deaths recorded in the country to , while other data showed that deaths had reached , (siobhán o'grady et al., a) . in the previous day (april ), it had reported cases and , new cases; the highest reported data in the region since the onset of the pandemic (who, a). as these occurred, president trump reported that hydroxychloroquine, an antimalaria drug, could be used against covid- (crowley et al., ) . in the middle east, the total number of confirmed cases in iran increased to , after more people tested positive. globally, the total confirmed cases stood at , , and the total deaths reported were , (who, s). in africa, where cases had started to increase significantlydreaching and deaths, it was reported that the former libya prime minister mahmoud jibril died of coronavirus the previous day after fighting the covid- disease for approximately weeks (aljazeera, d). in china, which reported new cases and only deaths, fears were that the country would be experiencing asymptomatic transmissions of the virus; hence, the increase in newly reported cases. these were experienced though the country had already introduced border restrictions with other countries, and following the fear of asymptomatic cases, the government vowed to tighten border control measures even further (zhang and munroe, ) . the most astonishing news reported on april was that of the positive testing for coronavirus of four tigers and three lions in the bronx zoo, bringing the total number of cats reported to have contracted the virus to (daly, ) . this raised alarms as no known research had shown that the virus could be passed from humans to animals. this far, a report by the who indicated that more than % (approximately . billion) of students globally have had to remain at home following the closure of school. to offer some intervention on this, the organization, together with unicef and the international publishers association, launched the "read the world" initiative to allow student access learning materials even during the difficult times (who, a). in the sporting sector, to safeguard the lives of participants and to comply with the health guidelines of social distancing and others, the open golf championship was called off; being the second time, it was canceled since during the ww (the open, ). after days of uncertainties, the chinese authorities finally lifted the lockdown on wuhan as promised, after the city successfully saw a reduction in the number of new confirmed cases for the coronavirus (aljazeera, a) . in fact, the report from the entire country was that the new cases that were being reported were all imported ones, and the government had vowed to take extra measures to control its borders (zhang and munroe, ) . the good news on the country is that on the previous day (april ), despite reporting new confirmed casesdwhich were all imported, the country reported zero death for the first time since it started publishing figures of the death related to covid- (who, b) . even on th, the number of new cases was only : all imported and only two deaths. the situation was, however, totally different in other regions, especially in europe where france, on a single day, reported new deaths to push its total death toll to , , while those whose tests turned positive in the past h increased by to take the country's total to , (who, t) . this total was however smaller compared to germany, italy, and spain whose totals had increased to , , , , and , cases, respectively. belgium and the netherlands also witnessed an increase in the number of deaths with and new recorded deaths, respectively (who, b). on this, despite germany having more confirmed cases, it had managed to keep the death toll relatively low, with reports showing that it is due to the mass testing initiative it had embarked on; thus, cases were getting identified before becoming critical (perrigo, ) . until eighth, it had only lost people, while its european counterparts were worse off (who, t) . the other nation that saw the number of casualty increase was the u.s, which, since a few days ago had started to witness numerous deaths, and confirmed cases. on this day, the country lost lives to the disease, and , were the newly confirmed cases taking the country's total to , , according to who data (who, t), but the uscdc reported the total number as , cases (cdc, ), where the difference could be due to difference in reporting time between who on central european time (cet) and us edt time zones. while this was happening, the country's president, donald trump who had frequently attacked the who for failing in its mandate in detecting the virus earlier threatened to withdraw funding to the agency (sevastopulo and manson, ) . he categorically said that the organization had withheld information about the virus and was wrong about the outbreak in china (davidson, a) . but, in a quick rejoinder, the who's director-general warned that it would be disastrous to politicize the fight against the pandemic (wise, ) . following the health situation in france, authorities announced that they were imposing a ban on daytime outdoor exercise in paris, which had allowed its citizens to enjoy despite the country being on lockdown (bbc, f). on the same day (april ), the egyptian authorities announced that the ban on mosques and churches would still continue even during the ramadan period as the country was still facing the challenge of coronavirus, with new cases increasing and more deaths being witnessed (egypt independent, ) . in the asian region, japan joined the list of many other countries who had declared a state of emergency as the number of cases in the country had started to rise in the month (rich et al., ) . in singapore, to contain the spread of the covid- , the health minister gan kim yong announced that the government was banning any form of social gathering whether at home or in public (zhang, ) . the ban came just a day after a motion to outlaw social gathering in the country was rushed and passed to the law in parliament the previous day. elsewhere, as the impacts of the virus continued to be felt, the wellness trust, on april , started an initiative aimed to raise a minimum of $ billion from the private sector by the end of april to fill the financial gap being experienced in search vaccines, drugs, and tests for covid- (wellcome, ) . a similar initiative dubbed "afrochampions initiative" was launched by african union and africa centres for disease control and prevention to raise over $ million for medical responses, with $ million required urgently for the same purpose (africanews, ) . after spending three nights in the intensive care unit, the uk prime minister boris johnson was finally discharged from the unit and transferred to a normal ward where he recovered. in fact, it was reported that he could manage short walks, though he needed some time to feel better (the sun, ) . while the report of his improvement was encouraging, it was not the case for over families in the country, from england who had lost their loved ones as a result of the disease on that particular day. in italy, a total of people lost their lives on th (who, h), while had died the previous day (who, u) . in total, the number of deaths in italy had reached a high of , by th with of these being doctors who had contracted the virus while in line of duty (aljazeera, g) . in spain, hopes of flattening the curve were high after the country saw a decline in the number of deaths for the third consecutive day (landauro and keeley, ) . however, the number of new cases in the country was increasing and had climbed to , cases after more cases were confirmed on th. the renewed hope was also being experienced in china after it continued to witness a reduced number of deaths in the country, as well as more recoveries, which had reached , (regencia et al., ) . while that was happening, the spread of the virus has gone as far as in deep rural areas where a -yearold boy from the indigenous tribe of yanomami found in brazil, amazon forest was confirmed with the coronavirus on t th, and on th, unfortunately, he passed away (phillips, ) . another rare place where the virus was reported was in the cook county jail in chicago where new cases involving inmates and staff were reported (whitcomb, a) . in america still, the number of death from covid- were increasing at an alarming rate, where new deaths were reported on th (who, h) and more had died the previous day (who, u). following these increases, with most of them coming from new york cities, the state had resulted in burials in mass graves as the numbers kept on increasing (anderson, ) , with its confirmed cases being more than any other country globally (bbc, d) . the situation in the united states prompted the german foreign minister to criticize the us handling of the virus (connor, ) . another rare place that was affected by coronavirus is a french navy airplane carrier, where servicemen tested positive to covid- (aljazeera, e) . as the cases globally increased to over . million people, the economic impact of the virus continued to bite. for instance, in the united states, another . million people filed for unemployment claims bringing the total number of those in the same predicaments into almost million in only weeks (long and dam, b) . in vietnam, it was reported that the country was in dire need of almost $ billion to caution its economy against the budget deficit that continued to widen (reuters, r) . albania was also trending on a tight economic path, prompting it to seek financial support from the imf, which extended a loan of $ . million (imf, ) . in zimbabwe, following the ban on all international flights in or out of the country, air zimbabwe sent its employees on leave, which was, unfortunately, unpaid (the herald, ). on the same continent, senegal adopted a directly opposite approach of protecting its workers against being laid off by companies in excuse of the covid- crisis (france-presse, ). on other news, there were reports that google and apple would cooperate to develop a mobile app that would help in tracking coronavirus spread (apple, ) . although that was good news especially coming from the tech world, in singapore, the use of technology faced concerns when online learning platforms that the government had initiated were suspended after the video conferencing zoom platform was hacked during a learning session, and the hackers displayed explicit images to the students (lee, ) . although the death tolls in at least four countries crossed the , mark, others have witnessed significant declines in the number of new cases being reported daily, and thus planning to ease lockdown stances and other strict measures that had been put in place. until april , countries like the united states ( , deaths), spain ( , deaths), italy ( , deaths), and france ( , deaths) were most affected, with the situation in the united kingdom worsening ( , ) (who, w). indeed, a report by the office for national statistics highlighted that uk numbers were underreported by %, the number in the country would be reading over , cases (bruce, ) . on this, it was highlighted that the number of deaths reported did not reflect the actual number represented in over care homes, where most of the elderly population were (mcintyre and duncan). although those numbers are many, there was hope in spain and italy as the number of deaths kept on decreasing each day, and from the report, these were optimistic that they would ease their stand on lockdowndspain by end of june (bbc, h). on this, other more countries including greece (by may ) (tugwell, ) , portugal (by may ), australia (had already started by then), pakistan, and austria (with already thousands of shops reopened (niesner and murphy, )) were considering this move (dw, ). although those countries were eager to lift lockdown measures, germany was considering reintroducing it after community infection cases over the past few days started to rise after the country had cautiously tried to ease the lockdown (mayberry et al., ) . in moscow, president putin strengthened the lockdown measures until may to counter the rising cases of infections (davidson, b) . similar measures were also being taken in china, in the heilongjiang province bordering russia, where cases were reported on april , where chinese nationals who had fled to russia tried to return home (wu, ) . following this, the cases in the province increased to , and chinese authorities in the province promised to reward locals who would report the "illegal migrants" (the straits times, b). georgia, on its part, was planning to lock down four of its largest cities, including its capital tbilisi for days as local transmission started to increase with the county's total cases reaching (who, w), an addition of more confirmed cases (who, v) . on the economic front, the imf warned that the global economy would shrink by approximately % following unprecedented measures like lockdowns, and ban on transportations, and closing down of manufacturing and other industries (rapperport and smialek, ) . in a way to ease the economic pressure, president donald trump started issuing stimulus checks to americans, amid some delays after he realized that his name did not appear on the checks (rein, ) . the situation of the economy is also pointed by activities in heathrow airports where passenger demands were expected to reduce by almost % this month (april), after having plummeted by % last month and cargo volume reduced by . % (rojas, ) . the economy was worsening also for the who after president trump retaliated that he was halting funds to the agency following its mismanagement of the coronavirus (mayberry et al., ) . on the societal fronts, the racial discrimination of blacks in china continued, with the mcdonald outlets in china forced to apologize after the store displayed posters banning black people from accessing the china store (folley, ) . elsewhere, turkey was planning to temporarily release over , inmates, after getting approval from parliament to ease overcrowding; thus, void the risk of coronavirus infection in the facilities (wilks, ) . as of th, covid- had spread to countries and territories across the globe with over , , confirmed cases and , deaths reported. of those cases and deaths, % were reported in europe, while more than % of the remaining cases ( , confirmed cases and , deaths) reported in regions of the americas. the eastern mediterranean region had , confirmed cases and deaths while the south-east asia region had a count of , confirmed cases and deaths. africa, which had started to experience some significant increase in infections, had , confirmed cases with of those newly reported and deaths of which of those occurred in a span of h (who, aa). still in africa, it was noted that out of the countries that had reported cases of covid- were drawn from the western and central part of the continent, and who officials reported that they had teams on the ground to establish the real reason why this was happening (who, ac) . of the total reported deaths, the highest number occurred in the united states with a high of deaths, while the united kingdom had the second tally of the day with cases. france had cases while italy and spain, two countries that had for past days shown remarkable improvement, had and cases, respectively (who, aa). on th, the french navy reported that an airplane carrier had soldiers affected, and in less than a week, on th, the number of those had increased to a total of (willsher and sabbagh, ). although reports in the health sector showed that the world was still unsafe, as had also been warned by the who, other issues were coming up in other sectors. for instance, in japan, whose total confirmed cases tallied to dwhich included three cabinet officialsd and the number of deaths increasing to , the prime minister shinzo abe declared a nationwide state of emergency (mccurry, ) . he also advanced a handout worth u , to every resident of the country regardless of their economic status to caution them during the period of this emergency (the japan times, a). in the united states, on the same day, . million people filed unemployment claims bringing the total number of those in this situation to more than million people in a period of only weeks (long, ) . following this, president donald trump unveiled guidelines aimed to help some economic activities in the country to resume, but he left the final decision of opening the economy, by easing the restrictions on each individual state, to the individual state governors (white house, ). in germany, after experiencing prior issues after easing lockdown measures resulted to increased number of confirmed cases, the government was planning to reopen the economy as from april by allowing some nonessential stores to open and also allowing schools to resume as from may (morris and beck, ). in brazil, president jair bolsonaro fired the minister for health after the minister insisted on strict social isolation guidelines, a move that the president was against. unfortunately, the president had been seen to have regularly downplayed the outbreak of the virus in his country despite the country having , confirmed cases and deaths by april (quinn, a) . in sports, following the situation in france, the tour de france scheduled for june and july was postponed to a tentative date between th august and th september, as the government banned public gatherings to reduce the spread of the coronavirus (tour de france, ). for world wrestling entertainment, the company was planning to lay off some employees including wrestlers and producers such as kurt angle (russell, ) . the move was to caution the company against the financial decline it was facing following the impacts of the pandemic. after months of extreme pressure, anxiety, and uncertainties, wuhan settled and revised its official data relating to the coronavirus. after the review, the death toll from the covid- pandemics increased by % meaning that its number increased from deaths to deaths, pushing the country total by april to deaths. the errors in reporting were attributed to delays, omissions, and incorrect reporting that are understandable following all the many things that are happening during that period (neuman, a) . the number of reported deaths also increased in the african continent after more deaths were reported, taking the total tally to , while new confirmed cases increased by , pushing the continent's total to , , according to africa centres for disease control and prevention (africa cdc) (xinhua, a) , but data by the who for the same period show a total of , cases and deaths (who, x) . death tolls were rising by higher margins in the european regions and had exceeded , deaths, from , , cases reported in the region. this increase, observed throughout spain and italy, however, continued to experience improvements with death rates reducing each day. the united kingdom, france, and belgium still reported increasing numbers of deaths ( , , and , respectively) . however, the united states still leads in the number of confirmed cases ( , on april ) and death tolls ( on the same day) per day for more than two consecutive weeks (who, x) . globally, the total number of cases had increased to a high of over . million, with the number of deaths exceeding , by april (who, x) . despite the risk of the disease being live, the orthodox churches in georgia were observed to flaunt the state of emergency declaration to hold easter masses, where hundreds of congregants attended (antidze, ) . in the united states, a day after the president had outlined a set of rules for reopening the economy, but left the final say on the hands of the governor, some protesters were observed in states of michigan, minnesota, and ohio and others, calling their governors to lift the restrictions in their states (gabbatt, ) . at the same time, texas governor is said to have signed an executive order to allow a reopening as from may (office of the texas governor, ). this was happening, even as the us secretary of defense extended the travel ban for one more month to void the earlier expiry scheduled for may ; thus, showing that the country was still not ready to ease the restrictions it had set. further south, in chile, the government started issuing "immunity cards" for all those who had infected and recovered from the virus. with the card, these could comfortably return to their work stations (thomson, ) . as this was happening, the number of cases in the country rose to , , taking the country as the third most affected in latin america (who, x) . however, the who, through dr. michael ryan, one of the executive directors, warned that there was no evidence that those who were recovering from covid- were developing any immunity that could prevent them from being reinfected (who, ab) . during the same press conference, the director-general emphasized that as chinese authorities allowed the wet market to reopen, they would ensure conformity to food safety and highest levels of hygiene and that the law banning any trade in wildlife for food was to be implemented strictly to save the world from future pandemics like the coronavirus (who, ab). elsewhere, more countries were slowly and cautious easing restrictions, with france allowing visitors to care homes, albeit some conditions (dodman, ). croatia also eased some restrictions allowing people to travel within their districts. in other countries such as the united kingdom and zimbabwe, the lockdown measures were to remain intact until when the government is confident the situations are controlled (today, b) . in saudi arabia, despite the start of ramadan, the top religious authorities of the country were recommending people to pray at home to reduce the spread of the virus among the faithful (reuters, p) . this was necessary, as already, deaths were not sparing countries' leadership. for instance, in nigeria, president buhari's chief of staff, abba kyari became the latest topranking official to die from covid- (akwagyiram, b) . a similar case was reported in guinea where a top official and ally of the president alpha condé also died (afp, a). within a period of h (april e ), the number of infections globally increased from . million cases to over . million, while the number of deaths increased to above , globally (reuters, g) . in europe, the number of those infected increased over this period to over . million, with a daily average increase of approximately , new cases, while the number of new deaths in the areas also increased by an average of above each daydtaking the total deaths in the region to , according to data by the who (who, y). in the american regions, cases increased by an average of , confirmations pushing the total confirmed cases to , cases (who, y). the number of deaths increased to approximately , by april , with a majority of these reported in the united states, which was the new epicenter for the coronavirus. the eastern mediterranean region had its total confirmed cases increase to , cases with people dying from covid- . the western pacific region had a total of , cases after new cases were confirmed while the number of deaths increased to . the south-east asia region and african regions, though have had their cases increase, have shown remarkable levels of resilience despite having some of the weakest health systems. their total confirmed cases increased to , and , cases, respectively, while the number of people who succumbed to the covid- in the regions increased to and deaths, respectively, (who, y) . following the unprecedented increase of infections in the united states, president donald trump had reported on april that he would be signing an executive order to suspend immigration to the united states for the next days. and, true to his word, on st, he signed the order meaning that green card recipients would be blocked from moving into the country, with only workers holding nonimmigrant visas allowed (nick miroff et al., ) . he supported his decision by arguing that the unprecedented effects on covid- had pushed many americans out of jobs, and he would wish to see them access the available job opportunities without having to compete with migrants (nick miroff et al., ) . and to ensure that job opportunities would be available, on the same day, his office and congressional leaders agreed on a $ billion small business and hospitals stimulus package, that now only awaited approval from the house of representatives (roberts, ) . in africa, south africa took a similar approach of bailing out the economy by unveiling a $ billion relief plan that would also aid the most vulnerable in the society during the period that the country was struggling with increasing cases of coronavirus and the lockdown measures (channelstv, ) . the economic struggles saw iran start to reopen its economy with major shopping centers in the capital tehran being the first (press, ) . similar actions were observed in israel, which eased lockdown restrictions to allow small shops and stores to open and allow people to move around, but on condition mask-wearing in public (haaretz, ) . this came as the number of recoveries in those countries started to increase, while the death toll contained (efrati and rabinowitz, ). in the poorest countries in the world, the world bank supported the pandemic bond, launched in , with an amount of $ . million to assist nations from the impacts of covid- (baker, ) . this came as the un world food programme warned that the impacts of covid- would result in the doubling of world hunger, representing a total of million people (anthem, ) . the economy was however not worsening for individuals only, but on this day, it was seen to have particularly worsened for us oil-producing economies and companies, with the prices per barrel going down to $ , a historical event that has never happened (suleymanova, ). the only sector that was seen to be doing well, especially in the united states is the gaming industry, probably due to the "stay-at-home" orders and also due to the closure of schools. but, while the market increased, there were fewer games produced due to the impacts sparked by the coronavirus (schreier, ) . the other sector that was seen to benefit from the impacts of covid- was the environment, where it was reported that following the reduced activities in the manufacturing and transport sector, less emissions ( % drop) are expected during the year (marchand and faigle, ). as over countries continued to fight the spread, and the impacts of covid- in their countries, the united states was the most hit with over , cases and over , reported deaths as up to april . the new cases in the country had increased at an average number of approximately , (cdc, ). as the number of cases continued to increase, and other more "staying-at-home" measures following the lockdowns in different states, the number of those filing for unemployment claims continued to increase with over . million additional claims reported over the past week, which was the fifth consecutive week since this trend started (lambert, ) . these new numbers raised the total of those who had filed for their employment benefits to over million people (chaney and guilford, ) . outside the united states, there were mixed responses to the virus. some were seen to ease and lift the lockdown and subsequent measures they had implemented, while others were seen to be in haste to institute those measures. for instance, in the netherlands, after experiencing some "relative calm" over the past weeks in respect to infections in the country, its number had started to rise with new deaths averaging over each day and reaching a high of deaths and , confirmed cases by april (who, z). to minimize further infection, a stern decision was taken to ban any form of public gather until september ( months extension) the first of such a ban globally. that means that events such as sports, music festivals, and religious grouping would not be resuming anytime soon (reuters, e) . but, it was not the only one extending such measure as in pakistan, the lockdown was extended by two more weeks until may , as the country was still experiencing increasing numbers of local transmission of the virus, and the lockdown extension would somehow reverse these trends (cherian, ) . indonesia also joined the list of those that were strengthening their measures after the government announced that it would be temporarily suspending nonessential domestic and international air and sea travel until the end of may to curtail the spread of the coronavirus in the country (bangkok post, ) . others that extended their lockdown include liberia (garda, ) , lebanon (reuters, k) , and czech, which sought parliamentary intervention to allow the extension of the state of emergency until may (xinhua, b) . on the same breath, some countries and states in the united states started easing restrictions to allow the reopening of their economy. these include states such as georgia, oklahoma, and others in the united states, which took these decisions despite the disapproval of president trump (smith, ) . belgium was ready to start reopening some businesses and schools as from may, but gradually and cautiously (martens, ) . although countries were reacting to covid- situations in different ways, some unfortunate news besides new cases and deaths were also reported. for instance, in the united kingdom, which had seen the confirmed cases increase significantly and the number of deaths rising to beyond , there were reports that among the dead were national health service personnel who had paid the ultimate price in a bid to save their country from the pandemic (express and star, ) . in bangladesh, it was reported that frontline doctors had tested positive for the coronavirus due to strains that the covid-i had put on the healthcare system, with most of those in the frontline experiencing a shortage of ppes, test kits, and hospital beds, among other basic essentials (mahmud, ) . elsewhere in japan, after a dreadful experience with the diamond princess cruise ship, on st, another fateful incidence involving an italian cruise ship (costa atlantica) happened in nagasaki shipyard (the japan times, b). it started when one of the crew members tested positive for coronavirus, while of his colleagues were also showing signs, especially high fever. the following day, after contact tracing from the first confirmed cases, another cases tested positive (the straits times, a). h later, another people tested positive (kaneko and kim, ) and by th, a total of people; all crew members were confirmed as testing positive (cna, b) . following this, the japanese government stated that it would test people, out of the who were on board the cruise ship and those who turned negative would be repatriated back to their home countries. as the fifth month, since the onset of the coronavirus, is almost over, the number of those confirmed to have contracted the virus increased to over million people globally, and at least , were reported to have succumbed to the disease. over the same period, of the million, over , patients had recovered (spotlight, ). one country where success against covid- have, and continued to be celebrated, was in wuhan, hubei province, china, where healthy officials reported that they had treated all cases and those who recovered were discharged from hospital (o'donnell, ) . in fact, as previously reported, the restrictions and lockdown in the provinces were lifted including in wuhan, on april (aljazeera, a) . following this, on rd, china pledged additional funding, amounting to $ million, to the who (shih, ) , as already, it could manage the few cases emerging, and the funds could help other areas that were experiencing high pressure from the pandemic. in the new cases, between th and th, china was reported to have only recorded a total of new cases and asymptomatic cases, but no death was reported in the days (who, i). as cases in china reduced, those in the european region seemed to have continued increasing reaching a high of , , cases after the addition of , new cases on th, and the number of deaths increased to , . in the american region, the number of cases was , , and , deaths reported, according to data by the who (who, i). the eastern mediterranean, western pacific, and south-east asia regions had , , , , and , confirmed cases, respectively. the african region continued to show high levels of resilience with only , confirmed cases and deaths reported (who, i). but as the number of cases continued to increase, reports of more countries planning to ease up the lockdown also increased. as of th, italy and spain, two of the most affected countries by the covid- pandemic, with cases of death in each exceeding , (italy , deaths, spain , ) unveiled plans of how they would open up their country as from may . in italy, according to prime minister giuseppe conte, the manufacturing industry would gradually open as from may , but schools would remain closed for three more months until september (kayali, ) . in spain, people would be allowed to walk out for physical activities, but social distancing will have to be observed, as infections in the country were still real (reuters, q) . saudi arabia is another country that considered lifting the nationwide curfew, except for mecca, which remained under -h curfew (aljazeera, h). in germany, after easing the restrictions a few days ago, it is reported that volkswagen was gearing to resume production in their wolfsburg factory on april (allan, ) , the same decision was also taken by bmw company and other companies such as mercedes, jaguar land rover, and others reopening in a few days (reuters, f) . this came as some protests were witnessed in berlin as people demanded the easing of lockdown measures to allow them to return to work (reuters, d) . in the united states, as some states started reopening businesses, the airline industry, which was yet to resume, received support funds amounting to $ . billion -taking their bailout total from the us treasury to $ . billion, with the first disbursement received on april (landay and shephardson, ) . although those countries were gearing to resume business, others insisted on strict measures as cases continued to rise. for instance, the united kingdom maintained the lockdown as it was still not safe from the virus infections (bbc, i). sri lanka also extended the lockdown in the country to counter the increasing number of cases (afp, b). honduras was even considering extending the lockdown by at least one more week until may (reuters, h) . in india, prime minister modi urged his citizens to piously adhere to the nationwide lockdown to contain the rising cases that came amid the month-long curfew (the straits times, d). by april , it became official that the number of covid- infection cases had exceeded the million (the who reported . million cases and , deaths) people and caused the death of over , people globally. of the confirmed cases, over million were reported in the united states while a quarter of reported deaths from the virus coming from the united states. the number of deaths in the country ( , ) even surpassed the total of those who died during the vietnam war between and , where nearly , people died (woodward, ) . in new york, it was reported that for every four people, one of them had contracted the coronavirus. this is after almost , cases had been confirmed, and more than , deaths were reported in the city alone. these numbers in the united states eclipse those of other reported regions. for instance, as of th, the total number of reported cases in africa ( , ) was far much less than the total number of deaths ( , ) reported in the united states on the same day. in addition, they were more than the total number of deaths ( , ) in italy and the united kingdom, which were leading in terms of reported deaths in europe (worldometer, ) . to reduce these unprecedented trends, some states in the united states started testing for asymptomatic residents such as delivery drivers, rideshare drivers, and others. this came as most of the states were planning to reopen by easing the lockdown restrictions, amidst opposition from president trump, and the latest, judge clay jenkins of dallas county, who mentioned the solution, for now, was to follow science and people to stay at home (holcombe, ) . besides the united states, other countries that are yet to experience some reductions in a number of cases include russia, where president vladimir putin stated that the country was bracing for a new and grueling phase of the pandemic (llyushina, ) . his statement came at a time when the number of confirmed cases had continued to stabilize, and the country performed significantly well to reduce casualties. italy, though determined to ease its lockdown restrictions, was experiencing some new cases, with its numbers reaching beyond the , mark. according to the who, africa, eastern europe, latin america, and parts of asia were still not yet out of risk; hence, caution was required even as some actions such as easing the restriction were being taken. this came as eu experts warned that the world would have to wait longer for the vaccine, which would not be ready until the end of , especially considering the cost implications, and other processes that have to be accomplished (cullen, ) . in relation to this, in new york city, it was reported that medical personnel had started testing famotidine; an over-thecounter heartburn medication could cure covid- (lentile, ) . this came as human trials had also started in germany where biontech, a pharmaceutical company was testing its vaccine on volunteers, with participants already having received the dose as from april (aljazeera, f) . as solutions for the covid- continued to be sought, some diplomatic disharmony was witnessed when india canceled orders for , rapid test kits from china after claiming that they were "faulty," and also went forth to withdraw some of the kits that were already in use in several states (bbc, b) . china responded harshly and claimed that it was unfair and irresponsible for india to label chinese products as "faulty" (business today, a). in sports, following the unseen end for the pandemic, french authorities stated that there was no hope for "big sporting affairs" to come back until september in the years, thus, throwing the french football season in disarray, thus, causing them to be canceled (aarons and lowe, ) . the cancellation came as other countries like germany had already announced that the football season (bundesliga) would return in a date to be confirmed albeit under closed doors (bassell, ) . regarding the olympics, the olympics international committee stated that it would cancel the event coming in the next year if the pandemic would not have ended (schad, ) . olympics must be delayed to ensure french football season will not resume but la liga has new hope of restart adb to provide $ million to support strained supply chains in fight against covid- african development bank launches record breaking $ billion "fight covid- leading member of guinea government dies from coronavirus sri lanka extends virus lockdown coronavirus e africa: african union and african private sector launch covid- response fund nigeria orders -day cessation of movement in lagos, abuja to fight coronavirus nigerian president's chief of staff dies from coronavirus china's wuhan ends coronavirus lockdown but concerns remain coronavirus: travel restrictions, border shutdowns by country curfew in mecca, medina extended to hours over coronavirus former libya prime minister mahmoud jibril dies from coronavirus france reports covid- cases on board aircraft carrier germany company begins human trials of coronavirus vaccine one hundered italian doctors have died of coronavirus saudi arabia partially lifts curfew except in mecca volkswagen restart production as european car factories reopen burials on new york island are not new, but are increasing during pandemic risk of hunger pandemic as covid- set to almost double acute hunger by end of hundreds of parishioners attend orthodox easter vigil in georgia apple and google partner on covid- contact tracing technology ( ) china to end lockdown of coronavirus-hit hubei province; wuhan to open next month moscow shuts down all non-essential shops & restaurants to stop spread of covid- trudeau says canada will return asylum seekers to fda authorizes -minute coronavirus test poorest countries finally set to get world bank pandemic bond funds indonesia bans air, sea travel until june over virus fears world bank group president malpass: remarks to the development committee bundesliga return in may now looks inveitable but fans far from happy coronavirus travel: china bars foreign visitors as imported cases rise coronavirus: india cancels order for "faulty coronavirus: italy extends emergency measures nationwide coronavirus: new york has more cases than any country bbc. ( e) coronavirus: new zealand announces lockdown coronavirus: paris bans daytime outdoor exercise coronavirus: prime minister boris johnson tests positive coronavirus: spain plans return to 'new normal' by end of coronavirus: uk must find 'new normal' to ease lockdown e raab formula e: china race called off amid coronavirus outbreak wuhan eases quarantine as coronavirus cases in us pass , singapore closes borders to keep virus at bay, but no shutdown bill & melinda gates foundation, wellcome, and mastercard launch initiative to speed development and access to therapies for covid- uk coronavirus death toll could be far higher than previously shown coronavirus crisis: china fumes over icmr clampdown on 'faulty' rapid testing kits, calls it 'unfair business today. ( b) coronavirus outbreak: india bans international flights till suspends season after player tests positive for coronavirus senate unanimously passes $ t coronavirus stimulus package it's a wreck': . million file unemployment claims as economy comes apart coronavirus disease : cases in the us china's first batch of schools reopens in qinghai province available at south africa president ramaphosa unveils $ bn covid- relief plan coronavirus: who head says nations must attack as 'pandemic is accelerating europe's hospitals among the best but can't handle pandemic pakistan extends lockdown for days, until may south korea throws up innovative tech solutions in coronavirus fight. available at: straitstimes. com/asia/east-asia/south-korea-throws-up-innovativetech-solutions china's imported covid- cases spike as fears grow of second wave covid- tally rises to on italian cruise ship in japan cruise ship stranded by covid- fears to dock in cambodia spain impose nationwide lockdown due to virus, closes all stores except groceries and pharmacies trump address sparks chaos as coronavirus crisis deepens germany's maas: trump coronavirus response took 'too long ignoring expert opinion, trump again promotes use of hydroxychloroquine coronavirus vaccine won't be ready until end of under "most optimistic seven more big cats test positive for coronavirus at bronx zoo donald trump stokes fresh coronavirus row as wuhan reopens global covid- cases near million as putin warns russia faces 'extraordinary' crisis devil's dilemma': france lifts ban on nursing home visits as some warn against relaxing rules coronavirus: what are the lockdown measures across europe? available at map: coronavirus and school closures israel reverses the trend: more coronavirus recoveries than new cases mosques to remain closed, charity iftar tables banned in ramadan negotiations intensify on capitol hill over massive stimulus legislation as coronavirus fallout worsens applications welcome from startups and smes with innovative solutions to tackle coronavirus outbreak commission publishes guidance on export requirements for personal protective equipment the nhs workers who have died during the coronavirus pandemic mcdonald's apologizes after store in china displayed sign banning black people trump signs historic $ trillion stimulus after congress passes it friday senegal bans layoffs during coronavirus crisis cuba closes borders to non-residents over virus: president lebanon announces two-week lockdown over coronavirus us anti-lockdown rallies could cause surge in covid- cases, experts warn cruise ship docks in mexico, passengers allowed to disembark after being denied entry in jamaica, cayman islands amied coronavirus fears cepi invests $ , into potential covid- vaccine from university of hong kong european union leaders denounce trumps coronavirus travel restrictions liberia: president extends lockdown measures for two weeks from april /update foreign travel advice: italy uk helps world's poorest countries withstand the economic disruption of coronavirus latest guard update: more than , troops mobilised for covid- response a fine for not wearing a mask: these are israel's new coronavirus regulations bolsonaro defies coronavirus to rally against congress coronavirus: iran release , prisoners in bid to tackle spread of virus trump invokes defense production act to force gm to make ventilators dallas county judge opposes governor's plan to reopen and calls for residents to follow science and stay home fact sheet: dhs notice of arrival restrictions on china, iran and certain countries of europe together we can with the war against covid- ) imf executive board approves us$ . million in emergency support to albania to combat italian cruise ship in japan has coronavirus cases italy and spain announce plans to ease coronavirus lockdowns further cepi approves $ . m in additional funding for coronavirus vaccine research, bringing total to $ how new york became the epicenter of america's coronavirus crisis ventilators are to this war what missiles were to world war ii un headquarters closes to public as coronavirus precaution real unemployment rate soars past %dand the u.s. has now lost . million jobs spain sees slowing coronavirus toll, holds virtual easter parades airlines receive extra $ . billion in payroll support singapore stops zoom for home-schooling after hacking report famotidine trial underway in nyc for covid- treatment trump declares national emergency e and denies responsibility for coronavirus testing failures russia will "face a new and grueling phase of the pandemic now has million unemployed, wiping out a decade of job gains america is in a depression. the challenge now is to make it short-lived america is in a depression. the challenge now is to make it short-lived hundreds of doctors in bangladesh infected with coronavirus coronavirus: beijing's ban on foreign travellers comes into force months after it criticised other countries for 'isolating china will coronavirus reduce emissions long term? available at belgium to gradually ease virus lockdown in first half of may covid- therapeutics accelerator awards $ million in initial grants to fund clinical trials coronavirus crisis 'like no other' as cases near m: live updates stocks plunge % in dow's worst day since japan declares state of emergency over coronavirus care homes and coronavirus: why we don't know the true uk death toll as virus spreads, africa gets medical supplies from chinese billionaire the dow jones had its biggest point drop in history monday angela merkel's first coronavirus test result is negative panama suspends domestic flights to curb the spread of coronavirus: civil aviation authority germany to reopen schools, shops after 'fragile' success egypt shuts mosques and churches over coronavirus fears china raises wuhan death stats by half to account for reporting delays and omissions goes into -week lockdown trump to suspend immigration to u.s. for days, citing coronavirus crisis and jobs shortage austria reopens thousands of shops in first loosening of coronavirus lockdown nearly % of americans have been ordered to stay at home office of the president. ( a) letter from president donald remarks by president trump and vice president pence at a coronavirus briefing with health insurers governor abbott issues executive order establishing strike force to open texas coronavirus-press-conference- apr .pdf?sfvrsn¼ bd c cd_ cuba is going under lockdown over coronavirus concerns italy joins germany in prolonging lockdown to quell outbreak covid- : how a phone app is assisting south korea enforce self-quarantine measures why is germany's coronavirus death rate so low? available at first yanomami covid- death raises fears for brazil's indigenous peoples spain to extend coronavirus state of emergency as deaths soar connecticut becomes latest state to postpone primary as coronavirus spreads iran begins to open its economy despite fears of second wave of infection brazil's populist president ousted his respected health minister while continuing to downplay the coronavirus pandemic italy's coronavirus death toll surpasses china's japanese pm and ioc chief agree to postpone olympics until ) i.m.f. predicts worst downturn since the great depression states have declared a state of emergency due to coronavirus global coronavirus death toll exceeds , : live updates unprecedented move, treasury orders trump's name printed on stimulus checks austrian joblessness hits record despite government bit to avoid layoffs bolivia postpones elections, announces nationwide -day quarantine to stem spread of coronavirus china's coronavirus epicenter reports just five cases, beijing tomb-sweepers urged to stay back dozens of protesters were arrested in berlin on saturday for flouting lockdown rules and staging a demonstration against lockdown measures dutch extend ban on major public events until sept europe restarts car factories amid uncertain demand global coronavirus cases pass . million as u.s. tally surpasses , honduras extends coronavirus curfew by one week to may italy's coronavirus lockdown measures to be extended to april : minister kazakhstan, uzbekistan close borders after first coronavirus cases kazakhstan-uzbekistan-close-borders-after-first-coronaviruscases-iduskbn ex lebanon advised to extend coronavirus lockdown to may mexico declares health emergency as coronavirus death toll rises nigeria closes land borders to fight coronavirus spread pakistan suspends internationa flights for two weeks portugal to treat migrants as residents during coronavirus crisis saudi top religious authority recommends home prayers in ramadan amid coronavirus spain to allow outdoor exercise if coronavirus cases contine to fall vietnam in talks to borrow $ billion as budget deficit seen widening japan declared a coronavirus emergency. is it too late? available at us senate approves $ bn funding for small businesses rockefeller foundation. ( ) the rockefeller foundation commits $ million in covid- assistance coronavirus: heathrow passenger demand to fall by more than % in april prince albert ii of monaco test positive for coronavirus the coronavirus map in spain: , dead and more than , infected wwe announces layoffs two days after florida deems company an 'essential business tokyo olympics will be canceled, not delayed, if coronavirus pandemic still poses threat in as italy covid- cases soar, who tackles ppe, test shortages gaming sales are up, but production is down donald trump threatens to freeze funding for who new york city public schools to close to slow spread of coronavirus trump extends social distancing guidelines through end of april china pledges additional $ million funding for world health organization india extends ban on international flights until airlines seek $ billion coronavirus aid package coronavirus deaths surpass , , hhs watchdog says american hospitals face 'severe' shortages of equipment, staff and tests china reports zero new local coronavirus infections; trump signs bill to ensure paid leave, other financial benefits too soon': trump disagrees with georgia governor's decision to reopen businesses coronavirus deaths in italy up by in a day to , global death toll from coronavirus tops greece to go into coronavirus-induced lockdown crash! us crude futures turn negative for first time in history white house expected to urge americans to wear face coverings in public to slow spread of coronavirus government bans exports of certain masks, ventilators, raw material for masks zimbabwe shuts borders after first coronavirus the herald. ( ) breaking: air zim sends workers on unpaid leave japan to declare nationwide state of emergency as virus spreads the japan times. ( b) one crew member tests positive, feverish on cruise ship in japan. available at the nation. ( ) pm announces pm to am nationwide curfew gives new guidelines, new york to close restaurants and schools and italian deaths rise the new york times. ( b) u.s. now leads the world in confirmed cases. available at statement from the r&a/the open in to be cancelled china reports newly imported covid- cases and one in wuhan cases-on-cruise-ship-docked-fo r-repairs. the straits times. ( b) china tightens russian border checks, approves experimental coronavirus vaccine china's imported coronavirus cases soar, no local transmission for third straight day the straits times. ( d) indian pm modi urges citizens to follow lockdown as coronavirus cases rise boris johnson's move from intensive care to a general ward is the lift britain needed world bank group increases covid- response to $ billion to help sustain economies world's first covid- immunity cards are coming to chile more than million people tested for coronavirus in us, but access varies from state to state georgia proposes -day lockdown of four cities including capital uk not thinking of easing virus lockdown measures yet: minister the tour de france greece to gradually start lifting lockdown measures on may . available at: greece to gradually start lifting lockdown measures on key cop climate summit postponed to 'safeguard lives secretary-general's remarks at launch of global humanitarian response plan for covid- african finance ministers call for coordinated covid- response to mitigate adverse impact on economies and society a global approach is the only way to fight covid- , the un says as it launches humanitarian response plan coronavirus: boston celtics guard marcus smart tests positive for covid- chinese businessman to donate , test kits and million masks to the world health organization has distributed . million coronavirus lab tests around the world global covid-zero initiative launched to fill $ bn shortfall for coronavirus response chicago's largest jail reports inmates, staff positive for coronavirus two los angeles lakers players have tested positive for covid- , team says president donald j. trump is beginning the next phase in our fight against coronavirus: guidelines for opening up america again australia bans overseas travel and extends social restrictions situation report - . available at who. ( b) coronavirus disease (covid- ): situation report - . available at who. ( c) coronavirus disease (covid- ): situation report - . available at who. ( d) coronavirus disease (covid- ): situation report - . available at who. ( e) coronavirus disease (covid- ): situation report - . available at who. ( f) coronavirus disease (covid- ): situation report - . available at who. ( g) coronavirus disease (covid- ): situation report - . available at who. ( h) coronavirus disease (covid- ): situation report - . available at who. ( i) coronavirus disease (covid- ): situation report - . available at who. ( j) coronavirus disease (covid- ): situation report - . available at who. ( k) coronavirus disease (covid- ): situation report - . available at who. ( l) coronavirus disease (covid- ): situation report - . available at who. ( m) coronavirus disease (covid- ): situation report - . available at who. ( n) coronavirus disease (covid- ): situation report - . available at who. 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( ac) opening statement who director-general's opening remarks at the media briefing on covid- e who director-general's opening remarks at the media briefing on covid- e who director-general's opening remarks at the media briefing on covid turkey to free one-third of its prisoners to curb coronavirus ebrd unveils v billion emergency coronavirus financing package inquiry after of french aircraft carrier's crew catch coronavirus coronavirus: nuggets report member of organization tested positive for covid- who chief warns against 'politicizing' coronavirus unless 'you want to have more body bags world food programme. ( ) statement from wfp executive director david beasley report coronavirus cases remote chinese city hit by coronavirus after weeks of feeling safe africa's covid- death toll hits , , as confirmed cases rise to czech parliament oks extending state of emergency until except wuhan, china's hubei reopens domestic flights what the u.s. needs to do today to follow south korea's model for fighting coronavirus how taiwan is containing coronavirus despite diplomatic isolation by china three members of the philadelphia ers organization test positive for coronavirus china sees rise in asymptomatic coronavirus cases, to tighten controls at land borders key: cord- -cxmw bfu authors: megna, rosario title: first month of the epidemic caused by covid- in italy: current status and real-time outbreak development forecast date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: cxmw bfu background: the first outbreaks of covid- in italy occurred during the second half of february in some areas in the north of the country. due to the high contagiousness of the infection, further spread by asymptomatic people, italy has become in a few weeks the country with the greatest number of infected people after china. the large number of severe cases among infected people in italy led to the hospitalization of thousands of patients, with a heavy burden on the national health service. methods: we analyzed data provided daily by italian authorities for the period from february to march . considering such information, we developed a forecast model in real-time, based on the cumulative logistic distribution. we then produced an estimate of the overall number of potentially infected individuals and epidemic duration at a national and regional level, for the most affected regions. results: we reported the daily distribution of performed swabs and confirmed cases, and the cumulative distribution of confirmed cases, of patients quarantined at home ( %), hospitalized in non-intensive care ( %), recovered or discharged ( %), deceased ( %), and hospitalized in intensive care ( %). the forecast model estimated a number of infected persons for italy of , about, and a duration of the epidemic not less than months. conclusions: once month after the first outbreaks there seems to be the first signs of a decrease in the number of infections, showing that we could be now facing the descending phase of the epidemic. the forecast obtained thanks to our model could be used by decision-makers to implement coordinative and collaborative efforts in order to control the epidemic. * correspondence: rosario.megna@ibb.cnr.it the outbreak of the novel coronavirus called covid- (or sars-cov- ), which originated at the end of december in the city of wuhan, in the hubei province, china [ ] , is having a dramatic global evolution, and was recently classified as a pandemic by the world health organization (who) on march [ ] . the disease, which can be diagnosed through the use of a nasopharyngeal swab, under the most severe forms can lead to bilateral pneumonia [ ] which can be lethal especially in elderly patients with comorbidities [ ] . the first outbreaks of covid- in italy occurred during the second half of february in some areas in the north of the country. despite the drastic restrictions imposed by the italian government in those areas (dl n. , february ) [ ] , several other outbreaks began in other areas of northern italy, forcing the authorities to extend the previously adopted restrictions to the entire national territory (dpcm of march et seq.) [ ] . due to the high contagiousness of the infection, further spread by asymptomatic people [ ] , in a few weeks italy has become the country with the greatest number of infected people after china [ ] . the large number of severe cases among infected people in italy led to the hospitalization of thousands of patients [ , ] , with a heavy burden on the national health service. in particular, the most affected regions are lombardy and emilia romagna, with more than half of the total cases. one month after the beginning of the epidemic in italy we report the situation and propose a forecast model in real-time to estimate its evolution. data on covid- used in our analysis are daily updates from the italian ministry of health managed by the civil protection department [ , ] . a report is released at : or : pm (cet), on the basis of information provided by national and regional local authorities. the most relevant variable is the number of confirmed cases. the other derivate variables to be considered are the number of hospitalized patients (in intensive or non-intensive care), individuals quarantined at home, patients who recovered or were discharged, and the number total deaths. we analyzed data used in this study using the r software, version . . (r foundation for statistical computing, vienna, austria). continuous variables were expressed as mean ± all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint standard deviation (sd) and categorical variables as percentages, while differences between groups were evaluated by χ . we developed a forecast model in real-time, based on the cumulative logistic distribution [ ] . the equation used is the following: where ( ) is the cumulative incidence on day , is the cumulative incidence at the end of the epidemic, is the parameter that governs the flexibility of the curve, is the logistic growth rate, and is the initial day of the epidemic analysis ( feb ). in order to determine the three parameters of the curve, we developed an algorithm based on the maximum statistical significance of and , according to their p-values (smaller values of pvalue indicate greater significance), and varying . nonlinear least squares (nls) function of r was used, normalizing ( ) → ( , = ) ≡ ′. the steps of the algorithm are the following: for n ( from min_n to max_n, step delta_n ) then continue else a = a( n- ); b = b( n- ); n = n- with min_n = , max_n = , and delta_n = for the national evaluation and min_n = , max_n = , and delta_n = for the regional evaluations. at the end of the last cycle, the algorithm provides parameters for best-fit. in order assess the % confidence interval (ci) of the fit values, bootstrap resampling were computed, through the ipec package of r. the graphics were obtained using the ggplot package of r. figure shows the daily distribution of performed swabs and confirmed cases. the percentages on the bars are related to the ratio between the two variables ( . % ± . %). until march , the total number of performed swabs is , . in figure we report the cumulative distribution of confirmed cases, of patients quarantined at home, hospitalized all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in in non-intensive care, recovered or discharged, deceased, and hospitalized in intensive care. in table is reported the number of confirmed cases and the patient categories (expressed in percentage) for at a national and regional (lombardy and emilia romagna) level. the confirmed cases are greater than , , while the number of deceased patients is significantly greater than the chinese [ ] (p < . ). in table we summarized the forecasted method results for at a national and regional (lombardy and emilia romagna) level. in figure we depict the cumulative logistic curve obtained by the forecast model in real-time for the national overview. one month after the outbreak in italy the situation remains complicated. despite the high number of performed swabs as compared to the confirmed cases, the epidemic has been growing with a very high rate. covid- is proving to have a high capacity for infection, probably reinforced by asymptomatic people, who represent a real danger for elderly and fragile individuals. in particular, the disease is showing to be lethal for the elderly ( % in patients aged ≥ ) and men ( %) [ ]. on the date we finalized this article ( march ), the trend of daily distribution of confirmed cases seems to show an initial decline of the growth of the epidemic. the total number of confirmed cases will eventually exceed those that occurred in china. the forecast model in real-time indicates a total number of national cases greater than , patients, with a figure of approximately , in lombardy only. in addition, duration of the epidemic was estimated of months about. since the theoretical cumulative curve has an asymptotic pattern (i.e. the maximum value is achieved for the t time towards infinite), considering % of the time from the beginning of the outbreak is a convention. therefore, if instead of % of time we considered , %, the overall number of days estimated for the epidemic to come to an end increases by % (i.e. ten more days need to be added to the calculation of time). moreover, several factors could affect the total number of cases and the duration of the epidemic. for example, a contribution to the spread of outbreaks in southern italy was caused by the movement of students and workers from northern to southern italy following the first governmental restrictions. on the other hand, more stringent restrictions imposed later on by the government could lower the expected number of total cases and reduce the number of days towards the end of the epidemic. another important factor is related to possible mutations of the novel coronavirus, which could have a positive or negative outcome on the trend of the epidemic. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in it is also necessary to consider the intrinsic limitations of this study. first of all, data was not always updated on a daily basis by each regional authority. moreover, we have to consider that the number of infected people is underestimated, since there are many undetected asymptomatic individuals. in addition to this, many individuals died without the possibility of checking if they were actually infected and therefore not recorded as such. finally, the factors that determine the trend of the epidemic could change without respecting the symmetry of the forecasted model. the epidemic caused by covid- in italy is having a dramatic evolution in terms of confirmed cases, hospitalized and deceased patients. once month after the first outbreaks there seems to be the first signs of a decrease in the number of infections, showing that we could be now facing the descending phase of the epidemic. the model presented in this article fits well with the data, therefore it is expected to be reliable in predicting the evolution of the epidemic. the forecast could be applied by decision-makers to take coordinative and collaborative efforts to control the epidemic. none. not applicable. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia covid- pneumonia: what has ct taught us? clinical features of deaths in the novel coronavirus epidemic in china president of the council of ministers a theory of growth key: cord- -e ey eo authors: patel, urvish; malik, preeti; mehta, deep; shah, dhaivat; kelkar, raveena; pinto, candida; suprun, maria; dhamoon, mandip; hennig, nils; sacks, henry title: early epidemiological indicators, outcomes, and interventions of covid- pandemic: a systematic review date: - - journal: journal of global health doi: . /jogh. . sha: doc_id: cord_uid: e ey eo background: coronavirus disease- (covid- ), a pandemic that brought the whole world to a standstill, has led to financial and health care burden. we aimed to evaluate epidemiological characteristics, needs of resources, outcomes, and global burden of the disease. methods: systematic review was performed searching pubmed from december , , to march , , for full-text observational studies that described epidemiological characteristics, following moose protocol. global data were collected from the jhu-corona virus resource center, who-covid- situation reports, kff.org, and worldometers.info until march , . the prevalence percentages were calculated. the global data were plotted in excel to calculate case fatality rate (cfr), predicted cfr, covid- specific mortality rate, and doubling time for cases and deaths. cfr was predicted using pearson correlation, regression models, and coefficient of determination. results: from studies of patients, . % of patients died, . % recovered, . % were admitted to icu and . % required ventilation. covid- was more prevalent in patients with hypertension ( . %), smoking ( . %), diabetes mellitus ( %), and cardiovascular diseases ( . %). common complications were pneumonia ( %), cardiac complications ( . %), acute respiratory distress syndrome ( . %), secondary infection ( . %), and septic shock ( . %). though cfr and covid- specific death rates are dynamic, they were consistently high for italy, spain, and iran. polynomial growth models were best fit for all countries for predicting cfr. though many interventions have been implemented, stern measures like nationwide lockdown and school closure occurred after very high infection rates (> cases per population) prevailed. given the trend of government measures and decline of new cases in china and south korea, most countries will reach the peak between april - , if interventions are followed. conclusions: a collective approach undertaken by a responsible government, wise strategy implementation and a receptive population may help contain the spread of covid- outbreak. close monitoring of predictive models of such indicators in the highly affected countries would help to evaluate the potential fatality if the second wave of pandemic occurs. the future studies should be focused on identifying accurate indicators to mitigate the effect of underestimation or overestimation of covid- burden. viewpoints research theme : there are confirmed cases worldwide with ( . %) deaths and ( . %) recovered cases [ ] . new york is the current epicenter of covid- ( cases and deaths) of united states of america (usa) ( cases, ( . %) deaths and ( . %) recovered patients) while italy ( deaths) and spain ( deaths) being worst affected countries [ , ] . globally, the epidemiological scenario of covid- is changing on a daily basis. the origin of severe acute respiratory syndrome coronavirus (sars-cov- ) virus was linked to a seafood market in wuhan from the handling and close contact with animals [ ] . in usa, the first case was reported on january , , with a recent travel history to wuhan [ ] . according to emerging literature, covid- symptoms can range from mild respiratory illness causing fever, dry cough, dyspnea, myalgia and fatigue to more severe manifestation of pneumonia, cardiac complications requiring intensive care unit (icu) admission and mechanical ventilation [ ] . the median incubation period is around days (range: - days), requiring prolonged monitoring in extreme cases [ , ] . real-time reverse transcriptase polymerase chain reaction (rt-pcr) of nasopharyngeal and/or oropharyngeal swabs are usually used to confirm the diagnosis [ , ] . preliminary demographic data of the infected patients suggests that most patients have mild disease, with older adults (≥ years) appearing to be more susceptible to severe illness requiring hospitalization [ , ] . covid- shows evidence of human to human transmission via respiratory droplets and from contact with contaminated surfaces or objects, with estimated median basic reproduction number (r ) of . (range: . - . ) [ ] , making the spread of the disease tough to contain. while recently published observational studies have provided insights on the epidemiology of this pandemic, their sample sizes are too limited for any definitive conclusions. hence, we sought to conduct a systematic review and analysis of all available studies comparing outcomes. primary aim of the study is to evaluate the epidemiological characteristics, needs of resources, and patients' outcomes. secondary aim is to evaluate the global burden and interventions. we evaluated epidemiological characteristics, risk factors, laboratory and imaging findings, complications and treatment utilized. we also calculated the mortality, recovery, and needs of resources like icu beds and mechanical ventilators. in order to evaluate the primary outcome, we performed a systematic review of these observational studies according to moose guidelines [ , ] . we searched the pubmed database for original observational studies that described any details on epidemiological characteristics on patients with covid- . the database was searched from december , , to march , . the search was conducted using the following keyword/mesh terms: ((covid- [title/abstract]) or coronavirus [title/abstract]) or sars-cov- [title/abstract] or -ncov [title/abstract]. all studies that compared outcomes of interest in covid- patients were included. any literature other than observational studies was excluded. non-english literature, non-full text, and animal studies were excluded. abstracts were reviewed, and articles were retrieved accordingly. two independent reviewers performed the search and literature screening (up, pm), with disputes resolved by consensus following discussion with a third author (cp). for the ease of understanding, we used a flow diagram to describe literature search and study selection process in figure s in the online supplementary document. a prespecified data collection excel sheet was used to collect the data relating to study characteristics and outcomes of interest by two authors (pm and cp), and discrepancies were solved by a discussion with a third author (up). the following study characteristics were extracted: publication year, country of origin, sample size, age, sex, direct exposure to infection, travel history, signs and symptoms, risk factors and comorbidities, laboratory and radiology findings, treatment utilized, and complications. data on the following outcomes were extracted: mortality, recovery, need for icu beds and mechanical ventilators. viewpoints research theme : covid- pandemic all analysis was done in excel (microsoft inc, seattle wa, usa) and sas . (sas institute, cary, nc, usa). the frequencies and percentages of epidemiological characteristics and outcomes were calculated. we evaluated the global burden of covid- including case fatality rates (cfr), strength of association between deaths and cases to predict cfr, case doubling time, covid- specific mortality rates, and control measures by governments to prevent spread among usa, china, italy, iran, spain, germany, india, and south korea. for this purpose, data were taken from the johns hopkins university corona-virus resource center [ ] , kff.org [ ] , world health organization-covid- situation reports [ ] , and worldometers.info [ ] up until march , . we evaluated changes in cases and deaths, cfr, created a predictive modeling for cfr, covid- specific mortality rate, and doubling time for cases and deaths. cfr was defined as the number of cases divided by the number of the diagnosed patients with covid- , and covid- specific mortality rate was defined by deaths due to covid- infections divided by total population of the country in , counted per population [ ] pearson correlation coefficient (r) was obtained to establish the strength of association between deaths and cases for individual countries. to predict cfr, we modelled the epidemic curves with simple linear regression, exponential growth, and polynomial growth models and used a coefficient of determination (r ) for model selection. the time of reporting the first death was used as the starting point for that country for all three models. we utilized government websites, national media, and other standard open sources to evaluate the governments' interventions during covid- pandemic, infection rate [(diagnosed cases/country' s population in ) per population] [ ] at the time of interventions like nationwide school closure and lockdown, and effects of such measures to predict the dates of peak number of cases in each country. our search resulted in studies, out of which non-human studies and other than observational studies, non-full text and articles with non-english language information were excluded. full-text studies were screened and studies with insufficient clinical information or outcomes-related information were excluded. full-text articles were assessed for eligibility. the final analysis included fulltext observational studies, presented in table , including a total of patients. [ ] china jan , -feb , young, mar [ ] singapore jan , -feb , - chang, feb [ ] china jan , -jan , wang, feb [ ] china jan , -jan , ng, mar [ ] singapore jan , -feb , - spiteri, mar [ ] europe jan , -feb , - covid- national incident room surveillance team, mar [ ] australia mar xu, feb [ ] china jan , -jan , . - bajema, feb [ ] usa jan , --ki, feb [ ] south korea jan , --chen, jan [ ] china jan , -jan , zhang, feb [ ] china jan , -feb , --yang, feb [ ] china figure s in the online supplementary document. several models, including a simple linear regression, exponential and polynomial (quadratic) growth models, were used to determine the type of association between cumulative deaths and cumulative cases to predict cfr ( table ) . the polynomial growth model had the best fit (higher r ) and indicates that for all countries the death rate increases with the number of cases, and this increase is steeper than a linear relationship. interestingly, while for the usa, italy, iran, spain, and india this association is always positive, for china, south korea, and germany the initial slope is negative but then is reversed as the number of cases continues to increase (figure ). figure s a in the online supplementary document). the daily covid- specific death rate is highest in spain (daily . deaths per population) and italy (daily . deaths per population) followed by usa (daily . deaths per population) ( figure s b in the online supplementary document). the county-specific timeline of doubling time for cases and deaths is shown in table and the increment in cases and deaths are plotted in (figure in the online supplementary document). march barred entry of foreign nationals who had been to european countries within last days [ ] march nationwide schools closed [ ] , lockdown in new york [ ] march a us$ trillion coronavirus stimulus bill was passed and signed by the president [ ] march more than half of us states underwent lockdown [ ] china: january response to public health emergency launched by hubei [ ] january the central government of china imposed a lockdown in wuhan and other cities in hubei province; public transport suspended. the wuhan airport, railway stations and metro were closed, not allowing residents to leave the city without permission [ ] ; public health emergency response announced by mainland province of zhejiang [ ] january mainland china has initiated public health emergency response [ ] ; quarantined whole hubei province [ ] ; curfew laws implemented in huanggang,wenzhou and other mainland cities [ ] south korea: an unlicensed covid- test authorized by the korea centers for disease control and prevention (cdc) [ ] ; travel denied to foreign nationals from hubei province into south korea [ ] february all kindergartens, elementary schools, middle schools, and high schools were announced to delay the semester start [ ] february entire country opened drive-through testing [ ] italy: january state of emergency declared, flights to and from china suspended [ ] february the council of ministers announced a new decree-law to quarantining more than people from different municipalities in northern italy [ ] march nationwide schools and universities closed [ ] march prime minister imposed nationwide quarantine lockdown [ ] march all commercial activities except pharmacies and supermarkets ordered to shut down [ ] ; € billion allocated by the government [ ] april drive-through testing began [ ] iran: all concerts and other cultural events cancelled for one week by ministry of islamic culture and guidance [ ] ; closure of educational institutions in several cities and provinces announced by the ministry of health and medical education [ ] march checkpoints placed between cities to limit travel [ ] march fatima masumeh shrine, jamkaran mosque in qom city, and imam reza shrine in mashhad closed [ ] viewpoints research theme : germany: new health security measures enacted to regulate air and sea travel that required passengers from china, south korea, japan, italy and iran to report their health status before entry [ ] ; federal police stepped up checks within km of the border [ ] march bavaria declared a state of emergency for days and measures to limit public movement and additional funds for medicine supplies were introduced [ ] ; all flights from iran and china stopped by german ministry of transport [ ] ; travelling in coaches, attending religious meetings, visiting playgrounds or engaging in tourism prohibited [ ] finance minister announced us$ billion stimulus package [ ] infection rate at the beginning of the major intervention (nationwide closure of school or major table mentions the predicted dates of the peak number of cases based on strict interventions. in china and south korea, it took - days and - days respectively in order to achieve the peak of the pandemic before the new number of cases began to decline. we have used a - days post-interventional model to calculate the peak of the pandemic keeping in mind the effect of china' s model of interventions. covid- has significantly impacted the entire world both socially and economically. the rapid human-to-human transmission has posed a great public health threat. across studies included in this review, we found confirmed cases of covid- with the majority of the published studies from china. % of the cases had a history of direct exposure or being exposed to the seafood market in wuhan, % were china residents and % had a travel history to china. initially the virus was limited to only wuhan and despite travel restriction, the virus continued to spread across the world at a rapid rate from china, likely due to asymptomatic transmission in the initial stages of the outbreak with a median incubation period of only days [ , ] , before travel restrictions. the covid- cases are increasing exponentially but underestimated due to mild symptoms in a portion of cases, long incubation periods, and shortage of testing kits. in concurrence with other studies [ , ] , we found that clinical characteristics of covid- are similar to those of sars and influenza virus. fever ( %), cough ( %) and myalgia or fatigue ( %) were the most prominent symptoms. % of patients reported dyspnea and sputum production/expectoration. major comorbidities were hypertension, smoking, diabetes mellitus, and cardiovascular disease. patients with these comorbidities are at high risk for complications including pneumonia, ards and cardiovascular complications. we found that patients had increased inflammatory markers including elevated crp in %, lymphopenia in % and elevated esr in % which is similar to other respiratory infections (sars, influenza). few studies [ , ] , have reported abnormal liver function in covid- patients, and we found % of patients had elevated alt and ast. additionally, increased ldh ( %), d-dimer( %) may indicate the severity of the disease [ ] . some studies have also reported elevated neutrophil count and cytokine storm induced by virus leading to coagulation activation and sustained inflammatory response [ ] associated with higher mortality [ ] . there is no proven therapy available as of now for covid- [ ] . large scale clinical trials for these drugs are under way. % patients received oxygen and antibiotics ( %), antivirals ( %) and steroids ( %) as supportive therapies. the prognosis of patients after receiving these treatments is not yet clear. in people with compromised immune systems such as older age, hiv, malignancy, diabetes, chronic pulmonary disease if treated promptly with antibiotics, convalescent plasma to increase the immune support might reduce the risk of complications and mortality [ ] . in our analysis, % of the patients required icu admission, % needed mechanical ventilation, % died and % recovered and were discharged from the hospital. these findings are consistent with guan et al. and wang et al that present similar rates [ , ] . currently in the usa, covid- is in the acceleration phase surpassing china and italy, and a national emergency was declared by the president, but the viewpoints research theme : duration and severity may vary depending on the virus characteristics and public health response [ ] . if confirmed cases continue to grow with this trend, soon the covid- pandemic will cause shortages of ventilators. as per institute for health metrics and evaluation (ihme) projections, on a peak day in the usa, there would be a shortage of icu beds by and a need of ventilators [ ] . the growing number of cases will place a burden on the current capacity of hospitals and hence it is essential to develop and implement strategies to mitigate the gap by increasing capacity and fair allocation of available resources. as of march , cfr in italy was . % and . % in china. according to onder et al. [ ] , cfr stratification by age, shows similar rates for - years ( %- . %) but higher in > years( %- . %). this difference might be due to high cfr reported in people > years in italy and no data from china for the same age group [ ] . other reasons might be demographics differences between two countries (≥ years population: italy- . % vs china- . %), overwhelming health care system, and shortage of icu beds and ventilators, which might lead to prioritizing treatment to younger and otherwise healthy patients over older patient [ ] . in our analysis cfr in italy increased from . % on february to . % on march , possibly due to the implementation of a strict policy of testing only suspected cases with severe symptoms [ ] . though widespread and drive-through testing is becoming more available in usa, cumulative tests conducted per million population lags behind compared to germany, italy, south korea, and spain. our data driven polynomial growth model predicts more deaths in future with an increase in cases in usa [ ] , italy, iran, spain, and india. as per our model predictions, doubling time of cases in the usa, germany and india is decreasing suggesting that they are inching towards the peak. different countries undertook interventions at different points in the timeline of spread of virus. the infection rates in the usa, italy, iran, spain, and germany were higher when they undertook substantial measures compared to china, south korea, and india, suggesting a delayed response and failure to undertake timely measures. the aforementioned timelines for peaks look optimistic because multiple other factors may influence the trajectory of spread, ie, population density, economy, demographics, health care, religious beliefs, and legislation. for instance, despite the growing number of cases, iran continued to keep its shrines open to pilgrims for a long time, but recently closed them, and no stringent curfew laws were imposed. also, many states in usa have still not implemented strict quarantine measures. such practices can seriously impede the efforts at containing the spread and skew the projection in many ways. restrictions have neither been homogeneously imposed nor simultaneously adopted throughout the country, making it difficult to predict the exact model of the spread. also, covid- testing capacity of the nations are limited and the true number of the infected people might have been higher than the estimated numbers at the time of our analysis. hence, an early phase covid- specific death rate would be a better estimate than cfr to compare the severity of the disease. many factors contribute to the accurate estimation of cfr such as testing capacity, care seeking and lack of understanding of the proportion of asymptomatic and pre symptomatic cases [ , ] . limited knowledge of these factors in the early covid- phase might have contributed to overestimation of cfr in our study. the use of serological testing for presence of igm or igg antibodies against sars-cov- will provide a better estimate of cumulative prevalence of covid infection [ ] . as recommended by who, measuring the seroprevalence of antibodies to covid- is crucial and will contribute to determine accurate cfr and help plan adequate public health response [ ] . the research on covid- is rapidly evolving and new publications are becoming available daily. the majority of the epidemiologic data are coming from single center with limited sample sizes. to overcome this limitation and provide a global view of the covid- pandemic, we have analyzed data on over patients from peer-reviewed studies. as a result, we provided more generalizable estimates of laboratory findings, clinical symptoms and complications of covid- patients. we have included data from several countries/regions; however, one limitation is that the majority of cohorts are from china, and as more data from other countries become available, additional meta-analyses would be essential. this is the first study rigorously tracking the timing of government interventions across multiple countries; however, as mentioned earlier, the adherence to those interventions could vary from one country to another, making the projections of the potential effectiveness challenging. we have not evaluated the duration of strict interventions in all these countries. the population prevalence data are based on the symptomatic patients with confirmed rt-pcr testing. since some patients can be infected and present mild or no symptoms, or have not undergone rt-pcr testing, serological antibody testing in the future may allow a viewpoints research theme : covid- pandemic more accurate understanding of the disease prevalence and death rates. despite all the limitations, this is the first study in our knowledge, highlighting and explaining epidemiological indicators, testing capacity, interventions, and expected burden of the covid at early phase. we have reviewed the burden of this pandemic and steps taken by the governments of different countries. though the governments can continue strict lockdowns, it is not a long-term solution. good hand hygiene, widespread testing, detection and isolation of new cases, rigorous contact tracing in low-prevalence settings, early vaccine development and its quick distribution, strengthening the overburdened health care system, and protecting frontline health care workers may help to gradually relax the strict lockdowns and cope with covid- pandemic. this would only be possible by a collective approach undertaken by responsible governments, wise strategy implementation, and receptive populations. the future studies should be focused on identifying accurate indicators to mitigate the effect of underestimation or overestimation of covid- burden. close monitoring of such indicators in highly affected countries is very crucial to evaluate the potential fatality if the second wave of pandemic occurs. who director-general' s opening remarks at the media briefing on covid- - coronavirus covid- global cases by the center for systems science and engineering (csse) at johns hopkins university. (jhu). first case of novel coronavirus in the united states clinical characteristics of coronavirus disease in china incubation period and other epidemiological characteristics of novel coronavirus infections with right truncation: a statistical analysis of publicly available case data the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease detection of novel coronavirus ( -ncov) by real-time rt-pcr clinical characteristics of coronavirus disease in china the novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) -china estimation of the reproductive number of novel coronavirus (covid- ) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis covid- ) situation reports countries in the world by population the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application clinical features of patients infected with novel coronavirus in wuhan a comparative study on the clinical features of covid- pneumonia to other pneumonias epidemiologic features and clinical course of patients infected with sars-cov- in singapore epidemiologic and clinical characteristics of novel coronavirus infections involving patients outside wuhan, china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china evaluation of the effectiveness of surveillance and containment measures for the first patients with covid- in singapore first cases of coronavirus disease (covid- ) in the who european region clinical findings in a group of patients infected with the novel coronavirus (sars-cov- ) outside of wuhan, china: retrospective case series persons evaluated for novel coronavirus -united states epidemiologic characteristics of early cases with novel coronavirus ( -ncov) disease in korea. epidemiol health epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of patients infected with sars-cov- in wuhan clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical features of cases with coronavirus disease in wuhan, china clinical characteristics of refractory covid- pneumonia in wuhan, china characteristics and outcomes of critically ill patients with covid- in washington state risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study statement from the press secretary regarding the president' s coronavirus task force trump declares coronavirus a public health emergency and restricts travel from china trump signs emergency coronavirus package, injecting $ . billion into efforts to fight the outbreak trump administration announces measures to speed coronavirus testing here' s what trump' s coronavirus emergency declaration does new york launches drive-thru testing site for covid- see which states and cities have told residents to stay at home dhs notice of arrival restrictions on china, iran and certain countries of europe map: coronavirus and school closures. house passes $ trillion coronavirus stimulus bill, which includes direct payments to americans and business loans notice of the people ' s government of hubei province on strengthening the prevention and control of pneumonia infected by new coronavirus china coronavirus: lockdown measures rise across hubei province zhejiang: newly diagnosed cases of new coronavirus infection and pneumonia were launched, and the first-level response to major public health emergencies was initiated all provinces in mainland china have initiated first-level response to public health emergencies xiangyang railway station is closed, and the last prefecture-level city "hubei" in hubei province ningbo have implemented the most restrictive order south korea learned its successful covid- strategy from a previous coronavirus outbreak: mers korea bars foreigners traveling from hubei province s ministry of education opening on march south korea pioneers coronavirus drive-through testing station italy suspends all china flights as coronavirus cases confirmed in rome ten lombard municipalities: thousand people forced to stay at home. quarantine at the milanese hospital in baggio. italy orders closure of all schools and universities due to coronavirus italy extends emergency measures nationwide merkel warns virus could infect two-thirds of germany coronavirus emergency, the government' s plan rises to billion. gualtieri to the eu: 'stimulus is needed'. we will make a day long holiday' to contain coronavirus, as sixth victim dies coronavirus: iran limits travel and urges banknote avoidance. shiite hardliners in iran storm shrines that were closed to stop coronavirus spread germany enacts new health security measures against coronavirus infections these rules apply in bavaria. germany halts flights from iran and china over coronavirus: bild. deutschland im shutdown-modus -die alternativlos-kanzlerin kehrt zurück. bayern impose curfew! contact bans on more than two people, hairdressers too -the federal and state governments have agreed on this spain prohibits all direct flights from italy until the community of madrid decrees the mandatory closure of bars, restaurants and clubs until spain to impose nationwide lockdown -el mundo marlaska suspends free movement and reestablishes border controls predictions and role of interventions for covid- outbreak in india coronavirus: all international arrivals to india to share travel history at airports. icmr to test for community transmission of covid- coronavirus: icmr recommends hydroxychloroquine for high-risk population. coronavirus: india enters 'total lockdown' after spike in cases global airlines have completely stopped flying scheduled flights due to travel bans, airspace closures, and low demand for travel fm nirmala sitharaman announces rs . lakh crore relief package for poor. to understand the global pandemic, we need global testing -the our world in data covid- testing dataset early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a comparative study on the clinical features of covid- pneumonia to other pneumonias remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial treatment of critically ill patients with covid- with convalescent plasma covid- ) -united states icu-days, ventilator days and deaths by us state in the next months case-fatality rate and characteristics of patients dying in relation to covid- in italy. jama. . online ahead of print italian doctors on coronavirus frontline face tough calls on whom to save the many estimates of the covid- case fatality rate evidence supporting transmission of severe acute respiratory syndrome coronavirus while presymptomatic or asymptomatic seroprevalence of immunoglobulin m and g antibodies against sars-cov- in china population-based age-stratified seroepidemiological investigation protocol for coronavirus (covid- ) infection key: cord- - iqc s authors: agostiniani, rino; bozzola, elena; staiano, annamaria; del vecchio, antonio; mazzone, teresa; greco, luigi; corsello, giovanni; villani, alberto title: providing pediatric well-care and sick visits in the covid- pandemic era: the recommendations of the italian pediatric society date: - - journal: ital j pediatr doi: . /s - - - sha: doc_id: cord_uid: iqc s pediatricians have observed a significant decrease in in-person child health visits during the covid- pandemic. in the post lockdown period, the coronavirus trend remains positive in italy but fears of a second wave have recently grown in italy due to active hotbeds of contagion. the pandemic may negatively affect the care of pediatric patients and overall children welfare as it may present with severe signs and symptoms or it may complicate. the italian pediatric society recommend to separate well visits from sick ones, to educate families and to promote hygienic strategies to provide an adequate pediatric assistance in case of a second pandemic wave. pediatric patients with coronavirus disease (covid- ) may experience flu-like signs or symptoms over the course of the disease, such as fever, cough, nasal congestion or rhinorrhea, sore throat, diarrhea, nausea or vomiting [ , ] . in children illness severity may range from an asymptomatic to a critical condition. clinical presentation may be as follow [ ] : asymptomatic mild (including fever, fatigue, myalgia, cough) moderate (such as pneumonia) severe (such as dyspnea, hypoxia) critical (such as acute respiratory distress syndrome, respiratory failure, shock, or multi-organ dysfunction). in italy, the pediatric cases are actually in children aged - years and in those aged - years, respectively , % and , % of the total cases [ ] . fewer cases of covid- among children compared to adult cases are reported, partially due to under diagnosis [ ] . in fact, most pediatric cases are asymptomatic or with mild-moderate symptoms [ ] . nevertheless, pediatric cases of covid- , caused by severe acute respiratory syndrome coronavirus (sars-cov- ), have been reported, as well as rare hyperinflammatory states (kawasaki-like syndrome). due to the recent onset of covid- pandemic disease, long term consequences are inevitably unknown at present. data suggest that toddler younger than months as well as underlying medical conditions represent severity risk factors [ ] . age-stratified analysis showed that the sars in children was . % compared with . % in adults and that the odds of infection in children was . times ( %ci . - . ) of that among the elderly [ , ] . although most pediatric cases are mild, pediatricians should maintain suspicion for sars-cov- infection in children, visit sick patients and monitor their clinical conditions for the risk of complications, in order to avoid the spread of the infection to other children, schoolmates caregivers, family members and physicians. in the post lockdown period, the coronavirus trend remains positive in italy but there are still active hotbeds of contagion, according to the monitoring report by the health ministry and the health institute (iss). these are small but they are also sufficient for the experts to say that the covid- epidemic in italy is not definitely over yet and that it is still necessary to keep the guard up and maintain social-distancing measures. some countries have experienced a second wave of cases, which consisted in a rise in infections after appearing to have the virus under control, such as israel and spain. fears of a second wave have recently grown in italy, relating next fall/winter season. pediatricians have observed a significant decrease in inperson child health visits during the covid- pandemic, which puts children's health at risk. moreover many parents have concerns about what to do if their child will be sick in the next months. the italian pediatric society suggests parent to contact the child's pediatrician/physician if they have concerns. it is important at this time to not immediately assume an emergency room visit if it is unneeded. on the contrary, in case of necessity, discourage families remaining at home for the fear of infection. as well as the american academy of pediatrics, the italian pediatric society strongly supports the continued provision of health care for children [ ] . on march , cdc posted guidance emphasizing the importance of routine well child care and immunization, particularly for children aged ≤ months [ ] . the current emergency might negatively affect the care of pediatric patients and overall children welfare. in particular, the fear of contracting covid- may determine a delayed access to pediatric emergency facilities as pointed out in a recent study. moreover, as parents are discouraged from entering hospitals even in case of severe clinical conditions, children were more frequently admitted in compromised situations [ ] . regarding healthcare providers, as covid is not yet over in italy, the italian pediatric society recommend to use strategies to separate well visits from sick ones. a suggestion may be to schedule routine appointments in the morning and sick visits in the afternoon. sanitation and hygiene services are an essential part of preventing and protecting people during infection outbreaks, such as the one caused by covid . promoting good handwashing behavior, hygiene, physical distancing and the use of face masks as the most effective strategies to prevent infection may part of daily communication activity to families. in the latest months, a significant drop in well-child visits has resulted in delays in vaccinations, delays in appropriate screenings. in some cases, pediatricians have rapidly adapted to assist children through telehealth. however, in most cases, a in-person visit is required to perform a physical exam. scheduling sick visits and well-child visits during different times of the day and/or different locations should help families not to miss appointments. physical and neurodevelopment screening should continue in order to provide both an early intervention service and a prompt assistance in case of pathological findings. in the meantime, healthcare providers should identify children who have missed routine controls and/or vaccinations and contact them to schedule in person appointments. even in pandemic, all newborns should be seen by a pediatric healthcare provider shortly after hospital discharge and should be evaluated for peri-natal problems such as jaundice, loss of weight, etc. .. as for routine children check, the italian pediatric society recommends: schedule appointments and fill a telephonic family interview to find out risks factors (for example exposure to and symptoms consistent with the coronavirus) as reported in table . reducing crowding in waiting rooms, planning appointment and asking patients to remain outside the medical office until they are called into the room for their appointment upon entering medical room, check temperature to the child and to the caregiver. both the patient, according to age, and the caregiver should wear a surgical mask and adopt hygienic practices (such as washing hands) inform families about the strategies already implemented in medical home office to assure safety. educate families and provide the information to avoid the spread of infections, by everyday preventive actions, as reported by who [ ] . explain health precautions put in place to help ensure the safety of the children during medical appointment (such as use facial mask and wash your hands before and after the medical visit) in case of sick children, the italian pediatric society recommends to identify those with signs or symptoms compatible with covid- and: schedule different appointments, separated from covid unrelated flu like patients. reducing crowding in waiting rooms, by asking patients to remain outside the medical office until they are called into the room for their appointment. it should be advisable to have just one caregiver with the patient inside the medical room. ask the patient, according to the age, and the caregiver to wear a facial mask and adopt hygienic practices (such as washing hands) discourage admission to hospital in case of mild symptoms like fever or viral symptoms unless severity or no other alternative arrangements can be made educate families and provide information to avoid the spread of infections in case of suspected covid- , healthcare providers should use specific personal and protective equipment rather than a facial mask [ ] . pandemics implicates many worries in the various settings of pediatrics, including primary, secondary and tertiary care. a major problems is that children are possibly less sick than adults, but most children live in large communities not capable to self management, including primary needs. in the latest years, people are searching for health online. a recent survey found out that more than half of the respondents ( %) used the search engine at least weekly to look up medical questions and symptoms and more than % use google as the only source of information on health. the results may have important repercussions on people' health, given that most of the time, "dr google" doesn't provide the right diagnosis [ ] . pediatricians should continue providing routine preventive and other nonemergency care, discussing with caregivers on the benefit of attending a in-person visit, immunizations and screenings to avoid both missing visits and wrong diagnosis trusting on-line information. the different clinical characteristics of corona virus disease cases between children and their families in china -the character of children with covid- epidemiology of covid- among children in china covid- disease severity risk factors for pediatric patients in italy the characteristics of household transmission of covid- household secondary attack rate of covid- and associated determinants. medrxiv guidance on providing pediatric well care during covid- effects of the covid- pandemic on routine pediatric vaccine ordering and administration -united states reluctance to seek pediatric care during the covid- pandemic and the risks of delayed diagnosis key messages and actions for covid- prevention and control in schools covid- -guidance for paediatric services the quality of diagnosis and triage advice provided by free online symptom checkers and apps in australia publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions be planned the study, ra coordinated the study, as and av analyzed the international literature, tm and lg analyzed the italian literature, gc and av were major contributors in writing the statement. all authors read and approved the final manuscript. availability of data and materials at bambino gesù children hospital, dr. bozzola's repository ethics approval and consent to participate not applicable. not applicable. received: july accepted: september key: cord- - war j authors: supino, m.; d'onofrio, a.; luongo, f.; occhipinti, g.; dal co, a. title: world governments should protect their population from covid- pandemic using italy and lombardy as precursor date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: war j the covid- pandemic is spreading worldwide. italy emerged early on as the country with the largest outbreak outside asia. the outbreak in northern italy demonstrates that it is fundamental to contain the virus' spread at a very early stage of diffusion. at later stages, no containment measure, even if strict, can prevent the saturation of the hospitals and of the intensive care units in any country. here we show that it is possible to predict when the intensive care units will saturate, within a few days from the first cases of covid- intensive care patients. using early counts of intensive care patients, we predict the saturation for lombardy, italy. governments should use the italian precursor to control the outbreak of covid- and prevent the saturation of their intensive care units to protect their population. the coronavirus disease (covid- ) is a respiratory infectious disease caused by sars-cov- (also known as -ncov), which originated in wuhan, china, in early december . on january rd , wuhan city shut down public transportation and airways; one week later, wuhan and other cities in the province of hubei, imposed strict social distancing measures (closure of school and non-essential work activities), combined with active search and isolation of infective cases and their contacts; on february th , all non-essential companies and manufacturing plants were closed. on february th , france reported the first death from covid- outside asia, while dozens of countries document cases of infection. on february th , three weeks after the lockdown of wuhan and other cities, cases in china have fallen from an average of , daily cases of two weeks before to cases. in the meanwhile, italy emerged as the country with the largest outbreak outside asia. on march th , italy imposed a lockdown of the whole nation. on march th , the world health organization (who) declared the pandemic state, with more than , cases in countries. on march th , while china announced that the incidence was brought to negligible levels and attempts to prudently restart normal life were all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in deaths, surpassing china. in europe, several countries experienced similar exponential growth of cases as italy, with just a few days of delay (fig. ). spain and france imposed a lockdown on march th and on march th , respectively. on march st , italy imposed a full lockdown of the nation, closing all nonessential companies and manufacturing plants. china was able to control the outbreak of covid- in about two months by implementing strong containments measures, such as lockdown of the population ( ). lockdown can appear as an extreme measure, but it is not. mild restrictions, such closure of schools and partial closure of workplaces, can lower the basic reproduction number of infection (r ) but not below the unit. with mild restrictions, the number of infections would grow at the same rate for a period equal to the incubation time (which is below days for % of covid- infections ( )), and would then grow exponentially at a lower rate. therefore, mild restrictions would slow down the epidemic, but not control it ( ) . in this work, we discuss the necessity and efficacy of lockdown measures for controlling the outbreak of covid- , analyzing data from italy, the country with the largest recorded outbreak of the disease today. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in temporally to superpose one on another, so that for all countries day zero represents the onset of covid- outbreak. japan and singapore were able to contain the outbreak at a the very early stage. the chinese region of hubei was able to contain diffusion at a later stage, by imposing a lockdown of the population on january th (red triangle), and a full lockdown of the population on february th (red diamond), closing all non-essential companies and manufacturing plants. italy, spain, france, uk, and the american states of california and new york (referred as us*) display exponential growth of confirmed cases. italy, spain, france and us* imposed a lockdown at similar (relative) times, indicated by the green, gold, blue and purple triangle, respectively. the four countries have comparable population sizes to hubei region, with a minimum of spain ( . m people) and a maximum of france ( . m). italy additionally imposed a full lockdown days afterwards (green diamond). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in while nhs are prepared to receive a certain number of icu patients distributed during the influenza season, which lasts several months, no nhs can manage an exponentially growing number of covid- patients. to avoid the saturation of the icus, governments need to impose strong containment measures, such as lockdown of the population. acting early is paramount: after containments measures are taken, the number of cases still grows exponentially for at least ten days, due to infections contracted before the measures ( ). the later these containment measures are taken, the stronger these measures need to be to contain diffusion of covid- , and could be anyways insufficient to avoid the catastrophic collapse of the nhs. for example, japan and singapore were able to avoid a lockdown of the population, because the governments implemented a range of measures at a very early stage of the outbreak (fig. ). from the early stage of the covid- outbreak (february th ), italy is providing statistics of the epidemic, through a daily bulletin and an open-access repository ( ). this repository contains daily counts of confirmed cases, hospitalized patients, icu patients, and deceased patients, at the national and regional level (fig. ). this repository represents an important and unique source of information for other countries that the pandemic will reach. the number of icu patients represents a more robust information compared to the number of infected people, which is subject to an under-reporting. the number of infected people strongly depends on the number of performed tests and on the strategy of sampling of the population (e.g. only symptomatic people, random people). testing capability and strategy might largely vary among different countries, while icu patients count is a routine operation performed by all nhss. icu counts are more reliable also compared to deaths counts, since most patients dying with covid- have comorbidities and ascertaining that covid- was the primary cause of death can be complicated. for example, germany reported no cases of death due to sars-cov- until there were all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in covid- outbreak in italy started in codogno, a town of , inhabitants in lombardy region. after ten days from the first icu patient (february, th ), the number of covid- icu patients was all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in ( ) all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in here we show that it is possible to predict the date of saturation of the icus in a region early on, by using the temporal information about the number of available icu beds. we focus on lombardy region. the number of icu patients in the region grew exponentially for the first ten days, starting from february th , and then slowed down as it reached the number of available icu beds. we can predict the date at which the icu beds became saturated by performing a linear regression of the logarithm of the number of icu patients, starting from the first four datapoints (fig. ) . this result shows that monitoring the icus statistics at the beginning of the epidemic allows countries to assess the date of possible saturation of the icu beds early on. monitoring the icus early in the outbreak is paramount: lombardy in italy has one the best nhs in the world ( , ), therefore most countries will face the saturation of their icu beds at earlier stages of the outbreak. it is worth to note that several factors can affect the time to saturate the icu beds. in particular, the saturation time depends on the connectivity of the population: the more people are connected within a region, the faster the infection diffuses ( ) . therefore, the risk of icus saturation is higher for the most developed and connected regions, and lombardy is the most connected region of italy. moreover, if a lock down measure is imposed, the saturation time depends on the incubation time of the disease and on the degree of adherence of the population to the lockdown. here we analyze the effects of the lockdown of the italian population. italy imposed two major containment measures: the lockdown on march th , and a full lockdown on march st , where all non-essential companies and manufacturing plant were closed. these measured helped to avoid the collapse of the whole national health system, yet they could not avoid the saturation of the icu beds in several italian regions (fig. ) . all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the effects of the confinement measures become evident with some delay. specifically, we expect the effect on the number of icu patients to appear within about two weeks (i.e. the maximum incubation time), and on the number of deaths to appear in about three weeks (i.e. the time from infection to death) ( ) . at the current moment, days have passed from the national lockdown, so we expect to see an effect only on the number of icu patients. because of the saturation of the icus in several italian regions, the number of icu patients in italy currently underestimates the number of cases that would require intensive care. therefore, we analyze the data of italy excluding the regions where the icus have saturated. this leaves us with of the regions and excludes about % of the italian population. we find that the recent growth of icu patients is consistent with a linear growth, rather than an exponential growth, suggesting that the lockdown measures have effectively reduced the spread of the infection (fig. ), as it has been for hubei region ( ). specifically, we can obtain a very good fit of the data using an exponential curve (icu patients(t) ∝ exp[r t], t = days), up to five days after the lockdown (equal to the median incubation time of covid- ( )) and a line (icu patients(t) ∝ b t, t = days) for later datapoints. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint the regional number of icu beds reported before the onset of the epidemics. this number has been increased during the epidemic. at the time we write this manuscript, days after the lockdown declaration, it is still not possible to discriminate between an exponential or logistic trend of the number of icu patients in time. therefore, it is not yet possible to offer a good estimate of the number of icu patients to expect in the near future. however, the growth of icu patients is slower than exponential. governments must consider stronger actions than lockdown, including immediate closing of non-essential companies and manufacturing plants (full lockdown), as italy did on march st . in this work, we analyzed the temporal evolution of covid- outbreak in italy up to march th and we discussed the effects of the national lockdown in containing the diffusion. the saturation of the icus in many italian regions suggests that containment measures were taken too late. using italy as a precursor, other countries should impose these confinement measures at earlier stages of the outbreak to be able to protect their population from covid- . we show that countries can predict the date of saturation of their icus early on, as soon as an exponential growth of intensive care patients is observed, as it was in lombardy region. the italian case demonstrates that a national lockdown is effective in reducing the growth of icu patients. the complete saturation of icus, thus the collapse of a nhs, would be a catastrophe that would affect the entire population of a nation. people would die if, for any reason, intensive care would be needed : no matter being positive or negative to covid- , the age, the richness, or the wellness of the patient. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in we strongly encourage any government to accurately share data, including icu patients; these data will significantly help the understanding of present and future evolution of the covid- pandemic. italy has been the first country in europe in which the pandemic outbreak has been observed, and it has adopted a politics of wide dissemination of open data with detailed spatial structure. unprecedented ms analyzed the data, conceived the manuscript and, together with fl, prepared the figures. all the authors contributed to the interpretation of the data and the discussion of the results. ms and adc wrote the manuscript, and all the authors reviewed it. the authors declare no competing interests. the italian covid- data are available through a github repository managed by the dpc (dipartimento della protezione civile -presidenza del consiglio dei ministri): https://github.com/pcm-dpc/covid- for figure , we used world covid- data published by the eu agency for disease prevention and control: all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted march , . . https://doi.org/ . / . . . doi: medrxiv preprint https://www.ecdc.europa.eu/en/publications-data/download-todays-data-geographic-distribution-covid- for figure , we also used the dataset maintained by the center for systems science and engineering evolving epidemiology and impact of non-pharmaceutical interventions on the outbreak of coronavirus disease the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation all rights reserved. no reuse allowed without permission preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted doi: medrxiv preprint and application information-related changes in contact patterns may trigger oscillations in the endemic prevalence of infectious diseases season ): strengths and weaknesses . results of a cohort study in two large italian hospitals measuring health system performance for countries italy: health system review epidemic processes in complex networks incubation period and other epidemiological characteristics of novel coronavirus infections with right truncation: a statistical analysis of publicly available case data this manuscript has been written with the hope that the heroic resistance showed by the city of bergamo, the lombardy region and the whole of italy will not be needed elsewhere.the authors want to thank all the italian nurses, doctors and health-care professionals that are fighting against an invisible enemy as war heroes in dark times. this manuscript is dedicated to the memory of doctor li wenliang, who first tried to warn the world about covid- , and all the healthcare professionals who died fighting against this virus. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in key: cord- -pua ogz authors: coker, eric s.; cavalli, laura; fabrizi, enrico; guastella, gianni; lippo, enrico; parisi, maria laura; pontarollo, nicola; rizzati, massimiliano; varacca, alessandro; vergalli, sergio title: the effects of air pollution on covid- related mortality in northern italy date: - - journal: environ resour econ (dordr) doi: . /s - - - sha: doc_id: cord_uid: pua ogz long-term exposure to ambient air pollutant concentrations is known to cause chronic lung inflammation, a condition that may promote increased severity of covid- syndrome caused by the novel coronavirus (sars-cov- ). in this paper, we empirically investigate the ecologic association between long-term concentrations of area-level fine particulate matter (pm( . )) and excess deaths in the first quarter of in municipalities of northern italy. the study accounts for potentially spatial confounding factors related to urbanization that may have influenced the spreading of sars-cov- and related covid- mortality. our epidemiological analysis uses geographical information (e.g., municipalities) and negative binomial regression to assess whether both ambient pm( . ) concentration and excess mortality have a similar spatial distribution. our analysis suggests a positive association of ambient pm( . ) concentration on excess mortality in northern italy related to the covid- epidemic. our estimates suggest that a one-unit increase in pm( . ) concentration (µg/m( )) is associated with a % ( % confidence interval: – %) increase in covid- related mortality. valley, an extension of flat river lands enclosed between the alps and apennines mountains, which causes the stagnation of pollutants due to low ventilation (giulianelli et al. ) . these factors help to characterize the po valley's peculiarity with respect to different european areas with comparable urban and industrial density levels (eeftens et al. ) . moreover, in addition to the urbanized and industrial areas, the remainder of the valley presents an intensive agricultural activity. local studies on emission sources highlight a varying composition of the final concentration values depending on the position of monitoring stations and with different sources acting as local or diffused ones (for instance having high emissions from traffic close to cities, while having background biomass burning diffused in the whole region) (bigi and ghermandi ; larsen et al. ) . indeed, given the eu ambient air quality directives that sets the air quality standards for the protection of health at μg/m for the averaging period of a calendar year, the po valley shows values consistently near or above the threshold. these values often range in the - μg/m interval with peaks of > μg/m , which in europe are only matched in southern poland and other smaller eastern european clusters (eea ). compared to its overall representation in the population, lombardy is disproportionately impacted by covid- related mortality, with approximately % of italy's covid- deaths as of april , (odone et al. ) . lombardy is also the most impacted italian region as far as the total number of deaths in excess in the first quarter of compared to the same period of the previous years. comparing the official covid- death data with registry deaths, it emerges that the latter is almost % larger than the former in lombardy, % larger in emilia-romagna and % and % in veneto and piemonte, respectively. it is, therefore, imperative to consider the role that pm may have played in such disproportionate covid- deaths in northern italy. there are a number of plausible pathways by which airborne pm may impact covid- related morbidity and mortality. existing data already finds a strong positive correlation between viral respiratory infection incidence and ambient pm concentrations (ciencewicki and jaspers ; sedlmaier et al. ). one plausible pathway for this phenomenon is the fate and transport of the virus itself within the environment. a recent position paper by the italian society of environmental medicine argues that pm may act as both a carrier and substrate of the virus and thus influence the virus' fate and transport in the environment and reaching susceptible receptors (setti et al. ) . another pathway is the increase in susceptibility to covid- mortality caused by long term exposure to pm. fine pm is already known to affect cardiovascular and respiratory morbidity and mortality (cakmak et al. ; jeong et al. ; mcguinn et al. ; yin et al. ) . moreover, among italian covid- patients who died in the hospitals, and for whom it was possible to analyze clinic charts, data showed substantial comorbidities including ischemic heart disease ( . %); atrial fibrillation ( . %); heart failure ( . %); stroke ( . %); hypertension ( . %), and chronic obstructive pulmonary disease ( . %) (istituto superiore di sanità ). biologically, long-term pm exposure may be responsible for a chronic inflammation status that causes the hyper-activation of the immune system and the life-threatening respiratory disorders caused by covid- (shi et al. ) . some preliminary evidence is now emerging about covid- that shows a positive relationship between air pollution and morbidity and mortality. beyond qualitatively describing the european air quality index for northern italy to argue the causal role of air pollution and the relatively high covid- mortality observed in that region, conticini et al. ( ) review the most recent existing toxicological and epidemiological literature. based on existing evidence from other empirical studies, they clarify the relationship between air pollution, prolonged inflammation and immune system hyper-activation and immune suppression, and the link between the latter and acute respiratory distress syndrome, and respiratory mortality. their paper is important in that it suggests a clinical and biologically plausible explanation to our analysis, but does not provide statistical evidence in support of the hypothesis. a separate empirical analysis by becchetti et al. ( ) finds preliminary evidence that confirms such a positive effect of air pollution on mortality in italy based on the analysis of death data at the province level. similarly, wu et al. ( ) show a positive association between long term pm exposure and covid- related deaths in us counties. ogen ( ) recently analysed data from administrative regions in france, spain, italy, and germany, and found that the highest covid- deaths in these regions were associated with five regions of northern italy that also corresponded with the highest levels of atmospheric nitrogen dioxide (no ). cole et al. ( ) estimate the same relationship using netherlands municipality data and find pm . positively associated with covid- cases, hospitalization, and deaths. in this paper, we follow this emerging stream of the empirical literature and test the hypothesis that a higher average long-term exposure to pm . is positively associated with the current extraordinarily high death toll in northern italy. we decided to focus on pm . because, given the complexity of air pollution, it is quite common in air pollution epidemiology studies to focus the analysis on a single pollutant (wu et al. ) , although multipollutant analyses are certainly warranted. we selected pm . for a variety of important reasons, including policy implications and evidence in the literature in terms of chronic health effects. regarding its policy implications, we selected pm . as opposed to pm because the former is more correlated with human activities than the latter, and it correlates with stronger health effects than pm does. with respect to respiratory mortality effects from the existing air pollution literature, the most robust evidence points to pm . as opposed to other gaseous air pollutants (bowe et al. ) . mortality data are collected at the municipality level for the period january-april . given that mortality data are not disaggregated by mortality cause, death counts are measured as the difference from the last five-years mean to reflect the abnormal number of deaths caused by the spreading of the pandemic. since pm . can be associated to generic mortality even in the absence of the pandemic outbreak (dominici et al. ; katsouyanni et al. ; samet et al. ) , we also estimate the impact of pm . on the excess mortality in the sample using data, a time in which the coronavirus epidemic had presumably not yet begun. data on pm . concentration at the municipality level refer to the years prior to account for long-term population exposure. we assign municipality pm . concentration by a set of different methods of spatial interpolation (kriging) of monitoring station data related to the years - . we estimate a negative binomial model of excessive deaths on historical pm . concentrations and a series of control variables that may plausibly affect both pm . concentration and mortality, including population density; the spatial concentration of the industrial manufacturing sites; climatic conditions observed during the first quarter of ; and the demographic composition of the municipal population among others. in addition, we consider spatial heterogeneity in the distribution of the number of deaths related to regional and local unobservable factors. we account for region-specific effects because regions, in italy, are the administrative units in charge of managing the health systems and the measures taken to trace and contrast the spreading of the pandemic varied greatly among even contiguous regions. we also account for local effects common to functionally linked clusters of municipalities (the local labour systems-lls). we deem this part of the identification strategy crucial because the relationship between pm . and covid- mortality may be confounded by several other factors, some of which were not observable or measurable, but are nevertheless intrinsically related to the geographical location of the observed units. the remainder of the paper is organized as follows. the next section introduces the empirical strategy and describes the dataset. the results are presented and discussed in section three, considering the total number of (excess) deaths. section four draws the conclusions and highlights the limitation of the study and the indications for future research. our analysis is restricted to the study area of northern italy (fig. ) , which encompasses the sub-regions of valle d'aosta, piemonte, liguria, lombardia, emilia-romagna, veneto, friuli-venezia giulia and trentino-alto adige/südtirol. official territorial data on covid- mortality in italy are available at the rather aggregate regional or provincial level, corresponding to the levels and , respectively of the european nomenclature units for territorial statistics (nuts). in addition, these official data refer to the deaths of patients tested positive for severe acute respiratory syndrome coronavirus (sars-cov ) only and do not include (potential) patients without covid- diagnosis because they were not tested and died at home or elsewhere. hence, the officially reported deaths are likely underestimated. because testing policies vary among regions in italy, the induced measurement error is also non-randomly distributed among the provinces. ciminelli and garciamandicó ( ) compare the official covid- fatality rates with historical death data and working under the assumption that covid- deaths are underestimated in italy, the choice is made in this paper to use the total deaths from the official registries, accordingly, and to scale the analysis at the municipality level, the smallest administrative units, to have a more granular representation of the spatial dimension of the phenomenon. since we are interested in excess deaths, we take the difference between the number of deaths in the period january -april , , and the average number of deaths in the same period of the previous years (exdeaths) and use this metric as the dependent variable in our statistical model. figure displays the geographical distribution of the above-described data among the municipalities for which data is available. the variable is assumed to follow a negative binomial distribution, a generalization of the poisson distribution that avoids the restrictive mean-variance equality of the latter, and is modelled as follows: where is the overdispersion parameter to be estimated and i is the municipality-specific expectation conditional on the value of the covariates. among the covariates, pm is the concentration of fine particulate matter in municipality i and is the associated parameter, which we expect positive and statistically different from zero; x is a vector of control variables that adjusts for the potential confounding effects and includes the (log of) total population as the offset while is a normally-distributed error term. our main source of pm . data is the european environmental agency's (eea) air monitoring database, which is provided to eea by the institute for environmental protection and research (ispra). ispra conducts ground-level air measurements of pm . air concentrations (µg/m ) collected at monitoring sites throughout italy. specifically, we use the eea's e a and e a datasets, which are primary validated assessment data and primary up-to-date assessment data reported by the european member states, respectively. although the measurements come both in hourly and daily averaging formats, we work ( ) does not include a time component, we further compute a six-year averaging time to obtain a metric of long-term (chronic) pm . concentration levels throughout different spatial units of northern italy. the number of years for the reference period is sufficiently long to account for long-term exposure while being not too long to be affected by the mobility of people among municipalities, and it is in line with existing literature assessing long-term effects of pm exposure (yorifuji et al. ). since the air monitoring stations provide only partial spatial coverage for municipality-level pm . concentration data, we impute missing observations using a spatial interpolation model. specifically, we fill in the gaps using a mean stationary ordinary kriging (see bivand et al. , p ) defined through an exponential covariance function with nugget, partial sill and range parameters estimated through (restricted) maximum likelihood methods. figure displays the resulting pm . concentration data. comparing figs. and , it is possible to visually appreciate a spatial coincidence between higher levels of excess mortality and higher levels of pm . , in particular in the lombardia region which notably is the region with both the highest particulate concentration and the highest number of excess mortality. the hypothesis that pm . concentration affected covid deaths, that is ̂> , is tested among several possible specifications. in model ( ) we include regional effects j . these effects are expected to capture the aspects related to the management of the outbreak, which may have systematically influenced covid- mortality and that are common to all the municipalities in the same region. italy has a national health system that ensures equal access to healthcare to all citizens. the system is managed by regions at the local level, and, in the specific case of this pandemic, regions were responsible for defining the testing and contact-tracing protocols and implementing the necessary measures to contain the outbreak, among which the measure to protect healthcare workers. in model ( ), we include lls-specific effects e k . lls are spatial clusters of contiguous municipalities related by commuting flows that share a common specialization in a specific sector of manufacturing production and correspond to the conceptualization of marshallian districts (becattini ) . the number of lls clusters per-region and the total number of municipalities belonging to clusters are reported in table , along with the minimum, maximum, and average cluster size. the use of lls captures the interlinkages within neighbouring municipalities that may have favoured the geographical spreading of coronavirus around specific hotspots. mortality data are then expected to vary among municipalities in different lls, but differences are expected to be non-systematic in this case. in model ( ) we include both the regional fixed effects and the lls random effects. control variables to be included in the model were chosen to avoid any potential spatial confounding effect and considering as well the emerging literature on the impact of pm on covid- related deaths (cole et al. ; wu et al. ). the population density and per-capita income account for urbanisation level. the most densely populated and wealthy municipalities are among the most polluted due to the spatial concentration of manufacturing and service activities but are also the places where the contagion could have been easier, with a potential impact on mortality. in addition to the density of population, the shares of municipality area occupied by industrial sites and the average size of manufacturing firms are included in the regression because they are related to pollutant concentration and possibly to mortality. national measures to stop the spreading of the viral infection (lockdown) involved the service sector to the largest extent while many manufacturing activities, being considered necessary, were left open and, in the absence of social distance and individual protection measures, the geographical concentration of these activities in a municipality with their complex logistics and transport interconnections, and the size of plants, may have influenced mortality. average temperature, for which an association with covid- deaths has also been found , is also included in the regression. moreover, covid- incidence has proven to be higher among men than women and people aged or more. hence these two variables are considered in the model, even though these aspects are not necessarily connected with the average pm . exposure in a municipality. underlying socioeconomic conditions can also play a role in covid- related mortality (goutte et al. ) . brandt et al. ( ) and mukherji (n.d.) have shown that, in the us, covid- is more threatening for ethnic minorities, and we believe that the share of migrants, identified as non-eu citizens, can control for this aspect influencing the observed excess mortality. on the other hand, mukherji ( ) and goutte et al. ( ) also find that places with a higher share of the population with a low level of education have higher deaths. in our paper, given the lack of updated data on education at the municipal level, we proxy it with the percentage of university students on the total population. the distance from the closest airport is a proxy for the functional and relational linkage between a municipality and a place of highly frequent national and international connections and potential sources of coronavirus spreading. finally, we consider the number of hospital beds as a proxy for the supply of health services to account for the fact that many people died at home without being diagnosed for coronavirus due to the shortage of beds in public structures. the full details of the variables in the model, including sources and summary statistics, are presented in table . having accounted for the confounding effect due to the omission of relevant information from the empirical specification, we exclude any other potential source of endogeneity considered in similar papers. in particular, we exclude endogeneity due to measurement error in the outcome variable and the main independent variable. concerning the outcome variable, the relationship between deaths and cases with fine pm could be spurious because more cases could be registered, and more individuals tested in highly polluted areas as people there are more likely to show covid- symptoms due to the chronic inflammation induced by pm. the high toll of deaths of people diagnosed with covid- would be a natural consequence of that. in contrast, the number of deaths in excess, used in this paper, is not affected by testing problems since it considers all the potential covid- deaths. concerning the pm variable, measurement errors are likely to occur when using satellite data or modelled data. we preferred to use pm . levels observed from monitoring stations to avoid such a measurement error. some caution is needed in the spatial interpolation because the method chosen to fill the missing data may underestimate the value in locations farther from the monitoring stations. with this concern in mind, we test the robustness of our results using pm . data obtained from different interpolation approaches. as indicated in table , the overall average of pm . for the study area between and is roughly µg/m , as most municipalities in norther italian regions belong to industrial and agricultural intensive locations. the average mortality between and for the period of interest (january -april ) was deaths, while it grew to in . that results in an average excess death of , with standard deviation four times as larger (see table ). estimation results from the negative binomial models are summarised in table for the four different specifications of the model ( -no geographical effects; -regional fixed effects; -lls random effects; -regional fixed effects and lls random effects). in the lower part of the table, the estimated overdispersion parameter, the akaike information criterion (aic), and the moran's test for the null hypothesis of absence of spatial autocorrelation in the residuals (moran ) are reported. the four specifications provide consistent results in terms of the direction and significance of pm . coefficients. the overall effect of pm . on covid- -related excess mortality is positive and statistically significant in all models. the estimated incidence rate ratios, reported in table with their confidence interval, for model , , and are . %, . %, . %, and . %, respectively. in model , the regional fixed effects coefficients are statistically significant. they indicate that other things being equal, the number of deaths in municipalities in lombardy and emilia romagna has been systematically higher compared to base category and in municipalities in veneto it has been systematically lower. the significance of the coefficient for emilia romagna, however, drops after including the random effects in the model. since the first three models are nested into model it is also possible to compare the models in terms of aic. model performs substantially better than the other three. in general, the inclusion of re in models and leads to a decrease in the value of the aic. in models and the residuals appear spatially autocorrelated, as the null hypothesis of no spatial autocorrelation is rejected in both cases (p < . ). the introduction of the lls random effects appears to solve the issue of autocorrelation. based on the estimates of model , we compute the expected value of excess deaths conditional on covariates (taken at the average level) in the average city for varying levels of pm . and show how the expected number of deaths by region varies at different concentration levels in fig. . notably, emilia-romagna and liguria are the regions in which a a reduction of average fine pm from the highest level to the lowest would have benefited the most. for robustness check of the pm . metric used in our study, we explored the influence that other alternate pm . metrics may have on the direction and magnitude of the observed associations. figure depicts the point estimates and the % confidence interval for table estimation result of main regressions, dependent variable: excess deaths during the period january -april , municipalities in northern italy ***p < . , **p < . , *p < . model ( ) model ( ) model ( ) model ( we find that while data from satellite elaborations (modis and dimaq ), and monitoring stations' interpolation eea pm . models result in irrs trending in the same direction, the point estimates for irrs are lower than our primary analysis which was based on a combination of ground monitoring and kriging. the lower irr point estimates are unsurprising because the underlying data for the alternate pm . metrics do not have the same temporal coverage as the ground-level monitoring data ( ) ( ) ( ) ( ) ( ) . this lack of temporal coverage contributes to non-differential exposure misclassification, which, in turn, would lead to suppressing effect estimates towards the null. despite this, it is encouraging to find that regardless of the pm . metric used, the direction of the observed associations remains, and so does statistical significance. as previously anticipated, we re-estimate model ( ) using different specifications of the kriging interpolator. in particular, we first relax the mean-stationarity assumption of ordinary kriging by modelling the mean function of the process through both a linear and a quadratic trend in latitude and longitude. next, we replace the simple exponential function with a spherical model and a more flexible matérn kernel with the characteristic parameter set at / (to preserve mean-square differentiability). all these specifications still assume covariance stationarity. figure and table in the appendix report the estimated incidence rate ratios (irr) regression coefficients for the pm variable in model ( ) under these multiple setups: both point estimates and % confidence intervals indicate that there are no substantive differences between using different trend or covariance models, indicating that our result is robust to alternative specification of the interpolation method. in each of the four specifications presented, the coefficient related to pm . is always of the hypothesized direction and statistically different from zero. precisely and consistently with previous results for the original sars-coronavirus during the outbreak (cui et al. ) , an increase in air pollution exposure is associated with increased mortality for covid- . the first panel in fig. , as well as table , suggests that, when using interpolated data from ispra monitoring stations, the increase in mortality rate due to a one-unit increase in pm . concentration varies between % (model -highest rate) and nearly % (model -lowest rate). the % confidence interval for the point estimate in model lies between roughly % and %. our findings fall in line with both wu et al. ( ) and cole et al. ( ) papers. specifically, both papers find a positive ecological relationship between pm . and covid- mortality. in relation to a µg/m increase in pm . , wu et al. find % change in covid- mortality, cole et al. find the same increase associated to additional covid- deaths (almost % if compared to their sample mean), and our paper finds % increase in covid- related excess mortality. despite this similarity in results, the two key differences between our study and the others relate to the exposure assessment method and the outcome assessment method. in our study we use a spatial interpolation method (kriging) from ground-level monitoring data, whereas these other two studies utilize pm . gridded surfaces such as chemical transport modelling in the case of cole et al. and a hybrid approach using chemical transport, aerosol optical depth and land use regression modelling in the case of wu et al. with respect to covid- mortality data, wu et al. use county-level data from the johns hopkins university, center for systems science and engineering coronavirus resource center, which is comprised of covid- deaths tabulated by the us centers for disease control and prevention and state health departments. in cole et al., researchers obtained covid- deaths by residential address and aggregated these to the municipality level. the obvious difference between their study and ours is that we used a surrogate excess mortality measure due to the issues of reliability for covid- death data, as we have already discussed. the other relevant difference between our study and the wu et al. and cole et al. studies is that we subsample the total cohort of italian municipalities to only regions with a very high mortality rate, which are also the regions most affected by the air quality problems. on the other hand, when satellite data are used, our estimate yields lower incidence ratios. although ground-level concentration metrics come with fewer measurement errors, satellite data proves nevertheless useful in corroborating both the direction and the significance of the effect of interest. this redundancy is particularly relevant in light of the relatively few stations capable of detecting the finest particulate. with reference to model ( ) and the remaining covariates, we observe no effect related to population density or income or the extent of industrial areas in the municipality. likewise, there is no evidence suggesting significant links between the share of non-eu residents, the female to male ratio (which disappears after we incorporate the random effects), and the level of education (proxy by the percentage of university students) on the dependent variable of interest. on the other hand, our results suggest a negative association between temperatures and mortality due to covid- . finally, as expected, we find that municipalities with higher shares of the population aged or more have been most affected. the distance from the closest airport, a measure of relational connectedness that also proxy for the exposure to the contagion process, deserves a last comment. we find that municipalities closer to an airport experience a higher number of deaths in excess. we speculate that the result could be related to a higher likelihood for these municipalities to become clusters of contagion in the initial phase of the pandemic, but a causal link cannot be inferred based on our result ad the topic needs more research to be addressed adequately. we conclude our analysis by checking the consistency of our results to different choices of the dependent variable. existing evidence (dominici et al. ; pascal et al. ; samet et al. ; yin et al. ) associates fine pm to severe cardiovascular and respiratory diseases and mortality. in european cities, in particular, an estimated increase in the number of daily deaths of . % is associated with an increase of µg/m of pm (katsouyanni et al. ) . this evidence suggests that long term pm exposure may have had an overall effect on deaths even before the outbreak in the sample municipalities, making it more difficult to isolate the real effect on covid- deaths. we thus run model ( ) using the total number of deaths in the same observation period of as the dependent variable to understand whether the effect of fine pm . on mortality has been more severe during the pandemic. we find no evidence of an effect of pm . on total deaths for in the sampled municipalities, suggesting that the effect of pm exposure on the mortality rate is closely connected to the novel coronavirus outbreak (see table in the appendix). however, since the dependent variable in this "placebo" regression cannot be directly compared to the excess mortality, we repeat the test using total mortality for the year . although the latter includes both covid- related and unrelated deaths, these two variables represent data generating processes of the same nature. as expected, both the regression coefficients and irrs calculated regressing total deaths in suggest a positive and statistically significant effect of exposure to fine particulate on mortality, even though its magnitude is greatly reduced if compared to the estimates in tables and (see table in the appendix). presumably, the effect of pm . concentration on covid- related mortality becomes muted by the noise introduced when accounting for other causes of death. this would also explain the non-significant pm . coefficient in the first "placebo" regression. italy is among the countries most severely affected by the new coronavirus, with more than thousand confirmed cases and more than thousand deaths as of the end of may. yet, the spatial distribution of confirmed cases and deaths suggest that the effects of the viral infection spreading largely vary across the regions of the country but also within regions. in this work, we examined the role of ambient pm . in explaining the spatial variation in deaths that occurred throughout the most extreme time period of the epidemic. the results in the paper, that suggest a positive relationship between pm . concentration and covid- related excess mortality, are robust to different specifications pm . and estimation strategies, even after controlling for additional confounder factors. coherently with previous findings in the literature, we highlight a strong positive correlation between viral respiratory infection incidence and ambient pm . concentrations and the increase in susceptibility to covid- mortality caused by long term exposure to pm . , consistent with evidence for the original sars-coronavirus during the outbreak. in fact, fine pm is already known to affect cardiovascular and respiratory morbidity and mortality. however, we are aware that the phenomenon and the cause and effect relationships are very complex and that our work can only address part of the problem. the cross-sectional nature of the dataset and the use of geographically aggregated information in the epidemiological model does not allow concluding a causal effect exists. in our opinion, the robust evidence in the paper shows that the relationship between pm . and covid- related excess deaths goes far beyond a simple geographical correlation, and further research is needed to explore the causal effect more in depth, when reliable time series data are available. in fact, our paper does not deal with the spread of contagion and the dynamics linked to it, also because, as we underlined, such analysis would require time-series data, a different econometric methodology, and the identification of the exogenous coronavirus insurgence in northern italy. to the latter purpose, the spread of the pandemic incorporates two different dynamics: ( ) on the one hand, the dynamics of the spread of the contagion requires further information to be investigated such as its genesis, the type of virus, and setting of the first outbreak; ( ) the effects of the lockdown changed (or partially blocked) the contagion in an asymmetric way. in addition to this, of course, there are other elements that should be investigated, such as additional variables about health data, mobility, and so forth. our results reinforce the need to adopt environmental policies that would not only reduce the impact of pollution on the health of citizens and workers but would contribute to smooth the negative effects of a (future) pandemics, avoiding collapses of health systems. indeed, recent studies show that in addition to chronic lung inflammation, environmental air pollutant concentrations can exacerbate the effects of increasingly frequent one-shot systemic shocks, which in turn are also caused by environmental factors. in this regard, sustainable and decarbonization policies such as the green new deal, conceived as long-term policies, should be accelerated. td-i (b) for the updated sample (column ) and td-i (c) for the latter release. it turned out that the number of istat excess deaths increased by % with this revision (compare column and column ). it further increased by % on april (compare column and column ) due to corrections, enlargement and new cases. d-pc stands for number of deaths with or from covid- reported by the national department protezione civile italiana (italian civil protection, which is a department of the presidency of the italian council of ministers) over january -march , (column ) and january -april , in column . the official d-pc data are available only at the regional level and they are officially released by the health departments of the regional administrations. in the period march and april , pc registered a % increase in covid- deaths. istat and pc department therefore collect data independently. the key difference between ed-i and d-pc lies in how pc recognizes fatalities as covid- related: only patients known to have tested positive to sars-cov- got registered under this nomenclature. on the other hand, ed-i is just a mathematical construct that takes into account all deaths in a given municipality, regardless the cause. due to difficulties in providing timely screenings and accurate testing during the peak of the pandemics, it is likely that official figures from pc might have been underestimated between early january and mid-april. this is particularly true for the most affected areas. consequently, the discrepancies between ed-i and d-pc can be either moderate or strong, as we observe for lombardy (where ed-i is roughly % higher than d-pc) and trentino alto-adige. last, the unlikely event of ed-i lying below d-pc is due the fact that istat initially collected figures for only a subsample of municipalities (i.e. those recording a percentage of excess deaths in greater than %) leaving out quite a lot of statistical units (for example, friuli-venezia giulia, which had initially the biggest discrepancy (ratio , ) reported data for a very small number of municipalities to istat). however, with some delay, istat has been upgrading the sample, such that in june it covered about all municipalities. in our paper, we use the updated data ed-i j- a (column ), disaggregated at the municipality level. column ( ) shows that the ratio of total ed-i and d-pc is about . , with mean ratio equal to . and standard deviation equal to . . underestimation of cases in the initial sampling base concerned all northern italian regions except emilia romagna, lombardy and veneto (the biggest regions in terms of population and covid- cases). the ratio of total ed-i (b) and d-pc (b) in column ( ) is about . , with a mean ratio equal to . and standard deviation . . one month later, the total ratio decreases to . column ( )-with mean ratio . and standard deviation . . in the latter case, a slight underestimation of the registered cases concerns only fvg and valle d'aosta. in any case, since lombardy has the biggest discrepancy on april (ratio , ) we also run our regressions excluding lombardy (i.e. taking all lombardy municipalities off) and the results are robust, reporting a significant higher-than- relative risk ratio for the exposure variable. the latter results are available upon request. from marshall's to the italian "industrial districts". a brief critical reconstruction understanding the heterogeneity of adverse covid- outcomes : the role of poor quality of air and lockdown decisions trends and variability of atmospheric pm . and pm - . concentration in the po valley, italy applied spatial data analysis with r burden of cause-specific mortality associated with pm . air pollution in the united states air pollution, racial disparities, and covid- mortality associations between long-term pm . and ozone exposure and mortality in the canadian census health and environment cohort (canchec), by spatial synoptic classification zone air pollution exposure, cause-specific deaths and hospitalizations in a highly polluted italian region air pollution and respiratory viral infection covid- in italy: an analysis of death registry data covid- 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comparison of receptor and chemistry-transport modelling results long-term concentrations of fine particulate matter and impact on human health in fine particulate air pollution and mortality in u.s. cities, - generation of avian influenza virus (aiv) contaminated fecal fine particulate matter (pm . ): genome and infectivity detection and calculation of immission evaluation of the potential relationship between particulate matter (pm) pollution and covid- infection spread in italy. sima position paper data integration model for air quality: a hierarchical approach to the global estimation of exposures to ambient air pollution covid- infection: the perspectives on immune responses global estimates of fine particulate matter using a combined geophysical-statistical method with information from satellites exposure to air pollution and covid- mortality in the united states long-term fine particulate matter exposure and nonaccidental and cause-specific mortality in a large national cohort of chinese men long-term exposure to fine particulate matter and natural-cause and cause-specific mortality in japan acknowledgements open access funding provided by università cattolica del sacro cuore within the see table . see table . see table . data comparison from different official sources. table reports the excess deaths data as reported by the italian bureau of statistics (istat) along with the official covid statistics as indicated by protezione civile italiana (pc). ed-i stands for excess deaths reported by istat, calculated as the sum of deaths (from all causes) between january and march (column ) or april . (column ) with respect to the average value in - (same months). the difference between column and column involves the sampling base. in fact, as long as istat was upgrading the deaths data, it was both enlarging the sample of municipalities and correcting the past figures; see column to : td-i (a) stands for total deaths reported by istat as the sum of total deaths from january to march , in the initial sample (column ), table estimation results for the placebo regression, dependent variable: total number of deaths during the period jan -april , municipalities in northern italy ***p < . , **p < . , *p < . model ( ) model ( ) model ( ) model ( ) estimate publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - opy z h authors: morelli, mara; cattelino, elena; baiocco, roberto; trumello, carmen; babore, alessandra; candelori, carla; chirumbolo, antonio title: parents and children during the covid- lockdown: the influence of parenting distress and parenting self-efficacy on children’s emotional well-being date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: opy z h on march , , italy went into lockdown due to the coronavirus disease- (covid- ) pandemic. the world health organization highlighted how the lockdown had negative consequences on psychological well-being, especially for children. the present study aimed to investigate parental correlates of children’s emotion regulation during the covid- lockdown. within the social cognitive theory framework, a path model in which parenting self-efficacy and parental regulatory emotional self-efficacy mediated the relationship between parents’ psychological distress and both children’s emotional regulation, and children’s lability/negativity, was investigated. a total of parents of children aged from to years completed an online survey that assessed their psychological distress, regulatory emotional self-efficacy, and parenting self-efficacy. parents reported also children’s emotional regulation and lability/negativity. a structural equation model (sem) using mplus . was tested. results showed that the hypothesized model exhibited excellent fit, chi-square ( ) = . , p < . , rmsea = . , cfi = . , tli = . , srmr = . . the influences of parents’ psychological distress and parents’ regulatory emotional self-efficacy on children’s emotional regulation and lability/negativity were mediated by parenting self-efficacy. the mediation model was invariant across children’s biological sex and age, and geographical residence area (high risk vs. low risk for covid- ). results suggested how parents’ beliefs to be competent in managing parental tasks might be a protective factor for their children’s emotional well-being. implications for intervention programs are discussed. the spread of the pandemic covid- in italy from february and the subsequent health emergency led to several restrictive measures. schools and universities have been closed at the end of february, and starting from march th, , a quarantine measure became necessary leading to a general closure of almost all public businesses and work activities. children and families have been deprived of their educational, work, and sport activities, but also from all their friendship and relational contacts. suddenly parents had to manage their children at home from school h a day and, at the same time, most of them had to start smart-working from home, still carrying out their children's school commitments. many parents also had to manage difficulties and pain related to having sick or dead relatives, having had wages reductions, or in some cases, having lost their work. it is easy to understand how italian families have been exposed to a very strong emotional and psychological stress. this situation had relevant repercussions on daily life of families, especially of children that have been deprived of their socialization and play spaces. the parents suddenly became the only point of reference for their children since the other references and educational figures were no longer available. as enlightened by the world health organization (who, a,b) , this situation could have long-term negative consequences on psychological well-being and there is a need to invest in mental health services and other services. a recent review stressed how people all over the world can show many different psychological disorder symptoms related to the pandemic (shahyad and mohammadi, ) . the who ( a) highlighted that children were also showing signs of mental illness. in fact, both international and domestic studies showed that, during the lockdown, children exhibited several problems, such as anxiety and emotional and behavioral disorders (jiao et al., ; spinelli et al., ; xie et al., ) . the european pediatric association-union of national european pediatric societies and associations (epaunepsa; jiao et al., ) has stressed the importance to address children's psychological needs during the pandemic due to the negative repercussions on their psychological well-being, highlighting the relevant protective role of parents in decreasing their fear and stress. research on psychological consequences of traumatic events, such as the terroristic attack of september , , showed how children can also experience long-term effects on psychological well-being, reporting mental disorders after months (hoven et al., ) . all these data highlight the importance of not underestimating the psychological risks that children and their families could face. in a report on may th, , the united nations also underlined how, during the covid- pandemic, the emotional problems of children and adolescents were exacerbated by family stress, social isolation, interruption of school and educational activity, and uncertainty for the future which occurred in critical moments of their emotional development (united nations, ) . understanding how to strengthen parents and families in this situation, with the aim to protect children, represents an important goal that researchers should have in this period because it is possible that other future pandemics will affect humanity (cluver et al., ) . the limited research conducted to date on the effects of the covid- pandemic on well-being of parents and their children both in china and in italy showed that covid- related risks, such as (a) living in a red zone (i.e., a high-risk zone like lombardia and veneto for italy), (b) being a parent positive to sars-cov- , (c) having relatives or friends positive to the sars-cov- or who died from the sars-cov- , and (d) living in a high-risk environment (i.e., not having an open space in the home during the lockdown, losing a job during the pandemic, having a low income, not having internet connection), did not have strong negative direct effects on families' well-being (spinelli et al., ) or on children's symptoms and problematic behaviors per se (jiao et al., ) . actually, the research conducted by spinelli et al. ( ) in italy showed that it was the parenting stress related to the health emergency, the pandemic, and the lockdown that increased children's psychological, emotional, and behavioral problems. in line with these findings, wang et al. ( ) suggested the need to deeply understand the family functioning and processes that can promote children's psychological wellbeing during the pandemic. for this reason, this study focused on identifying which parental psychological variables can mediate the relationship between parents' psychological distress during the pandemic and the lockdown and their children's emotional regulation, in order to understand which possible intervention should be implemented to ameliorate families' well-being. two recent meta-analyses highlighted the relevant role that the parent-child relationship can have in promoting children's effortful self-regulation (pallini et al., ) and in decreasing children's behavioral problems, specifically attention problems (pallini et al., ) . the stress of quarantine can affect psychological well-being of adults, as confirmed in a recent review (brooks et al., ) , and might also have long-term effects (liu et al., ) . a study conducted on parents and children quarantined in during the h n influenza showed that the high-stressful isolation increased parents' psychological distress that in turn had an impact on their children's well-being (sprang and silman, ) . children who have parents with high levels of stress showed more externalizing problems and developed less emotion regulation (deater-deckard and panneton, ) . as reported by leary and hoyle ( ) , psychological distress upsets the ability to self-regulate (tillema et al., ; scott and cervone, ) but regulatory emotional self-efficacy is crucial in the self-regulation of relationships and behavior (bandura et al., ) . according to bandura ( ) , psychological distress, such as lack of social support or parental depression, can affect parenting self-efficacy, which is the belief that parents have to be able to manage their parental tasks successfully and that it is, in turn, related to children's adjustment (jones and prinz, ) . some previous studies showed that the relationship between parental mental health and children's emotional and behavioral well-being is mediated by positive parenting strategies (giallo et al., ) . according to eisenèberg et al. ( ) , parents' positivity and warmth can promote effortful control in children, reducing their externalizing behaviors. likewise, self-efficacy, specifically parenting self-efficacy, can function as a mediator between environmental variables or psychological conditions related to an external situation (e.g., the stress related to the pandemic) and parenting competence. in fact, environmental aspects might also indirectly affect parents' belief to be competent in managing parental tasks, and this could lead to less psychological well-being of the children (jones and prinz, ) . for this reason, it is important that parents have a good parenting self-efficacy in order to display positive parenting strategies that can foster adaptive functioning and emotion regulation in children (stack et al., ) . within the theoretical framework of social cognitive theory (bandura, ) , the present study aimed to investigate a path model in which parenting self-efficacy and parents' regulatory emotional self-efficacy (related to covid- lockdown) mediated the relationship between parents' psychological distress and both children's emotional regulation, and children's lability/negativity, in line with a previous study that stressed how parenting self-efficacy can mediate the relationship between parents' psychological distress and children's adjustment (giallo et al., ) . additionally, in our model parents' psychological distress was also predicted by being exposed to several risks related to covid- quarantine and the pandemic. moreover, the second aim was to assess whether children's biological sex, children's age, and geographical area (northern italy, which is the most at-risk area for the spread of the pandemic and for the risk of contagion, vs. the rest of italy) moderated the structural paths of the model. there is evidence that children's biological sex can affect parents' way to respond to children (sanders and morawska, ) and that parenting selfefficacy can change over time (deater-deckard and panneton, ) , growing during early childhood (weaver et al., ) , and decreasing when children become adolescents (glatz and buchanan, ) . conversely, we did not expect to find any differences regarding living (or not living) in a high at-risk zone for the covid- (i.e., northern italy), as found by recent italian and chinese studies (jiao et al., ; spinelli et al., ) . the present study was conducted in italy, via an online survey, during april when there was a lockdown related to the health emergency due to the covid- pandemic. specifically, italian parents with a child aged between and years were recruited through a snowball sampling procedure to complete the online survey. at the time of data collection, italy had been in quarantine for more than month. a link to the survey was shared among parents using different social networks (e.g., facebook, whatsapp), also asking parents to share the link among their contacts. overall, parents have had access to the survey, and a total of valid questionnaires were used in the present investigation, yielding a response rate of . %. parents' age ranged from to years old (m age = . , sd age = . ) and the recruited sample mostly consisted of mothers (n = ; . %). as abovementioned, children were aged between and years (m age = . , sd age = . ) and were almost equally distributed for biological sex ( % were boys and % were girls). parents' socioeconomic status (ses) was predominantly medium-high ( . %; n = ). . % (n = ) lived alone at home with children during the quarantine. % (n = ) of parents were from northern italy, the italian area most affected by the pandemic, and . % (n = ) were from central and southern italy which were areas less affected by the pandemic (although they were also put in lockdown). regarding their work situation, parents ( . %) continued to work and earn as before the quarantine, while parents ( . %) lost their works or have had wage reductions or layoffs. . % (n = ) were health workers and . % (n = ) were health workers in a hospital department that treated sars-cov- -positive patients. . % of parents (n = ) did not have any relative tested positive for the sars-cov- , and . % (n = ) had at least one relative that tested positive for the sars-cov- . . % (n = ) have not-hospitalized relatives due to sars-cov- , and . % (n = ) have at least one hospitalized relative. finally, . % of parents (n = ) did not have any acquaintance or a loved one that tested positive to sars-cov- , and . % (n = ) had at least one acquaintance or a loved one that tested positive for sars-cov- . each parent gave his/her consent by clicking "yes, i accept to participate in the study" on the first page of the survey. this study was approved by the ethics committee of sapienza university of rome, department of developmental and social psychology, protocol number: , april , . two power analyses were conducted to determine the recommended minimum sample size: ( ) for detecting a significant bivariate effect and ( ) for conducting a structural equation model (sem; cohen, ) . a moderate effect size of . was anticipated with a power level set at . and a significant alpha level set at . . the minimum sample size necessary to detect a significant bivariate effect was n = . regarding the sem, with five latent and fifteen observed variables, using the software developed by soper ( ) , results indicated that the required minimum sample size to run a sem and detect a significant effect was n = . using a similar procedure as used by spinelli et al. ( ) , we created an ad hoc index that assessed risks related to the covid- pandemic. specifically, a composite index was created given one point for each of the following risk factors, if present: (a) relatives that tested positive for sars-cov- , (b) friends or acquaintances that tested positive for sars-cov- , (c) hospitalized relatives due to sars-cov- , (d) living in northern italy, which was the most at-risk area for the spread of the pandemic and for the risk of contagion, (e) being a health worker, and (f) being a health worker in hospital departments that treated sars-cov- positive patients. again, using a similar procedure as used by spinelli et al. ( ) , we created an ad hoc index that assessed risks related to family situation during the quarantine and the pandemic. specifically, a composite index was created given one point for each of the following risk factors if present: (a) a lower ses, (b) a worsened working situation during the quarantine, and (c) being a single or divorced parent who had to manage her/his own children at home alone during the quarantine. both the family risk index and the covid risk index are intended as summative rating scales that were created ad hoc for this research. parents' psychological distress during the lockdown was evaluated using the perceived stress scale (cohen et al., ; italian validation by mondo et al., ) . parents were asked to think about the last month. the scale is composed of items that parents rated on point-likert scales from (never) to (very often). an example item is "during last month how do you usually feel nervous and stressed?" the scale showed a good reliability and validity also in the italian validation (mondo et al., ) . in the present sample, the measure showed a good reliability, cronbach's alpha of . . the regulatory emotional self-efficacy scale (caprara et al., b ) is a -item scale that evaluates the belief of parents to be able to manage with their negative emotions (i.e., anger, sadness, fear, and guilt) during the covid- lockdown on a point-likert scale from (not able) to (able). the scale was modified asking parents to think about the quarantine period related to covid- health emergency, and the following item was added to the scale "how do you feel able to manage the anxiety caused by hearing the news about coronavirus that is given on tv or that you read on the internet?" the scale showed good validity and reliability (caprara et al., a,b) . in the present sample, the scale showed a good reliability: cronbach's alpha of . . parents completed the parenting self-agency measures (dumka et al., ; baiocco et al., ) which is an -item scale that evaluates the belief of parents to be able to manage with daily parental demands (i.e., feeling to be a good parent, working to face and solve difficulties with their children) during the month of lockdown on -point likert scales from (seldom) to (always). the scale was modified, asking parents to think about the quarantine period related to covid- health emergency, and three items were added to the original scale. these three items asked parents how they feel able to reassure their children about the health emergency, to organize their children's daily life during the quarantine, and to explain to their children what is happening. the scale showed good validity and reliability (baiocco et al., . in the present sample, the scale showed a good reliability: cronbach's alpha of . . parents were asked to think about their child during the quarantine and to complete a short version of the emotion regulation checklist (molina et al., ) . this is a -item scale that evaluates two sub-dimensions, namely, emotional regulation (i.e., positive emotions, being able to give voice to his/her negative emotions) and lability/negativity (i.e., anger, disruptive behaviors, excessive exuberance) of children during the covid- lockdown on a point-likert scale from (almost never) to (almost always). the scale showed good validity and reliability (molina et al., ; di maggio et al., ) . in the present sample, both emotional regulation and lability/negativity scores showed acceptable reliability, respectively cronbach's alpha of . and . . firstly, bivariate correlations among variables were calculated along with descriptives. afterward, a mediation analysis with latent variables was performed via sem, employing a parceling strategy (e.g., bagozzi and heatherton, ; little et al., ) . a parcel represents an aggregate of different items measuring a specific construct (little et al., ; coffman and maccallum, ) . two or three parcels were constructed for each of the latent variables using the "item-to-construct" balance approach (little et al., ) , which means building each parcel by examining the item-construct relationships as represented by factor loadings in the item-level factor analyses (for a detailed description of this procedure, see little et al., ) . in such a way, parcels typically contained a balanced number of items and had comparable reliabilities. therefore, our model comprised three latent variables with three parcels each and two latent variables with two parcels each. summative indexes (such as the cri and fri) were treated as manifest variables. model fit was evaluated with the following indices: (a) the comparative fit index (cfi); (b) the tucker-lewis index (tli); (c) the root mean squared error of approximation (rmsea); (d) and the standardized root mean square residual (srmr). in general, for tli and cfi, values between . and . are considered acceptable (e.g., bollen, ; byrne, ; marsh et al., ) and values above . are deemed to be very good (hu and bentler, ) . on the other hand, rmsea and srmr values smaller than (or equal to) . indicate a good fit (e.g., bollen, ; browne and cudeck, ; hu and bentler, ; marsh et al., ) . in order to evaluate the statistical significance of indirect effects, which represented the "mediated" effects, the bootstrapping procedure was used employing samples with replacement from the full sample to construct biascorrected percent confidence intervals (ci) (preacher and hayes, ; hayes, ) . mediation typically occurs if the indirect effect is significant, that is, the zero value is not included in the ci (preacher and hayes, ; hayes, ). finally, to test possible moderation effects of children's biological sex and age, and living in a geographical area with high covid- risk, a multigroup approach within sem was employed as suggested by baron and kenny ( ) . in this procedure, the invariance of the structural parameters of the proposed model was tested separately for (a) boys and girls; (b) different levels of children's age; (c) geographical residential area, that is, living (vs. not living) in northern italy. a detailed description of the procedure will be given in the "results" section (see also sauer and dick, ; cattelino et al., ) . all analyses were run with statistical software spss and mplus . . the family risk index was positively related with parents' psychological distress and positively with lability/negativity. parents' psychological distress was negatively related with parenting self-efficacy, parents' regulatory emotion self-efficacy, and children's emotion regulation and was positively related with children's lability/negativity. parenting self-efficacy was positively related with parents' regulatory emotion self-efficacy, and children's emotion regulation, and was negatively related with children's lability/negativity. parents' regulatory emotion selfefficacy was positively related with children's emotion regulation and negatively related with children's lability/negativity. finally, children's emotion regulation was negatively related with children's lability/negativity. correlations, means, and standard deviations are reported in table . a sem was employed to test the hypothesized mediation model in which parenting self-efficacy and parents' regulatory emotional self-efficacy (related to the covid- lockdown) mediated the relationship between parents' psychological distress and both children's emotional regulation and children's lability/negativity. moreover, parenting self-efficacy mediated the relationship between parents' regulatory emotional self-efficacy and both children's emotional regulation and children's lability/negativity. in the present paper, the mediation analysis strategy recommended by james et al. ( ) was followed. in the first step, the mediation model was tested (i.e., model without the direct effects, indicated with m med ). in the second step, a full model, including all the direct effects, was tested (indicated with m full ). the two nested models were compared via the chi-square difference test, contrasting m med with m full ( χ , satorra and bentler, ) . a non-significant χ would reveal that the full model does not significantly increase the fit and therefore the mediation model is to be preferred since it is more parsimonious. the mediation model (m med ) showed an overall good fit, chisquare ( ) = . , p < . , rmsea = . , cfi = . , tli = . , srmr = . . the full model including direct effects (m full ) did not apparently improve the model fit, chisquare ( ) = . , p < . , rmsea = . , cfi = . , tli = . , srmr = . . in fact, the two models were contrasted, and the chi-square difference test was not significant, χ ( ) = . , p = . . therefore, the mediation model (m med ) should be preferred due to being more parsimonious compared to the full model. in figure , all measurement and structural parameters of the mediated model (m med ) are reported. parents' psychological distress (ppd) was significantly, although modestly, affected by both covid and family risk indexes. in turn, parents' psychological distress significantly and negatively affected both parents' regulatory emotional self-efficacy (pser) and parenting self-efficacy (pse). parents' regulatory emotional self-efficacy significantly and positively affected parenting self-efficacy. finally, parenting self-efficacy positively and significantly influenced children's emotional regulation (er_c) and negatively children's lability/negativity (ln_c). more importantly, parenting self-efficacy mediated the effect of parents' psychological distress and parents' regulatory emotional self-efficacy on both children's emotional regulation and children's lability/negativity. with the exception of those involving the covid and family risk indexes, all other indirect effects were statistically significant, supporting the mediated model (see table ). in table , a full decomposition of total and specific indirect of the mediated model (m med ) are reported. the covid and family risk indexes displayed no significant effects on the other variables except for the aforementioned influence on parents' psychological distress. moreover, referring to the full model (m full ), we also decomposed total, direct and indirect effects (see table ) with the aim to report the ratio of indirect to the direct effect and the proportion of mediated effect (mackinnon et al., ) . in regard to the relationship between ppd and er_c, the ratio of indirect to the direct effect was . (− . /− . = . ), while with respect to the relationship between ppd and ln_c the ratio was . ( . / . = . ). in regard to the relationship between ppd and er_c, about the . % of the effect was mediated (− . /− . = . ), while with respect to the relationship between ppd and ln_c about the . % of the effect was mediated ( . / . = . ). in regard to the relationship between pser and er_c, the ratio of indirect to the direct effect was − . ( . /− . = − . ), while with respect to the relationship between pser and ln_c the ratio was − . (− . / . = − . ). more problematic was to estimate, in the same fashion, the amount of mediated effect regarding the relationship between pser with both er_c and ln_c since in those cases the indirect and the direct effects have opposite signs (as can be seen in table ) , and therefore, they tend to suppress each other, resulting in a reduced non-significant total effect (pser er_c: . = − . + . ; pser ln_c: − . = − . + . ). with respect to non-significant total effect, scholars have pointed out that mediation can occur also in the absence of a detectable total effect if the indirect effect is significant (e.g., mackinnon, ; all effects are standardized coefficients. if the zero value is not included in the bootstrap % ci, the effect is significant at p < . . ppd, parents' psychological distress; pser, parents' regulatory emotional self-efficacy; pse, parenting selfefficacy; er_c, emotion regulation of children; ln_c, lability/negativity of children. hayes, ) . this is apparently the case. in this perspective, although it cannot be absolutely claimed that effects were totally mediated and despite the presence of null total effects, it is worth to note that indirect effects were significant and that mediation has occurred. overall, we can conclude that the hypothesized mediation model (m med ), reported in figure , is consistent with the data. all effects are standardized coefficients. if the zero value is not included in the bootstrap % ci, the effect is significant at p < . . ppd, parents' psychological distress; pser, parents' regulatory emotional self-efficacy; er_c, emotion regulation of children; ln_c, lability/negativity of children. moreover, the mediation did not fit significantly worse than the full model (m full ) and therefore it was retained since it is more parsimonious (james et al., ) . additionally, all indirect effects of the mediated model (m med ) were significant, indicating that mediation has occurred (e.g., preacher and hayes, ; hayes, ). within sem, the test for a moderator effect can be performed using a multigroup analysis of the model in which the structural parameters are constrained equal across groups. firstly, the structural parameters are freely estimated across groups to test for the baseline model. secondly, the structural parameters are constrained to be equal across groups to test for the invariant model. in order to compare the fit of the two models, the chi-square difference test was used (satorra and bentler, ) . a non-significant chi-square indicates that the parameters cannot be ruled out to be equal, then the invariant model should be retained and no moderation occurs. instead, if the chi-square difference between the invariant and the baseline models is significant, which would mean that the invariant model fits significantly worse. therefore, parameters are not equal across the groups and there is a moderation effect. results of chi-square difference tests of multigroup analyses with sem are reported in table . regarding children's biological sex, the fit of the baseline model was chi-square ( ) = . , p < . , rmsea = . , cfi = . , tli = . , srmr = . , whereas the fit of the invariant model was chi-square ( ) = . , p < . , rmsea = . , cfi = . , tli = . , srmr = . . the chi-square difference test was not significant showing that the invariant model could not be rejected (table ). this finding suggests that biological sex of the children was not a moderator variable. in regard to children's age ( - y.o. vs. - y.o.) , the fit of the baseline model was chi-square ( ) = . , p < . , rmsea = . , cfi = . , tli = . , srmr = . ; conversely, the fit of the invariant model was chi-square ( ) = . , p < . , rmsea = . , cfi = . , tli = . , srmr = . . the chi-square difference test was not significant showing that the invariant model could not be rejected (table ) . therefore, children's age did not appear to moderate the mediational effects tested in our model. finally, turning to geographical area (northern italy vs. rest of italy), the fit of the baseline model was chi-square ( ) = . , p < . , rmsea = . , cfi = . , tli = . , srmr = . . instead, the fit of the invariant model was chisquare ( ) = . , p < . , rmsea = . , cfi = . , tli = . , srmr = . . the chi-square difference test was not significant, showing that the invariant model did not fit significantly worse and therefore could not be rejected (table ). this result suggested that living in a high at-risk area for covid- , as it was northern italy at the time of data collection, did not significantly affect the structural parameter of our hypothesized model. the health emergency related to the covid- pandemic and the consequent restrictive measures of quarantine have upset our lifestyles and our daily life. in particular, families with children had to face an unprecedented and completely new situation in which parents suddenly remained the only reference point for their children. results of the present study, indeed, showed that the covid risk index and the family risk index partially contributed to the psychological distress of parents, although their impact was modest in terms of accounted variance. specifically, parents with higher levels of psychological distress tendentially had a lower ses, had seen their working situation worsened during the quarantine, and were single or divorced parents who had to manage their children at home alone during the quarantine. furthermore, regarding the covid- risk index, parents with more psychological distress more likely had relatives, friends, or acquaintances tested positive for the sars-cov- , had hospitalized relatives because of the sars-cov- , lived in northern italy which was the most at-risk area for the spread of the pandemic and for the risk of contagion, were health workers, and worked in hospital departments that treated sars-cov- -positive patients. however, our sem showed that parents' psychological distress impacted on the emotional regulation and lability/negativity of their children passing through the mediators' effect of parenting self-efficacy and parents' regulatory emotion self-efficacy. these findings suggested that what could have a positive effect on children's well-being and positive emotional regulation was not just being exposed to low level of parents' psychological stress, but it was the fact that parents felt able to manage and carry out their parental role and the related tasks. our results suggest that self-confident parents can successfully activate many personal resources that in turn seem to prevent their children's emotional dysregulation, even in emergency situations such as the pandemic that increased their levels of psychological distress. furthermore, three multigroup analyses were performed to test the possible moderation effects of children's biological sex and age and of geographical area (i.e., living or not living in northern italy, which is the most at-risk area for the spread of the pandemic and for the risk of contagion). the multigroup analyses showed that the hypothesized model was robust and invariant across children's biological sex, and age, and living (or not living) in northern italy. thus, in line with spinelli et al. ( ) , parents' and children's psychological distress was not affected by living in the high at-risk zone for covid- (vs. not living in the high at-risk zone). we can speculate that, regardless of living in a more risky area, relationships among variables remained stable because this unprecedented situation characterized by the isolation and quarantine measures was perceived in the same way throughout italy. alternatively, it is also possible that our study did not have enough power to detect differences in parameters between groups. parents should be supported to improve their strengths and to feel able to manage their parental role and their emotions. during the quarantine, parents were the unique reference point for their children aged between and years who rely much on their parents in this life stage. it is important that parents know that they can protect their children, preventing their emotional dysregulation, using their strengths and self-confidence, even if they are experiencing fear and severe stress for the health emergency. moreover, even if parents are exposed to high levels of stress, they can still promote a positive emotional functioning in their children if they feel able to reassure their children about the health emergency, to organize their children's daily life during the quarantine, and to explain them what is happening. despite these important findings, this study had some limitations. we collected a convenience sample that was not representative of the italian population. moreover, emotion regulation and lability/negativity of children were reported by parents and this could be less informative. however, many other scholars have used this type of data collection which is very common in this kind of studies (e.g., trumello et al., ; spinelli et al., ) . moreover, our data are correlational and it is also conceivable that parental distress and self-efficacy could be affected by children's lack of emotion regulation and lability/negativity. furthermore, we assessed the parent's own judgment of their children's well-being and it is possible that parents who experience (according to themselves) a lot of distress also tend to judge their children's well-being more negatively irrespective of the children's actual well-being. finally, it is not possible to infer causal relationships among variables because of the correlational nature of data. future longitudinal study can be conducted in order to deeply test the possible long-term effects of parents' psychological distress related to the health emergency on their children's psychological well-being and the possible reverse causation effect. however, despite these limitations, the present study presents many implications for prevention and intervention programs. in order to prevent children's distress, intervention programs should start from family and parents. this programs should be aimed at increasing parents' regulatory emotional self-efficacy and parenting self-efficacy, by activating their adaptive strategies and resources to deal with daily tasks and reinforcing their strengths. these parents' skills could be taught and learned, representing an important resource even in emergency situations such as a pandemic, in which parents remain the only points of reference and education for their children. these prevention programs should be primarily addressed at (but not limited to) parents who are health workers, who lived alone with children during the quarantine, who have sick relatives, and who have a low ses and a worsened work situation, in order to prevent the impact of their psychological distress on their children, reinforcing their belief to be able to face this difficult situation and to manage both their parents tasks and their unavoidable negative emotions. these findings suggest how clinicians should give psychological support to parents remotely during a lockdown, reinforcing their personal strengths and working on effective parenting and regulatory efficacy strategies. indeed, parents with beliefs of self-efficacy in parenting behaviors and emotional regulation have children more emotionally regulated and psychologically healthy. likewise, the present results can be used to implement psychological and educational intervention for parents in order to prevent their children's psychological distress. these results can also give pediatricians and psychologists important indications on how to specifically support families during the quarantine due to a global pandemic, providing advice to parents who in this period turn to pediatricians or psychologists to understand what to do to improve the well-being of their children. telling parents that, even if they experience negative emotions, they can do a lot to help their children could empower parents, activating their skills and strategies. intervention programs should be aimed to explain parents how to communicate to their children what is happening in the world around them. using the correct words is more probable when parents have high levels of parental self-efficacy and emotional regulation self-efficacy (jones and prinz, ) , and this could be very useful for parents' and children's well-being. talking about the fear and the negative emotions related to the pandemic and the isolation would represent an important protective factor for families' well-being. if parents understand which is the right way to communicate about the pandemic with their children, they can probably feel more self-confident in managing their parental tasks and their children's emotion, and this aspect can have in turn positive effects on their children's positive adjustment. a general approach to representing multifaceted personality constructs: application to state self-esteem same-sex and different-sex parent families in italy: is parents' sexual orientation associated with child health outcomes and parental dimensions? same-sex and different-sex parent 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internet addiction policy brief: covid- and the need for action on mental health mitigate the effects of home confinement on children during the covid- outbreak parenting self-efficacy and problem behavior in children at high risk for early conduct problems: the mediating role of maternal depression mental health and psychosocial considerations during the covid- outbreak substantial investment needed to avert mental health crisis mental health status among children in home confinement during the coronavirus disease outbreak in hubei province the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by the ethics committee of the department of developmental and social psychology, sapienza university of rome. written informed consent was not provided because data were collected via an online survey and participants were recruited via a snowball sampling. thus, participants gave their informed consent by clicking "yes, i accept to participate to this study" on the first page of the online survey. mm, ac, ec, and rb conceptualized the study and organized the data collection. mm, ac, ec, rb, ct, ab, and cc collected the data. ac and mm run the analyses and wrote the methodological and results section. mm wrote the first draft of the manuscript. ec, ct, ab, and cc contributed to revision of the final version of the manuscript. all authors contributed to the article and approved the submitted version. key: cord- -m o yw authors: di ciaula, agostino; palmieri, vincenzo o.; migliore, giovanni; portincasa, piero title: covid‐ , internists and resilience: the north‐south italy outbreak. date: - - journal: eur j clin invest doi: . /eci. sha: doc_id: cord_uid: m o yw according to data from the world health organization, italy has been particularly affected by the ongoing covid‐ pandemic. on april (st) , italy gained, at a world level, the highest number of total confirmed cases (n= , ) and deaths (n= , ) since the beginning of the outbreak. the number of cases raised exponentially, reaching a total of , infected subjects and , deaths on may, . the distribution of infected subjects and deaths, however, was not homogeneous, being respectively about ‐times and ‐times higher in northern‐ than in southern regions according to data from the world health organization, italy has been particularly affected by the ongoing covid- pandemic. on april st , italy gained, at a world level, the highest number of total confirmed cases (n= , ) and deaths (n= , ) since the beginning of the outbreak [ ] . the number of cases raised exponentially, reaching a total of , infected subjects and , deaths on may, . the distribution of infected subjects and deaths, however, was not homogeneous, being respectively about -times and -times higher in northern-than in southern regions [ ] . besides monitoring the outbreak, epidemiologic data should be analyzed to test the adequacy of strategies adopted to contain the pandemic, to better face the rising cases of covid- in other countries, and to use lessons from this pandemic for future possible outbreaks. from a clinical point of view, evidence points to a multi-organ involvement secondary to the sars-cov- infection [ ] . in this rapidly changing and novel context, the role of the specialist in internal medicine represents a winning factor for optimal management of critically ill adults with suspected or diagnosed covid- . the role of the internist provides a precious support to specialists working in different settings, namely emergency room (er), infective disease, respiratory, and intensive care units (icu). here, we analyze the main phases of the covid- outbreak in italy. in addition, while discussing some pathophysiological implications of this complex disease, we briefly describe the events that we experienced -following the covid- outbreak-at our academic division of internal medicine in the largest regional hospital of apulia. epidemiologic data were searched from public available databases from the world health organization [ ] , and the italian ministry of health [ ] . the rates of infected subjects and deaths in italy (whole country and regional level) per , residents were calculated considering the official number of residents derived from the national institute of statistics (istat). correlations were tested using the pearson coefficient and plotted using linear regression. data updated to may , . this article is protected by copyright. all rights reserved the spread of the covid- pandemic is not homogenous worldwide. most rapid and extensive outbreak occurs in a few countries (i.e. usa, italy, spain, china, germany, france, iran) [ ] . in italy, the first cases of infection (a couple of chinese tourists) occurred on january , at the italian experimental institute "lazzaro spallanzani" in rome. figure summarizes the daily progression in the cumulative number of covid- positive subjects and in the incidence of deaths related to covid- in southern and northern italy, since the start of the outbreak. table lists the containment strategies adopted by the central government. the spread of infection followed divergent trends between northern and southern italy. since the average incubation period for covid- is - days and, in its early stages, the epidemic double in size every . days [ ] , the initial outcome of the country lockdown was expected by two-three weeks. as shown in figure , this hypothesis was confirmed by the analysis of the absolute daily number of new cases of infection in northern and southern italy. a total of , , subjects live in northern italian regions. the first covid- case in a northern region was noticed in codogno, a small town in lombardy, km south-east from milan. in codogno a local quarantine started on february . after two weeks (march ), the whole northern italy was quarantined. on the national "lockdown" day (march ), in northern italy there was a total of , infected patients and covid- related deaths. on may , infected subjects were , and the number of deaths increased to , . a total of , , subjects live in southern italian regions. the first three cases in this area occurred only five days after the first case in northern italy. however, on the "lockdown" day, the total number of infected patients was , about -fold lower than in northern italy. the covid- related deaths were , about -folds lower than in northern italian regions. on may , infected subjects were , and covid- related deaths were , , about -and -fold lower, respectively, than in northern italy. in most southern regions, travelers were obliged to inform the local authorities upon their arrival to track possible newly diagnosed infections. thus, before the whole country was on lockdown, accepted article the containment measures in southern italy were only on a voluntary basis (i.e., social isolation was advised). in apulia (southern italy, about million inhabitants), the regional authorities estimated that at least , subjects travelled from northern italy from february to march . in this region, the incidence of infected subjects increased from . (february ) to . per , residents (may ) being, on the same day, slightly higher than the rate observed in southern italy ( . per , residents). this value, however, remained about -fold lower than the incidence recorded in northern italy (i.e., . per , residents). on may , a total of , confirmed cases and covid- related deaths were recorded in apulia [ ] . the median age of infected subjects was years, and about % of deaths were recorded in subjects with or more years of age. at a regional level, a total of , patients were isolated at home, and % of cases were admitted in covid- dedicated hospitals [ ] . of note, only a minority of patients had a severe ( %) or critical ( %) clinical presentation [ ] . the majority of patients remained asymptomatic ( %) or had a non-critical clinical presentation ( %) requiring, after an initial triage, home isolation or clinical management in non-intensive care units. following the covid- outbreak, the local apulian government firstly increased the number of available beds in the units of intensive care, pneumology, and infectious diseases across the region. afterwards, the local health care system was re-modulated by creating "covid- " and "non-covid- " dedicated hospitals. the policlinico, the second largest hospital in southern italy, hosts about , beds, with all medical specialties. it is the main hospital in bari (capital city of apulia), with a population of , inhabitants, raising to , inhabitants in the urban area and to . million inhabitants in the metropolitan area. before the outbreak, the clinica medica "a. murri" was a typical academic division of internal medicine in the tertiary referral hospital (i.e., beds with admissions/year, day services/year, and , outpatients, including an active ultrasound service). all non-urgent outpatient activities, however, had been this article is protected by copyright. all rights reserved hours, the medical and paramedical staff from both divisions (including residents in geriatrics and internal medicine) were properly trained in the correct use of personal protection devices. the staff was therefore re-located in the so-called "grey zone" equipped with up to beds, occupying the whole th floor of the "asclepios" block, a -floor building entirely dedicated to covid- patients (figure ) . as internists, our specific mandate was the screening of a wide range of (noncritical, non-surgical) patients received from the er, and suspected of having covid- infection. the division was quickly arranged to accommodate covid- positive, negative or suspected subjects, with clear separate pathways (dedicated rooms, corridors, elevators, doors, routes). after admission, patients not requiring icu support are assessed and immediately transferred to a room with single bed, where they undergo the nasopharyngeal swab to obtain a specimen for testing by rt-pcr. if negative, a second test is repeated h later, at the local laboratory of public health [ ] . confirmed negative patients are either transferred to specific covid- negative wards or discharged, with a nasopharyngeal swab repeated (outpatient basis) weeks later. persistently positive patients are managed accordingly. one option is the transfer to other covid- positive wards in the "asclepios" block, depending on the clinical evolution. a second option is the transfer to post-acute facilities in the territory. a third option (if asymptomatic), is to be placed in quarantine at home, after being full instructed about isolation requirements. two weeks later, a special hospital unit will perform two nasopharyngeal swabs at home, with intervals of hours. this organization facilitates the initial management of non-covid- and covid- -related clinical presentations, while decreasing the burden of patients entering other covid- dedicated wards. on april , a total of patients had already been screened at our unit. covid- infection may remain asymptomatic [ ] in a still undefined number of patients. otherwise, a -stage classification system of increasing severity has been proposed for covid- infection, namely stage i (early infection), stage ii (pulmonary phase), and stage iii (hyperinflammation phase) [ ] . thus, the clinical symptoms range from fever, dry cough, headache to dyspnea, fatigue, to ards, shock, and cardiac failure [ , ] . the multi-organ involvement secondary to sars-cov infection is therefore a possibility. gastrointestinal symptoms (i.e. diarrhea, nausea, vomiting, and abdominal pain) may be observed early [ , ] . about half of patients show elevation of liver tests, alt, ast and ggt during this article is protected by copyright. all rights reserved disease progression, and liver injury is possible in severe cases of covid- , possibly due to a direct viral infection of vascular endothelium, cholangiocytes, to immune-mediated inflammation or to drug hepatotoxicity [ ] . diabetes mellitus can increase the risk linked with covid- infection, negatively affecting the progression and prognosis of the disease [ , ] . furthermore, relationships exist between covid- and myocardial injury [ ] , heart failure [ ] , vascular inflammation, myocarditis, cardiac arrhythmias [ ] , and hypoxic encephalopathy [ ] . (ii) extra-pulmonary systemic hyperinflammation syndrome (in the minority of infected patients): the "cytokine storm" is associated with increased cytokine levels (il- , il- , il- , il- and tnfα), granulocyte-colony stimulating factor, interferon-γ inducible protein , monocyte chemoattractant protein , macrophage inflammatory protein -α, tumour necrosis factor-α, lymphopenia (in cd + and cd + t cells), decreased ifnγ expression in cd + t cells [ ] [ ] [ ] . d-dimer , troponin and n-terminal pro b-type natriuretic peptide (nt-probnp) can also increase, together with altered coagulation function [ ] [ ] [ ] in those patients with more severe disease [ ] . available epidemiologic evidence also point to more severe clinical presentations and higher risk of death in the elderly and/or in fragile patients with several co-morbidities [ ] [ ] [ ] [ ] . in this context, the internist is typically trained to manage the complexity of systemic diseases, and to coordinate a multidisciplinary approach in the case of severe presentation. the spread of covid- has rapidly reached a pandemic dimension within few weeks, imposing a rapid remodulation of health systems to contain and adequately face the outbreak. the management of the disease also requires the transmission of adequate, evidence-based information and optimal decision-making [ ] . this article is protected by copyright. all rights reserved italy was the first european country extensively affected by the outbreak, with national control measures (air traffic embargo) adopted the same day of the first diagnosed case in rome, and with strict control measures (lockdown) about two weeks after the first local outbreak was noticed in northern italy. the critical analysis of the ongoing italian scenario, will help to manage the spread of the pandemic in other countries, and also to face the outbreak by optimizing the locally available resources. italy extends for a length of approximately , km, and a different pandemic burden occurred in southern vs. northern italy, as also observed in other geographical areas at a global level [ ] . the difference in population density between northern and southern italy ( . and . residents/km , respectively) seems too small to fully explain the divergent epidemiologic burden of covid- in these two areas. thus, geographical differences in the incidence of infected subjects and covid- related deaths point to the existence of possible specific and local environmental factors which need to be urgently identified, and that can play a major role in determining the spread and the outcomes of the infection. besides economic, socio-cultural and lifestyle-related factors, a role is possible for climate change variations [ , ] , air pollution [ ] [ ] [ ] , and the effects of endocrine disrupting chemicals on the onset and progression of several comorbidities [ ] able to influence the individual vulnerability to covid- [ ] [ ] [ ] [ ] . at a world level, a °c temperature rise above pre-industrial levels is linked with new and emerging infectious diseases [ ] . there might be relationships between climate change, marked reduction in biodiversity, and transfer by species-crossing (from wild species to humans) of several viruses, namely ebola, sars coronavirus , middle east respiratory syndrome (mers) coronavirus, nipah virus and hendra virus [ , ] . climate changes are also associated with increased incidence of respiratory infections [ , ] . a study evaluating the effects of temperature on mortality in communities from country/regions, described italy as one of the countries with the highest temperature-related mortality risk [ ] . an analysis on administrative regions in italy, spain, france and germany showed that longterm exposure to nitrogen dioxide, a common air pollutant mainly produced by anthropogenic processes, may be one of the most important contributors to fatality caused by sars-cov- infection. this effect occurs mainly when the air concentrations of this pollutant are combined with downwards airflow which prevent efficient dispersion [ ] . some regions in northern italy (mainly lombardy and emilia romagna) are among the most polluted european geographical this article is protected by copyright. all rights reserved areas and, at the same time, have the highest world level of virus lethality [ ] . positive associations have been also described between newly covid- confirmed cases and short-term exposure to particulate matter (i.e. pm . , pm ), nitrogen dioxide, and ozone [ ] . lastly, individual outcomes from covid- are strongly dependent on the presence of comorbidities [ ] [ ] [ ] [ ] . chronic exposure to both air pollutants and endocrine disruptors introduced with contaminated food and beverages or through dermal contact, might play a role in increasing individual vulnerability and frailty, mainly in elderly subjects [ , ] . further analyses are therefore required to adequately identify the major environmental determinants regulating the spread of sars-cov- infection, and the prognosis of covid- . italian epidemiological data confirm the efficacy of early and stringent measures of primary prevention, as observed in china [ ] . about two weeks after the drastic containment measures legally imposed by the lockdown, a trend towards a reduction of new cases clearly emerged in the whole country. in the absence of vaccines or specific therapeutics of confirmed efficacy, the only useful tool to govern and limit the epidemic remains the social distancing, quarantine, community containment and all other public health measures aimed at the primary prevention [ , ] . in addition, we should be aware that rapid and aggressive outbreaks can easily originate from care homes for elderly people. a worrying aspect emerges from a last study showing that rapid and widespread transmission of sars-cov- occurred in skilled nursing facility, where more than half of residents with positive test results were asymptomatic at the time of testing and most likely contributed to transmission [ , ] . the problem involves the residents, their family members and medical, nurse personnel if protection protocols are poorly implemented [ ] . the italian national health system was established with the law n. , on december , . this law was based on the equity of care and underlined the precious role of primary prevention, parallel to adequate health assistance and rehabilitation. as clinicians working in the public healthcare system, we argue that the system should be prepared to limit everywhere morbidity and mortality deriving from this and future pandemics. in the most affected regions (northern italy, mainly lombardy) serious concerns existed about the effective capacity of the national health system to adequately face the burden of disease. this capacity was challenged by the total and simultaneous number of cases, in particular of those requiring intensive care. in this respect, systemic resources required to adequately manage such a critical situation might be insufficient this article is protected by copyright. all rights reserved (i.e. personnel, number of available beds, overcrowding, intensive care facilities) [ , ] . the structural limits deriving from insufficient resources has also generated ethical concerns regarding the prioritization of patients to be treated (i.e. younger patients prioritized over the elderly), and the equity of care [ ] . a prospective data collection of critical care bed number in european countries from july to july showed a marked variability between countries. italy had . critical care beds per , capita of population, a number slightly over the continental average ( . ) but about half than that of germany, the european country with the highest value ( . ) . of note, several eu countries downsized their curative bed capacity in the past years furthermore, evidence show that outcomes are strongly dependent on age (i.e. worst outcome in the elderly), on the extent of fragile patients and on the presence of co-morbidities [ ] [ ] [ ] [ ] . in the next future, severe health damage from further similar outbreaks could be strongly prevented by measures focusing on successful aging and healthspan, rather than merely on increasing lifespan. this approach should include a better management of all the environmental factors affecting noncommunicable diseases, thus contributing to primary prevention of diseases and to decreased frailty, as well as to increased number of years lived without diseases or disability [ ] . this article is protected by copyright. all rights reserved "the discipline of internal medicine is involved in scientific and educational activity, as well as dedicated health care in the field of medical pathophysiology, instrumental and functional medical semeiology, medical methodology, evidence-based medicine, general clinical medicine and medical therapy, with specific aspects of emergency medicine, geriatrics and gerontology, allergology and clinical immunology; further fields of interests include clinical and metabolic aspects of vascular diseases, clinical nutrition, sport medicine, and thermal medicine" in this complex scenario, and according to our experience, internists are called to serve prevention measures, front-line selection of patients, management of the disease and complications, tight coordination with other specialties, according to specific requirements. in our opinion, this is an outstanding example of professional resilience to sudden, and unexpected events which endanger the public health. the sars-cov- infection became an unprecedented clinical challenge. the rapid pandemic spread has strongly stressed the national policies and health systems worldwide, highlighting critical issues and capacity of resilience. the dramatic experience deriving from the covid- pandemic should be maximized to adequately manage the present outbreak in other countries, but also to provide useful tools for future, potential pandemics. an additional goal should be the improvement of the overall health systems, oriented to adequately fulfill public needs. the best approach derives from a combination of public health measures aimed at primary prevention, an adequate allocation of financial resources needed to efficiently face public health needs (in terms of personnel, processes, and tools), and an appropriate clinical management of affected individuals. this last goal, in particular, relies on the evidence that the infection by sars-cov- generates a complex and systemic disease, with multi-organ involvement. in this context, specialists in internal medicine play an undisputed pivotal role. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved [ ] . dots and lines indicate the incidence of cases per , residents in southern and northern italian regions. world health organization. coronavirus disease (covid ) covid- monitoring in italy clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china geneva: world health organization italian ministry of health. covid- italia -monitoraggio della situazione early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia regional health agency (aress) -apulia. epidemia covid- . bollettino epidemiologico regione puglia - aprile laboratory diagnosis of emerging human coronavirus infectionsthe state of the art incidental findings suggestive of covid- in asymptomatic patients undergoing nuclear medicine procedures in a high prevalence region covid- illness in native and immunosuppressed states: a clinicaltherapeutic staging proposal diagnosis and management of covid- disease covid- : gastrointestinal manifestations and potential fecal-oral transmission novel coronavirus infection and gastrointestinal tract liver injury in covid- : management and challenges covid- and diabetes mellitus: unveiling the interaction of two pandemics diabetes is a risk factor for the progression and prognosis of covid- covid- and the cardiovascular system potential effects of coronaviruses on the cardiovascular system: a review clinical characteristics of deceased patients with coronavirus disease : retrospective study single-cell rna-seq data analysis on the receptor ace expression reveals the potential risk of different human organs vulnerable to -ncov infection single cell rna sequencing of human tissues identify cell types and receptors of human coronaviruses angiotensin-converting enzyme is a functional receptor for the sars coronavirus sars-cov- : a storm is raging covid- : consider cytokine storm syndromes and immunosuppression clinical features of patients infected with novel coronavirus in wuhan, china accepted article this article is protected by copyright. all rights reserved prominent changes in blood coagulation of patients with sars-cov- infection abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia disseminated intravascular coagulation in patients with -ncov pneumonia coronavirus disease in elderly patients: characteristics and prognostic factors based on -week follow-up estimates of the severity of coronavirus disease : a model-based analysis prevalence of underlying diseases in hospitalized patients with covid- : a systematic review and meta-analysis comorbidity and its impact on patients with covid- in china: a nationwide analysis coronavirus disease : the harms of exaggerated information and non-evidencebased measures the report of the lancet countdown on health and climate change: ensuring that the health of a child born today is not defined by a changing climate climate change and respiratory infections assessing nitrogen dioxide (no ) levels as a contributing factor to the coronavirus (covid- ) fatality rate can atmospheric pollution be considered a co-factor in extremely high level of sars-cov- lethality in northern italy? environmental pollution association between short-term exposure to air pollution and covid- infection: evidence from china diet and contaminants: driving the rise to obesity epidemics covid- is possibly a consequence of the anthropogenic biodiversity crisis and climate changes impacts of biodiversity on the emergence and transmission of infectious diseases global variation in the effects of ambient temperature on mortality: a systematic evaluation the environment as a determinant of successful aging or frailty evolving epidemiology and transmission dynamics of coronavirus disease outside hubei province, china: a descriptive and modelling study public health and ethical considerations in planning for quarantine interventions to mitigate early spread of sars-cov- in singapore: a modelling study presymptomatic sars-cov- infections and transmission in a skilled nursing facility asymptomatic transmission, the achilles' heel of current strategies to control covid- detection of sars-cov- among residents and staff members of an independent and assisted living community for older adults covid- and italy: what next? the lancet this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord- -erntrh p authors: gisondi, p; piaserico, s; conti, a; naldi, l title: dermatologists and sars‐cov‐ : the impact of the pandemic on daily practice date: - - journal: j eur acad dermatol venereol doi: . /jdv. sha: doc_id: cord_uid: erntrh p since the first case of “pneumonia of unknown aetiology” was diagnosed at the wuhan jinyintan hospital in china on december , what was recognised thereafter as “severe acute respiratory syndrome coronavirus ” (sars‐cov‐ ) has spread over the four continents, causing the respiratory manifestations of coronavirus disease‐ (covid‐ ) and satisfying the epidemiological criteria for a label of “pandemic.” the ongoing sars‐cov‐ pandemic is having a huge impact on dermatological practice including the marked reduction of face‐to‐face consultations in favour of teledermatology, the uncertainties concerning the outcome of covid‐ infection in patients with common inflammatory disorders such as psoriasis or atopic dermatitis receiving immunosuppressive/immunomodulating systemic therapies; the direct involvement of dermatologists in covid‐ care for patients assistance and new research needs to be addressed. it is not known yet, if skin lesions and derangement of the skin barrier could make it easier for sars‐cov‐ to transmit via indirect contact; it remains to be defined if specific mucosal or skin lesions are associated with sars‐cov‐ infection, although some unpublished observations indicate the occurrence of a transient varicelliform exanthema during the early phase of the infection. sars‐cov‐ is a new pathogen for humans that is highly contagious, can spread quickly, and is capable of causing enormous health, economic and societal impacts in any setting. the consequences may continue long after the pandemic resolves, and new management modalities for dermatology may originate from the covid‐ disaster. learning from experience may help to cope with future major societal changes. since the first case of "pneumonia of unknown aetiology" was diagnosed at the wuhan jinyintan hospital in china on december , what was recognised thereafter as "severe acute respiratory syndrome coronavirus " (sars-cov- ) has spread over the four continents, causing the respiratory manifestations of coronavirus disease- and satisfying the epidemiological criteria for a label of "pandemic," broadly defined as the uncontained spread of an infection in multiple regions [ ] . as of march , more than , cases has been identified worldwide, and almost , deaths have occurred. after china, the area more severely affected nowadays is europe, with italy having the dubious record of the number of deaths [ ] . sars-cov- is a zoonotic single-stranded rna virus of the coronaviridae family, which has crossed species to infect humans, as previously occurred for the virus of the "severe airborne spread, per se, is not a major route of transmission. faecal shedding has been demonstrated in some patients, but oro-fecal transmission is not recognised as a relevant driver of infection [ ] . transmission of sars-cov- mainly occurs in households and other close settings. nosocomial outbreaks have been reported [ ] . disease presentation can range from no symptoms to severe pneumonia and death ( table ). the mean incubation period is - days (range - days) and the virus can be isolated from the nasopharynx - days prior to symptom onset. about % of people have mild disease and recover [ ] . a small proportion has a severe or critical condition. the proportion of truly asymptomatic people remains to be determined. transmission during an asymptomatic stage does not seem to play a relevant role in spreading the virus. individuals at higher risk for severe disease include people aged over years and those with underlying conditions such as hypertension, diabetes, cardiovascular disease, chronic respiratory disease and cancer [ , ] . the disease in children is unapparent or mild. to date, the management of infection has been largely supportive [ , , ] . case this article is protected by copyright. all rights reserved fatality rates, i.e., the proportion of deaths from the disease compared to the total number of people diagnosed during a certain period of time, varies, among the others, with location, intensity of transmission, demography of the population, health service organization, and modalities adopted to identify cases. the estimates range from . % in germany to about % in italy [ ]. sars-cov- is a new virus to humans, and no immunization exists in the population at large. hence, the virus spreads with astonishing speed. the basic reproduction number (i.e., the number of cases one infected individual generates), r , has been estimated to range between . to . according to the mitigating measures adopted, and the epidemic in the early phase in china was doubling every . days [ , , ] . some model predictions indicate that millions of people may be infected by the end of . there is evidence that public health interventions can reduce and even interrupt transmission. these measures must fully incorporate immediate case detection and isolation, rigorous close contacts tracing with quarantine, and direct population engagement (table ) [ ] [ ] [ ] . contents of educational interventions should include social distancing measures and personal protection strategies such as hand hygiene. the on going pandemic is having a huge impact on dermatological practice. we are making specific reference to italy, but the situation is similar to what is happening in other countries (personal communications and [ ]). a significant reduction of outpatient dermatological visits both in public hospitals and in private practice offices is registered in most countries. in italy, the reduction of dermatological consultations is approximately - %. such a marked reduction also applies to medical specialities other than dermatology. most hospitals have postponed appointments for elective surgery and non-urgent visits to avoid people moving form home and to focus on covid- management. in italy, the reduction is also one of the consequences of the legislative decree of the italian prime minister #iorestoacasa (translated: i stay at home) recommending the closure of any commercial activities all over italy, except for pharmacies, groceries and other essential services [ ] . citizens are not allowed to leave their homes unless there is the need of going to work (such as in the case of health workers), for urgent health reasons or the purchase of necessary goods. triage is made before any patient attends a health consultation looking for any respiratory symptom or fever. in case of symptoms the patient is put in a separate area. this article is protected by copyright. all rights reserved in this situation, remote working is boosted, and telemedicine -that is defined as ''the remote diagnosis and treatment of patients by means of telecommunications technology'' -could be very appropriate [ ] . by practicing teletriage, which prioritizes in-person clinic visits for patients with conditions associated with greater morbidity or mortality and uses telemedicine for the rest, the delivery of dermatologic care can be pursued during the pandemic [ ] [ ] [ ] [ ] . dermatology forum and the american academy of dermatology guidelines [ ] ). the safety of initiating immunosuppressant medications during the pandemic is questionable [ ] . the benefit-to-risk ratio of any immunosuppressive therapeutic intervention should be carefully weighted in dermatological patients on a case-to-case basis. individuals over the age of years and/or patients with comorbid conditions including cardiovascular diseases, diabetes, metabolic syndrome, chronic obstructive pulmonary disease, chronic kidney diseases, and cancer have a higher risk of developing more serious infections [ , ] . as of now, there is insufficient evidence to determine how sars-cov- infection will impact the clinical course of chronic inflammatory conditions such as psoriasis and to estimate potential risks associated with systemic treatment [ ] . in covid- patients, but also in sars and mers patients, inflammatory cytokines assume a double role: in the first place they stimulate the activation of an effective immune response, while at a later time, in case of failure of the adaptive immunity (mainly th -polarized), they mediate the development of an exaggerated systemic inflammation [ ] [ ] [ ] . this "cytokine storm" is this article is protected by copyright. all rights reserved both ineffective toward the pathogen and detrimental for the body, eventually leading to acute respiratory distress syndrome (ards) and potentially to death [ ] . (tnf)-alpha elevation plays a key role in the development of the detrimental inflammatory response correlated to both coronavirus and non coronavirus viral pneumonia [ ] . in a model of immunosuppressed macaques infected with mers-cov, significantly higher levels of mers-cov replication in respiratory tissues and viral shedding, was found. however, despite increased viral replication, pathologic changes in the lungs were significantly lower in immunosuppressed animals [ ] . mers-cov virus itself caused little damage to the cells that it infected and the tissue damage might be attributed to the overactive inflammatory response. therefore, it has been hypothesized that treatment for patients with symptomatic covid- would benefit from additional therapy that lessens the inflammatory response, and not be based solely on therapies that are aimed at controlling virus replication. in this context, agents blocking tnf or il pathways could have the potential to improve covid- 's aberrant immune response and ards-related mortality [ ] . the exaggerated inflammatory responses (cytokine storm) and increased damage of tissues seen in sars, mers and covid- might also be enhanced by patient's comorbidities, namely diabetes, or even by ageing per se. older adults have elevated levels of pro-inflammatory cytokines, and the term 'inflamm-ageing' was coined to describe this phenomenon. this could partially explain the association between patient's older age and some comorbidities with a worse outcome [ ] . intriguingly, during sars outbreak in and mers and covid- (so far), no death was reported in transplanted patients or under immunosuppressive treatments, (e.g. cyclosporine, methotrexate, azathioprine) at any age. in a recent letter, d'antiga reported immunosuppressed children in bergamo, italy, who were tested positive for sars-cov- but only had a mild disease without any pulmonary involvement [ ] . published data on sars, mers and sars-cov- seem to suggest this article is protected by copyright. all rights reserved that patients with drug-induced immunosuppression are not at particularly increased risk of severe pulmonary disease compared to the general population. the role of dermatologists during the pandemic may not be limited to their dermatology clinics. in the areas of italy severely affected by the coronavirus outbreak, extra beds have been created in internal medicine wards for covid- patients; dermatologists, along with doctors from other medical subspecialties, have therefore been asked to provide assistance to covid- patients in these departments due to the shortage of doctors. for those dermatologists who are now fighting in the front line against covid- , a number of challenges need to be faced on a daily basis, the most crucial one being personal protection. as of th march, . health workers were infected with covid- in italy, representing about % of the total number of positive patients [ ] , and physicians died due to sars-cov- infection [ ] . such appalling data underscore the importance of the availability of protective equipment like medical masks, gloves, eye protection and gowns for medical personnel dealing with infected patients. shortage of medical masks for health workers due to "panic shopping" from the population may jeopardize the safety of physicians dealing with covid- patients. a further area where dermatologists are involved is with damages to the skin from personal protective equipment of healthcare professionals and with procedures such as frequent hand washing. clinical manifestations include acute and chronic irritant dermatitis, secondary infections and possible aggravation of pre-existing cutaneous disorders. recommendations for preventing these adverse effects have been published [ ] . recommendations for preventing irritant hand dermatitis include avoiding harsh soaps because while these will clear any infectious agent from the skin, at the same time, they destroy the protective barrier that protects us from other pathogens. we need to compensate for the damage we're inflicting on our skin with frequent hand washing or alcohol-based hand sanitizers by putting moisturizer on damp skin [ ] . this article is protected by copyright. all rights reserved there are several unaddressed issues of sars-cov- infection for dermatologists. first, dermatologists from wuhan suggested that skin lesions and derangement of the skin barrier could make it easier for sars-cov- to transmit via indirect contact [ ] . however, no clear evidence is currently available pointing to any modes of transmission other than inhaling droplets and aerosols, or contact with a contaminated surfaces followed by touching mouth, nose, or eyes [ ] . secondly, it remains to be defined if specific mucosal or skin lesions are associated with sars-cov- infection. recalcati s. disease's severity [ ] . from thailand, joob b et al. reported a case presenting with a skin rash with petechiae associated with low platelet count, initially diagnosed as dengue, suggesting that also vascular lesions may be early signs of the infection [ ] . we observed a diffuse papular eruption in a woman with covid- febrile infection, as reported in figure . finally, data are currently lacking, as already discussed, concerning the outcome of covid- infection in patients with inflammatory and malignant skin conditions. the course of covid- in patients with immune-mediated diseases like psoriasis receiving different systemic therapies, is completely unknown and may be of special interest to guide the future management of these patients and, more in general, to understand the role of immune response in covid- outcome [ ] . covid- is a new pathogen for humans that is highly contagious, can spread quickly, and is capable of causing enormous health, economic and societal impacts in any setting. dermatological care is already deeply impacted by the pandemic. the consequences may continue long after the pandemic resolves, and new 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of chinese experts on protection of skin and mucous membrane barrier for healthcare workers fighting against coronavirus disease emergency management for preventing and controlling nosocomial infection of novel coronavirus: implications for the dermatology department epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (covid- ) during the early outbreak period: a scoping review cutaneous manifestations in covid- : a first perspective covid- can present with a rash and be mistaken for hypothesis for potential pathogenesis of sars-cov- infection--a review of immune changes in patients with viral pneumonia this article is protected by copyright. all rights reserved key: cord- -d t bzio authors: fiore, josè ramòn; centra, michele; de carlo, armando; granato, tommaso; rosa, annamaria; sarno, michelina; de feo, lucia; di stefano, mariantonietta; d' errico, maria; caputo, sergio lo; de nittis, rosella; arena, fabio; corso, gaetano; margaglione, maurizio; santantonio, teresa antonia title: results from a survey in healthy blood donors in south eastern italy indicate that we are far away from herd immunity to sars‐cov‐ date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: d t bzio here we present results from a survey on anti‐sars‐cov‐ seroprevalence in healthy blood donors from a low incidence covid‐ area (apulia region, south eastern italy). among subjects tested, only in cases ( . %) antibodies against sars‐cov‐ were demonstrated. all the seropositive patients were negative for the research of viral rna by rt‐pcr in nasopharyngeal swabs. these data, along with those recently reported from other countries, clearly show that we are very far from herd immunity and that the containment measures are at the moment the only realistic instrument we have to slow the spread of the pandemic. this article is protected by copyright. all rights reserved. herd immunity is a concept in epidemiology that describes how people can collectively stave off infections if some percentage of the population has immunity to it. when most of a population is immune to an infectious disease, this provides indirect protection, or herd protection, to those who are not immune ( ) there are two ways to achieve herd immunity: a large proportion of the population either gets infected or gets a protective vaccine. based on early estimates of sars-cov- infectiousness, it should be likely needed at least - % of the population to be immune to have herd protection ( ) . however, more recently mathematical models suggest that lower thresholds could be enough to place populations over the herd immunity threshold once as few as % ( ) or even - % of its individuals are immune. ( ) it is therefore important and urgent to evaluate the extent of circulation the virus and of immunity to sars-cov- in the general populations of affected countries because this information should guide the extent of reduction or increasing of preventive measures such as social distancing etc. italy registered the first imported cases of infection in january , and after one week the first local case. after that, a dramatic burden of infections was diagnosed: . cases (with . deaths) as for june , , with main clusters in northern italy. as a whole, infections/ . inhabitants. in foggia (apulia region, south eastern italy) the first case was observed in march the st and as for june , cases were diagnosed with an incidence of cases/ . inhabitants ( )). this article is protected by copyright. all rights reserved. although the total number of diagnosed infections is moderate, we miss clear information regarding the number of individuals in the general population that became immune, possibly acquiring the virus with no or mild symptoms. studies on blood donor cohorts are useful to evaluate the prevalence, incidence and natural course of infectious diseases in the general population and may thus help to assess both the viral circulation and the evolution of the covid- outbreak. we therefore studied a group of healthy blood donors from foggia province for the presence of igm and igg to antibodies to sars-cov- to examine the circulation of the virus in the general population three months after the local start of the epidemic. the main study cohort was composed of blood donors, who were apparently healthy subjects, aged - years. exclusion criteria were active infection or medical conditions, recent surgical procedures, stay in endemic areas, reported risk factors for parenterally acquired infections, chronic degenerative conditions, diagnosis of cancer or high risk of cardiovascular events. all donors underwent clinical examination, medical history evaluation and biochemical testing. all subjects should had been free of recent symptoms possibly related to covid- , nor had close contact with confirmed cases, symptoms free during the preceding days, nor had contacts with suspected cases. each blood donor signed written informed consents allowing for testing for communicable diseases, storing anonymized data and biological materials for diagnostic/research purposes, and use of their anonymous data for clinical research. a total of blood donors, referring to the transfusional center at the "ospedali riuniti" university hospital (foggia, italy) were included in the study and subjected to the search for anti-sars-cov- antibodies, in the period may - . in the case of positivity, subjects were re-called and rt-pcr for detection sars-cov- from nasopharyngeal swabs was performed. anti-sars-cov- igg and igm were analyzed by using a chemiluminescent analytical assay (clia) commercially available kit (new industries biomedical engineering co., a total of subjects were enrolled in this study ( males and females) ranging from to years (table ) . among them, tested positive for antibodies to sars- no statistical differences were observed in the rate of antibody detection according to the sex and the age. alterations; all of them tested negative for rt-pcr research of sars-cov- rna in two nasopharyngeal swabs performed on two consecutive days. herd immunity is the resistance to the spread of a contagious disease that results if a sufficient proportion of a population is immune due to natural infection or mass vaccination. because an effective covid- vaccine is not yet available, herd immunity can be established if a large proportion of immune persons exist in a population to confer indirect protection from infection to susceptible individuals. the breadth of the barrier required to achieve herd immunity depends in large part on viral spread and infectivity. in the nomenclature of epidemiology, the basic reproduction number or r and the classical formula for calculating a herd immunity threshold is - /r . the higher the r , the higher the threshold required for achieving herd immunity. other important factors in calculating herd immunity thresholds include the number of social interactions and their durations, innate differences in individual immune responses, and divergent exposures to the infectious microbe ( ) people who recover from a covid- coronavirus infection, at least for some time, develop immunity to the virus ( ). the disease-induced herd immunity threshold for sars-cov- , according to various epidemiologists, is believed to be around to percent ( ) . more recently, some researchers have reported lower thresholds: from % to just to % of the population ( , ) , suggesting that we could reach herd immunity thresholds by natural infections in the setting of covid- pandemic. recently, data from spain, france and italy ( ) ( ) ( ) countries that adopted strict lockdown measures indicate a very low seroprevalence in the general population ( . %, % and . %, respectively). noteworthy, even in sweden, a country that decided for a herd immunity strategy, with very light restrictions on daily life, antibodies to sars-cov- were detectable in only . % of the general population in stockholm ( ). in other geographical regions, serosurveys in healthy individuals demonstrated a very low rate of positivity for sars-cov- ( ) ( ) ( ) ( ) ( ) . in our study, we confirm a low rate of antibodies against sars-cov- on a group of blood donors from a geographical region with a moderate incidence ( cases/ . inhabitants vs the national data of infections/ . inhabitants); our results are at variance with those observed in other regions of italy. in fact, a higher rate of anti-sars-cov- antibodies has been reported in studies conducted in a region with high incidence of infection such as lombardy, the most affected region in italy, where the seroprevalence accepted article in blood donors was . %, a number that is justified by the wide circulation of the virus in that area during the pandemic a limitation of our study is that the enrolled population ( - years old) is representative of only a part of the general population, since in italy % of the individuals is aged > years ( ) however, presented data are relevant in that they refer to the more acting/interacting group of the population, also important from a productive point of view. certainly, we are far away from herd immunity and even from the more optimistic projections of threshold ( to % of the population) to adopt more relaxed strategies. in conclusion, strengthening herd immunity to control the covid- epidemic is not a viable option as large numbers of people are expected to become infected and many may die from covid- . preventive measures, including physical distancing, remain essential to contain the spread of infection until herd immunity can be safely acquired with the vaccine. health authorities should take in account these considerations when facing the public health measures to be adopted throughout the covid- transition phases. herd immunity": a rough guide herd immunity: understanding covid- the disease-induced herd immunity level for covid- is substantially lower than the classical herd immunity level individual variation in susceptibility or exposure to sars-cov- lowers the herd immunity threshold. medrxiv: the preprint server for health sciences presence of sars-cov- reactive t cells in covid- patients and healthy donors medrxiv consumo y bienestar social estudio ene covid : primera ronda estudio nacional de sero-epidemiologia de la infeccion por sars-cov- en espana estimating the burden of sars-cov- in france sars-cov- seroprevalence trends in healthy blood donors during the covid- milan outbreak covid- seroprevalence rate in healthy blood donors from a community under strict lockdown measures medrxiv . . seroprevalence of sars-cov- in hong kong and in residents evacuated from hubei province, china: a multicohort study lancet microbes seroprevalence of igg and igm anti-sars-cov- among voluntary blood donors in rio de janeiro, brazil scielo electronic library online preprint seroprevalence of sars-cov- -specific antibodies among adults #:~:text=struttura% della% popolazione% dal% ,anziani % % anni% ed% oltre author's contribution jose ramon fiore planned and supervised the project and the manuscript writing; michele centra, michelina sarno and lucia de feo selected blood donors, collected and stored samples; armando de carlo, tommaso granato, annamaria rosa, mariantonietta di stefano, rosella de nittis performed serological and biomolecular assays; maria d errico, sergio lo caputo and teresa antonia santantonio contributed to project planning and manuscript preparation;, fabio arena, gaetano corso and maurizio margaglione supervised laboratory assays and contributed to manuscript writing the authors declare no conflicts of interest key: cord- -zisujjsx authors: sabat, iryna; neuman-böhme, sebastian; varghese, nirosha elsem; barros, pedro pita; brouwer, werner; van exel, job; schreyögg, jonas; stargardt, tom title: united but divided: policy responses and people's perceptions in the eu during the covid- outbreak date: - - journal: health policy doi: . /j.healthpol. . . sha: doc_id: cord_uid: zisujjsx to understand the public sentiment toward the measures used by policymakers for covid- containment, a survey among representative samples of the population in seven european countries was carried out in the first two weeks of april . the study addressed people's support for containment policies, worries about covid- consequences, and trust in sources of information. citizens were overall satisfied with their government's response to the pandemic; however, the extent of approval differed across countries and policy measures. a north-south divide in public opinion was noticeable across the european states. it was particularly pronounced for intrusive policy measures, such as mobile data use for movement tracking, economic concerns, and trust in the information from the national government. considerable differences in people's attitudes were noticed within countries, especially across individual regions and age groups. the findings suggest that the epidemic acts as a stressor, causing health and economic anxieties even in households that were not directly affected by the virus. at the same time, the burden of stress was unequally distributed across regions and age groups. based on the data collected, we draw lessons from the containment stage and identify several insights that can facilitate the design of lockdown exit strategies and future containment policies so that a high level of compliance can be expected. the outbreak of covid- triggered a wide range of responses from governments in the european union. given that the disease was new and effective medical countermeasures did not exist in early , governments had to adopt non-medical measures aiming at the containment and mitigation of covid- . with the aim of "flattening the curve," these policies included bans on public gatherings, closures of academic institutions and public places, national and international mobility restrictions, confinement, and several others [ ] . italy was the first country in europe to apply intervention measures from the beginning of march in response to the severity of the covid- outbreak. other eu countries followed soon afterward, using similar countermeasures around mid-march [ ] . the adoption of these policies varied in their scale, stringency, and pace across countries. while most european states implemented confinement measures, the extent of limitations of people's freedoms differed across individual countries. lockdowns were usually strictest where the pandemic was deadliest (italy, spain, and france), imposing severe limitations on population movements. some governments chose less stringent versions of confinement or no lockdown at all, for instance, "an intelligent lockdown" in the netherlands or "freedom under responsibility" in sweden [ ] . forced to react swiftly to the unfolding epidemic situation, policymakers in every country tried to balance the implementation of containment policies against numerous important factors with the priority mostly given to the protection of the population's health. consequently, there has been a lot of debate in every society about whether measures taken by the government were appropriate or not. some parts of the population have been voicing support for more severe containment policies to minimize the spread of the virus. such attitudes were likely fueled by people's worries about their health and the potential of their national healthcare system to withstand the epidemic. meanwhile, others expressed their concerns about the social and economic consequences of such policies, thereby advocating for less severe containment measures [ ] . as the pandemic began to abate, governments started designing the lockdown exit strategies and restarting their economies. however, the risk that the new wave of the epidemic may happen did not disappear, especially given that the vaccine development takes a long time, and herd immunity was not achieved [ ] . in this light, the issue of lifting lockdowns has become a new subject of public debate across and within european countries raising discussions about the appropriateness of timing, risks, and potential consequences of ending the confinement [ ] . lifting lockdown restrictions creates acute dilemmas to the policymakers since the economic and human costs of any exit strategy seem to be closely linked together. taking a utilitarian approach in this situation could backfire if the society's understanding is not preliminarily secured or expectations are not fulfilled. policymakers and public health experts have to persuade their citizens to make behavior changes and respect future containment interventions while facing the difficulty of enforcing such regulations. therefore, it becomes crucial to understand people's worries about the pandemic and their perceptions of the effects of containment policies, so that the design of further policies and contingency measures is well-informed, and a high level of compliance can be expected from the population. moreover, trust in the government and social institutions may become central to achieving a successful implementation of future measures, whereas lack of it may turn detrimental to the fight against the pandemic. hence it is of paramount importance to understand who people trust most so that public health messages can be amplified using correct means of communication. we provide a timely description of the current situation and draw lessons from the containment stage to inform the design and implementation of the lockdown exit policies. in order to understand the public sentiment towards the covid- containment measures and to inform future policy development, we collected information on people's support for these policies, their worries in relation to the unfolding epidemic, and their trust in different sources of information. we surveyed over , people representative of the adult population in seven european countries: denmark, france, germany, italy, portugal, the netherlands, and the uk. the fieldwork was conducted online during april - , , using multi-sourced online panels provided by the market research company dynata. to ensure that the sampling frame was representative given the online nature of the study, the company applied diverse recruiting procedures to reach the general population (through open recruitment, loyalty programs, affiliate networks, mobile apps). it then used quotas to match the national census shares in each country. the questionnaire was designed by the authors of the study except for the worry items that were adopted from the world health organization (who) covid- snapshot monitoring project [ ] . the questionnaire was carefully translated into six other languages by native speakers and then implemented using the qualtrics platform first as a pilot ( % of the sample in every country) and next as a large-scale survey. the data from the pilot study were included in the total sample. in each country, we collected data from a sample of , respondents representative of the national population in terms of region, age, gender, and education. given that the italian region lombardy was the most severely hit by the covid- outbreak, we collected additional responses in this region representative in terms of age and gender. learning about perceptions and attitudes of people who reside there could provide essential insights to researchers and policymakers. the extra data collected from lombardy were not included in the representative sample of italy. thus, no weighting was used as the additional lombardy sample was analyzed separately and denoted as lombardy in the results section. we assessed people's approval of policy measures that were taken (or were likely to be taken) by their national government in response to the covid- outbreak. in particular, we covered such issues as school closures, bans on public gatherings, border closures, bans imposed on the export of medical equipment, fines for quarantine violations, random temperature checks, curfews, public transport suspensions and utilization of mobile phone data for tracking covid- cases and their contacts. on average, % of people in the seven european countries approved of the policies taken in their country in response to the pandemic, implying considerable public support. nevertheless, the extent of approval differed by country and by policy measure. the most approved measures were fining -day quarantine violations, ban of public gatherings, and border closures (each supported by % of respondents). by the time of the survey's fieldwork, restrictions on public gatherings had been adopted in all countries covered by the study, whereas international travel controls had been imposed to a certain extent everywhere, except the uk [ ] . prior to complete border closures in mid-march , some countries (for example, italy, france, germany, denmark) had been requiring screening and -day quarantine for arrivals from high-risk regions already since february. in contrast, other countries, such as portugal and the netherlands, started later and turned directly to strict measures, such as banning arrivals from high-risk areas and imposing partial border closures. the latter typically implied either limitation on entries of nonresidents or closure of only certain types of borders (land, sea, air), while ensuring "green lanes" for freight vehicles transporting goods. however, complete border closures occurred haphazardly and led to disrupted commerce and stranding citizens. among countries covered in our study, denmark was the first to close all borders in mid-march, whereas the uk did so only in the second half of may . moreover, at the time of fieldwork, the uk did not have routine screenings at its airports or quarantine requirement for travelers [ , ] . thus, the results for the uk showed the extent of public support that these measures would have received, had they been implemented earlier. meanwhile, the most opposed containment policies were public transport suspension ( % of respondents against it), ban of medical export, use of mobile phone data for tracking, and the imposition of a curfew (each disapproved by approximately % of respondents). these trends might reflect within-country regional and age structure of the population. for example, older individuals and those living in remote areas tended to be the most strongly opposed to public transport suspension. in fact, among countries covered by the survey, public transport suspension was implemented only in italy, whereas its volume was reduced in all other states except for germany [ ] . the stay-at-home orders were most significantly opposed by the youngest respondents aged below . this measure was enforced in all countries covered by the survey except for denmark, where it was introduced as a recommendation [ ] . overall, a north-south gradient could often be noticed in the eu regarding policy support: people living in the southern states (portugal, italy, and france) tended to approve of the containment policies more than residents in the northern countries (denmark, germany and the netherlands). noteworthy, the largest share of supporters for every containment measure was noticed among the residents of italy and particularly in lombardy. here, on average, % of the population approved of the government's response to the pandemic. interestingly, the most significant share of the population who explicitly opposed each of the containment policies taken by their government was identified in denmark. here, for example, % of respondents disapproved of school closures and % disapproved of the imposition of a curfew. in comparison, the average disapproval of these measures in other countries was around % for schools and % for curfews. the most polarizing opinions were observed concerning the use of mobile data for tracking covid- cases and their contacts. the most significant share of people explicitly opposing such policy was identified in denmark ( %), the netherlands ( %), and germany ( %). it was particularly disfavored by the youngest age group ( % of respondents aged below against it). this policy received significant media attention as some countries and the european commission started the collaboration with telecom providers to access individual geolocation data for prediction and surveillance of covid- spread [ , ] . as of march , deutsche telekom provided german authorities with the anonymized data on the movement of its users. in italy, vodafone, windtre and telecom italia offered aggregated user data provision to the government for the same purpose. authorities in the lombardy region used mobile phone data to check compliance with the lockdown restrictions [ , , ] . other countries either initiated the development of their own mobile phone tracking apps or cooperated on the creation of common software, such as the pan-european privacy-preserving proximity tracing (pepp-pt) project led by germany. however, the launch of the pepp-pt was delayed at the end of april due to the data protection concerns voiced by experts and even some of the project participants [ ] . while proponents of the contact-tracing measures claim that using mobile data is of paramount importance in response to the covid- pandemic, many people worry about the government's use of technology due to possible privacy violations, thereby raising debates about the appropriateness of such social control measures [ , , ] . according to our data, people in some european countries expressed considerable reluctance about supporting such policy, which therefore makes future compliance questionable. moreover, such privacy disputes, as in the case of the pepp-pt project launch, might trigger higher reluctance among the potential users to use any contact-tracing app in the future, which could be detrimental for the implementation of a viable tracing technology [ ] . to better understand public opinion on certain policies, it is essential to look at the big picture and place obtained results into the national contexts. people's attitudes were likely based on their perceptions of the general state of affairs in their country, particularly in terms of the epidemic situation and restrictions they were subject to at that moment. in view of that, table summarizes the scale of the pandemic and the stringency of government's response in seven european countries at four points of time spaced around april (when the survey's fieldwork was % complete in every country). the public health situation in each state is described using total confirmed cases of covid- and total deaths attributed to covid- , both measured per million people and reported by the european centre for disease prevention and control [ ] . the stringency of government's response is measured with the covid- government response stringency index, a composite measure of containment policies ranging from to , where a higher value denotes a stricter response [ ] . at the time of the survey's fieldwork, the epidemic situation was worst, and the stringency index was highest in italy and france [ , ] . clearly, there was a north-south gradient in the stringency of government response: italy, france and portugal imposed more demanding policies than denmark, germany, the netherlands and the uk. nevertheless, although people in southern countries were exposed to more severe containment measures, they approved of them more than people residing in northern states, who experienced less stringent restrictions. turning now to within-country variations, we observed considerable heterogeneity of attitudes towards many policy responses within individual countries with particularly marked differences between regions and age groups in italy, france, and the netherlands. hereinafter, we grouped regions based on the severity of the covid- outbreak distinguishing between the most and the least affected areas. noteworthy, lombardy denotes the extra sample collected in italy and was analyzed separately from the representative italian sample. overall, we did not find significant differences in policy support between lombardy and the rest of italy. to illustrate within-country differences, fig. . reflects regional and age-related heterogeneity of public opinions in france and italy toward banning the export of medical equipment, such as masks. in fact, this measure was briefly undertaken by germany and france at the onset of the pandemic in early march , leading to political tensions between the eu member states. germany declared that the reason was to avoid shortages of masks, gloves and safety glasses within the country, whereas france argued that the ban was needed for the assessment of inventory and storage capacity [ ] . following the call for solidarity, both countries lifted the within-eu export ban on equipment in mid-march [ ] . while support for this policy tended to be similar in the most and the least severely affected parts of italy and france, the approval of the export ban conspicuously differed across age groups. older individuals approved more of this policy than younger people, which, besides other factors, may be related to the levels of worry people in these age categories have about the risks that covid- poses to their health. we found that % of french and % of italian respondents aged above perceived risks to their health from covid- as high or very high, while the corresponding share among people aged below equaled % in france and % in italy. to address the mental health implications of the covid- outbreak and subsequent containment measures, we assessed levels of worry prevailing in european societies over several domains (health, economic, emotional, work, and future). more specifically, we addressed concerns about losing a close person, becoming unemployed, health system getting overloaded, school closures, small companies running out of business, recession, restricted access to food supplies, blackouts, and society getting more egoistic. these items were adopted from the who covid- snapshot monitoring project, which will allow future comparisons with similar data collected for other countries and at different points in time [ ] . we found that the mean trend was similar in all countries: people worried most of all about the health system getting overloaded so that the capacities could become insufficient to cope with the surge in covid- cases. we observed that even in case of households that had not been directly hit by the novel coronavirus (above % of respondents in the total sample), the pandemic might have acted as a stressor causing health and economic anxieties. fig. presents people's worry about selected issues across seven eu countries (measured on a likert scale from -not worry at all to -worry a lot), where the higher intensity of color reflects a larger share of the population who worry "quite a bit" or "a lot". cross-country differences look substantial, and a north-south divide in the worry caused by the covid- outbreak is conspicuous. fig. . the proportion of respondents who worry "quite a bit" or "a lot" for instance, % of respondents in portugal and % in italy mentioned that they worried "quite a bit" or "a lot" about the national health system becoming overloaded, while the corresponding shares in denmark and germany were % and %, respectively. these health concerns might have reflected the development of the pandemic. as showed in table , the progress of the epidemic had a north-south pattern with more covid- cases and deaths per million of the population in southern states than in northern. the exception was the uk, where the epidemic was third deadliest after italy and france, but government response was less strict than in countries with a better epidemiological situation [ , ] . similarly, more people in portugal and italy were concerned with the economic consequences of the pandemic than in other european countries. for example, % of portuguese and % of italians were worried about losing their jobs, while respective shares in the netherlands and denmark were % and %, correspondingly. these cross-country differences in economic anxieties may be related to people's perceptions of the economic and financial countermeasures taken by their national government and the eu. during the pandemic, european countries implemented several fiscal and monetary measures to mitigate the economic impact of the covid- outbreak. these policies typically included support of wages under the reduced-hour scheme, postponement of tax payments for companies, direct financial supports and grants to small enterprises and self-employed, the extension of unemployment benefits, provision of capital buffers to banks, etc. [ ] . nevertheless, there were substantial variations in the timing and specific content of these countermeasures across the states. to briefly overview the scale of economic support provided by the government in each of the seven countries, table summarizes values of the economic support index, a composite measure reflecting income support and debt/contract relief provided by the national government to households [ ] . it is measured on a to scale, where a higher value refers to a more substantial economic assistance. at the time of the survey's fieldwork, all countries provided some type of economic relief to their residents. nevertheless, the extent of such support was conspicuously different: france and the uk ranked highest, while denmark, germany, and italy ranked lowest [ ] . hence, it may be possible that higher levels of economic concerns in some countries indicated people's beliefs in the insufficiency of the government's response, which will be subject to the analysis in the next waves of the survey. moreover, the composition of employment varies across the eu, especially in terms of informal and temporary employment. temporary contracts provide lower levels of social protection and job security to employees, but their prevalence has increased over the last years, particularly in the netherlands, italy, and france. as of , the share of temporary employees in the total number of employed was highest in southern european countries: portugal ( . %), france ( . %), and italy ( . %). in contrast, it was significantly lower in northern states: the uk ( . %), denmark ( . %), and germany ( . %). the only exception was the netherlands, where temporary workers constituted . % of all employees [ ] . thus, such differences in the employment composition may be in part responsible for the cross-country dissimilarities in economic concerns. we also observed differences in the levels of concern within individual countries. fig. shows the extent of worry about the health system and a recession in italy. we grouped regions based on the severity of the covid- outbreak and distinguished the levels of anxiety across age categories. higher intensity of the color reflects a greater extent of worry. overall, the level of worry in the highly affected regions of the country was not higher than elsewhere in italy, except for the youngest age group. however, economic concerns tended to be unequally distributed across the age groups. for instance, worries about the recession and small companies running out of business were higher among older individuals than younger age cohort. this pattern was similar in all countries covered by the survey. we asked people about the main sources of information from where they received news about covid- . the data show that overall % of respondents closely followed the news on the situation with covid- , implying a high level of public awareness. regarding the sources of information, % of respondents mentioned receiving updates from the tv and % additionally searched for information on the internet. presumably, reliable information presented through the television emerged as the best channel to reach the population at large. next, we assessed the extent of people's trust in the information received from various sources in the context of the covid- situation. the trust in the following information sources was addressed: national government, the eu, the who, hospitals and gps, national news channels and newspapers, social media, relatives and friends. fig. shows mean values of trust in information from six selected sources across seven european states (measured on a likert scale from -no trust at all to -trust very much). higher intensity of the color reflects a higher level of trust in the information from a specific source. the data show that overall people had the highest levels of trust in information from hospitals, family doctors, and the who, followed by information from the national government and main national news channels. this ranking of sources by trust was similar in all countries covered by the survey, except for france, where citizens had a high level of confidence only in healthcare providers and placed relatively little trust in all other sources. moreover, a north-south divide could be noticed in the level of trust in information from the national government. trust was highest in denmark and the netherlands (more than % of respondents trusted "much" or "very much"), whereas it was lowest in france ( % of respondents had a high level of trust). furthermore, a similar north-south gradient was observed concerning the trust in the eu: trust was highest in denmark ( %), germany ( %), the netherlands ( %) and the uk ( %), whereas it was lowest in italy ( %) and france ( %). portugal was an exception to this case since the corresponding value here constituted %. finally, we also observed considerable regional heterogeneities in levels of trust within countries with particularly noticeable differences across individual regions in italy, france, and germany. fig. shows people's trust in information from the national government in the context of covid- in germany and france as an example, where the higher intensity of the color indicates a greater extent of trust. while trust did not differ significantly between regions grouped with respect to the covid- severity, it was heterogenous across the age groups. although the survey asked about the level of trust in information from different sources in the context of the covid- situation and not about the overall trust in institutions, these two are likely to be related. generally, trust reflects people's perceptions of whether institutions are doing what is right. thus, trust in the information they provide can be considered an indicator of the confidence that citizens have in these institutions [ ] . the covid- pandemic raised new challenges for policymakers across the eu. the imminent threat to public health at the onset of the pandemic led most governments to impose a lockdown on society. however, as the peak of the pandemic abated, the focus of attention turned to the social and economic consequences of the containment measures. given that without acquired herd immunity the risk of a new wave of the epidemic remains high, and the production and distribution of vaccines may take to months [ ] , governments must try to strike the right balance between effects on public health, social life and the economy when considering possible exit-strategies from the current lockdown situation. in the absence of medical intervention, policymakers and public health officials must resort to non-medical behavioral interventions. lifting the lockdown requires that citizens support and adhere to the policy measures that aim to contain the spread of the virus as social and economic activity gradually restarts. given the difficulty of enforcing such regulations, future measures need to be both well-designed and well-communicated to the public. the more people are willing to comply voluntarily with the new measures, the less enforcement and supervision will be needed to achieve high compliance. for this, people's perceptions and attitudes need to be factored in at the policy-design and implementation stages. our survey sought to capture the public sentiment toward measures previously taken by policymakers to contain covid- and addressed people's support for policies, worries about the consequences of covid- , and trust in different sources of information. the first insights obtained from the data showed that containment and mitigating policies undertaken by national governments in response to the initial stages of the covid- pandemic were generally wellreceived by the population in all countries covered by the survey. nevertheless, the extent of approval varied across states and specific policy measures. several lessons can be drawn for the design and implementation of policies for the prolongation or gradual removal of lockdown restrictions. first, we observed a north-south divide in people's perceptions, worries and trust across the european countries. this finding suggests that further containment measures and lockdown exit strategies need to be balanced against the factors that worry people in each specific country. one noteworthy example is the level of importance that people in european countries attribute to the concepts of individual freedom and privacy. using mobile data for tracking covid- cases and their contacts may be a controversial decision to take even though it is believed by many experts to be a useful tool to manage the covid- outbreak. the effectiveness of this policy critically depends on a sufficient level of adoption of the technology by the population [ ] . our data suggest that this may not be achieved easily in some european countries. a clear takeaway is that an open dialogue with society on this matter is needed. explaining the need for and the advantages of such intrusive policies through trusted means of communication, while addressing people's concerns explicitly and being open about the risks of using such policy measures may help raise the support and compliance in society to a sufficient degree. another critical issue is the balance between saving lives and saving livelihoods. according to the survey, people in southern european countries are substantially more concerned about the economic aspects of the covid- outbreak than people in northern european countries. economic anxieties, if left unaddressed, may have adverse effects on the mental health and wellbeing of the population, as well as cause downward adjustments in consumption behavior, thereby exacerbating the economic situation in a country if the recession indeed happens. second, we found considerable heterogeneities in people's approval of policies within individual countries. this tendency was particularly noticeable in france and italy. one possible determinant of regional differences in public support could be the extent of the devolution of decision-making in the country. on the one hand, devolution could enable regional or local authorities to make better decisions due to their better awareness of region-specific circumstances. on the other hand, it could harm the coordination of policy responses between the central and regional authorities within individual countries. thus, it is crucial to understand the determinants of such differences and address them to secure public support of future policies and ensure high compliance with government measures. furthermore, our results showed that the burden of stress tended to be unequally distributed across and within countries. even in case of households that were not directly hit by covid- , the pandemic may have acted as a stressor causing health and economic anxieties. such worries may be detrimental to individual mental health and wellbeing, and they may become further exacerbated by the imposition of self-isolation policies. thus, it may be reasonable to consider an asymmetric approach to the design of exit strategies taking region-specific levels of support and worry into account. this includes the identification of vulnerable categories of the population not only in terms of health risks but also with respect to social and economic activities, and addressing their concerns satisfactorily. third, during a pandemic, public trust in the government and the information it provides is of paramount importance. to expect high compliance over extended periods of time, policymakers need to adopt effective strategies and means of communication whereby securing a sufficient level of trust and confidence from the society. as our results suggest, some countries were more successful in this respect than others. society needs to be well-informed about the dilemmas faced by policymakers, and for this, the communication between the government and the citizens must be clear and transparent. the data showed that % of respondents closely followed the news on the situation with covid- mainly using television to keep themselves updated. thus, television emerged as the best channel to reach the population at large, suggesting that presenting reliable information through this means is an effective strategy to follow. nevertheless, given that the data show regional and age-related heterogeneities in trust and policy support, it may be worth tailoring messages and means of communication to specific groups of the society. for example, cooperation with public figures and well-known experts can be used to deliver government and public health messages in a simple language, or local voices could be used to amplify such messages in individual regions of the country. overall, information provision, public education and effective communication strategies should be among the key guidelines for policymakers when implementing exit strategies and designing future containment measures so that these policies have public support and high compliance. additional waves of the survey are scheduled in june and august . this will allow us to investigate in more detail how the population copes with the health, social and economic consequences of the covid- pandemic as the situation evolves. declarations of interest: none estimating the number of infections and the impact of non-pharmaceutical interventions on covid- in european do low-trust societies do better in a pandemic? lockdown fatigue hits as europe enforces coronavirus restrictions answering the right questions for policymakers on covid- . the lancet global health the new york times covid- snapshot monitoring (cosmo): monitoring knowledge, risk perceptions, preventive behaviours, and public trust in the current coronavirus outbreak oxford covid- government response tracker. blavatnik school of government temporary reintroduction of border control tracking and tracing covid: protecting privacy and data while using apps and biometrics how will governments know when to lift restrictions? european mobile operators share data for coronavirus fight deutsche welle on the responsible use of digital data to tackle the covid- pandemic complete our world in data covid- dataset seeks solidarity as nations after intense discussions, i welcome that de and fr now allow for export of #covid protective equipment. i will continue to follow supply to it closely. no single eu country can win this battle alone. #cooperation #solidarity temporary employees as percentage of the total number of employees trust in government, policy effectiveness and the governance agenda the race against covid- this project has received funding from the european union's horizon research and innovation programme under the marie skłodowska-curie grant agreement no , the work was supported by funding under the excellence strategy by the german federal and state governments, as well as by the university of hamburg, erasmus university rotterdam, and nova school of business & economics lisbon -chair bpi | "fundação la caixa" on health economics. we thank our colleagues for their feedback and work on the adoption of the survey to national contexts: helen banks, joana pestana, maarten husen, laurie rachet jacquet, nicolai fink simonsen. key: cord- -mr z o authors: zhan, choujun; tse, chi k.; lai, zhikang; hao, tianyong; su, jingjing title: prediction of covid- spreading profiles in south korea, italy and iran by data-driven coding date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: mr z o this work applies a data-driven coding method for prediction of the covid- spreading profile in any given population that shows an initial phase of epidemic progression. based on the historical data collected for covid- spreading in cities in china and the set of parameters of the augmented susceptible-exposed-infected-removed (seir) model obtained for each city, a set of profile codes representing a variety of transmission mechanisms and contact topologies is formed. by comparing the data of an early outbreak of a given population with the complete set of historical profiles, the best fit profiles are selected and the corresponding sets of profile codes are used for prediction of the future progression of the epidemic in that population. application of the method to the data collected for south korea, italy and iran shows that peaks of infection cases are expected to occur before the end of march , and that the percentage of population infected in each city will be less than . %, . % and . %, for south korea, italy and iran, respectively. the coronavirus disease (covid- or sars-cov- ) is a highly contagious disease, which began to spread in china in mid december [ ] , and as the volume of intercity travel escalated around the lunar new year period, the number of infected individuals began to soar in mid january . with no travel restriction in place due to the low level of vigilance or unawareness of the disease during the early phase of the outbreak, the spreading of the disease had gone almost unobstructed. travel restriction began to be implemented throughout china since january , , which has proven to be effective in curbing the spread of the virus. however, international traffic has not ceased and infectious city's population), respectively, whereas the number of infected individuals in other cities in south korea would be fewer than , i.e., less than . % of city population. for italy, we predict that lombardi and amelia romagna would eventually have about , and , infected cases (i.e., . % and . % of region's population), respectively, and the number of people eventually infected in other cities in italy would be below (< . % of city population). moreover, iran would have around , infected individuals, i.e., . % of its population, of which around , and , will be expected in tehran and zanjan (i.e., . % and . % of the city's population). in addition, the number of people infected in most other cities would be fewer than , (< . % of the city's population). from the progression trends of the epidemic these three countries, provided control measures continue to be in place, the epidemic would come under control before the end of april . in the remainder of the paper, we first introduce the official daily infection data used in this study. the augmented seir model is briefly reviewed, mainly to introduce the parameters of the model used for prediction of spreading profiles. the key procedure for matching historical profiles and prediction of future spreading profiles will be explained. results of application of the proposed method to prediction of the peaks and extents of outbreaks in south korea, italy and iran will be given. finally, we will provide a discussion of our estimation of the propagation and the reasonableness of our estimation in view of the measures taken by the authorities in controlling the spreading of this new disease. the world health organization currently sets the alert level of covid- to the highest, and has made data related to the epidemic available to the public in a series of situation reports as well as other formats [ ] . our data include the number of infected cases, the cumulative number of infected cases, the number of recovered cases, and death tolls, for individual cities and regions in south korea, italy and mazandran, from february , , to march , . data organized in convenient formats are also available elsewhere [ , , ] . samples of data for daegu, gyeongsang north road (south korea), lombardi, amelia romagna, tehran and iran are shown graphically in figure . it should be noted that the data obtained for south korea, italy and iran correspond to initial stages of the epidemic progression as the number of infected cases are still climbing, as of march , . the travel-data augmented seir model [ ] describes the spreading dynamics in terms of a basic fourth-order dynamical system with consideration of intercity travel in china. consider a city j of population p j . the states of the model are the number of susceptible individuals i j (t), the number of exposed individuals (infectious but without symptom) e j (t), the number of infected individuals i j (t), and the number of recovered or removed individuals r j (t). the model takes the following form in discrete time [ ] : t is the state vector on day t, f aseir (.) is the traveldata augmented function, m j is the set of inflow and outflow travel strengths for city j, and µ j is the set of parameters for city j, i.e., where β j is the rate at which a susceptible individual is infected by an infected individual in city j, α j is the rate at which a susceptible individual is infected by an exposed individuals in city j, κ j is the rate at which an exposed individual becomes infected in city j, and γ j is the recovery rate in city j, k i is the possibility of an infected individual moving from one city to another, and δ j is the eventual percentage of the population infected in city j. moreover, the eventual infected population in city j is given by n s j = δ j p j . to facilitate comparison and matching of profiles, we introduce the normalized states as . thus, ( ) can be represented in normalized form as where ≤ |x j (t)| ≤ . since the above model has taken into account the human migration effect as well as the necessary transmission mechanism, we may consider the basic set of parameters to represent the characteristics of the propagation profile of city j. the complete set of parameters have been identified for cities in china [ ] , which will serve as a set of codes for various propagation profiles of covid- so far obtained. for brevity, we do not repeat the results here. while two different cities may have different population size and percentage of eventual infected population, the rates of infection and recovery should be similar across a group of cities, i.e., µ i ≈ µ j . thus, in normalized form, we have for cities i and j within a group of cities having similar parameter sets. this also means for the group of cities having similar rates of infection and recovery. thus, provided the historical archive has adequately covered the possible dynamical profiles, we are able to perform fast prediction for any city o, by fitting an incomplete set of data (corresponding to an early outbreak stage in city o) and using the model parameters already obtained previously, as detailed in the following subsection. the proposed data-driven prediction algorithm is based on the set of historical data of the spreading profiles of covid- in cities in china, namely, sets of normalized time series of the form:Ī . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . where i = , , · · · , , and k i is the length of the data recorded in city i. superscript "(c)" denotes data of chinese cities. now, suppose an outbreak occurs in city o, and only k o days of data have been obtained in normalized form asĪ where k o < k i . then, assuming the spreading profile of city o is related to that of city i in the historical archive, as permitted by virtue of the validity of ( ), we formulate the following optimization problem to predict the epidemic progression in city o: where n is the number of chinese cities in the historical archive, w i and w j are weighting coefficients, µ l and µ u are the lower and upper bounds of the searching space, respectively. by solving the the nonlinear optimization problem, we can find the most closely resembling growth curve from the historical profiles, e.g., city i. then, we apply the the augmented seir model with the profile code given in the parameter set for city i to predict the future spreading trend of city o. furthermore, we can choose the top n best candidates with the smallest error as the candidate set for prediction, giving an average predicted propagation profile and a deviation range based on n best-fit profile codes. we apply the aforedescribed procedure to the data obtained so far for cities in south korea, italy and iran. these data represent the early stage of progression of the epidemic spreading, as the trends clearly show that the numbers of infected cases in most cities are still climbing, as of march , . for each city or region, we identify a group of profiles of best fit from the historical archive, and retrieve the corresponding sets of profile codes for generating the propagation profiles in the coming days. using these profiles, we produce an average progression profile, which is also accompanied by a deviation range at % confidence level. other iranian cities will see less than , eventual infected cases. our prediction gives a total of ± individuals eventually infected, which is . % ± . % of the country's population. . provided the authorities continue to impose strict control measures, the epidemic will come under control by the end of april and is expected to end before june , and as the quality of treatment improves, more rapid recovery will be expected. our prediction on the south korean cities has revealed a very rapid progression of the epidemic, with , infections emerged within days and peaks to be expected in most cities or regions within about weeks. the korean authorities have managed to test an overwhelmingly large number of people ( , ) within a short time, thus preventing a large number of infected and infectious individuals not being quarantined in time [ ] . this strategy has an obvious advantage of offering a clear picture of the extents and locations of the infected individuals in the country at the early phase of the epidemic progression. the epidemic progression is found to be more rapid than typical, reflecting on the effectiveness of the control measures being taken. italy has the second highest death toll after china, reaching on march , [ ] . the fatality rate is about %, which is the highest in the world. with infection cases soaring to , (as of march , ), italy had implemented control measures to contain the spread of the virus by shutting down schools and suspending public events in regions where outbreaks were reported. the epidemic is expected to progress in a typical pace (with the present set of parameters), unless more stringent measures are in place. the situation in iran is also critical, with the number of infected cases escalated to over , in less than weeks. iran has reported death of two lawmakers as of march , , and has been struggling to control the contagion, which has spread to provinces [ ] . the progression profile is again typical, however, expecting to peak in around weeks. like italy, most cities show typical spreading profiles, and the peaks and subsequent decline are . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint not expected to advance sooner unless more stringent measures are implemented to control the contagion. finally, depending on the effectiveness of treatment, recovery rates vary, and judging from the predicted trends shown in figures , and , the epidemic progressions for the three countries are expected to subside by the end of may, with south korea expected to recover sooner than the others. the spreading of the new coronavirus disease (covid- or sars-cov- ) has evolved from a contagion originally confined within wuhan, china, in december , rapidly to a global contagion, which has spread to countries within two months. the numbers of confirmed infection cases in south korea, italy and iran have surged in the last two weeks, reaching , , , and , , respectively, on march , . the global fatality rate, however, remains below %. in this study, we build on the result of our previous work [ ] that establishes a library of parameters of an augmented seir model, corresponding to the historic spreading profiles of cities in china. this library forms a set of profile codes that cover a variety of possible epidemic progression profiles. by comparing the early incomplete data of epidemic progression collected for a specific population with the historic profiles, we select a few candidate profiles from the historic archive using a nonlinear optimization procedure. the corresponding profile codes of the selected historic progression profiles can then be used to produce estimates of the future progression for that specific population. we apply this method to predict the spreading of covid- in south korea, italy and iran. results show that the three countries will soon see infection peaks in most cities in the coming to weeks, with south korea's cases reaching their peaks slightly earlier than the others. the percentage of population eventually infected will be less than . %, . % and . % for south korea, italy and iran, respectively. the epidemic is expected to end before june , and depending on the effectiveness of treatment, particular cities may see full recovery or zero infection sooner or later than others. it is worth noting that the epidemic progression in south korean cities are found to be more rapid than typical, implying that the authorities might have taken effective measures to control the spread. the predicted progressions for italy and iran, on the other hand, are found to display profiles that are typical of those in the historical archive, and unless more stringent measures are taken, the peaks and subsequent decline of the infection numbers will unlikely come sooner or more rapidly than the predicted trajectories. finally, we should stress that the proposed data-driven coding method is applicable to predicting epidemic progression in any given population and the accuracy of prediction will depend on the adequacy of the available data in allowing a reliable match to be identified from the historical archive. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint feb feb feb feb feb mar mar (d) amelia romagna feb feb feb feb mar mar mar (e) tehran feb feb feb feb mar mar mar . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint feb feb mar mar mar mar (f) gyeongsangnam road feb feb mar mar mar mar mar (j) king kong road feb feb mar mar mar mar apr . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint feb feb mar mar mar mar (j) king kong road feb feb mar mar mar mar apr . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint feb feb mar mar mar mar (j) campania mar mar mar mar mar mar apr . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint feb feb mar mar mar mar (g) sicily feb mar mar mar mar mar apr (j) campania mar mar mar mar mar apr apr . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint feb feb mar mar mar mar (c) semnan feb mar mar mar mar mar apr (e) lorestan feb mar mar mar mar mar apr (i) kum feb mar mar mar mar mar apr (j) isfahan feb feb mar mar mar mar apr (k) gillan mar mar mar mar mar apr apr . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint feb feb mar mar mar mar (e) lorestan feb mar mar mar mar mar apr (i) kum feb mar mar mar mar mar apr (j) isfahan feb feb mar mar mar mar apr (k) gillan mar mar mar mar apr apr apr . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . early transmission dynamics in wuhan china of novel coronavirus-infected pneumonia risk of transportation of novel coronavirus disease from wuhan to other cities in china the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak modeling and prediction of the coronavirus disease spreading in china incorporating human migration data clustering model for transmission of the sars virus: application to epidemic control and risk assessment super-spreaders and the rate of transmission of the sars virus mathematical tools for understanding infectious disease dynamics world health organization. coronavirus disease (covid- ) outbreak coronavirus outbreak in south korea coronavirus cases by country, territory, or conveyance covid- ) cases in italy by region virus testing blitz appears to keep korea death rate low covid- : italy death toll soars to another iranian mp dies of covid- key: cord- -c ezmshe authors: bartolini, barbara; rueca, martina; gruber, cesare ernesto maria; messina, francesco; carletti, fabrizio; giombini, emanuela; lalle, eleonora; bordi, licia; matusali, giulia; colavita, francesca; castilletti, concetta; vairo, francesco; ippolito, giuseppe; capobianchi, maria rosaria; di caro, antonino title: sars-cov- phylogenetic analysis, lazio region, italy, february–march date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: c ezmshe we report phylogenetic and mutational analysis of severe acute respiratory syndrome coronavirus virus strains from the lazio region of italy and provide information about the dynamics of virus spread. data suggest effective containment of clade v strains, but subsequently, multiple waves of clade g strains were circulating widely in europe. we analyzed nasopharyngeal swab (n = ) and bronchoalveolar lavage (n = ) samples from patients with covid- to perform sars-cov- whole-genome reconstruction and mutational analysis. we collected samples in late february and early march, (table ) . at sampling time, all patients reported symptoms such as fever, sore throat, cough, or other respiratory symptoms. two sequences were identical, so we included only of them in the analysis, resulting in total sequences. we named the sequences inmi -inmi for their detection at national institute for infectious diseases and analyzed them together with the previously published inmi and inmi ( ) , along with all the sequences from italy posted to gisaid database by april , . we performed next-generation sequencing (sars-cov- panel) on ion torrent platform (thermo fisher scientific, https://www.thermofisher. com) using shotgun approach for inmi - and amplicon approach for inmi - . after quality control, we generated a median number of . × reads for each shotgun sample and . × for each amplicon sample (ranging from . × to . × ). the sequencing mean depth of sars-cov- ranged from -fold in inmi to , -fold in inmi . we submitted consensus sequences to gisaid. we used the proposed phylogenetic lineage classification (a. rambaut we report phylogenetic and mutational analysis of severe acute respiratory syndrome coronavirus virus strains from the lazio region of italy and provide information about the dynamics of virus spread. data suggest effective containment of clade v strains, but subsequently, multiple waves of clade g strains were circulating widely in europe. according to gisaid phylogenetics, as reported ( ), and clade b ; the clade includes other sequences from eu countries, but no additional sequences from italy. all other inmi sequences cluster with the gisaid g clade, and with the b clade; we focused subsequent analysis on clade b (figure) . the clade b inmi sequences are distributed in main clusters, one including most of the northern italy strains and the other including sequences mainly from central italy. in particular, inmi , which was epidemiologically linked to bergamo (lombardy region), clusters with sequences from central italy (abruzzo region). the other inmi sequences cluster with strains from northern italy. of note, in both clusters the sequences from italy are intermixed with sequences from other eu countries, which can also be seen in the broader phylogenetic analysis on gisaid, in which more eu sequences are analyzed. we have identified synonymous and nonsynonymous substitutions distributed along the whole genome (table ) . each patient showed several amino acid substitutions ranging from to . the g clade-specific single-nucleotide polymorphism a g led the amino acid change d g in the s protein. we observed one additional mutation in this protein, that of c t (l f) in inmi , which is detected in few other sequences in gisaid, interspersed among different non-g clades (m. chiara et al., unpub. data, https://doi.org/ . / . . . ). its location in a marginal region of the gene and the sporadic distribution in different clades indicates repeated occurrence not followed by fixation, consistent with no evolutionary advantage. the s protein in the sars-cov- virus is a chief determinant of the host range and pathogenicity. the virion attaches to the cell membrane by binding the s protein with the host ace receptor ( ) . the d g mutation, located in the putative s -s junction region near the furin polybasic cleavage site (rrar), might have an effect on priming by host cell proteases; however, the real impact of this high-frequency mutation is unclear. the variants c t, c t (located in the noncoding region) and c t (in open reading frame ab, orf ab) were present in all inmi -inmi sequences. these mutations have been detected in several sars-cov- isolates throughout europe and are characteristic of clade g (c. yin, unpub. data). a nonsynonymous substitution d g in membrane glycoprotein was detected in inmi sequence. we detected nucleotide changes in inmi , located in a high variable region of the gene, in adjacent codons of the nucleocapsid (n) gene, two -amino acid changes, r k and g r. n protein, responsible for the formation of helical nucleocapsid, can elicit humoral and cell mediated immune response and has potential value in vaccine development. however, none of the observed mutations has been so far associated with changes in viral pathogenicity or transmissibility. the phylogenetic reconstruction we report suggests possible multiple introduction of sars-cov- virus in italy, supporting previously reported analysis conducted on a more limited number of sequences ( ) ( ) ( ) . the analysis consistently places the strains described in this study in distinct clusters in b clade. no other sequence from italy clusters in b (or gi-said v) clade, indicating the positive effect of containment measures established by health authorities in both italy and china to limit viral transmission directly from china. the same measures were unable to contain a wave of subsequent multiple introductions in italy of strains that were widely circulating in europe, all clustering with clade b . the inclusion of the viral sequences from infections occurring in the lazio region helps to demonstrate the dynamics of virus circulation in italy. in particular, a small number of mutations have been detected in these strains, but the real impact and role that these mutations may have on the pathogenicity and transmissibility of sars-cov- remains to be determined. a limitation of our research is that only a portion of viral sequences, including the sequences from italy, have been published as of april , ; phylogenetic analysis could substantially change when more sequences are made available. continued genomic surveillance strategies are needed to improve monitoring and understanding of current sars-cov- epidemics, which might help to lessen the public health impact of covid- . furthermore, increased sequencing capacity is necessary for contact tracing and enhanced surveillance activity. coronavirus disease (covid- ) situation report- european centre for disease control and prevention (ecdc) a doubt of multiple introduction of sars-cov- in italy: a preliminary overview on behalf of iss covid- study group. whole-genome and phylogenetic analysis of two sars-cov- strains isolated in italy in genomic characterization and phylogenetic analysis of sars-cov- in italy molecular characterization of sars-cov- from the first case of covid- in italy characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov we thank the contributors of genome sequences of the newly emerging coronavirus (the originating and submitting laboratories) for sharing their sequences and other metadata through the gisaid initiative, on which this research is based. we thank salvatore conti and alessandro albiero for their support in ngs sequencing and analysis. dr. bartolini is a senior scientist at microbiology laboratory and infectious diseases biorepository at the national institute for infectious diseases "l. spallanzani."her primary research interests are next-generation sequencing and emerging and reemerging infections. t t t t t t t t noncoding utr c t noncoding utr c t t t t t t t nucleocapsid protein *nucleotide positions refer to the wuhan-hu- reference genome (genbank accession no. mn ). orf, open reading frame; syn, synonymous substitution; utr, untranslated region. key: cord- -e whegrf authors: guglielmetti, lorenzo; chiesi, sheila title: covid- in italy - passing through bitter waters date: - - journal: eur respir j doi: . / . - sha: doc_id: cord_uid: e whegrf the covid- epidemic in italy has shown many shortcomings of the national health care system but it also represents a historic opportunity to reinforce the central health care governance and reduce inequalities across the country the catastrophic impact of the coronavirus disease (covid- ) epidemic in italy has been described previously. [ , ] in a recent editorial, nava and co-authors pay a well-deserved tribute to the almost health care workers who succumbed to covid- in the country in less than months since the first case was reported. [ ] we would like to contribute with some considerations on the shortcomings of italy's response to the pandemic. around the first covid- cluster in northern italy, between the regions of lombardy and emilia-romagna, the explosion in the caseload has been abrupt. [ ] soon, resources became insufficient to provide adequate assistance to all patients who required it. [ ] as the system got progressively overwhelmed, the death toll started to rise. during these weeks working among the communities paying the highest price to the epidemic, we have witnessed the relentless commitment of health care personnel at all levels. it was at once moving and disheartening to observe them struggling to cope with unbearable workloads in a system which was not prepared to support them. faced with exhausting shifts and often without adequate personal protective equipment, health care workers have been unsurprisingly affected by high rates of infection and death. [ ] indeed, as shown by nava and co-authors, % of the casualties among medical doctors occurred in the two aforementioned regions. [ ] an obvious reason for italy's inadequate outbreak response can be found in years of neglect for the public sector, increased private expenditure, and health care budget cuts by governments of all political affiliations. [ ] other major shortfalls stem, in our opinion, from the extreme regionalization of health care, which has led to fragmentation of the decision-making process, increased inequalities, and lack of national coordination. [ ] for instance, covid- testing policies vary widely in italy, where some regions perform mass contact tracing and outreach activities, while others prioritize severely-sick patients. the role of general practitioners in outbreak response activities is also differing substantially across the country. this heterogeneity, coupled with ambiguous communication, has generated inefficiencies and confusion. resource allocation in the emergency was similarly impacted, in particular for the management of intensive care unit needs. as previously highlighted, italy already had a modest number of intensive care unit beds. [ ] during the last years, this number has been progressively tapered due to budget necessities, ignoring repeated warning by experts, [ ] and leading to complete unpreparedness to handle the burden of patients who required intensive support. major differences between regions, even neighboring ones, in terms of respiratory equipment availability were obvious on everyday news. nevertheless, inter-regional collaboration was limited. most patient transfers to relieve overwhelmed intensive care units were indeed performed inside the same region, or towards foreign countries. finally, the lack of standardization also affected patient management, which was guided by regional -not national -guidelines. moreover, participation to clinical trials and access to investigational drugs were easier for bigger centers and university hospitals, where greater research experience and manpower facilitated their implementation. quoting a famous colleague, doctor van helsing from bram stoker's dracula, we will have to pass through the bitter waters before reaching the sweet. once there, though, this historic opportunity for reform should be seized: strengthening our public health care system, reinforcing a central governance, reducing inequalities across regions, and going back from "aziende ospedaliere" (hospital companies) to just hospitals. only then, we will be ready to react efficiently to next pandemic threat while protecting adequately our health care workers. [ ] at the epicenter of the covid- pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation facing covid- in italy -ethics, logistics, and therapeutics on the epidemic's front line an italian sacrifice to covid- epidemic covid- and italy: what next? the toughest triage -allocating ventilators in a pandemic integrated surveillance of covid- in italy the italian health system and the covid- challenge economic crisis, decentralisation and health inequalities: the case of italy, universal health coverage. aida isabel tavares intechopen what other countries can learn from italy during the covid- pandemic cassandra's curse and covid- : why do governments listen to businesses over doctors? key: cord- -i n t authors: candela, massimo; luconi, valerio; vecchio, alessio title: impact of the covid- pandemic on the internet latency: a large-scale study date: - - journal: nan doi: . /j.comnet. . sha: doc_id: cord_uid: i n t abstract the covid- pandemic dramatically changed the way of living of billions of people in a very short time frame. in this paper, we evaluate the impact on the internet latency caused by the increased amount of human activities that are carried out on-line. the study focuses on italy, which experienced significant restrictions imposed by local authorities, but results about spain, france, germany, sweden, and the whole of europe are also included. the analysis of a large set of measurements shows that the impact on the network can be significant, especially in terms of increased variability of latency. in italy we observed that the standard deviation of the average additional delay – the additional time with respect to the minimum delay of the paths in the region – during lockdown is ∼ − times as much as the value before the pandemic. similarly, in italy, packet loss is ∼ − times as much as before the pandemic. the impact is not negligible also for the other countries and for the whole of europe, but with different levels and distinct patterns. at the time of writing, the coronavirus disease (covid- ) pandemic is still ongoing and billions of people are under some form of lockdown. the restrictions faced by citizens are more or less stringent, depending on the resolutions adopted by the different governments, but in many cases non-essential activities have been shut down and a large fraction of people is confined at their homes. many activities that are normally carried out in physical presence are now taking place on-line. as a consequence, the amount of traffic on the internet increased significantly during the last months. in this paper, we analyze the impact of the covid- pandemic on the latency of the internet. latency is one of the major properties of the network and it is becoming every day more important, as several internet applications are particularly sensitive to its fluctuations. ex-amples include on-line videogames [ , ] , video calls, voip [ ] , and ip geolocation [ , , ] . we analyzed a large set of measurements, collected by means of the ripe atlas platform [ ] , to better understand the effects on the network caused by this major change in the way we live. the analysis focuses on italy which, in april , has been under lockdown for more than a month, experiencing some of the strictest limitations enforced by authorities: all schools, universities, and non-essential shops are physically closed, and people are authorized to leave their homes only for undeferrable necessities. distance learning and remote working were applied whenever possible, with a significant increase in usage of virtual-meeting and video-conference applications [ , ] . table summarizes the most important events which could have had an impact on the italian internet latency. as can be noticed, limitations to citizens have been introduced progressively. for this reason, the changes caused by the italian lockdown are studied, in the remaining of this paper, by comparing the situation status of the network, as it is antecedent to all restrictive measures; the latter, on the contrary, comes just after the most restrictive limitations. the period in between corresponds to a transitory phase, where partial lockdowns start to impact the network performance. hereafter, we will use w to indicate the baseline week, and w for the week just after the major lockdown event. besides italy, we include a brief analysis also concerning spain, france, germany, sweden, and the whole of europe. spain, france, and germany have been characterized by restrictions similar to the italian ones. sweden instead decided not to impose a mandatory national lockdown. for spain, france, and germany w is shifted according to their major lockdown event (shown in table ). for the entire europe the situation is more heterogeneous, as some countries were less hit by covid- and thus adopted milder restrictions. for sweden and europe, w corresponds to the week, the last of our observation period which goes, overall, from february through march . results show that the impact is not always the same across the considered countries and on a european scale. the contribution of this paper can be summarized as follows: • some statistics have been recently released by internet service providers (isps), and other players of the internet ecosystem, about the increased amount of traffic they have been exposed to because of the covid- pandemic. however, a picture that leaves aside the very specific points of view of the single operators is still missing. this study provides a more global view, not polarized by the single operator's perspective. in addition, most of the statistics released by operators concern the amount of traffic, with limited (or absence of) information about latency. • the amount of measurements analyzed is large, thus providing solid foundations for the included statistics. moreover, besides the sheer number, we decompose the impact on delay according to the most relevant factors, including the time of the day, the type of target (belonging to a content delivery network or not), and the version of the internet protocol. in addition to icmp-based delay, we provide information also on packet loss and path changes, as they are, more or less, related with internet latency, and on http-based latency. • besides italy, results also concerning spain, france, germany, sweden, and the whole of eu-rope are included. since measurements have been collected using a single platform, results obtained for the different countries can be compared without the possible bias introduced by the adoption of multiple and heterogeneous systems. results show that, in italy, lockdown impacted the latency of the network, especially in terms of increased variability. effects are more evident during the evening, suggesting that latency is more negatively affected by the increased traffic due to recreational activities rather than remote working or distance learning. for the other european countries included in the study, the impact is milder in germany and france, and similar to the italian one in spain and sweden. the remaining of the paper is organized as follows: in section , we summarize the most significant work concerning the detection and analysis of large anomalies occurred in the internet; section describes the data collection phase; in section , the method we followed to compute the performance indexes is explained; the main characteristics of the datasets are illustrated in section , together with a preliminary analysis; section contains the results on the italian internet latency from different perspectives (type of measurements, hour of the day, ipv vs ipv , etc), whereas section shows the results concerning the above-mentioned countries and the whole of europe (with less details compared to italy); section concludes the paper. there is an extensive body of literature about anomalies on the internet. however, the focus is, usually, on the design of techniques aimed at detecting the occurrence of an anomaly (for instance [ , , , ] ). in this paper, we do not try to define another detection technique, as the impact of the covid- pandemic on the network is evident. we strive to provide a view of the effects of the pandemic at a large-scale, including a quantitative evaluation of its main characteristics as seen from the perspective of latency. when natural events assume catastrophic proportions, their effects might be observed also on the performance and reliability of the internet. during the last years, several studies have focused on the impact on the internet of a few catastrophic natural events, such as earthquakes and hurricanes. in [ ] , authors studied the effects of the taiwan earthquake on the asian internet, from the viewpoint of interdomain routing and traffic in research and educational networks. the earthquake damaged submarine fiber cables causing the fail- [ ] . the study analyzes the impact on the internet as seen by a local isp, analyzing both traffic and routing. due to link failures, the internet experienced traffic drops and subsequent peaks mainly due to the reconfiguration of content distribution networks. however, thanks to planned backup and redundancy, the internet proved to be resilient to such an event, and was affected only locally and with minimal damage. another study analyzed the latency variations due to the same event [ ] , as seen from pinger monitors [ ] . the study shows that, after the earthquake, latencies from some monitors experienced a significant increase for a limited amount of time. these studies point out the importance of maintaining redundant internet routes, even if at a cost, as they can be extremely useful when disruptive events occur. similar work was conducted on the effects caused by severe weather conditions, such as the hurricane sandy. in [ ] , authors used ping to test the reachability of edge networks, to discover that the number of outages in the areas affected by the hurricane sandy significantly increased during and after the hurricane. from the interdomain routing point of view, aben shows that a significant portion of traffic was rerouted around the affected area, again demonstrating the internet resilience and the importance of a redundant structure [ ] . similar outages can be observed also at a smaller scale, as pointed out in [ ] . the study uses ping to show that reachability issues can occur at residential hosts in case of moderately bad weather conditions (e.g. thunderstorms). however, unlike large-scale outages, on the small scale the degree of redundancy is generally not sufficient to cope with these events. the vast majority of the studies concerning the impact of natural events on the internet found in scientific literature tackle the problem from a reachability, routing, or traffic point of view. very few analyzed the experienced latency increase in such events as we do for the covid- pandemic. however, we believe that studying latency is of paramount importance, as it can give an indication of the perceived quality of service by end users. in addition, none of the previously studied events reached the size of the covid- pandemic, in both space and time. at the time of writing, the covid- pandemic has been impacting the lives of billions of people all around the globe for several months, while in the other analyzed natural events the impact of the internet was limited in time or circumscribed to a relatively small geographic area. since the covid- pandemic has started only few months ago, there is still little scientific literature focusing on its impact on the performance of the internet. however, several network operators, content providers, and internet exchange points (ixps), released reports about the increased usage of their infrastructure, which we review in the following. cloudfare reported statistics about the traffic increase towards their servers placed in seattle, northern italy and south korea [ ] . in particular, cloudfare reports an increase of % of the overall traffic in northern italy and a reduction of the traffic coming from fitness trackers perhaps reflecting the scarce mobility induced by social distancing. similarly, fastly reported traffic and download speeds towards their servers [ ] . various countries were considered, including italy, which had a . % increase in terms of traffic and a . % decrease in terms of download speed on average. de-cix, one of the world's biggest ixps, reported a new traffic world record of . tbps [ ] , as well as a % increase in video conferencing traffic and % of social media traffic. finally, the organisation for economic co-operation and development (oecd) released a report that aggregates the various traffic increase information reported by internet operators in a single document [ ] . such report highlights some important numbers, among which: (i) an increase up to % of traffic reported in many ixps and isps; (ii) an increase up to times higher of the volumes of traffic for video conferencing platforms. all these reports focus on the increase of internet traffic and not on latency. we believe that latency is extremely important as, without information about capacities, traffic itself cannot be used for an estimation of the perceived performance of the internet by end users. moreover, as already mentioned, all the currently available studies are limited to the boundaries of the organizations that provided them. one of the few papers focusing on this topic, from a scientific perspective, is the one by favale et al. [ ] , where the impact of the covid- pandemic is observed from the campus network of an italian university. favale et al. highlight a time decrease in incoming traffic and an increase of . times in outgoing traffic, as a consequence of remote learning activities. moreover, using passive measurements collected using tstat [ ] and application logs, they studied the fruition and performance of their in-house distance learning system. the raw latency data used for this study was collected by ripe atlas [ ] . we then filtered and enriched such data as detailed in the following subsections. ripe atlas is a globally distributed internet measurement platform that produces more than measurements per second [ ] . among the open platforms aimed at measuring the internet, ripe atlas is the one with the largest number of vantage points [ ] , and it has a massive presence in europe. ripe atlas automatically carries out anchoring measurements (ams), where the set of targets is pre-defined. in particular, a large set of devices called probes periodically perform measurements towards other devices called anchors. anchors are usually hosted in ixps, in the operation centers of isps, or in data centers. hence, they enable monitoring of the core infrastructure of the internet. the results produced by ams have been extensively used for both research (see, for instance, [ ] , [ ] , [ ] , [ ] , [ ] ) and operational purposes (for example dnsmon [ ] , a service aimed at monitoring the worldwide core dns infrastructure). additionally, ripe atlas allows its users to collect measurements towards arbitrary targets. results of user-defined measurements (udms) are stored in a database from where they are accessible to the public (access is not restricted to the experimenters who triggered them). ripe atlas, for its measurements, relies on classical network tools. the latency from an atlas node to a target is estimated by means of the ping tool which, as known, makes use of icmp echo requests and echo replies. for ams, ping is launched to collect three round trip time (rtt) values. for udms, the default number of collected rtt values is again three, but this number can also be changed by the experimenter. our analysis of the impact of covid- pandemic relies on the results generated by both ams and udms. starting from the dates of the events reported in table , we considered all ams comprised in the interval from the th of february to the th of march . the set of probes and anchors involved in the measurements is stable, with little variations caused by the possible temporary unavailability of probes. since ams are performed periodically and for the entire period of study, they provide information on the internet latency from a stable point of view. however, to be sure to eliminate measurements occurring in a short time frame and thus not covering appropriately the observation period, we further filtered the selection to contain only the measurements concerning source-target pairs that produced successful results in at least different days ( % of the total time frame). the position of source and target nodes is fundamental to analyze the impact on a country-level basis. for ams, the position of both source and target nodes is well-known, as such information is provided for each node participating in the platform. we use such information to select a subset of the ping measurements having both source and target in europe. from now on we will refer to such a subset as the am-derived dataset (amd). amd is composed of more than . billion rtt values generated by source-target pairs during the monitored time period. users of the ripe atlas platform can define their own latency measurements according to their needs and in-terests. they can select the set of targets to be probed, define the periodicity of probing, and set the time-span of their measurement activities. in udms, targets frequently include the servers of major internet companies, dns servers, or privately owned network resources. as a consequence, the set of targets involved in udms is heterogeneous. sources are always a subset of ripe atlas nodes, but the subset can be different from user to user. for our analysis, we are interested in a subset of udms where measurement activities were possibly scheduled before the covid- outbreak in europe and repeated periodically throughout the observation period. to obtain such a subset, we adopted the following strategies. first, we extracted only periodic latency measurements, i.e. configured to be automatically repeated after a certain amount of time. second, we discarded the measurements configured to collect less than three latency values per ping execution. third, similarly to amd, we filtered the selection to contain only the measurements concerning source-target pairs that produced successful results in at least different days. fourth, we restricted measurements to the ones targeting ip addresses in europe. it is important to notice that in udms only the location of the source nodes is wellknown, as it is provided by ripe atlas. hence, for this step, we estimated the position of the targets by using ripe ipmap [ ] . ripe ipmap uses active geolocation, and it has been reported to be % accurate at continent level, . % at country level [ ] , and . % at city level [ ] . additionally, we used maxmind ge-olite [ ] as a fallback tool, in case of failed ip geolocation with ripe ipmap. measurements where the target was not successfully geolocated using these two tools were discarded. finally, in some cases, we had to limit the amount of extracted information due to the almost unmanageable volume of data in the repository. in particular, when the number of targets in the geographic area of interest was too large, we randomly selected targets and the analysis was restricted to them. even when we had to limit the number of targets, the number of sources from which measurements were started was not subject to any limitation. from now on we will refer to the dataset built according to the above-described procedure as the udmderived dataset (udmd). udmd is composed of more than billion rtt values generated by sourcetarget pairs during the observation time period. let d be the delay of a given internet path. it can be roughly expressed as d = d tra + d pro + d que , where d tra is the transmission delay, d pro is the propagation delay, and d que is the time spent because of queues and processing at intermediate routers and target host. in a wide-area scenario like the one considered, d pro amounts to a significant fraction of the overall delay, as signals travel at approximately km/ms in fiber. moreover, the only component that is going to be affected by increased traffic is d que . to isolate d que from the other terms, the d tra + d pro component can be estimated as the minimum delay observed in a set of latency measurements collected on a given path. the larger the number of collected samples, the better the quality of the estimate: intuitively, the collection of more samples increases the probability of finding lightly loaded network conditions. this approach is not novel and it has been followed in other studies to characterize latency variations on a large scale. in [ ] , for each hostpair the difference between the maximum and minimum rtt observed in a time bin was calculated. then, the evolution of the obtained difference values was used to investigate on transient congestion. the use of the minimum observed rtt as an approximation of the fixed delay associated with a path was adopted also in [ ] , to study the variability in tcp connections. delay variation metrics are discussed in rfc [ ] , where one of the most widely implemented formulations is based on the use of the packet with the minimum delay in the sample as the reference packet. in particular, packet delay variation is defined in rfc as the one-way delay of the considered packet minus the one-way delay of the packet with the lowest value for delay over the current test interval. using the minimum delay as the basis for delay variation is the preferred method also according to the itu-t y. recommendation [ ] (albeit when considering -point packet delays, i.e. one-way delays computed by means of the absolute arrival time at destination minus the departure time at the source host). starting from the above considerations, we defined the following method. each source node, say a, measures the rtt towards a target, say b, using the ping command at time t, which produces a list of delay values d ab t = {rt t , rt t , ..., rt t n }. let us also define d ab min,t = min d ab as the minimum, average, and maximum value found in the execution of the ping command at time t from a to b. first, we found the global minimum value observed for each source-target pair as where o is the entire period of observation. since the observation period we considered is relatively long, it is possible that some path changes occurred, i.e. that the set of traversed routers was not always the same. this is not a problem, provided that some of the above concepts are reformulated appropriately. in a scenario where path changes occur, m ab is an estimate of the transmission and propagation delay of the best path between a and b, among all the paths followed during the observation period. then, we used m as a baseline to estimate the additional time experienced for every single pair of nodes. in particular, we computed for each couple ab of nodes, and for each t ∈ o. the values of q min,t , q avg,t , and q max,t of all source-target pairs in the region of interest were then grouped in buckets with a duration of minutes and averaged. this last step originates from our interest in evaluating the impact of the changed style of life on a large scale, for instance at the country level. more formally, let us call r min,k , r avg,k , and r max,k the average values obtained in the kth bucket t k : r min,k = avg q ab min,t r avg,k = avg q ab avg,t r max,k = avg q ab max,t with t ∈ t k and across all ab pairs located in the region of interest. in practice, r represents the average additional time with respect to the best path ever followed during the whole observation period. note that the average is computed for all measurements occurred in that specific bucket and across all source-target pairs located in the region of interest (e.g. all source-target pairs in italy); as a consequence, the value of r is generally greater than zero. to have r min,k equal to zero, all considered ab couples should experience, almost simultaneously, their best rtt (m ab ) during the kth bucket, which is unlikely. the smallest observed delay found for a source-target pair (m) provides an indication of the "uncompressible" component of the delay for such pair and, as such, can be excluded in a study aimed at evaluating the impact of the pandemic. in this section, we provide a picture of the datasets in terms of geographical extension and overall rtt dynamics. figure a shows the distribution of distances of source-target pairs in italy, for both amd and udmd. in detail, the distances between hosts are computed as great circle distances (gcds), i.e. the length of the shortest path on the surface of the earth. actual distances travelled by packets are generally larger than the corresponding gcds because of circuitousness of internet routes (deviations from shortest physical paths can be significant [ ] ). distances span a couple orders of magnitude, from few kilometers, when source and target are in the same city or metropolitan region, to almost one thousand kilometers. figure b shows the distribution of distances of european source-target pairs limited to targets (but using all sources). for the european data, we had to limit the number of source-target pairs, because to compute the distance both source and target have to be located at city level, and active geolocation is expensive. for both italian and european data, the distances reported in figures a and b are the ones for which we have been able to compute the position of the involved nodes at city level (approximately - % of the considered couples). we believe that the distributions shown in figure are reasonably close to the ones of the complete datasets. the distributions of minimum rtt value observed for each source-target pair (i.e. m) located in italy and in the whole of europe are shown in figure c and d, respectively. the wide range of values, from few milliseconds up to ∼ ms in italy, and up to ∼ ms in europe, originates from the geographical extension of the considered areas. figure a shows the raw rtt values collected in italy, ams, before any processing. more in detail, the scatterplot has been produced using just : of the values to make the image readable. the largest values tend to be slightly higher after lockdown events. however, a trend does not clearly emerge from raw values, probably because of the large amount of values and because they are related to paths with very different lengths. the red curve that is also shown in figure a case, increased variability can be observed in the righthand side of the figure because of lockdown. the period of fluctuations suggests that daily activities may play a role and for this reason they are further analyzed in section . . figure b shows the absolute values, again for the italian ams, but now grouped according to the method defined in section . the d min values have been grouped in buckets and averaged, similarly to r min , but without subtracting m. let us call such values s min . the curve is approximately the same shown in figure a , but with less spikes as the size of buckets is minutes. figure c shows the same s min curve, but now together with r min . the scale is different to have a better view of the phenomenon. the r min curve is very similar to the other one, but translated downwards, as now the m value found for each pair is subtracted. figure d shows the th percentile of q min , again grouped in buckets of minutes. it is thus computed similarly to s min , but now using the th percentile instead of the average. variability of delays is even more visible in this case. we do not provide an analogous discussion about the european dataset and/or udmd for the sake of brevity. in the remaining of this paper, we base the analysis mostly on r, as we are interested in evaluating the impact of the pandemic on the variable component of latency (with the exception of section . where we also include s). the phenomenon is principally observed in its evolution along the line of time, as we wish to understand if, and to what extent, the lockdown measures, enforced in italy and in the other considered european countries, had an impact on the network. we studied the impact of the covid- pandemic on the latency of the italian internet from different perspectives: when both source and target are located in italy or just one of the two, when considering the time of the day and workweek/weekend, and when taking into account the version of the internet protocol. we also studied the observed latency when the target is part of a content provider network. beside icmp-based latency, an evaluation of latency as seen at the http level is provided. finally, a subsection also shows the path changes occurred at the as-level as they can be related to the phenomenon under observation. we start our analysis from the measurements in amd with both source and target in italy. we first analyze the values of s measured in italy. we recall that s is the average of the d values in each bucket, as defined in section . figure a shows the evolution of s min , s avg , and s max for the whole observation period. we notice that all values progressively increase over time. since italy experienced some partial lockdown events before the most restrictive one, there is no step-like increase, rather a continuous one. however, approximately from february , when schools were closed in northern italy, delays start to grow and higher peaks can be observed. the largest increase can be observed on march , which is the date of the complete italian lockdown. the increase appears to be quite significant, for example if we consider the s min line, the peaks during the lockdown are approximately % higher than the ones before the lockdown. besides the generally increasing value of all the three curves corresponding to the three s variations, the higher variability of latency is also evident. to evaluate the increased variation we compared the value of s min in w and w . the average s min experiences a . % increase in w , and the s min standard deviation experiences a . % increase. the measurements in udmd show a similar pattern, but more accentuated, as can be seen in figure b . in fact, in w , the average s min is . % higher than in w , and the s min standard deviation is . % higher. it has to be noticed that the s values in udmd show a little drop around march , which lasts some hours. we investigated on this aspect, and found that one of the targets of the measurements was unreachable in that interval. the target involved multiple source-target pairs, corresponding to approximately / of the measurements, which usually were providing relatively high absolute delays. in other words, this is an artifact due to a temporary lack of measurements, which can happen when measuring real world devices. similar patterns can be observed when analyzing the evolution of r min , r avg , and r max for both amd and udmd, which can be found in figures c and d . in this case however, the curves are translated downwards, as expected. this reflects on the increment of the average r min value in w with respect to w , which is . % in amd and . % in udmd, but not on the increment of the r min standard deviation value, which remains almost unchanged: . % in amd and . % in udmd. similar considerations can be made about measurements with sources in italy and targets spread all over europe (excluding italy), as shown in figures a and b for amd and udmd respectively. also in this case, the generally increased variability during lockdown is clearly visible. when considering measurements with sources in europe and targets in italy, as shown in figures c and d , the situation is a bit different between amd and udmd. in particular, for amd the pattern is approximately the same as in figure a . for udmd, instead, the pattern is quite different. an increase of the overall variability is still noticeable, however the three curves appear to be much more squeezed on top of each other, i.e. the r min and r max curves are much closer to each other than in the other scenarios. it is also worth to notice that, especially for amd, the local minima of the r min curve tend to get higher during the the transitory period but then they start to get lower. this is particularly visible in figures a and c . the local minima (the troughs) correspond to night hours, when the network is lightly loaded. this phenomenon could be explained by the infrastructural enhancements introduced by network operators to respond to the crisis. for example, during the transition period, tim (the italian incumbent) started peering again in public peering lans of italian ixps for the first time since the end of [ ] . also, ixps reported an increase of traffic of - %, which pushed them to introduce upgrades in the capacity of their peering lans, as reported during the italian network community meeting held for the occasion [ ] . such improvements could also jus- tify the situation in figures a and c , where the troughs during the lockdown reach smaller values compared to the one of the first analyzed week. we estimated the impact of the lockdown in terms of packet loss, as the fraction of unsuccessful echo request/reply. figure shows the evolution of the packet loss for the whole observation period, for both amd and udmd. both datasets show an increase of packet loss over time, especially after the lockdown start. this is consistent with the previous results, and indicates a generally increased congestion due to the lockdown. we computed the average packet loss in w and w . the increase is significant: from . e- to . e- (+ . %) for amd, and from . e- to . e- (+ . %) for udmd. also the standard deviation of the packet loss rate in w increases significantly compared to w : from . e- to . e- for amd (+ . %), and from . e- to . e- for udmd (+ . %). we notice that in amd there is an increase of the packet loss that lasts approximately days, around february -march . we investigated this behaviour and found that the increase is not attributable to a narrow subset of the source-target pairs. we did not register any disconnections of sources nor targets, and the same packet loss pattern can be seen in measurements directed to % of the targets, although with different intensities. these targets are geographically spread all over italy. in particular there are two targets that contribute in a significant manner, which are located in milan (at the milan internet exchange) and monopoli (puglia region), respectively (we point out that measurements towards one target involve multiple source-target pairs). the temporary increase of packet loss is not reflected in the r curve, as for the involved source-target pairs the packets that are correctly delivered experience a negligible increase of the rtt. the three curves shown in figures and show repeated peaks and troughs. this is particularly evident during lockdown, and suggests that latency gets more influenced by weekly or circadian rhythms. to evaluate the influence of the day of the week, we separated the italian measurements in those run during work days and those run during weekends for w and w . results can be seen in figure , which shows the boxplot of r min in w and w for work days and weekends. in both w and w the weekend values are slightly higher and show higher variability. in addition, a significant increase can be observed in w compared to w . to evaluate the influence of the time of the day on latency, we divided italian measurements in one-hour slots and aggregated them when executed in the same hour. then, we calculated for each slot the ratio between the average r values collected during w and during w . results are represented in figure a form across the time of the day. night hours show no considerable increase. this is not surprising, as human activity is very limited at night, thus also the congestion of the internet. morning hours show some little increase. afternoon hours show a more evident increase, but the highest increase occurs between : utc and : utc, with a peak between : and : utc. this is interesting, as, combined with the results of weekend highlighted in figure , it suggests that remote working and distance learning have some impact on the italian internet latency, but the major effects can be attributed to leisure activities which typically occur in the afternoon/evening and in weekends, such as gaming or video streaming (the reader has to keep in mind italy is utc+ in the analyzed period). this could be due to the lack of other recreational activities during the lockdown. to deepen on this aspect we computed the empirical cdfs of the r min values in peak and off-peak hours for w and w (figures a and b) . for peak hours, we considered the interval : - : , for off-peak we considered the interval : - : . we can notice that in both peak and off peak hours the cdfs for w and w are clearly separated, and the cdf for w shows the highest values. however, it is clearly visible that while in off-peak hours the two cdfs are very similar, in peak hours the cdf for w shows a substantial increase of the r min values (note that the x axis scale in the two figures is different). in section . , we highlighted as in night hours the values of r min are particularly low during the lockdown, hour of the day hour of the day figures a and c the local minima during the lockdown seem lower than the ones before the lockdown. to evaluate this effect we computed the ratio between w and w for the two cases depicted in the aforementioned figures. the results are shown in figures b and c , respectively. in both cases the ratio goes slightly below one during night and morning hours. especially surprising is figure c where the the ratio goes below one for the entire morning. this could happen as in figure c we consider sources outside italy, thus the access network is still not involved in a lockdown phase, and targets in the italian infrastructure, which as mentioned has been improved to cope with the traffic increase. during night and morning hours the load on the network is still light, so in this particular configuration the performance could increase. to conclude, in evening hours the increased internet us- age during lockdown generally produces larger delays, but in periods of lighter load the network is sometimes more efficient than before the lockdown. we further analyzed amd taking into account the version of the ip protocol. to perform a fair comparison we only selected measurements run by dual stack probes (i.e., probes which have both ipv and ipv connectivity). this means that for both ipv and ipv we consider measurements run between the same sources and targets. we found . million ipv rtt values and . million ipv rtt values with both source and target in italy. figures a and b shows the three r variations for ipv and ipv , respectively. ipv latency, in italy, seems to be characterized by larger variability compared to ipv latency, independently from the lockdown period. such variability increases even more during the lockdown period. however, for ipv a reduction of minimum r min values is particularly evident, probably due to the network improvements introduced by operators. in particular, a significant drop around march is visible. we investigated on this aspect and found that a subset of measurements were initially originated by source-target pairs which shared a common upstream provider and were flowing through non national paths (mainly via germany, switzerland, and netherlands). in correspondence of the observed drop, the measurements involving these source-target pairs start to flow through local paths via other providers. these paths show considerably smaller latencies. after few days, the original configuration is restored. we further studied the latency experienced between : utc and : utc (the peak hour previously identified) by the two protocol versions. in particular, we compared the values collected during w with the values during w . for ipv , values of the r min average show a . % increase in w . for ipv instead, the increase is more modest, . %. both show a similar increase in variability in w : . % ipv , and . % for ipv . to conclude, ipv in italy is characterized by a generally higher variability than ipv , but in peak hours the former has been impacted less than the latter by stayat-home orders. this is not surprising, as ipv and ipv are generally served by different infrastructures, and follow different paths [ ] . in addition, ipv is not as common as ipv in domestic connectivity, and this could justify the minor impact of the lockdown on latencies observed in ipv measurements. the same analysis was not repeated using udmd, as the relatively limited amount of ipv -based udms does not allow us to produce statistically sound results. since a large fraction of traffic is nowadays directed towards content providers, which we suspect also being related to most of the evening traffic (e.g. video entertainment), we investigated the impact of the lockdown on the latencies towards youtube. we collected measurements towards the youtube content delivery networks (cdns), which is used to serve video content. youtube operates elaborated server selection strategies [ ] which could lead to inaccurate results. to avoid this, we operated as follows: (i) we first mapped the names associated with the ad-hoc youtube cdns (googlevideos.com and ggpht.com) to the ip addresses that are used to serve con- tent in italy. for this purpose, we used all the ripe atlas probes in italy to run multiple dns queries in order to obtain the ip addresses associated with the youtube servers. (ii) after obtaining all the visible ip addresses of the youtube's servers ( addresses), we used the anycast detection service offered by ripe ipmap, which uses active measurements from ripe atlas probes to detect if an ip is anycast or not. (iii) then, we used again ripe ipmap to geolocate the ip addresses of the servers. the servers not in italy were discarded. (iv) finally, we used these ip addresses to extract ripe atlas measurements targeting them. we only selected icmp ping measurements (which are not subject to http or application layer redirects). we found approximately million rtt values towards youtube's servers located in italy. figure shows the three r variations we obtained. an increase of the overall latency and its variability is visible during the days of the lockdown. the standard deviation of r min is approximately . % higher in w with respect to w . the average r min instead increases by . %. our hypothesis that the increase of rtt registered in the evening hours is due to people forced to stay home and using the internet for entertainment, is thus strengthened by the results of the measurements towards youtube. figure a shows measurements collected between : utc and : utc while figure b shows measurements collected between : utc and : utc, for the entire observation period. also in this case, the r min during night hours slightly improves during the lockdown. the r min in the evening gets moderately higher during the transitory period and abruptly increases after the first day of complete lockdown. we acknowledge that youtube is not fully representative of all content providers networks, in fact our initial purpose was to collect measurements towards facebook and netflix as well. however, we did not find in ripe atlas enough measurements towards facebook and netflix servers to cover the whole observation interval and obtain statistically significant results. to further improve our analysis, we looked for anomalous patterns in the level of path changes between the sources and targets of our measurements. we consider path changes at the autonomous system (as) level, as such level is the one impacting the most on the geography of the paths, while the ip level is subject to various known artifacts and intra-as load balancing [ ] . from ripe atlas, we collected icmp traceroute measurements to match the italian sourcetarget pairs of our analysis. for amd, this was rather straightforward as, besides icmp ping measurements, ams also include other types of measurements such as icmp traceroutes. for udmd, instead, we could not find enough traceroute measurements to match the source-target pairs of the ping measurements, thus we restricted our analysis to the amd source-target pairs. to extract the as paths from the traceroute ip paths, we use the following methodology: initially, we match all the ip addresses against the internet topology data kit [ ] produced by caida. this step converts different ips belonging to the same router to a single one. then, we remove the first ip hop inside the network of the probe's host, as it is usually fixed. this step allows us to avoid most of the artifacts introduced by the icmp rate limiting happening close to the probes [ ] . if an ip path includes series of wildcards (i.e., non responding hops), these are squashed into a single one. then, each ip is converted to the as number originating the prefix such ip belongs to. for this step we use the longest-prefix matching on ripe ris data [ ] . some addresses cannot be mapped to an as because they are not available in the ripe ris data (e.g. not announced publicly), or, more frequently, they are part of the private address space (rfc ) [ ] . we discard such unmappable addresses. this approach has been previ-ously described by hyun et al. [ ] . more sophisticated approaches are of difficult application due to the size of our dataset. moreover, our main interest is to quantitatively estimate the variation of the path changes over time, and not to analyze the internet as-level topology. we then divided the observation period in buckets of one day each, and for each bucket we computed the fraction of source-target pairs that incurred in at least one change of as path in that bucket. figure shows the fraction of path changes over time for amd. the fraction of source-target pairs experiencing path changes each day is rather uniform on all the observation period, between . and . , which means that each day just - % of source-target pairs experienced a change of as path. however, by comparing w and w from a numerical point of view, we found that the average fraction of path changes per day is . % higher in w . thus, there seems to be a slight increase of the number of path changes per day due to the impact of lockdown. in the previous sections, we reported results obtained via ping measurements based on icmp echo requests. this type of traffic is used for network diagnostics and sometimes routers can treat it differently from traffic generated by end users, even if they usually behave similarly in terms of rtt [ ] . even if the trends highlighted in the previous sections by icmp show an evident increase of the network congestion caused by the covid- lockdown, we also analyze the latencies produced by ripe atlas http anchoring measurements (based on tcp). these kinds of measurements are quite different from the icmp measurements which we analyzed above. in particular, each http measurement collects just one sample (instead of at least three for the icmp ping ones). in addition, the latencies that can be extracted from this measurements include the time for sending an http request to a server and waiting for the http response body to arrive. this time includes the time needed for the server to build the response. the time needed for the dns resolution of the server address, instead, is not included. for this reason, it must be noted that the latencies extracted from http measurements are not directly comparable with the latencies obtained by means of icmp ping. we analyzed the latencies extracted from http measurements with the methodology described in section , however, being each measurement composed by just one sample, we will not have d min , d avg , and d max , but just d, and therefore just q and r. figure a shows the results for measurements with source and target located in italy. the figure shows an increase in the latency due to the lockdown. like in the previous results, the increase is progressive and not step-like, as we recall that italy experienced multiple partial lockdowns before entering in the strictest one. a step-like increase is noticeable in correspondence of the increase of the packet loss showed in section . . in this case the packet loss affects the performance of http as, differently from icmp, the packet loss triggers retransmissions of the tcp protocol. consequently, the end-toend latency of the tcp connection increases. besides this anomaly, the overall increment is noticeable also by comparing average and standard deviation values of r, which are respectively . % and . % higher in w with respect to w . in figure b , the failure rate of http measurements is shown. such failures are due to a combination of network errors which include connection timeout, host unreachable and network unreachable problems. also in this case an increment of the failure rate can be noticed as long as the italian lockdown becomes more restrictive. these results allow us to con-firm that the increased latency, in italy, during lockdown is not limited to network diagnostic traffic but also to end user traffic. in this section, we show how the latency in other european countries was affected by the covid- pandemic. we considered spain, france, and germany, which had their major lockdown events respectively five, seven, and twelve days after italy. in addition, we considered sweden, as an example of a country that adopted a less formal lockdown policy. finally, we considered all european countries together. overall results are depicted in figure . table reports the increment of the average and standard deviation of r min and packet loss after stay-at-home orders. to obtain the increment, for each country we compared the first week of measurements (w ) and the first week of lockdown (w ). for germany, the considered periods are four day long instead of seven: germany was put on lockdown at the end of our observation period and a full week was not covered by the collected data. since there is not a unique date for lockdown in the whole of europe, and since some countries did not even enter a lockdown phase, for sweden and europe we compared the first and the last week of the observation period. the two weeks used for comparison are highlighted in light grey in figure . in the following we analyze the considered countries in detail. spain shows high variability of latencies also before the lockdown. however, an increase due to enforced restrictions is noticeable in amd. similarly, circadian patterns become more evident (figure a ). this is confirmed also by the summary statistics, which show an in-crement of . % of the r min average, and of . % of the r min standard deviation, in w compared to w . the visual analysis of udmd shows a progressive increase of latency which starts much earlier than the lockdown, with a generally spread variability (figure b ). in fact, results in table show a significant increment in the average r min of . %, but a modest . % for the standard deviation. on march , a temporary increase can be observed in figure b . we investigated this phenomenon and found that it is due to a considerable increase in the latency towards one target, observed from multiple sources. we believe this to be an anomalous behavior due to the target itself or the network in its proximity. the analysis of the packet loss shows opposite behaviours between amd and udmd. in amd, the packet loss decreases during the lockdown, while in udmd increases greatly. in france, the situation is different, as shown in figures c and d, which show amd-and udmd-based results respectively. in amd, the overall increase of latency is barely noticeable. lockdown seems to accentuate the periodic fluctuations due to circadian rhythms. in fact, the summary statistics included in table show even a decrement of r min average and standard deviation in w compared to w , in the amd. however, it must be noticed that the first week of measurements in amd appears as particularly noisy if compared to the other pre-lockdown weeks. udmd instead show much higher variability, which is the attribute that shows the most evident impact of the lockdown. in fact, the analysis shows an increment of just . % for the r min average and . % for the standard deviation of r min . the packet loss increases during lockdown for both amd and udmd, in both average and standard deviation. in germany the situation is different from both spain and france. in amd, the effect of lockdown is noticeable only in terms of amplified circadian patterns (figure e) . the analysis confirms this, by showing just a . % increase of the r min average, and a . % increase of the r min standard deviation. in udmd, a more significant increase of latency is visible (figure f) , which corresponds to a . % higher value for the r min average and a . % higher value for the r min standad deviation in w compared to w . it is worth noticing that in germany the degradation of latency starts more than one week before the lockdown. this happened because germany, like italy, proceeded to some partial lockdowns and school closures before the major restrictions. the average packet loss slightly increases for udmd, and decreases for amd. as mentioned, sweden adopted less formal restrictions. for this reason, results shown in figures g and h are definitely interesting. both amd and udmd show a progressive increase of r and of its variability in the considered time period. this can mean either that swedish people autonomously increased social distancing and implemented stay-at-home policies as suggested by the swedish government, or that the performance of the swedish internet infrastructure has been affected by the lockdown imposed in other countries. also the comparison of w and w show a significant increase of r min average and standard deviation: . % and . % for amd and . % and . % for udmd. the packet loss increases just for amd, but decreases for udmd. figure i and figure j show respectively the amd-and udmd-based results for all of europe. additional latency is generally smaller than the individual countries we analyzed. the response to national lockdowns seems to be fairly good even if we notice an accentuation of the variability due to the circadian activities, starting from the italian lockdown, but not a significant increase of the overall latencies. this is confirmed by the statistics reported in table : the r min average is subject to a modest increase, equal to . % and . % in amd and udmd respectively, while the r min standard deviation experiences a significant increase, equal to . % and . %. these results seem to indicate that, on a continent-level scale, the impact of lockdown is still noticeable but without dramatic changes in observed performance. the packet loss instead decreases for amd and is almost unchanged for udmd. it is worth to notice that, after analyzing the packet loss in the different countries, we cannot conclude that an increase in latency is coupled with an increase in packet loss. in fact, in some cases we found increasing latency and decreasing packet loss, and vice-versa. it is well-known that computer viruses may cause a slowdown of the internet [ , ] . in we all learned that also biological viruses may affect the global internet performance, because of the changes they bring in the way we live. in this paper, we analyzed the impact of the covid- pandemic on the latency of the internet on a large scale. latency is particularly important not only because it has a profound effect on some classes of applications, but also because it is, by itself, an excellent indicator of the health status of the network. results, which have been obtained from the analysis of a large amount of measurements, show that the impact of the increased on-line activities is relevant, especially in terms of higher variability. the major changes have been observed in the evening, the time of the day when most of the on-line activities are related to entertainment. this suggests that distance learning and remote working contributed to a lesser extent in terms of additional network latency. results obtained for the considered countries show relevant differences, which can be due to the resilience levels of their network and/or to the non-uniform restrictions imposed by authorities. we believe that the provided numbers and the related analysis, despite being limited to a portion of the internet, definitely help in better understanding this previously unseen event in the history of the internet. latency and player actions in online games the effects of latency on player performance in cloud-based games assessing voice quality in packet-based telephony geolocation of internet hosts: accuracy limits through cramér-rao lower bound smartphone-based geolocation of internet hosts using ripe atlas for geolocating ip infrastructure ripe atlas: a global internet measurement network our commitment to customers during covid- zoom cfo explains how the company is grappling with increased demand detecting anomalies in network traffic using maximum entropy estimation on the detection of network traffic anomalies in content delivery network services measurement of large-scale bgp 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measurement platforms and related standardization efforts vantage point selection for ipv measurements: benefits and limitations of ripe atlas tags pinpointing delay and forwarding anomalies using large-scale traceroute measurements disco: fast, good, and cheap outage detection joint minimization of monitoring cost and delay in overlay networks: optimal policies with a markovian approach systems for characterizing internet routing visualization and monitoring for the identification and analysis of dns issues tracing cross border web tracking ripe ipmap active geolocation: mechanism and performance evaluation why is the internet so slow?! proceedings of the rd acm sigcomm conference on internet measurement, imc ' , association for computing machinery packet delay variation applicability statement internet protocol data communication service -ip packet transfer and availability performance parameters on the spatial properties of internet routes italian networking community meeting summary measuring the deployment of ipv : topology, routing and performance dissecting video server selection strategies in the youtube cdn proceedings of the th acm sigcomm conference on internet measurement, imc ' periodic path changes in ripe atlas routing information service (ris) traceroute and bgp as path incongruities, tech. rep., cooperative association for internet data analysis (caida) on evaluating the differences of tcp and icmp in network measurement the internet worm program: an analysis worm epidemics in high-speed networks alessio vecchio is an associate professor at the university of pisa, italy. he has been involved in several national and eu-funded projects (congas, neutmon, mecperf). he is currently serving as associate editor of pervasive and mobile computing and ieee access. he co-launched and co-organized five editions of the permoby workshop (an ieee percom workshop focusing on human mobility). he has been tpc chair of the seventh ieee international workshop on sensor networks and systems for pervasive computing (ieee percom persens) and in the technical committee of many other international events. key: cord- -pg zc o authors: delogu, mauro; cotti, claudia; lelli, davide; sozzi, enrica; trogu, tiziana; lavazza, antonio; garuti, giacomo; castrucci, maria rita; vaccari, gabriele; de marco, maria alessandra; moreno, ana title: eco-virological preliminary study of potentially emerging pathogens in hedgehogs (erinaceus europaeus) recovered at a wildlife treatment and rehabilitation center in northern italy date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: pg zc o simple summary: most of the newly emerging infections arise from animal reservoirs, frequently represented by wildlife species. western european hedgehogs (erinaceus europaeus) are mammalian hibernators, mainly nocturnal and insectivorous, living in natural open and green spaces as well as artificial, rural and urban, areas. they are generalist predators of macro-invertebrates, but they may also eat meat, bird eggs and on occasion pet food. these ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possibility of intra- and interspecies interactions accounting for the possible involvement of e. europaeus in the ecology of several potentially emerging pathogens, including coronaviruses. using pcr-based and virus isolation methods, we found that . % of hedgehogs’ fecal samples were pcr-positive for erinaceus coronaviruses (ericovs). we did not observe any clinical disease related to the ericov infection in hedgehogs. however, the high mutation rates characterizing members of the coronaviridae family and their potential successful interspecies host jumps—as that likely occurred in the novel coronavirus ( -ncov) emergence—should be considered in the management of hedgehogs admitted to multi-species wildlife rehabilitation centers, recommending their return back to the original recovery areas. abstract: the western european hedgehog (erinaceus europaeus) is one of the four hedgehog species belonging to the genus erinaceus. among them, e. amurensis is extant in east asia’s areas only, whereas e. europaeus, e. roumanicus and e. concolor are mainly found in europe. e. europaeus is endemically distributed from western to central and southern europe, including italy. western european hedgehogs’ ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possible involvement of e. europaeus in the ecology of potentially emerging viruses, such as coronaviruses, influenza a and influenza d viruses, canine distemper virus, pestiviruses and aujeszky’s disease virus. we examined e. europaeus individuals found injured in urban and rural areas of northern italy. of the fecal samples collected and tested for the above-mentioned pathogens by both pcr-based and virus isolation methods, were found pcr-positive for betacoronaviruses belonging to lineage c and related to the known erinaceus coronaviruses (ericovs), as determined by partial sequencing of the virus genome. our findings suggest that hedgehogs could be considered natural reservoirs of covs, and also act as chronic shedding carriers of these potentially emerging rna viruses. western european hedgehog (erinaceus europaeus) is one of the four hedgehog species belonging to the genus erinaceus, biologically classified in the order eulipotyphla [ ] . among them, e. amurensis is extant in east asia's areas only, whereas e. europaeus, e. roumanicus and e. concolor are mainly found in europe. in particular, e. europaeus-from here on also simply called hedgehog-is a terrestrial mammal endemically distributed from western to central and southern europe; this species is also present in parts of fennoscandia, in north-western european russia, the british isles, azores and several mediterranean islands [ ] . e. europaeus, protected by the italian law / [ ] , is abundantly distributed throughout the italian peninsula, in sicily, in sardinia and in some smaller islands, without any reported evidence of a rapid decline [ ] . hedgehogs are mammalian hibernators, with different plesiomorphic features in their morphology, physiology and behavior. they are mainly nocturnal and insectivorous, build nests and use hearing and smell as a dominant sense, and possess peculiar specializations such as spines and highly developed back muscles allowing them to roll up [ ] . hedgehogs' suitable habitats include natural open and green spaces as well as artificial, rural and urban, areas with marked preference for lowlands and hilly areas providing abundant food supply and plenty of grass, trees and fallen leaves. they also need diversity of habitats as those found in edges of deciduous woodlands or in man-made ecotonal interfaces (i.e., parks and garden with hedges) [ ] . despite its solitary lifestyle [ ] , e. europaeus has a highly variable home range, ranging in italy from . to . ha [ ] . as generalist predators of macro-invertebrates, hedgehogs consume most frequently caterpillars, earthworms, earwigs, slugs, millipedes and beetles (including coprophagous species); they may also eat meat (e.g., in the form of carrion or preyed small vertebrates such as mice, snakes and birds), bird eggs and pet food that is frequently found in gardens and shared with dogs and cats. the above-mentioned ecological and feeding habits, along with their high population densities, notable synanthropic attitudes and frequent contacts with sympatric wild and domestic species, including humans [ ] , implicate intra-and interspecies interactions [ ] accounting for the potential involvement of e. europaeus (e.g., as a maintenance host, bridge host or dead-end host) in the ecology of several potentially emerging pathogens, as those reported below. coronaviruses (covs) can infect productively a wide range of animal species and cause respiratory, enteric and neurological diseases of variable severity. as shown during the last two decades, potentially zoonotic covs can pose massive public health threats such as those related to the severe acute respiratory syndrome coronavirus (sars-cov) [ ] , middle east respiratory syndrome coronavirus (mers-cov) [ ] and novel coronavirus ( -ncov) [ ] emergences. two of the four recognized genera of covs (i.e., alphacoronavirus and betacoronavirus) are probably perpetuated in bat populations [ , ] , recently recognized as the evolutionary origins of the mers-cov, emerged in in humans [ ] . the recent detection of mers-related covs in hedgehogs from germany [ ] , france [ ] and great britain [ ] suggests that e. europaeus, which is belonging to a chiroptera-related order [ ] , represents a wild reservoir of betacoronaviruses known as erinaceus covs (ericovs). the wild bird influenza a virus (iav) gene pool poses significant risks for both animal and human health because of its ability to colonize a wide variety of animal species (included in the mammalia, aves and reptilia classes) in which iav can cause variable outcomes of infection, with possible high morbidity and fatality rates [ ] . the newly identified influenza d virus (idv), also circulating in italian livestock [ ] , is known to infect cattle, which are the main reservoir of the virus, and small ruminants, swine, horses, camels, ferrets and humans. both iav and idv exhibit respiratory and fecal shedding and the broad iav host range includes wild and pet species sympatric or potentially sympatric with e. europaeus (e.g., dogs, cats, ferrets, land-based birds) [ ] . canine morbillivirus (canine distemper virus, cdv) causes a highly contagious and systemic disease often involving the respiratory, gastro-intestinal and nervous systems of canids and poses worldwide a relevant health risk for domestic and wild species of carnivores [ ] [ ] [ ] . its wide range of naturally infected hosts includes rodentia, primates, artiodactyla, proboscidea and eulipotyphla species, and a morbillivirus antigenically related to cdv was also isolated in fecal samples obtained from a sick e. europaeus [ ] . pestiviruses include eleven known pestivirus species along with related unclassified emerging viruses that can infect domestic and wild ruminants, pigs and wild boars [ ] , rats and bats. pestiviruses can cause a wide variety of symptoms ranging from mild to severe disease and even death. transmission of pestivirus infection can occur through nasal secretion, urine, feces, contact with fomites and by vertical routes [ ] . to the best of our knowledge, no pestivirus infection has been ever reported in hedgehogs. suid alphaherpesvirus (shv- ) is the etiological agent of the aujeszky's disease and may infect a very broad range of domestic and wild mammalian species, including hedgehogs [ ] , but not humans and tailless apes. shv- replicates in the central nervous system and other organs, such as the respiratory tract, and only pigs and wild boars can survive productive infections, thus playing the role of a natural reservoir in which the virus can persist latently after clinical recovery [ ] . occasional consumption of wild boar carrions could expose hedgehogs to shv- infection, which is quite frequently detected in populations of this wild suid species in italy [ ] . the aim of this study is to investigate the presence of the above-mentioned viral pathogens in fecal samples of hedgehogs to better clarify the role of e. europaeus in the ecology of such viruses. this research could also provide epidemiological information useful for the health management of hedgehogs admitted to wildlife rescue centers, where different animal species, including humans, can interact. fecal material was collected non-invasively (i.e., by collecting freshly deposited feces, avoiding manipulation of the animals) from hedgehogs found injured in both urban areas (cities, towns, villages and their suburbs) and rural areas in three provinces of the emilia-romagna region (northern italy) (table ) , and then recovered at a wildlife treatment and rehabilitation center (wtrc). when admitted to the wtrc, hedgehogs were kept in isolation until sample collection (within days of admission) to reduce the possibility of nosocomial infections. between november and january , the samples were collected individually and stored in transport medium ( : pbs:glycerol with potassium penicillin, streptomycin sulfate, gentamicin sulfate, polymyxin b, mycostatin) at − • c until laboratory examinations. different real time pcrs were performed to detect iav [ ] , idv [ ] , cdv [ ] , and shv- [ ] . pestiviruses were tested by a pan-pestivirus assay targeting most of known species and related unclassified viruses [ ] . a pan-coronavirus nested pcr for the rna-dependent rna polymerase (rdrp) gene was carried out to obtain a bp pcr amplicon [ ] , which was sequenced by using a big-dye terminator cycle sequencing kit (applied biosystems, foster city, ca, usa) and the same primers used for amplification. sequencing was performed using the inner primers only and the final pcr product analyzed by sequencing was bp long. positive and negative controls used were the betacov bovine field strain bov/italy/ / and feces from spf chickens, respectively. virus isolation was attempted by inoculation of fecal samples in confluent monolayers of vero cells bs cl (african green monkey kidney) according to lelli et al. [ ] . fisher's exact test was used to test significant differences in virus-detection prevalence between urban and rural areas (epistat . , epistat services, richardson, tx, usa). phylogenetic tree was constructed using the iq-tree . . software under the tim + f + g model with a bootstrap analysis of replicates [ ] . only bootstrap values > % were reported. the cov rdrp gene sequences obtained from italian hedgehogs (e. europaeus species) were compared with those originated from hedgehogs (including e. europaeus and e. amurensis sequences), bats, humans and camels. sequences were named according to a pattern including: genbank accession number, virus name, virus strain, host species, country of isolation and year of isolation. of the hedgehogs examined, ( . %) tested positive for erinaceus covs, named ericovs, by rt-pcr: attempts of isolation on cell culture did not give any positive results. all samples were negative for influenza a and influenza d viruses, canine distemper virus, pestiviruses and aujeszky's disease virus when tested by both molecular and virus isolation methods. no significant difference was found between percentages of hedgehogs found to be cov-positive in rural ( %) and urban ( . %) areas. phylogentic analysis was based on partial sequencing of rdrp gene of italian sequences compared with those of alpha and betacovs originated from hedgehogs, bats, humans and camels. results showed that ericovs from italian hedgehogs ( figure ) grouped together with mers-like betacoronaviruses obtained from western european hedgehogs sampled in germany and great britain (> . % of nucleotide identity) [ , ] , forming a subclade within the betacov lineage c. this lineage, recently renamed as merbecovirus subgenus [ ] , included mers and mers-like cov sequences. in particular, the italian hedgehog viruses were related to mers-cov, showing an identity from . % to . % with some mers-cov representative strains. table for details). table for details). since the first detection of a novel betacoronavirus in hedgehogs [ ] , e. europaeus has been indicated as a possible additional wild reservoir of emerging mers-like covs with potential public health implications. in the present study, an overall prevalence of ericov infection ( . %) was detected in sampled hedgehogs with no significant difference between percentages found in rural ( %) and urban ( . %) areas. indeed, this may be associated with a sustained circulation of viruses in their wild recovery areas likely due to frequent and consistent overlaps of home ranges reported in this species [ ] . the detected prevalence was higher than that observed by saldanha et al. [ ] in great britain ( . %), but in accordance with data reported in germany ( . %) and france ( %), respectively [ , ] . from an epidemiological point of view, the high ericov prevalence observed suggests that, in the study area, e. europaeus could play the role of natural reservoir, competent for infection, replication and excretion of this virus. such high prevalence observed in this study also suggests that hedgehogs could represent chronic shedding carriers of the virus, as reported for alphacoronaviruses infecting bats [ ] and cats [ ] . it is also noteworthy that most of our samples were collected during a physiological phase of reduced immune function [ ] that occurs during hedgehogs' hibernation period, consisting of four or five months of "winter dormancy" in which these mammals may alternate phases of sleep and arousal [ ] . as previously reported [ , ] , we did not observe any clinical disease related to this infection in hedgehogs, that were all reintroduced into the wild within april . nevertheless, the high mutation rates characterizing members of the coronaviridae family and their possible successful interspecies host jumps [ ] should be considered in the management of hedgehogs admitted to multi-species wildlife rehabilitation centers [ ] , recommending their return back to the original recovery areas. phylogenetic analysis showed that ericovs from e. europaeus clustered together, whereas, interestingly, novel betacoronavirus sequences, recently detected in amur hedgehogs from china [ ] , were placed in a different group (nucleotide identity to e. europaeus sequences ranging from . % to . %) within the same lineage, closely related to bat betacov hku detected in china. a limit of this study is that we cannot definitively exclude hedgehogs' involvement in the ecology of the viral pathogens that tested negative. in fact, the prevalence detection threshold value, which is related to the sample size ( hedgehogs), did not allow us to be % certain of detecting at least one positive sample, when the virus circulation prevalence was lower than % [ ] . future studies will be pointed to increase the number of sampled hedgehogs as well as expand the number of urban and rural areas sampled in order to strengthen the results here reported. overall, we provide new information about distribution areas of ericov infection in e. europaeus populations. our preliminary observations suggest that this species might act as a reservoir for ericov in northern italy's areas. however, future research will be needed before confirming the role of e. europaeus as competent reservoir of ericov. a more in-depth molecular analysis, based on a whole genome characterization, will also be required to provide more information about ericovs from italy. molecular phylogenetic evidence confirming the eulipotyphla concept and in support of hedgehogs as the sister group to shrews of - - -ordinary supplement n. ). [legge febbraio , n. . norme per la protezione della fauna selvatica omeoterma e per il prelievo venatorio erinaceus europaeus italian iucn committee, ministry of the environment and protection of the territory and the sea, federation of parks urbanization and disease emergence: dynamics at the wildlife-livestock-human interface the effects of sex and season on home range in european hedgehogs at the northern edge of the species range movements and activity patterns of hedgehogs (erinaceus europaeus) in mediterranean coastal habitats. zeitschrift für säugetierkunde bridge hosts, a missing link for disease ecology in multi-host systems the aetiology, origins, and diagnosis of severe acute respiratory syndrome mers: emergence of a novel human coronavirus the continuing -ncov epidemic threat of novel coronaviruses to global health-the latest novel coronavirus outbreak in wuhan bats and viruses: a new frontier of emerging infection diseases global epidemiology of bat coronaviruses 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canine distemper virus as an emerging multihost pathogen in wild carnivores in northwest italy paramyxoviruses of the morbilli group in the wild hedgehog erinaceus europeus pestiviruses in wild animals itcv, international committee on taxonomy of viruses. virus taxonomy. genus: pestivirus aujeszky's disease aujeszky's disease (infection with aujeszky's disease virus) long-term surveillance of aujeszky's disease in the alpine wild boar (sus scrofa) development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h and h hemagglutinin subtypes development and evaluation of a new real-time rt-pcr assay for detection of proposed influenza d virus detection of canine distemper virus nucleoprotein rna by reverse transcription-pcr using serum, whole blood, and cerebrospinal fluid from dogs with distemper molecular survey of latent pseudorabies virus infection in nervous tissues of slaughtered pigs by nested and real-time pcr validation of a real-time rtpcr assay for sensitive and specific detection of classical swine fever avian coronavirus in wild aquatic birds detection of coronaviruses in bats of various species in italy iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies persistent infections support maintenance of a coronavirus in a population of australian bats (myotis macropus) persistence and evolution of feline coronavirus in a closed cat-breeding colony hibernation: the immune system at rest? genetic recombination, and pathogenesis of coronaviruses hedgehog zoonoses identification of a novel betacoronavirus (merbecovirus) in amur hedgehogs from china. viruses livestock disease surveys: a field manual for veterinarians this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank all the staff of the "otus" wildlife treatment and rehabilitation center (lipu-bologna), in particular nadia caselli for their valuable assistance during sample collection. the authors declare no conflict of interest.animals , , key: cord- -vew uxu authors: mohanty, s. k.; dubey, m.; mishra, u. s.; sahoo, u. title: impact of covid- attributable deaths on longevity, premature mortality and daly: estimates of usa, italy, sweden and germany date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: vew uxu in a short span of four months, the covid- pandemic has added over . million deaths worldwide, which are untimely, premature and unwarranted. the usa, italy, germany and sweden are four worst affected countries, accounting to over % of covid- attributable deaths on longevity, years of potential life lost (ypll) and disability adjusted life years (daly) in usa, italy, germay and sweden. data from united nation population projection, statista and centre for disease control and prevention were used in the analyses. life expectancy, ypll and daly were estimated under four scenarios; no covid- deaths, actual number of covid- deaths as of nd may, and anticipating covid- death share of % and % , respectively. the covid- attributable deaths have lowered the life expectancy by . year each in usa and sweden, o. year in italy and . year in germany. the loss of ypll was . , . , . and . million in usa, italy, germany and sweden. comapression in life expectancy and increase in ypll and daly may intensify further if death continues to soar. covid- has a marked impact on mortality. reduction in longevity, premature mortality and loss of daly is higher among elderly. key words: covid- , mortality, life expectancy, italy, usa, germany, sweden covid- attributable deaths are soaring each day in most of the countries with uncertainties over projected numbers, infection fatality ratio, development of a vaccine and possible end of pandemic. globally, with over million confirmed infections and additional deaths of over thousand by end of may, , the covid- attributable deaths accounts for % of total all-cause mortality. if the covid- mortality continues with same pace, the life expectancy would begin to shrink by end of the year though the survival threat is more among the elderly and the chronically ill. rapid spread of the infection as well as its associated fatality may well be due to novel disease, lack of medical know how, ill-prepared health care system, crowding in urban cities, administrative inefficacy, demographic and social determinants etc. the case fatality ratio (cfr) is a crude measure of mortality, underestimate the mortality impact of covid- . an alternative cfr with days' delay depicts at least twice higher mortality than cfr [ ] . the mortality impact of covid- is higher than many other disease [ ] . the standardized metrics such as disability adjusted life years (daly) and years lost due to disability (yld) are suggested to infer infection fatality by age [ ] . considerable attempts are made on tracing future trajectories, estimation of infection and fatality rate and risk factors of covid- [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . demographic structure, co-morbidities and health-care burden explain covid- attributable mortality to some extent [ ] [ ] [ ] . most common observation made as regard covid- fatality is its greater risk among elderly and people with comorbidities including hypertension, diabetes, cardiovascular disease, myocardial injury [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the diamond princes cruise ship study of japan, a standard estimate of infection, estimated the overall case fatality ratio of . % as against the same being % among the older aged and above [ ] . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint inadequate testing and misclassification of deaths by cause underestimate the extent of covid- deaths. in usa, the excess deaths due to pneumonia and influenza raise an apprehension as regard miss-classification of covid- deaths in the absence of adequate testing [ ] . in italy, % deaths were attributed to covid- making a case for misclassification of cause of death. the covid- attributable mortality has potential to reduce life expectancy in india and seasonal life expectancy in italy [ ] [ ] . in united states, million deaths from covid- would increase mortality by one-third and reduction in period life expectancy by . years in [ ] . mortality impact of covid- is higher in urban counties and the social determinants are significant predictors of its mortality [ ] . high and low fatality due to covid- attributed to density and age structure in terms of elderly in uk [ ] . demographic vulnerability of covid- mortality is lower in younger countries in sub-saharan africa than the industrialized countries [ ] . the spread of infection and mortality depends on containment measures, health system response and micro-management of epidemic which may alter reproduction number [ ] . by april , the case fatality rate varied from . % in south korea to . % in italy. usa, italy, sweden and germany were worst hit countries by the pandemic. by end of may , usa had over . million confirmed cases and over thousand deaths. about % of deaths occurred among adults aged years or more [ ] . in italy, the cfr increased from . % to . % within days and % of the change was due to increasing age specific case fatality rates [ ] . in italy, usa and germany, estimated cases of infections are times, times and . times higher than the number of confirmed cases, respectively [ ] . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . existing studies of the pandemic on fatality is limited. given its rise in intensity it becomes pertinent to gauge impact of covid- attributable mortality on longevity, premature mortality and daly. this will answer questions like "would additional deaths due to covid- reduce longevity and increase premature mortality and daly". we have analysed four worst affected countries; namely usa, italy, sweden and germany that the total deaths obtained from un projection are estimated deaths in the absence of covid- infection. the age specific covid- attributable deaths for usa is collected from centres for disease control and prevention [ ] and that for italy, germany and sweden is taken from statista [ - ] . the total number of confirmed cases and deaths for each country is collected from worldmeter website [ ] . we have redistributed the total deaths available from worldmeter as per age distribution of deaths for which age data was available. under the assumption that the estimated deaths without covid- and deaths due to covid- are mutually exclusive, we have added these deaths to derive age specific death rate (number of deaths per population). the age specific case fatality ratio (ascfr) was computed for italy and sweden from given data. in case of germany, the age group of number of infections were not uniform and deaths were available for - , - , - , - and - . we have redistributed the deaths as per population distribution in -year age group. in case of usa, . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint we have used the ascfr of diamond cruise study that had constant rate ( . ) till age beyond which we have taken the age group close to nearest age group [ ] . scenario considers covid- deaths accounting for % of total deaths while scenario would increase the death share to % of total deaths by the end of the year. expected deaths due to covid- are distributed in accordance with the age distribution of covid- as of date. a brief description of ypll and daly estimation is given below. the ypll is a summary measure of premature mortality that estimates the average years a person would have lived had he or she not died prematurely. it gives higher weight to the deaths occurring at younger ages and lower weight to the deaths at higher ages [ ] [ ] . ypll is estimated as: where, . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint l i is the life expectancy at age i and d i is the number of deaths at age i. the deaths are weighted by life expectancy at each age. daly measures the health of a population by combining data on mortality and non-fatal health outcomes into a single number. the daly measures health gaps as opposed to health expectancies. it measures the difference between a current situation and an ideal situation where everyone lives up to the age of the standard life expectancy, and in perfect health. it combines in one measure the time lived with disability and the time lost due to premature mortality: where, yll= years of life lost due to premature mortality and yld= years lived with disability. we have calculated yll and yld with discounting rate of % where discounting health with time reflects the social preference of a healthy year now, rather than in the future. the value of a year of life is generally decreased annually by a fixed percentage. for many years, a discount rate of % per annum has been standard in many economic analyses of health and in other social policy analyses, but recently environmentalists and renewable energy analysts have argued for lower discount rates for social decisions. the world bank disease control priorities study and the gbd project both used a % discount rate, and the us panel on cost-effectiveness in health and medicine recently recommended that economic analyses of health also use a % real discount rate to adjust both costs and health outcomes [ ] . the yll is estimated as: where, n= number of deaths . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint germany. at % share, the reduction in life expectancy would be . years for usa, years for both italy and germany and . years for sweden (appendix ). appendix (a) presents life table probability of death with and without covid- in italy, germany and sweden and appendix (b) presents the same for usa (due to dissimilarity in age group). the probability of death without covid- was lowest in sweden followed by italy and germany. the covid- attributable deaths have disproportionately increased the probability of death in + age group in all these four countries. the pattern is also similar for usa. estimates from life table with and without covid- for these four countries exhibit the changing age-specific survival patterns ( table the additional deaths due to covid- results in a rise in cdr from . to in italy and this would rise to . with the covid- death share rising to %. in case of usa, it has also increased from . to . and the pattern is similar in germany and sweden as well. age specific assessment of years of potential life lost (ypll) under varying scenario of covid- death share is presented in table is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint italy, germany and sweden. higher age-groups ( years and above) are contributing more than % of ypll in all the countries. the estimated daly at current share of attributable covid- deaths is . million in usa, among all the four countries, the population years and above account more than threefourth contribution in daly while younger ages have relatively low contribution in all the scenarios. the covid- pandemic is one of the worst ever misery posed to mankind. while epidemics in the past have gripped limited geographical boundaries, the covid- has engulfed the entire world within a brief period of four months with a reasonable degree of spread potential. apart from threat to human life, its containment measures have led to economic loss and generated psychological scare among individuals, households, community and the nation at large. the covid- pandemic has paralysed the economic activities, deepened the global recession and has assumed a crisis proportion worldwide. given the scale and intensity of this pandemic, this is first attempt in our knowledge to assess the mortality attributed to covid-. cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . these findings are markers of tragedy experienced in countries ranked high in the level of human development, higher income level and are said to be having a better health care system. hence the failure of preparedness to confront this pandemic by the developed world exposes our vulnerability to emerging infection of similar kind in future. in the absence of a vaccine as well as no systematic medical intervention, the only way out is the containment of its spread . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint or developing a herd immunity in due course. at present great efforts are made by national and local government for management and control of pandemic by diverting the resources (financial and physical) for health care and lock down measures. we acknowledge that the covid- attributable deaths are to some extent underestimated due to lack of comprehensive testing, under-reporting and misclassification of covid- deaths in these countries. despite these limitations, these estimates of mortality pattern do signals about its long-term implications towards structural and compositional balance of population across world regions. though it is very early to gauge its final impact on population structure and composition, its persistence with its virulence unless curbed by introduction of an effective vaccine and means of cure may well change the world order to a significant extent. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . reduction in life expectancy due to covid- death share of % . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . real estimates of mortality following covid- infection. the lancet infectious diseases risk factors for predicting mortality in elderly patients with covid- : a review of clinical data in china estimated inequities in covid- infection fatality rates by ethnicity for aotearoa new zealand. medrxiv case-fatality rate and characteristics of patients dying in relation to covid- in italy estimates of the severity of coronavirus disease : a model-based analysis global epidemiology of coronavirus disease : disease incidence, daily cumulative index, mortality, and their association with country healthcare resources and economic status covid- : death rate is . % and increases with age, study estimates demographic science aids in understanding the spread and fatality rates of covid- epidemiologic and clinical characteristics of novel coronavirus infections involving patients outside wuhan, china diagnosis and treatment of novel coronavirus infection in children: a pressing issue clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid- infection clinical characteristics of coronavirus disease in china estimating the infection and case fatality ratio for coronavirus disease (covid- ) using age-adjusted data from the outbreak on the diamond princess cruise ship estimating the early death toll of covid- in the united states covid- and mortality: india's perspective, working paper no news from the front: estimation of excess mortality and life expectancy in the major epicenters of the covid- pandemic in italy demographic perspectives on mortality of covid- and other epidemics (no. w ) social determinants of covid- mortality at the county level the contribution of age structure to the number of deaths from covid- in the uk by geographical units covid- death rates by age and sex and the resulting mortality vulnerability of countries and regions in the world estimating excess -year mortality associated with the covid- pandemic according to underlying conditions and age: a population-based cohort study monitoring trends and differences in covid- case fatality rates using decomposition methods: contributions of age structure and age-specific fatality a demographic scaling model for estimating the total number of covid- infections /statistics/ /coronavirus-covid- -deaths-by-gender-germany) /statistics/ /number-of-coronavirus-deaths-in-sweden-by-age-groups years of potential life lost (ypll)-what does it measure? epidemiology years of potential life lost for six major enteric pathogens halys and qalys and dalys, oh my: similarities and differences in summary measures of population health. annual review of public health disability weights for the global burden of disease study key: cord- -lt t shi authors: gilad, vered; masoero, giovanni title: treatment of covid- patients in italy: a physician’s experience and insights date: - - journal: rambam maimonides med j doi: . /rmmj. sha: doc_id: cord_uid: lt t shi the outbreak of coronavirus disease (covid- ) in italy, the first western country hit by the pandemic, seriously impacted the italian healthcare system and social and economic environment. this perspective piece focuses on the main challenges faced by italian hospital managements: hospital overcrowding; the need for urgent reorganization of the country’s healthcare systems; the lack of data regarding covid- diagnostics, clinical course, and effective treatment; individual and collective consequences of the crisis; and the importance of disease containment measures and early treatment strategies. february , marked the beginning of the worst healthcare system crisis of the last years in italy. when the first case of "communityacquired" coronavirus disease (covid- ) was diagnosed, italy-the first western country facing the new virus outbreak-was caught unprepared and did not entirely and properly foresee the subsequent events. the lack of understanding of the asymptomatic or pre-symptomatic carrier state of the virus, , together with an underestimation of its virulence, an exaggerated concern related to public opinion, and the potential for economic damage, led to the loss of precious time and widespread dissemination of the infection. written by two physicians working within an italian hospital, this paper provides an important perspective based on first-hand experience, regarding the challenges faced by italy's healthcare system during the covid- crisis. shortly after the first patient was diagnosed with covid- , italy witnessed a rapidly increasing number of emergency department referrals; many of the patients were in a critical condition, needing intensive treatment and mechanical ventilation. recognizing that the number of patients requiring hospitalization was much higher than hospital capacities led to a rapid reorganization of activity. all nonurgent and elective procedures were immediately suspended and routine outpatient clinic activity cancelled, leaving many chronic patients without adequate medical assistance. the number of dedicated covid- hospitalization departments grew rapidly, with some hospitals becoming fully dedicated to treatment of only new pandemic victims. within a few days, the number of hospital intensive care beds was at least doubled, operating rooms became small intensive care units (icus), and emergency departments were transformed into sub-icus, enabling non-invasive and mechanical ventilation of increasing numbers of patients. due to a shortage of healthcare workers (hcw) in many hospitals, specialists from different departments and resident doctors were transferred to covid- departments, and external specialists were hired to help with the growing workload. despite the eventual adoption of the above measures, in areas with early spread and high contagion rate of the virus, treatment was frequently inadequate due to extreme overcrowding. many hospitals had an insufficient number of icu beds due to the rising demand, forcing doctors to make difficult ethical decisions, and an unknown number of patients remained undertreated. many patients were sent home despite displaying evident signs of pneumonia, and many others died before reaching the hospitals. treatment delay led to advanced pulmonary and multi-organ damage and was often fatal, contributing to the high number of deaths in italy- , patients as of july , corresponding to about % of diagnosed cases. virus spread was highly variable among the regions in italy, resulting in significant divergence in covid- and overall mortality rates. for example, bergamo, a city where the virus spread widely in the early stage, had a prevalence of . % with a . % covid- mortality rate, and , total deaths between february and march , , corresponding to a mortality rate that increased by % compared to the same time period in previous years ( - ). , conversely, during the same time, rome, as a result of early lockdown, had a prevalence of . %, and significantly lower mortality rates, with covid- deaths ( . %) and an overall decrease in mortality (- %) compared to the same time period in - . , however, the tragedy extended far beyond numbers; owing to local precautionary isolation measures, patients often faced the disease completely alone. contact between cities and regions was limited, and many families remained separated for many weeks. throughout italy, during lockdown relatives were not allowed to enter the hospitals, and information about clinical status and communication with patients were possible only via telephone and electronic technology. families lost their loved ones without any possibility to say farewell, nor to even hold funerals, which were suspended until the beginning of may (and for which attendance was then strictly limited to only a small number of relatives, no more than people). the incidence of anxiety and stress disorders was high among hcw; a recent cross-sectional study reported a high prevalence of post-traumatic stress symptoms ( . %), depression ( . %), anxiety ( . %), and insomnia ( . %) among , italian hcw during the outbreak. incidences of psychological distress were higher among frontline hcw, similar to data published from china. the main factors associated with stress disorders included supporting the challengingly high number of critically ill patients, scarcity of intensive care beds, unfamiliar treatment strategies, feelings of inadequacy, uncertainty about pandemic duration, the risk of infection and deficiency of personal protective equipment, high-workload shifts, and physical distress related to the need to use heavy protective uniforms. [ ] [ ] [ ] contagion was high among hcw, especially during the first weeks of the outbreak, due to scarcity and non-optimal use of personal protective equipment. to date, more than , hcw have been infected and doctors have died, many of them working on the frontlines. moreover, in light of concerns regarding infection transmission to family members, many health workers decided to stay away from their homes, further complicating the challenges related to the lockdown, such as closed schools and separation from grandparents and other relatives. while the number of covid- patients was rapidly increasing, italy saw a drastic decrease in hospital admissions for other pathologies, since patients were afraid of contracting the infection at the hospital. recent italian reports document an almost % decrease in myocardial infarction admissions for acute coronary syndromes compared to equivalent periods in previous years, with a subsequent increase in severe cardiac complications and a -fold higher cardiovascular mortality rate. facing a novel virus, the primary challenges were related to the lack of data regarding diagnostic criteria, disease management, and treatment options. diagnostic challenges due to the limited availability of tests, slow processing of samples, and low reliability of some diagnostic kits created anxiety among patients and hcw. to avoid nosocomial spread of the virus, "grey" departments were established for patients with high clinical suspicion; nevertheless, it was almost impossible to prevent inhospital spread. given the limited clinical experience and lack of evidence-based data, new channels for relaying updates emerged; doctors who first started seeing covid- patients primarily shared their experience and findings through internet pathways. we followed daily updates by attending videoconferences and open webinars to discuss emerging findings, including new clinical signs, typical radiologic patterns, laboratory findings, common complications, and possible treatment strategies. additionally, open groups on the social networks and dedi-cated forums were created, reaching up to , doctors who used these platforms for consultation and exchange of essential real-time information. as clinical experience advanced, knowledge accumulated about the clinical course of covid- and the essential role played by cytokine storm and coagulopathy for disease progression and complications. [ ] [ ] [ ] given the novelty and the rapid spread of the virus in italy, most pharmacological treatments were initially based on compassionate and "offlabel" use, with wide heterogeneity between centers. as clinical evidence solidified, protocols became more uniform and included a combination of antimicrobial drugs (antiviral, antibacterial, and antimalarial), anti-inflammatory and immunomodulatory drugs, anticoagulants, and, later, convalescent plasma. a comprehensive management algorithm was recently published by galluccio et al., summarizing the timing indication for each treatment, based on clinical features and laboratory and imaging findings. although no targeted treatment is yet available, following the first weeks of outbreak it became clear that recognition of early "warning signs," and thus early treatment, could drastically reduce disease progression, complications, and mortality. in fact, many critically ill patients developed initial symptoms several days prior to hospital admission and exhibited tardive dyskinesia and sudden clinical deterioration associated with a hyper-inflammatory state. , furthermore, many patients presenting with only mild symptoms were found to have a significantly reduced oxygen saturation level and extensive pulmonary damage. in view of the importance of identifying patients in the early stages of disease, an open letter signed by more than , italian doctors was sent to the italian ministry of health, asking for a strengthening of community assistance and the telemedicine infrastructure to allow home surveillance and care of covid- patients. from the end of march , many italian regions started building dedicated medical teams (called "usca"-"unità speciale di continuità assistenziale"), assigned to monitor covid- patients at home through daily calls or home visits. they evaluated clinical parameters (e.g. symptoms, fever, hemodynamics, and neurologic state), and in selected patients laboratory exams and lung ultrasound, which led to hospital admissions earlier in the course of the disease, resulting in a significant decrease in the number of critically ill patients. in view of the concrete risk for a second wave, we believe that there should be more emphasis placed on community assistance, focusing on clinical surveillance and early pre-hospitalization treatment, in order to prevent repeated overcrowding of hospitals and healthcare system collapse. a schematic representation of the covid- management approach-initially, in the midst of the pandemic, and looking ahead-is shown in figure . the covid- outbreak led to an unprecedented health crisis in italy, as for the rest of the world, with severe consequences on personal and public health, substantial psychological, social, and economic repercussions, and considerable challenges for governments and hcw. although errors were undoubtedly made at the beginning of the outbreak that significantly contributed to extended illness and the high number of deaths, the italian government, the national healthcare system, and italian citizens have reacted with purpose and made important decisions that allowed italy to gradually recover from the severe crisis. the decision to impose extensive and national lockdown measures, despite the psychological, social, and economic impact, has permitted significant containment of the virus spread, preventing additional pressure on the healthcare system (which had already exceeded its maximum capacity in many places), and limiting further high morbidity rates and numerous deaths. moreover, primary importance is attributed to the cooperation of citizens in following the rules, fast reorganization of hospitals in response to the extraordinary workload, broad and real-time sharing of medical findings and updates, strong cooperation between doctors, and the community's sense of solidarity. in italy, there is still much to be done to prevent recurrences, and focus should be on preventing new outbreaks and reinforcing early treatment. social distancing measures, extensive testing, fast and comprehensive tracing of new cases, and isolation of covid- -positive patients in dedicated recovery centers can prevent further widespread diffusion of the virus and the need for more stringent actions. resources should be invested to strengthen the national healthcare system with an emphasis on community assistance, and digital and telemedicine infrastructures. we believe that it is essential to extend and reinforce outpatient care to prevent clin- * executed by dedicated medical teams, "usca-unità speciale di continuità assistenziale," and independently organized by the regional local health public body. ical deterioration of many patients, reduce hospital admissions, and avoid hospital overcrowding as well as the related consequences on the public healthcare system, non-covid- patients, and hcw. the covid- outbreak in italy demonstrated to the world the destructive effects that a highly contagious virus can have on unprepared healthcare and social systems. the high number of patients needing hospital treatment can lead to the overcrowding of hospitals and a very real consequent risk of inadequate treatment of both covid- and non-covid- patients. containment measures are fundamental to prevent disease dissemination, while monitoring and early treatment are essential to prevent complications and reduce mortality. italy introduced serious lockdown measures and is now gradually recovering, but the deep wounds incurred by the pandemic have also provided important lessons and insights. we hope that the lessons learned will contribute to the prevention of similar tragedies in the future. a novel coronavirus outbreak of global health concern presumed asymptomatic carrier transmission of covid- presymptomatic transmission of sars-cov- -singapore siaarti recommendations for the allocation of intensive care treatments in exceptional, resource-limited circumstances covid- integrated surveillance data in italy impact of the covid- epidemic on total mortality of the resident population first quarter covid- : daily update mental health outcomes among frontline and second-line health care workers during the coronavirus disease (covid- ) pandemic in italy mental health survey of medical staff in a tertiary infectious disease hospital for covid- the emotional impact of covid- : from medical staff to common people personal protective equipment and intensive care unit healthcare worker safety in the covid- era (ppe-safe): an international survey online ahead of print. crossref . editorial staff. list of doctors who died during the covid- epidemic reduced rate of hospital admissions for acs during covid- outbreak in northern italy reduction of hospitalizations for myocardial infarction in italy in the covid- era sars-cov- and covid- group for doctors only. private facebook group covid- illness in native and immunosuppressed states: a clinical-therapeutic staging proposal prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia isth interim guidance on recognition and management of coagulopathy in covid- a brief note on randomized controlled trials and compassionate/off-label use of drugs in the early phases of the covid- pandemic letter to the italian health ministry from , doctors key: cord- - vucm s authors: franzo, giovanni; tucciarone, claudia maria; moreno, ana; legnardi, matteo; massi, paola; tosi, giovanni; trogu, tiziana; ceruti, raffaella; pesente, patrizia; ortali, giovanni; gavazzi, luigi; cecchinato, mattia title: phylodynamic analysis and evaluation of the balance between anthropic and environmental factors affecting ibv spreading among italian poultry farms date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: vucm s infectious bronchitis virus (ibv) control is mainly based on wide vaccine administration. although effective, its efficacy is not absolute, the viral circulation is not prevented and some side effects cannot be denied. despite this, the determinants of ibv epidemiology and the factors affecting its circulation are still largely unknown and poorly investigated. in the present study, ibv qx (the most relevant field genotype in italy) sequences were obtained between and from the two main italian integrated poultry companies. several biostatistical and bioinformatics approaches were used to reconstruct the history of the qx genotype in italy and to assess the effect of different environmental, climatic and social factors on its spreading patterns. moreover, two structured coalescent models were considered in order to investigate if an actual compartmentalization occurs between the two integrated poultry companies and the role of a third “ghost” deme, representative of minor industrial poultry companies and the rural sector. the obtained results suggest that the integration of the poultry companies is an effective barrier against ibv spreading, since the strains sampled from the two companies formed two essentially-independent clades. remarkably, the only exceptions were represented by farms located in the high densely populated poultry area of northern italy. the inclusion of a third deme in the model revealed the likely role of other poultry companies and rural farms (particularly concentrated in northern italy) as sources of strain introduction into one of the major poultry companies, whose farms are mainly located in the high densely populated poultry area of northern italy. accordingly, when the effect of different environmental and urban parameters on ibv geographic spreading was investigated, no factor seems to contribute to ibv dispersal velocity, being poultry population density the only exception. finally, the different viral population pattern observed in the two companies over the same time period supports the pivotal role of management and control strategies on ibv epidemiology. overall, the present study results stress the crucial relevance of human action rather than environmental factors, highlighting the direct benefits that could derive from improved management and organization of the poultry sector on a larger scale. a total of qx sequences were included in the final dataset. of those, belonged to "company a" and to "company b". the sampled farm location is reported in fig. . overall, farms were mainly located in the "pianura padana" region (central area of northern italy) and, to a lesser extent, in north-eastern, north-western, central and southern italy. although the two companies tend to operate in different italian regions, a clear overlapping was present in the high densely populated poultry area of northern italy (fig. ). qx-population genetics parameter estimation. all considered field sequences formed a monophyletic group including only italian strains ( supplementary fig. ). tempest investigation revealed that the positive correlation between genetic divergence and sampling time (i.e. r = . ) was suitable for phylogenetic molecular clock analysis . the tmrca of the overall qx population in italy (i.e. qx genotype introduction) was estimated in . [ hpd: hpd: . - . ] using the structured coalescent approach. almost identical results were obtained including a third "ghost" deme (i.e. an estimated deme for which no sequences were available, representative of other unsampled companies and farms) in the analysis or using the "traditional" coalescent approach. when strains collected from integrated poultry companies were considered independently, the tmrca was predicted in . [ hpd: [ hpd: . - for company a and in . [ . - . ] for company b. the viral population dynamics evidenced a substantially constant ne*t (effective population size * generation time, or relative genetic diversity) with the remarkable exception of the period between mid- and mid- , when a sudden fluctuation was observed. however, a quite different scenario was demonstrated between the two integrated poultry companies. in fact, company a was featured by a substantially constant population size, with a minor decrease affecting particularly the period - . however, the ne*t hpd were relatively broad and at odds with the significance of the observed variations. on the contrary, a much more changeable pattern was observed in company b (fig. ) . migration among companies. the structured coalescent model fitted with the two company, evidenced the presence of separate clades (fig. a) for the companies, with only exceptions, represented by strains sampled in company b but clustering in the company a clade, thus suggesting the migration of strains from company a to company b. accordingly, the migration rate from company b to company a was . * − [ hpd: . * − - . * − ], while the one from company a to company b was . * − [ hpd: . * − - . ]. the figure . location of farms from which samples have been obtained. different companies have been color coded. samples collected in company b but clustering with company a clade have been colored in red (herein named "imported"). farm location has been jittered using an internal routine of ggplot library to guarantee anonymity. the map was generated in r (version . . ), using the library ggmap . phylogeographic analysis. all the samples phylogenetically belonging to company a but collected from company b originated from farms located in the high densely populated area of northern italy (fig. ) . the continuous phylogeographic analysis reconstructed a spreading pattern originating from a single introduction in emilia romagna region (company a), followed by a progressive expansion and persistence at high level in the pianura padana region. more rarely, spreading episodes toward other italian regions were observed (fig. ) . after qx introduction, the infection wave front increased slowly approximatively until , when a rapid expansion led to the final distribution range by the middle of (fig. ) . accordingly, the dispersal velocity progressively increased in the first years after qx genotype introduction, peaking in the period - and then remaining essentially constant, despite some fluctuations (fig. ) . the presence of a high dispersal velocity after , when no further increase in wave front was observed, suggests that ibv continued to circulate at high rate after its first establishment in a region. the analysis of the effect of different environmental factors on qx genotype dispersal velocity led essentially to negative results (i.e. absence of significant correlation). the only exception was represented by the poultry density www.nature.com/scientificreports www.nature.com/scientificreports/ sl model, which was positively and significantly correlated to viral dispersal velocity: d = . , percentage of d with p-value < . = %. despite the economic relevance, the epidemiology of ibv and the factors affecting its behavior have been only partially investigated. even if a huge amount of knowledge and literature has accumulated over time, most of the reports are anecdotal or based on the analysis of single clinical outbreaks , , . although relevant pieces of information could be obtained, the risk of being biased by personal believes or the particular condition under www.nature.com/scientificreports www.nature.com/scientificreports/ investigation is high. a certain caution is thus required when inferring and extending the same conclusion on a broader/general scale. moreover, most of the available studies are focused on avian influenza and, to a lesser extent, newcastle disease and infectious laryngotracheitis , , . the aim of the present study was to construct an objective and statistically sound framework to understand ibv field strains behavior, the effect of control measures and the factors conditioning their epidemiology. the field of phylodynamics, and all related extensions, provides an invaluable tool for the study of viruses and particularly of rapidly evolving ones, whose evolution can be measured in "real time", over the course of an epidemic . ibv qx genotype is the most relevant field strain in italy , , and despite a relatively long circulation and the efforts devoted to its control, it still remains one of the main menaces for poultry industry profitability. therefore, the understanding of the forces shaping its epidemiology would be of remarkable relevance in order to prevent the induced damages, rather than try to control them. remarkably, the italian ibv strains appear to originate from one introduction events only, as previously reported . therefore, it was possible to reconstruct ibv italian strain evolution and epidemiological pattern without the biasing effect of strains recently introduced from other counties. the implemented approach allowed to reconstruct the migration history of the qx genotype over time. the estimated introduction, in emilia romagna region, shortly predates the first detection, posing in favor of the effectiveness of the italian monitoring and early detection systems. all the analyses, independently of the underlying statistical model, support that company a was the first introduction site (fig. ) . thereafter, the virus circulation was limited to farms belonging to this company for years, until approximatively , when company b became involved. contextually, a progressive increase in diffusion speed was noticed (fig. ) , not unexpectedly considering the rising number of involved farms (especially at the border between veneto and lombardy regions, where most farms are located) and thus the increase in spreading potential and opportunity. the high farm density of this area has been described as a risk factor for different infectious diseases , and ibv seems to be no exception. interestingly, the viral population size remained relatively constant in this time period, evidencing that, even if qx strains were able to effectively spread from farm to farm, their replication was adequately controlled, likely by effective vaccination strategies. actually, a certain slowdown in dispersal velocity was noticed in - , potentially because of a progressive decrease in naive populations availability. a dramatic change was observed in , when a new spreading wave (fig. ) and an increase in diffusion rate (fig. ) and population size (fig. ) were detected. a more detailed analysis demonstrated that this variation affected company b only (fig. ) . a previous study has ascribed this episode to a change in the vaccination scheme adopted by this company, which moved from a heterologous mass+ b based vaccination to a mass only vaccination leading to an increased viral circulation and clinical outbreaks number . moreover, experimental studies demonstrated a significant reduction in r in vaccinated groups compared to unvaccinated ones . it can therefore be speculated that the increase in infectious pressure within-farm and the higher flock susceptibility to infection could have enhanced the risk of ibv spreading to other farms and regions. in support of this hypothesis, the geographical spreading affected mainly northern italian farms (where company b is located). moreover, when a new double vaccination was implemented, the decrease in viral population size was mirrored by a reduction of dispersal velocity. www.nature.com/scientificreports www.nature.com/scientificreports/ continuous phylogeography showed that the areas interested by a more intensive viral circulation were those featured by a higher poultry density, and this evidence was confirmed by a statistically significant correlation between poultry density and dispersal velocity. the association between spatial proximity and farm infection is probably the most consistently reported risk factor for poultry infectious diseases , , . although an airborne transmission has been proposed for ibv, its occurrence has rarely been demonstrated experimentally . however, the spatial proximity likely increases the likelihood of a greater number of horizontal contacts between farms, including the movement of people, vehicles and fomites between farms, as well as sharing similar risk factors (e.g. environmental conditions, climate, presence of wild animals, etc.) , . based on these premises, the presence of segregated poultry companies should represent an effective obstacle to viral shedding and the obtained results partially confirm these evidence. the strains from different poultry companies formed two independent clusters, which suggests the effectiveness of independent production flow/chain in protecting farms from exogenous introductions. additionally, the application of adequate biosecurity measures, enforced also by the italian legislation, likely contributed in limiting new strain introduction. the exceptions to this general rule were farms located in the high densely populated poultry area of northern italy, where an overlap between the two companies occurs. the unidirectionality of the viral flux from company a to company b implies that other factors, besides spatial proximity, must be in place. a detailed survey could shed some insights into relevant factors like different biosecurity measures, structural factors, vaccination strategy etc.. the mediation of other "actors" cannot also be excluded. in fact, the analysis of just two companies, however predominant they are on the italian poultry sector, cannot be considered an accurate depiction of the italian situation. remarkably, the inclusion of a third deme (representative of other unsampled companies and farms) in the analysis model highlighted that several transmission events could be mediated by smaller entities operating in the same region. actually, the high migration rate estimated between company b and this ghost deme poses in favor of its pivotal role in maintaining an active ibv circulation. even if the idea of modeling demes for which no sequences are available could seem counterintuitive, previous studies showed that the structured coalescent can provide meaningful estimates even in absence of samples from one population and this approach has already been applied and proven effective for other diseases, including ebola . since also company a was evaluated in the same analysis run, the absence of relevant links between this company and the ghost deme further supports the analysis reliability, posing in favor of an actual interaction between company b and the ghost deme rather than a mere low specificity of the method. a less effective control of ibv infection could be speculated for small companies, whose management capability and resources are limited compared to big-integrated companies. in fact, all italian farms have to follow national legislation dictating the minimum biosecurity measures to be applied. however, integrated poultry farms, part of major companies, enforce additional managerial practices to increase biosecurity levels. personnel and veterinarian formation, internal audits and periodic controls guarantee a higher level of application of the required standards, compared to most of small non-integrated farms. the higher spatial overlap and the likely sharing of some infrastructures (e.g. streets, accessory personnel, services and infrastructures) could nevertheless have a negative indirect effect on the major companies, especially in northern italy where company b is located. however, differences between company a and company b in the application of biosecurity measures and production flow management could also explain the different ibv epidemiology, as demonstrated by the dissimilar patterns in viral population fluctuations in the two companies (fig. ) . a further risk factor that would deserve further investigation is the presence of the rural sector, which is highly concentrated in the densely populated poultry area of northern italy. this sector is characterized by a complex mix of growers, dealers and backyards flocks, often applying poor biosecurity measures and linked together by a poorly traceable contact network . although interactions with industrial poultry farming is hardly discouraged, illegal/indirect interactions have been documented and multiple epidemiological connections could result in a bidirectional transmission between the two sectors, as demonstrated in the italian low pathogenicity avian influenza (ai) outbreaks occurred in - . after these episodes, a stricter legislation has been developed, imposing limits to animal movements and more active surveillance in the rural sector. nevertheless, no measures were taken for the monitoring and control of ibv in these enterprises, and therefore their role as sources of encroachment in intensive farming cannot be excluded. other environmental factors do not seem to play a relevant role in affecting viral dispersal. while climatic conditions like temperature, humidity and wind could actually affect viral viability and spreading, their effect could be circumvented by a transmission mediated by "fast-moving" vectors like trucks, personnel and, potentially, wild species , . more surprising could be the non-significant role of road density. however, it must be stressed that the available raster reported the overall density of roads, which could significantly differ from those preferentially used for live animal or their byproduct transportation, hindering the detection of an otherwise plausible risk factor. therefore, the mapping of the live animal transportation pathways could provide remarkable benefits in ibv (and other infectious diseases) epidemiology understanding and control. the present study demonstrates that ibv spreading potential is mainly affected by farm and poultry density overall, which can be reasonably claimed as a major risk factor. other environmental/climatic variables do not seem to affect ibv epidemiology, stressing the pivotal role of human action and thus highlighting the direct benefits that could derive from an improved management and organization of the poultry sector on a larger scale. actually, the integration of poultry production seems to provide a relevant constrain to ibv circulation, even though some differences were noted between the two considered companies. in fact, despite differences in management and applied control strategies likely playing a role, the presence in the same area of other minor poultry companies seems to represent a major issue, probably due to the less effective infection control ascribable to the sometimes lower organization capability and resources of small enterprises. the present study results emphasize the need of an active sharing of sequences and related molecular epidemiology data originating from all the actors in poultry production, allowing a proper depiction of the viral exchange dynamics, based on actual data rather www.nature.com/scientificreports www.nature.com/scientificreports/ than estimations. the obtained information would represent a fundamental substrate for the implementation of effective and shared efforts for the infection control on a broad regional scale. ibv strain sampling, diagnosis and sequencing. samples were collected for routine diagnostic purpose in the period - from poultry flocks belonging to the two main poultry companies (here named company a and company b) operating in italy, which account together for about % of italian poultry production. samples were obtained mainly from outbreaks of respiratory disease, following a standard protocol that enforced the collection of a pool of tracheal swabs from randomly selected birds. for each sampling, collection date and farm localization were recorded. all considered samples had been performed in the context of routine diagnostic activity and no experimental treatments or additional assays were implemented during the study. therefore, no ethical approval was required to use specimens collected for diagnostic purpose. additionally, several samples from company a were already sequenced using the same protocol and published in franzo et al. . when detailed information on sampling farm and time could be traced back, these samples were included in the study. the permission to use the collected samples for research purpose was obtained from each company. swab pools were resuspended in ml of pbs and vortexed. thereafter, rna was extracted from µl of the obtained eluate using the high pure viral rna kit (roche diagnostics, monza, italy) kit. diagnosis was performed by amplification and sanger sequencing of the hypervariable region of the s region using the primer pair described by cavanagh et al. . obtained chromatograms quality was evaluated using finchtv (http://www. geospiza.com) and consensus sequences were generated using cromaspro (cromasproversion . ). sequence dataset preparation. all obtained sequences plus the reference dataset provided by valastro et al. ( ) were aligned using mafft and a phylogenetic tree was reconstructed using iq-tree selecting as the best substitution model the one with the lowest akaike's information criterion, calculated using jmodeltest . the strains clustering with the gi- lineage (previously known as qx genotype) were selected and further evaluated for the presence of recombination in the considered region using rdp and gard : to limit the computational burden the sequences were clustered using a % identity threshold using cd-hit and a single representative sequence for each cluster was selected. these sequences plus the valastro et al. ( ) references were re-aligned and recombination analysis was performed. recombinant sequences, including the ones belonging to the same cluster, were removed from the dataset. finally, the dataset was re-expanded to the original size and sequences identical or closely related (p-distance < . ) to the qx-based vaccines administered in italy were also excluded. to evaluate the distribution of italian gi- strains in the international scenario, an extensive dataset of s ibv sequences was downloaded from genbank and a phylogenetic tree was reconstructed as previously described. to reduce computational complexity and increase interpretation easiness (without losing information), only one sequence representative of all identical ones was selected using cd-hit and included in the analysis. the presence of an adequate phylogenetic signal was assessed by a likelihood mapping analysis performed with iq-tree. tempest was used to preliminarily evaluate the temporal signal of the italian qx phylogeny and therefore the applicability of molecular clock-based methods . strain migration among integrated poultry companies. ibv qx strain migration among companies was evaluated using the structured coalescent-based approach implemented in the multitypetree extension of beast . according to this model, the considered population is divided in a series of demes, which can be imagined as different islands, featured by their own populations size and interconnected by a certain migration rate among them. in the particular italian qx scenario, the serially sampled (i.e. with known collection date) strains were used to infer the migration rate and history between the two integrated poultry companies (i.e. considered as different demes) over time. additionally, the bayesian approach implemented in beast allowed to contextually estimate other population parameters, including the time to most recent common ancestor (tmrca), evolutionary rate and population size. accounting for the presence of other farms and companies operating in the italian poultry sector, which could take part in or mediate the viral transmission among the investigated major companies, a third "ghost" deme (a deme for which no sequences were available) was added to the model . the priori of the ghost deme size was set to one tenth of the other demes, according to the estimated poultry population distribution. however, broad priori distribution (i.e. relatively uninformative priori) was chosen to avoid constrains or biases in the parameter posterior estimation. for all analyses, the best substitution model (tn + g ) was selected based on the bayesian information criterion, calculated using jmodeltest , while the relaxed lognormal molecular clock model was selected based on marginal likelihood calculation and comparison using the path sampling and stepping stone method . the final estimations were obtained performing a million generation markov chain monte carlo run, sampling parameters and trees every twenty thousand generations. results were visually inspected using tracer . and accepted only if mixing and convergence were adequate and the estimated sample size was greater than for all parameters. parameter estimation was summarized in terms of mean and % highest posterior density (hpd) after the exclusion of a burn-in equal to % of the run length. maximum clade credibility (mcc) trees were constructed and annotated using treeannotator (beast package). results consistency was also evaluated performing a "traditional" serial coalescent analysis in beast . . . the same substitution and clock model of the structured coalescent analysis were selected, while a nonparametric skyline population model was chosen to reconstruct the viral population dynamic over time . independent ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ analysis for each integrated company were also performed using the same approach but generating two new datasets including only the sequences collected from a specific company. however, sequences introduced from one company to the other were excluded from the company-specific analysis since they did not share a common evolution history. continuous phylogeography and determinants of ibv spreading. the history of qx dispersal was reconstructed over time using the continuous phyogeographic approach described by lemey et al., using beast . . . substitution and clock models were selected as previously described. similarly, the gamma relaxed random walk was preferred over the other phylogeographic continuous diffusion models based on the marginal likelihood calculation and comparison using the path sampling and stepping stone method , . the final estimations were obtained performing a million generation markov chain monte carlo run, sampling parameters and trees every twenty thousand generations. results were visually inspected using tracer . and accepted only if mixing and convergence were adequate and the estimated sample size was greater than for all parameters. the reconstruction of qx movements over time within italian borders was obtained using spread , summarizing and visualizing the full posterior distribution of trees obtained in continuous phylogeographic analyses . pattern and determinants of viral spreading were evaluated as described by (dellicour et al.) , using the seraphim r library . the history of lineage dispersal was recovered from the posterior trees generated using beast and annotated with ancestral longitude and latitude reconstruction. particularly, the distance, duration and velocity of spatial dispersal were recoded as vectors and used to generate different summary statistics of viral spreading, including dispersal velocity and maximal wave front distances (measured from the location of the tree root). several environmental/social variables were considered to determine if they were associated with the dispersal rate of ibv lineages. the environmental rasters describing the variables of are shown in supplementary fig. . more in detail, the values in the raster (i.e. altitude, population density, poultry density, temperature, etc.) were used to associate a weight to the abovementioned vector. two models of spatial movements were considered: ( ) "straight line (sl) path" model, assuming a straight movement between the starting and ending locations of each branch (i.e. the branch weight is computed as the sum of raster cells through which the straight line passes); ( ) "least cost (lc) path" model, using a least cost algorithm (i.e. the branch weight is computed as the sum of the values of cells transition values between adjacent cells along the least-cost path). in this model, the analyzed environmental variable can be considered both as a conductance (i.e. enhancing viral dispersal through the cells with higher values) or resistance factor (i.e. allowing an easier dispersal through cells with lower values). both instances were evaluated for each considered factor. the obtained "environmental" weights were used to calculate a regression with the branch duration and the corresponding coefficient of determination (r env ) was obtained. a null coefficient of determination (r null ) was also calculated assuming the null raster (i.e. when only the spatial distance of each movement is assumed to affect branch duration). the statistic d = r env -r null was selected as final outcome, and describes how much the regression is strengthened when the spatial variation in the environmental variable is included. to account for the phylogenetic uncertainness, the d statistic was calculated for each tree of the posterior distribution. however, for computational constraints, the number of posterior trees was down-sampled to after discharging a % burn-in. only the environmental variables with more than % of d statistics > were considered for further analysis. particularly, the significance of d statistic of those variables was assessed against a d null distribution obtained by randomizing times the phylogenetic nodes location under the constraint that branch length remained equal. a p-value was generated for each initial tree, therefore a percentage of the trees with p-value < . could be calculated, which can be interpreted as a posterior probability of observing a significant correlation between lineage movements and considered environmental variable. according to , a percentage of p-value < . greater than % was considered a strong evidence that the environmental variable is associated to viral movement speed . molecular evolution and emergence of avian gammacoronaviruses s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification review of infectious bronchitis virus around the world effect of different vaccination strategies on ibv qx population dynamics and clinical outbreaks sjaak) & cook, j. k. a. factors influencing the outcome of infectious bronchitis vaccination and challenge experiments infectious bronchitis virus variants: a review of the history, current situation and control measures age-dependent immune responses and immune protection after avian coronavirus vaccination a novel variant of the infectious bronchitis virus resulting from recombination events in italy and spain porcine circovirus type (pcv ) evolution before and after the vaccination introduction: a large scale epidemiological study genetic data provide evidence for wind-mediated transmission of highly pathogenic avian influenza modelling the wind-borne spread of highly pathogenic avian influenza virus between farms avian influenza virus infections. ii. experimental epizootiology of influenza a-turkey-wisconsin- virus in turkeys characterisation of influenza a viruses isolated from turkeys in england during avian influenza in caged laying chickens isolation of avian influenza virus in texas assessing the probability of introduction and spread of avian influenza (ai) virus in commercial australian poultry operations using an expert opinion elicitation transmission parameters of highly pathogenic avian influenza (h n ) among industrial poultry farms in northern italy in - risk maps for the spread of highly pathogenic avian influenza in poultry explaining the geographic spread of emerging epidemics: a framework for comparing viral phylogenies and environmental landscape data exploring the temporal structure of heterochronous sequences using tempest (formerly path-o-gen) risk factors for the introduction of high pathogenicity avian influenza virus into poultry farms during the epidemic in the netherlands in risk factors for highly pathogenic h n avian influenza virus infection in poultry during the - epidemic in italy a cross-sectional survey of australian chicken farms to identify risk factors associated with seropositivity to newcastle-disease virus wind-borne transmission of infectious laryngotracheitis between commercial poultry operations phylogenetic and epidemic modeling of rapidly evolving infectious diseases continued use of ibv b vaccine needs reassessment after its withdrawal led to the genotype's disappearance think globally, act locally: phylodynamic reconstruction of infectious bronchitis virus (ibv) qx genotype (gi- lineage) reveals different population dynamics and spreading patterns when evaluated on different epidemiological scales transmission of infectious bronchitis virus within vaccinated and unvaccinated groups of chickens control of avian influenza in poultry studies on australian infectious bronchitis virus. iv. apparent farm-to-farm airborne transmission of infectious bronchitis virus estimating population parameters using the structured serial coalescent with bayesian mcmc inference when some demes are hidden new routes to phylogeography: a bayesian structured coalescent approximation proroga e modifica dell' ordinanza agosto e successive modificazioni, concernente: «misure di polizia veterinaria in materia di malattie infettive e diffusive dei volatili da cortile epidemiology and control of low pathogenicity avian influenza infections in rural poultry in italy coronaviruses in avian species -review with focus on epidemiology and diagnosis in wild birds coronaviruses in poultry and other birds longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions mafft multiple sequence alignment software version : improvements in performance and usability w-iq-tree: a fast online phylogenetic tool for maximum likelihood analysis jmodeltest : more models, new heuristics and parallel computing rdp : detection and analysis of recombination patterns in virus genomes gard: a genetic algorithm for recombination detection cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences efficient bayesian inference under the structured coalescent improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty bayesian coalescent inference of past population dynamics from molecular sequences phylogeography takes a relaxed random walk in continuous space and time spread : interactive visualization of spatiotemporal history and trait evolutionary processes seraphim: studying environmental rasters and phylogenetically informed movements spatial visualization with ggplot this research was partially founded by the grant (bird / ) from the department of animal medicine, production and health, university of padua. g.f., a.m. and m.c. planned the study, g.f., c.m.t., m.l., t.t., r.c., p.p. performed laboratory work and generated the sequences obtained in the present study, c.m.t. and m.l. curated the sequences dataset, g.f. analyzed the data, m.c., a.m., p.m., g.t., g.o., l.g. supervised the respective research groups, g.f. wrote the manuscript, c.m.t., m.l., m.c. revised and improved the manuscript. all authors reviewed and agreed on the current version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to g.f.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -l zxic y authors: volpato, stefano; landi, francesco; incalzi, raffaele antonelli title: a frail health care system for an old population: lesson form the covid- outbreak in italy date: - - journal: j gerontol a biol sci med sci doi: . /gerona/glaa sha: doc_id: cord_uid: l zxic y nan italy is one of the oldest countries in europe and worldwide with an average life expectancy at birth of . years, an old age index of , and % of the population being and older ( ). on the second half of february , lombardy, one of the richest regions of the country, and northern emilia-romagna experienced the beginning of one of the largest and most serious clusters of covid- in the world. despite aggressive containment efforts, the disease continued to spread, and the number of affected patients has been rising steeply. according to the bi-weekly bulletin of the italian national institute of health (istituto superiore di sanità) as to march , , italian people ( % men) have been recognized as affected by reverse transcriptase-polymerase chain reaction (rt-pcr) testing over the country ( ) . of them, % were older than years and about % were older than years. since most of the italian regions decided to test only symptomatic patients, these figures clearly show that most cases of covid- affect the geriatric population. older people also have a worse clinical course, confirmed by case fatality rates by age group, showing a steeper increase in mortality from . % in the - age group to . % in the - group. furthermore, comparison with other european countries suggested an excess mortality in italy as compared, for example, with france ( . %) and germany ( . %) ( ) confirmed also in age-stratified analysis comparing italy with china demonstrating an excess mortality rate in italy after the age of ( ). according to the data released by the italian ministry of health, mortality rates varied across different regions of italy. for example, considering the four regions with more than deaths, mortality rate was . % for the veneto region, . % for piemonte, . % for emilia-romagna, and . % for lombardia. although age-stratified mortality rates across different regions are not available now, it is likely that regions with higher total mortality had even higher mortality rates for older people. higher mortality rates of older patients are expected as complicated covid- is characterized by severe interstitial pneumonia followed by acute respiratory distress syndrome, thromboembolic events, and eventually multiorgan failure, a cascade of negative events that is obviously more likely in older frail patients, those with elevated multimorbidity and reduced functional reserve. although it remains unclear how the different mortality rates vary across different regions of the country, the first spontaneous question is whether the italian health care system is well suited to respond to an unexpected and so huge health emergency. first, the italian health system, regarded as one of top ten in the world ( ), is equipped with . hospital beds for , persons, with extremely high bed occupancy rate, that is far below the european union average . / , and less than half of what is available in germany ( . / , ) ( ). the sudden explosion of the covid- outbreak, with almost % of infected patients having respiratory failure and requiring mechanical ventilation has immediately saturated the acute care beds availability of lombardy and northern emilia-romagna, including intensive care units' (icu) beds. the lack of availability of icu beds and mechanical ventilators has forced italian physicians to make quick and dramatic decisions on who had to be ventilated, intubated, and admitted to icu wards and who not. the guidelines of italian college of anesthesia, analgesia, resuscitation, and intensive care (siaarti) ( ) did not suggest that age should be the only factor determining resource allocation, but the document acknowledged that an age limit for icu admission may ultimately needs to be set. although chronological age was not the only criterium for selection, decision was certainly not based on a comprehensive geriatric assessment and therefore, it is likely that older patients and particularly geriatric patients, even with limited multimorbidity and good functional reserve, have had very limited access to icu and mechanical ventilation. as a matter of fact, it has been reported that several older patients have been dying at home, often alone, because of the lack of hospital beds' availability ( ) . a second important issue is that, because of the italian regionalization of the health care system, the availability of home care service and long-term care for older adults is substantially different across regions. home care and long-term care are cornerstones for continuity of care and prevention in older patients, particularly for those socially disadvantaged. from this point of view, lombardy has a very robust hospital network, but a limited long-term and home care organization that might have hampered early recognition of covid- cases among the oldest patients and might have postponed the access to care. third, according to the world health organization recommendation, laboratory testing for sars-cov- virus detection from nasopharyngeal swab or sputum sample has been performed only on symptomatic individuals or persons who had a close unprotected contact with confirmed patients. this policy may have limited the benefits of social isolation because asymptomatic people, including caregivers and health care personnel, might have carried the virus to older persons, particularly the most disabled and dependent and those living in nursing homes. from this point of view, it is striking that the veneto region that is experiencing the lowest mortality rates in italy (about %) performed the highest level of laboratory testing ( . % of the population), whereas, lombardy that tested only % of the total population is having the highest mortality rate (almost %). finally, it is possible that because of the higher burden of multimorbidity and consequent atypical presentation of covid- in older people, a diagnostic delay may have occurred in some cases leading to more severe pneumonia and worst prognosis. overall, these data suggest that italian national health care system was not suited and prepared to respond to this sudden and dramatic outbreak. first, our system, like our older frail patients, is lacking functional reserve able to increment the overall efficiency and maintain the system homeostasis under stressful conditions. the immediate shortages of icu beds and ventilators are one of the major causes of the incredibly high mortality rates observed in italy. second, our health system in probably well oriented to the cure and prevention of multifactorial conditions very common in western countries like cancer, cardiovascular diseases, and metabolic conditions but it is no longer used to counteract infection diseases outbreaks. from this point of view, the italian tragedy suggests that a prompt population mass testing for detecting asymptomatic infected people along with immediate, widespread, draconian measures of social isolation, along with contract tracing and quarantine, might have more strongly reduced the rate of covid- transmission, with the greatest benefit for frail patients, more prone to a complicated course of the disease. third, retirement homes for the older adults and nursing home should develop and implement specific strategies and protocols to prevent external contamination and widespread of the infection between their residents. fourth, social isolation might be particularly negative for older people and might be associated with worsening of many chronic conditions. it is of paramount importance to guarantee indirect contact with older patients and their caregivers, like a teleconsultation service for older persons. geriatric care teams must be integrated along with infectious disease specialists, pneumologists, and anesthesiologists into the management of this sars-cov- infection crisis and must develop tailored management strategies for older patients and should promptly investigate and describe the peculiar clinical presentation of covid- in older patients in order to facilitate immediate recognitions of the disease and start available therapy to prevent the most severe form of pneumonia complicated by respiratory failure ( ) . in this perspective, the gerocovid initiative of the italian society of gerontology and geriatrics aims at expanding the knowledge of the disease and its impact on health status and needs of care as well as at promoting health care strategies tailored to multiple different settings and different available resources. any geriatric society or working group can voluntarily join the gerocovid group at www.sigg.it. this would allow explore the effects of covid pandemic on the older population through the same protocol and, thus, have comparable data. each geriatric society or geriatric working group will use its own data besides contributing to the whole study. we hope that such a working method will make geriatricians able to recognize the disease, help their patients, and provide valuable information to the health authorities. case-fatality rate and characteristics of patients dying in relation to covid- in italy italy's health performance, - : findings from the global burden of disease study clinical ethics recommendations for the allocation of intensive care treatments, in exceptional, resource-limited circumstances troppi malati lasciati a casa. non c'è più la sanità pubblica the new challenge of geriatrics: saving frail older people from the sars-cov- pandemic infection key: cord- -o dp ag authors: marín‐hernández, daniela; schwartz, robert e.; nixon, douglas f. title: epidemiological evidence for association between higher influenza vaccine uptake in the elderly and lower covid‐ deaths in italy date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: o dp ag the italian covid- epidemic may finally be slowing, although the virus has spread from the north in lombardy throughout the rest of the country. while there have been more than , confirmed cases, and a mortality rate estimated around %, italy will now navigate an exit from lockdown with continued testing, monitoring, and contact tracing of any new infections. this article is protected by copyright. all rights reserved. epidemiological evidence for association between higher influenza vaccine uptake in the elderly and lower covid- deaths in italy to the editor, the italian covid- epidemic may finally be slowing, although the virus has spread from the north in lombardy throughout the rest of the country. although there have been more than , confirmed cases and a mortality rate estimated around %, italy will now navigate an exit from lockdown with continued testing, monitoring, and contact tracing of any new infections. the higher mortality rate in italy compared with china has been attributed to a higher prevalence of elderly people living in italy and a different strain of sars-cov- . however, as the virus has now spread throughout the country, regional differences in mortality rate from covid- have been the mdck cell line is made available to vaccine manufacturers from cell banks that have produced the cell line in accordance with good manufacturing practice guidelines, and has been tested for purity, identity, and for the absence of contaminating viruses as required by food and drug administration, emma, and world health organization guidelines. all three forms of the influenza vaccine were available for potential prescription and use. we used publicly available data to compute a pearson productmoment correlation for assessment of the relationship between the percentage of vaccinated adults greater than years old and the percentage of covid- deaths from each region in italy up to may . we calculated a simple linear regression to predict the percentage of covid- deaths based on the percentage of adults greater than years old vaccinated against influenza. we found a moderate to strong negative correlation (r = −. , n = , p = . ), meaning that where there were higher influenza vaccination rates, less deaths from covid- occurred. then, we as covid- cases, deaths and fatality rates surge in italy, underlying causes require investigation first cell-based quadrivalent influenza vaccine approved for use in europe adjuvanted influenza vaccine for the italian elderly in the / season: an updated health technology assessment how influenza (flu) vaccines are made safety of mdck cell culture-based influenza vaccines vaccinazione antinfluenzale in italia: coperture vaccinali nella popolazione generale (per abitanti) coronavirus deaths by region statista. italy coronavirus deaths by reg.|stat. sars-cov- coinfections: could influenza and the common cold be beneficial key: cord- -fy le ar authors: bellizzi, saverio; pichierri, giuseppe; napodano, catello mario panu; picchi, sara; fiorletta, serena; panunzi, maria grazia; rubattu, edoardo; nivoli, alessandra; lorettu, liliana; amadori, antonella; padrini, susanna; ronzoni, anna rita title: access to modern methods of contraception in italy: will the covid- pandemic be aggravating the issue? date: - - journal: journal of global health doi: . /jogh. . sha: doc_id: cord_uid: fy le ar nan t he strain on health system imposed by the current covid- pandemic is undoubtedly impacting the sexual and reproductive health of women and girls of many countries in the world. reconfiguration of health systems to cope with the epidemic has led to the forced closure of health services deemed not essential as well as diversion of health care workers to fulfill other needs. in addition, physical distancing and travel bans are having adverse effects on access to and supply of contraceptive commodities. manufacturing of specific pharmaceutical components is, for instance, causing a shortage of condoms [ ] . a mathematical model exercise done by the guttmacher institute has estimated a % proportional decline in the use of short-and long-acting reversible contraceptive methods in low-and middle-income countries (lmics) due to reduced access [ ] . other pieces of very recent research have predicted much worse scenarios, with decline in sexual and reproductive health services up to % [ ] . access to contraception is not just an lmics issue: the contraception atlas initiative, powered by the european parliament forum for sexual and reproductive rights (epf) in partnership with a group of experts in sexual and reproductive health and rights, showed that for many european countries, ensuring that people have choice over their reproductive lives is not a priority [ ] . specifically, the atlas tracks government policies on access to contraceptive methods, family planning counselling and the provision of online information on contraception in european states. in the european contraception atlas, italy ranked just th out of the total countries. access to modern methods of contraception in italy: will the covid- pandemic be aggravating the issue? access to contraception is not just a low-and middleincome countries issue: in the european contraception atlas, italy ranked just th out of the total countries for various reasons such as the very limited provision of free contraception as well as under-resourced and understaffed family planning centers. despite the disruption due to the current covid- pandemic, it is essential to ensure that women can have control over their bodies and their sexual and reproductive life, and protect the access to modern contraceptives and family planning services for both men and women. in order to analyze the bad performance of italy in terms of access to contraception, a national atlas has been promoted in order to understand also the impact of the regional differences. despite the fact that access to contraception is regulated at the national level, in italy the organization and implementation of contraceptive services are left to the regional governments. for this reason, only regions out of have introduced the provision of free contraception [ ] . the italian contraception atlas indicates two main results which have to be considered when the effect of covid on contraception is taken into account in terms of advocacy and emergency policies. the first is the huge differences among regions with the historical divide between north and south (with the only exception of puglia which is the first region that has introduced free contraception at regional level). such regional differences are multi-factorial and likely driven by cultural and social aspects. the second is a widespread tendency to fund programs which handle the access to the information of contraceptive methods more than those directed to their provision. this is as a result of the scarcity of public resources as well as the strict manner in which related budgets can be spent. information programs are preferentially funded as they are less expensive than providing actual contraceptive methods. the fragmentation of the policies has an impact on the equal opportunity to have free access to contraception in italy. there is evidence that a lack of general direction at national level generates gaps among territories which could be addressed with several initiatives, as the introduction of a sort of minimum level of assistance and the vocational training of medical professionals, thus facilitating integrated programmes on contraception and reproductive health, or by improving what has been already implemented as the web page on the portal of the ministry of health dedicated to contraception. despite the establishment of the counseling centers under the law n. in [ ] , with the aim of supporting "responsible parenthood" through providing information on contraception and by protecting the health of women and their children, institutionalized provision of appropriate information on contraception is still broadly lacking: % of boys and % of girls seek for information related to sexual and reproductive health in the internet [ ] . on the other hand, % of boys and % of girls have never been in a counseling center [ ] . in italy, family planning centers are often under-resourced and understaffed; as a consequence, individual counselling is rarely provided by interdisciplinary teams, which might affect the quality of the service depending on users' needs [ ] . moreover, few centers are directly linked to hospitals or specialized services, so that women are not always referred to specialists when appropriate. importantly, family planning centers are not easily accessible outside the larger cities [ ] . in the north-eastern regions plus lombardy and molise there is less than family planning center per women. this picture is worse even compared to southern regions (where the average is to ) as well as to other regions in the north and the center (where the average is to ) [ ] . as far as the use of contraception is concerned, % of adolescents use withdrawal as preferred method of contraception while % relies on standard days and % do not use any contraceptive method [ ] . this non-optimal situation is compounded by the largely stigmatizing vision on reproductive health, specifically around abortion and some modern methods of con-photo: a young italian couple engaging in discussion about contraception (from saverio bellizzi's collection, used with permission). traception like the pill. in fact in italy, despite the legalization of voluntary interruption of pregnancy under the law n. in [ ] , conscientious objection remains one of the main obstacles, with a percentage of . % of gynecologists who avail themselves of this option [ ] , preventing women in italy from accessing the human right to have control over their bodies and the legal right to terminate their pregnancies, often forcing them to seek treatment abroad. financial accessibility represents another key bottleneck: the intrauterine device (iud), a very effective contraceptive method with a long-term effect on avoiding or delaying pregnancy, could be as costly as euros, which is well beyond the reach of all women and girls belonging to vulnerable social group who cannot afford this method. as for the pill, the italian pharmaceutical agency has classified in the "c" category, which makes it almost completely in charge of women and requires prescription. one of the main recommendations of the atlas report refers to the need for financially strengthening counseling centres, which should be much more widespread in the territory ( every inhabitants instead of the current every ). the counseling centres, perform key function in providing information, support and promotion of women's health. data collected during an iss (istituto superiore di sanita') survey carried between - , showed that more than % of the counseling centres involved in the survey provide support and care related to sexuality, contraception, vip' s pathway, preconceptional health, family planning, birth pathway, sexually transmitted diseases, cancer screening, menopause and postmenopause [ ] . furthemore, % of the overall counseling centres in italy offer free contraceptives [ ] . sexual education in the schools is another crucial recommendation. despite the disruption due to the current covid- pandemic, it is essential to ensure that women can have control over their bodies and their sexual and reproductive life, and protect the access to modern contraceptives and family planning services for both men and women. in line with the call made by the un secretary general, access to contraception must be streamlined, even without prescription and free of charge. when regular health care services are disrupted, access to long-acting and emergency contraceptives becomes even more essential. how will covid- affect global access to contraceptives -and what can we do about it? devex estimates of the potential impact of the covid- pandemic on sexual and reproductive health in low-and middle-income countries. guttmacher institute methodology for calculating impact of covid- european parliamentary forum on population & development (epf). contraception atlas associazione italiana donne per lo sviluppo (aidos) legge luglio , n. . istituzione dei consultori familiar ministero della salute, istituto superiore di sanita'. principali risultati del progetto "studio nazionale fertilita women' s access to modern contraceptive choice -barometer legge maggio , n. . norme per la tutela sociale della maternità e sull'interruzione volontaria della gravidanza legge , la relazione annuale al parlamento con i dati consultori: indagine dell'istituto superiore di sanita', strutture censite no funding was received for this report.authorship contributions: all authors contributed to writing and conceptualizing the manuscript. the authors completed the icmje unified competing interest form (available upon request from the corresponding author), and declare no conflicts of interest. key: cord- -ioph vsp authors: tobías, aurelio title: evaluation of the lockdowns for the sars-cov- epidemic in italy and spain after one month follow up date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: ioph vsp from the end of february, the sars-cov- epidemic in spain has been following the footsteps of that in italy very closely. we have analyzed the trends of incident cases, deaths, and intensive care unit admissions (icu) in both countries before and after their respective national lockdowns using an interrupted time-series design. data was analyzed with quasi-poisson regression using an interaction model to estimate the change in trends. after the first lockdown, incidence trends were considerably reduced in both countries. however, although the slopes have been flattened for all outcomes, the trends kept rising. during the second lockdown, implementing more restrictive measures for mobility, it has been a change in the trend slopes for both countries in daily incident cases and icus. this improvement indicates that the efforts overtaken are being successful in flattening the epidemic curve, and reinforcing the belief that we must hold on. the first confirmed cases of sars-cov- in italy and spain were identified a week apart in late february (saglietto et al., ) . since then, italy has become the third most affected country worldwide ( , cases, in april th) after the united states and has the highest number of deaths due to the sars-cov- pandemic ( , deaths, in april th). meanwhile, spain is the second most affected in the number of diagnosed cases ( , ) and mortality ( , ) (our world in data, ). since march th, widespread lockdown measures have been in place in italy. specific measures restricting social contact were first introduced in the northern regions, where most cases had occurred, then extended to the whole country on march th. furthermore, science of the total environment j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / s c i t o t e n v italy tightened these measures extending the lockdown on march st: all businesses were closed, with the exception of those essential to the country's supply chains. similarly, spain imposed a lockdown on march th , with social distancing measures similar to those established in italy. two weeks later, on march th, spain also implemented a more restrictive lockdown, aimed at reducing the mobility and non-essential industrial activity countrywide (mitjà et al., ) . these restrictive measures have mainly been focused on flattening the epidemic curve. most of the results reported daily by governments, official agencies, and the media are aimed at describing cumulative epidemic data. conversely, little attention has been paid to describe, quantify, and compare the lockdown effects within and between countries from an epidemiological point of view using incident data. we collected data between february th and april th from the websites of the italian and spanish ministries of health (dipartamento della protezione civille, ; instituto de salud carlos iii, ). we have analyzed the trends of the daily incident diagnosed cases, deaths, and intensive care units (icu) admissions for sars-cov- in italy and spain before and during their respective national lockdowns, using an interrupted time-series design (bernal et al., ) . the data was analyzed with quasi-poisson regression, using an interaction model to estimate the change in trends (bernal et al., ) . the data was analyzed using stata, release (statacorp, college station, tx, ). fig . shows the observed daily incident data and the estimated trend slopes for italy and spain since february th. before the lockdown, the daily percent increase of all the incidence outcomes was higher in spain ( . % for diagnosed cases, . % for deaths, and . % for icu admissions) than in italy ( . %, . %, and . %, respectively) ( table ) . during the first lockdown period, both countries show similar daily trends ( . %, . %, and . % in italy; and . %, . %, and . % in spain). thus, during the first lockdown the daily increase in incident data was considerably reduced. in italy, the diagnosed cases decreased by . %, deaths by . %, and icu admissions by . %. this reduction was even higher in spain, where the diagnosed cases decreased by . %, deaths by . %, and icu admissions by . %. however, although the slopes have been flattened for all outcomes, the trends kept rising (fig. ) . during the second and more restrictive lockdown, currently ongoing, both countries show some positive signs, indicating that trends may be changing (fig. ) . specifically, in italy all outcomes start declining; diagnosed cases if − . %, daily deaths of − . %, and icu admissions of − . %. similarly, spain also shows decline trends in daily diagnosed cases of − . %, deaths of − . %, and icu of − . %. lockdown, including restricted social contact and keeping open only those businesses essential to the country's supply chains, has had a beneficial effect in both countries. the trend slopes were considerably reduced for all the outcomes. however, this was not enough to change the rising trend of the epidemic. for this reason, more restrictive actions were suggested. the second lockdown, still ongoing, shows how the trends have changed, with a reduction of daily incident cases, deaths, and more significantly in icus. these are of similar magnitude in both countries, although italy carries a week ahead of spain. however, mortality still shows a small increase, probably because it follows incidence trends with a delay of - weeks. we should acknowledge that this figures could change during forthcoming weeks. analysis of trends using daily incident data may be a useful complement to more conventional approaches used to monitor the sars-cov- epidemic by testing and reporting changes overtime after a public health intervention (i.e., lockdown, confinement or quarantine). interrupted time-series regression models are relatively easy to implement using readily available data by health authorities (bernal et al., ) . this is a descriptive analysis without predictive purposes. for an easy interpretation, and comparison of the effectiveness of lockdown measures between countries, a linear trend is assumed before and during the lockdown periods. the changes in the definition of diagnosed cases have not been taken into account, nor has the reduction in the susceptible population because of the lockdown. therefore, the incident cases are modeled directly instead of the incidence rate, assuming that the entire population is at risk. timely indications for public health authorities and governments are essential to slow down the sars-cov- epidemic and relieve the pressure on overburdened health services. although available data is still limited, and findings must be interpreted with caution, we believe that real-time detection of pattern changes are essential to evaluate the current measures of control and design future ones. in this sense, the positive signs already shown by the decreasing trend slopes after a more restrictive lockdown in italy and spain could indicate an optimistic and encouraging forecast for those countries that in late march also announced restrictive lockdown measures for flattening the sars-cov- epidemic curve (e.g., the united kingdom on march th or ireland on march th). these results show that the sacrifices that our society is making are gaining us valuable time, which is essential to get ready to face the future pressures that this epidemic will bring forth. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. interrupted time series regression for the evaluation of public health interventions: a tutorial situación de covid- en españa experts' request to the spanish government: move spain towards complete lockdown covid- ) -statistics and research. oxford martin school, the university of oxford, global change data lab covid- in europe: the italian lesson to milena maule (department of medical sciences, university of turin) for her useful comments helping to improve the contents of the manuscript. . ( . , . ) − . (− . , . ) key: cord- - jursf h authors: diaferio, lucia; parisi, giuseppe fabio; brindisi, giulia; indolfi, cristiana; marchese, giuseppe; ghiglioni, daniele giovanni; zicari, anna maria; marseglia, gian luigi; miraglia del giudice, michele title: cross-sectional survey on impact of paediatric covid- among italian paediatricians: report from the siaip rhino-sinusitis and conjunctivitis committee date: - - journal: ital j pediatr doi: . /s - - - sha: doc_id: cord_uid: jursf h background: there is ample evidence that covid- is significantly less severe in children than in adults and asthma and allergy, the most common chronic disorders in children, are not included in the top comorbidities related to covid- fatalities. nevertheless, concerns about asthma and allergy are still high.. in order to evaluate the impact of paediatric covid- among italian paediatricians, we sent a -questions anonymous internet-based survey to italian paediatricians with particular address to allergic symptoms and those affecting the upper airways. methods: the questionnaire was conceived and pretested in april , by a working group of experts of the italian paediatric society for allergy and immunology (siaip), and structured into different sections of categorized and multiple choice questions. the first part included questions about epidemiological data follows by a second part assessing the way to manage a suspected covid- infection and personal experiences about that. the third part concerned questions about patients’ clinical characteristics and clinical manifestations. the survey was emailed once between april and mid-may . results: a total participants had participated in our survey and provided responses to our electronic questionnaire. the distribution of patients reported per month varies significantly according to the geographical area (p = . ). data confirmed that in the north part of italy the rate of patients referred is higher than in the rest of italy. almost all respondents ( %) reported caring for up to a maximum of infected children and the last % more than twenty. among these patients, according to the % of responders, a maximum rate of % were affected by allergic rhino-conjunctivitis and in particular in the north of italy while in the centre and in the south there was a higher incidence (p = . ). almost the same applies for asthma, % of responders declared that up to a maximum of % of affected children were asthmatic, from to % for the , % of responders and from to % for the last , %. as for the allergic conjunctivitis also for asthma, we found a higher incidence in the centre and in south than in the north (p = . ). conclusions: this study is the first to provide a comprehensive review of covid- knowledge and impact among paediatricians in italy about allergic asthma and upper airway involvement. from our point of view, it provides important information clearly useful for improving a good practice. in march , the world health organization (who) declared the covid- pandemic. the novel coronavirus, sars-cov- , a top threat to global health, emerged in wuhan (china) in december last year and rapidly spread worldwide [ ] . globally, at the end of august the total confirmed cases of covid- have reached over , , with over , deaths and daily data shows continuous increases in new covid- cases [ ] . in italy, we have experienced serious outbreaks linked to the first cluster, in south lombardy, with about , confirmed cases and more than , deaths [ ] . research shows that covid- causes symptoms including fever, dry cough, dyspnoea, fatigue, lymphopenia and in more severe cases, severe acute respiratory syndrome (sars) and even death [ ] [ ] [ ] [ ] . every age may be affected but childhood seems to be safeguarded by severe covid- , due to comorbidities associated with lethal covid- infection (obesity, diabetes and chronic heart disease) [ ] . it has been reported that asthma and allergy, the most common chronic disorders in children, are not included in the top comorbidities associated with covid- fatalities [ ] . nevertheless, there would seem that the concerns about asthma and the risk of disease and related outcomes are still high [ ] . actually, data on covid- in italian children are limited and almost certainly underestimated, since they are frequently asymptomatic or presenting mild or moderate infection, similar to common cold. in order to evaluate the impact of paediatric covid- among italian paediatricians, we sent a -questions anonymous internet-based survey to italian paediatricians with particular address to allergic symptoms and those affecting the upper airways. the questionnaire was conceived and pretested in april , by a working group of experts of the italian paediatric society for allergy and immunology (siaip) based on their personal clinical experience and on the extensive review of most relevant international literature on covid- infection searched on medline, embase and scopus. the prior revised and confirmed paper version of the questionnaire was finally converted in a web-based survey with google-drive (google drive™,© google inc. all rights reserved), a free internet platform applied for the creation of internet-based survey forms which allows to have real-time digital archiving of collected data, real-time presentation of survey results, and simple download of all data of registered anonymised participants in excel© format for statistical analysis. the questionnaire was structured into different sections of categorized and multiple choice questions. the first part included questions about epidemiological data follows by a second part assessing the way to manage a suspected covid- infection and personal experiences about that. the third part concerned questions about patients' clinical characteristics and clinical manifestations. finally, the last part focused on the knowledge in the field and educational priorities of participants. the language of the questionnaire was the national one. the reported time to complete the survey was approximately min. the survey was emailed once between april and mid-may to about members of the italian paediatric society for allergy and immunology (siaip). participants were allowed to complete only a single survey, duplicate entries were avoided and responses were scrupulously monitored. informed consent was not obtained, given that the participation was voluntary. no financial incentive was offered. the ethics committee of the university of bari (italy), was contacted and no special permission was deemed to be required because the study design satisfied the criteria of an activity audit. once the questionnaire results were obtained, they were statistically processed. answers were converted in different categorical variables. differences in categorical variables were evaluated with chi square and fisher exact tests as appropriate.-sas® university edition (cary, nc: sas institute inc) was used for all analyses. data are expressed as percentage, p < . were considered statistically significant. a total participants had taken part in our survey and provided responses to our electronic questionnaire by may th, . the characteristics of the survey participants are detailed in table . among responders, % practiced in a place where there is not a children hospital dedicated to covid care. % of respondents reported that in a month referred to them up to patients for suspected sars-cov- and up to for the % (more than just for the %), starting from february according the majority of them ( %). in particular, the distribution of patients reported per month varies significantly according to the geographical area (p = . ). data showed that in the north part of italy the rate of patients referred is higher than in the rest of italy. moreover, we found that only the infectious disease specialist reported that in a month referring more than patients for suspected sars-cov- (p < . ). the diagnosis of covid- is made once a month according to % of participants, once a week for % of participants, once in months for %, once in months for %, and once a day for % of participants. almost all respondents ( %) reported they had in charge up to a maximum of infecting children and the last % more than twenty. among these patients, according to the % of responders, a maximum rate of % were affected by allergic rhino-conjunctivitis and in particular in the north of italy while in the centre and in the south there was a higher incidence (p = . ). almost the same applies for asthma, % of responders declared that up to a maximum of % of affected children were asthmatic, from to % for the , % of responders and from to % for the last , % (fig. ) . as for the allergic conjunctivitis also for asthma, we found a higher incidence in the centre and in south than in the north (p = . ) ( table ) . on average, these children are ≤ years old according to % of participants, from ≤ to years old for the % of responders as well from ≤ to years old, until years old for the % and more than years old for the last %. of the respondents, % agreed immediately isolation in a proper place and to alert the public health service system was the first step in case of a suspected infection, % declared to suggest just the isolation without any geographical differences. eleven percent of respondents would refer out patients to the emergency department and the last % leading to an emergency call. however, % of participants clarified that confirmed cases of sars-cov- infection had nasopharyngeal and oropharyngeal swab sampling, % reported that it was not performed in suspected cases and not yet for % of responders. in particular, we found that the rate of not performed nasopharyngeal and oropharyngeal swab sampling is higher in the centre and in the south than in the north (p = . ). regarding signs and symptoms suggestive of sars-cov- infection, the majority of respondents ( %) recognized fever, cough ( %) and gastrointestinal disease ( %) as main symptoms. interesting, olfactory and gustatory dysfunctions in children are rare (fig. ) . finally, the majority of italian paediatricians ( %) declared to have a good knowledge about covid- , however they'd all be interested to increase knowledge about the impact of covid- on italian children. this cross-sectional survey provides information on the impact of covid- among paediatricians. . a good level of knowledge in the field is linked to a successful practice. so that, evaluating knowledge, attitude and practice among paediatricians is of considerable practical importance. it should be noted that responders were allocated evenly among italy in order to guaranteed information from all italian regions. in addition, no significant difference has been identified with regard to management of a suspected covid- case among italy. regarding signs and symptoms suggestive of covid- , our results showed that in children, unlike adults, olfactory and gustatory dysfunctions are not prevalent. these findings are in line with a recent meta-analysis which included research performed in china (just one clinical case in singapore) [ , ] . allergic rhino-conjunctivitis and asthma, according to our data, seem not to be a risk factor to developing more severe covid- . however, since the role of asthma in increasing the severity of covid- is still unclear, it remains a great concern for patients and paediatricians. the diagnosis and management of covid- in children is still difficult due to the mild or moderate clinical course. moreover, asymptomatic infections were not infrequent [ ] with the risk of unconfirmed disease. this seems to be a frequent problem in daily clinical practice. nevertheless, our data showed that italian children have good chances to be tested for sars-cov- , indicating the importance of an accurate diagnosis, which will facilitate appropriate treatment options and preventive measures. our study shows some limitations. although almost participants completed our survey, only those with access to the internet and only those with available email addresses were recruited. other limitations are related to our pilot survey and include the use of a nonstandardised questionnaire. however, to the best of our knowledge, standardised and validated surveys on this issue are not available. some selection bias includes the recruitment methodology; those who felt more interested about covid- may have been more inclined to complete the survey. this study is the first to provide a comprehensive review of covid- knowledge and impact among paediatricians in italy about allergic asthma and upper airway involvement. from our point of view, it provides important information clearly useful for improving a good practice. our data confirmed that comorbidities as asthma or rhino-conjunctivitis cannot represent a risk factor for more severe covid- disease. moreover, symptoms such as anosmia and ageusia are rare in the paediatric population. epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan,china: a descriptive study clinical features of patients infected with novel coronavirus in wuhan clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical characteristics of novel coronavirus infection in china severe acute respiratory syndromecoronavirus (sars-cov- ) infection in children and adolescents: asystematic review asthma is absent among top covid- risk factors, early data shows consensus statement of the italian society of pediatric allergy and immunology for the pragmatic management of children and adolescents with allergic or immunological diseases during the covid- pandemic sars-cov- infection in children upper airway involvement in pediatric covid- publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank all the paediatricians who took part in this survey.authors' contributions mmdg and glm conceived the survey, ld, gfp, gb, ci, gm, az, dgg conceptualized and designed the survey. gfp and mmdg collected and analysed the data. ld wrote the manuscript. gfp, gb, ci, gm, az, dgg reviewed the manuscript. mmdg and glm performed a critical revision of the manuscript and offered precious technical advice on how the study might be improved. all authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work. the authors did not receive any funding for the research. the italian paediatric society for allergy and immunology (siaip) covers the articleprocessing charge. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable. not applicable. the authors have no conflicts of interest to disclose that could be perceived as prejudicing the impartiality of the research reported. key: cord- - b n un authors: spousta, martin title: parametric analysis of early data on covid- expansion in selected european countries date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: b n un we analyze the early data on covid- expansion in selected european countries using an analytical parametric model. a description of the time dependence of the disease expansion and a method to evaluate trends of the expansion are proposed. several features are observed in the data, namely a high predictability of the expansion of disease in italy and a convergence of the “pushback” parameter towards a limiting value in all the countries where restrictive measures have been adopted. basic predictions for the evolution of the disease expansion are made for selected countries with a stable evolution in the parametric space of the model. the findings presented here should contribute to the understanding of the behavior of the disease expansion and the role of the restrictive measures on the evolution of the expansion. the outbreak of new coronavirus sars-cov- causing severe respiratory tract infection in humans, known as covid- , is a global health concern. restrictive measures have been adopted in many countries in order to mitigate the impact of the spread of the disease on public health system [ ] . in this paper we propose a straightforward analytic description of the time dependence of the disease expansion under the restrictive measures and a method allowing to identify trends in the expansion and make predictions. the analytic parametric modeling can be used as an alternative to complex models for the disease expansion such as those reviewed e.g. in ref. [ ] . in the first section of this paper, we introduce the model, analyze the data which are available as of st of march from selected european countries and discuss several features seen in the data. in the second section, we explain calculations for predictions and we make predictions for three countries which appear to be on a predictable trajectory in the parametric space of the model. data used in this work are taken from [ ] , cross-checked using information system described in ref. [ ] , and from [ ] . email address: martin.spousta@mff.cuni.cz (martin spousta) number of newly infected people from one infected person has a power-law dependence on time. in general, the total number of infected people in time t can be fully characterized by a function where α i and a i are unknown parameters. the sum goes trough individual centers of the disease expansion. these centers carry constant parameters α i and a i only over a limited time interval which is characterized by a step function χ i (t). the full description of the outbreak by a function ( ) can be replaced by exponential dependence with time dependent parameter a(t), that is by the function where the right-hand side of ( ) represents a taylor expansion of the time dependent parameter a. if one assumes that a(t) is constant or monotonically decreasing with time, one may assume that |b i | > |b i+ | and higher order coefficients of the taylor expansion will contribute less to f (t). one may therefore start the description of the total number of infected people at a given time t with three approximations, approximation ( ) is a pure exponential distribution which we shall observe if no human measures are taken to control the spread of the disease and no immunization of the population is assumed. approximation ( ) is modified exponential expansion where parameter b , if negative, characterizes the "pushback" resulting from human measures to control the disease. approximation ( ) allows to control the validity of |b i | > |b i+ | assumption and a need for the higher order terms to model the spread of the disease. data can be fitted by ( ), ( ), and ( ). the quality of the fits then decides which of the approximation serves as the best description of the data. when fitting the data over the full available time range from three representative european countries, namely italy, france, and czechia, we found that indeed |b | |b | |b | and that approximation ( ) has slightly worse χ /ndof than approximation ( ) (typically by %). we may therefore conclude that approximation ( ) and higher order terms are not needed for parameterizing the data. to evaluate the evolution of the spread of the disease as a function of time, one can study the time dependence of parameters b , b , and n . this can be done by fitting the data in time windows or by fitting the data between the time of the beginning and a given time t. the former method is more susceptible to fluctuations of the data which may occur within the time windows while the latter method may be biased by the change of the parameters in time. we tested both of these methods as well as various sizes of the time window with the conclusion that fitting from the beginning leads to less fluctuating values of parameters which exhibit the same trends and tend to converge to the same values as the values of parameters analyzed by fits in time windows. from now on, we therefore evaluate the evolution of parameters b , b , and n by fitting from the beginning up to a time t indicated on the x-axis of plots. the evolution of parameters b , b , and n is shown in upper left and lower panels of figure . one can see that parameters are highly oscillating at the beginning but then for times greater than days since the first registered the parameter b can be interpreted as a parameter quantifying the uncontrolled spread of the disease. as such, it will be assumed that it is universal for all the european countries since they share approximately the same default density of interactions among inhabitants (cities of similar density, similar level of public transportation, similar default . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. cultural behavior). consequently, for subsequent studies, the parameter b was fixed to a value . representing a value preferred by a minimum χ condition for the fit in the country with the most stable evolution which is italy. the time evolution of n and b parameters in the case of fixed b is shown in figure . one may identify several features seen from the behavior of parameters: . parameter b starts from zero (no restrictive measures), then decreases as expected for all the countries, and then it becomes constant for countries where restrictive measures are in place for a longer time (namely italy and france). . all the countries seem to tend to the same value of the parameter b . . parameters b and n are consistent with a constant over the last ∼ days in italy. this means that the situation in italy is highly predictable as further discussed in section . . parameter n is much higher for the case of italy than for other countries. the features − , if confirmed by other studies, open questions for a detailed epidemiological analysis: what is the reason for the existence of a limiting efficiency of applied measures which is indicated by the data? is it e.g. due to the high-efficiency for the disease to spread inside closed communities? or is it e.g. due to having a certain fraction of the society failing to follow correctly the measures? the analysis presented here obviously cannot answer these questions. at the same time, it allows us to assess the efficiency of applying the existing measures. for example, if we observe that the parameter b achieves a limiting value of italy even in countries where more restrictive measures have been applied, such as mandatory usage of masks in czechia, then this indicates that the use of these measures does not bring further reduction of the spread of the disease . since n reflects the number of cases at the time zero, feature implies that the infection was likely present in italy even before the time zero. this may also explain the excessive values of parameter b observed for first ∼ days. we should note that when fixing b , the values of parameters n and b still remain correlated. in some cases the correlation coefficient is greater than . . this may limit the predictive power of the model and a straightforward interpretation of the parameter n as the number of cases at time zero. a way to reduce these correlations needs to be further studied. so far we have tested alternatives to ( )-( ), e.g. and with b i , t i , and c being free parameters and k = , . none of these more ad-hoc functional forms provided a better description of the data than ( ). while n and b remain correlated, one can notice that the values of the n and b start to be constant in time for italy and france even if the number of cases evolve with time. this builds a confidence in the predictive power of the model which is discussed in the next section. when being in the situation where all the parameters tend to constant values, one can predict the behavior of the spread of the disease using simple analytic formulae. an estimate of the time when at the same time it would obviously not prove that this particular measure is not very efficient in the case when no other measures are taken. additionally, if the b value for czechia goes below the b value for italy, it would indicate that more restrictive measures applied in czechia may increase the efficiency in reducing the spread of the disease. . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the restrictive measures (b ) outperform the uncontrolled spread (b ), t max , that is the time when no new cases of the disease should be registered is given by a derivative of ( ) with respect to time, which also allows to calculate the total number of cases, an estimate of the time when the number of new cases should stop to grow, t ∆ , is an inflection point of f (t) and it is given by a second derivative of ( ) with respect to time, the results of the predictions using equations ( )-( ) are shown in figure for countries with constant parameters of the fit which is italy and france, and for czechia which seems to be converging to a constant value of parameters as well. we do not attempt to introduce systematic uncertainties on the predictions at this point. instead, we use evaluation of the distribution using two methods: using fits in time windows of seven days and the fits over the full distribution. as already mentioned before, the later method brings results less susceptible to fluctuations. left and middle panel of figure show the prediction for the t max values and n (t max ) values, respectively, as a function of time since the first registered case. one can therefore see what prediction one would make in the past. the current prediction is represented by the last point of the distribution. one can again see that the prediction does not change over the last ∼ and ∼ days in the case of italy and france, respectively. as said before, these countries seem to be on a highly predictable trajectory which can be illustrated for italy by evaluating the prediction for the total number of cases for today which one would make days ago. the prediction would be cases while data for today ( th of march) show cases. for the case of czechia, one can see an evolution towards more optimistic scenario as the time increases. at the same time, the prediction remains relatively stable over the last few days. the prediction for the t ∆ is given in terms of the distribution of newly indicated cases as a function of time from today. one can see that the maximum of new cases should already been achieved in italy, while in france and czech republic it is expected in about and − days, respectively. we proposed a straightforward method for evaluating the temporal dependence of spread of covid- disease. the analysis of the data indicate several features, namely the high predictability of the expansion of disease in italy and a convergence of the "pushback" parameter towards a limiting value in all the countries where restrictive measures are applied. predictions of the evolution of the spread of the disease are made for three countries that appear to be on a predictable trajectory in the parametric space of the model, namely for italy, france, and czechia. the proposed model and analysis method represent an alternative to complex modeling which is simple to implement and independently verify, and which can be quickly extended towards more complete description of the situation in many different countries affected by the covid- outbreak. world health organization -coronavirus disease (covid- ) situation reports dynamically modeling sars and other newly emerging respiratory illnesses worldometer -collection of online world statistics an interactive web-based dashboard to track covid- in real time official statistical web site of minister of health of the czech republic i'd like to thank jiří dolejší for useful discussions and suggestions and for careful reading the manuscript. key: cord- -mtcqawxc authors: ruggieri, p.; trovarelli, g.; angelini, a.; pala, e.; berizzi, a.; donato, d. title: covid- strategy in organizing and planning orthopedic surgery in a major orthopedic referral center in an area of italy severely affected by the pandemic: experience of the department of orthopedics, university of padova date: - - journal: j orthop surg res doi: . /s - - - sha: doc_id: cord_uid: mtcqawxc background: according to the required reorganization of all hospital activities, the recent covid- pandemic had dramatic consequences on the orthopedic world. we think that informing the orthopedic community about the strategy that we adopted both in our hospital and in our department of orthopedics could be useful, particularly for those who are facing the pandemic later than italy. methods: changes were done in our hospital by medical direction to reallocate resources to covid- patients. in the orthopedic department, a decrease in the number of beds and surgical activity was stabilized. since march , it has been avoided to perform elective surgery, and since march , non-urgent outpatient consultations were abolished. this activity reduction was associated with careful evaluation of staff and patients: extensive periodical swab testing of all healthcare staff and swab testing of all surgical patients were applied. results: these restrictions determined an overall reduction of all our surgical activities of % compared to . we also had a reduction in outpatient clinic activities and admissions to the orthopedic emergency unit. extensive swab testing has proven successful: of more than people tested in our building, only three covid- positives were found, and of over more than surgical procedures, only two positive patients were found. conclusions: extensive swab test of all people (even if asymptomatic) and proactive tracing and quarantining of potential covid- positive patients may diminish the virus spread. the recent covid- pandemic had dramatic reflexes on the organization of the health care system and hospitals worldwide; of course, it also had consequences on the orthopedic world and it required reorganization of all the hospital activities. we decided to report the experience in our department of orthopedics and orthopedic oncology at the university of padova in italy for several reasons. first of all, italy was the first country in europe suffering from the covid- pandemic. second, padova is one of the major towns in veneto, and the region veneto in italy has been one of the most heavily affected by the covid- pandemic. besides these, chronologically, the two first areas, where unfortunately covid- began in europe, were in our two italian regions lombardia and veneto. more precisely, the first person that died of coronavirus infection was on february in a small village named vo' euganeo, which is very close to padova. the other focus of the beginning infection in italy was in codogno, another small town close to milan in lombardia. finally, ours is the major hospital in padova, and it was elected since the start as the referral center for all covid- patients. in our hospital, thanks to an appropriate political organization when the infection began, actions were performed to try to face the virus spread. we think that informing the orthopedic community about the strategy that we adopted both in our hospital and in our department of orthopedics could be useful, particularly for those who are facing the pandemic later than italy. this is a report of the current experience in orthopedics during the covid- pandemic at the department of orthopedics of the university of padova, veneto, italy. our orthopedic department is part of a huge hospital in padova that includes about beds, collected in two areas: one university hospital of over beds and another smaller hospital of about beds, called sant'antonio hospital. changes were done in the orthopedic department as well as in other departments of the university hospital by medical direction (d.d., one of our co-authors, our medical director) in order to minimize resources reallocating anesthesiologists, nurses, and medical assistants to dedicate to covid- patients in new icus, semiintensive units, and recovery units. since the start, it was decided that sant'antonio hospital is covid free, whereas the university hospital could accept patients who were probably positive. of course, areas were identified into the university hospital to be fully dedicated to covid patients: these areas and these units were increasing over the times, according to the need. also, the strategy involved a relevant increase in the number of beds of the intensive care unit (icu): we used to have beds, while beds were added, so going to a total number of beds available during covid- pandemic. also, part of the cardiac surgery unit was adapted to become an icu during the covid- pandemic. another crucial decision was to divide the emergency unit into two areas: one a covid area for all suspected cases and another covid-free area. consequent to this, there was the reorganization of the orthopedic department (directed by p.r., the first author) that is part of the university hospital. usually, our department had beds. however, a dramatic decrease in the number of beds and reduction of the surgical activity was necessary during this covid- pandemic: it was decided that the number of beds had to be . we usually have three operating rooms (ors) available, if required, even h daily; now, with the restrictions in the covid- infection, we have two ors working h a day with one or available h for emergencies. since march , it has been avoided to perform elective surgery. consequently, despite the activity of our department before covid- including all types of orthopedic surgeries, most of these required to be cut off and our current activity includes only trauma surgery (such as fractures) and musculoskeletal tumor surgery (such as malignant bone tumors, soft tissue sarcomas, metastases, and pathologic or impending fractures) ( table ) . there was also a reduction in outpatient clinic activity: since march , our medical direction abolished nonurgent outpatient consultations, allowing only acute evaluation (e.g., oncologic check and post-op check) to reduce the virus spread. this activity reduction was associated with a careful evaluation of staff and patients. on one side, extensive swab testing with periodically retesting of all healthcare staff (physicians, nurses, sanitary assistants, etc.) was applied. on the other side, every day, all patients attending consultation are scanned with a thermometer and have to wear surgical masks during their permanence in the hospital. moreover, extensive swab testing of all surgical patients was also performed. summarizing, our procedure for admission in our department of surgical patients during covid- was based on five categories: department: always nasopharyngeal swab in urgent modality. a room in the ward is reserved for patients waiting for the result of the nasopharyngeal swab. . patients requiring surgical treatment in an emergency: nasopharyngeal swab is done in urgent modality, but the patient has surgery before having the results. patients are treated as potential covid- positive: all staff have to use ffp instead of a surgical mask, and or is sanitized after surgery. . patients transferred from other hospitals: need to have nasopharyngeal swab done with the result before our admission . pediatric patients: same as point , , and but also their parent/parents "caregiver" need to have nasopharyngeal swab. . patients coming from their home for planned surgeries in priority class (e.g., tumors): they need to have had nasopharyngeal swab before admission or if not possible at admission (see ). these restrictions determined an overall reduction of all our surgical activities of % compared to (table ). we initially also had a relative reduction in the number of hip fractures. however, there were no actual differences in terms of low-energy fractures since they occur in elderly patients following an accidental fall at home. in contrast, high-energy fractures dramatically decrease by %. tumor surgery, once we excluded basically benign tumors, was decreased by over % (fig. a, b) . since march , it has been recommended to reduce elective surgery: we performed elective surgery until march when restrictions were applied (fig. a, b) . moreover, a decrease in surgical activity was also due to delay in intervention depending on the need to obtain swab test results before surgery and elongation of operating timing for patients treated in an emergency before having the results. we also had a reduction in outpatient clinic activities (fig. a, b) and admission to the orthopedic emergency unit (fig. a, b) . philosophy of our current approach on swab testing has proven successful since, of more than people tested in our building, only three covid- positives (all asymptomatic) were found. they were quarantined, and all their potential contacts were identified and periodically checked by swabs (all remained negative). besides, of over more than surgical procedures, an extremely low incidence of positive patients was found. only two patients were found covid- positive after discharge ( and days, respectively). however, their positivity was evident after they were referred to other hospitals for rehabilitation, so possibly they could have been affected by the virus outside our department. overall, the evolution and spread of the virus in italy were dramatically heavy; in fact, the number of diagnosed cases requiring treatment increased rapidly up to today (april ). over a population of more than , , people, there were , confirmed covid- positive cases, with , deaths, , recovered, and , with active disease, according to data reported by the italian public safety committee [ ] . in our opinion, it is particularly essential to retrospectively review the experience in two regions first affected by covid- pandemic: lombardia and veneto (fig. ) . lombardia accounts for about a double number of population than veneto; however, there were a quarter of positive cases in veneto, less than a tenth of deaths, and a similar number than in lombardia of swab tests performed. basically, from this data, it is possible to deduce that different strategies were used. in particular, veneto strategy was, on one side, an extensive swab test of all people (even if asymptomatic), and on the other, a proactive tracing and quarantining of potential covid- positive patients. on the contrary, in lombardia, swab tests were performed only in severe symptomatic cases, increasing the risk that asymptomatic patients (possibly covid- positive) could spread the virus in the community. it is particularly true if we consider that all the healthcare staff (physicians, nurses, and sanitary assistants) and all people working in hospitals in veneto (especially in padova) were tested and periodically retested with nasopharyngeal swabs. it certainly helped to protect both the healthcare staff and the patients, and it could be the best way up to now, following the experience of south korea, singapore, and hong kong [ , ] . again, when we compare the experience in italy overall and veneto, we also may see that the mortality rate in veneto was lower, as well as the need for hospitalization (fig. ) . it is probably due to the better denominator of swab testing. indeed, the swab test's extensive use gave a more reliable definition of the real situation of the positive patients, leading to a limitation of virus spread from asymptomatic patients [ ] [ ] [ ] [ ] [ ] and the possibility of an earlier start of the treatment. there were fewer cases of covid- patients in the south of italy: probably it was because chronologically, the first areas affected by covid were in regions of the north of italy. so, the italy lockdown on march was able to prevent virus spread in south italy; moreover, it could be possible, as suggested by some virologists, that a different climate could play a role in virus spread, although this is not confirmed. nevertheless, the overall results in italy were very poor as they are now in the uk and spain, and it is known that-even with a very different population-it is becoming delicate, if not dramatic, in the usa. our hospital strategy was based firstly on reallocating resources to the treatment of covid- patients and secondly on extensive swab testing. these first action points are similar to those performed worldwide [ ] : leading in some hospitals to a redeployment of orthopedics staff (physician and resident), while in others, as well as in ours, to a reduction in orthopedic surgical activities, with the recommendation to perform only acute cases (urgent or emergent), postponing or avoiding elective procedures [ , [ ] [ ] [ ] [ ] . changes performed by our medical direction lead to a reduction in our surgical activities. this was also related to a decrease in admission to the orthopedic emergency unit; it was initially determined by the fact that patients voluntarily no longer went to the hospital after the first death in vo' euganeo for fear of contagion (to avoid the risk of virus exposition). then, the italian government limited activities and mobility of the people, prohibiting people from having their normal social life and leaving home (italy lockdown on march ), with a remarkable reduction of car accidents, bike accidents, etc. the second action point of our strategy appears successful in minimizing spread to healthcare staff and patients treated in our orthopedic department during this period. the key points were the following: first, we treat every suspected case as a potential covid- positive from the start. one or has been chosen with a specific path and procedure for covid- patients, similar to those reported by other orthopedic departments [ , ] . on the contrary, in other italian hospitals, swab tests were done only late, showing an incidence of positivity of people working in the orthopedic ors close or superior to % (personal communication, unpublished), similar to what appended in wuhan at the beginning of the pandemic, where incidence ranged from . to . % [ ] . what does this mean to us? what is the important message that could come to all orthopedic community from our experience? probably the key to minimizing the extent of the disease and the number of deaths now (in the absence of pathognomonic signs [ ] , appropriate treatment [ ] , or of a vaccine) is highlighting all positive patients, social distancing, and quarantining positive patients. we feel that, if the covid- pandemic persists, in every single hospital, it could be possible to continue the orthopedic surgical activity and also to restart elective surgery [ ] , using a strategy that implies testing all the healthcare staff and all the patients (possibly before the admission or surgical treatment). this can remarkably contribute to control or minimize the risk of infection spread in the hospital. the covid- pandemic is undoubtedly a dramatic situation spreading worldwide, and it is not only affecting people who get infected but also have grave psychological as well as economic consequences in many countries. the future strategy to control the disease or minimize the damages will involve adopting systems to identify possible asymptomatic positive patients and maybe also use apps and digital tools to help recognize positive individuals. extensive swab test of all people (even if asymptomatic), and a proactive tracing and quarantining of potential covid- positive patients may diminish the virus spread. the now starting antibodies testing may contribute (doubts still exist about their validity!), and also, at present, it is not known if patients who successfully went through and were cured by infection may even again be re-infected. it is undoubtedly a significant "war" probably for the healthcare world, and we would like to conclude quoting a sentence from sir winston churchill, "success is not final, failure is not fatal: it is the courage to continue that counts…." abbreviations icu: intensive care unit; or: operating room covid- bollettino della protezione civile novel coronavirus and orthopaedic surgery: early experiences from singapore survey of covid- disease among orthopaedic surgeons in wuhan, people's republic of china covid- pandemic in italy comparing italy overall and veneto according to different categories of patients (quarantined at home, admitted in hospital substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sarscov ) transmission of -ncov infection from an asymptomatic contact in germany presumed asymptomatic carrier transmission of covid- transmission interval estimates suggest presymptomatic spread of covid- . medrxiv fair allocation of scarce medical resources in the time of covid- limiting spread of covid- in an orthopaedic department-a perspective from spain. j surg case rep preparing to perform trauma and orthopaedic surgery on patients with covid- a review of state guidelines for elective orthopaedic procedures during the covid- outbreak covid- : all non-urgent elective surgery is suspended for at least three months in england what we do when a covid- patient needs an operation: operating room preparation and guidance musculoskeletal symptoms in sars-cov- (covid- ) patients covid- -the real role of nsaids in italy joint statement: roadmap for resuming elective surgery after covid- pandemic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable the following authors have designed the study (pr, gt, ab, dd), gathered the data (ep, aa, ab), analyzed the data (pr, ab, gt, aa), wrote the initial drafts (pr, gt, aa, dd), and ensured the accuracy of the data and analysis (ep, ab, dd). all authors read and approved the final manuscript. ethics approval and consent to participate not applicable the authors declare that they have no competing interests received: may accepted: june key: cord- - cji tzq authors: dowd, jennifer beam; andriano, liliana; brazel, david m.; rotondi, valentina; block, per; ding, xuejie; liu, yan; mills, melinda c. title: demographic science aids in understanding the spread and fatality rates of covid- date: - - journal: proc natl acad sci u s a doi: . /pnas. sha: doc_id: cord_uid: cji tzq governments around the world must rapidly mobilize and make difficult policy decisions to mitigate the coronavirus disease (covid- ) pandemic. because deaths have been concentrated at older ages, we highlight the important role of demography, particularly, how the age structure of a population may help explain differences in fatality rates across countries and how transmission unfolds. we examine the role of age structure in deaths thus far in italy and south korea and illustrate how the pandemic could unfold in populations with similar population sizes but different age structures, showing a dramatically higher burden of mortality in countries with older versus younger populations. this powerful interaction of demography and current age-specific mortality for covid- suggests that social distancing and other policies to slow transmission should consider the age composition of local and national contexts as well as intergenerational interactions. we also call for countries to provide case and fatality data disaggregated by age and sex to improve real-time targeted forecasting of hospitalization and critical care needs. governments around the world must rapidly mobilize and make difficult policy decisions to mitigate the coronavirus disease (covid- ) pandemic. because deaths have been concentrated at older ages, we highlight the important role of demography, particularly, how the age structure of a population may help explain differences in fatality rates across countries and how transmission unfolds. we examine the role of age structure in deaths thus far in italy and south korea and illustrate how the pandemic could unfold in populations with similar population sizes but different age structures, showing a dramatically higher burden of mortality in countries with older versus younger populations. this powerful interaction of demography and current age-specific mortality for covid- suggests that social distancing and other policies to slow transmission should consider the age composition of local and national contexts as well as intergenerational interactions. we also call for countries to provide case and fatality data disaggregated by age and sex to improve real-time targeted forecasting of hospitalization and critical care needs. covid- | demography | age structure | mortality g overnments are rapidly mobilizing to minimize transmission of coronavirus disease (covid- ) through social distancing and travel restrictions to reduce fatalities and outstripping of healthcare capacity. the pandemic's progression and impact are strongly related to the demographic composition of the population, specifically, population age structure. demographic science can provide new insights into how the pandemic may unfold and the intensity and type of measures needed to slow it down. currently, covid- mortality risk is highly concentrated at older ages, particularly those aged + y. in china, case fatality rate (cfr) estimates range from . % for those y to y jumping to . % for those + y ( ). this age pattern has been even more stark in italy, where, as of march , , the reported cfr is . % for those y to y, and . % for those > y, with . % of deaths occurring in those aged y and over ( ) . current cfrs are likely overestimated due to underascertainment of cases. in south korea, with broader testing and strong health care capacity (only deaths), the current cfr for those + y is still an alarming . % ( ). the importance of age structure for covid- transmission and fatality rates population age structure may explain the remarkable variation in fatalities across countries and the vulnerability of italy. the deluge of fatal covid- cases in italy was unexpected, given the affected region's health and wealth. italy is one of the oldest populations, with . % of its population over y, compared to % in china ( ) . italy is also characterized by extensive intergenerational contacts, supported by a high degree of residential proximity between adult children and parents ( ) . even when intergenerational families do not coreside, daily contacts are frequent. many italians prefer to live close to extended family, with over half of the population in the northern regions commuting ( ) . intergenerational interactions, coresidence, and commuting may have accelerated the outbreak in italy through social networks that increased the proximity of elderly to initial cases ( ) . the age structure of initial cases, along with early detection and treatment, likely explains the low numbers of fatalities in south korea and germany. the korean outbreak was concentrated among the young shincheonji religious group ( ), with only . % of cases thus far falling into the > -y group ( ) . this contributed to a low overall cfr in south korea relative to italy ( . % vs. . %). germany has, likewise, few deaths ( out of , cases to date), with the median age of confirmed cases at y compared to y in italy ( ) . covid- transmission chains that begin in younger populations may go undetected longer ( ), with countries slow to raise the alarm. the initial low cfr in england may have reflected the relatively young age structure of early infections, including greater london, which has a small fraction of residents over y compared to more rural areas ( ) . covid- was only detected in king county, wa, once it reached the life care center in kirkland, where out of of the state's first reported covid- deaths occurred, with virus genetic sequence estimates suggesting it circulated for several weeks prior ( ) . once community transmission is established, countries with high intergenerational contacts may see faster transmissions to high-fatality age groups, as seen in italy and spain, leading to higher average cfr ( ) . the overall burden of serious cases and mortality reflects linkages between the age distribution of early cases, age structure of the population, and intergenerational connections. fig. contains population pyramids to illustrate how population age structure interacts with high covid- mortality rates at older ages to generate large differences across populations in the number of deaths, holding constant assumed rates of infection prevalence ( %) and age−sex-specific cfrs (italy) ( ) . adjusting assumptions changes the total number of expected deaths but not the relative comparisons across countries with different age structures. for example, assuming that cfrs, by age, are half of current italian rates would reduce the numbers of expected deaths by half. fig. , top considers two countries, italy and south korea, with very different population age structures. the larger number of expected fatalities is clearly visible in fig. , top right for italy ( , ) versus korea ( , ). in fig. , bottom, we consider two countries with similar population sizes but very different age distributions. brazil has . % of its population over age + y, with our simulated scenario leading to dramatically more deaths ( , ) compared to nigeria ( , ), where only . % are + y. visualizes expected deaths by age groups in countries with different population age structures: italy (older), united states (middle), and nigeria (younger). we see stark implications of an older age structure for higher fatalities, amplified at higher population infection rates. si appendix, fig. s animates differences by infection rate ( to %). demographically informed projections will better predict the covid- burden and inform governments. while population age structure is crucial for understanding those at the highest risk of mortality both across and within countries, it is also vital for understanding social distancing measures to reduce critical cases that overload the health system-aka "flattening the curve." our illustrations show that countries with older populations must take aggressive protective measures. for these to be effective, special attention should be devoted to high-risk population groups and intergenerational contact. within countries, mapping of agerelated spatial clustering can improve hospital and critical care forecasts ( ) . consideration of population age structure also necessitates understanding the interlinkage of policy measures and how policies might create unintended consequences. while schools may be a hub of virus transmission, school closures may inadvertently bring grandparents and children into contact if grandparents become the default carers. in aged populations with close intergenerational ties, governments need to facilitate childcare solutions that reduce contact. in a pending decree, the italian government introduced a special leave for parents with children at home from school, and a voucher for babysitting. the age structure of populations also suggests that the squeezed "sandwich" generation of adults who care for both the old and young are important for mitigating transmission. beyond introducing sick pay for those who need to self-isolate or care for family members, joint government and industry emergency policy measures should seek to counter family economic crises, particularly for vulnerable and precarious workers who are less able to comply with policies that allow social distancing. the rapid spread of covid- has revealed the need to understand how population dynamics interact with pandemics. population aging is currently more pronounced in wealthier countries, which, mercifully, may lessen the impact of this pandemic in lower-income countries with weaker health systems but younger age structures. it is plausible that poor general health status and coinfections such as hiv and tuberculosis will increase the danger of covid- in these countries, along with intergenerational proximity and challenges to physical distancing. thus far, the lower than expected number of cases detected in africa (despite extensive trade and travel links with china) suggests that the young age structure may be protective of severe and thus detectable cases. beyond age structure, demography can shed light on the large sex differences in covid- mortality that need to be understoodwith men at higher risk. distributions of underlying comorbidities such as diabetes, hypertension, and chronic obstructive pulmonary disease will likewise refine risk estimates. until more nuanced data are available, the concentration of mortality risk in the oldest old ages remains one of the best tools to predict the burden of critical cases and produce more precise planning of availability of hospital beds, staff, and other resources. few countries are routinely releasing their covid- data with key demographic information projections assume % population infection rate and current age−sex-specific case fatality rates from italy (dataset s ). such age, sex, or comorbidities. we call for the timely release of these disaggregated data to allow researchers and governments to nowcast risk for more focused prevention and preparedness. data and analysis. data to produce figs. and are from https://population. un.org/wpp ( ), and age−sex-specific case fatality rates are from italian data (https://www.epicentro.iss.it/coronavirus/bollettino/bollettino-sorveglianzaintegrata-covid- _ -marzo- .pdf), accessed march , ( ). for figs. and , total number of expected deaths by age group was derived by multiplying the total number of people in each age−sex group and country by an assumed population infection rate of . (and . for fig. ) and italian age−sex-specific fatality rates (movie s ) (march , ). data analysis is in r using the packages (ggplot ). the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) in china age specific mortality rates covid- why are korea's covid- death rates so low? asia times a comparative perspective on intergenerational support spostamenti quotindiani e nuove forme di mobilità [daily shifts and new forms of mobility social contacts and mixing patterns relevant to the spread of infectious diseases the updates on covid- in korea as of march coronavirus disease (covid- ) daily situation report for germany association of public health interventions with the epidemiology of the covid- outbreak in wuhan, china covid- : track coronavirus cases cryptic transmission of novel coronavirus revealed by genomic epidemiology monitoring trends and differences in covid- case fatality rates using decomposition methods: contributions of age structure and age-specific fatality how will country-based mitigation measures influence the course of the covid- epidemic? predicting peak hospital demand: demographics, spatial variation, and the risk of "hospital deserts" during covid- in england and wales key: cord- -r bxop t authors: romani, lorenza; chiurchiù, sara; santilli, veronica; bernardi, stefania; lombardi, mary haywood; scarselli, alessia; villani, alberto; ciofi degli atti, marta luisa; campana, andrea; d’argenio, patrizia title: covid‐ in italian pediatric patients: the experience of a tertiary children’s hospital date: - - journal: acta paediatr doi: . /apa. sha: doc_id: cord_uid: r bxop t coronavirus disease (covid‐ ) caused by the novel sars‐cov‐ has spread worldwide since its onset in wuhan in december . in italy covid‐ rapidly increased in february and by may , . % of the confirmed cases were under years and . % of those had been hospitalized. this case series report reviews the demographic characteristics, clinical course, laboratory findings, radiologic features and treatment of children admitted with covid‐ to a tertiary care hospital in italy. its onset in wuhan in december . in italy covid- rapidly increased in february and by may , . % of the confirmed cases were under years and . % of those had been hospitalized ( ). this case series report reviews the demographic characteristics, clinical course, laboratory findings, radiologic features and treatment of children admitted with covid- to a tertiary care hospital in italy. table . thirty-eight on forty-three children belonged to a family cluster. in / ( %) of cases the family member was a health care worker. the symptoms at the admission are reported in table . on patients, children ( %) developed new symptoms during hospitalization including: respiratory symptoms requiring supplemental oxygen for three days in a years-old-boy, mild diarrhoea with spontaneous resolution of ileal thickening detected on ultrasound in a -years-old girl, conjunctival hyperemia without sars-cov- detected on ocular surface in a years-old-boy and the hyperinflammatory syndrome in -and- years old boys. these two patients were admitted with fever and cough at the onset of the disease; after two days and three weeks from the admission respectively, they developed abdominal pain, diarrhoea, high fever associated to lymphopenia, high levels of inflammatory indexes (c -reactive protein, ferritin, d-dimer) and a progressive mild heart failure. the transfer to the picu was necessary. the clinical course and treatment of these two patients will not be discussed in this brief report. patients laboratory findings at the admission are presented in table . and that its effect is reinforced by azithromycin ( ). antiviral treatment has been used in only one immunocompromised patient but data on its efficacy in children with covid- are missing ( ). pending on a pediatric clinical trial we included lopinavir-ritonavir in our guidelines. chest x ray was performed in / ( %) patients while only / ( %) children were subjected to chest ct scan. in / ( %) children over the age of years the ct scan showed more extensive lung involvement than the x-ray, with ground glass opacities: one monolateral and one bilateral because of the co-administration of drugs that potentially could prolong qt, routine ecgs were performed every - hours we observed bradycardia (hr - bpm) likely related to drug-drug interaction between the two drugs (hcq and lopinavir-ritonavir)and misdiagnosed. multiple hypothesis have been proposed to justify the different clinical presentation of covid- between adults and children; still today there is no a univocal explanation. noteworthy, although a small percentage is reported, pediatric patients may develop a hyperinflammatory syndrome that needs to be carefully evaluated for a prompt treatment. notably, adults have a much higher prevalence of increased c-reactive protein, suggesting a much milder immunological response and less immune-mediated tissue damage in children ( ) epidemiology for public health, istituto superiore di sanità (iss) systematic review of covid- in children shows milder cases and a better prognosis than adults clinical and epidemiological features of children with coronavirus disease (covid- ) in zhejiang, china: an observational cohort study hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial clinical and epidemiological characteristics of pediatric sars cov- infections in china: a multicenter case series key: cord- -dabjcvno authors: poli, piero; boaga, jacopo; molinari, irene; cascone, valeria; boschi, lapo title: the coronavirus lockdown and seismic monitoring of anthropic activities in northern italy date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: dabjcvno in march/april the italian government drastically reduced vehicle traffic and interrupted all non-essential industrial activities over the entire national territory. italy thus became the first country in the world, with the exception of hubei, to enact lockdown measures as a consequence of the covid- outbreak and the need to contain it. italy is also a seismically active area, and as such is monitored by a dense permanent network of seismic stations. we analyse continuous seismic data from many stations in northern and central italy, and quantify the impact of the lockdown on seismic ambient noise, as a function of time and location. we find that the lockdown reduces ambient noise significantly in the – hz frequency range; because natural sources of seismic noise are not affected by the lockdown, the seismic signature of anthropic noise can be characterised with unprecedented clarity, by simply comparing the signal recorded before and after the lockdown. our results correlate well with independent evaluations of the impact of the lockdown (e.g., cell phone displacements), and we submit that ambient-noise seismology is a useful tool to monitor containment measures such as the coronavirus lockdowns. noise is of interest to geoscientists, as it can be used at relatively small scales, for instance in mapping and monitoring efforts , . while earlier studies have attempted to characterise high-frequency seismic noise , [ ] [ ] [ ] [ ] , the current lockdown of industrial activities and reduction in road and train traffic in italy is an unprecedented opportunity to discriminate it from ambient noise of natural origin. italy is a highly industrialized and urbanized country, densely covered with non-stationary noise sources , such as traffic and industry-induced vibration . this is particularly true in its northern regions, which account for % of the country's entire industrial output, and where lockdown measures have been enacted earlier than everywhere else in europe. we analyse continuous data from an array of broadband seismic stations, located in the vicinity of known industrial districts in lombardy, emilia-romagna and tuscany (fig. ) ; we identify the spectral signature of the march lockdown, and take advantage of the lockdown to quantify and evaluate the spectral signature of anthropic activities. importantly, measuring the overall reduction in seismic energy associated with the lockdown is also a way to quantify its effects; this is relevant to governmental entities, wishing to monitor the effectiveness of the measures being taken. we downloaded publicly available, continuous, three-component seismic recordings from a set of permanent broadband stations, part of the italian national seismic network operated by the istituto nazionale di geofisica e vulcanologia . all instruments have a flat response at frequencies between ~ . and ~ hz, or broader; we remove ("deconvolve") instrument response from the data prior to our analysis. the locations of stations employed in most of our study are shown in fig. . stations were selected based on their proximity to industrial districts; in particular, miln is located near the city of milano, with a particularly high concentration of vehicle traffic and industrial activities. the seismic signature of the containment measures in italy is apparent from a relatively simple analysis of continuous recordings at station miln, located within the city limits of milano, in a busy area near the university of milano campus and the lambrate train station. we compute spectrograms (fig. ) by fourier-transforming -hour-long segments of continuous signal, with a -minute overlap between subsequent segments; for each calendar day, all segments are then averaged, and the squared modulus of the resulting average fourier transform is computed: this way, a single "power-spectral density" (psd) function is obtained, for each station, component (east-west, north-south, vertical) and calendar day. figure shows clearly that the lockdown has a relevant impact on recorded seismic noise over a broad frequency range; its effect disappears at frequencies below hz, where anthropic noise is weaker. the energy drop associated with the lockdown is comparable with that occurring every weekend and during the winter break, both in / and / . interestingly, loss of energy is gradual over time, starting with the first lockdown measures on february , and increasing with time until a plateau is reached around march (interruption of non-essential industrial activities). a trend similar to that seen in fig. has also been found through the analysis of cell phone displacements . this suggests that vehicle traffic, which was significantly reduced (particularly in and around milano) already with the february measures, contributes significantly to the entire spectrum of anthropic noise; there is also episodical evidence from the press that a number of factories were closed based on the unilateral decision of their owners, before the government-imposed lockdown. the analysis applied to station miln is repeated for all seismic stations of fig. , and the results are illustrated in figs www.nature.com/scientificreports www.nature.com/scientificreports/ lockdown measures apparently impact all stations under consideration, but the character of their effects changes in various ways with station location. in the case of fir, located in the city of florence, the signature of the winter break is almost negligible, while the february/march lockdown still has a prominent effect; it might be possible to interpret this observation through the analysis of anthropic activities usually taking place in the area (e.g., tourism, which is presumably not reduced by the holiday). the drop is gradual at all stations, with no specific governmental decree standing out with respect to the others. at station prma, a slight increase in ambient noise occurs after february and before march . we next characterise anthropic noise by evaluating variations in the spectra of seismic ambient noise before and after the implementation of lockdown measures. we compute the ratios of the psd measured (as described in sec. ) on tuesday march , to that measured at the same station on tuesday december . we carry out this calculation separately for each component, and for all stations analysed thus far; the results of this exercise are shown in fig. . the energy associated with ambient signal is clearly reduced for all stations, at all frequencies in the range of interest. at each station, psd ratios change with frequency almost exactly in the same way for all components. near hz, all stations show a more or less rapid decline in the psd ratio, with ambient noise being more effectively reduced as frequency grows. this trend continues all the way to hz at stations euct and prma, while other stations show a more complex behaviour. above hz station miln stands out, its psd ratio growing quickly with frequency. anthropic noise is known to be relevant at frequencies above hz, and to consist of a range of different excitation mechanisms , , , . natural sources such as rain, wind and sea/ocean waves are typically characterized by frequencies below hz, and are obviously not affected by the lockdown. we infer that, by taking the ratio of noise spectra before and after the lockdown, an estimate of the spectral character of anthropic noise is obtained, and the spectra in fig. can help us estimate the nature of anthropic noise in the region of interest, independent of the lockdown; the frequencies where the psd of ambient signal is most reduced by the lockdown are those where, in normal times, the contribution of anthropic activities to seismic ambient noise is most important. the fact that most energy loss associated with the lockdown is at frequencies between - hz is coherent with what is known of the typical signature of industrial activity and vehicle traffic . seismic data recorded during the lockdown might be particularly useful in identifying sources of anthropic noise, which could be employed by geophysicists, after the lockdown, e.g. to characterise the upper subsoil by cross correlation of ambient signal , . we further analysed the relationship between ambient noise recorded on different components, finding the "h/v" ratio between the psds of horizontal-component and vertical-component signals: first, the psd of each component of signal recorded on a given day is averaged in the frequency range - hz; then the arithmetical average of the resulting east-west-component and north-south-component values is taken; finally, the ratio of the resulting horizontal psd to the vertical one is computed. the procedure is iterated for each station and for each day between december and march , and the results are shown in fig. . in general, the value of h/v is related to how seismic energy in the ambient-noise field is distributed in the form of compressional, shear and surface waves , , ; changes in h/v after vs. before the lockdown would reveal whether the reduction in anthropic noise affects one of these seismic phases/components more or less importantly than the others; in other words, whether traffic and industry-induced vibration can be associated to one particular constituent of the seismic field. figure shows that the lockdown measures have no effect on h/v, and we infer that, while anthropic noise is reduced significantly by the lockdown (fig. ) , the relative contributions of compressional, shear and surface waves remain approximately constant: the noise wave field is stable in the fig. are plotted here on a single graph, for comparison; for each station, the average value of the psd observed in the time interval of interest is subtracted from the corresponding curve, prior to plotting, as this can change significantly from station to station, but is not relevant to our analysis. each colour corresponds to one station, as specified. again, the dates of mentioned governmental decrees are highlighted as in fig. . the italian territory is densely covered by seismic instruments, and by repeating our analysis on the entire network of available stations we are able to quantify the spatial dependence of anthropic noise reduction. for each station, for each day, the psd of signal recorded am to pm is computed, and averaged over different frequency bands. in practice, we employ the direct fourier method , as implemented in the obspy package , : this is standard procedure to identify artefacts related to station operation, episodic cultural noise, overall station quality and level of earth noise at each site. to emphasize the change in ambient noise with the lockdown, we plot the difference between the values so obtained on three dates in , and reference values obtained conducting the same calculation on data recorded for five months until the lockdown, and averaging. we include as supplementary material s an animated version of fig. , showing the psd at the same stations, october , through april , ; through this time-dependent visualization, the drastic effects of the lockdown are further emphasized. our main result, that noise be strongly reduced after the lockdown in the "cultural" frequency range, is confirmed by fig. , and extended to most of northern italy. between - hz, the lockdown effects are more pronounced in the lombardy and veneto regions than in central italy and along the apennine range. the most important reductions in ambient noise are recorded by stations along the alpine arc, near torino, milano and verona, and in the city of florence. we have analysed continuous data from northern italy, and quantified the effects of the march coronavirus lockdown on the seismic ambient noise field. we confirm that this effect is significant, and easily observed in our data: see in particular figs. and . the italian government first imposed a reduction of people (and therefore vehicle) movement, on march ; we find that this date marks the beginning of a gradual loss in ambient-noise energy at all frequencies, which we attribute to the reduction of road and railroad traffic in the region of interest. depending on the station, the energy curve flattens out, or starts to decline more slowly towards the beginning of april, despite the more stringent measures imposed at that time (interruption of all non-essential industrial activities). a similar trend has been found from cell-phone displacement data . one implication of our observations is that seismic data could be useful for governmental institutions to monitor the effectiveness of measures involving a reduction or interruption of human activity in a given area. it is understood that the lockdown only reduces noise of anthropic origin; it follows that by comparing the fourier spectrum of seismic ambient noise before and after the lockdown (fig. ) , one can attempt to characterise anthropic noise. we find that, confirming earlier estimates , , anthropic noise becomes dominant at frequencies coronavirus lockdowns have changed the way earth moves seismic imaging and monitoring with ambient noise correlations stationary-phase integrals in the cross-correlation of ambient noise a theory of the origin of microseisms seismic noise in fennoscandia, with emphasis on high frequencies variations in broadband seismic noise at iris/ida stations in the ussr with implications for event detection the nature of noise wavefield and its applications for site effects studies: a literature review emergence of broadband rayleigh waves from correlations of the ambient seismic noise faster, better: shear-wave velocity to meters depth from refraction microtremor arrays rain and small earthquakes maintain a slow-moving landslide in a persistent critical state characterization of and correction for cultural noise h/v ratio: a tool for site effects evaluation. results from -d noise simulations on the stability and reproducibility of the horizontal to vertical spectral ratio on ambient noise: case study of cavola, northern italy cultural noise and the night-day asymmetry of the seismic activity recorded at the bunker-east (bke) vesuvian station maninduced low frequency seismic events in italy a catalogue of non-tectonic earthquakes in central-eastern italy italian seismological instrumental and parametric database (iside) the reduction of social mixing in italy following the lockdown observations and modeling of seismic background noise recent advances in seismology spectral analysis of seismic noise induced by rivers: a new tool to monitor spatiotemporal changes in stream hydrodynamics sources of long range anthropogenic noise in southern california and implications for tectonic tremor detection shear wave structural models of venice plain, italy, from time cross correlation of seismic noise train traffic as a powerful noise source for monitoring active faults with seismic interferometry observation of equipartition of seismic waves seismic velocity change patterns along the san jacinto fault zone following the m . el mayor-cucapah and m . collins valley earthquakes an algorithm for the machine calculation of complex fourier series obspy: a bridge for seismology into the scientific python ecosystem seismic noise analysis system, power spectral density probability density function: stand-alone software package. united states geological survey open file report seismic noise level variation in south korea global oceanic microseism sources as seen by seismic arrays and predicted by wave action models rete sismica nazionale (rsn) mediterranean very broadband seismographic network (mednet) north-east italy seismic network. international federation of digital seismograph networks regional seismic network of north western italy. international federation of digital seismograph networks the generic mapping tools version we downloaded and analysed continuous seismic data provided by the istituto nazionale di geofisica e vulcanologia, the osservatorio geofisico sperimentale, the university of genova. the generic mapping tools were used to generate the map in figure . piero poli was supported by the european union horizon research and innovation programme (grant agreements, -monifaults). the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to p.p.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -mnlzj ly authors: barattucci, massimiliano; chirico, alice; kuvačić, goran; de giorgio, andrea title: rethinking the role of affect in risk judgment: what we have learned from covid- during the first week of quarantine in italy date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: mnlzj ly due to covid- spreading in italy, on march the prime minister of italy declared a lockdown and imposed severe restrictive measures impacting citizens’ freedom at several levels. people were required to stay at home and go out only to satisfy basic needs. several risk models have postulated a link among online searching behavior, affect, anxiety, and complaints by individuals toward government restrictions (gr), which emerged as also related to an increased perception of knowledge toward risk. however, to date, no study has addressed how these key risk-related aspects (i.e., affect, anxiety, perceived knowledge on risk, and risk dimensions) can act jointly to orient online health information-seeking behavior, and people’s complaints toward gr imposed during the lockdown. this study investigated the mechanisms underlying online health information-seeking behavior and people’s complaints toward the government’s restrictions during a covid- emergency in the italian population. drawing from the health belief model (hbm), which postulates a link between sociodemographic variables, risk, and affect dimensions in emergency, we assumed risk factors as predictors of affect and anxiety, which, in turn, were posited as mediators between risk dimensions, online health information-seeking behavior, and complaints toward gr. participants ( , ) were involved during the first week of the quarantine (march – ) and completed an online survey composed of (i) an adapted version of the italian risk perception questionnaire; (ii) the italian positive (pa) and negative affect (na) schedule (panas- ); (iii) the state anxiety scale (stai-y ); (iv) ad hoc personal knowledge measure about novel coronavirus; (v) ad hoc item measuring information search behavior regarding the novel coronavirus; (vi) ad hoc measure of the complains regarding gr; and (vii) sociodemographic questions. general linear models and structural equation modeling (sem) were carried out to test the model. sociodemographic and cognitive factors predicted the participants’ affect and anxiety, which, in turn, motivated and fully mediated both information search behavior and complaint toward gr. this research can offer useful suggestions for policy-makers during the covid- emergency, and it advanced the knowledge on the risk–emotion link in emergency situations. in december , a cluster of pneumonia cases of unknown etiology was detected in the city of wuhan, hubei province, central-eastern china. this initial phenomenon turned into a novel coronavirus (zhu et al., ) , which is named sars-cov- (i.e., severe acute respiratory syndrome), and which caused a disease named covid- (qu et al., ) . even though symptomatology has been defined clearly, it is still hard to define how long it will last and if a cure is possible (porcheddu et al., ; wangping et al., ) . recently, the infection has caused enough deaths to be considered as a pandemic by the world health organization (who) (onder et al., ; sohrabi et al., ) . in italy, the outbreak spread on february , and after an ad hoc decree of the president of the council of ministers (dpcm), a lockdown was imposed on italians (i.e., days after the first recognized patient). all italians were required to stay home if they were not involved in jobs or tasks involved in other people's survival. since march in italy, restrictive and severe measures have been gradually implemented (from march to ) (de giorgio, a,b) . according to the health belief model (hbm) (janz and becker, ; carpenter, )-well-established theoretical frameworks in health-related behavior research-often, the psychological counterpart of disease-related emergencies can entail an increased risk perception (bults et al., ) modulated also by sociodemographic variables (e.g., vaughan, ; clifton et al., ) . this cognitive perception of risk can have significant implications on individuals' emotional states on the short and on the long-term (cafagna and barattucci, ) . moreover, it would be closely related to the intention to adopt protective behaviors (leppin and aro, ; goodwin et al., ) as well as to personal susceptibility (lin et al., ) . however, hbm has never been used to investigate the mechanisms underlying all these variables in a pandemic situation. moreover, no data on the italian population's risk perception have been reported yet. crucially, no studies have investigated the impact of risk cognition and emotional response on research behavior and compliance with government actions. this last aspect can be far more relevant if considering that cognitive perception of risk is sensitive to peculiar emergencyrelated environmental factors. for instance, italians were forced to stay home, thus changing their normal habits related to work and leisure activities. confined at home, italians tended to rely more on the internet to remain up-to-date on pandemic progress in a safe way. crucially, online information searching regarding health issues is not a neutral task since it can influence people's affective states, especially anxiety (jutel, ) . to investigate the joint impact of cognitive risk dimensions, affect, and anxiety on online searching behavior and compliance toward government restrictions (gr), in the peculiar context of the italian pandemic emergency, we drew from the hbm to formulate and test a novel explicative model. first, we posed the first day of lockdown (march , ) as the trigger event and the online health information on covid- searching behaviors as the main outcome. then, we built and tested a novel model including sociodemographical factors, risk cognitions, behaviors, and affect as mediators between the trigger event and the main outcome of the online health information searching behaviors (figure ) . elucidating this mechanism can be crucial also because information-seeking behaviors can influence the population' general compliance with government decisions (clifton et al., ) . therefore, these data can provide the government with useful indications regarding which online communication strategies would be the most effective in an emergency situation (liao et al., ) . the term "risk" represents the possibility of suffering damage connected to foreseeable circumstances. in essence, it is consequently a variable connected to the frequency (or probability) of the occurrence of the damage and the magnitude that the latter can cause in the individual (slovic, ) . this universally recognized definition may look as reducible to a mere mathematical formula. however, its subjective dimension suggests a deeper complexity. indeed, a plethora of approaches have been developed to capture all the key aspects related to risk perceptions, as well as its main consequences on people' behavior. among the main subjective dimensions of risk, cognitive factors emerged as playing a key role (slovic, ; leppin and aro, ) . risk perception would be determined by a complex series of cognitive factors: (i) the perceived possibility of having damage to health; (ii) the subjective importance that the damage is more or less possible; (iii) by personal uncertainty associated with the exposure to a specific risk factor (slovic et al., ) . in the case of general risk or infection or disease, personal knowledge negatively affects the perception of risk danger (shook et al., ) . in turn, risk perception impacts behaviors (sjöberg, ) , specifically between different risk dimensions regarding infection, perceived fatality, severity, vulnerability, and uncontrollability, and are proven to have effects on protective conduct . in regard to pandemic-related risk perceptions, two main factors emerged as relevant, that is, vulnerability (a person's subjective perception of the risk of acquiring an illness or disease) and severity (a person's feelings on the seriousness of contracting an illness or disease) of harm (carpenter, ) . however, despite that it has been repeatedly shown that risk perception can affect behavior (brewer et al., ; vaughan, ; shook et al., ) , the underlying mechanism still needs to be elucidated. specifically, antecedents of cognitive dimensions of risk should be still clarified. with this regard, demographic factors/variables emerged as playing a key role in shaping pandemic risk perception and subsequent behaviors (see e.g., vaughan, ) . for instance, women resulted as more avoidant, fearful, and vulnerable in terms of pandemic risk perception, with lower risk acceptance scores when compared to men (see e.g., de zwart et al., ) . conversely, age often leads to an increased perception of control on infection risk, lower susceptibility, avoidance, and higher acceptance of risk (see e.g., clifton et al., figure | the first week of lockdown in italy: epidemiological and variable trends. all variables have been standardized. due to the numerical difference of epidemiological data between northern and southern italy, we divided the real values by , and have thus reported them in the y-axis. ). conversely, the level of education was negatively related to the risk of infection and contagion (i.e., vulnerability) (gidengil et al., ) . lower income and urbanization positively affected vulnerability and perceived infection risk (brewer et al., ; de zwart et al., ; gidengil et al., ) . another factor, which would act as a mediator, should be included between risk perception and behavior, that is, affect. affect, such as fear, is related to a general amplification of the perception of the danger of risky events, while anger would be significantly associated with underestimation of dangers (slovic, ; brown, ) . moreover, the degree of emotional involvement in the perceived consequences of different risks, or specific personality dimensions that determine emotional attitudes, is associated with different aspects of risk perception (among all, vulnerability, and severity) (slovic, ; brown, ) . crucially, among the stimuli triggering emotional states, also online searching information should be included, which could also lead to a phenomenon of large-scale emotional contagion (hatfield et al., ) . emotions expressed via the internet, and mainly through social media, can lead to a longterm psychological impact (arapakis et al., ; fowler and christakis, ; coviello et al., ; kramer et al., ; ferrara and yang, ; mui et al., ) including also a simple health information search (gadahad et al., ) . specifically, both general and specific discrete emotional states can orient people's online search for information on health issues (wissow, ; myrick and willoughby, ) . emotions and affect act as motivators of specific survival behaviors (frijda et al., ) , and this definition could hardly be more appropriate than in this worldwide emergency. in this case, one key survival behavior motivated by affect could consist of online health information seeking or avoidance (savolainen, ) . while positive affect (pa) resulted in determining people's attitudes toward information avoidance, negative affect (na) predicted individuals' attitudes toward information seeking (yang and kahlor, ) . on the other hand, searching for information about symptoms or specific illnesses can increase people's distress and anxiety about their health (graffigna et al., ) . crucially, na and anxiety have often resulted in closely positively intertwined affective states (crawford and henry, ) , even though they can be considered as clear, distinguishable constructs (watson and kendall, ; clark and watson, ) . according to the tripartite model of anxiety and depression, high levels of na underlie both anxiety and depression, while na would act as a central risk factor of anxiety (clark and watson, ) . na has also often been considered an early predictor of anxiety in several domains (crawford and henry, ; cisler et al., ) . during the lockdown, the internet became one of the most important sources of health-related information; thus, it would be crucial to analyze antecedents of this behavior as well as its potential impact on compliance with gr. to date, the literature regarding risk perception and behavior on worldwide pandemics has focused mainly on general population's or on healthcare workers' punctual psychological responses immediately after the end of isolation (wilder-smith and freedman, ). acute stress/posttraumatic disorders, as well as higher propensity to live state anxiety, emerged as serious issues (leppin and aro, ) . crucially, no data on the italian population's risk perception have been reported yet. moreover, no studies have investigated the role of risk cognition and emotional response to research behavior and compliance with government actions. in this study, we aimed to advance previous studies on covid- at two levels. first, we elucidated the link between cognitive and emotional risk dimensions in a pandemic, then, we built and tested a novel model linking cognitive, emotional, and sociodemographic factors to a peculiar behavior enacted in this emergency, which would be probably increasingly adopted in the future, that is, online searching behavior of health-related information. moreover, we also used the hbm, for the first time, as a general explicative framework in a pandemic situation. health belief model posits a cognitive appraisal framework, in which perception of the risk for individual health affects emotions and protective behavior (roseman, ) . more specifically, when referring to the hbm framework (janz and becker, ) and adapting recent theoretical models (watson and spence, ; keller et al., ; lemée et al., ) , the present research model considers sociodemographics as antecedents of risk cognition and emotion as a buffering factor between risk perception and behavior (figure ). this novel model proposes that two different risk cognition aspects have independent effects on pa and na. perceptions regarding specific pandemic and perceived knowledge of risk (champion and skinner, ; carpenter, ) can act differently on contingent affect, which can have an impact on both information search behavior and complaints regarding government action. in a situation of physical and social constraint, i.e., quarantine, the sudden perceptions of the risk would depend mainly on mass media, social media, and word of mouth information (jung et al., ) . this growing information impacting the emotional state can, in turn, act as both a search trigger for further infection information and a facilitator of compliance with the government's restrictions (goodwin et al., ; rolison and hanoch, ) . in line with hbm and literature, major evidence linking cognitive risk dimensions and affect keller et al., ) , this study aimed to explore the following hypotheses: sociodemographic factors have an impact on risk perception and perceived risk knowledge (hp ); more precisely, the research expects that age (hp a) and education (hp b) will negatively affect risk perception and positively risk knowledge; thus, it is hypothesized that women will have a worse perception of pandemics and less perceived knowledge compared to men (hp c). the research assumed that risk perception would positively impact on na (hp a) and negatively on pa (hp b); on the contrary, it expected that risk knowledge would negatively impact on na (hp a) and positively on pa (hp b). moreover, the research intends to elucidate whether the differential effect of na (hp a) and pa (hp b) on search behavior and a complaint is mediated by state anxiety (hp ). in order to test all the mentioned hypotheses thoroughly, we tested this novel model by means of structural equation modeling (sem) (figure ). one thousand thirty-one participants from italy voluntarily took part to this study (mean age = . ; sd = . , range = - ). after removing the data of the participants who did not answer all the survey questions, we analyzed participants, of which were females (mean age = . ; sd = . ) and were males (mean age = . ; sd = ). their marital status was as follows: . % were engaged in a relationship, . % were married, . % were single, . % were divorced, . % were widowed; . % reported living in central big city areas, . % were living in the suburb of a big city, . % reported living in a small town (i.e., less than , inhabitants), and . % reported living in the countryside; . % resided in northern italy, . % resided in central italy, . % resided in south italy, and . % resided in the islands; . % were students, . % were retired, . % were freelance, . % were temporary workers, and . % were full-time employees with a permanent position; and . % reported having no children, . % reported having two children, . % reported having one child, and . % reported having more than three children. regarding schooling, . % reported having a middle school diploma, . % declared having a high school degree, . % reported having a bachelor's degree, . % reported having a master's degree, and . % reported having a ph.d. this study was conducted in accordance with apa ethical standards and with the declaration of helsinki. participants: (i) were fully informed in regard to institutional affiliations of the researchers and research scope; (ii) continued the survey only if they were adult (> years old); (iii) gave information that could not allow their identification; (iv) had the right to refuse to participate in the study and withdraw at any time; (v) filled an anonymous questionnaire and confirmed the understanding of instructions and voluntary participation. participants completed an online survey between march , the first day of quarantine and national lockdown, and march . the research design relied on snowball sampling (chain referral process). participants were recruited through flyers, social networks, and by word of mouth. the questionnaire answering began in the evening (march ) when the dpcm decree was issued. first, participants completed the part of the questionnaire created to gather sociodemographic information. second, the following questionnaires were then completed: . italian risk perception questionnaire (cafagna and barattucci, ) : originally developed by savadori et al. ( ) . based on literature indications (keller et al., ) , the study deduced that the pandemic risk could generally be identified as terrifying, uncontrollable, fatal, and dangerous for future generations, and widespread in terms of exposure. hereupon, the researchers built a tool that measures five single-item dimensions of risk infection, on a seven-point scale ranging from to : severity, vulnerability, uncontrollability, terror, and danger for future generation (item examples: "considering the scale below ( 'not fatal' to 'fatal'): in your opinion, when the virus infects a subject, how likely are the consequences of being fatal?" "considering the scale below ( 'not exposed' to 'totally exposed'), in your opinion, to what extent do you think you are exposed to the coronavirus risk?" . italian short version of the positive and negative affect schedule (terraciano et al., (agresti and kateri, ) . moreover, regarding the residence area, we operationalized the "residence area" into two different variables. the former "residence area" refers to how far from the city center a person lives: (i) city center; (ii) suburb of a city; (iii) town; (vi) countryside, and (v) the latter, "residence area in italy" refers to a zone of residence from the north to the south of italy and islands, which also coincides with the distance from the first epidemic center of diffusion (i.e., codogno) in northern italy. a comprehensive structural equation model with amos was used to test the proposed theoretical model and the main hypotheses. commonly reported fit statistics were: comparative fit index (cfi), tucker-lewis index (tli), normed fit index (nfi), goodness of fit index (gfi), incremental fit index (ifi), root mean square error of approximation (rmsea), plus standardized root mean square residual (srm) for measurement model fit. research has sought to reduce response bias and common-method variance problems utilizing suggested methods (podsakoff et al., ) : scales were visually divided, and different formats and endpoints were used for each different measure. psychological impact on anxiety, negative affect, risk dimensions, and search behavior all factors are reported in table with significant regression coefficients and wald statistics. all omnibus models were residency: countryside note. "male" as category of "gender"; "single" as a category of "status"; "ph.d,/ms" as a level of "education"; "three or more children" as "number of children"; "city center" as a level of "residency"; "islands" as a level of "residency in italy" were not reported since they are a redundant levels. marital status was transformed into a dummy variable (engaged vs. single). a the predictor was significant, but the omnibus model was not. ci , conventional % confidence interval; pa, positive affect; na, negative affect; sb, search behavior; knw, knowledge; vul, vulnerability; con, control; sev, severity; ter, terrifying; dfg, damage for future generations; com, compliance. significant except for the model with "knowledge, " i.e., a risk dimension, as the predicted variable. we reported results for each of the dependant variables (anxiety, pa, na, sb, knw, vul, con, sev, ter, dfg, com) in relation to all predictors taken together (gender, marital status, education, number of children, residency, residency in italy). only b values useful for explaining results were reported in order to avoid redundancies. younger , not engaged, significantly positively predicted the perception of being vulnerable against risk. a lower education (beta decreased positively from high school to elementary school) and being older (b = . ) significantly positively predicted the perception of control over the risk associated with the pandemic. being female, with a lower level of education, and with no to one child and older led to a significantly higher perception of risk severity. being less educated and younger led to a significantly higher perception of risk as terrifying. being female, with lower education, and an increasing number of children (from no to two children) led to a significantly higher perception of risk damage associated with new generations. females with lower education and younger tended to report more compliance toward the government's measures. descriptive statistics for all the measures and zero-order correlations between them are described in table . with the aim of exploring a measurement model and construct validity, a confirmatory factor analysis (cfa) was conducted comparing four nested models from one factor to a final model composed of the four principal latent factors (risk perception, na, pa, and anxiety). table represents chi-square and goodness of fit indices for the four measurement models developed. considering that risk perceptions were all measured with single items, and despite the final cfa indexes not being optimal, there was an evident amelioration of all indices from the first to the final model. therefore, the measurement model can be profitably used in further testing of the proposed structural model. thus, we tested through sem the proposed structural model (figure ) : the five risk perception dimensions (fatality, vulnerability, uncontrollability, terrifying) and risk knowledge as (correlated) antecedents, with direct relationships with both na and pa as intermediate variables, which themselves have direct links with state anxiety that fully mediates information search behavior. the proposed model exhibited optimal goodness of fit: chi-square = . (df = ; p < . ), rmsea = . , cfi = . , ifi = . , nfi = . , gfi = . , tli = . . consequently, we tested the same model deleting nonsignificant relationships (severity, vulnerability, and terrifying with pa; risk knowledge with na) and some correlations between antecedents (vulnerability and danger for future generations, with terrifying risk, risk dimensions, and risk knowledge). consistent with our hypothesized relationships, the model showed excellent goodness of fit: chi-square = , (df = ; p < . ), rmsea = . , cfi = . , ifi = . , nfi = . , gfi = . , tli = . , with all significant relationships (p < . ). regression weights are presented in table , while the path diagram of the final model is shown in figure . as hypothesized (hp a), each dimension of risk perception is positively related to na. in contrast, only two dimensions (uncontrollability and danger for future generations) are significantly linked to pa, not confirming what was expected (hp b); conversely, confirming hypotheses hp a but not hp b, risk knowledge is only positively related to pa. results confirmed that the expected differential effect of na (hp a) and pa (hp b) on search behavior and on a complaint is fully mediated by state anxiety (pa indirect effect on search behavior: β = − . , p < . ; na indirect effect on search behavior: β = . , p < . ). overall, the relationships expressed in the model explained % of the variance for na, % for pa, % for state anxiety, % for search behavior, and % for complaint in government actions. the present research carried forward the literature regarding the fact that cognitive factors predict population affect that, in turn, motivate and fully mediate information search behavior and complaints about government actions, overturning contributions that proposed that risk behavior is driven by affects (kahan, ; leppin and aro, ; wu et al., ) . considering our sample of participants, results showed that being female and younger with a lower level of education led to more anxiety, na, a higher risk perception as terrifying, and higher complaint regarding gr. moreover, pa increased significantly in older males and those with a decreasing number of children (less than two). younger people or those who were not engaged or married tended to look for information online about the covid- more frequently. older females with a lower education level (from middle to elementary school) were more prone to perceive themselves as competent regarding their acquired knowledge on covid- . being engaged or married acted as a protective factor regarding the perceived vulnerability against covid- . older people with lower levels of education (from high school to elementary school degree) tended to feel more able to control the gravity of risks associated with this pandemic. older females having from one to no children, and with high-school to elementary school degree, tended to perceive the covid- -associated risk as more severe. females who have a higher number of children (from no child to two children) and with a lower level of education (this effect increased from bachelor to elementary school) tented to perceive an increased risk associated with covid- for future generations. younger females with lower education tended to report more compliance toward the government's measures. in this study, we elucidated two crucial phenomena in emergency: general na and its link with anxiety. na and anxiety have often resulted in closely positively intertwined affective states (crawford and henry, ) even though they can be considered as clear, distinguishable constructs (watson and kendall, ; clark and watson, ) . the tripartite model of anxiety and depression confirmed that high levels of na underlie both anxiety and depression, while na acts as a predictor of anxiety (clark and watson, ) . specifically, na has been often considered as an early predictor of anxiety in several domains (crawford and henry, ; cisler et al., ) . the model tested in this study confirmed the direction of this link. emotions and affect also act as motivators of specific survival behaviors (frijda et al., ) , and this definition could hardly be more appropriate than in this worldwide emergency. in this case, affect can trigger behaviors such as online health information seeking or avoidance (savolainen, ) . while pa resulted in playing a pivotal role in determining people's attitudes toward information avoidance, the negative one predicted individuals' attitudes toward information seeking (yang and kahlor, ) . on the other hand, searching for information about symptoms or specific illnesses can increase people distress and anxiety about their health following a reinforcing spiral to the extent that a new term has been coined to refer to this condition, i.e., "cyberchondria" (te poel et al., ) . indeed, people with high health anxiety (i.e., fears stemming when individuals exaggerate in interpreting their bodily symptoms as an indicating severe illnesses) (mcmullan et al., ) trend to increase their negative responses related to the likelihood of suffering from a given disease now and in the future (baumgartner and hartmann, ) . the present result showed that anxiety triggered by na acted as a strong predictor of people's searching behavior regarding health. in other words, italians were motivated by anxiety stemming from na and triggered by their risk perception on the controllability and vulnerability regarding sars-cov- spread and health searching behavior. overall, results provided support for the cognitive appraisal framework in risk perception keller et al., ) and the main hypotheses. risk perception and knowledge acted with different mechanisms on emotions: risk perception mainly contributed to having an effect on negative affect, while knowledge influenced only positive affect. furthermore, in line with our hypotheses, emotions fully mediated the relation among risk cognition, complaint, and information search behavior (champion and skinner, ; carpenter, ; jung et al., ) . risk perception and affective response to pandemics can be crucial factors for managing population behaviors, thus ensuring the best adherence to prescription and safety norms (poletti et al., ; merino, ; shook et al., ) . moreover, the efficiency of prevention behaviors in pandemics by the government is related to population cooperation, which is highly related to risk perception (leppin and aro, ; goodwin et al., ) . exploring risk perception during pandemics is fundamental because misperceptions can often cause inadequate responses (poletti et al., ; merino, ) . in particular, perceptions regarding infection can lead people to take safer actions, to reduce exposure, and to increase protective conducts (e.g., vaccination, social distancing, hygiene, search for information; shook et al., ) . these individual behaviors can significantly influence the disease progression at a system level (de zwart et al., ; jiang et al., ) . since emotion and behavior are closely related (loewenstein et al., ; slovic and peters, ; brown, ) , beliefs and perceptions regarding risk represent core predisposing factors to predict people reactions. therefore, it would be crucial to promote public order and right risk communication and to prevent counterproductive behaviors linked to bad information and fake news (brug et al., ; voeten et al., ; shook et al., ) . the risk controllability is one of the most important factors that need to be considered since pa can reduce anxiety and, consequently, affect complaint and informational search behavior. in italy, especially during the first days of the epidemic (from the end of february), there was too much conflicting information (e.g., "this virus is very similar to normal flu."/"please, pay attention, it is a very dangerous virus; it is not like normal flu."). it is crucial to evidence that too much information, especially if conflicting (or worse, fake news), can cause confusion in the population, and this, in turn, can affect emotional states (bawden and robinson, ). politicians should act on proper information dispersion procedures regarding specific risk, as perceived knowledge may act on search behavior and complaint. our results can suggest more tailored strategies of communication for prevention to be implemented by the government, not just in pandemic emergency (smith, ) . research regarding the way the population appraised hazards acquired significant scientific attention, and different approaches and paradigms to the perception of risk have been discussed (leppin and aro, ; keller et al., ) . recent contributions have conformed on the emotional appraisal of risk perception (loewenstein et al., ; wu et al., ) . thus, results from our study could offer evidence in favor of the hypothesis that the analytic system (i.e., risk judgment) would precede the emotional one, at least in a pandemic emergency. covid- is having, and is predicted, to have a substantial impact on the world economy, both due to the effect on national health systems, and on the slowdown of business activities through lockdowns and measures of social distancing. the economic impact would be even more substantial in developing countries, due to both difficulties related to social distancing in the slums and in the suburbs, as well as for the absence of stable health systems, welfare measures, and smart-working policies, and for the access to the various forms of institutional communication and to the mass media. the literature concerning the other pandemics has clearly shown that the perception of risk has a strong cultural component; thus, communication strategies should be tailored according to the peculiarities of each country . in this regard, the proposed model can indicate a priority of all the variables capable of influencing preventive behavior or adherence to restrictions directly, which must be taken into account when planning communication to the general public. for instance, accurate and clear communication should clarify the danger for future generations, the terror aroused, and the degree of exposure to the pandemic (van bortel et al., ) . furthermore, the proposed model evidenced also perceived knowledge of risk as another key variable to be considered in mass communication. finally, communication in developing countries should consider that people living in precarious economic conditions could give less weight to the health consequences of covid- , in a cost-benefit assessment process that could overestimate economic costs to the detriment of those for health and economics (leppin and aro, ). given the novelty and relevance of this study, some limitations should be discussed. first of all, the cross-sectional design of the research limits the generalizability of its findings. although results should be interpreted, especially concerning the specificity of both the contagion risk and the quarantine situation, useful indications on the mechanism that operates between cognitions, emotions, and behaviors in situations of high stress and forced captivity can be provided. moreover, due to the recruitment type (i.e., online), and despite a large number of participants, this sample cannot be considered as fully representative of the italian population ( % males, % in northern italy). almost % of the participants filled out the survey in the first days (maybe caused by people's reactions to the lockdown). therefore, this distribution does not allow for longitudinal analysis. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. the patients/ participants provided their written informed consent to participate in this study. categorical data analysis affective feedback: an investigation into the role of 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sánchez-montesó, laura; maramao, fabio stefano; toussaint, loren title: coping with the covid- pandemic in italy and spain: lessons in response urgency date: - - journal: journal of global health doi: . /jogh. . sha: doc_id: cord_uid: z t csg nan in italy, on friday, february , , the first cases of infection were reported in the province of lodi, km from milan. codogno, a town of inhabitants, placed the so-called "patient " of italy, a -year-old male, on the world covid- map [ ] . until that moment the coronavirus was geographically distant. within just hours a "red zone" was established isolating villages. a penal sanction of up to years in prison was established for anyone who violated the isolation. in a little more than two weeks, the mayor of codogno, francesco passarini, showed his satisfaction because there were no infections [ ] . nevertheless, on february , the italian prime minister giuseppe conte signed a ministerial decree with the "confinement" measures for the regions of lombardy and veneto [ ]. on saturday, march , [ ] , a ministerial decree was approved by which the whole country was in a "red zone," an area that was quarantined and had strict social distancing rules. a rumor about the decree having been leaked or hours before the official announcement allowed thousands of inhabitants of the affected area in the north of the country, especially lombardy, to hastily return to their homes in other areas of the country with the risk of spreading the epidemic. for giuseppe conte, italy was experiencing the most serious crisis since the second world war [ ]. march , italy closed all universities of all grades and majors and began teaching over the internet [ ] . it took the italian government just two weeks to react firmly with the laws enacted on march to establish a "red zone" throughout the country, which is when it closed all shops, except pharmacies and food stores, and banned entry and exit from the country. on march , italian citizens were banned from leaving their homes except for a justified reason. the ministry issued a document to be carried by each citizen to justify any departures. on march all but the most basic services such as hospitals, food stores, banks, kiosks and tobacconists were closed [ ] . indeed, italy had already recorded cases and deaths as of march , and it had become the european focus of the outbreak [ ] . an overview of the timeline of events in italy can be found in figure , panel a. coping with the covid- pandemic in italy and spain: lessons in response urgency pilar montesó-curto , laura sánchez-montesó , fabio stefano maramao , loren toussaint both the language and the response to the pandemic were more direct and forceful in italy, as they perceived the focus of northern italy as a great threat. spain documented its first case of covid- on january , . however, it wasn't until march that spain took significant action. in fact, not quite one week prior, madrid hosted the " th international women's day" which italian professor walter ricciardi, a who executive and advisor to the comte government, described as madness due to italy's previous experience [ ]. on march , the catalan government, chaired by torra, ordered the closure of educational centers to stop the coronavirus epidemic. at the same time, the president of the catalan government asked for more measures, as those already taken seemed insufficient to him. at that time, positive cases of covid- had been reported. all educational centers were closed, including universities, where teaching would be provided virtually, as well as civic centers and meeting centers for retired people, and also toy libraries. on march , by order of the catalan ministry of health, bars, restaurants, and all commercial spaces that are not necessities (ie, not dedicated to the sale of food) were ordered closed. also the ski slopes, gyms, and entertainment venues such as discos were ordered closed. to contain the pandemic. then, on march , the government of catalonia decreed the "confinement" of several municipalities due to a cluster within the epidemic that caused an exponential increase in cases of covid- in igualada, vilanova del camí, santa margarida de montbui i Òdena [ ] . the departure of people who were not permanent residents in these affected municipalities was restricted. the march decree arrived late, the measures were announced on friday, march , they were approved on saturday, march , so many citizens "escaped" in search of other homes and villages of origin. a "state of emergency" in spain was finally declared through a royal decree ( / ) [ ] on march for a period of calendar days. to carry out all the measures involved, it required a high degree of coordination in all its health policies, since very diverse regions were included. the "state of emergency" was declared by the spanish government appealing to the unity of the spanish state affirming the harshness and difficulty that would be required to stop the spread of covid- . to this end, it announced the mobilization of "all the resources" of the state, public, private, civil, and military entities. article . of the constitution empowered the government to apply this measure, using a decree agreed upon by the council of ministers, and for a maximum period of days. on march , all face-to-face educational activities were suspended, leaving the home was allowed only for the purchase of basic necessities and work in activities declared essential. for the approval of the extension of the "state of emergency", the state needs the authorization of the congress of deputies. for this reason, on march , sánchez met with the presidents of the autonomous communities or regions by video conference. he was accompanied by the three ministers who formed the delegated authority in this crisis; the head of health, salvador illa, the head of defence, margarita robles, and the head of the grande-marlaska interior and transport and mobility and urban agenda, josé luís Ábalos [ ] . as of april , spain was the second country in the world in infected people after the usa and the second in deaths after italy with deaths [ ]. with a total of deaths recorded on april , spain recorded the lowest number of deaths per day for a week. the president of the government sánchez called pablo casado, leader of the opposition, to inform him rhetoric surrounding the pandemic has been important in each countries response. recognizing the threat and talking about it very realistic, grounding ways can be a key motivator for stimulating behavioral change that promotes health, decreases infectious spread, and limits peak demands within healthcare. of his intention to extend "the state of emergency" from april . an overview of the timeline of events in spain can be found in figure , panel b . spain and italy have had two different responses to the same crisis. the spanish government' s response has been considered late by many [ ] . in contrast to the direct, swift, and decisive action taken by the italian government, the response in spain was much more protracted. however, in the catalan region of spain the catalan government attempted to implement policies before the royal decree by the government of spain chaired by pedro sánchez ( / ) [ ] to curb the spread of covid- . as of march , , spain had cases of covid- , the fourth highest number in the world, and deaths, the second highest in the world. the spanish government held a plenary session in congress on march to address the covid- crisis. the aim was to extend "the state of emergency" for an additional days [ ] . meanwhile, italy, which was ahead of spain at the time of infection with covid- and with the harshness of its policies in the face of the great impact of northern italy, proposed a "confinement." this confinement period was longer than the days of the quarantine ordered in spain, until may and then a progressive return to normal life little by little from may [ ] . the united states, which entered the covid- pandemic later, claimed that new york was experiencing a "near-war atmosphere." also trump recognized that "this is not a flu, this is ruthless. we are going to have a very hard two weeks. it is going to be painful, very painful for two weeks" [ ] . did the united states learn lessons about message framing and urgency of response? perhaps, but were those lessons learned too late? the usa had the dubious distinction of leading the world in infections as of april , , and continues to lead the world in cases at present. italy' s rhetoric was much more direct and discomforting and focused on "war" and that they must find the "ammunition," that is, the resources to resolve it. in short, italy used metaphors that reflect the danger detected and the necessary ways of managing the pandemic. other countries should understand that the language that defines their policies is related to the actions that accompany them. governments should be attentive to their rhetoric and metaphors and learn from previous experiences. response urgency is crucial and should be reflected in government response and discourse. el mapa del coronavirus: más de . casos y más de . muertos en países. [the coronavirus map: over , cases and over el govern decreta el confinament dels municipis d'igualada, vilanova del camí, santa margarida de montbui i Òdena. [the government establishes the confinement of the municipalities of igualada the resilience of the spanish health system against the covid- pandemic el congreso debatirá el de marzo si se prorroga el estado de alarma sánchez llamará a casado para informarle de la prórroga del estado de alarma coronavirus en italia hoy abril: . casos de covid- . el plural el coronavirus provocará entre . y . muertes en ee uu, según las proyecciones de la casa blanca. el país the authors completed the icmje unified competing interest form (available upon request from the corresponding author), and declare no conflicts of interest. pilar montesó-curto avda remolins - , tortosa spain mariapilar.monteso@urv.cat key: cord- - zhabgkt authors: savoia, e.; gori, d.; argentini, g.; neri, e.; piltch-loeb, r.; fantini, m. p. title: factors associated with access and use of ppe during covid- : a cross-sectional study of italian physicians date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: zhabgkt objectives during the course of the novel coronavirus (sars-cov- ) pandemic, italy has reported one of the highest number of infections. nearly ten percent of reported coronavirus infections in italy occurred in healthcare workers. this study aimed to understand physicians access to personal protective equipment (ppe) and to information about their use, risk perception and strategies adopted to prevent contracting the infection. methods we undertook a cross-sectional, online self-reported survey implemented between march and april of italian physicians. results responses were received from physicians, only % of which reported to have access to ppe every time they need them. approximately half of the physicians reported that the information received about the use of ppe was either clear ( %) or complete ( %). risk perception about contracting the infection was influenced by receiving adequate information on the use of ppe. access to adequate information on the use of ppe was associated with better ability to perform donning and doffing procedures [or= . % c.i. . - . ] and reduced perception of risk [or= . , % c.i. . - . ]. conclusions results from this rapid survey indicate that while ramping up supplies on ppe for healthcare workers is certainly of mandatory importance, adequate training and clear instructions are just as important. globally, as the novel coronavirus (sars-cov- ) pandemic has evolved there has been a shortage of personal protective equipment (ppe) available to the healthcare workforce. as the world health organization has warned since the beginning of march, disruption to the global supply of ppe, has left frontline healthcare workers ill-equipped to care for their patients. since the start of the epidemic, guidance on the usage of such equipment has continued to evolve, and has emphasized conservation of resources rather than optimizing protection of workers. the coronavirus pandemic has taken a dramatic toll worldwide and especially in italy. as of the beginning of april, italy has reported one of the highest number of infections and the highest number of deaths of any european country. media reports from across italy have shone a light on the burden that the coronavirus is placing on health workers. nearly ten percent of reported coronavirus infections in italy occurred in healthcare workers. as of april , . cases and , deaths attributed to covid- were confirmed in the country, and the number of healthcare workers infected and those that lost their life due to covid- was , and respectively. many of these infections are likely due to occupational hazard; workers becoming infected while caring for patients suggesting the shortage or inappropriate use of ppe may be at the root of part of these infections. the use of ppe has been identified as one of the biggest physical and psychological challenges experienced by physicians while responding to covid- . for example, physical burdens related to ppe include repeated donning and doffing of equipment and extended hours wearing uncomfortable masks and respirators, while psychological burdens include challenges communicating with peers and patients when wearing ppe on and operating under changed practice standards. because of ppe shortages, healthcare workers, who may have been trained on how to don and doff ppe to maximize protection from infection, have had to make ad hoc adjustments on what piece of equipment to use and when, that are not reflected in any training they have received. the additional burdens created by a shortage whereby processes for using ppe are continuously changing, has not been explored. the italian healthcare system is regionally based and organized at the national, regional, and local levels, with each region having the autonomy of managing the delivery of the healthcare services based on local needs. italian national health service system certifies healthcare workers and requires continuing education and quality and standards of care are set by the regions and hospitals. training procedures for the healthcare workforce are also left to the regions and local hospitals, specifically regarding the management of ppe. such differences are expected given local needs and hospital settings differ by localities, however such differences may also have caused inconsistencies and confusion on the appropriate use of ppe in a rapidly evolving situation such as the covid- outbreak. currently, there is lack of literature on how the healthcare workforce in italy has adapted during the novel coronavirus pandemic in the use of equipment. this study aimed to understand physicians' access to ppe, reception of information about their use, ability to perform donning and doffing procedures, risk perception and strategies adopted to prevent contracting the infection. we believe the results of our work may be helpful in the development of policies and training related to the use of ppe in italy as well as in other countries. all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. we undertook a cross-sectional, self-reported survey, of physicians working in italy during the response to covid- . we disseminated an online survey by the use of two social media groups (via facebook and whatsapp) created by physicians engaged in the response. we obtained an institutional review board approval to conduct this study by xyz (name omitted for blind review purposes). the survey was implemented between march and april . the population of interest included physicians aged ≥ years with a valid medical license (criteria to join the social media group) and working in italy during the emergency. the questionnaire was developed through a series of meetings between the researchers and practitioners in charge of infectious control procedures and ppe training activities at the hospital level, the practitioners provided feedback on the content validity and comprehensiveness of the survey instrument before implementation. questions were designed to inform the development of training and policies in response to the crisis and included questions about the physician's work experience (years of experience, specialty, experience in covid- units and geographic area of work), and questions related to the use of ppe divided in four parts: ) access to ppe and strategies to cope with shortage, ) information received on the use of ppe, ) self-reported ability to perform donning and doffing procedures, and ) risk perception of contracting the disease. our analysis examines four dependent variables: ) access to ppe, ) use of ppe, ) selfreported ability to perform donning and doffing procedures and ) risk perception in the work all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. response options were coded as follows: ( =i do not know how to don or doff the piece of equipment, = i am not sure, =i know how to do it). we combined all responses for each piece of equipment into a scoring system to create a new variable named "ability to perform donning and doffing procedures" , we then dichotomized the variable into high ability when the score was th percentile and less than high when below the th percentile). finally, to measure risk perception we asked ) what do you believe is your risk of contracting covid- in the work setting in the next days? physicians rated their perception using a scale ranging from =no risk to =high risk, responses were coded as follows: = low risk (≤ th percentile), =medium risk ( th- th percentile) and =high risk (≥ th percentile). we first performed descriptive statistics for each variable. we then applied ordered logistic regression to the three ordinal variables access to ppe, information on ppe use, and donning and doffing ability and logistic regression to the variable risk perception. we tested for bivariate associations between each predictor (years of experience, geographic region, type of position, working in a dedicated covid- unit) and the dependent variables using a p-value ≤ . as cut-off as inclusion criteria for the multiple regression model. we tested the parallel regression assumption by means all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint of the brant test for the ordered logistic model which resulted not statistically significant and as such the ologit command was used to run the analysis. qualitative analysis was conducted on open questions to identify how physicians coped with ppe shortages and what strategies they adopted to reduce the risk of infecting their family members. we used stata software (stata, college station, tx) to analyse the data. the study was found exempt by harvard longwood medical area institutional review board. responses were received from physicians, the majority of respondents were in the age category - ( %), working in the hospital setting as employees of the national healthcare system ( %), and the most frequently reported category for years of experience was - ( %). physicians from all italian regions and the republic of san marino were included in our survey, most respondents were from the lombardia region ( %), the most impacted by the emergency. over medical specialities were reported by the respondents, the most frequent of which being pediatrics ( %), primary care ( %) and anesthesiology/intensive care ( %) and cardiology ( %). details on the sample characteristics are provided in table . when asked if they had access to ppe when they needed it, ( %) of the physicians said they rarely or never did, ( %) sometimes and ( %) always did. ffp and ffp (equivalent to n- and n- in the usa) were the pieces of equipment most frequently reported as lacking by % and % of physicians respectively. other pieces of equipment were also reported as lacking but by a lower percentage of respondents: gown ( %), hair cups ( %), all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint surgical masks ( %), gloves ( %). lack of ppe forced % of physicians to come up with strategies to cope with the shortage. such strategies included using the same n- for long shifts ( hours and beyond), disinfecting the respirator with alcohol, adding a surgical mask either under or on top of the n- , re-using the same mask for multiple shifts, exposing the respirator to "the sun" as reported to some of them or ozone, making masks on their own at home, or buying respirators of unknown certification. in the bivariate analysis of factors that related to ppe access; working in a covid- unit, in the table . when physicians were asked how frequently they had received adequate information regarding the use of ppe to protect themselves from contracting covid- , ( %) reported that they always did, ( %) sometimes, ( %) rarely or never. approximately half of the all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint physicians reported that the information received to date about the use of ppe was either clear ( %) or complete ( %) and approximately one quarter was unsure about clarity ( %) or completeness ( %), leaving only % satisfied with the information they received. when asked if the information received was useful to them, opinions were equally split between three groups: those who found it useful ( %), those who did not ( %), and those who were unsure about its usefulness ( %). as a result of the bivariate analysis years of experience, working in a covid- unit and in a primary care setting were associated to the dependent variable, while table . when asked if they believed they could correctly execute donning and doffing procedures for specific pieces of ppe, respondents felt mostly unprepared for putting the respirators and gowns on ( % and % respectively) or unsure if they were doing it correctly ( % and %). in regards to doffing, once again, taking off the respirator and the gown were the procedures they all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint did not know how to do correctly ( % and %) or were unsure about ( % and %). see table . as part of the survey we also presented the physicians with scenarios of activities that would require the use of different types of ppe and asked them, based on their knowledge, what was the most appropriate piece of equipment for each activity. the activities included transportation of presumptive and positive covid- patients within the hospital or by ambulance, activities in the triage area, routine physical examination of patients with respiratory and without respiratory symptoms and administrative activities with direct contact with clients. overall respondents assigned to each activity a level of protection higher compared to what is currently recommended by current guidance. for example over % of physicians said that a face shield is appropriate when conducting physical examination on a covid- positive patient while a surgical mask is what is typically recommended. similarly over % physicians reported that a face shield is needed when conducting the same routine examination in any patient with respiratory symptoms. interestingly gloves were reported as appropriate by over % of physicians for the examination all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . of any patient even if without respiratory symptoms. and over % reported as appropriate the use of a surgical mask when performing administrative duties. see table . when physicians were asked to rate their perceived risk, on a scale from to , of contracting the infection in the healthcare setting, they attributed a mean value of (sd= ) to such risk, table . interestingly, among all specialties physicians in dentistry, otolaryngology, occupational all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint medicine and pneumology were the ones that showed the highest risk perception based on the descriptive analysis. the lowest level of risk perception was reported by those holding a specialty in hygiene and preventive medicine which is consistent with the fact that these professionals are typically in healthcare management positions and do not directly assist patients. see figure . our results present some of the first evidence on how italian physicians experienced lack of ppe, and what factors influenced their understanding of ppe procedures and use. while our results are not generalizable to the all population of italian physicians, and certainly derived from a group of physicians with high level of interest in covid- , group differences within our sample rather than general group estimates by extrapolation, can be useful to understand predictors of behaviors and specific challenges in access and use of ppe. the majority of those surveyed reported not to have access to ppe every time they need it and at least one third of them reported not having received adequate information on the use of the equipment, nor were they consistently comfortable with donning and doffing procedures, in particular when using respirators and wearing gowns. working in a covid unit made a difference in multivariate analysis of both having access to ppe, adequate information on their use, feeling comfortable with donning and doffing procedures, and perceived risk. this likely reflects training efforts focused on educating this subset of the workforce, those actually at the highest risk of contracting covid- based on occupational risk. however, given the difficulties of creating % covid- free clinics as many patients may present to a clinic in a pauci-symptomatic status, the current variation in access and knowledge about ppe use, may put at a disproportionate risk those working outside covid- units. more specifically, our results all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint indicate how primary care physicians may have been neglected from informational initiatives posing them at high risk of contracting the infection. with the advancement of testing and treatment options in the months ahead more and more covid- patients will be diagnosed and cured outside the hospital setting. therefore, additional attention is needed to provide ppe and ppe training for this group of providers and all those working outside covid- units. interestingly, respondents consistently overestimated the level of ppe needed to interact with a non-symptomatic patient; reflecting they either had inadequate understanding of current guidance or regardless of the guidance they were fearful of becoming infected themselves and/or infecting the patient when a diagnosis was not confirmed. the ongoing changes to ppe guidance provided by international, national and regional public health agencies, in particular in regards to the use of respirators, likely made it more challenging to make sense of which equipment to use. standards of use evolved in mid-march, a couple of weeks prior to our survey, as a result of ppe shortages and lack of logistics planning within hospitals. limited training as well as pre-existing professional norms that lacked a culture of ppe use may have been factors that shaped challenges in developing adequate training and information material. we suggest that future efforts should be made to include ppe training in the medical curriculum so that in times of crisis physicians can better adapt to their use and differences in knowledge and practices would be less evident across categories. methods for just-in-time training including the use of video trainings may be one mechanism to improve donning and doffing procedures. in times of crisis, an overuse and gauging of ppe by concerned physicians may cause as much harm as lack of supplies. we found ppe perceptions and use were also tied to perceived risk of contracting the infection in the work environment. overall, risk perception was high, but both adequate access and ppe training decreased such perception. of concern, is also the fact that many physicians took actions in their personal lives to protect their families, limiting physical interactions and in some cases renting separate apartments. in the long term, these actions will certainly affect their emotional well being. results from this rapid survey indicate that while ramping up supplies on ppe for healthcare workers is a necessity, adequate training and clear instructions are just as important. to the extent possible instructions need to be consistent overtime and across regions, include recommendations not only on the overall safety of the workers in the healthcare setting but also on strategies to maintain their overall physical and emotional health and the health of their loved ones. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . risk perception by specialty* *for n within each category see table . all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint covid- : the crisis of personal protective equipment in the us shortage of personal protective equipment endangering health workers worldwide rational use of personal protective equipment for coronavirus disease covid- : phe upgrades ppe advice for all patient contacts with risk of infection covid- ) situation report - nearly in of italy's infected are health care workers: cnn; the italian coronavirus disease outbreak: recommendations from clinical practice. anaesthesia the italian health care system. international health care system profiles: the commonwealth fund medical masks vs n respirators for preventing covid in health care workers a systematic review and meta analysis of randomized trials. influenza and other respiratory viruses indicazioni ad interim per un utilizzo razionale delle protezioni per infezione da sars-cov- nelle attività sanitarie e sociosanitarie (assistenza a soggetti affetti da covid- ) nell'attuale scenario emergenziale sars-cov- a randomized trial of instructor-led training versus video lesson in training health all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder we would like to thank for their support the administrator and all of the moderators of the facebook private group "coronavirus, sars-cov- e covid- gruppo per soli medici -https://www.facebook.com/groups/coronavirusmediciitaliani/" we are also grateful to all of the colleagues of the group which decided to compile the questionnaire giving their suggestions and point of view on this topic". key: cord- - xusxrij authors: licastro, danilo; rajasekharan, sreejith; dal monego, simeone; segat, ludovica; d’agaro, pierlanfranco; marcello, alessandro title: isolation and full-length genome characterization of sars-cov- from covid- cases in northern italy date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: xusxrij in december , the novel coronavirus severe acquired respiratory syndrome sars-cov- emerged in the city of wuhan in the hubei province, people's republic of china, as the etiologic agent of coronavirus disease (covid- ), which has hence spread worldwide causing a global pandemic ( - ).…. available at - - ( ) . bootstrap support values were calculated from pseudoreplicate trees of the whole data set (fig. ) . despite a high burden of covid- in italy, very little information is available to date from full-length high-quality sequences. the first sequences deposited in gisaid (epi_isl_ and epi_isl_ ) were collected in rome from a chinese tourist from hubei province who got infected before visiting italy, and another one (epi_ isl_ ) was from a test-positive italian citizen returning from china. only two sequences were reported from the lombardy cluster (epi_isl_ and epi_ isl_ ). in this report four additional sequences from cases epidemiologically linked to northern italy have been examined. all infected individuals were connected to the city of udine; s and s were from the same cluster of closely related cases, while s got infected probably in lombardy and s visited udine from a neighboring city (table ) . sequence analysis showed a good coverage along the sars-cov- genome for all four isolates (fig. ) . based on the marker variant s d g, all four sequences grouped in the bavarian rooted subclade g, which is dominant in europe, including the sequence from lombardy, but distinct from the three sequences mentioned above originating directly from china ( ) . intriguingly, the new isolates were more closely related to epi_isl_ , while epi_isl_ was more distant (fig. ) . no evidence could be found for the putative -nucleotide (nt) deletion in orf detected in singapore, which has been proposed to indicate an attenuated phenotype ( ) . these findings strongly urge the need for comprehensive studies that combine genomic data with epidemiological data and clinical records of symptoms from patients with covid- . data availability. the longer sequences were named hcov- /italy/fvg/icgeb_s , _s , _s , and _s and were deposited in gisaid (https://www.gisaid.org/) with accession numbers epi_isl_ , epi_isl_ , epi_isl_ , and epi_isl_ , respectively ( ) . a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china world health organization. . coronavirus disease (covid- ) anonymous. . dipartimento della protezione civile-emergenza coronavirus: la risposta nazionale detection of novel coronavirus ( -ncov) by real-time rt-pcr mega x: molecular evolutionary genetics analysis across computing platforms the first two cases of -ncov in italy: where they come from discovery of a -nt deletion during the early evolution of sars-cov- gisaid: global initiative on sharing all influenza data-from vision to reality work was financed through intramural funds of the icgeb, university of trieste, asugi and the argo, miur-mise-region fvg agreement protocol signed by miur-mise-region fvg and managed by area science park.we thank elena samar, fabia petronio, and ilaria sandron for their excellent technical assistance.the key: cord- -nukv fh authors: bartoszek, krzysztof; guidotti, emanuele; iacus, stefano maria; okrój, marcin title: are official confirmed cases and fatalities counts good enough to study the covid- pandemic dynamics? a critical assessment through the case of italy date: - - journal: nonlinear dyn doi: . /s - - -w sha: doc_id: cord_uid: nukv fh as the covid- outbreak is developing the two most frequently reported statistics seem to be the raw confirmed case and case fatalities counts. focusing on italy, one of the hardest hit countries, we look at how these two values could be put in perspective to reflect the dynamics of the virus spread. in particular, we find that merely considering the confirmed case counts would be very misleading. the number of daily tests grows, while the daily fraction of confirmed cases to total tests has a change point. it (depending on region) generally increases with strong fluctuations till (around, depending on region) – march and then decreases linearly after. combined with the increasing trend of daily performed tests, the raw confirmed case counts are not representative of the situation and are confounded with the sampling effort. this we observe when regressing on time the logged fraction of positive tests and for comparison the logged raw confirmed count. hence, calibrating model parameters for this virus’s dynamics should not be done based only on confirmed case counts (without rescaling by the number of tests), but take also fatalities and hospitalization count under consideration as variables not prone to be distorted by testing efforts. furthermore, reporting statistics on the national level does not say much about the dynamics of the disease, which are taking place at the regional level. these findings are based on the official data of total death counts up to april released by istat and up to may for the number of cases. in this work, we do not fit models but we rather investigate whether this task is possible at all. this work also informs about a new tool to collect and harmonize official statistics coming from different sources in the form of a package for the r statistical environment and presents the “covid- data hub.” abstract as the covid- outbreak is developing the two most frequently reported statistics seem to be the raw confirmed case and case fatalities counts. focusing on italy, one of the hardest hit countries, we look at how these two values could be put in perspective to reflect the dynamics of the virus spread. in particular, we find that merely considering the confirmed case counts would be very misleading. the number of daily tests grows, while the daily fraction of confirmed cases to total tests has a change point. it (depending on region) generally increases with strong fluctuations till (around, depending on region) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] march and then decreases linearly after. combined with the increasing trend of daily performed tests, the raw confirmed case counts are not representative of the situation and are confounded with the sampling effort. this we observe when regressing on time the logged fraction of positive tests and for comparison the logged raw confirmed count. hence, calibrating model parameters for this virus's dynamics should not be done based only on confirmed case counts (without rescaling by the number of tests), but take also fatalities and hospitalization count under consideration as variables not prone to be distorted by testing efforts. furthermore, reporting statistics on the national level does not say much about the dynamics of the disease, which are taking place at the regional level. these findings are based on the official data of total death counts up to april released by istat and up to may for the number of cases. in this work, we do not fit models but we rather investigate whether this task is possible at all. this work also informs about a new tool to collect and harmonize official statistics coming from different sources in the form of a package for the r statistical environment and presents the ''covid- data hub.'' keywords covid- Á coronavirus Á r language Á data mathematics subject classification - Á n in december , the first cases of pneumonia of unknown etiology were reported in wuhan city, people's republic of china. analyses of patients' samples collected from their respiratory tract revealed that a novel coronavirus, later named as ''severe acute respiratory syndrome coronavirus '' (sars-cov- ) is the pathogen responsible for infection [ ] . the disease, officially called covid- by world health organization (who), is characterized by higher transmissibility and infectivity but lower mortality than middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars) caused by other coronaviruses [ ] . apart from the source of infection, the spread of the virus depends on the transmission route and general susceptibility of the population. sars-cov- is believed to be transmitted mostly by close contact (and further carry-over to the mucous surfaces of the body) and inhalation of aerosol produced by an infected person. the presence of the virus was also reported in samples from the gastrointestinal tract [ ] , but the potential role of the oral-fecal route of infection is unknown. the evidence of asymptomatic carriers who may unintentionally transmit the virus together with relatively long incubation period up to days [ ] increase the risk of viral spread worldwide and make prevention measures difficult. on the other hand, separation of identified cases, prior immunity to sars-cov- or cross-reactivity of human antibodies naturally risen against other viruses would act as a barrier for virus transmission. the latter is probable as rna sequences of sars-cov- are in % identical to the sequences of sars-cov responsible for the previous pandemic in far east countries in and % identical to mers-cov [ ] . all above- fig. cumulative confirmed cases and case fatalities for all the regions of italy. right: cumulative case fatalities divided by confirmed cases, left: cumulative confirmed cases divided by the cumulative number of tests mentioned issues would act as confounding factors for any modeling of pandemic progression. except of the city of wuhan where the first reports of covid- were announced in december , there was another outbreak of disease, which took place in january-february on the diamond princess cruise ship with more than people onboard. as such a great number of people were locked in a confined space using common facilities, air-condition systems, restaurants, etc., and once the chronology of infections, symptoms and undertaken health measures are known [ , , ] , one can consider this as a unique, naturally occurring epidemiological study useful for prediction of mortality, disease spread and other parameters of the covid- pandemic. since the virus has spread across the world appeared, especially on the arxiv preprint server. they are usually concerned with connecting the pandemic with various epidemiological models (e.g., [ , , , ] following a brief survey of arxiv at the start of april ). however, such models of course require data concerning the infected individuals. furthermore, the media are bombarding today with two basic numbers (for each country)-the number of confirmed cases and the number of case fatalities. given that supposedly the vast majority of people are asymptomatic and testing is not done as random sampling of the population but due to particular protocols these values by themselves might be misleading. we can only second [ ] in despite millions of tests having been performed, there are still no results from statistically well founded samplingbased testing programmes to establish basic epidemic quantities such as infection fatality rate and infection rates. in the absence of such direct data, epidemic management has to proceed on the basis of data produced largely as a side effect of the clinical response to the disease. as a motivating example, we present fig. from which we can see that in italy the case fatality to confirmed ratio is constant, while the confirmed cases to number of tests has been decreasing since around march . indeed, the time period since march is longer than the median time of . days of infection till death [ ] , so one should already start observing some drop in the case fatality to confirmed ratio. through the case of italy, this paper tries to investigate the following issues: -with each country having their own reporting standard and testing strategy are these raw numbers comparable across countries? -do these data actually mean what they are being said to be and are they appropriate for model fitting at all? clearly, the curves presented in fig. suggest that a more in-depth look at the raw numbers is required and that there is a need to put the data in a correct perspective before trying to fit any epidemiological model to them, especially because the viral dynamics are starting to be inferred from reported case fatalities [ , , ] . in this work, we approach these issues by looking in detail at the available infection data for individual italian regions (sect. ) and present the r [ ] acquisition process and the subsequent analysis. section contains a discussion on what other data would be useful (if of course possible to collect for the already overworked public services), in understanding the dynamics of the pandemic. most regional analyses are contained in the appendices. italy is a country which is being very extremely hardhit with the covid- pandemic. it is currently (as of may ) as a whole in lockdown and the medical services are extremely strained. however, due to this situation it has also very detailed epidemiological data that has been made publicly available. its constantly increasing infected and case fatality count has lead us looking in greater detail into this data, especially as it is used for curve-fitting of epidemiological models (e.g., [ , , , ] following brief survey of arxiv) and presented in public media. the first hurdle that one comes across is what do the presented counts actually represent. this seems to be region dependent. furthermore, any deceased whose test result is found positive is classified as a covid- case fatality, regardless of any past or underlying diseases, and this methodology has been consistently applied in italy since the beginning [ ] . it is important to point out that different countries seem to have different testing strategies and classification systems of deaths-hence, raw counts between countries might not be comparable. given the huge amount of tests performed in italy , , (as of th may , [ ] ) an important question is: ''what fraction of them were serological tests?'' as there is no official data on this. a serological test may not distinguish between a person actively infected with the virus and a person that was exposed to the virus in the past. alternatively, serological test may not detect person actively infected with still low viral titer of anti-virus antibodies. on the other hand, if the protocol is to test only people exhibiting symptoms and medical personnel, then given that it is hypothesized that the vast majority of cases are asymptomatic, such a raw count might not be representative of the scale of the epidemic. given the above uncertainties, we set out to see how the italian regional data could be presented in a standardized manner. furthermore, we see how the data of each region compares to the diamond princess' data. we focus on the two values that are being presented everywhere-the confirmed case count and the case fatalities count. however, these should be scaled. we scale the confirmed case count by the total number of tests performed. scaling the case fatalities is more problematic. a common way is to present them as the case fatality ratio, but these may be misleading when estimated during an epidemic [ ] . furthermore, assuming that the vast majority of cases are asymptomatic-hence, not tested and not inside given the lack of hard data, another objective approach would be to compare the daily count of case fatalities to the total deceased count for the day. to the best of our knowledge, such statistics are not centrally reported in italy in real time. daily deceased counts (from nearly all of the italian municipalities-see discussion) are available though for the period january- april . hence, for this time period we are able to plot the weakly ''nearly''-desired ratios (see sect. ). we aggregate per week to remove daily fluctuations, which obscure the picture. furthermore, the same data source provides deceased counts for the years - (for the same time period). this allows us to also visualize the excess mortality (with respect to the per week average from the past five years). beyond this time interval, it is impossible to provide such curves. however, having daily case fatalities counts and past mortality (this is taken as a constant value equaling the average number of deceased for th april) we are able to plot the (per week) ratio of case fatalities to previous average mortality. this provides some indication of the magnitude of excess mortality. however, it is worth noticing that when looking at the current excess mortality it could be appropriate to compare with past mortality peak (e.g., for uk death toll, the / and / peaks, , , , , in appendix b'' we compare the current deceased peak with the seasonal start of the january one. we should remark that perhaps more focus should be on the cumulative positive test fraction instead of the daily positive test fraction. this is because the daily fraction is extremely noisy and furthermore, it sometimes happens that this fraction, in the official data source for italy, exceeds . for similar reasons, we plot the weekly scaled deaths and cumulative scaled deaths. the daily counts are extremely noisy as well. we plot the scaled daily and cumulative positive test count and scaled case fatalities next to the cumulative positive tested fraction of passengers on the diamond princess. here, we present the graphs from two special regions in italy: lombardy and veneto. the remaining regions are presented in ''appendix a.'' lombardia is the center of the epidemic, where the cases and deaths counts are the highest. veneto seems to be a region where the pandemic's dynamics are special. it was a region that very early on undertook population-wide testing and drastic lockdown measures. on all of the graphs, the curve labels have the following meaning. veneto) on the log-scale graphs, the cumulative case to tests ratio curve seems the peak around or below the diamond princess' cumulative case curve and then start dropping. the scaled death curves exceed this curve. when looking at the graphs of the number of tests per day, two things can be seen. firstly, the number of positive cases closely follows the number of tests (this is clearly visible on the log-scale graphs and supported by the regression study). we look at this issue in detail and present for each region and italy the confirmed cases with respect to the total tests carried out. we also regress the logð(daily confirmed cases)/ (daily total tests)) on time (in days) and logð(cumulative confirmed cases)/(cumulative total tests)) on time (in days). the slope of such a regression can be presented in terms of the half-lives, if it is negative. such a presentation in terms of effect sizes is important; otherwise, it is difficult to assess if the raw slope is big or small. the linear model approach means that the proportion of infected behaves exponentially dailyðcumulativeÞtotaltests then to get the half-life (for a negative, t [ t ) one takes for a [ , one will obtain the doubling time in the same way as ðt À t Þ ¼ logð Þ=a. it is important to point out that this is a rather rule-of-thumb approach-our aim is not to model the dynamics of infections, but rather to visualize and understand what the data in front of us are. these regressions were not performed from the first day, as initially there seems to be a lot of noise in the tests, the starting time considered is visible in each graph-where the fitted line with prediction confidence band is fitted. we performed a regression for both the daily and cumulative counts. for some regions (molise, valle d'aosta), no regression is performed as the daily counts seem to noisy. secondly, one can very clearly identify days when something must have changed due to the testing methodology in the emilia romagna region-there are huge dips in the numbers of tests performed. hence, for this region the dates - march were removed for the regression estimation. in the basilicata and calabria regions, spikes to can also be observed (these are also removed, as on the log scale would result in infinite values which cannot be handled by the regression procedure in lm(). however, such dips require careful investigation. the directly plotted death toll in figs. , , , , , and shows that in the regions emilia-romagna, lombardia and valle d'aosta, p.a. bolzano combined with p.a. trento, there is a larger current (spring ) mortality peak than the past december/january ( - are plotted separately) maximum one. in the regions liguria, marche and piemonte, such a larger current peak is present for men only. in the other regions for all age groups and both men and women, the current ''covid- peak'' seems to be approximately of the same height, or lower, than past december/january ( - ) maximum ones. looking at italy for men and both sexes combined, it is higher, but women seem to have the same peak height. however, it must be stressed that this is only considering the peak's height, not the total amount of deceased during the current peak and the december/january ones. we used the, available on cran, covid r package for the purpose of obtaining the data. the package unifies covid- datasets across different sources in order to simplify the data acquisition process and the subsequent analysis. covid- data are pulled in real time and merged with demographic indicators from several trusted sources including but not limited to: johns hopkins university center for systems science and engineering (jhu csse); world bank open data; world factbook by cia; ministero della salute, dipartimento della protezione civile; istat -istituto nazionale di statistica; swiss federal statistical office; open government data zurich. besides worldwide data, the dataset includes fine-grained data for the diamond princess, switzerland and italy. at the time of writing, these include the number of confirmed cases, deaths and tests, total population, population ages - , - and þ (% of total population), median age of population, population density per km , population mortality rate. depending on the data provider, the data are available at the country level, state level, or city level. for non-r users, the combined datasets are available in csv format. discussion or should we use these data to calibrate epidemiological models? in this work, we analyzed in depth the two statistics that are commonly reported for the currently ongoing covid- pandemic-the number of confirmed cases and the number of case fatalities for the different regions of italy. we found significant variability between regions but also some common insights. in particular, the number of confirmed cases is clearly related to the number of tests and their ratio seems to be decaying for some time now in all regions. this is confirmed when looking at the log-scale plot. the difference between the logarithm of the cumulative number of tests and the logarithm of the cumulative number of confirmed seems to be (visually) dropping linearly (apart from the below, extremely noisy ones) regions and italy as a whole. furthermore, for a number of regions (molise, valle d'aosta), on the log scale, the tests, total, positive and difference behave very chaotically, suggesting rather various test handling situations, than any pattern. such oscillations can be visible in all regions at the initial stages, but they settle down (apart from the previously mentioned three regions). however, in regions with seemingly well-behaved curves individual huge dips can be observed (emilia-romagna, marche). therefore, reports claiming the growth of the epidemic based only on the increasing number of confirmed individuals will not be catching its dynamics. furthermore, studying only daily positively tested counts could be misleading. on a number of days, we found (for some regions) that this count was greater than the number of tests performed. this can certainly be understood, as the result of reporting procedures, in a crisis situation. however, this also implies that any statistical analysis or modeling of such data has to be done very carefully. we find that the cumulative positively tested fraction behaves much more stably, even though in the official cumulative counts decreases can be observed. more importantly, using the raw confirmed case counts one could risk combining the sampling effort with the actual disease spread. in our regressions, for the logarithm of the ratio confirmed cases to total tests on time the fitted slopes are all negative (indicating that the virus is receding and this was observed also by [ ] ). furthermore, these slopes are steeper than the slopes of the logarithm of the raw confirmed case counts on time. with the exceptions of lazio, p. a. bolzano the % confidence intervals for these two slopes do not overlap, or overlap very slightly. the ratios of the two slopes lie between . (p. a. bolzano) and . (piemonte). we report these ratios alongside the slope estimates in the captions of figs. , , , , , , , , , , , , , , , , , , , and . this means that the number of confirmed cases will be confounded by the number of performed tests and cannot be analyzed without them as a point of reference. hence, the raw confirmed case counts are not representative of the virus' infection dynamics. the logarithm of the fraction of confirmed cases to total tests is modeled well by a linear function with an increasing number of daily tests being performed and has a steeper slope than the logarithm of the confirmed case counts. drawing conclusions from raw confirmed case data would seem to be mixing-in the study of the sampling effort (it is important to stress that we do not make any statements here concerning the interpretation of the confirmed cases to tests fraction). therefore, calibrating model parameters for this virus's dynamics should not be done based solely on confirmed case counts, but maybe rather also on case fatalities or hospitalization data (given that classification protocols are taken into account) as, e.g., [ , , ] do. in fact, already [ ] , critised (as [ ] later also did following them) looking at case counts and postulated a focus on the ''observed deaths'' while [ ] writes that ''the cumulative number of deaths can be regarded as a master variable.'' [ ] developed an estimation methods based on the cumulative reported number of case fatalities. on the other hand, we also looked at the ratio of case fatalities to the number of deceased per day. this has the analytical advantage, of referring to something certain and well measured, and detailed records are collected (sooner or later) on the exact number of deceased in a given time period. here, there is hardly any chance of missing asymptomatic (of being dead) people. if the assumption, mentioned in the introduction, that a significant proportion of the tests is serological is true, then the ratio of case fatalities to all deceased should be telling us something about the cumulative proportion of infected individuals. our graphs (especially on the log-scale) do not contradict this, while the cumulative proportion of confirmed cases changes very slowly, the ratio of case fatalities to total deceased per day seems to look like an epidemic growth curve. since italy has very high-quality data on the case fatalities, this data could be further studied to assess the dynamics of the pandemic (e.g., [ ] uses the raw death counts for assessing the dynamics of the pandemic, albeit at the country level). this seems to be supported by that if one compares the curves to a potential ''gold standard''-the cumulative fraction of confirmed cases on the diamond princess, then the case fatalities ratio seems to shadow this curve (on the initial part when the epidemic was taking place on the cruise ship and for some regions like emilia-romagna or lombardia) but exceeds it. one could hope that once all curves would flatten at the same level, then the epidemic will reach the plateau. unfortunately, at the level of some (e.g., emilia-romagna or lombardia) of the regions, the scaled case fatalities grew and exceeded both the diamond princess and cumulative fraction of confirmed cases. we also compared the regional results to the same curves for the whole of italy, fig. . on the one hand, the same patterns are visible-the number of confirmed cases are related to the testing effort, the case fatalities exceeding the diamond princess' cumulative confirmed cases and the confirmed cases fraction seems to be stabilizing around the diamond princess' and then dropping. however, these graphs completely miss the regional variation. this is particularly visible when looking at the total death tolls directly figs. , , , , and . combined italy shows a visible increase in the death toll during the march-april period compared to previous years and the seasonal december/january peak. however, this peak is driven by particular regions emilia romagna, lombardia and piemonte (liguria, marche, valle d'aosta, p.a. bolzano combined with p.a. trento also shows a big increase, but in raw numbers are much lesser than the other three). all the other regions' peak is on the same level or lower than the december/january one and for some the death toll is on similar levels to the march-april one from previous years. furthermore, looking just at epidemiological country level data would be especially misleading for italy as lombardy acted differently from veneto in terms of their testing strategies. we believe that our presented view on the italian regional data gives some insights how the pandemic data reporting can be improved (if of course given the difficult situation, it would be possible in practise). for the confirmed cases count, a breakdown should be provided, how many of these were medical personnel, how many had symptoms, how many were seriously hospitalized before, how many were tested for other reasons (e.g., after contact). similarly for the number of tests carried out and their type (serological or not). the case fatalities counts should also be put in perspective with a report of how many people died in total on the given day and how many deceased were tested negatively. this would allow for estimating excess mortality (crudely compared to previous years' average or more exactly if number of deaths for the given time period are available) and for correct scaling to compare to other ratios. in fact, in the time period january- april, we are able to visualize the excess mortality directly-the number of deceased (in each week) in to the average from the past five years. the dataset is based on the , italian municipalities. to the best of our knowledge, the presented here counts are at the moment the best available data that can be used for scaling and putting the deceased counts in italy in perspective. the death counts seem to be collected in a consistent manner, both the number of case fatalities and the (used here) population death counts. this means that such counts could be used as a proxy for monitoring the dynamics of the virus. it is also a question whether the diamond princess can be considered as a gold-standard. certainly at the beginning it seems to behave like the other presented here curves. however, the data very quickly end, when the passengers were disembarked. we do not know if it reached the plateau or would have still grown. the confirmed case ratio seems to usually stay below/ around this curve, slightly go above and then drop. scaled case fatality curves exceed the curve. finally, the counting methodology should be made readily available for easy comparison between different countries. while of course each country is free to follow their own protocol, without putting numbers into context one can analyze data in an overpessimistic or over-optimistic way. the effect of different counting methods is pointed out by [ , , ] , when fitting parameters to the confirmed case counts (in lombardy, bergamo and brescia), one has a change of coefficients following march and march, and the latter can be possibly due to containment measures, but the former the authors are convinced is due to a change in the counting methodology. we have also abstained here from fitting any models to the data (the regression performed does not have as an aim modeling but formally testing what the respective curve could be telling us). it is known that due to different protocols between regions and changes in the protocols with time, the data are not homogeneous. in order to fit any model, one would have to obtain documentation what were the measurement strategies for each region in the time periods. in fact, when [ ] modeled the cumulative number of infections in italy through time (obtained using the covid r package), they performed fits to date separately in different time intervals which corresponded to various government introduced confinement measures. the covid r data package is available from https://cran.r-project.org/web/packages/covid / . the r script used to generate the graphics is available from https://github.com/krzbar/covid . responsibility for the content of this document lies with the authors. conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. on the uncertainty of real-time predictions of epidemic growths: a covid- case study for china and italy presumed asymptomatic carrier transmission of covid- what we know, and what we don't, about the true coronavirus death toll why estimating populationbased case fatality rates during epidemics may be misleading. medrxiv basic estimation-prediction techniques for covid- , and a prediction for stockholm basic prediction methodology for covid- : estimation and sensitivity considerations. medrxiv la prima foto del virus a milano: la mappa dei contagi in città e nell'hinterland editor: tracking covid- excess deaths across countries: official covid- death tolls still under-count the true number of fatalities report -estimating the number of infections and the impact of non-pharmaceutical interventions on covid- in european countries coronavirus death toll in uk twice as high as official figure covid- data hub. datahub clinical features of patients infected with novel coronavirus in epidemiological study of novel coronavirus (covid- ). arxiv e-prints genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor 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evidence simple models for covid- death and fatal infection profiles , missing deaths:tracking the true toll of the coronavirus crisis evidence for gastrointestinal infection of sars-cov- . gastroenterology estimation of the reproductive number of novel coronavirus (covid- ) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study some numerical observations about the covid- epidemic in italy publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we would like to thank marco picariello (miur-iiss) and paola aliani (cognizant) for sharing their preliminary work on data analysis of the veneto region with us. we would like to thank pierpaolo malinverni (jrc) for his critical reading of an earlier version of the manuscript. all key: cord- -jqm hxps authors: nan title: abstract date: - - journal: eur biophys j doi: . /s - - - sha: doc_id: cord_uid: jqm hxps nan standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organisation of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. we have combined two existing methodologies to tackle these challenges: tandem affinity purification (tap) and nanoflow esi-ms. we use methods designed to maintain non-covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. structural models will be presented for three oligomeric protein complexes of unknown structure: the yeast exosome and the human u snrnp and eif complexes. these models will then be examined within the context of their function. recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross-section. using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross-sections. very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase . this enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. in vivo molecular sensing: fluorescence beyond labeling f. beltram scuola normale superiore, i- pisa, italy fluorescent molecules are powerful reporter tools that have much extended the impact of optical microscopy, particularly thanks to the flexibility of genetically encoded tags. detection can now target single molecules even in the complex environment of intact live cells offering unprecedented insight on biological processes in real time in live cells and tissues. fluorescent labels, however, can do more that this. our increased ability to tailor molecule and, more in general, nanosystem properties allows us to design, produce and exploit intelligent tags that can actually analyze the cellular environment. today multifunctional nanosystems can be produced that provide a signal dependent on the value of a specific biochemical parameter. importantly these nanosystems can target specific subcellular domains and have the ability to be used also in the case of live organisms. recent results will be discussed that highlight the impact of nanobiotechnology in this context with a particular emphasis given to methods suitable for in vivo studies that can be transferred to the biomedical world. far-field optical nanoscopy s. w. hell max planck institute, göttingen, germany the resolution of a far-field optical microscope is usually limited to d = λ/ ( n sin α) > nm, with n sin α denoting the numerical aperture of the lens and λ the wavelength of light. we will discuss lens-based fluorescence microscopy concepts that feature a resolving power on the nanoscale. all these concepts share a common basis: exploiting selected (pairs of) states and transitions of the fluorescent marker to neutralize the limiting role of diffraction. specifically, the fluorophore is switched on and off, that is, between a bright and a dark state, to detect the emission of adjacent features sequentially in time. the first viable concept of this kind was stimulated emission depletion (sted) microscopy in which the fluorescence ability of the dye is switched off by stimulated emission. in the sted microscope, the extent of the region in which the molecule is able to fluoresce follows d ≈ λ/ n sin α + i/i s , meaning that fluorophores that are further away than d can be separated. i is the intensity that drives a fluorophore from the bright fluorescent state to the dark ground state by stimulated emission. i s depends (inversely) on the lifetime of the states. for i/i s → ∞, it follows that d → , meaning that the resolution can be molecular). altogether, far-field optical 'nanoscopy' is a fascinating development in optics with high relevance to the many areas of sciences, in particular the life sciences. since it has already been a key to answering important questions in biology, and owing to its simplicity and commercial availability, we expect far-field fluorescence 'nanoscopes' to enter most cell biology and many nanoscience laboratories in the near future. grabbing the cat by the tail: discrete steps by a dna packaging motor and the inter-subunit coordination in a ring-atpase c. bustamante, j. moffitt university of california, berkeley, california, u.s.a. as part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid. bacteriophage ϕ packages its . mm long double-stranded dna into a nm dia. x nm high capsid using a multimeric ring motor that belongs to the asce (additional strand, conserved e) superfamily of atpases. a number of fundamental questions remain as to the coordination of the various subunits in these multimeric rings. the portal motor in bacteriophage phi is ideal to investigate these questions and is a remarkable machine that must overcome entropic, electrostatic, and dna bending energies to package its genome to near-crystalline density inside the capsid. using optical tweezers, we find that this motor can work against loads of up to ∼ piconewtons on average, making it one of the strongest molecular motors ever reported. we establish the force-velocity relationship of the motor. interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to dna compression attaining the value of ∼ megapascals at the end of the packaging. we show that the chemical energy of atp is converted into mechanical work during phosphate release. using ultra-high resolution optical tweezers, we determined the step size of the motor and established the coordination of the polymerases around the ring. we propose a comprehensive model of the operation of this motor. watching proteins function in real time via timeresolved x-ray diffraction and solution scattering p. a. anfinrud laboratory of chemical physics/niddk, nih, bethesda, maryland, u.s.a. to generate a deeper understanding into the relations between protein structure, dynamics, and function, we have developed x-ray methods capable of probing changes in protein structure on time scales as short as ps. this infrastructure was first developed on the id b time-resolved x-ray beamline at the european synchrotron and radiation facility, and more recently at the id b biocars beamline at the advanced photon source. in studies of ligand-binding heme proteins, a picosecond laser pulse first photolyzes co from the heme, then a suitably delayed picosecond x-ray pulse passes through the protein and the scattered x-rays are imaged on a d detector. when the sample is a protein crystal, this "pump-probe" approach recovers time-resolved diffraction "snapshots" whose corresponding electron density maps can be stitched together into movies that unveil the correlated protein motions that accompany and/or mediate ligand migration within the hydrophobic interior of the protein. when the sample is a protein solution, we recover timeresolved small-and wide-angle x-ray scattering patterns that are sensitive to changes in the size, shape, and structure of the protein. scattering studies of proteins in solution unveil structural dynamics without the constraints imposed by crystal contacts; thus, these scattering "fingerprints" complement results obtained from diffraction studies. this research was supported in part by the intramural research program of the nih, niddk dc-sign is a trans-membrane protein expressed on antigen presenting cells and recognizes pathogens like hiv- , hepatitis c virus and ebola. by electron microscopy and near-field optical nanoscopy, we demonstrated that at the plasma membrane dc-sign is organized in well-defined nanodomains of - nm in diameter. intensity-size correlation analysis revealed remarkable heterogeneity in the nanodomains molecular packing density. we constructed and characterized several dc-sign mutated forms lacking specific molecular domains. by immunogold labeling and spatial point pattern analysis, we show that the extracellular neck domain is essential for dc-sign nanoclustering. finally, we present a model that describes the probability of a cell to have a certain number of receptors joining the contact site in the initial encounter with an external object. monte carlo simulations subsequently define the parameters that are determinant in the object-cell encounter. our results show that receptor nanoclustering is of particular importance for binding objects of sizes comparable to the nanocluster size, indicating that the nanoscale spatial organization of dc-sign is optimized for binding to virus-sized objects. imaging of mobile stable lipid rafts in the live cell plasma membrane m. brameshuber , j. weghuber , v. ruprecht , h. stockinger , g. j. schuetz johannes kepler university linz, austria, medical university of vienna, austria the organization of the cellular plasma membrane at a nanoscopic length scale is believed to affect the association of distinct sets of membrane proteins for the regulation of multiple signaling pathways. based on in vitro results, conflicting models have been proposed which postulate the existence of stable or highly dynamic platforms of membrane lipids and proteins. here we directly imaged and further characterized lipid rafts in the plasma membrane of living cho cells by single molecule tirf microscopy. using a novel recording scheme for "thinning out clusters while conserving stoichiometry of labeling" , molecular homo-association of gpi-anchored mgfp was detected at • c and ascribed to specific enrichment in lipid platforms. the mobile mgfp-gpi homo-associates were found to be stable on a seconds timescale and dissolved after cholesterol depletion. having confirmed the association of mgfp-gpi to stable membrane rafts, we attempted to use an externally applied marker to test this hypothesis. we used bodipy-gm , a probe that was recently reported to be enriched in the liquid-ordered phase of plasma membrane vesicles. when applied to cho cells at different surface staining, we found that also bodipy-gm homo-associated in a cholesterol-dependent manner, thus providing further evidence for the existence of membrane rafts. [ ] appl phys lett , ( ) . synapsin knock-out mice as an in vitro model of human epilepsy studied with multi-electrode arrays d. f. boido, p. farisello, p. baldelli, f. benfenati department of neuroscience and brain technologies, italian institute of technology, genova, italy mutant mice lacking synapsins (syn), a family of synaptic vesicles (sv) proteins implicated in the regulation of neurotransmitter release and synapse formation, are epileptic. the attacks appear after the third month of age and severity increases with age. several mutations of syn genes have been found in families of patients with epilepsy. we used micro-electrode arrays (meas) to study spontaneous and chemically evoked epileptiform activities in cortico-hippocampal brain slices obtained from wild-type (wt) and synko mice. -months old synko mice show sporadic ictal (ic) events in the entorhinal cortex. a potassium channel blocker, aminopyridine ( ap), elicits ic and inter-ictal (i-ic) events in both wt and synko slices. in the hippocampus of young synko ( -days old) mice, ap induces i-ic events at higher frequencies than in wt mice. also the frequency of ic events, mainly observed in the cortex, is higher in synko. the analysis of adult ( -year old) mice, revealed a clear age-related aggravation, which paralleled the increase in the severity of the epileptic phenotype observed in vivo. many slices from adult synko mice showed an ic event, while wt slices were refractory at this age to experience ic activity. synko mice are useful to study how neuronal network hyperexcitabilty due to mutations in sv proteins leads to the development of epileptiform activity. meas proved themselves to be useful tools to characterize the epileptic signals foci and patterns of propagation. high electron mobility transistor (hemt) structures were used to bridge the gap between the analysis of biological reactions and biophysical characterization. the combination of nanotechnological measurement approaches with biological reactions provides new possibilities for living cell examinations after exposure to ionizing radiation and basically during the irradiation experiments itself. in this transdisciplinary approach experimental data and handling of biological material enables the identification and specification of systems properties of biological responses to ionizing radiation at different hierarchical levels. gan/algan-heterostructures form a hemt with a gate very sensitive to ph-value changes and potential changes in general. to record cell membrane potentials and ion fluxes during and after irradiation experiments living cells are cultivated on the functionalised biocompatible chip surface. here, we present results of x-ray stimulated cell responses grown on gan-chip surfaces. we recorded transistor signal changes of . µa within s caused by an irradiated cell monolayer. to measure cell potentials, not only after irradiation experiments but also during the irradiation itself expands the examination restrictions in an enormous way. measuring diffusion by spatial-cross-correlation e. gratton, m. a. digman laboratory for fluorescence dynamics, university of california, irvine, u.s.a. fluorescence correlation spectroscopy (fcs) has emerged as a very powerful method to study the motions of proteins both in the interior and exterior of the cell. it provides information at the single molecular level by averaging the behavior of many molecules thus achieving very good statistics. single particle tracking (spt) is also a highly sensitive technique to measure particle movement. however, the fcs method suffers in spatial resolution while the spt technique only allows for the tracking of isolated molecules. here we propose a change of paradigm in which using spatial pair cross-correlation functions we can overcome this limitation. our method measures the time a particle takes to go from one location to another by correlating the intensity fluctuations at specific points on a grid independently on how many particles are in the imaging field. therefore we can trace the average path of the particles. for example, our method could be used to detect when a protein passes the nuclear barrier and the location of the passage. this information cannot be obtained with the frap (fluorescence recovery after photobleaching) technique or the image correlation spectroscopy method. the interaction of the bax c-terminal domain with membranes j. c. gomez-fernandez, s. sanchez-bautista, a. perez-lara, s. corbalan-garcía departamento de bioquimica y biologia molecular. universidad de murcia, murcia, spain the c-terminal domain of the pro-apoptotic protein bax (bax-c) acts as a membrane anchor during the translocation to the membrane of this protein leading to programmed cell death. we have used static and mas-nmr techniques to show that the interaction of bax-c with membranes is modulated by the presence of a negatively charged phosphatidylglycerol. the width of the resonance peaks were considerably more increased by bax-c, in the presence of phosphatidylglycerol. bax-c substantially decreased the t relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol but they were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. c-mas-nmr showed that t values were decreased when bax-c was incorporated and, when phosphatidylglycerol was also present, the decrease in t affected considerably more to some carbons in the polar region. these results indicate that bax-c interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of bax-c with the membrane determines the membrane disposition of this domain. fluorescence spectroscopy showed that the trp residues of bax-c were located in a microenvironment more hydrophobic when phosphatidylglycerol was present. h. g. franquelim , l. m. s. loura , n. santos , m. castanho instituto de medicina molecular, univ. lisboa, portugal, faculdade de farmácia, univ. coimbra, portugal since the efficacy of hiv fusion inhibitors was previously reported to be related to an ability to interact with membranes, we studied the interaction of the hiv fusion inhibitor sifuvirtide, a aa negatively charged peptide, with lipid vesicles. since this peptide has aromatic residues, fluorescence spectroscopy techniques were used with no need for attached probes. results showed no significant interaction with both zwitterionic fluid phase and cholesterol-enriched membranes; however extensive partition to fluid phase cationic membranes were observed. in the dppc gel phase, however, an adsorption at the surface of these membranes was detected by using a differential quenching approach with lipophilic probes, as well as by fret. moreover, the interaction with gel phase membranes seems to be specific towards pc vesicles, since no significant interaction was retrieved for membranes composed by shingomyelin and ceramide. besides fluorescence, atomic force microscopy and zeta-potential were used to further investigate this issue. our results show a selectivity and specificity of the peptide towards rigid domains, where most of the receptors are found, and help explain the importance of the interaction with membranes in the improved efficacy of sifuvirtide compared to other fusion inhibitors, by providing a local increased concentration of the peptide near the fusion site on both cellular and viral membranes. the study of molecular dynamics at the single-molecule level with fluorescence far-field optics offers new detailed insights into scientific problems, especially in living cells. unfortunately, the resolution of common far-field techniques is limited to about nm in the lateral direction by diffraction. in recent years, several concepts such as stimulated emission depletion microscopy (sted) have been successfully applied to overcome the diffraction barrier by exploiting the photophysical properties of fluorescent labels. we present the combination of high resolution sted microscopy with different fluorescence fluctuation techniques providing the unique ability to study molecular dynamics with high spatial (< nm) and temporal resolution (< ms) in living cells. using fluorescence correlation spectroscopy (fcs) and general single-molecule analysis, we were able to explore single-molecule dynamics in up to -fold reduced focal volumes on two-dimensional samples such as lipid membranes with excellent signal-to-noise ratios. special attention is drawn to inhomogeneous lipid diffusion on the plasma membrane of living cells . by extending the available spatial scale of standard single-molecule fluorescence far-field spectroscopy techniques, our experiments outline a new way of approaching scientific problems. modulation of the properties of membrane microdomains as a control mechanism in cellular physiology p. o'shea cell biophysics group, institute of biophysics, imaging & optical science, school of biology, university of nottingham, nottingham ng rd, u.k. this presentation will outline the molecular-physical rationale of how membrane microdomains may modulate the behaviour of membrane receptor systems as a controlling mechanism in cell signaling. a number of external factors that modulate these properties will be indicated that have a bearing on controlling cellular behaviour. some of key questions will be considered such as the factors that control the assembly and disassembly of microdomains, the size and numberdensity of the microdomains and the lifetime that they exist within the membrane. the technical challenges that these questions identify will also be outlined with some possible solutions. throughout this presentation, correlations will be made between theory and experiment as well as between model membrane systems and real cellular systems. structural and dynamic properties of caveolin- and - fragments at the membrane interface c. le lan , j. gallay , m. vincent , j.-m. neumann , b. de foresta , n. jamin cea, ibitecs, sb sm, & ura cnrs , gif-sur-yvette, france, ibbmc, université paris-sud, umr -cnrs, ifr , orsay, france caveolins are major protein components of caveolae, microdomains of the plasma membrane involved in a large number of biological functions, including signal transduction, cholesterol homeostasis and transport. the consensus topological model of caveolin- includes a small central intramembrane region ( - ) flanked by two cytosolic amphiphilic domains ( - and - ) which probably constitute in-plane membrane anchors. we investigated the interaction of the cav- ( - ) juxta-membrane segment with various membrane mimics, using fluorescence, cd and nmr. this segment partitioned better in dpc and in dm/anionic lipids micelles than in dm micelles and this partitioning was coupled with the formation of an amphipathic α-helix. this amphipathic helix was located in an average shallow position, in the polar head group region of the dpc micelle, as shown by fluorescence data and intermolecular noes, with the aromatic doublet w -f probably pointing towards the inside of the micelle on average. the peptide encompassing the homologous sequence of cav- was also localized to the dpc micelle polar head group region, in which it adopted a more stable helical conformation than cav- ( - ) . these data brings experimental support for the role of this segment as an interfacial membrane anchor. resveratrol (trans- , ', -trihydroxystilbene) , a phytoalexin present in grapes and its analogue piceatannol (trans- , , ', '-tetrahydroxystilbene) are biologically active compounds and possess potential chemopreventive and anticancer properties. the activity of resveratrol and piceatannol can be mediated by membrane effects since structure of lipid membrane domains may play an important role in cell signalling pathways. drugs interactions with dmpc bilayers was investigated using a combination of esr spectroscopy and differential scanning calorimetry. spin probes used in epr experiment were located in different part of lipid bilayer. study was performed at temperatures below and above phase transition temperature (t m ). epr spectra were simulated and displayed with ghost condensation method. the values of ϑ and ϕ (the main and asymmetry cone angles of wobbling spin probe, respectively) were taken for free rotational space parameter (Ω) calculation. the decrease of Ω values was observed in the presence of both compounds and the effect was more pronounced in lipid gel phase. order parameter and correlation time were also determined and presented in form of ghost patterns. using this approach the differential influence of studied compounds on membrane heterogenity was revealed. separating hydrostatic pressure from cellular strain: development of an in vitro model system r. sulley , j. whatmore , c. p. winlove , a. shore , j. tooke , r. ellis , k. gooding peninsula medical school, school of physics, university of exeter, uk endothelial cells (ecs) line blood vessels & are constantly subjected to haemodynamic and mechanical stresses and strains. these stimuli are known to influence ecs, modifying their morphology, intracellular signalling & gene expression. most reported systems exposing ec to mechanical forces in vitro alter pressure & strain simultaneously, making it impossible to distinguish the two potentially independent stimuli. this distinction is particularly relevant when examining the interaction of haemodynamic forces on microvascular ecs, which are exposed to low hydrostatic pressure but significant strains. this research aims to create an in vitro system that can independently examine the effects of pressure and strain, over a range experienced by ecs in the microvasculature. human ecs are seeded ( x ˆ cells/cmˆ ) on to the inner surface of compliant mm diameter tubing. which is mounted on a perfusion rig inside a sealed, fluid-filled chamber. a continuous sinusoidal cyclical strain of - % is created by a pump attached to the external chamber. lumenal pressure is generated using two hydrostatic pressure heads. validation experiments show that pressure & substrate strain can be independently varied & controlled over a physiological range. the system is now being used to investigate the effects of pathophysiological haemodynamic abnormalities on ec function. membrane potential dynamics of living cells in response to femtosecond laser irradiation n. i. smith , j. ando , k. fujita , s. kawata photonics advanced research center, osaka university, suita, osaka - , japan, dept of applied physics, osaka university, suita, osaka - , japan the ultrashort pulsed near-infrared femtosecond laser has had a large impact in biomedical research fields and in microscopy, where it has enabled new imaging methodologies. at high intensities, the focused beam of a femtosecond laser has been used to irradiate specific locations inside a cell, often beneath the cell membrane, exploiting the inherent penetration and localized absorption that comes from the multiphoton absorption physics. this has been applied in photobleaching, photouncaging, laser surgery and other experiments where the light is used not merely for observation but is instead an integral tool to interact with the dynamics of cells, to probe and perturb the cell condition. in this talk i will discuss biological and mechanical effects that can be generated by short exposures to femtosecond laser irradiation, such as calcium waves, membrane hyperpolarization, and cell contraction. this talk will concentrate on the changes in membrane potential that can occur when the cell is subjected to focused femtosecond laser beams. both depolarization and hyperpolarization of the membrane potential could be evoked, depending on the laser parameters and on the position of the laser focus. these results have implications for the use of laser beams in microscopy, optical gene transfection, and laser nanosurgery. in a recent study we showed that the melting behavior of supported lipid bilayers (slbs) on mica can be influenced by the solution ionic strength and by the slb preparation temperature [ ] . by changing these parameters we could control the coupling between the two bilayer leaflets obtaining a coupled or decoupled melting behavior. thus, we could provide evidence that the slb model system is also suited for the study of lipid/protein interactions which had been questioned in the past. then we investigated the mutual interactions between the membrane lipids (pope:popg : ) and the kcsa potassium ion channel by studying kcsa proteins reconstituted in slbs. in particular, we studied the melting behavior of the slb and the ion channel distribution relative to the different membrane phases by temperature controlled atomic force microscopy (afm). by decreasing the temperature we found that the proteins underwent diffusion so to be excluded from the growing solid ordered regions. further, the ion channels tended to accumulate at the domain boundaries or they aggregated in the liquid disordered phase. both effects have been suggested to affect protein function. when we started from a low temperature at which the membrane was mainly in the solid ordered phase the membrane melting processes started in the vicinity of the included ion channels. we report fluorescence recovery after photobleaching (frap) measurements performed at variable spot radius for t -egfp-hmop receptors on sh-sy y neuroblastoma cells in the presence of ligands. two different agonists, damgo and morphine, caused markedly different changes to receptor diffusion as compared to the basal state. like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joint semi-permeable domains, but with smaller domain size and diffusion coefficient. this effect was inhibited by pertussis toxin, suggesting that this dynamic behaviour is associated with early steps of signaling. in the presence of damgo, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. hypertonic sucrose buffer suppressed this effect which we attribute to receptor entry into clathrin-coated pits. it is likely that the observation of distinct receptor dynamics in the presence of damgo and morphine involves the agonist-selective phosphorylation of the receptor. the alteration of the physiological transcriptional program is one of the constant features of cancer cells. however, the characterization of the chromatin changes at single-gene level requires going beyond the diffraction limit affecting conventional fluorescence microscopy. the ability of molecular biology techniques to obtain a detailed view of the chromatin status at a sub-promoter resolution has to pay instead the price of averaging over a cell population. we report here the application of an approach based on high-resolution cytometry, chromatin immuno-precipitation and transcriptional profiling (dna microarray) for the characterization of the transcriptional and chromatin changes induced by the oncogenic transcription factor pml/rarα. the presentation will focus on the imaging protocol employed to observe the effects on the chromatin status and the extent of the deregulation induced on transcriptional activity in acute promyelocytic leukemia cells. this multiple-approach examination provides a further step towards the comprehension of the hierarchy of chromatin modifications leading to the establishment of a malignant transcriptional program. dna is rigid negatively charged polymer and in solution exists in extended conformation. i n vivo, volume occupied by dna must be reduced to fit to tiny space of cell nucleus. to condense dna, dna-dna electrostatic repulsion must be cut off that is achieved by interaction with cationic ligands. in binding to dna, oligocations compete with salt cations (k + , na + , mg + ). description of salt dependence of oligocation-induced dna condensation is still lacking. we studied dna condensation by model oligocations, ε-oligo(l-lysines), with variation of charge from + to + . combination of light scattering, uv-monitored precipitation assay and isothermal titration calorimetry allowed covering wide range of dna (c dna ) and salt (c kcl ) concentrations. salt dependence of dna condensation efficiency of the ligand, ec (ligand concentration at the transition midpoint) displays two regimes: salt-independent at low c kcl and salt-dependent at higher c kcl (steep increase of ec with c kcl ). simple formula describing ec as function of ligand charge, c dna and dissociation constant of ligand-dna complex (k d ), was proposed. in the salt-independent regime ec is defined by c dna . salt-dependence of ec is rooted in the variation of k d with c kcl earlier described in ligand-dna binding studies. importance of our findings for description of chromatin is discussed. interaction between proteins from linker region of nucleosome in presence/absence of dna in solution i. b. kipenko , e. v. chikhirzhina , a. m. polyanichko faculty of physics, saint-petersburg state university, russia, laboratory of cell biochemistry, institute of cytology, ras, saint-petersburg, russia interactions in the linker region of the nucleosome play a key role in the structural organization of the chromatin. the most fascinating and least understood is the interplay between non-histone chromatin protein hmgb and a linker histone h . it is known that both h and hmgb bind the linker region of the dna in vivo. however it is still a matter of debate as to whether these proteins assist each other or compete upon binding. the main attention in this work is paid to the investigation of the interactions between the hmgb and h proteins in physiological environment. using circular dichroism (cd) spectroscopy we have studied the interactions between hmgb and h at various hmgb /h ratios (r). it has been shown that there is a cddetectable interaction between the proteins h and hmgb at r < . we have demonstrated that the interaction between these proteins results in changes of their secondary structure. cd indicates that the structural impact of the unordered fragments decreases while the net α-helicity of the proteins increases upon the interaction. we have also shown that large higher order structures are formed in solution. in this work we have also discussed the dna-binding properties of the hmgb and h proteins. the work was supported by a rfbr grant ( - - ) and the government of staint-petersburg. t. u. rodionova , a. m. polyanichko , v. i. vorob'ev faculty of physics, saint-petersburg state university, russia, institute of cytology of the russian academy of sciences, saint-petersburg, russia hmgb is a nonhistone chromosomal protein. data regarding the structure of the hmgb-proteins obtained so far are rather different. thermodynamic experiments reveal predominant α-helical structure of the proteins only at temperatures below + • c, i.e. under physiological conditions they are mainly disordered. despite a lot of experimental data biological role of hmgb still remains unclear. it is believed that the proteins perform structural functions in chromatin and participate in various regulatory processes in cell. using circular dichroism spectroscopy and dna melting analysis we have shown that hmgb changes its structure upon binding to dna. it was shown that at room temperature only about % of amino acid residues form α-helices, while in dna-hmgb complex the degree of the α-helicity of the protein increases to approximately %. based on the data obtained we estimate the size of hmgb binding site as - b.p. we have also demonstrated that despite of strong dna-bending properties of hmgb its binding to dna results in increase of the double helix termostability. the authors are grateful for the financial support from the russian foundation for basic research (grants - - , - - ) and the government of saint-petersburg. structural organization of supramolecular complexes of dna with chromosomal proteins hmgb and h a. m. polyanichko , h. wieser faculty of physics, saint-petersburg state university, russia, dept. of chemistry, university of calgary, canada a combination of uv and ir absorption and circular dichroism spectroscopy together with atomic force microscopy was applied to investigate the structure and formation of large supramolecular dna-protein complexes. this combination of techniques was used to overcome limitations of uv-cd spectroscopy due to considerable light scattering in such solutions. based on the analysis of ftir and uv circular dichroism spectra and afm data the interaction of dna with highmobility group non-histone chromatin protein hmgb and linker histone h was studied. it is believed, that hmgb-domain proteins perform both structural and regulatory functions in chromatin. however, the particular mechanisms of it functioning remain unclear/ our data show that histone h facilitated binding of hmgb to dna by interacting with the sugar-phosphate backbone and binding of asp\glu amino acid residues of hmgb . acting together, hmgb and h stimulated the assemblage of supramolecular dna-protein structures. the organization of the ternary complexes is modulated by the interactions between hmgb and h molecules. the dna-proteins interactions in the presence of metal ions were different, causing prominent dna compaction and formation of large intermolecular complexes. the work was supported by rfbr (grant - - ). biophysical properties and mechanisms of phage dna ejection t. mdzinarashvili , m. khvedelidze , a. ivanova , t. partskhaladze , n. shengelia i. javakhishvili tbilisi state university, tbilisi, georgia, institute of molecular biology and biophysics, tbilisi, georgia to determine the requirements for phage adsorption on bacterial cell and for the realizing resources of following dna ejection thermodynamic and hydrodynamic methods were employed. the temperature, bacterial membrane fragments and receptors had been chosen as such external factors. the phages with short and long tail, both contractile and noncontractile have been studied. our viscometric studies of the phage dna ejection induced by receptor by the example of t phage and its receptor fhua have shown that the minimum protein-to-phage ratio necessary for complete dna release is to . the viscometric study of ddvi phage dna ejection induced by membrane fragments obtained from its host cells has shown that the environmental conditions play significant role in ejection process. both methods show that the thermally induced phage dna ejection for all investigated by us phages have shown that this process is nonenthalpic. finally from our experimental results we conclude that the start of the dna ejection process from the phage particle occurs without additional energy from either a physical or chemical source. we thank gnsf for the financial support. nitroxides induce apoptosis through caspase- activation and collapse of mitochondrial potential k. matczak , a. koceva-chyla , k. gwozdzinski , z. jozwiak department of thermobiology, department of molecular biophysics, university of lodz, lodz, poland nitroxides are new class of antioxidants that have been proved to show high reactivity toward free radicals. they act as superoxide dismutase mimics dismutating superoxide anions, but can also exert pro-oxidative properties. in view of their possible dual activity nitroxides could be of great importance in medicine. we have investigated pro-apoptotic activity of pyrroline and pyrrolidine nitroxides pirolid (pd) and pirolin (pl) in human breast cancer cells. in cancer, it is the failure of malignant cells to undergo apoptosis that is crucial. using microplate fluorescence methods, we estimated kinetics of changes in mitochondrial transmembrane potential and caspase- activity in breast cancer cells mcf- treated with pirolin or pirolid. these features are connected with induction of apoptosis in some type of cancer cells. we observed steady-state increase in caspase- activity up to h of postincubation that was followed by a decrease in the enzyme activity at h. caspase- activation was considerably greater in cells treated with pirolid. both nitroxides also caused notable decrease in mitochondrial transmembrane potential, which suggest that they can induce apoptosis in breast cancer cells through mitochondrial pathway. o. chernyavskiy , l. vannucci , p. bianchini , f. difato , l. kubínová dept. of biomathematics, inst. of physiology, as cr, prague, czech republic, dept. of immunology, inst. of microbiology, as cr, prague, czech republic, lambs, dept. of physics, university of genoa, italy, dept. of neuroscience and brain technologies, the italian institute of technology, genoa, italy the second harmonic generation (shg) imaging along with confocal laser scanning microscopy in reflectance mode can be applied to imaging unstained tissues in vivo, so it can be considered as a fast and non-invasive tool for in vivo studies. murine b f melanoma cells after subcutaneous inoculation in syngeneic mice were let to develop into tumor up to - mm in diameter. microscopic images were taken before and after microwave hyperthermia treatment (mwht). the microscopic images were acquired by -photon imaging in reflectance mode, shg imaging and -photon imaging of tissue autofluorescence. the evaluation of changes in the images after mwht of the tumor demonstrated changes in the architecture and organization in both the tumor capsule and tumor mass. the presented study was supported by the academy of sciences of the czech republic (grant iaa , institutional research concepts no.av z , av z ), and ministry of education, youth and sports of the czech republic (research program lc ). intracellular delivery and fate of peptide-capped gold nanoparticles: towards cellular biosensors y. cesbron , v. sée , p. free , p. nativo , d. g. spiller , m. r. h. white , m. brust , b. lounis , r. lévy liverpool institute for nanoscale science, engineering and technology, liverpool, uk, université bordeaux i / cnrs, bordeaux, france gold nanoparticles (nps) have extraordinary optical properties that make them very attractive single molecule labels. although understanding their dynamic interactions with biomolecules, living cells and organisms is a prerequisite for their use as in situ sensors or actuators. while recent research has provided indications on the effect of size, shape, and surface properties of nps on their internalization by living cells, the biochemical fate of nps after internalization has been essentially unknown. here we show that peptide-capped gold nps enter mammalian cells by endocytosis. we demonstrate that the peptide layer is subsequently degraded within the endosomal compartments through peptide cleavage by the ubiquitous endosomal protease cathepsin l. preservation of the peptide layer integrity and cytosolic delivery of nps can be achieved by a combination of cathepsin inhibition and endosome disruption. this is demonstrated using a combination of distance-dependant fluorescence unquenching and photothermal heterodyne imaging. these results prove the potential of peptide-capped gold nps as cellular biosensors. current efforts focus on in-vivo labeling of nps, nanoparticle-based real-time sensing of enzyme activity in living cells, and the development of photothermal microscopy for single nanoparticle imaging in living cells. towards intravital two photon microscopy study of lymphocytes mobility in lymphonodes m. caccia , l. sironi , m. collini , i. zanoni , t. gorletta , m. di gioia , g. francesca dipartimento di fisica, università di milano bicocca, italy, dipartimento di biotecnologie e bioscienze, università di milano bicocca, italy during the last years the edge between optical fluorescence based microscopy and the world of bio-medical research has become thinner and thinner and two photon laser scanning microscopy (tplsm) is one of the most powerful tool for immunological and medical research. one of the most limiting step in intravital microscopy is the preparation of the animal model and the number of animals to be sacrificed to get good statistics. alternative routes must be searched. we employ tplsm to explanted lymphnodes. two photon excitation and a non descanned detection mode allow to increase, respectively, the excitation and detection efficiency while the explanted organs are kept very close to the condition they experience in live animals by means of an home-made temperature controlled box surrounding the microscope and a system for the flux of physiological fluids. experiments performed on explanted lymphonodes, kept under constant flux of co -o saturated buffers and at o c, agree with literature for what concern t-cell homing and motility. this seems to confirm that t-cells behavior in explanted organs mantained in physiological conditions is very similar to that observed in live animals. we then believe that our tplsm microscope would allow to study cell behaviors in in-vivo with high efficiency and a little technical effort. a generalized quantitative frap method with no restriction on the size of the photobleached area heterochromatin protein in dna damage response -recruitment or dissociation from repair sites? j. w. dobrucki division of cell biophysics, faculty of biochem. biophys. and biotechnol., jagiellonian university, kraków, poland we studied recruitment of dna repair proteins to damage sites in live cells, by microscopy approaches, using a new method of inflicting local, sublethal damage in nuclei of live cells. oxidative damage, which was inflicted by exciting dna-intercalated ethidium with focused green light, triggered recruitment of base excision repair enzymes. surprisingly, an epigenetic regulator, heterochromatin protein (hp ) (zarebski et al., ) was recruited to damage as well. hp is a constitutive component of heterochromatin, and plays an important role in transcriptional repression and regulation of euchromatic genes, however it was not known to be required for repair of oxidative damage. the finding of hp recruitment is particularly puzzling, since in another study hp was shown to dissociate from chromatin as a result of dna damage (ayoub et al. ) . technical aspects of live cell imaging that may explain these contradictory results will be discussed. in this presentation, we report a method for determining both the presence and the stoichiometry of protein complexes at pixel resolution and apply it to disassembling focal adhesions. the method is derived from fluorescence fluctuation methods that have single molecule sensitivity and is based on our previously described n&b (number and brightness) method that measures the number and brightness (aggregation state) of fluorescent molecules in every pixel of a confocal microscope image. the new method exploits the correlation of fluorescence amplitude fluctuations for two colors and detects the presence of molecular complexes and their stoichiometry. while the original n&b method was developed for one color, i.e., a single molecular species, the new method, ccn&b, extends the analysis to two colors and introduces the concept of cross-variance. this method is similar in concept to the two-color pch analysis. however, the covariancebased ccn&b method also generates pixel resolution maps of protein complexes and can be used on commercial confocal microscopes. the method is highly sensitive and has relatively high temporal resolution. we have applied this method to adhesion complexes in cells. in addition of their structural role, to link the extracellular substratum to actin filaments, they also serve as signaling centers that regulate many cellular processes including their own assembly and turnover, migration, gene expression, apoptosis, and proliferation. spatio-temporal analysis of membrane lipid remodeling during phagocytosis s. de keijzer , d. kilic , c. g. figdor , s. grinstein , a. cambi department of tumor immunology, radboud university of nijmegen, nijmegen, the netherlands, department of biochemistry, university of toronto, toronto, canada the constant threat posed by pathogens and cell debris is tackled by phagocytosis, the process through which cells engulf and destroy dangerous material. the signaling, targeting and trafficking during phagocytosis is dependent on cytoskeleton rearrangements and membrane remodeling. it is becoming increasingly evident that lipids play an important role and can affect the phagocytic response. they assemble microdomains which can act as signaling platforms and confer charge and curvature to the membrane surface promoting electrostatic attraction and retention of proteins. little is known about the mechanism(s) regulating membrane lipid remodeling during phagocytosis. here we used fluorescently labeled biosensors based on k-ras and h-ras proteins to obtain spatio-temporal information on phagosomal membrane lipid remodeling during fcreceptormediated phagocytosis. the results show that cell activation by cytokines modulates the kinetics of anionic lipids thus affecting the membrane charge and the recruitment of cytosolic proteins to the phagosomal membrane. our data emphasize the fundamental role of lipids in the generation and transduction of signals in phagocytosis, and we believe this can be extrapolated to many important processes in a cell. fluorescence correlation spectroscopy (fcs) is a useful technique for characterizing the mobility and concentration of fluorescent molecules both in vitro and in vivo. we utilize two-photon fcs to characterize the concentration and mobility of fluorescent molecules within living cells of bacillus subtilis. autocorrelation functions were measured in bacteria expressing green fluorescent protein (gfp) under the lac promoter in both nutrient rich and nutrient poor culture medium. although considerable heterogeneity was evident from cell to cell, on average, both intracellular concentration and mobility were found to be dependent upon culture medium. we also investigated bacteria expressing gfp under control of native promoters for involved in the regulation of the carbon metabolic cycle in bacillus subtilis. the gfp concentration, which should be related to promoter activity, was investigated for single cells and cell populations under different metabolic conditions. some photobleaching was observed during the course of the measurements as a decrease in the average fluorescence intensity. this is due to the small size of the bacteria (∼ fl) and low basal expression levels of gfp (∼ nm) in the absence of iptg. methods to take this into account during data analysis are discussed. a. fassio , s. congia , p. baldelli , f. benfenati dimes, university of genoa, genoa, italy, dnbt , iit, genoa, italy several mutations have been discovered in the synapsin i (syn) gene in families with epilepsy, but the mechanism inducing the epileptic phenotype is unknown. syn is a protein associated with synaptic vesicles (svs) that control sv trafficking and neurotransmitter release. the syn mutations subject of this study are a non sense (ns- ) and of two missense (ms- , ms- ) . syn knockout (ko) hippocampal neurons were transfected with the either wild type (wt) or mutated syns. all syns presented a common punctate pattern of expression at the level of axonal arborizations but, while ns- syn targeted to synapses as wt-syn, ms- and ms- syns reached the presynaptic terminal less efficiently. we next set up a live cell imaging experiment using synaptophysin-phluorin and evaluated the effects of ns- , ms- and ms- syns on the size of sv pools. restoring wt-syn in syn i ko terminals led to a increase of all sv pools. restoring ms- and ms- syns resulted in a phenotype not significatively different from syn i ko background whereas restoring ns- syn caused a decrease of all sv pools as compared either with wt-syn or syn i ko background. these data suggest an alteration in the subcellular distribution and function of syn in patients carrying ms- e ms- mutation and a more severe effect on synaptic activity in patient carrying ns- mutation. imaging dynamics of dc-sign at the plasma membrane of hiv- -stimulated dendritic cells o understanding how viruses interact with host receptors is essential for developing new antiviral strategies. dendritic cells (dcs) can efficiently capture and take up hiv- through multiple attachment factors, such as the c-type lectin dc-sign. however, the initial interactions between hiv- and this receptor on dcs are not fully understood yet. in this work, we have used single-molecule epi-tirf microscopy in combination with fluorescently labelled hiv- virus like particles (vlps) and dc-sign-specific antibodies to image the dynamic interaction between hiv- and dc-sign nanoclusters at the plasma membrane of dcs. by tracking individual trajectories of dc-sign on the cell surface of living dcs we have found heterogeneity in the modes of motion of dc-sign: some clusters are immobile whereas others move very quickly, with a few ones showing a directed motion on the cell membrane. to investigate how such motion might be correlated to dc-sign function as virus attachment factor, we have developed glass platforms functionalized with hiv- vlps to locally stimulate living dcs. these virus platforms have allowed us to measure dc-sign diffusion rates and motion modes over the cell surface of stimulated dcs and represent a powerful tool for studying the dynamic interactions between hiv- and the dc membrane. a. esposito , t. tiffert , j. mauritz , s. schlachter , j. n. skepper , v. l. lew , c. f. kaminski dept. of chemical engineering and biotechnology, univ. of cambridge, u.k., dept. of physiology, development and neuroscience, univ. of cambridge, u.k. plasmodium falciparum (pf ) causes the most lethal form of malaria in humans. early research exposed two paradoxes: ) during its intraerythrocytic cycle pf permeabilizes the host cell so much that a comparably permeabilized healthy red blood cell (rbc) would lyse prematurely, and ) pf digests far more hemoglobin than needed for its metabolism. a model of the homeostasis of a pf infected rbc suggested a common explanation of both puzzles: excess hemoglobin digestion is required to reduce the colloidosmotic pressure within the host cell thus ensuring its osmotic stability to the end of the pf asexual cycle. we investigated these predictions with direct measurements of [hb] and volumes of parasitized rbcs. reliable volume and morphological data was obtained by confocal microscopy and quantitative surface reconstruction. furthermore, we developed a new fret-based method to measure hemoglobin molecular crowding by exploiting the reduction in fluorescence lifetime of a donor fluorophore loaded in the rbc cytosol. fret imaging techniques are powerful tools for probing the biophysics of living cells. these tools provided a first validation of the colloidosmotic hypothesis and a deeper understanding of the homeostasis of the intraerythrocytic stage of pf. hiv- assembly and release occur at the plasma membrane of infected cells and are driven by the gag polyprotein. using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated assembly of fluorescently labeled hiv- at the plasma membrane of living cells with high time resolution. gag assembled into discrete clusters corresponding to single virions. after their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over - hours. using a photoconvertible fluorescent protein, we determined that assembly was nucleated by membrane bound gag molecules, while both membrane-bound and cytosol derived gag polyproteins contributed to the growing bud. assembly kinetics were rapid and three phases are observed. in phase i, the number of gag molecules at a budding site increases following a saturating exponential with a rate constant of ∼ x − s − . hence, gag assembly is complete in ∼ s. in phase ii, a plateau in fluorescence intensity is observed with no exchange of gag protein. the fluorescence intensity decays in phase iii. this decay, in some cases, corresponds to the release of a virion. the time scale from the onset of assembly to release of extracellular particles was measured to be ∼ , +/- s. fast d chromatin dynamics studied in living yeasts using a novel lab on chip technology h. hajjoul , m. dilhan , i. lassadi , k. bystricky , a. bancaud laas-cnrs, université de toulouse, france, lbme, cnrs umr , toulouse, france we present a novel lab-on-chip technology for d particle tracking yeast abased on v-shaped mirrors, which are used to observe fluorescent specimens from multiple vantage points, providing stereo-images that can be recombined for d reconstruction. our technology is based on v-shaped mirrors, which are fabricated by wet etching of silicon wafers, and used as optical and fluidic components. after rigorous optical optimization of the device in vitro, the lab-on-chip is applied to study chromatin dynamics in vivo using budding yeasts as a model system. yeasts cells are visualized using gfp fused to the associated repressor protein. we confirm earlier observations that telomeric sequences, i.e. located close to chromosome ends, accumulate at the nuclear periphery, whereas genes found midway along chromosome arms are mostly present in the nuclear lumen. the dynamics of these sequences is followed in d with an unpreceded temporal resolution of ms, showing that chromosome dynamics is non linear in the short time regime and switches to a linear regime at larger timescales. notably, this behavior is reminiscent of universal responses observed in polymer solutions, and is related to the confinement and the structure of chromosomes. this technique shows a great potential for studying dynamic processes in small living organisms. retrovirus induced remodeling of the host cell actin architecture m. gladnikoff, i. rousso department of structural biology, weizmann institute of science, rehovot, israel retrovirus budding is a key step in the virus replication cycle. yet, despite substantial progress in the structural and biochemical characterization of retroviral budding, the underlying physical mechanism remains poorly understood, primarily due to technical limitations preventing visualization of bud formation in real time. using atomic force-, fluorescence-and transmission electron microscopy we find that both hiv and moloney murine leukemia virus (mlv) remodel the actin cytoskeleton of their host cells and utilize the forces it generates to drive their assembly and budding. highly dynamic actin-filamentous structures which varied in size over the duration of budding appeared to emanate from the assembled virion. these actin structures assemble simultaneously or immediately after the beginning of budding, and disappear as soon as the nascent virus is released from the cell membrane. analysis of sections of cryo-preserved virus infected cells by tem reveals similar actin filaments structures emerging from every nascent virus. substitution of the nucleocapsid domain implicated in actin binding by a leucine-zipper domain resulted in budding of virus-like particles that was not accompanied by remodeling of the cell's cytoskeleton. notably, budding of viruses carrying the modified nucleocapsid domains was an order of magnitude slower than that of the wild type. the results of this study show that retroviruses utilize the cell cytoskeleton to expedite their assembly and budding. t. gensch institute of structural biology and biophysics (isb- ) cellular biophysics, research centre jülich, germany the determination of ion concentrations in cells -in particular in neurons -is very important for understanding cell function and life. ca + is an ubiquitous messenger in almost all cell types, cl − has several roles, e.g. plays an important role in some neuronal signaling pathways including olfaction, nociception and vision. fluorescence lifetime imaging (flim) is of advantage over intensity based fluorescence microscopy, when comparisons between micro-domains of one cell or between different cells of one cell type are performed. several (organic chromophores and fluorescent protein based) ca + -and cl − -sensors have been tested in culture cells with respect to their applicability in flim studies. the ca + -and cl − concentration in rodent olfactory sensory neurons, dorsal root ganglion neurons and neurons of the retina is investigated by time-resolved flim with two-photon excitation. viscosity is one of the main factors which influence diffusion in condensed media. in a cell viscosity can play a role in several diffusion mediated processes, such as drug delivery and signalling. previously, alterations in viscosity in cells and organs have been linked to malfunction; however, mapping viscosity on a single-cell scale remains a challenge. we have imaged viscosity inside individual cells using novel fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent. , this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to × higher than that of water. we have also shown that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt). we have demonstrated the effect of such viscosity increase on intracellular reactions by directly monitoring the rates of formation and decay of a short lived toxic intermediate, crucial in pdt, called singlet molecular oxygen, in light perturbed cells. characean algae, close relatives of higher plants, represent a convenient model for studying the effect of propagating electrical signals, action potentials (ap) on photosynthesis. illuminated chara corallina cells produce coordinated spatial patterns of chlorophyll fluorescence (chl fl) and extracellular ph. photosynthesis is higher in the cell regions adjacent to acid zones compared to alkaline zones. under physiological conditions, the electrically induced ap differentially and reversibly suppresses photosynthesis in the alkaline and acid regions. in this work we examined the effects of an artificial psi acceptor, methyl viologen (mv), on chl fl with imaging-pam technique. mv is a divalent cation and poorly permeates through biological membranes. the presence in the medium of mv had no effect on fl as well as on p + absorbance signals until the application of a single excitatory stimulus. once an ap was generated in the presence of mv, it induced irreversible inhibition of native (nadpdependent) electron flow and a strong non-photochemical fl quenching all over the cell. this indicates that ap redirects electron flow from the main pathway to the artificial acceptor. we concluded that ap generation opens access for permeation of mv from the medium to the chloroplast stroma across two membrane barriers, plasmalemma and chloroplast inner membrane. we suggested that mv might enter chara cell via plasmalemma ca + -channels activated during ap. evaluation of cell damage after photodynamic and sonodynamic treatment h. kolarova, k. tomankova, s. binder, p. kolar, r. bajgar, m. strnad department of medical biophysics, faculty of medicine and dentistry, palacky university, hnevotinska , olomouc, czech republic photodynamic therapy (pdt) and sonodynamic therapy (sdt) is a new, combined therapy for treating cancer. the basis of the therapy is to administer a small amount of photosensitizer and sonosensitizer, which are selectively taken up by cancer cells, and then expose the body to light and ultrasound to activate these sensitizers. when the sensitizers absorb light of an appropriate wavelength, it may cause their excitation with subsequent energy transfer to oxygen; the oxygen then becomes highly reactive in cancer cells and produces reactive oxygen species (ros). the resulting damage to organelles within malignant cells leads to tumor ablation. sdt uses an agent that is sensitive to ultrasound, allowing deeper penetration and destruction of abnormal cells. changes in human melanoma cells were evaluated using fluorescence microscope and atomic force microscopy.we focused on obtaining pictures of the topography and pictures involving elastic properties of cell surface. the production of ros was investigated with the molecular probe cm-h dcfda, and morphological changes in cells were evaluated using fluorescence microscope. the quantitative ros production changes in relation to phthalocyanine concentration, irradiation doses and ultrasound intensity were measured by a fluororeader. activity correlation imaging: visualizing function and structure of neuronal populations s. junek , t.-w. chen , m. alevra , d. schild department of neurophysiology and cellular biophysics, university of göttingen, germany, dfg research center for molecular physiology of the brain (cmpb), university of göttingen, germany understanding a neuronal network relies on knowledge about both the function and the structure of its neurons. while he simultaneous observation of hundreds of neurons is possible by densely staining brain tissue with functional dyes, the low contrast of these stainings does not allow the identification of neuronal processes from the raw fluorescent images. however, as neurons are known to exhibit complex temporal patterns of activity, fluctuations in signal intensity over time could be exploited to generate contrast in densely stained tissue. we demonstrate that the uniform calcium signals within individual neurons of the olfactory bulb can be exploited to visualize the morphology and projection patterns of these cells in tissue slices. as different neurons exhibit distinct time patterns, it is possible to generate a highcontrast multi-color visualization of the network's active neurons, solely based on the specificity of temporal fluctuations of a single-color calcium dye. it is thus possible to use other spectral channels for additional labelling, for example using cell-type or protein specific markers. the ability to map function and structure of neuronal populations online opens up a number of intriguing applications, such as selecting cells or cell pairs with certain functional or projection profiles for targeted recordings, ablation or stimulation. -live cell imaging - investigation of the dynamics of redox elements in live cells by using fluorescence ratio microscopy g. maulucci , g. pani , v. labate , m. mele , e. panieri , m. papi , g. arcovito , t. galeotti , m. de spirito istituto di fisica, università cattolica s. cuore, roma, italy, istituto di patologia generale, università cattolica s. cuore, roma, italy oxidative stress or signaling events can affect cellular redox environment, which act simultaneously as regulator and indicator of key cellular functions in both physiological and pathological settings. by using a redox-sensitive protein (rxyfp), employed ratiometrically, it is possible to generate high resolution redox maps of cells. the spatial distributions of oxidized and reduced elements have been discriminated in human embryonic kidney cells by the deconvolution of the histograms of redox maps. by transfecting cell with glutaredoxin v (grx-v), a significant shift towards more reduced state with respect to that recovered from non-transfected cells is observed. despite such large differences, a common behaviour in the spatial distribution of reduced and oxidized couples can still be observed: oxidized population shows a pronounced localization on the cell borders, near the plasma membrane, while reduced population appears as a collection of well separated spots homogeneuosly distributed thoroughly the inner part of the cell with a mean dimension of um. furthermore we observe that the role of grx-v consists in causing a shift towards reduced values of the highly reduced region, while leaving unaltered the redox-balance of the intracellular side of the plasma membrane. fluorescence resonant energy transfer (fret) is a popular approach to studying molecular interactions. fluorescence lifetime imaging (flim) provides a robust read-out of fret but has largely been restricted to fixed cells owing to relatively long acquisition times. we present a highspeed wide-field multidimensional fluorescence microscope for flim fret in live cells on second timescales for high content analysis and studying fast dynamics. the system uses a nipkow disc for optical sectioning and a time-gated image intensifier for wide-field flim. a spectrally selected supercontinuum excitation source facilitates versatile realtime flim of live cells transfected with fluorescent proteins. for cell signalling studies we have developed a multiplexed fret approach. ras activation at the plasma membrane is demonstrated using flim to read out tagrfp-raf rbd interaction with mplum-h-ras. simultaneously, a spectral ratiometric read-out is used for a second fret (cfp/yfp cameleon) probe to monitor the downstream calcium flux. to further develop multiplexed fret as a tool for imaging multiple components of cell signalling networks, we are working to include polarisation-resolved imaging for steady-state and time resolved fluorescence anisotropy measurements. monitoring gene expression via novel nucleic acid and delivery methods k. lymperopoulos , c. spassova , a. seefeld , h. s. parekh , d. p. herten bioquant institute,heidelberg university, germany, school of pharmacy, university of queensland, australia application of single-molecule and high-resolution fluorescence methods to monitor gene expression in living cells increase the demand on novel probes and delivery methods.they require fluorophores with high photostability and quantum yield and highly-efficient delivery methods that ensure the minimum interference with cell processes such as metabolism and signal transduction. we used a novel class of dendrimers with varying generations and branching factors and different number of positive charges due to different moieties and functional groups.these different properties were tested for their efficiency to transfect eukaryotic cells with fluorescent oligonucleotides (odns).different parameters (temperature,concentration of dendrimers, ratio of dendrimers and odns) were evaluated and optimised.we utilised these established optimal conditions to deliver a modified concept of smartprobes to mammalian cells targeting mrnas involved in signal pathways.we tested different mrna targets and we optimised the fluorescence signal by varying a range of parameters,namely the fluorescent label and the intrinsic properties of the smartprobe (length of the loop and stem, conformation and number of guanosines).in the near future,we plan to use these probes for monitoring gene expression levels using diffusion imaging microscopy. we present a novel near-field scanning microwave microscope (smm) capable of providing surface impedance measurements of samples with nanometric resolution. the instrument is the integration of a microwave vector network analyzer (vna) and a scanning probe microscope (afm/stm). a key point is that our software, controlling and synchronizing both the instruments, creates simultaneously images of the sample at several frequency points. this can be used to extract several features of the sample depending on the frequency. moreover, close frequencies show the same features, added to random noise. exploiting this redundancy of information, we have achieved remarkable results. we have been working on the optimization of this system for biological applications, to detect functional characteristics of cells generating a variation of their dielectric properties. this instrument offers the possibility of performing local impedance measurements on a single live cell and, if correctly calibrated, it provides also quantitative information (e.g. absolute measurements of membrane permittivity). the system was demonstrated to work on saccharomyces cerevisiae. a better model for a full test of the potentialities of the new technique is given by excitable cells, characterized by a greater variability of dielectric properties. a challenging objective could be directly imaging ion channels. hiv- to efficiently complete a replication cycle has to integrate its genome into the host cellular dna.after hiv- enters target cells,neosynthesized viral dna forms along with other proteins the pre-integration complex (pic).pics are then transported into the nucleus where integration,catalyzed by the viral integrase,takes place.hiv- viral particles engineered to incorporate integrase fused to egfp have proven effective to study pics within nuclei of infected cells.in this study we report the live imaging analysis of nuclear pic dynamics obtained by time-lapse microscopy.intranuclear trajectories of in-egfp-labeled pic were collected in three dimensions and examined by both mean squared displacement (msd) and cage diameter (cd) analysis.in cd the maximum distances measured between two positions occupied by a pic in a time window of minutes were calculated while in our msd analysis -minute long trajectory segments were considered.remarkably,msd revealed the presence of an underlying active transport mechanism.to test the possible role of actin filaments,pic nuclear trafficking was analyzed in cells treated with latrunculin b (actin polymerization inhibitor).preliminary results suggest that the disruption of actin function impairs the active nuclear movement of pics. second harmonic generation microscopy reveals sarcomere contractile dynamics of cardiomyocytes n. prent , c. a. greenhalgh , r. o. cisek , j. aus der au , s. elmore , j. h. van beek , j. squier , v. barzda department of physics and institute for optical sciences, university of toronto, toronto, canada, department of physics, colorado school of mines, golden, usa, department of molecular cell physiology, vrije universiteit, amsterdam, netherlands cardiomyocytes, like other striated muscles, exhibit strong inherent second-order nonlinear optical properties that make them exemplar for live cell dynamic studies with second harmonic generation (shg) microscopy. laser scanning shg microscopy with an incident wavelength around µm, enables fast imaging for extended periods of time with negligible tissue damage. strong shg signal originates from the anisotropic (a-) bands which are comprised of regularly arranged myosin molecules, while actin molecules, the main constituent of the isotropic (i-) bands, produce negligible shg. consequently, the alternating bands along the myofibril are clearly visualized, therefore, enabling the determination of individual sarcomere lengths and the study of sarcomere length dynamics during macro-scale contractions. shg intensity is shown to positively correlate to sarcomere length, which leads to the development of real-time inherent force sensors for in vivo myocytes. rich sarcomere dynamics can be observed during myocyte contraction, which can be used for medical diagnostic purpose of muscular degenerative diseases. imaging cellular communication in insulin secretion ex vivo and in vivo d. w. piston vanderbilt university, nashville, tennessee, u.s.a. the islet of langerhans is the functional unit responsible for glucose-stimulated insulin secretion (gsis), and thus plays a key role in blood glucose homeostasis. the importance of the islet is demonstrated by the proven ability of islet transplants to reverse type i diabetes pathologies in human patients. we are interested in understanding the multicellular mechanisms of islet function, and their role in the regulation of blood glucose under normal and pathological conditions. in many ways, the islet appears to function as a syncytium, which exhibits synchronous behavior of membrane action potentials, ca + oscillations, and pulsatile insulin secretion across all β-cells in the islet. in other ways, the islet works as individual cells, especially in the regulation of gene transcription. using our unique quantitative optical imaging methods and novel microfluidic devices, the dynamics of these molecular mechanisms can be followed quantitatively in living cells within intact islets. these investigations utilize transgenic and tissue-specific knock-out mouse models with demonstrated phenotypes, as well as traditional biochemical and molecular biological approaches. do retinal rod outer segments carry out oxydative phosphorylation? i. panfoli , p. bianchini , d. calzia , s. ravera , a. morelli , a. diaspro biology dept., university of genova, italy, lambs-microscobio res.centre, university of genova, italy, neuroscience and brain technology dept., i.i.t. genova, italy visual transduction in vertebrate retinal rod outer segments (os) is very energy demanding. however, atp supply in os, that are devoid of mitochondria, is still puzzling. by a proteomic analysis we identified in purified bovine os disks, proteins involved in vision, as well as the respiratory chain complexes i to iv and f f o -atp synthase, whose activity was comparable to that of mitochondria and sensitive to specific inhibitors. rhodamine fluorescence quenching experiments showed the presence of a proton potential difference across disks. disks consumed oxygen. confocal laser scanning and transmission electron microscopy showed that cytochrome c oxydase and atp synthase are localized on disks. mitochondrial vital dyes stained os ex vivo, and disks. rhodopsin and mitotracker fluorescence co-localized in os. data, suggestive of an aerobic metabolism in os, point to the existence of "mitochondrial inner membrane-like membranes" ectopically producing atp through oxidative phosphorylation, with respect to mitochondria. new scenarios open on the patho-physiology of many retinal diseases associated to mitochondrial dysfunction. -live cell imaging -abstracts unmixing using lifetime-excitation multidimensional confocal fluorescence microscopy data s. schlachter , s. schwedler , a. esposito , a. d. elder , g. s. kaminski schierle , c. f. kaminski cambridge university, u.k., universität bielefeld, germany fluorescence imaging provides a powerful tool to probe biological systems, enabling the high resolution investigation of the localization, interaction and biochemical modification of biomolecules. multidimensional imaging, in particular, is a growing application of confocal and other forms of fluorescence microscopy. it seeks to measure not only fluorescence intensity in three spatial dimensions, but also other features of fluorescence emission, such as lifetime and fluorescence spectra. although multi-dimensional fluorescence microscopy has been demonstrated before, little attention has so far been paid to the problem of data interpretation and representation when dealing with such large datasets. we present here an instrument capable of recording fluorescence lifetime and excitation data by combining tcspc for lifetime determination and a supercontinuum excitation source for extracting excitation spectra. these technologies permit the acquisition of d and d images with lifetime and excitation spectrum information at every pixel. we demonstrate a method whereby the multidimensional datasets acquired with this instrument are processed to yield biologically relevant parameters, (e.g. unmix fluorophores in a multiply labelled sample). our method relies on a global analysis approach and uses ab plots for displaying multidimensional data. we demonstrate the instrument & processing method on dye and multiply labelled biological samples. advanced neuroimaging with diffractive optical elements p. saggau baylor college of medicine, houston, texas, usa studying neural systems is complicated by the large number and small size of its cellular elements. the traditional way of exploring neuronal function by electrical monitoring with micropipettes is increasingly replaced by molecular imaging. fluorescent molecules allow optical monitoring of neuronal signaling with cellular and often subcellular resolution. in addition to monitoring activity, analyzing the functional properties of individual neurons or neuronal populations requires their controlled activation. optical approaches are well-suited, including molecular photolysis of inert precursors and light activation of engineered proteins that control ionic membrane currents. to fully utilize optical techniques for exploring neural systems requires more than adequate spatial resolution to distinguish neuronal elements and sufficient temporal resolution to induce and/or monitor neuronal signaling. because of the non-linear and non-stationary nature of the studied system it is necessary to access many neuronal sites simultaneously. in modern imaging, wide-field illumination and imageformation are often replaced by patterned excitation and nonimaging photon collection. in many instants, diffractive illumination schemes can substitute for conventional reflective or refractive designs. in particular, the availability of programmable diffractive optical elements has made this an attractive alternative, since they permit highly versatile microscope designs and support a significant increase in the overall spatio-temporal resolution. i will present an advanced imaging approach using diffractive optical elements to analyze structure and function of live neurons in brain slices and intact cortex. efficient evaluation of fret in image cytometry with acceptor photobleaching and ratiometric methods j. roszik, d. lisboa, j. szöllősi, g. vereb department of biophysics and cell biology, university of debrecen, debrecen, hungary fluorescence resonance energy transfer (fret) is a powerful technique that can be applied to study nanoscale intra-and intermolecular events and interactions of molecules in situ in biological systems. a robust, easy to use, self-controlled fret method, independent of donor and acceptor concentration and stochiometry, is acceptor photobleaching fret, which requires only simple image mathematics. another approach with more complicated calculations is the intensitybased ratiometric method, which is not based on destroying the acceptor fluorophores, making it applicable for following molecular interactions in live cells. as the need for using fret in image cytometry for evaluating molecular interactions increases, we have undertaken to develop softwares for these methods involving the calculation and usage of all correction factors needed to obtain reliable energy transfer efficiencies. correction possibilities of the acceptor photobleaching method include unwanted photobleaching of the donor, fluorescent photoproduct of the acceptor after photobleaching, cross-talk of unbleached acceptor into the donor channel and partial photobleaching of the acceptor. in the case of intensity-based ratiometric fret, we can correct for all channel cross-talks and calibrate the fret efficiency calculations. both programs provide registration and semiautomatic processing, and they are freely available. the pendrin (slc a ) gene is responsible, when mutated, for the pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid disfunctions. pendrin is an anion exchanger. the way it works and its role in different tissues, owing to the lack of known isoforms, is matter of wide research and debate. we focused on a still unexplored pendrin function, that is most important in the inner ear: cellular volume control and cl − fluxes regulation. we used hek cells over-expressing wild type pendrin or a mutated isoform together with the eyfp. we challenged cells with hypo-osmolar solutions and followed their volume variations in time. taking advantage of the confocal optical sectioning we independently measured cell volume and fluorescence intensity. in this way, given the dependence of eyfp fluorescence from [cl − ] and ph (measured with snarf ) we could estimate at the same time cl − fluxes, volume and ph variations. the contemporary measurements of the three variables, not yet reported in living cells, allowed to assess the role of pendrin in volume regulation and evidenced its participation to cl − fluxes as compared to the mutated isoform or controls. particle tracking inside the cell largely benefits of the ability to spatially and temporally mark specific structures to follow their "signalling" over a "dark" background as made possible since the advent of the photo-activatable markers. in terms of spatial confinement of the photo-activation process, the use of multiphoton excitation provides several favourable aspects compared to single photon confocal microscopy in photomarking biological structures to be tracked: the confined excitation volumes, of the order of magnitude of subfemtoliter, due to the non-linear requirements provide a unique control of the excitation and consequently photoactivation in the d space. in this context photoactivation experiments can be used to assess quantitative information about the binding kinetics of a macromolecule expressed in different cellular compartments. in this work we extended to photoactivation procedures and models originally developed for the quantitative analysis of frap experiments and we evaluated, for different proteins of medical interest (rac-pagfp), the diffusive behaviour in the cytoplasm and the binding kinetics at the large endosomes. the results are compared with standard photobleaching experiments, in order to evidence the gained sensitivity obtained with photo-activatable proteins. motile plaques involved in stress fiber assembly revealed by high-speed spm for living cells k. tamura, t. mizutani, h. haga, k. kawabata division of biological sciences, graduate school of science, hokkaido university, kita-ku, sapporo, japan stress fibers, which are contractile actin cables aligned in a highly-ordered manner, are important for cell migration, mechanical support of plasma membranes and extracellular matrix organization. although stress fibers are dynamically disassembled and assembled again in cells, it is poorly understood how actin filaments are organized into cables with highly-ordered alignment for stress fiber assembly. for investigation of actin cytoskeletal dynamics during actin cable formation, we performed time-lapse observation of actin cytoskeleton in lamella of living fibroblasts by using scanning probe microscopy (spm). high-speed spm observation revealed that motile plaques defined front-side ends of new actin cables and that preexisting mesh-form actin networks were remodeled into new actin cables. directional order analysis of movement of plaques and pharmacological experiments clarified that plaques were driven by myosin-ii-based retrograde actin flow, indicating that plaques are associated with actin cytoskeleton. immunofluorescence experiments showed that plaques were localized on foci of vinculin, a component of cell-substratum adhesions, suggesting that plaques can bind to extracellular substrata via vinculin. based on these results, we propose a model for actin cable formation that motile plaques initiate remodeling of preexisting actin networks into actin cables aligned in a direction of actin flow by associating with extracellular substrata. abscisic acid (aba) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. recently, aba was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells and by human and murine pancreatic β cells. aba autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin (ptx)-sensitive receptor/g-protein complex, cyclic amp, cd -produced cyclic adp-ribose and intracellular calcium. here, the aba receptor on human granulocytes and on rat insulinoma cells is identified as the lanthionine synthetase c-like protein lancl . co-expression of lancl and cd in the human hela cell line reproduces the aba-signaling pathway. the ptx-sensitive g protein coupled to lancl is identified as g i by transfection of cd + /lancl + hela with a chimeric g protein (gα q/i ). identification of the mammalian aba receptor will enable the screening of synthetic aba antagonists as prospective new anti-inflammatory and anti-diabetic agents. investigation of post-thaw damage of s.cerevisiae yeasts using fluorescent dyes -dab and -dab t. s. dyubko , i. a. buriak , v. d. zinchenko , i. f. kovalenko state scientific institution "institute for single crystals", national acadey of sciences of ukraine, kharkov, ukraine, institute for problems of cryobiology and cryomedicine, national academy of sciences of ukraine, kharkov, ukraine we investigate applicability of the fluorescent probes -dab and -dab available from seta biomedicals (www.setabiomedicals.com) to study the post-thaw damage of saccharomyces cerevisiae yeast cells. the leaving cells stained with these dyes have bright fluorescence in the yellow and red region, respectively. however, the freezing followed by post-thawing causes cell damage, which results in a change of fluorescence intensity. in the native living cells the dyes are preferably localized in the cell membranes and organelles which are highly fluorescent. partially damaged cells have even brighter fluorescence as compared to the living cells. however, their cell ultra-structure is not well-distinguished in the fluorescence mode. ultimately destroyed cells are almost non-fluorescent: the cell membranes are not visible in the fluorescent mode and their organelles are only weakly fluorescent. the number of intensively fluorescing cells with partially destroyed membranes, which were obtained by freezing to - • c, is lower compared to those frozen at - • c. -dab and -dab enable not only to distinguish damaged and undamaged cells, but also allow quantitative estimation of the extent of damage in membranes by cryogenic effects. the thylakoid membrane is a structured network in higher plants which is organised into stacked granal thylakoids that are interconnected by single 'stromal' lamellae. we have studied the mobility of a resident protein of the stromal lamellae, hcf , part of the tat protein translocase. hcf -green fluorescent protein fusion (gfp) was targeted into thylakoids and studied using photobleaching approaches. we show that small regions fail to recover significant levels of hcf -gfp fluorescence within minutes after photobleaching. autofluorescence from the photosystem ii light-harvesting complex (lhcii) in granal stacks likewise fails to recover over this time scale. although the thylakoid membrane is a single continuous entity, these data show that both hcf -gfp and lhcii are constrained within this network. since the hcf homologue, tatb, is highly mobile in the escherichia coli plasma membrane, we believe that the stromal lamellae take the form of distinct domains that are effectively constrained by boundaries within the thylakoid network. cytomechanical modifications induced by drugloaded carriers uptake by breast cancer cells the novel opportunities offered by the nanotechnologies have attracted great interest in the development of novel biomaterials for targeted drug delivery in cancer research. the desired features of pharmaceutical drug delivery for intravenous administration are their small size, biodegradability, high drug content, prolonged circulation in the blood and the ability to target required areas. in this work we have compared efficacy of different type of carriers having complementary properties for pharmaceutical delivery in cancer therapy. drug nano-colloids encapsulated by combination of layer by layer (lbl) techniques and ultrasonication, phytochemical encapsulated artificial oleosomes and drug-loading clay/carbon nanotubes have been used for uptake into breast cancer cells. analysis of viscoelastic response of neoplastic cells induced by cargo-carriers uptake has been carried out by a combination of high resolution optical and scanning force microscopy techniques. furthermore, the effects of drug-reservoir carriers on the cytoskeleton (re)organisation of neoplastic cells were further investigated by confocal microscopy using different fluorescent probes. mapping diffusion by raster image correlation spectroscopy (rics) with analog detection time lapse and flow cytometry data integrated in a common cell cycle proliferation model p. ubezio, v. colombo, m. lupi, f. falcetta istituto di ricerche farmacologiche "mario negri", milano, italy we present a cell cycle simulation tool, connecting the basic proliferation process to the cell population data obtained by time lapse and flow cytometry. the computer program is a general framework within which cell cycle progression can be interactively modeled at the desired level of complexity, including g /s/g m cell cycle phases with variable duration, g phase, cell subpopulations belonging to different generations or differentiation stages, distinct block and cell death parameters for each phase. in this way, we achieved a detailed rendering of cell proliferation of normal or tumor cell populations, additionally including cytostatic and cytotoxic effects of treatments. the program gives as output simulated measures, reproducing those obtained by absolute cell counting, growth inhibition tests, flow cytometric dna histograms and cell cycle percentages, pulse continuous-labeling studies, time-lapse intermitotic times and generation-wise analyses. each technique provides a piece of information related to the underlying proliferation process, catching only some of the phenomena in play, and the measures often cannot be univocally interpreted. we used the cell cycle simulator to fit together time lapse and flow cytometry time courses measures, to achieve a detailed reconstruction of the cell cycle proliferation of an ovarian cancer cell line in vitro after x-ray exposure. -live cell imaging - detection of functional modes in protein dynamics j. s. hub , b. l. de groot deptartment of cell & molecular biology, uppsala university, sweden, computational biomolecular dynamics group, max-planck-institute for biophysical chemistry, göttingen, germany proteins frequently accomplish their biological function by collective atomic motions. yet the identification of a collective motions related to a specific protein function from, e.g. a molecular dynamics trajectory, is often non-trivial. here, we propose a novel technique termed`functional mode analysis' that aims to detect the collective motion that is directly related to a particular protein function. based on an ensemble of structures, together with an arbitrary`functional quantity' that quantifies the functional state of the protein, the method detects the collective motion that is maximally correlated to the functional quantity. the functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. two different correlation measures are applied. first, the pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. the new method is illustrated using various biomolecules, including a polyalanine-helix, t lysozyme, trp-cage, and leucine-binding protein. logic estimation of the optimum source neutron energy for bnct of brain tumors boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron- . a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding conditions on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating different source neutron energies, between . ev and . mev. mcnp b monte carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. the results were discussed. v. l. cruz, j. ramos, j. martinez-salazar instituto de estructura de la materia. csic cannabinoid receptors are an important class of g protein coupled receptors. in particular cb and cb have received considerable interest because they mediate a variety of physiological responses in the central nervous and immune systems. their tertiary structure is still unknown. experimental information suggests the presence of both, an active and an inactive conformation. cb and cb share a common structural framework consisting in a seven transmembrane α-helix bundle connected by three extracellular and three intracellular loops. however, the knowledge of structural differences between both receptors may serve to design new ligands to activate/deactivate selectively only one of those receptors. in the present research, we report a multi-nanosecond molecular dynamics simulation of both receptors in solution, starting from a structure obtained by homology modelling using the x-ray determined bovine rhodopsin protein. we look for differences in the behaviour of cb and cb during the simulation process to shed light about those structural features which can be important for ligand selectivity. in this sense, we observed that cb tend to present a more flexible and opened helix bundle than cb . thus, it is expected than the former receptors would present less steric hindrance for ligand binding. we expect these results will be useful to design more selective ligands. self-assembly and equilibration of bola-lipids membranes studied by molecular dynamics m. bulacu, s. j. marrink groningen university, the netherlands bola-lipids consist of two monopolar, twin-tailed lipids that are held together by chemical linkage between one or both ends of the tails from one lipid and the corresponding ends from the other one. membranes formed by these lipids or by their mixtures with monopolar lipids are known to have additional mechanical stability while retaining membrane fluidity. this is traditionally attributed to the fact that, in the membrane phase, the bola-lipids have predominantly spanning configuration (the two polar heads are positioned at opposite membrane-water interfaces) in detriment to looping configuration (both head groups are located in the same membrane-water interface). we perform molecular dynamics simulations, using the coarse grained martini forcefield. we start with self-assembly simulations of bola-lipids followed by bilayer equilibration. an artificial pore is created in the membrane that significantly increases the flip-flop mobility of the lipids and hasten equilibration. the membrane properties are characterized (area per lipid, thickness, order parameter, pressure profile) with emphasis on the spanning/looping ratio. our study can help designing new artificial membranes, with higher stability under extreme conditions. -multiscale simulation - evidence for proton shuffling in a thioredoxinlike protein during catalysis d. narzi , s. w. siu , c. u. stirnimann , j. p. grimshaw , r. glockshuber , g. capitani , r. a. böckmann theoretical and computational membrane biology, center for bioinformatics, saarland university, germany, institute of molecular biology and biophysics, eth zürich, switzerland, structural and computational unit, embl, heidelberg, germany, paul scherrer institute, villigen psi, switzerland proteins of the thioredoxin (trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. all members of the trx family with thioldisulfide oxidoreductase activity contain the characteristic cys-x-x-cys motif in their active site. here, using poisson-boltzmann-based protonation-state calculations based on -ns molecular dynamics simulations, we investigated the catalytic mechanism of dsbl, the most oxidizing protein known to date. we observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. these results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. a consistent catalytic mechanism for dsbl, probably conferrable to other proteins of the same class, is presented. our results suggest a functional role of hydration entropy of active-site groups . the role of water in zn(ii)-abeta( - ) complexes beta-amyloid (aβ) peptides are the main component of amyloid fibrils detected in the brain of alzheimer patients. fibrils display an abnormal content of cu and zn ions whose binding to aβ-peptides has been recently studied by x-ray absorption spectroscopy [ ] and interpreted in terms of ab initio simulations. in order to perform such simulations it is of the utmost importance to find a compromise between the need of having a realistic description of the actual physical system and the difficulty of dealing with too many atoms and electrons. what is usually done is removing solvent (water molecules), thus studying the system in the so called "gasphase". in this work we investigate the relevance of water in the zn-aβ − coordination mode. relying on a combination of classical [ ] and quantum chemistry [ ] methods we find a significant difference in the zn coordination geometry depending on whether water is present or not. this information is exploited in building a full model system for subsequent car-parrinello simulations where two aβ peptides in water are in interaction in the presence of zn. [ although experiments which can directly probe the mechanical properties of proteins have only been performed recently, it is clear that the complex folds of polypeptide backbones lead to very heterogeneous mechanical behavior. this heterogeneity is likely to play an important role in protein function and interaction and it would be useful to be able to predict what mechanical (and dynamical) properties will result from a given structure. we have been using coarse-grain elastic network models to investigate this question and have found that these models are able to link specific mechanical properties to a number of functional features including enzymatic active sites, folding nuclei and changes in behavior due to point mutations. these models are also adapted to looking at complex formation between proteins and how specific recognition is achieved, while being fast enough to be applied to very large numbers of interactions. refinement of protein model structures using biasing potential replica exchange simulations s. kannan, m. zacharias school of engineering and science, jacobs university bremen, d- bremen, germany comparative protein modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. frequently, the quality of the target-template sequence alignment is non-uniform along the sequence: parts can be modeled with a high confidence, whereas other parts differ strongly from the template. in principle, molecular dynamics (md) simulations can be used to refine protein model structures but it is limited by the currently accessible simulation time scales. we have used a recently developed biasing potential replica exchange (bp-rex) md method (kannan, s. zacharias, m. proteins , , - ) to refine homology modeled protein structure at atomic resolution including explicit solvent. in standard rex-md simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. in a bp-rexmd simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. the method requires much fewer replicas for efficient sampling compared with standard temperature rexmd. bp-rexmd simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional md simulations. m. s. p. sansom, p. j. stansfeld, c. l. wee, k. balali-mood department of biochemistry, university of oxford, u.k. coarse-grained molecular dynamics simulations may be used to probe the interactions of membrane proteins with bilayers and their component lipids on an extended (∼ µs) timescale ( ) . conversion to atomistic resolution allows more detailed protein/lipid interactions to be examined. this multiscale approach will be examined via three examples: (i) interactions of a small ion channel toxin (vstx ) we present a new competitive approach for the treatment of biomolecular flexibility to provide an alternative to the limitations of current methodologies such as molecular dynamics and normal mode analysis. this method, called static mode method, is based on the "induced-fit" concept and is aimed at mapping the intrinsic deformations of a biomolecule subject to any external excitations: direct mono or multi-site contact, electrical etc... the algorithm allows obtaining a set of deformations, each one corresponding to a specific interaction on a specific molecular site, in terms of force constants contained in the energy model. such a process can be used to explore the properties of single molecular intrinsic flexibility, as well as to predict molecular docking or molecule/surface interactions. from a modelling point of view, the interaction problem can be expressed in terms of reactive sites between the interacting entities, the molecular deformations being extracted from the pre-calculated static modes of each separated ones. the first applications of our method have focused on the intrinsic flexibility of biomolecules like nucleic acids and proteins. more recently, this new methodology allowed us to investigate the folding of the region - of the amyloid β-peptide, via the docking of a zinc ion on the reactive sites of the molecule. conformational study on a myelin basic protein fragment: molecular dynamics simulations in membrane e. polverini , g. harauz dipartimento di fisica, università di parma, parma, italy, department of molecular and cellular biology, university of guelph, guelph, ontario, canada myelin basic protein (mbp) is a multifunctional protein of the central nervous system whose principal role is in maintaining the compactness and integrity of the myelin sheath, the multilamellar membrane wrapped around nerve axons. however, mbp also interacts with other proteins such as cytoskeletal and signalling proteins, adapting its structure to the different roles. mbp is a candidate autoantigen in the human demyelinating disease multiple sclerosis. this study investigated at atomic detail the conformation of a highly conserved central fragment of mbp, constisting of two consecutive regions with different relevant functionalities. the first one is associated with the membrane and comprises the primary immunodominant epitope in multiple sclerosis; the second one was predicted to be a ligand for sh -domains of signalling proteins. molecular dynamics simulations were perfomed in the presence of dodecylphosphocholine micelle, starting from a structure extrapolated from experimental data (harauz and libich, curr. protein pept. sci., ). the results confirm the experimetal hypothesis, showing, in the micelle, a stable alpha-helix anchored to the membrane for the first region and, for the proline-rich second one, a poly-proline type ii helix pointing outwards, ready to interact with the signalling proteins. multiresolution modelling of drug and hormone permeability through a lipid bilayer traditional atomic-level (al) modelling of biomembranes is time-consuming, and hence limited in the range of systems and phenomena that can be simulated. to alleviate this problem, we designed a coarse-grain (cg) representation where each lipid molecule, in reality consisting of more than atoms, is modelled with only cg sites. our cg technique proves two orders of magnitude less demanding of computational resources than traditional al methodology. a unique feature of our approach is that the cg potentials are directly compatible with standard al models, thus facilitating the simulation of multiresolution systems, where the "chemically-sensitive" components (e.g., the solutes in membrane permeation studies) are modelled atomistically, while the surrounding environment is coarse-grained. in this contribution, we present a summary of our multiscale methodology, together with its application to the permeation of large molecules -drugs and steroid hormones. the calculated permeabilities are compared to the available experimental measurements and al simulation data. molecularlevel insights regarding the permeation mechanism are obtained and rationalised. -multiscale simulation - the processes of life involve a variety of events that occur on different scales, ranging from a fewÅ/ps of the triggering steps of the biochemical reactions, up to their macroscopic effects in cells and organs. intermediate steps involve the structural rearrangement of the bio-molecules (∼nm/ - ns), their aggregation, folding (∼ nm-µm/ µs-ms), internal cell diffusion and dynamics (µm-mm/ms-hours), evidently requiring a multi-scale modeling approach. here the multi-scale approaches are first briefly illustrated with a particular attention to the issue of matching the different resolutions, which is essential to achieve a coherent descripition. the focus is then fixed on the coarse grained (cg) models, typically spanning the nm-µm and µs-ms scale, and in particular on their minimalist -simplest and computationally cheapest -versions [ , ] . a parameterization strategy that combines accuracy and predictive power within these models is presented here and applications are shown to relevant cases including the proteins involved in the hiv replication, the green fluorescent proteins and examples of macromolecular complexes. [ ] controlled degradation of collagen is an important process in tissue remodeling and wound healing. collagenase cleaves fibrillar collagens about three quarters of the distance from the amino-terminus. even though the determination of the cleavage site and the collagenase structure took place decades ago, the mode of action of collagenase on collagen is not clear. to understand the mechanism of collagenase activity on collagen, the structure, stability and dynamics of collagen, its conformation around the cleavage site and the possibilities of conformational rearrangements between the two domains in collagenase was explored using md simulation. the results of principal component (pca) and normal mode (nm) analysis of the collagen and collagenase suggests that the c-terminal domain of collagenase recognizes the collagen, and then the n-terminal catalytic domain undergoes rearrangement on the substrate (with the help of linker regions). the sda software for carrying out brownian dynamics simulations of protein-protein diffusional association with the help of biochemical constraints is being used to predict the mode of interaction of collagen and collagenase. different conformations obtained from pca and nm analysis are being used for the docking. the predicted structures of the complex will help us to understand how collagenase recognizes and binds collagen specifically. acknowledgment: daad, germany, csir-srf india, and the klaus tschira foundation. membrane aoration by antimicrobial peptides combining atomistic and coarse-grained descriptions d. sengupta, a. j. rzepiela, n. goga, s. j. marrink university of groningen, the netherlands antimicrobial peptides (amps) comprise a large family of peptides that include small cationic peptides, such as magainins, which permeabilize lipid membranes. previous atomistic level simulations of magainin-h peptides show that they act by forming toroidal transmembrane pores. however, due to the atomistic level of description, these simulations were necessarily limited to small system sizes and sub-microsecond time scales. here, we study the long-time relaxation properties of these pores by evolving the systems using a coarse-grain (cg) description. the disordered nature and the topology of the atomistic pores are maintained at the cg level. the peptides sample different orientations but at any given time, only a few peptides insert into the pore. key states observed at the cg level are subsequently back-transformed to the atomistic level using a resolutionexchange protocol. the configurations sampled at the cg level are stable in the atomistic simulation. the effect of helicity on pore stability is investigated at the cg level and we find that partial helicity is required to form stable pores. we also show that the current cg scheme can be used to study spontaneous poration by magainin-h peptides. over-all, our simulations provide a multi-scale view of a fundamental biophysical membrane process involving a complex interplay between peptides and lipids. the varkud satellite (vs) ribozyme is the largest of the nucleolytic ribozymes, and the only one for which there is no crystal structure. we have determined the overall architecture of the complete vs ribozyme using small-angle x-ray scattering in solution. the substrate stem-loop docks into the tertiary fold of the ribozyme, allowing an intimate loop-loop interaction to occur. this brings two key nucleobases a and g into close proximity of the scissile phosphate, and we believe that these nucleobases are involved in general acid-base catalysis of the phosphoryl transfer reactions. this is supported by functional group substitution, and the ph dependence of the reaction rate for the natural and modified rna. although possessing totally different folds, the functional elements of the vs and hairpin ribozymes are topologically and mechanistically very similar if not identical. this has probably arisen by convergent evolution. other nucleolytic ribozymes have diversified to employ hydrated metal ions and even small molecules to participate in genera acid-base catalysis. by contrast, the larger ribozymes seem to have adopted a different, metalloenzyme, catalytic strategy. why these different groups have evolved different catalytic chemistries is an interesting challenge to our understanding of biocatalysis. comparison of dna and sirna binding and nuclease protection by non-viral vectors for gene delivery j. lam, k. witt, a. j. mason, l. kudsiova, m. j. lawrence pharmaceutical science division, king's college london, uk the biggest obstacle for the success of gene therapy is delivery. with the discovery of rnai, the use of sirna to regulate gene expression as potential therapy has attracted much attention recently. in contrast to dna, sirna delivery does not require nuclear entry. with one less barrier to overcome, the delivery of sirna seems to be easier. it is frequently assumed that comparable delivery strategy could be used for both dna and sirna. in fact the two types of nucleic acids are fundamentally different with distinct properties, which impact their delivery strategies. in the present study it was found that most non-viral delivery vectors including polymers, peptides and lipids were generally more efficient in binding with dna than sirna as shown in gel retardation assay. the inferior sirna binding is possibly due to the rigid structure of sirna, resulting in weaker electrostatic interaction with the cationic vectors. surprisingly, it was observed that all the vectors studied offered better nuclease protection for sirna than dna despite poorer sirna binding. either the nuclease protection for sirna is easier to achieve due to its small size, or the gel retardation assay did not truly reflect the binding efficiency as the weaker sirna complexes may dissociate during electrophoresis. not only the delivery strategy, the results between dna and sirna study must also be carefully adapted and interpreted. single molecule studies of spliceosomal rnas u and u z. guo, d. rueda department of chemistry, wayne state university, detroit, michigan, u.s.a. splicing is an essential step in the maturation reaction of mrna, in which intervening introns from exons. the spliceosome is a dynamic assembly of small nuclear rnas and > proteins that catalyzes splicing. u and u are the only snrnas strictly required for splicing. major conformational changes are expected to take place during spliceosomal assembly and catalysis. we have developed a single-molecule fluorescence assay to study the structural dynamics of a protein-free u -u complex from yeast. our previous data have revealed a -step large amplitude conformational change of the u -u complex. the st step is a mg + -induced conformational change where helix iii and the u -isl are in close proximity in low mg + and separated in high mg + . the nd step corresponds to the formation of the highly conserved helix ib. we now examine the role of the highly conserved bases in the folding dynamics of the u -u complex. the data show that u and the acagag loop play an important role in stabilizing the interaction between helix iii and the u -isl. we hypothesize that u flips out of the u -isl and interacts with the acagag loop to bring them in close proximity. our results raise the interesting possibility that this interaction plays an important role in bringing the ' splice site and the branched a into close proximity of u , which may be critical for catalysis. a structure-based approach for targeting hiv- genomic rna dimerization initiation site s. freisz, s. bernacchi, p. dumas, e. ennifar ibmc -cnrs, strasbourg, france all retroviral genomes consist in two homologous single stranded rnas. hiv- dimerization initiation site (dis) is a conserved hairpin in the ' non-coding region of the genomic rna and essential for viral infectivity. the dis initiates genome dimerization by forming a kissing-loop complex, further stabilized into an extended duplex upon interaction with the ncp nucleocapsid protein. x-ray structures of the dis kissing-loop and extended duplex revealed similarities with the bacterial s ribosomal rna a-site, which is the target of aminoglycoside antibiotics. as a result, aminoglycosides also bind the hiv- dis as shown by our x-ray structures of the dis kissingloop bound to aminoglycosides. using fluorescence, uvspectroscopy and microcalorimetry, we further characterized hiv- dis/aminoglycosides interactions. we found that the affinity of aminoglycosides for the dis was higher than for their natural target, the s a site. they strongly stabilize the dis kissing-loop, blocking its conversion into the duplex form. finally, we also solved x-ray structures of the dis duplex bound to aminoglycosides, revealing an important conformational change following drug binding. these structures show that it is possible to target the hiv- dis dimer before and after the ncp -assisted rna maturation with the same molecule. altogether, our studies create the basis for a rationally driven design of novel potential drugs targeting the hiv- genome. the core protein of hepatitis c virus is a multifunctional protein of aa, consisting of a hydrophilic n-terminal domain with three basic domains (bd -bd ) responsible for the interactions with rna and a hydrophobic c-terminal domain. the n-terminal domain exhibits nucleic acid chaperone properties similar to those of the nucleocapsid protein from hiv. here, we characterized the mechanism of the chaperone properties of a peptide e corresponding to the bd and bd clusters of the n-terminal domain. to this end, we monitored the promotion by this peptide of the annealing of dtar, the dna analogue of the transactivation response element to its complementary sequence, ctar dna, taken as models. the annealing involves two second-order kinetic components that are activated by at least three orders of magnitude by peptide e. this activation was correlated with the ability of peptide e to destabilize the lower half of dtar stem. using, ctar and dtar mutants, the two kinetic components were assigned to two pathways which are connected with the fast annealing of the terminal bases of ctar to dtar and slow extended duplex formation, limited kinetically by the nucleation of central segments of ctar and dtar stems. structure and conformational dynamics of a unique dead box helicase m. rudolph , m. linden , r. hartmann , d. klostermeier hoffmann-la roche, basel, switzerland, biozentrum, univ. of basel, switzerland, univ. of marburg, germany dead box helicases couple atp hydrolysis to rna structural rearrangements. t. thermophilus hera consists of a helicase core and a c-terminal extension (cte) with a putative rnase p motif. crystal structures show that the cte is bipartite, forming a highly flexible dimerization motif with a novel fold and an additional rna-binding module. we provide a first glimpse on the orientation of an rna-binding domain outside the helicase core. in a structure-based model for the complete hera dimer, the rna-binding sites of the helicase cores face each other, allowing for inter-subunit communication. the plasticity of the dimerization motif allows for drastic changes in the juxtaposition of the helicase cores within the dimer. in single molecule fret experiments we identified fragments of the s rrna and rnase p rna as substrates for hera. both substrates switch the hera core to the closed conformation and stimulate the intrinsic atpase activity. rna binding is mediated by the cte, but does not require the putative rnase p motif. atp-dependent unwinding of a short helix in s rrna suggests a specific role for hera in ribosome assembly, in analogy to the e. coli and b. subtilis helicases dbpa and yxin. in addition, the specificity of hera for rnase p rna may be required for rnase p rna folding or rnase p assembly. simultaneous action of two hera subunits on the same rna molecule may be important for efficient rna remodeling in vivo. structural basis for the encapsidation process of turnip yellow mosaic virus m. petersen , j. hansen , s. s. wijmenga nucleic acid center, department of physics and chemistry, university of southern denmark, odense, denmark, physical chemistry/biophysical chemistry, radboud university, nijmegen, the netherlands formation of hairpins with internal loops with c c and c a mismatches in the ' untranslated region is a common characteristic among the plant viruses belonging to the tymovirus genus. turnip yellow mosaic virus possesses two such hairpins, hp and hp . hp , and in particular the presence of the c c and c a mismatches in its internal loop, is important for initiation of the encapsidation of the viral genome. the encapsidation occurs under acidic conditions at the neck of invaginations of the chloroplast membrane. we have now determined the d structures of revertants involved in the evolutionary pathway using nmr spectroscopy. these structures reveal how the grooves in the hairpin become increasingly positively charged in successive generations of evolution. the similarity between the ccca mutant and the wild-type hairpin (hp ) is striking and explains why this mutant gives rise to a viable virus. in addition, a characteristic tilt of the backbone is observed upon occurrence of a protonated c c base pair. both the positively charged major groove and the kink in the rna backbone appear to be crucial for interactions with the viral capsid. at neutral ph, the structure of hp resembles the watson-crick base paired mutant which explains why encapsidation only occurs under acidic conditions. a. percot , j. vergne , m.-c. maurel , s. lecomte ladir, umr , thiais, france, lbeam, upmc, paris, france, cbmn, umr , pessac, france the existence of "rna world" as an early step in the history of life increases the interest for the characterization of these biomolecules. the studied hairpin ribozyme is a self-cleaving/ligating motif found in the minus strand of the satellite rna associated with tobacco ringspot virus. surface-enhanced raman spectroscopy (sers) was successfully used to detect sub-picomole quantities of nucleic acids. sers takes advantage of the strongly increased raman signals generated by local field enhancement near metallic (typically au and ag) nanostructures. sers spectra of dna or rna are strongly dominated by stockes modes of adenine. through an interaction of adenyl residues with silver colloid, adenyl raman signal is times increased compared to raman scattering. in controlled conditions, sers signal is proportional to the amount of free residues adsorbed on the metal surface. upon rna cleavage, residues are unpaired and free to interact with metal. in the present study, we proposed to follow the cleavage reaction of hairpin ribozyme (hpr ) using the sers signal of the liberate adenyl residues. as the sers signal is proportional to the adenyl residues, reactivity of hr was monitored by measuring the raman intensity of the fragment liberated during the cleavage of hairpin. the results obtained were compared with the electrophoresis method performed on the same sample and similar results were obtained. -rna world - eur biophys j ( ) the development of arrays for biomolecular recognition for a broad range of applications in biomedical diagnostics is receiving a constantly increasing attention. the design of efficient protein biochips, however, requires the optimization of protein immobilization protocols for improving the device sensitivity. moreover, innovative platforms for in-vitro detection in highly diluted, small volume samples need to be developed, for which standard micro-fabrication techniques are not suitable. therefore, the development of nano-scale platforms for protein and antibody detection is essential. we report here on a novel approach for the fabrication of multiple protein nanosensors using atomic force microscopy based nanografting and dna-directed immobilization (ddi), which takes advantage of the specific watson-crick hybridization of oligonucleotide-modified proteins to surfacebound complementary oligomers. using nanografting, single-stranded dna nano-structures with well defined local environments were fabricated on a flat surface. dna-protein conjugates were then anchored on the engineered binding sites by ddi in a single chemical operation and detected by the corresponding topographic height increase of the relevant patches. immunological assay were used to demonstrate the biochemical functionality of the immobilized proteins, proving the specificity of biomolecular recognition of our nanodevices, in the micro-molar to pico-molar concentration range. dna accessible surface area and indirect protein-dna recognition: study by bioinformatical approach o. p. boryskina , m. y. tkachenko , a. v. shestopalova , m. y. tolstorukov institute of radiophysics and electronics nas of ukraine, harvard-partners center for genetics and genomics, usa revealing the mechanisms of protein-dna recognition is essential for understanding the regulation of many cellular processes. there is growing evidence that recognition through sequence-specific contacts (direct readout) can be enhanced by recognition via dna sequence-dependent deformability (indirect readout). the role of changes in dna accessible surface area (asa) in distorted dna configurations in complexes with proteins is not fully understood yet, even though such changes and related changes in polarity of dna surface are among key factors of indirect readout. to fill this gap we have developed a publicly-available internet database of protein-dna complexes, which integrates the data on dna conformational parameters with information on asa of dna atoms in the minor and major grooves, protna-asa. the database has been used to analyze the effect of changes in dna backbone configuration on asa of dna atoms in major and minor grooves. we observe that sugar puckering and conformation of torsion angle γ affect the accessibility of dna atoms in both grooves to a noticeable extent. we also report sequence specific preferences of the nucleotides for structural domains of γ. these results can shed new light on the mechanisms of indirect protein-dna recognition. a. arakelyan biology dpt., yerevan state university, yerevan, armenia the influence of ligand, irreversibly binding with dna, on the isotherm of adsorption of ligand binding with dna reversibly has been modeled theoretically. the isotherm of adsorption of etbr on dna in presence of cis-ddpt has been considered at low concentrations of cis-ddpt and etbr.. the isotherm of adsorption of etbr on dna has linear form in scatchard coordinates at low degrees of occupation. the comparison with experimental isotherm permits to estimate the parameters of etbr binding with dna. with taking into account the pseudo-ring structures formation with partially molten regions we consider two types of binding regions, linear and ring at low degrees of occupation. the isotherm of adsorption of etbr on dna and also variation of the number of bounded etbr molecules are calculated with taking into account these two types of binding regions. it was shown that at low concentrations cis-ddp, bounding with dna, changes the isotherm of ligands adsorption. the linear in scathcard coordinates isotherm of adsorption transforms into non-linear isotherm. the degree of transformation depends on the fraction of dna in the ring regions, on the ratio between number of etbr binding cites in the ring and linear regions, and also on the binding constants for these regions. it was shown that low concentrations of cis-ddp affect the dependence of dispersion on the concentration of ligands, changing both the magnitude of maximum and its position. tunable nanoconfinement structures for dna manipulation e. angeli, l. repetto, g. firpo, c. boragno, u. valbusa nanomed labs, advanced biotechnology center and physics department, university of genoa, italy nanostructures, such as nanochannels [ ] or nanoslits [ ] , have been successfully used to confine and stretch dna molecules, offering interesting opportunities of investigation on conformational changes induced by confinement, physical and biological properties, etc. the integration of these nanostructures on lab-on-chip systems has shown their great potential for applications such as dna sieving or single molecule manipulation [ ] , [ ] . arrays of nanochannels fabricated, using a focused ion beam (fib) on a silicon master, are replicated using elastomeric materials, such as poly(dimethylsiloxane) (pdms), and soft-lithography techniques. the cross-section of these flexible polymeric nanoconfinement structures can be reversibly and dynamically tuned, in order to vary biomolecules transport characteristics and confinement conditions of trapped dna molecules. moreover, these nanochannels, with tunable cross-section, are used to study and exploit the sieving mechanism of "entropic recoil" [ ] for the separation of long dna chains. [ ] mannion jt, et al., ( ) biophys. j., , : - . [ ] jo k, et al., ( ) pnas, , : - . [ ] fu j, et al., ( ) nat. nanotechnol., : - [ ] huh d, et al., ( nat. mater., : - . the nucleocapsid protein ncp of hiv- is characterized by two conserved zinc fingers and plays crucial roles in the virus, through its binding to nucleic acids. ncp is required for efficient proviral dna synthesis, by promoting the initiation of reverse transcription and the two obligatory strand transfers. using fluorescence techniques as well as fcs and nmr, we investigated the chaperone properties of ncp on the primer binding sites (pbs) and transactivation response elements (tar) sequences involved in the two obligatory strand transfers. we showed that ncp binds mainly to the (-)pbs loop, which results in an extension of the loop and a destabilization of the upper base pair of the stem. these structural changes chaperone a kissing complex with the complementary (+)pbs loop and its further conversion into an extended duplex. in contrast, the ncp -promoted annealing of ctar-tar results from the destabilization of the bottom of ctar stem, which favors the invasion of the tar stem. by developing new fluorescence methodologies to site-specifically characterize these interactions, we further showed that ncp slows down the ps to ns conformational fluctuations of its nucleic acid targets and freezes the local mobility of the bases contacted by the zinc fingers. exploring dna orientation in flow e. l. gilroy, m. r. hicks, a. rodger department of chemistry, university of warwick, uk dna is one of the most important biomolecules. in order to undertake its biological role the dna needs to fold and unfold for which its structure and flexibility are crucially important. we have studied the characteristics of dna in flow to probe its structure. flow aligned linear dichroism (ld) is a technique that uses light polarised parallel and perpendicular to an orientated sample. it can be used to measure how aligned a sample is, and the orientation of any interacting molecules. to create this alignment, the sample is placed between two concentric cylinders where one is spun to create a shear flow. the longer and more rigid the molecule is the better the orientation and the signal. as the sample is in flow there are other factors than need to be considered when using ld. these include viscosity and temperature. there are many methods to measure the viscosity of a sample of dna at varying temperatures. these include viscometer, rheometer and dynamic light scattering. the effects temperature has on viscosity and the sample itself need also to be considered. the findings of all these studies have been reported and show the significance that viscosity and temperature have on dna ld measurements. possible applications of using ld to study dna have also been discussed, showing the importance of the use of ld and in the results shown. a.-m. florescu, m. joyeux laboratoire de spectrométrie physique, université joseph fourier grenoble , france we present a dynamical model for simulating non-specific dna protein interactions, which is based on the "beadspring" model for dna with elastic, bending and debye-hückel electrostatic interactions, and where the protein interacts with the dna chain through electrostatic and excluded-volume forces. we study the properties of this model using a brownian dynamics algorithm that takes hydrodynamic interactions into account and obtain results that partially agree with experiments and predictions of kinetic models. for example, we show that the protein samples dna by a combination of three-dimensional diffusion in the solvent and one-dimensional sliding along the dna chain. this model evidences the presence, in a certain range of values of the effective protein charge, of facilitated diffusion, i.e. a combination of the two types of diffusion that leads to faster than -dimensional diffusion sampling of dna. moreover, the analysis of single sliding events shows that the number of base pairs visited during sliding is comparable to those deduced from single molecule experiments. in contrast to kinetic models, which predict an increase of the number of different base pairs visited by the protein proportional to the square root of time, our model however suggests that this number increases linearly with time until it reaches a value that is close to the total number of dna base pairs in the cell (published in j. chem. phys. , ( )). use of md simulation to identify the critical radiation-induced lesions of a dna binding protein n. chalabi, s. goffinont, n. garnier, d. genest, orléans cedex , france a key step in the regulation of gene expression, dna structuring and dna repair is the binding of some proteins to specific dna sequences. we have shown previously that ionizing radiation destabilizes such dna-protein complexes mainly through damage to the protein. for the typical lactose operator -repressor system we have shown by fluorescence measurement and mass spectrometry that upon irradiation, all the four tyrosine residues of the dna binding domain (called headpiece) are oxidized into , dihydroxyphenylalanine (dopa). a circular dichroism study revealed a global conformational change and the destabilization of the headpiece. to decide which lesion is critical for the induction of these effects, a molecular dynamics simulation study was undertaken in parallel with a site-directed mutagenesis one. each tyrosine residue of the headpiece was replaced by another amino-acid that mimics the damaged tyrosine. the most common amino-acid used in site-directed mutagenesis being alanine, we have replaced one tyrosine ( , , or ) in the nmr-based structure of the headpiece from pdb databank ( lqc) by an alanine. the conformational stability of each tyr→ala mutant has been studied by molecular dynamics simulation (md) and compared to that of the native sequence and of the different tyr→dopa mutants. the mammalian high mobility group protein at-hook (hmga ) is a nuclear protein associated with mensenchymal cell development and differentiation. disruption of its normal expression pattern is directly linked to oncogenesis and obesity. our laboratory has utilized a variety of biochemical and biophysical methods to investigate the molecular mechanisms of hmga recognizing at-rich dna. using a pcr-based selex procedure, we discovered that hmga is a sequence-specific dna-binding protein and recognizes the following two sequences: '-atattcgcgawwatt- ' and 'atattgcgcawwatt- ', where w is a or t. using a double-label emsa assay, we found that hmga binds to at-rich dna as a monomer. using isothermal-titrationcalorimetry, we demonstrated that hmga binds to at-rich dna with very high binding affinity whereby the binding of hmga to a-tracts is entropy-driven and to alternate at sequences is enthalpy-driven. this is a typical example of enthalpy-entropy compensation in which the hydration difference between hmga -dna complexes is a main reason for the compensation. interestingly, the binding of hmga to different at-rich sequences is accompanied with a large negative heat capacity change indicating an important role of solvent displacement and charge-charge interaction in the linked folding/binding processes. partition of gibb´s free energy of drug-dna complexation we report a computation methodology, which leads to the ability to partition the gibb's free energy for the complexation reaction of aromatic drug molecules with dna. using this approach it is now possible to calculate the absolute values of the energy contributions of various physical factors to the dna binding process, whose summation gives a value that is reasonably close to the experimentallymeasured gibb's free energy of binding. application of the methodology to binding of various aromatic drugs with dna provides an answer to the question 'what forces are the main contributors to the stabilization of aromatic ligand-dna complexes' ? we report the effects of khz ultrasound irradiation of double-stranded dna solutions under conditions of transient cavitation. a new method was developed for studying these effects which is based on combination of two procedures: ultrasound irradiation of the solutions of double-stranded dna fragments and subsequent analysis of the high resolution denaturing gel electrophoresis data. statistical treatment of the data on the mobility of '-end-labelled restriction fragments with known sequence allowed us to discover the phenomenon of sequence specific ultrasonic cleavage of dna. our analysis results in the following conclusions: ) all steps with '-ward cytosine [ '-d(cpn)- '] have significantly higher cleavage rates than others; the intensity of cleavage diminishes in the order cpg > cpa ≈ cpt > cpc; ) the cleavage rates of all steps depend on the type of flanking base pairs; ) the cleavage rates of the complementary base pair steps are not identical. thus, subtle sequence specific conformational and physical-chemical variations modulate the reaction of sugar-phosphate single bonds on the ultrasound exposure. a theory of the mechanisms on the simultaneous binding of two aromatic drugs to dna it has long been recognized that certain combinations of dna-binding aromatic drugs, x +y, lead to synergistic biological effects. considering x as a basic ligand and y as an added ligand, the change of the integral biological response of x in the presence of y has been interpreted in terms of two mechanisms: the interceptor and protector action of y on x. this mechanisms have been characterized by two criteria, r d and a d , reflecting the removal of x from dna by y (biophys. chem., , vol. , pages - ) . in this work we develop the theory of the interceptor-protector action of a mixture of two biologically-active dna-binding aromatic drugs. the theory is based on solution of a general system of mass balance equations in the three-component system x -y -dna with respect to the two factors, r d and a d . the outcome is a set of expressions enabling estimation of the change in biological response of x on addition of y as a function of equilibrium parameters under different restrictions. the results are in good agreement with known in vitro data on caffeine+antibiotic action in leukemia cell lines. the influence of mn + ions on the structure of natural calf thymus dna was studied by raman spectroscopy. measurements were done at room temperature and ph . ± . , in the presence of the physiological concentration of mm na + ions, and in the presence of mn + concentrations that varied between and mm. no condensation of dna was observed. dna backbone conformational changes were not detected in the whole concentration range of mn + ions as judging from the raman spectra. no evidence for dna melting was identified. binding of manganese(ii) ions to the charged phosphate groups of dna, stabilizing the double helical structure, is indicated in the spectra. as judged from the marker band of dc near cm − , altered nucleoside conformations in dc residues are supposed to occur, in the mn + concentration range of - mm. binding of divalent ions to n of guanine and, possibly, in a lesser extent to adenine was observed as judging from the raman marker bands at , , and cm − . toward rapid dna sequencing -ab initio study of nucleotide sandwiched between au( ) plates c. morari, d. bogdan, i. turcu national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania recently a new technique for dna sequencing based on measurement of transversal conductive properties of a single strained dna molecule has been proposed. such a method would allow single-base resolution by measuring the electrical current perpendicular to the dna backbone. until now, it is still not clear if the electrical signals obtained for the four nucleotide can be clearly distinguished by a hypothetical experimental setup. several factors -like the influence of the pentose group or the presence of water -may influence quite strongly the electrical signatures of the four bases. therefore, in order to obtain a working device, the understanding and detailed description of the conduction mechanisms through dna bases connected to a metallic electrode is essential. the goal of theoretical studies in this field is to describe the electric signatures of dna bases from a theoretical point of view. our study is focused on the detailed description of the electronic structure of dna's base pairs "squeezed" between two au plates. while such a geometrical model closely mimic the sequencing devices proposed in the literature, it allows us to compute meaningful physical properties such as density of states, charge transfer and orbital localizationby using "ab initio" methods. the results allow us to give qualitative prediction over the potential use of such a device in the dna's sequencing technology. multinuclear platinum complexes represent a new class of anticancer agents, distinct in dna binding and antitumor activity from their mononuclear counterparts. bbr as a first representative of this class has undergone phase ii clinical trials for treatment of ovarian and lung cancers. the structure of this trinuclear pt drug consists of two trans-ptcl(nh ) units bridged by a trans- the main lesions formed by multinuclear pt complexes in dna are long-range intra-and interstrand cross-links (cls) bridging two guanines separated by up to four base pairs. since interstrand cls can prevent dna strand separation interfering with critical cellular events they represent a serious obstacle in cell survival. in order to contribute to understanding the biological effects of dna interstrand cls of bbr , we analyzed the effect of the single, site-specific , -interstrand cl formed by this metallodrug between two guanine bases on opposite strands in the '- ' and '- ' direction on the thermal stability and energetics of short dna duplexes. the results demonstrate that , -interstrand cls of bbr in both '- ' and '- ' directions exist as two distinct conformers that are not interconvertible and affect thermodynamic stability of the dna differently. side-by-side and end-to-end attraction of doublestranded dna c. maffeo , b. luan , a. aksimentiev university of illinois at urbana-champaign, urbana, usa, ibm watson research center, yorktown heights, usa genomic dna is densely packed inside cell nuclei and viral capsids. such close packing suggests that electrostatic repulsion between negatively charged dna in the condensed states is balanced by counterion-induced attraction. several theoretical models have been proposed to explain dna attraction, however, specific microscopic mechanisms are not known. here, we report all-atom molecular dynamics simulations of the effective force between double-stranded dna in side-by-side and end-to-end orientations. in the side-by-side orientation, the dna molecules were found to form a bond state in the presence of magnesium ions. in the bond state, the dna molecules contact each other via their negatively charged phosphate groups, bridged by magnesium ions. the maximum attractive force in the side-by-side orientation is about pn per dna turn. in the end-to-end orientation, a strong ( > pn) attractive force was observed at short (< . nm) end-to-end distances regardless of the electrolyte concentration. the presence of a phosphate group at the 'ends of the fragments was found to direct dna end-to-end self-assembly and produce bound states resembling a continuous dna molecule. the computed potentials of the mean force suggest that the end-to-end attraction, rather than being mediated by counterions, is likely caused by hydrophobic and van der waals interactions between terminal nucleobases of the fragments. doxorubicin (dox) is an anticancer antibiotics with a four-membered ring system containing an anthraquinone chromophore, and an aminoglycoside. it has good anticancer activity against a wide spectrum of tumors and is one of the most extensively used antitumor chemotherapeutic compounds currently in clinical use. interestingly, conversion of dox to pyrrolinodoxorubicin analog (p-dox) exhibits - times higher toxic effects in human breast cancer cell line (nagy, a. et al., pnas, ( ) . molecular mechanisms responsible for this enormously enhanced cytotoxicity have not been entirely clarified. there is good evidence that a key component of the mechanism of action of dox is its intercalation into dna and the formation of dox-dna adducts. therefore, we have examined in detail, using the methods of molecular biophysics, dna interactions of p-dox in cell-free media and compared these results with the same studies focused on the parental dox. we find distinct differences between dna interactions of dox and p-dox and suggest that these differences are responsible at least in part for different biological effects of these two anticancer drugs. design of a microfluidic devices for the detection of oligonucléotides by sers designing fast and efficient analytical tools allowing the detection of free dna or rna at very low concentration within small volumes, without specific molecular labeling, remains a major issue of significance to perform diagnostic or to detect pathogen agents. our strategy is to use surface-enhanced raman scattering (sers) to probe selectively the spectral signature of each base in polynucleotides. sers takes advantage of the strongly increased raman signals of species when adsorbed on adapted silver colloids. we already demonstrated that adenyl raman signal of pa in presence of silver colloids is times enhanced compared to bulk signal. we plan to use nanoliter droplets of uniformed size that form spontaneously in microchannels when two immiscible fluid streams merge. these tiny droplets are almost ideal reactors as they create homogeneous control condition. we will design an optimized channels network platform that result in droplets production hosting both nucleotides and silver colloids: internal fluids recirculation provide fast and efficient mixing, favoring base adsorption on silver nanoparticles. sers will be used to determine the chemical composition of the droplets. nicks represent the most common damage in dna which occurs naturally in living cells. structural properties of nicked dna fragments have been an object of numerous studies due to its special role in reparation processes. here we report experimental results covering ultrasound irradiation of nicked dna solutions. several single-stranded nicks were produced into one strand of dsdna fragments by the nicking enzyme bst i. we have quantitatively estimated the ultrasonic cleavage rates in nicked dna fragments with known sequences using the polyacrylamide gel electrophoresis. computer analysis of the cleavage pattern in the '-end labeled and primarily intact strand reveal cleavage enhancement in the regions of about b. p. up and down the nicks which were initially produced into complementary strand. the intensity of cleavage near the nicks is (in average) about times higher than cleavage in the same sites of the intact dsdna fragments. at the same time, the cleavage rates in positions beyond the regions of the nick markedly grow weak even comparing to the sequence-specific cleavage of intact double-stranded dna fragments. thus, the presence of the nick serves as an expressive structural indignation, which exceeds modulation of the structure caused by the base-pair sequence and is capable of absorbing mechanical stresses applied to the nearby sites of the molecule. comparing the native and an irradiated lactose repressor-operateur complex by md simulation g. naudin, n. garnier, d. genest, rue charles sadron, orléans cedex , france the function of the e. coli lactose operon requires the binding of a protein, the tetrameric repressor, to a specific dna sequence, the operator. the formation of this complex involves the interaction of at least one protein dimer with the operator sequence. this occurs via the dna-binding domains (called headpieces) of the two constitutive monomers. we have previously shown that upon irradiation with gamma rays the complex is destabilised mainly because the repressor losses its dna binding ability. radiation-induced lesions were identified that may be responsible for this deleterious effect: all tyrosine residues of the headpieces are oxidized into , -dihydroxyphenylalanine (dopa). in order to unravel the mechanisms leading to the observed destabilization of the operator-repressor complex, we compare by md simulations two complexes: -the native complex formed by a dimer of two headpieces and a fragment of dna with the operator sequence and -the damaged complex in which all tyrosines are replaced by dopa. analysis of these trajectories shows a loss of stability and binding energy as well as changes in the structure of the damaged complex with respect to the native one. by comparing precisely the evolution of the two complexes we can explain how the oxidation of the tyrosine residues of the headpieces into dopa may trigger the destabilization of the complex. local conformation of confined dna studied using emission polarization anisotropy in nanochannels with dimensions smaller than the dna radius of gyration, dna will extend along the channel. we investigate long dna confined in nanochannels with dimensions down to * nm, using fluorescence microscopy. studies of the statics and dynamics of the dna extension or position in such confinements as a function of e.g. dna contour length, degree and shape of confinement as well as ionic strength has yielded new insight into the physical properties of dna with relevance for applications in genomics and fundamental understanding of dna packaging in e.g. viruses. our work extends the field by not only studying the location of the emitting dyes along a confined dna molecule but also monitoring the polarization of the emission. we use intercalating dyes whose emission is polarized perpendicular to the dna extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched dna, information on the local spatial distribution of the dna backbone can be obtained. we will discuss results in shallow ( nm) and deep ( nm) channels and describe how the technique can be used to investigate non uniform stretching of a single dna molecule. raman spectroscopy of dna modified by new antitumor nonclassical platinum complexes o. vrana, v. kohoutkova, v. brabec institute of biophysics as cr, královopolská , cz- brno, czech republic platinum anticancer agents (cisplatin, carboplatin, oxaliplatin) are in widespread clinical use especially against testicular, ovarian and head and neck carcinomas. there is a large body of experimental evidence that dna is the critical target for the cytostatic activity of cisplatin. platinum complexes form several types of adducts, which occur in dna with a different frequency and differently distort the conformation of dna. clinically ineffective trans isomer of cisplatin (transplatin) also covalently binds to dna bases. the trans geometry in dichloridoplatinum(ii) complexes was activated by various ways. the replacement of at least one amine ligand by planar amine ligand represents example of such activation. raman spectroscopy is powerful technique for examining both structural and thermodynamic properties of nucleic acids in solution. interactions of cis-and trans-pt(ii) complexes having nonplanar heterocyclic amine ligand such as piperidine, piperazine and -picoline with dna have been investigated by laser raman spectroscopy. raman difference spectra reveal that the pt(ii) complexes induce great structural changes in b-dna and indicate disordering of b-dna backbone, reduction in base stacking and base pairing and specific metal interaction with acceptor sites on purine residues. acknowledgement: this work was supported by grant as cr, iqs . a. v. vargiu , a. magistrato , p. carloni , p. ruggerone cnr-infm-slacs and physics dept., university of cagliari, cagliari, italy, cnr-infm-democritos and sissa/isas, trieste, italy, iit and sissa/isas, trieste, italy the minor groove of dna is the target of several anticancer agents, which interfere with replication and translocation processes, leading to cell death. the molecular recognition event is a key step to achieve detailed knowledge of the interactions behind selectivity and affinity of a ligand towards a particular nucleic acids sequence. recognition is a multiroute process which can involve many steps before the formation of the most stable adduct. in particular, many studies have pointed out the importance of events like sliding along the groove and dissociation (which is a relevant step in the translocation among different sequences) for the affinity and the specificity of minor groove binders. despite this, no studies on the dynamics of this event were reported in the literature. in this contribution i present our recent work on the subject. umbrella sampling and metadynamics were used in the framework of classical md to characterize mechanisms andfree energy profiles of molecular recognition routes by the antitumoral agents anthramycin, duocarmycin and distamycin. our results are in very good agreement with the available experimental data, and provide insights on the influence of factors like size, charge and flexibility on the molecular recognition process. amplification of oligonucleotide sequence recognition using bioresponsive hydrogels s. tierney, b. t. stokke biophysics and medical technology, dept. of physics, the norwegian university of science and technology, ntnu, no- trondheim, norway we describe development and characterization of oligonucleotide functionalized hydrogels for amplifying the molecular recognition signal occurring on hybridization between dioligonucleotides. the µm radius hemispherical hydrogels were integrated on a high resolution interferometric fiberoptic readout platform supporting determination changes in the optical length of the hydrogel with nanometer resolution. the hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network as network junctions in addition to the covalent crosslinks or oligonucleotides grafted to the network chains. the probe oligonucleotide destabilizing the junction point by displacement hybridization yielded an optical signal about five times as large as for binding within the hydrogel design with a comblike grafted oligonucleotide. the optical signal was found to be dependent on the concentration of the probe, the sequence and matching length between the probe and sensing oligonucleotide. concentration sensitivity applied as specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. the current design support detection in excess of x sequences. amplification of the molecular recognition employing the developed oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequence designed as aptamers for determination of other types of molecules are discussed. tracing t-cells by paramagentic nanoparticles in the brain of the rat model of als d. bataveljic , g. vanhoute , g. bacic , p. andjus inst. for physiology and biochemistry, univ. of belgrade, serbia, bio imaging lab, univ. of antwerpen, belgium, school of physical chemistry, univ. of belgrade, serbia amyotrophic lateral sclerosis (als) is a devastating neurological disorder affecting upper and lower motoneurons. since immune disbalance is known to be an important manifestation of the disease we were particularly interested in following the labeled immune cells in the familial als rat model, hsod- g a . a t -or t -weighted mri protocol was used with a mini surface coil placed over the skull of the anesthetized animal in a . t wide bore magnet. in order to compare this approach to standard high field small animal imaging a . t mri system was also used. there was a congruence of images of lesions in the brainstem at the two field strengths. it was confirmed with gd-dtpa contrast that the blood brain barrier (bbb) is compromised at the interbrain level. in order to study immune cell infiltration rats were i.v. injected with magnetically labeled antibodies against helper cd + or cytolytic cd + killer t cells. by combined t , t and t * weighted imaging cd + lymphocyte infiltration was observed in the brainstem-midbrain region while the cd + cells were more confined to the brainstem region. comparison of mri of labelled cd + vs. cd + lymphocytes reveals the relevant cellular inflammatory mechanism in als. the appearance of the mri signal from the latter t cell type points to the mechanism of bbb disruption as suggested from a recent study on the role of cd + t cells in a model of multiple sclerosis. emodin uptake study in u- mg cells using optically trapped surface-enhanced raman scattering probes s. balint , s. rao , p. miskovsky , d. petrov icfo -the institute of photonic sciences, barcelona, spain, department of biophysics, university of p.j.Šafárik, košice, slovakia emodin ( , , -trihydroxy- -methylantraquinone) is a photosensitizing pigment present in plants of herbal laxatives. emodin inhibits nuclear transcription factor-κb activation and induces free radical production in human mononuclear cells resulting in its antiviral and anti-cancer activity. the uptake and distribution of emodin inside the u- mg cell line is studied by combining optical tweezers and surfaceenhanced raman spectroscopy (sers). sers greatly enhances the spectrum of an otherwise weakly scattering material which is achieved by attaching nano-sized silver colloids to micron-sized dielectric beads. the distribution of emodin in the cell is studied by simultaneously trapping and exciting the sers bead and scanning it across the membrane while recording the emitted light. secondly, the beads are statically placed inside the cell and excited at certain intervals in order to track the migration of emodin through the membrane. the results give new insight in to the metabolic pathways of emodin and demonstrate a new imaging and detection technique that is fast and less invasive than current standards. acknowledgement: miin fis - (spain), fundació cellex barcelona, nadacia spp (slovakia), apvv- - (slovakia) amphotericin b (amb) is a polyene antibiotic that has been widely used for treatment of systemic fungal infections. the main mechanism of biological mode of action of amb is considered to be associated with formation of ionic membrane pores or channels in the lipid membranes. the aim of this work was to study the influence of the k + and na + ions on the aggregation process of amb in aqueous medium. the analysis of electronic absorption and fluorescence spectra of amb shows that the increasing k + concentration have influence on the level of aggregation of the drug much more than the same amount of na + ions. this effect is especially noticed at neutral ph values. the rls technique was used to study aggregation of amb in solution, in the environment of the k + and na + ions. the application of this technique makes it possible to study the electronically coupled chromophores, especially molecular aggregates. the results of the atr-ftir and raman spectroscopic studies also support this conclusion. these results provide a better understanding of the interaction between k + and na + ions and antibiotic which has not been previously considered to be significant for biological action of amb. g-quadruplexes: combining theory with experimental spectroscopic methods guanine-rich oligonucleotides can form unique tetrameric structures with four coplanar guanine bases, known as gquadruplex motifs. the g-quadruplexes have been found in vivo in the terminal parts of telomeres and other genomic regions. ligands, specifically binding to the g-quadruplex regions inhibit telomerase activity and thus can play an important role in the cancer therapy. most recently, the potential use of these structures has been tested in biosensors and nanotechnology industry. in the present work we use the infrared spectroscopy, including the relatively novel technique of the vibrational circular dichroism (vcd), in a combination with molecular dynamics and quantum chemistry computational methods to investigate the structure and spectroscopic response of the quadruplexes formed by the d(g) and selfassociated dgmp. we obtained a good agreement between the computed and experimental spectra, confirming that the proposed geometrical models are realistic. the vcd technique appears especially convenient for the studies as it can detect the liquid-crystalline phases of the g-quadruplexes by an anomalous enhancement of the signal. j. bogdanovic pristov, a. mitrovic, k. radotic, i. spasojevic institute for multidisciplinary research, belgrade, serbia fructose, due to its high antioxidative capacity, represents a significant component of non-enzymatic defense of some plants against cold-provoked oxidative stress. in the present study, we have investigated role of fructose in seasonal adaptation of picea omorika (pančić) purkinye to cold. this endemic coniferous species is exposed to subfreezing temperatures that range from - to - • c during the autumn/winter and high temperatures exceeding • c during the summer. characteristic epr signal of free or weakly bound mn + was used as an indicator of oxidative status of needles, since coldrelated oxidative damage leads to mn + release from photosystem ii. it was observed that prooxidative conditions developed in the autumn, at the beginning of cold season, which corresponded to significant increase of fructose level. total sod, as well as mnsod activity also rose significantly higher in the autumn. observed activation of antioxidative system (non-enzymatic and enzymatic) led to adaptation of needles to cold, as oxidative status during winter was decreased and similar to the status of needles in cold-free seasons. calyx of held: sted nanoscopy of a glutamatergic terminal p. bingen , t. m. staudt , c. kempf , h. horstmann , j. engelhardt , t. kuner , s. w. hell german cancer research center / bioquant, heidelberg, germany, max planck institute for biophysical chemistry, göttingen, germany, institute for anatomy and cell biology, university of heidelberg, heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of < nm inside µm thick tissue sections. three-dimensional shim and pem to study collagen arrangement and crimping pattern p. bianchini , m. franchi , l. leonardi , a. diaspro lambs-ifom microscobio research centre and department of physics, university of genova, genova, italy., italian institute of technology (iit), genova, italy, department of human anatomical sciences and physiopathology of the locomotorapparatus, university of bologna, italy ligaments have been generally described as multifascicular structures with collagen fibre bundles cross-connecting to each other or running straight and parallel with crimps. a different collagen array and crimping pattern in different ligaments may reflect a different mechanical role. aim of this study was to relate the d collagen arrangement and crimping pattern by backward and forward second harmonic imaging microscopy (shim) and -photon excitation microscopy ( pem). shim on a laser-scanning system is a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of tissue architecture. although it is a coherent process the multiple scattering through the tissue give us the capability to acquire signal in both backward and forward direction [ ] . shim and pem were combined in a dual-mode nonlinear microscopy to find out collagen fibre arrangement and crimping pattern. both polarization dependence and differences between forward and backward signals allowed to yield information on local structure [ ] . currently used tcspc flim systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. the systems are, however, restricted to count rates on the order of a few mhz. in the majority of applications, such as fret or autofluorescence, the photostability of the samples limits the count rate to much lower values. the limitation of the count rate is therefore no problem. however, if flim is used for ion concentration measurements or imaging of chlorophyll in plants the available count rates can exceed the counting capability of a single tcspc channel. we therefore developed a flim system that uses eight fully parallel tcspc channels. by using a polychromator for spectral dispersion and a multichannel pmt for detection we obtain multi-spectral flim data at a rate of several frames per second. we will demonstrate the application of the system to dyamic changes of the fluorescence lifetime of chlorophyll in living plants. -imaging and spectroscopy - scattering effects on non linear imaging of thick biological samples f. cella , z. lavagnino , a. diaspro lambs-ifom, microscobio research center, university of genoa, italy, iit, italian institute of technology, genoa, italy non linear optical scanning microscopy became a useful tool for living tissue imaging. biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. while performing imaging of biological scattering tissues in two photon excitation ( pe) regime, the localization of the maximum pe intensity was found to shift closer to the surface and the imaging depth limit appears strongly limited by near surface fluorescence . in this work we computed illumination and photobleaching intensity distribution in order to characterize the effects induced by scattering. simulations of pe illumination and photobleaching intensity profiles have been performed for different scattering coefficients and at different focus depth. furthermore imaging of fluorescent immobile sample (polyelectrolyte gel) allowed to perform an experimental test on thick turbid media. results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface. [ ]ying et al., appl. opt. , ( ) . [ ] theer p. and denk w, j. opt. soc. am. a. ( ) [ ]mazza d. et al., appl. opt. ( ) . biospectroscopic probes for real time measurement of hydrogen-deuterium exchange p. carmona , m. molina instituto de estructura de la materia (csic), madrid, spain, escuela universitaria deÓptica, madrid, spain isotopic exchange has long been used for the analysis of biomolecular structure and dynamics. hydrogen-deuterium exchange rates depend on ph, temperature and biomolecular environment. this is due to hydrogen bonding, low solvent accessibility, and steric blocking. time resolved measurement of hydrogen-deuterium exchange for subsequent d correlation spectroscopic analysis can, then, be very useful to obtain structural information from the said viewpoint. we have developed a microdialysis quartz cell for use in conjunction with raman spectroscopy to investigate hydrogen-isotope exchange reactions of biomolecules. the system requires only µl volumes of the initial substrate and perturbing effluent solutions. we have obtained a d o efflux rate of k d = . ± . min − with the greatest mwco ( kda) used here, which involves that an exchange rate of . min − is the limiting rate that could be resolved with the said cell system. analogous results have been obtained using an infrared biospectroscopic microdialysis probe. the use of the method described here has the advantage of avoiding sample dilution (and subsequent signal loss) involved in the known stop flow methods. acknowledgements: the authors gratefully acknowledge financial support from the spanish ministerio de ciencia e innovación (project ctq - /bqu). polarized transient absorption to resolve electron transfer between tryptophans in dna photolyase photoactivation of dna photolyase comprises electron transfer through the chain fadh • -w -w -w . photo-excited fadh • abstracts an electron from the tryptophan residue w in ∼ ps (monitored by transient absorption spectroscopy). the subsequent electron transfer steps (from w to w •+ and from w to w •+ ) are difficult to resolve experimentally, because electron transfer between chemically identical species does not give rise to net absorption changes. to overcome this difficulty, we make use of the fact that polarized excitation (pulse laser) induces a preferential axis (that of the excited flavin transition) in the system (photoselection), and that w and w form different angles with that axis (known from the crystal structure). thus, polarized detection should allow distinguishing between them. using polarized "classical" transient absorption on a nanosecond time scale and the pump-probe technique on a picosecond scale, we demonstrate the feasibility of the method and provide evidence that electron transfer from w to w •+ is faster than the ps time constant of the initial electron transfer from w to excited fadh • . synchrotron based fourier transform infrared (sr-ftir) microspectroscopy was applied to investigate apoptotic death of u- mg cells induced by the photosensitizer hypericin (hyp), in using different transport systems (hyp alone vs. hyp/ldl complexes) and incubation protocols. the differences between ir spectra of non-treated and hyp treated cells are mainly manifested in the positions of amide i and amide ii vibrational bands of proteins. these vibrational shifts are attributed to the protein structure changes from dominantly alfa-helix, in the non-treated cells, to beta-sheets and random coil structures, which prevail h and h after photodynamic treatment, respectively. the observed conformational changes of proteins can be explained as the consequences of the processes leading to apoptosis as was verified by flow cytometry experiments. the results confirm suggestion that ir spectroscopy can be successfully applied for the detection of early apoptotic processes. a. k. de, d. goswami indian institute of technology kanpur, india molecular fluorescence has been an indispensable tool in modern day optical imaging. one of the state-of the-art challenges in fluorescence microscopy is having better depth resolution as embodied by the confocal and multi-photon laser-scanning microscopic techniques. however, each technique bears its own limitation in having sufficient out-of-focus signal for the former while the low non-linear photon absorption cross-section for the latter. for confocal microscopy using one-photon excitation, we have shown how the clever choice of pulsed illumination instead of continuous-wave excitation leads to a gigantic enhancement in fluorescence that also has immediate applications in microscopy. moreover, single-photon illumination with ultrafast pulses leads to a novel way of achieving axial resolution along with numerous other advantageous applications e.g. reduced photo-bleaching of the chromophore. on the other hand we have thrown new insight demonstrating that the use of mode-locked laser pulses in multi-photon microscopy induces severe solvent-induced photo-thermal damage and prescribed methods to get rid of it. besides, the use of pulse pair excitation in multi-photon microscopy leads to probe and control the dynamics of fluorophores which has crucial role in selective excitation of fluorophores from quantum control perspectives. all these cutting edge research works will be presented in addition to our recent work on application of laser pulse shaping in multi-photon microscopy. interkingdom signalling in pseudomonas aeruginosa b. davis , r. jenson , p. williams , p. o'shea institute of biophysics, imaging and optical science, university of nottingham, u.k., institute of infection, immunity and inflamation, university of nottingham, u.k. quorum sensing is the process through which some bacterial species coordinate cell-cell communication. pseudomonas aeruginosa; the pathogen responsible for over % of chronic lung infections and the leading cause of mortality in cystic fibrosis patients, expresses two major classes of quorum sensing molecules characterized as n-acyl homoserine lactones and -alkyl- -quinolones. these compounds, in addition to their signalling roles have also been found to possess virulent properties, not only against competing species of bacteria such as staphylococcus aureus but also eukaryotic cells such as t-lymphocytes. the process of bacterial quorum signalling molecules influencing eukaryotic cell activity is termed 'interkingdom signalling'. to date it has been suggested that the quorum sensing molecules outlined above act on eukaryotic cells through interactions with an as yet unidentified plasma membrane or cytosolic receptors. this project is directed towards developing an understanding of how these compounds elicit eukaryotic response through a combination of membrane based interactions at physiologically relevant concentrations. particular emphasis is placed on studies of ligand binding with membrane microdomains in and the consequent downstream signalling are also considered. this work is significant as it will not only lead to a better understanding of pseudomonas infection, but may also lead to the discovery of new classes of agents for the treatment of infective diseases. a xas study of the sulphur environment in human neuromelanin and its synthetic analogues neuromelanin is a complex molecule accumulating in the catecholaminergic neurons that undergo a degenerative process in parkinson's disease. it was shown to play an either protective or toxic role depending on whether it is present in the intraneuronal or extraneuronal milieu. in the present study x-ray absorption spectroscopy is employed to investigate the sulphur binding mode in natural human neuromelanin, synthetic neuromelanins and in certain structurally known model compounds, namely cysteine and trichochrome c. based on comparative fits of human and synthetic neuromelanin spectra in terms of those of model compounds, the occurrence of both cysteine-and trichochrome-like sulphur coordination modes is recognized and the relative abundance of these two types of structural arrangement is determined. data on the amount of cysteine-and trichochrome-like sulphur measured in this way indicate that among the synthetic neuromelanins those produced by enzymatic oxidation are the most similar ones to natural neuromelanin. c. cremer kirchhoff-institute for physics, university of heidelberg, germany here we report on "spectral precision distance/position determination microscopy (spdm) with physically modifiable fluorochromes (spdm phymod ) to analyse the spatial distribution of single nuclear proteins and dna sequences at the macromolecular optical resolution level. like other methods of "spectrally assigned localization microscopy" (salm), spdm phymod is based on labelling 'point like' objects (single molecules) with different spectral signatures, spectrally selective registration and high precision localization monitoring by far field fluorescence microscopy. the intranuclear spatial location of single molecules was determined up to a density up to ca. molecules/µm of the same type, and distances down to - nm were nanoscopically resolved. quantum dots (qds) are semiconductor nanoparticles with increasing application as fluorescent markers in biology.we investigated structure of the cell walls of different species complexed with cdse qds using fluorescence microscopy, fluorescence spectroscopy and ftir techniques. in the experiments we used the cell walls isolated from three distinct plant species: arabidopsis thaliana, acer sp. and picea omorika. we studied both unlabeled and cdse-labeled cell walls. fluorescence spectroscopy and microscopy were used for detection of qds alone or complexed to the cell walls. emission spectra were deconvolved using the nelder-mead algorithm in matlab . . we calculated approximate probability distribution (apd) for positions of spectral component maxima. there was certain difference between unlabeled cell walls and those complexed with qds. the ftir spectra also show some difference between the complexed and pure cell walls. the results show that structure was changed, but not significantly in reaction with cdse qds. these results are promising in context of use of qds as labels in cell wall studies. the characterization of the complex of cell wall structure with qds is a part of the study of nanoparticles application in investigations of plant materials. modulating the response of single neurons and neuronal networks with biophysical stimuli f. difato, a. maccione, l. berdondini, f. benfenati, a. blau italian institute of technology, department of neuroscience and brain technologies, genoa, italy during differentiation, cell processes create connections with other cells to form tissue capable of performing complex tasks. biophysical constraints provide necessary inputs for cellular organization in living organism . to better understand how biophysical conditions influence tissue development, it is necessary to bridge the gap between experiments on single cells and complex tissues , . to achieve this goal we pair optical tweezers with electrophysiology measurements . by adopting neuronal networks as a biological model, neuronal signal transmission can be recorded either by patchclamp electrophysiology or microelectrode arrays (meas). dissociated neurons will be cultured on meas to record neuronal network activity at different sites of the network while applying spatio-temporally defined biophysical stimuli to individual neurons. a. diaspro , k. cortese , p. bianchini , c. gagliani , c. tacchetti iit -italian institute of technology, morego, genova, italy, microscobio, university of genoa, italy correlative light/electron microscopy (clem) is becoming increasingly frequent in molecular and cellular biophysics. we successfully applied the method to analyze the d structure of rough and smooth russell bodies used as model systems. the major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibilitˆto perform d correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of the high data analysis capability of flm with the high precisionaccuracy of transmission electron microscopy in a clem hybrid morphometry analysis. we have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic d reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach. the relevance of the presented approach lies in two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques. multidrug resistance is a well known phenomenon which limits effectiveness in treating malignancy with chemotherapy by modifying the internalization and/or externalization flow of the drugs through the cancerous cells. combined chemotherapies, such as mvac, are therefore currently used in bladder cancer treatment. however, about % of patients do not respond this chemotherapy because of inherent or acquired drug resistance. we developed a non invasive predicative test on urinary cells to estimate the chemotherapy effectiveness before treatment, based on the fluorescence emission of mvac. we first studied the mvac photophysical properties in solution and using five cell lines: a drug sensitive cancer cell line mgh-u s, its multidrug resistant subline mgh-u r, a not tumorigenic cell line sv-huc- , its tumorigenic counterpart mc-sv-huc t- and a cell line from transitional cell carcinoma t . the results revealed a penetration and localization of the drug depending of the cell line type, allowing us to find a specific fluorescence signature for the identification of mvac resistant cells. similar data have been obtained for cytospined fixed culture cells and patients urinary cells. -imaging and spectroscopy -abstracts multilayered photoresist system as a ghost model for biological samples in confocal microscopy. l. ferrari, f. cilloco, f. r. bertani, s. selci istituto dei sistemi complessi cnr rome we have realized a bilayer photoresist system as a model to perform optical characterization in the vis-ir range of multilayered complex biological specimen. our goal is to obtain structural and spectroscopic reference parameters from the identification of reflectance pattern features within a novel project granted by miur (skintarget, ideas firb). our model is composed by a nm thick layer (shipley photoresist ) with a refractive index n= . - . , wavelength dependent, and a nm tick hsq (hydrogen silsesquioxane) layer with a refractive index n around . , both spin-coated over a glass substrate. we present the analysis of reflectance pattern that can be obtained by a confocal laser reflective system as compared to the expected values as coming from analytical calculations, using a matrix approach, or a microscopic electromagnetic finite element analysis. interfacial roughness and consequent optical scattering are analyzed using a neural network approach. translational biophysics: multimode imaging for preclinical and clinical applications d. l. farkas cedars-sinai medical center, los angeles, usa our focus is where light and patient meet, and improvements yielding better outcomes. surgery is moving towards minimally invasive intervention, where biophotonics represents a major area of hope and growth. the translation of useful laboratory-derived knowledge into clinical practice has been hampered by the difficulty of detecting, characterizing and monitoring molecules and cells in the human body, especially dynamically. advanced bi ophotonic imaging is best suited for studying such entities, but has been lagging in clinical acceptance, in spite of major advances, and a clear need for the kind of resolution (spatial, temporal, spectral) and specificity that it alone can offer. biophysics-based new strategies are needed to address this challenge. the development of biophysical methods for translational medicine will be reviewed, with emphasis on our recent advances. our approach is a multimode one -combining methods to achieve early, quantitative detection of abnormalities. with imaging fulfilling its dual role of better describing anatomy and physiology, intrasurgical histopathologyequivalent molecular and cellular imaging is achievable in vivo, as is a closer spatio-temporal connection between imaging and intervention. some application areas to be covered: cancer (early detection by spectral reflectance/autofluorescence; progression quantitation by oct; nano-and targeted chemotherapy assessment in vivo); neurobiology (imaging fast calcium transients and alzheimer's plaques); hyperspectral mie scattering imaging for in situ displasia; design and use of an advanced multimode imaging endoscope with in vivo delineation of hirschsprung's disease for better intervention; monitoring of stem cell fate in vivo. immobilization of liposomes in a sol-gel matrix: a fluorescence confocal microscopy study r. esquembre , s. n. pinto , j. a. poveda , m. prieto , c. r. mateo ibmc, universidad miguel hernández, elche, spain, cqfm, instituto superior técnico, lisbon, portugal immobilization of liposomes shows interesting applications in protein biology, membrane biophysics and biosensor technology. previous fluorescence spectroscopy works revealed that the entrappement of zwitterionic phospholipid liposomes in a silica sol-gel matrix alters the thermodynamic properties as well as the fluidity of the lipid bilayer. interactions between the polar head of phospholipids and the porous surface of the host matrix could be responsible of such behaviour. in order to get more insight into this possibility we have immobilized, for the first time, giant unilamelar vesicles (guvs) and the shape and size of these structures as well as the possible existence of lipid domains have been visualized through fluorescence confocal microscopy. this technique allows for direct observation of the effect of encapsulation on an individual liposome, in contrast to the averaged information given by macroscopic spectroscopic techniques. liposomes composed of pure popc or dopc as well as mixtures dopc/dppc were labelled with the fluorescent probes bodipy, rhd-pe and rhd-dope. preliminary results shows that only the smallest guvs ( - µm) survive to the encapsulation process but often with a slight loss of its sphericity, probably due to pressures suffered during the matrix gelation. however no change in the gel/fluid phase proportion has been observed for immobilized dopc/dppc guvs, regarding to solution. solvent fluctuations play a key role in controlling protein motions and functions. here, we have studied how the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (por) couple with solvent dynamics (g. durin et al, biophysical j. ( ) , . to simultaneously monitor the catalytic cycle of the enzyme and the solvent dynamics, we designed temperature-dependent uv-visible microspectrophotometry experiments, using flash-cooled nano-droplets of por. the temperature-dependant formation of the first two intermediates in the por reaction were measured, together with the solvent glass transition temperature (t g ) and the build-up of crystalline ice. we find that formation of the first intermediate occurs below t g and is not affected by solvent dynamics, whereas formation of the second intermediate occurs above t g and is influenced by solvent dynamics. these results suggest that internal protein motions drive the first step of the por reaction whereas solvent slaved motions control the second step. we propose that the concept of solvent slaving applies to complex enzymes such as por. amphotericin b (amb) is one of the main polyene antibiotics widely used to treat deep-seated fungal infections. the mechanism of biological action of amb is most probably directly related to the ability of the drug to form hydrophilic pores in the membrane core, thus affecting physiological transport of ions. the effects of amb-cu + complexation are demonstrated by the electronic absorption and fluorescence spectra. the absorption spectra of amb in water (ph= ) after the injection of water solution of copper(ii)sulfate display a complex structure with hypsochromic-and bathochromic-shifted bands indicative of formation of molecular aggregates of the drug. formation of the electronically coupled chromophores of amb, especially aggregates, was analyzed at different cu + concentrations by the rls (resonance light scattering) technique. intensity of the fluorescence emission spectrum (characteristic of the dimeric form of amb) decreases after the amb-cu + complex formation. this effect of the formation of the amb aggregated structures by amb-cu + are different from the spontaneous molecular aggregation process, as deduced from the spectroscopic analysis. morfo-functional asymmetry of the olfactory receptors of the honeybee apis mellifera l e. frasnelli , g. anfora , f. trona , f. tessarolo , r. antolini , g. vallortigara cimec, centre for mind/brain sciences, university of trento, italy, iasma research and innovation center, fondazione e. mach, s. michele all´adige (tn), italy, biophysics and biosignals lab., dept. of physics, university of trento, italy lateralization, i.e. the different functional specialisation of the left and right side of the brain, has been documented in many vertebrate and, recently, invertebrates species. in the honeybee apis mellifera l. olfactory memory seems to involve at first the use of the right antenna. the present study investigated physiological and anatomical differences between left and right antennae of honeybees. electroantennographic responses (eag) were recorded from the left and right antennae of honeybees to linalool, a floral volatile compound, and isoamyl acetate, an alarm pheromone, at doses (from , to µg). the number of sensilla on the left and right antennae was recorded by scanning electron microscopy (sem). each antenna segment, from insects, was observed from different viewpoints in order to image the whole antenna surface and compute the number of sensilla. the tested compounds induced higher eag responses on the right than on the left antenna at every dose. sem showed that the placoidea olfactory sensilla were slightly more abundant on the right antenna surface than on the left one. results suggest an asymmetry in the peripheral odour perception mechanism in the honeybee a. mellifera. super-resolution imaging of dna through single molecule switching of intercalating cyanine dyes c. flors, c. n. j. ravarani, d. t. f. dryden school of chemistry, university of edinburgh, u.k. a growing trend in far-field super resolution fluorescence microscopy involves the replacement of photoactivatable fluorophores by common dyes such as cy, atto or alexa [ ] . it has been shown that these dyes can blink in useful timescales for single-molecule based imaging by adding suitable buffers. this strategy greatly simplifies the sample preparation and imaging scheme, enabling its application to a wider range of biological systems. we have explored if a similar approach might be useful to study dna topology using intercalating cyanine dyes such as yoyo- . there are two main advantages of this approach: i) dna labelling with intercalating dyes is straightforward, and ii) the free dye in solution is essentially non-fluorescent, greatly reducing the fluorescence background. we show that yoyo- can blink in the absence of oxygen and in the presence of cysteamine, which allows its application to nanoscale imaging. we exemplify its use by imaging λ-dna and a puc plasmid. we also explore the compatibility of several intercalating dyes with biological systems such as enzymes or cells. our results suggest that dna intercalating dyes are a promising option for fluorescence super-resolution studies of dna topology. seeing more in total internal reflection fluorescence (tirf) microscopy r. fiolka, a. stemmer nanotechnology group, eth zürich, zürich, switzerland total internal reflection fluorescence (tirf) microscopy is an effective widefield imaging tool that selectively excites a very thin sample layer within the evanescent excitation field at the glass-water interface. lateral resolution of standard tirf microscopy is limited to approx. nm using green emission, which can be insufficient for a large class of biological investigations. additionally, the evanescent excitation field is prone to light scattering, creating out of focus blur in the final image. we present several techniques that address these mentioned shortcomings of standard tirf microscopy. using evanescent standing waves, the lateral resolution in tirf microscopy can be extended by a factor of . , reaching nm. we further show techniques to reduce the blur induced by light scattering of the evanescent field. finally, we demonstrate optical sub-sectioning capabilities in tirf microscopy by acquiring several images with different penetration depth of the evanescent field and applying suitable post processing algorithms. thereby the obtainable z-resolution exceeds the classical limit of widefield microscopy, and object structures lying within the evanescent field can be reconstructed. the natural photosentizer hypericine (hyp) exhibits potent properties for tumor diagnosis and photodynamic therapy. evidences of hyp release from ldls prior to passive diffusion within cells are addressed in this study. fluorescent properties of hyp have been used for dynamic studies of its interaction with low-density lipoproteins (ldls) and u glioma cells. subsequent non-specific staining of intracellular membranes compartment were observed by mean of colocalization fluorescent imaging studies. it was shown, that monomers of hyp are only redistributive forms. increasing of hyp concentration leads to the formation of non-fluorescent aggregates within ldls as well as within the u cells, and can preclude its photosensitizing activities. in all experiments, hydrophobic character of the molecules appears as the driving force of its redistribution process. acknowledgment: this work was supported by the slovak res. and dev. agency contracts no. lpp- - . we also kindly thank the synchrotron soleil for using the detection system of the disco beamline. ledgf/p switches from a dynamic to a tight chromatin interaction upon binding to hiv- integrase j. hendrix , z. debyser , y. engelborghs laboratory of biomolecular dynamics, katholieke universiteit leuven, belgium, laboratory of molecular virology and gene therapy, katholieke universiteit leuven, belgium human transcriptional co-activator ledgf/p is hijacked by hiv- integrase (in) during the replication of hiv. little is still known about the molecular complex of these two proteins in the living cell. in this work we first studied the cellular chromatin interaction of egfp-tagged ledgf/p with tunable-focus fluorescence correlation spectroscopy (tf-fcs) and show that ledgf/p is in equilibrium between a free brownian motion and a very slow movement on the chromatin. being dependent on the size of the laser focus, this slow movement represents a continuous associationdissociation-reassociation process that is governed by diffusion. concentration-dependent continuous photobleaching measurements (cp) furthermore revealed the existence of high-affinity chromatin binding sites. next, we co-expressed mrfp-tagged in and confirmed its intracellular interaction with ledgf/p by fluorescence cross-correlation spectroscopy (fccs). interestingly, cp and fluorescence recovery after photobleaching (frap) indicated that the affinity of this complex for chromatin is exceptionally high. by twophoton fluorescence lifetime imaging ( p-flim) we verified if the cellular stoichiometry was altered when the proteins were expressed together. we believe that this work is useful for the understanding and targeting of hiv-replication. biophysical identification of orf from clavulanic acid biosynthesis cluster as a cyp l. s. goto , c. o. hokka , j. f. lima , o. r. nascimento , a. p. u. araújo grupo de engenharia bioquímica, ufscar, br, grupo de biofísica molecular "sérgio mascarenhas", usp, br streptomyces clavuligerus produces the clinically important β-lactamase inhibitor clavulanic acid. biosynthesis related genes reside in three gene clusters, one of these, named clavulanic acid gene cluster, includes most of the known clavulanic acid biosynthetic enzymes. the penultimate step along clavulanic acid biosynthesis remains unclear. required transformation involves at least two events: oxidative deamination and double epimerization of ( s , s )-clavaminic acid into ( r, r)-clavaldehyde. downstream the known part of clavulanic acid cluster lays orf , a putative gene encoding a tentative cytochrome p -like protein which knockout has been proven deleterious to clavulanic acid biosynthesis. should such protein exist, it would be candidate to fulfill the clavulanic acid pathway missing step. in this work, orf encoded protein is characterized aiming to place it as a real p . for this task, molecular cloning and recombinant expression of orf were accomplished. purified protein was submitted to spectroscopic measurements such as circular dichroism and electron paramagnetic resonance which indicate p features, including catalytic relevant heme iron redox states and homolytic peroxide scission mechanisms. further, peroxide reaction adducts were characterized by spin trapping. this work is supported by fapesp. muscle structure and gabaergic innervations in the limbs of barnacle cyprid l. gallus , s. ferrando , c. gambardella , a. diaspro , p. bianchini , p. ramoino , v. piazza , g. tagliafierro libiom, dibio, università di genova, italy, ifom-lambs/microscobio, difi, università di genova, italy, dipteris, università di genova, italy, ismar cnr, venezia, veneto, italy balanus amphitrite is a sessile crustacean that settles at the larval stage of cyprid. in this stage we studied the gabaergic innervation of limb striated muscular fibers, by immunohistochemistry. the second harmonic generation (shg) and -photon excitation ( pe) microscopy were used to set out the muscle structure and its relationship with nerve terminal. sections were observed at a multimodal nonlinear microscope composed by the leica clsm. the laser system used is a ti:sapphire chameleon-ultra (coherent inc, santa clara, ca, usa), tunable between nm and nm and characterized by a pulse width of fs delivered at a repetition rate of mhz by means of a homebuilt set-up (bianchini p. and diaspro a. j biophotonics : - ). the z-stacks were performed in order to obtain the -dimensional distribution of the muscular fibers. in the posterior ganglion gad immunoreactive (ir) motor neurons were arranged in clusters near the emergence of the limb nerves. gaba and gababr ir neuromuscular junctions (nj) were localized in the limb muscle fibers; vgat ir cells surrounded each limb muscles. these results suggest that gaba plays a key role in the regulation of limbs movement. the shg was very useful to outline the relationship between nerve terminals and limbs muscle fibers. giant unilamellar vesicles (guvs) are very useful model membrane systems to study many aspects of lipid-lipid and lipid protein interactions, particularly employing fluorescence microscopy related techniques (bagatolli ) . the use of this model system can be particularly useful to study aspects related with the lateral structure of bacterial membranes using the aforementioned approach (i.e. fluorescence microscopy). bacterial cells have a size close to the resolution limit of optical microscopy and details about the organization of their membranes are not easy to achieve using such technique. recently a new electroformation method to prepare guvs composed of compositionally complex mixtures under physiological conditions was developed in our laboratory (montes ). in the present work we further extended this electroformation method to prepare guvs composed of bacterial lipid extracts and lipopolysaccharide (lps). in our experiments we used e.coli lipid extract to prepare small vesicles containing various lps species, from smooth strains and rough mutants. suvs were used as starting point to electroform guvs using various types of buffers with high ionic strength. the successfully obtained bacterial-guvs were used to study the interaction of these membranes with known lps-binging proteins (lung surfactant protein d, sp-d) and peptides (polymyxin b). our results remark the usefulness of this particular bilayer models to perform studies mimicking bacterial membranes. mechanical properties of polymeric membranes probed by afm m. kocun , i. mey , w. müller , m. maskos , a. janshoff georg-august universität, institute for physical chemistry, göttingen, germany, johannes gutenberg universität, institute for physical chemistry, mainz, germany many biological cell functions are dependent on the mechanical properties of the membrane. polymeric membranes that mimic native cell membranes are valuable research tools which can be used to better understand the physics of biological membranes. we have investigated free standing artificial membranes prepared from polybutadiene-b-polyethylene oxide (pb-b-peo). the membranes were prepared from vesicles ruptured on porous silicon substrates. these polymeric membranes were studied by confocal laser scanning microscopy (clsm) and atomic force microscopy (afm). force indentation curves were performed on the membranes and theoretical models were used to extract elasticity constants from the results. the study of polymeric membranes can give insight to the function of biological membranes, furthermore polymeric membranes can be used to create new hybrid systems by incorporating biological (lipids, proteins), artificial (polymers, dyes) and inorganic (nanoparticles) components. site-directed spin labeling study of the lightharvesting complex cp the topology of the long n-terminal domain of the photosynthetic light-harvesting complex cp was studied using electron spin resonance (esr). cp is a minor antenna complex of the photosystem ii (psii), a multisubunit protein complex. wild-type cp protein containing a single cysteine at position and nine single cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. in all cases the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. the spin label esr spectra were analyzed in terms of a multi-component spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. these results permit to trace the structural organization of the long n-terminal domain of cp . we took the crystal structure of light-harvesting complex ii (lhcii), major antenna complex of psii available in pdb as a starting point and constructed a model for cp based on esr data. present opportunities and future developments in soft x-ray transmission and emission microscopy b. kaulich, a. gianoncelli, v. babin, m. kiskinova elettra -sincrotrone trieste, s.s. km . in area science park, i- trieste-basovizza, italy soft x-ray transmission and emission imaging and spectromicroscopy are bridging the gap to other microscopy techniques in terms of lateral resolution, penetration depth and chemical sensitivity. the novel soft x-ray spectromicroscopy approach of the twinmic instrument at the elettra synchrotron radiation facility is combining several imaging modes for morphology characterization, such as scanning, projection and full-field imaging with several contrast techniques including brightfield, darkfield, differential phase and interference contrast at sub- nm lateral resolution. complementary chemical information is provided by chemical imaging and micro-spectroscopy using the photon-in/photon-out xray absorption and x-ray fluorescence. unique for twinmic is the low-energy x-ray fluorescence setup operated at - ev photon energies, which allows simultaneous analysis of the morphology, and the distribution of light elements on cellular and sub-cellular level. in the presentation the principles of the methods used in the twinmic instruments the performance and potentials of the instrument will be demonstrated by several examples of applications in the field of human, animal and plant biology, biophysics and chemistry, physiology and genetics. the potential impact of microscopy techniques using free-electron x-ray lasers on biophysics will be illustrated by the diproi project at fermi@elettra. -imaging and spectroscopy - fluorescence anisotropy and afm used as tools to characterized porin´s reconstituion in luv´s s. c. lopes, i. sousa, p. eaton, p. gameiro requimte, faculdade de ciências, universidade do porto, rua do campo alegre, - porto, portugal a major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that assure, not only, a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. for this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. in this study we report the reconstitution of ompf in preformed dmpc and e. coli liposomes using two different techniques for detergent removal: ( ) exclusion chromatography and ( ) incubation with detergent adsorbing beads. the incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine ompf effect on the structural order of membrane lipids. these results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. moreover more anisotropy and atomic force microscopy studies will allow a better characterization of bacterial model system membranes. the wavelength dependence of the luminescence for different sized aunp by -photon clsm k. li, m. schneider pharmaceutical nanotechnology, saarland university, campus a , d- saarbrücken, germany gold nanoparticles (aunp) with different sizes can exhibit original luminescent properties if excited with pulsed nearinfrared laser light which makes them a suitable object to be detected in biological tissues. the main obstacle is to distinguish between the object of interest (emitted light) and the autofluorescence from the sample which limits the scope of application of aunp. therefore, our aim is to characterize the luminescent properties of such nanoparticles regarding their excitation and emission. in this study, the excitation and emission spectra of aunp with different sizes below nm in an excitation range of nm to nm were investigated. our study shows the emission spectra curves of aunp are broadband spectrum and vary with the changing of excitation wavelength from nm to nm. the results also suggest the minimum laser power necessary to trap the aunp depends on particle size and excitation light wavelength and a maximum power above which the particles are destroyed. in all the experiment above, to avoid the simultaneous effects from slide and cover slip is a must. rocking, tumbling, and sliding: real-time nanomotion of a membrane-bound virus p. kukura , h. ewers , c. mueller , a. renn , a. helenius , v. sandoghdar laboratory of physical chemistry, eth zurich, ch- zurich, switzerland, institute of biochemistry, eth zurich, ch- zurich, switzerland the interaction of a virus with its receptors in the plasma membrane is decisive for its infection of cells. optical studies have revealed that after binding, virus particles move laterally on the membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings. here, we examine a model system, in which single simian viruses (sv ) interact with their gm ganglioside receptors in supported lipid bilayers. we employed scattering interferometry and single molecule fluorescence localization to visualize the vectorial rotational motion of virions. at low receptor concentration, we observed sliding and tumbling of single virions during rapid lateral diffusion. in contrast, at increased receptor concentration the virions repeatedly underwent periods of standstill, reminiscent of their behavior prior to endocytosis. by an unprecedented combination of millisecond time and nanometer spatial resolutions, we revealed that during these immobile periods, the virions rock back and forth among nanoscopic spots separated by nm. our insights, together with the structure of the viral capsid, suggest aggregation of receptors in nanodomains and recurrent swap of binding between receptor molecules and neighboring viral protein pentamers. herein, we have developed different membrane probes binding spontaneously to the outer plasma membrane leaflet and showing sensitivity to membrane properties such as surface charge and phase state. the first two types of probes were based on -hydroxyflavone fluorophore. one of them showed a high sensitivity to the surface charge and to the phase state of lipid bilayers, while the other was only sensitive to the surface charge. the third type, which was based on nile red fluorophore, was sensitive only to the phase state. surprisingly, the probes that were sensitive only to the surface charge did not respond to apoptosis, while the other two types of probes showed significant spectroscopic response to it. moreover, the latter exhibited response to cholesterol depletion, which was similar to that observed on apoptosis. thus, according to our data, the intact living cells present a remarkable fraction of the cholesterol-rich domains, while apoptotic loss of the transmembrane asymmetry decreases it dramatically. these probes represent a new tool for quantification of surface charge and cholesterol-rich domains in cell membranes. -imaging and spectroscopy - due to its porosity and unique optical properties porous silicon (psi) is an attractive template to develop biomaterials and biosensors. porous silicon microcavity (psimc) structures were prepared then functionalized for covalent protein attachment of glucose oxidase (gox) or solubilized bacteriorhodopsin (br). functionalization and protein infiltration was monitored by specular reflectometry sensitive to change in refractive index, when a molecule is attached to the large internal surface of psi. protein infiltration into the porous scaffold was confirmed by edx spectroscopy and the structures were imaged by biphoton microscopy. second harmonic generation and enhanced two-photon excited fluorescence emission from porous silicon was observed when resonantly exciting the structures. in addition, when the microcavities were infiltrated with gox or br, the proteins acted as a very efficient internal two-photon-excited fluorescence emitter, hence protein infiltration enabled the in-depth visualization of the porous structure by taking advantage of the optical sectioning capacity inherent to the non linear optical microscopy technique. patterning of bio-molecules: methods for characterization of neuron-substrate interfaces we investigated how to use and improve micro printing techniques to obtain molecules patterns on cell culture substrates. with micro contact printing (µcp), we generated geometrically defined depositions of poly-d-lysine (pdl) using poly-dimethylsiloxane stamps and optimized neuronal culturing conditions. rat and mice hippocampal neurons grown on those patterns showed to be alive and functional. we then applied µcp to study axonal development by combining cell culture assays with atomic force microscopy (afm) to investigate in more detail the molecule deposition on the surface and to measure morphological changes in the growth cone (gc) during the early phases of neurite development. we found distinct shapes of the gcs depending on whether they were growing on l adhesion molecule patterned by indirect-µcp or on pdl coated surfaces. we also attempted to transfer such patterns on multi electrode arrays in order to constrain neuronal cell bodies on the electrode area and to improve electrophysiological recordings from neuronal networks. other patterning techniques were therefore explored using a nano-drop printing system. patterned surfaces were analyzed with afm and scanning electron microscopy to combine different approaches aimed to the improvement and characterization of the printing techniques. insights on the mechanism of electron transfer in complex i a. l. maniero , c. bergamini , m. bortolus , r. fato , s. leoni , g. lenaz università degli studi di padova, padova, italy, università degli studi di bologna, bologna, italy complex i (nadh dehydrogenase) plays a central role in cellular energy production, transferring two electrons from nadh through a series of iron-sulfur clusters (fes) to ubiquinone (coenzyme q); the electron transfer is coupled to the translocation of protons across the membrane. the fes center n is the last acceptor in the electron-transfer chain, but the mechanism through which the enzyme couples the e − reduction of the fes centers to the e − reduction of ubiquinone (q→sq→qh ) is unclear [ ] . in our experiments, submitochondrial particles were treated with different inhibitors, in the absence or in the presence of different quinone analogues, and nadh addition initiated the electron transfer. we assess by epr (electron paramagnetic resonance) spectroscopy the relative abundance of the reduced n center and of the semiquinone radical, and coupled epr data to enzymatic activity assays and to fluorescence measurements on the effect of inhibitors on reactive oxygen species (ros) production. we identify two different classes of inhibitors showing different effects on ros production. moreover the redox state of the complex has shown to depend both on the inhibitors and on the quinone analogues. a possible mechanism of the electron transfer, that can explain the experimental findings, will be presented. conventional fluorescence microscopy suffers from a resolution limit imposed by the diffraction of light. stimulated emission depletion (sted) microscopy overcomes this resolution barrier without being limited by the wavelength by taking advantage of the photophysics of the observed sample into the image formation process, and has proven to be a powerful approach for exploring relevant biological issues. the outstanding resolution of sted microscopy is achieved by drastically minimizing the spatial extent of the focal region from which fluorescent molecules can emit signal in the sample. so far, mainly complex and relatively expensive lasers systems providing pulsed beams have been used to inhibit the fluorescence in the outer area of the focal region. here we report on the development of a new setup based on a fast beam scanning confocal microscope using compact turn-key and inexpensive continuous wave lasers. the great potential of this simple configuration is demonstrated for a selection of commonly used fluorescent markers. characterization of hepatitis b antigen particles by atomic force microscopy each year, over one million people die from hepatitis b virusrelated chronic liver disease, including cirrhosis and hepatocellular carcinoma. the major surface antigen of hepatitis b virus (hbsag) is a cysteine-rich, lipid-bound protein with amino acids. recombinant hbsag produced in yeast can self-assemble into -nm immunogenic spherical particles that are used in licensed hepatitis b vaccines (protein/lipid ratio is / in mass). hbsag size and shape have been mainly investigated by transmission electron microscopy after negative staining of the particles. however, under these conditions, no details of the particle surface can be obtained because of the shadowing effect due to the uranium salts. here we describe new structural insights of hbsag particles using atomic force microscopy (afm) performed under physiological conditions. we applied atomic force microscopy to define structural details of the surface organization with a resolution in the nanometer range. as expected, the diameter of hbsag particles is , ± , nm in average. the surface of these particles clearly shows the presence of protuberances that most probably correspond to proteins. indeed, reduction and alkylation induces the disappearance of the protuberances. the number of the protuberances estimated from afm micrographs is about per spherical particles. a biophysical study of equine herpesvirus- entry into cells g. mckenzie , p. o'shea , j. kydd , c. rauch school of veterinary medicine and science, university of nottingham, uk, institute of biophysics, imaging and optical science, university of nottingham, uk equine herpesvirus- (ehv- ) can cause respiratory disease in young horses, varying degrees of paralysis and abortion during the later stages of pregnancy. furthermore, there has been a recent increase in the number of outbreaks involving paralysis of thoroughbred horses in training. virus disseminates rapidly after initial infection via cell associated viraemia. controlling this may prove crucial in combating the pathogenicity of the disease. we have investigated the cellular interactions of ehv- . initially, binding of ehv- to fluorescein phosphatidylethanolamine (fpe)-labelled phospholipid vesicles of various compositions was examined. a variety of microscopy techniques were then employed to study the events surrounding the binding of virus to equine peripheral blood mononuclear cell (pbmc) membranes. confocal microscopy images have highlighted possible colocalisation of ehv- with membrane 'rafts'. total internal reflection fluorescent microscopy was then used to identify viral binding at the membrane with high contrast images, able to observe single virus particles. establishing the viral entry pathway into pbmcs would allow drugs that target this process to be employed, reducing clinical viraemia. the interpretation of in-vivo binding rate measurements allows inferring the molecular interactions that regulate cellular functions. fluorescence recovery after photobleaching (frap) is a widely used tool to quantify binding reactions in vivo. however the lack of "golden standards" for these measurements requires the measured binding rates to be validated with other techniques. we present a new fluorescence correlation spectroscopy (fcs) method to measure the in vivo bound fractions and residence times for molecules that interact with an immobile substrate. we apply this method to measure binding of mutants of the transcription factor vbp (vitellogenin binding protein) to the dna. comparison of fcs with frap results in comparable estimates of the measured diffusion constants and bound fractions. however, fcs provides an estimate of the vbp residence time at the dna, while frap does not. this limitation in the analysis of vbp is due to the larger photobleaching volume used in frap, if compared to the observation volume of an fcs experiment. in sum, we present a method to measure binding rates with fcs. substantial agreement of this method with frap is shown. however, further validation on tightly bound molecules will be necessary to assess if frap and fcs agree in the measurement of residence times. -imaging and spectroscopy - we have investigated the changes in the mechanical properties of the zona pellucida (zp), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. the response of the zp to mechanical stress has been recovered according to a modified hertz model. zp of immature oocyte's shows a pure elastic behaviour. mature and fertilized oocyte's zps evidence, instead, a transition from a purely elastic behaviour, which occurs when low stress forces are applied, towards a plastic behaviour has been observed. the high critical force necessary to induce deformations, that well supports the non-covalent long interactions lifetimes of polymers, increase after the cortical reaction. afm images show that oocytes' zp surface appear to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles more wrinkled surface characterize marure oocytes with respecto to immature and fertilized oocytes from a mechanical point of view, the transition of the mature zp membrane toward fertilized zp, through the hardening process, consists in the recovery of the elasticity of the immature zp, while maintaining a plastic transition that, however, occurs with a much higher force with respect to that required in mature zp. dynamical behavior of ribose and deoxyribose supercooled water solutions s. e. pagnotta , s. cerveny , a. alegria , j. colmenero centro de fisica de materiales, centro mixto csic-upv/ehu, san sebastián, spain, departamento de fisica de materiales, universidad del pais vasco (upv/ehu), facultad de quimica, san sebastián, spain, donostia international physics center, san sebastián, spain ribose and '-deoxyribose are probably the most widespread monosaccharides in nature. they can be extensively found in ribonucleic acid (rna) and '-deoxyribonucleic acid (dna), respectively, where they form, together with a nitrogenous base and a phosphate group, a peculiar building-block structure called nucleotide. in the present work, the relaxation dynamic of ribose and deoxy-ribose water solutions at different concentrations has been studied by broadband dielectric spectroscopy and differential scanning calorimetry in the temperature range of - k. two relaxation processes are observed for all the hydration levels; the slower (process i) is related to the relaxation of the whole solution whereas the faster one (process ii) is associated with the reorientation of water molecules in the mixture. as for other polymeric water solutions, dielectric data for process ii indicate the existence of a critical water concentration above which water mobility is less restricted. moreover, according with these results, atr-ftir measurements of the same sugar solutions showed an increment in the intensity of the oh stratching sub-band close to cm − as water content increases. the regulation of the formation of cytoskeletal protein complexes by actin-binding proteins m. nyitrai, a. vig, t. kupi, z. ujfalusi, s. barkó, g. hild university of pécs, faculty of medicine, department of biophysics, pécs, hungary in living cells various groups of proteins are associated to supramolecular actin filament structures, often in a nucleation factor dependent manner. for example, actin structures associated with formins can bind tropomyosin and profilin, while those polymerised by the nucleation of the arp / complex bind cofilin and myosin i. the molecular mechanisms underlying the regulation of the formation of these protein complexes is still ambiguous. we have shown recently that formins can bind the actin filaments and change their conformational state. subsequent binding of other actin-binding proteins, such as tropomyosin and myosin, can reverse these changes. it appears that the reversal effect assumes that the actin-binding protein binds the filaments in a well-defined and specific binding site. the altered conformational state of the actin filaments observed after the binding of these proteins provides a possible explanation for the modified affinity of the filaments for other-actin binding proteins. based on the results available so far we assume that the affinities are modified differently by different nucleation factors, and the conformational changes introduced to actin by actin nucleation factors can serve as the molecular bases for the regulation of the formation of actin based intracellular protein complexes. experiments are currently in progress to test and further corroborate the existence of such regulatory mechanisms in living cells. correlation between sub-cellular distribution of photoactive drug hypericin (hyp), determined by applied delivery systems (hyp vs hyp/ldl), and mode of the cell death is addressed in this study. co-localization of hyp with mitochondria, lysosomes and golgi apparatus in u- mg glioma cells was determined by confocal laser scanning microscopy in using organelle specific fluorescent dyes as well as by time resolved fret experiments. flow cytometry experiments were realized to study a photodynamic effect of hyp ( nm/ jcm − ) on cells. significant differences in the proportional representation of live, apoptotic and/or necrotic cells were observed for different types of delivery systems of hyp hours after hyp ( x − m) photoactivation. conclusions: i) sub-cellular distribution of hyp depends on using delivery systems, ii) the mode of cell death depends more on concentration of hyp inside cells, than on different type of delivery systems (for non selective wide-field cell illumination), iii) fluorescence lifetime is sensitive parameter to study sub-cellular distribution of hyp. novel time-resolved spectroscopic methods have been used to investigate the interactions between a fluorescently-labelled mutant of the peptide melittin and supported lipid bilayers, formed by self-assembly at a silica-water interface via vesicle deposition. time-resolved evanescent wave-induced fluorescence spectroscopy (trewifs) is a surface-selective technique in which the evanescent field from a pulsed laser source is used to photoexcite fluorescent species at an interface. the resulting fluorescence decay kinetics, measured using time-correlated single-photon counting, report on the micro-domains experienced by those fluorescent species at an interface. extending trewifs to time-resolved evanescent wave-induced fluorescence anisotropy measurements (ew-trams) provides dynamic rotational information of a fluorescent species, reporting on its mobility at an interface. presented here are trewifs and ew-trams data obtained from the fluorescence of an alexa -labelled melittin mutant interacting with a dipalmitoylphosphocholine bilayer at room temperature, physiological ph and ionic strength. the results provide new insights into the conformation, location and motion of cytolytic peptides interacting with cell membranes. optical tweezers are used to controllably apply forces to red blood cells and the resulting chemical and structural changes are monitored using raman spectroscopy. the forces are applied in vivo and mimic that which the cell undergoes mechanically as it passes through vessels and smaller capillaries. the first result presented will be spectroscopic evidence of a transition between the oxygenation and deoxygenation states of hemoglobin that is caused by the stretching of the red blood cell. the transition is due to mechanically induced enhancements of hemoglobin-membrane and hemoglobin neighbor-neighbor interactions. the latter, lesser known effect is further studied by modeling the electrostatic binding of two of the protein structures using molecular dynamics methods. secondly, polarized raman spectroscopy is utilized to study the packing and ordering of the hemoglobin proteins in the red blood cell as it is stretched. depolarization ratios for a number of heme group modes change, indicating that the applied force additionally packs and orders the proteins inside the cell which further demonstrates the role of cell deformation in the oxygen transport kinetics. acknowledgements: this work was supported by miin fis - (spain) and fundació cellex barcelona fret microscopy is widely used to study protein-protein interactions in living or fixed specimens. currently, most commonly used visible fluorescent proteins for live-cell fret studies are the cerulean and venus variants of the cyan and yellow fluorescent proteins. even though this fret pair appears to be ideal for monitoring protein-protein interactions, the most commonly used fixed laser wavelengths do not excite cerulean at peak absorption. recently, we characterized an ideal donor, the monomeric teal fluorescent protein (mtfp), which is excitable using the commonly available ( ) opt. june/july, ). we used teal as a donor for various red fluorescent proteins as acceptors including tdtomato, mko , morange , mtagrfp, mkate. we have employed a "fret standard" genetic construct to minimize variability in separation distances and positioning of the fused donor and acceptor fps. using spectral fret imaging and fluorescence lifetime measurements in living cells expressing the fused proteins, we have characterized both sensitized acceptor emission and the change in the donor lifetime distribution as a result of quenching for each of the fused fp pairings. our results indicate that some red fps are better acceptors than others in terms of quenching the teal donor and sensitizing the emission of the acceptor indicating a fret event. s. m. perepelytsya, s. n. volkov bogolyubov institute for theoretical physics, nasu, -b metrologichna str., kiev, , ukraine stability of the dna double helix is determined by na + counterions, neutralizing the negatively charged phosphate groups of the macromolecule backbone. but under spatial conditions they may be replaced by much heavier ions, for example cs + . to determine the influence of heavy counterions on internal dynamics of the double helix the conformational vibrations of na-and cs-dna are studied. for this purpose the model of conformational vibrations of dna with counterions is used [perepelytsya s.m., volkov s.n., eur.phys.j. e. , , ] . as the result the frequencies and amplitudes of vibrations for b -dna with na + and cs + counerions are calculated. the frequencies of internal modes of the double helix are about , , and cm − . the frequencies of ion-phosphate modes are about and cm − for na-and cs-dna respectively. the calculated amplitudes of vibrations show that light counterions not disturb the dna internal dynamics, but heavy counterions make move all structure elements of the double helix. using the valence-optic approach the intensities of the dna vibrations in raman spectra are calculated. the calculations show that the ion-phosphate mode in cs-dna spectra is prominent, in contrast to na-dna spectra, where it has very low intensity. obtained results describe the intensity increase of the band cm − in cs-dna spectra that was observed in [bulavin l.a., et al., arxiv: . v ]. s. soria , f. quercioli , r. mercatelli , f. bianco , i. cacciari , g. righini centro studi e ricerche "e. fermi", p. del viminale , rome, italy, ifac-cnr, istituto di fisica applicata "n. carrara", via madonna del piano , sesto fiorentino (fi), italy, isc-cnr, istituto dei sistemi complessi, via madonna del piano , sesto fiorentino (fi), italy we report on the application of a simple white light source based on the supercontinuum generation from commercial photonic crystal fibres to confocal fluorescence microscopy and fluorescence lifetime imaging (flim) microscopy. the coherent white light can be tuned by varying the wavelength and intensity of the pump, a ti:sapphire laser. there are several advantages jn the use of sc sources: spatially coherent white radiation, tuning ranges of approximately nm, high brightness, a robust compact system (potentially all-fibre) and relatively low cost. being pulsed, sc sources are suitable for flim. we have used this system for measuring foerster resonance energy transfer (fret) in order to study interactions between ions channels and proteins of membrane within lives cells resolving the quaternary structure of plague f capsular antigen a. soliakov , j. r. harris , m. r. hicks , a. rodger , r. woody , a. watkinson , j. h. lakey cell and molecular biosciences inst., newcastle univ., uk, chemistry dept., univ. of warwick, coventry, uk, biochemistry and molecular biology dept., colorado state univ., usa, pharmathene uk, billingham, cleveland, uk most gram-negative pathogens express multi-subunit fibres on their surfaces that mediate host cell attachment, biofilm formation, invasion of host defenses and protection against phagocytosis. here we have studied capsular antigen fraction (or caf ), secreted through the conserved chaperone/usher pathway by the plague agent yersinia pestis [ ] . caf is highly immunogenic and is used in a recombinant subunit vaccine against plague. recent immunological studies indicated vaccines containing polymeric caf have higher protective efficacy than vaccines containing its monomeric variant. this difference in protective efficacy was attributed to the quaternary structure. however the quaternary structure of caf has not been characterized [ ] . in our study we have used transmission electron microscopy of negatively stained specimen and linear dichroism spectroscopy to determine the quaternary structure of recombinant caf . whereas electron microscopy revealed morphology of caf fibres bound to the surface of a carbon coated grid, linear dichroism gave the orientation of subunits in flow aligned caf fibres in solution. our results show recombinant caf comprises extended linear fibres and circularized fibres, both of which have high degree of conformational freedom. despite the vast application of lambert-beer law in biology, this empirical law cannot accurately describe the light absorption process of molecules with a long-lived excited state. this family of molecules includes most fluorescent molecules, which are very important in biology. lambert-beer law is in fact only valid at very low intensity of incident light and very low concentration of chromophore. if the system doesn't meet these conditions, it falls into a nonlinear regime when it is affected by a phenomenon which we call "dynamic photobleaching": the depletion of chromophores from the first layers, due to their transition to the excited state, leads to a sub-exponential propagation of light in the medium [ ] . this phenomenon leads to the necessity of a new formula for the light absorption dynamics which depends on the lifetime of the excited state of the chromophore. the predictions of the theory were successful in describing the absorption dynamics of azobenzene [ ] , but now they have been tested also on a biologically relevant molecule like chlorophyll. the results indicate that the absorbance is affected by the intensity of the incident light, and it is therefore a non reliable way of determining, for example, the concentration of the molecule. annular pupil filter to improve spatial highfrequency signal to noise ratio in linear and non linear microscopy e. ronzitti , v. caorsi , a. diaspro lambs, microscobio, department of physics, university of genoa, genoa, italy, semm, ifom-ieo, university of milan, milan, italy, neuroscience and brain technologies department, iit, genoa, italy shot-noise significantly affects and deteriorates the imaging capabilities of typical two-photon excitation and confocal laser scanning microscope, especially in biological applications where the detected signal can be remarkable slight [ ] . in particular, shot-noise substantially influences the spatial high-frequency range inducing a remarkable reduction of the optical transfer bandwidth. the insertion of an annular filter on the microscope objective lens in the illumination light pathway is here proposed to retrieve the high frequencies information loss [ ] .the electromagnetic interference effect induced by the filter insertion, gives a redistribution of the optical transfer function. in particular, the microscope frequency response in filter scheme exhibits an enhancement of signal to noise ratio at the high frequencies able to recover the high frequencies hampered by shot-noise [ ] . the goal of biomarker studies is to develop simple noninvasive tests that identify disease states. focus is beginning to shift from identification of individual biomarkers to identification of biomarker panels comprising multiple targets of different molecular species. there are, however, no current technologies available that allow for a comprehensive and simultaneous analysis of the expression levels of multiple cellular components, i.e. proteins and rna. surface plasmon resonance (spr) polaritons are surface electromagnetic waves that propagate in a direction parallel to the interface between a metal surface and an external medium e.g., liquid. these oscillations are very sensitive to any change of this boundary, a phenomenon that has been exploited to facilitate label-free, real-time detection of biological interactions, e.g. protein binding interactions. we are utilising the power of spr to develop technologies that facilitate diagnostic procedures for complex diseases such as alzheimer's disease and chronic obstructive pulmonary disease, through identification and detection of patterns of biomolecules indicative of disease. our approach will facilitate better disease characterisation, improve early detection strategies and aid drug discovery. surface generated fluorescence detection by supercritical angle confocal microscopy d. verdes, m. rabe, s. seeger institute of physical chemistry, university of zürich, winterthurerstrasse , zürich, switzerland a two channel confocal microscope for surface and simultaneously in solution fluorescence detection is presented. the microscope's core element is a parabolic mirror objective that collects the fluorescence above the critical angles for total internal reflection (tirf) of the water/glass interface. an aspheric lens incorporated into the parabolic mirror is used for diffraction limited focusing and collecting the fluorescence at low angles with respect to the optical axis. this low angles excitation approach is technically straightforward and gives an advantage over high numerical objectives that require very high angles for tirf illumination. by separating the collection of the fluorescence into supercritical and subcritical angles, two detection volumes highly differing in their axial resolution are generated at the interface. the surface selectivity of the detection volume is obtained on the basis of the dipole emission profile near a dielectric interface. its angular distribution is highly anisotropic and consists of a superposition of traveling and evanescent waves, which both are detected using the parabolic mirror objective. unlike with objective tirf microscopy, the parabolic mirror objective achieves easily diffraction limited excitation/detection volume at the water/glass interface. the objective optical performance is shown by measuring the actin cytoskeleton of cultured cells, fret energy transfer within adsorbed clustered proteins and single molecules detection. differential polarization laser scanning microscopy. anisotropy in biological samples g. steinbach , i. pomozi , g. garab biological research center, hungarian academy of sciences, szeged, hungary, pi vision bt., budapest, hungary differential polarization (dp) spectroscopy provides unique information on the anisotropic organization of biological samples [tinoco et al. ann rev biophys biophys chem : ]. however, anisotropy is often a microscopic property. the dp-lsm, constructed in our laboratory enables us to image the main dp quantities: linear and circular dichroisms (ld&cd), also in fluorescence detection ( . further applications include: periodic structure of isolated amyloids, anisotropy variations in cell walls related to drought resistance, and strong anisotropy of 'artificial chlorosomes', nanorods of synthetic porphyrins. dp-lsm might thus represent a novel tool in the better understanding of highly organized molecular macroassemblies. t. m. staudt , p. bingen , c. kempf , h. horstmann , j. engelhardt , t. kuner , s. w. hell german cancer research center/ bioquant, heidelberg, germany, max planck institute for biophysical chemistry, göttingen, germany, institute for anatomy and cell biology, university of heidelberg, heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of < nm inside µm thick tissue sections. -imaging and spectroscopy - r. worch, t. weidemann, p. schwille biotec, biophysics group, technische universität dresden, germany interleukin- (il- ), a small soluble protein, is a principal regulatory cytokine, playing an important role during the maturation and clonal expansion of antigen specific b-cells in mammals. at the plasma membrane, il- is recognized by a receptor that consists of two single spanning transmembrane proteins: a high affinity il- r alpha chain, and a low affinity il- r gamma chain. it is still controversial by which molecular mechanism the signaling complex is fully activated: dimerization of chains, large conformational change upon il- binding or a combination of both. moreover, the influence of the lipid environment in which the activation takes place is poorly characterized. to address these issues we aim to reconstitute the receptor component in artificial membrane systems to study the various mutual interactions by means of fluorescence-based techniques, mainly fluorescence correlation spectroscopy. due to reduced background in a chemically defined system this may provide details not yet accessible in the living cell. k. wicker, s. sindbert, r. heintzmann randall division of cell and molecular biophysics, king's college london, se ul london, u.k. fluorescence confocal microscopy, an indispensable tool of modern biology, allows the imaging of live fluorescent specimen with high lateral as well as axial resolution. through the introduction of a sufficiently small confocal pinhole, the lateral resolution can be enhanced compared to that of a wide field microscope. however, this gain in resolution comes at the cost of a decrease in detection efficiency, as light blocked by the pinhole is lost. we present a method for improving the lateral resolution (extending work of sandeau et al. [ ] ) and detection efficiency of scanning microscopes by adding an interferometer with image inversion in one of its arms to the detection pathway [ ] . this surpasses the lateral resolution achievable in a conventional confocal microscope (closed pinhole) while increasing the detection efficiency substantially. point spread function measurements for a uz-interferometer (uzi) are shown. the light in this setup follows threedimensional u-and z-shaped paths and relies on reflections off planar surfaces only in order to achieve image inversion. we achieved an interference contrast of % for white light, and excellent agreement with theoretical predictions. g. vicidomini, a. schönle, j. keller, r. schmidt, c. von middendorff, s. w. hell max planck institute for biophysical chemistry, department of nanobiophotonics, göttingen, germany fluorescence far-field microscopy is a indispensable tool in modern life science. however, the resolution of its standard variants is limited by diffraction to ∼ nm in the focal plane and ∼ nm along the optical axis. to overcome this limit, a new class of super-resolution microscopy techniques has been designed. one example is pi microscopy. to obtain isotropic resolution, pi uses interference of the wave fronts from two opposing lenses. the point spread function (psf) of pi system is characterized by multiple maxima/sidelobes, which replicate the object in the image. therefore image restoration is mandatory to render unambiguous imaging. the situation is further complicated because the positions and the relative heights of the multiple maxima/sidelobes of the psf depend on the phase difference (pd) between the two wave fronts at the focus. if the refractive index of the sample varies in space, this pd becomes a function of position and pi image formation process looses its shift invariance. the pd function (pdf) may not even be known a-priori and must then be estimated from the image, leading to a blind image restoration problem. here, we propose a maximum a-posteriori based method to solve the problem. we either assumed a mathematical model for the pdf that depends on a small number of parameters or allowed for an arbitrary pdf but introduced a smoothing constraint. designer multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from to times more efficient than binary complexes usually employed for gene delivery purposes [ ] [ ] [ ] . here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid dna to escape from endosomes with extremely high efficiency [ ] . endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid dna throughout the cytoplasm and in the nucleus [ ] . fluorescence spectroscopy and stopped-flow technique were utilized for the study of the kinetics of incorporation of hypericin (hyp), a natural photosensitizing pigment, into lowdensity lipoproteins (ldl). triphasic kinetics of hyp association with ldl was observed when solutions of hyp and ldl were mixed together. the most rapid phase of hyp incorporation is completed within tens of msec, while the slowest one lasts - min. the most of hyp molecules are incorporated into ldl in the slowest phase. the kinetics of the incorporation of hyp into ldl particles pre-loaded with hyp were also investigated. the observed decrease of the lifetime and total intensity of hyp fluorescence with the increase of the incubation time of hyp with hyp/ldl complex is a sign of the formation of aggregates and the dynamic quenching of singlet excitation state of hyp inside ldl. to study the kinetics of a transfer of hyp molecules between ldl particles, the time evolution of the stopped-flow and time-resolved fluorescence experiments were investigated after the mixing of the complex hyp/ldl= : with appropriate amounts of free ldl. for each final hyp/ldl ratio the increase of the lifetime and total intensity of hyp fluorescence was observed. the half-time of this process is similar to that one of the slowest phase of hyp incorporation into free ldl. a. bosca , r. magrassi , g. firpo , l. repetto , c. boragno , u. valbusa italian institute of technology, genova, italy, nanomed labs (difi-cba), genova, italy the technique of choice to measure the electrophysiological activity of neuronal cells is the so called patch-clamp method because of its precision and sensitivity; this procedure could play a major role also in the investigation of the behavior of a biological neuronal network and so represents an important tool for understanding its functionality and for screening the effects of drugs and compounds on it. the final aim of this project is the development of a planar patch clamp device suited to measure simultaneously the electrical activity of cultured neurons associated in a network. this device will be made of polymeric disposable material and will include a microfluidic perfusion system. in order to create a smooth micro sized pore in a thin polymeric membrane we exploited the prototyping capabilities of focused ion beam etching and we extended the air moulding technique by combining it with soft moulding, obtaining micro structured substrates with the requested features. we also subjected our substrates to a chemical treatment capable of rendering its surface stable and hydrophilic and we verified that it makes them suitable for neuron culturing. use of lipoamino acids for nasal delivery of therapeutic proteins c. bijani , s. sice , j. elezgaray , c. degert , o. broussaud , e. j. dufourc physica pharma, pessac, france, umr cbmn, cnrs-université bordeaux , iecb, pessac, france most of the therapeutic proteins are clinically administered through an intravenous injection several times a day / week. because the repeated injections are not convenient and cause pain in patients, an alternative route of administration is desirable such as oral or nasal. unfortunately, proteins are easily degraded by proteolytic enzymes in the gastrointestinal tract and, therefore, have a low bioavailability when administered via oral route. physica pharma has gained experience in forming sprayable solutions combining lipoamino acids (laa) and small therapeutic organic compounds with the aim to improve their intranasal absorption. in the present work we investigated the ability of laa to complex large molecule such as human erythropoietin, human growth hormone and salmon calcitonin in order to form easily sprayable colloids. these three proteins are used respectively to treat anaemia, growth problem and hypercalcemy. circular dichroism and dynamic light scattering were used to further characterize the laa-protein colloids. a specific molar ratio of laa versus protein was found for wich the proteins keep their secondary structure and have an overall isotropic size slightly increased. molecular dynamics show that proteins are indeed coated with laa. such a complex is shown to pass very easily through a culture of nasal cells growth at confluence. in this study we describe two systems based on soft matter designed for the drug delivery and for the replacement of synovial fluid in osteoarticular pathologies. (i) a new class of temperature sensitive hydrogels pva/poly(ma m nipaam n ) shaped as microspheres obtained with a water-in-water emulsion photocrosslinking reaction. microparticles of pva/poly(ma m nipaam n ) with m:n theoretical molar ratios equal to : ; : , : have been studied in terms of average size and responsiveness to temperature characterized by confocal laser scanning microscopy (clsm), dynamic light scattering (dls) and differential scanning microcalorimetry (dsc). (ii) a physical network based on hyaluronic acid with a small extent (degree of substitution: %) of hydrophobic moiety grafted on the backbone, hyadd , has been characterized in order to account for the influence of thermal treatment on the stability of the hydrogel. dynamic light scattering (dls) and small angle neutron scattering (sans) have been used for dynamic-structural characterization of hyadd hydrogels. diffusion of macromolecular probes has been studied by fluorescence recovery after photobleaching (frap) to study the mesoscopic texture of the hydrogel and molecular dynamic (md) simulations were used to approach the time evolution of the physical junction points and of chains clusters. multi-scale estimation of water soluble diffusivity in polysaccharide gels g. eisele , s. bertini , d. paganini , l. piazza ronzoni institute, milan, italy, distam, university of milan, italy diffusion properties in gels play important role in food and biotechnological applications. an attractive goal is to design gels in such a way that the active molecules are delivered by the material according to specific release sequences. the transport of macromolecules within polymeric gels depends: on the obstruction effects of the surrounding gel strands, on the molecular interactions between the gel and the solute, on the interactions between the solute molecules themselves and the interactions between the solute and the solvent. physical polysaccharide gels were evaluated in this work to probe diffusion over both microscopic and macroscopic distance scales. physical gels from agar and starch were investigated by high and low resolution nmr techniques in order to characterize their structures. obstruction effects of the surrounding gel strands were considered by studying diffusion of glucose. local diffusion, due to brownian motion, was quantified by low resolution nmr spectroscopy. the fickian diffusion coefficient was measured by modelling experimental concentration-distance curves obtained by means of a two-compartment diffusion-cell. diffusion coefficients depend on the viscoelastic properties of the gel matrix and on water-polysaccharide interactions. j. l. cuellar, g. koehler, m. fischlechner, e. donath medizinische institut für physik und biophysik, leipzig, germany rather than being just pathogens, from the view point of biotechnology and materials science viruses can be regarded as nanocontainers in which nucleic acids have been enclosed in a protective self assembled protein cage. in some viruses an additional lipid-protein envelope wraps the capsid shell. the capsid is constituted of several copies of one single protein subunit or just a few, arranged in a regular fashion showing icosahedral symmetry. the mechanical properties of the cage are important for understanding the infection mechanism involving the release of the encaged genome. by means of atomic force microscopy, it becomes possible to probe such material properties by nanoindentations of single viral particles. it is very interesting to learn how strong or brittle a virus can be. here we have studied the mechanical properties of empty rubella virus particles (rlps) due response of external applied forces. we found that rlps are extremely soft comparable to that of some rubber materials. a peculiarity of the rubella virus is that the capsid is considerably smaller than the surrounding shell filling only a fraction of the lumen provided by the envelope. the envelope in rubella has a distinguishable response on the material properties of the virus. deformation and fracture of the capsid requires comparatively larger forces. our results indicate that the ph is a major factor influencing on rubella particle material properties. this can be related to the infection mechanism. pentavalent antimony (pa) (glucantime, sanofi-aventis or glaxo) are the mainstream agents of choice for leishmaniasis treatment. in therapeutic doses the pa treatment has cardiac side effects like electrocardiographic (ecg) alterations, that include qt segment prolongation, t wave flattening or inversion, inversion of st segment, p, r and t waves amplitude reductions, torsade de pointes arrhythmias and sudden death by cardiac arrest. the objective of this study was to characterize the arrhythmogenic potential of pa. we used guinea-pig to assess the chronic effects of pa therapeutic dose on corrected qt interval, qt dispersion, ventricular action potential (ap) amplitude and duration at % and % of maximal repolarization and survival rate. guinea-pig received daily mg/kg pa or saline for days. eight lead ecg were recorded before and in the last treatment day. at the end the animals were killed and the left ventricle papillary muscles excised for ap recording with the intracellular microelectrode technique. our results of chronic pa treatment showed significant increase of qrs complex duration, qt interval duration, qt dispersion and incidence of t wave flattening or inversion and arrhythmias. the ap analysis demonstrated prolongation at % duration. the treatment was lethal in % of the animals. we concluded that pa is a proarrhythmic drug that upon chronic use may causes arrhythmias and mortality by disturbances in the ventricular repolarization process. m. m. khvedelidze , t. j. mdzinarashvili , t. partskhaladze , n. nafee , m. schneider ivane javakhishvili tbilisi state university, tbilisi, georgia, institute of molecular biology and biological physics, tbilisi, georgia, biopharmaceutics and pharmaceutical technology, saarbrücken, germany, pharmaceutical nanotechnology, saarbrücken, germany we have studied the thermodynamical properties of chitosan-coated nanoparticles (cnp) and non-coated nanoparticles (np) and have gained some insights about plga nanoparticles' properties using supersensitive differential microcalorimetry. the experiments show that in a wide ph interval the changes in transition temperature did not take place. it was shown that such nanoparticles could be used in acidic surrounding for drug transfer. stability and their other properties are less depended on either the particles were in bidistilled or deionized water, or the suspension of particles were located in buffer. to determine the interaction of plga nanoparticles with dna. in the case of dna presence in cnp solution the calorimetric experiments show that the heat absorption peak is constricted, what biophysically means that interaction between them takes place. for more exact determination the contribution of cnp in spectrum, we have compared the spectra of pure dna with the spectrum of the same concentration dna plus cnp. the optimal ratio for dna loading onto the cnp was found to be : . protein-based "epoxy-like" physical hydrogels for stem cell transplantation s. c. heilshorn, c. wong po foo, j. s. lee materials science and engineering, stanford univ., u.s.a. stem cell transplantation has emerged as a promising therapy for multiple injuries and diseases; however, cell survival after transplantation is often poor and unpredictable. we hypothesize that co-injection of stem cells encapsulated within an optimized physical hydrogel will enhance viability. whereas, current physical hydrogels require a shift in environmental conditions (e.g., ph, temperature) to initiate the sol-gel phase transition during encapsulation, our newly designed molecular-recognition gels do not require environmental triggers. instead, these "physical epoxy-like" gels consist of two components that undergo a sol-gel transition upon mixing due to specific hydrogen bonding. the gel viscoelasticity is predictably tuned through precise variation in the molecular-level design of the two components, created using recombinant protein techniques. the design of the two components is based on simple polymer physics considerations and utilizes bio-mimimcry. adult neural stem cells or mesenchymal stem cells are encapsulated within these gels with high viability at constant physiological conditions. the gels promote the growth and differentiation of neural progenitors into neuronal phenotypes, which adopt a d-branched morphology. the gels are further optimized for use in the central nervous system by tethering neuroprotective peptides to the gel through molecular-recognition sites. these peptides are released-on-demand by cells through the action of proteolytic enzymes. we consider the integration of the portal protein of the bacteriophage virus φ into lipid bilayers of giant unilamellar vesicles (guvs) membranes with the aim of constructing a functional cargo-device able to transport dna and later translocate it outwards. our nano-engineering plan consists of growing guvs from bilayer membranes built up from proteoliposomes previously prepared by extrusion. we have designed two alternative chemical routes for integrating the portal protein in the lipid bilayer, the first considering the native protein and the second a mutant modified with a hydrophobic belt made of histidine-tags. in our contribution we will present details on the different nano-engineering strategies and experimental evidence about the integration of the portal protein in the membrane with an orientation adequate to allow for functional dna translocation. p.-h. guelluy , m.-p. fontaine-aupart , m. hoebeke biomedical spectroscopy, ulg, belgium, laboratory of molecular photophysics, univ. of paris-sud, france ppme is a second generation photosensitizer (ps), and a promising candidate for photodynamic therapy (pdt) treatment. we have previously demonstrated that ppme can be easily and efficiently encapsulated in dmpc liposomes, used as ps-vector. we therefore compared the photophysical and photochemical properties of free and encapsulated ppme incubated with human carcinoma cells. absorption and fluorescence microspectroscopy as well as flim analysis allow evaluating the aggregation state of ppme inside the different cellular organelles and the extracellular medium. confocal microscopy established undoubtedly the colocalization of ppme, by robust probabilistic exclusion method, within mitochondrion (central siege of apoptosis). after ps activation ( h and h), the balance apoptosis-necrosis was double-estimated by facs device and fluorescence confocal microscopy. quantification of hydroxyl radical was purchased by spin trapping-esr spectroscopy and quenching technique. all these techniques required peculiar settings because of the fluorescence activity of ppme. these results allow to ascertain that the vectorization of ppme affords a better cellular penetration and a monomeric state of ppme. the presence of ppme inside mitochondrion orientated the cellular death in apoptosis h following ps-activation. but necrosis is the major actor h after treatment. active substrates to study mechanotransduction j. le digabel, p. hersen, b. ladoux laboratoire matière et systèmes complexes, université paris diderot -cnrs, paris, france cellular processes imply an important coordination of interactions with the extracellular medium. accumulating evidences demonstrate that cell functions can be modulated by physical factors such as the mechanical forces acting on the cells and the extracellular matrix, as well as the topography or rigidity of the matrix. these extracellular signals can be sensed by mechanosensors on the cell surface or in the cell interior to induce various cell responses. we have developed an original approach based on micro-fabricated substrates of polydimethylsiloxane (pdms) to study cell migration. we used a closely spaced array of flexible micropillars (diameter∼ ?m) to map the forces exerted by cells on their substrates. in this case, the micropillars act as passive force sensors. here, we propose to analyze the cell response to an external applied stress by a well-controlled actuation of the substrate. to do so, we used magnetic pillars. such substrates allow us to modify dynamic adhesion conditions of cells and to better understand the coupling phenomena between mechanical sensing and biochemical activity of a living cell. using polyacrylamide hydrogels doped with ferromagnetic iron oxide particles or ferrofluids, we can make magnetic pillars with diameters of to microns while a magnetic field can be locally applied with a magnetic needle. such a technique will be helpful to study the mechanical response of cells to an external force or to local changes in their microenvironment. glucose scavenging activities of pamam dendrimers m. labieniec , t. gabryelak , c. watala university of lodz, deptartment of general biophisics, lodz, poland, glycation is a spontaneous, non-enzymatic modification of biomacromolecules with hexoses, mainly glucose. in terms of its pathophysiological relevance, it targets predominantly proteins, but also nucleotides and phospholipids, and is of major importance to both physiology (ageing) and pathology (diseases with a metabolic background, like diabetes). earlier we demonstrated in vivo that the administration of pamam g to diabetic rats resulted in a significantly reduced blood glucose, as well as the early (hba c ) and late (ages) protein glycation products. in this study we investigated the ability of pamam g ( surface nh groups) and g ( nh ) to inhibit glycation of proteins in plasma, and a model protein -bsa. pamam g and g competed chemically with protein nh for the binding of glucose, and hampered protein glycation. in a high-glucose medium they underwent an excessive glycation themselves. this modification was more effective in pamam g , in which surface nh were more mobile and accessible. pamam modification with glucose rendered these dendrimers less polycationic in aqueous solutions. pamams neither affected bsa conformation nor formed stable complexes with a protein. we conclude that pamams are very effective glucose scavengers. thus, even less toxic pamams of lower generations, like g , may appear useful in further medical applications as the agents attenuating the detrimental effects of sever hyperglycaemia on biomacromolecules. selective drug delivery and novel drug approaches by polyelectrolytes s. krol cbm-cluster in biomedicine, area science park, s.s. , . km; trieste, italy the use of polyelectrolytes in the past was mainly related to targeted drug delivery and nanoparticle preparations for medical applications. but rarely the polyelectrolytes were investigated for the own features as a drug. in our present work we use a special physical feature of cancer cell membranes as a target for a specific polycation. we found that the polycation is selectively up-taken by cancer cells (leukemia, hepatocarcinoma, cancer stem cells) while normal cells remain unaffected. another interesting application of polyelectrolytes are in form of a multilayer coating on the surface of nanogold particles. for this topic, two new approaches were developed for a drug delivery through the blood brain barrier. in the first case, high amounts of creatine were bound to the gold particles and delivered as protective agents for ischemic stroke (viota et al., j colloid interface sci. , ( ): - ). in the second case coated multifunctional gold particles were prepared as a drug for neurodegenerative disease on the basis of protein aggregates. the use of gold as core for coated nanoparticles offers the possibility to study our systems as "theranostics", system which are modified to recognize selectively diseased cells and carry the moieties to treat or destroy the malfunctioning cells. possible treatments can be local hyperthermal therapy by using the particles as amplifier and enhancer or photodynamic therapy with gold as a drug. m. koneracka , v. zavisova , m. muckova , p. kopcansky , a. jurikova , n. tomasovicova , g. lancz , m. timko , m. fabian institute of experimental physics, slovak academy of sciences, košice, slovakia, hameln rds a.s., modra, slovakia, institute of geotechnics, slovak academy of sciences, košice, slovakia the aim of this study was to develop biodegradable and biocompatible paclitaxel loaded magnetic plga nanospheres (nps) suitable for biomedical applications. biodegradable poly(d,l-lactic-co-glycolic acid) (plga) was used as a capsulation material and the magnetic fluid was used as a magnetic carrier. incorporation of magnetic particles and drug in the plga polymer matrix was confirmed by infrared spectroscopy (ftir) and differential scanning calorimetry (dsc). the release of the drug from the prepared nps to the surroundings under the different conditions was studied also. the prepared magnetic plga nps with encapsulated paclitaxel (spherical shape, size - nm) have good stability in the presence of high nacl concentration at • c, the toxicity of prepared samples declared times higher value of lethal dose ld in comparison with pure paclitaxel (ld = mg/kg) and showed the significant response to external magnetic field which is useful from the point of view to achieve pharmaceutically acceptable drug delivery systems for tumour treatment. success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety. many cationic polymers have been used to condense dna by electrostatic interaction into small particles (polyplex), for protecting the dna from degradation and enhancing uptake via endocytosis. polyethylenimine (pei) appears to be one of the most advanced delivery system that can condense dna efficiently forming pei-dna polyplex complex. the physicochemical properties of different molecular weights of pei, such as condensation ability, buffer capacity, time kinetics, ftir and surface charges of the pei-dna complexes may be important factors to obtain a higher transfection efficiency of the polycation vectors. our intent in this study was to characterize pei-dna complexes to see whether these physicochemical properties have any influence on their disposition characteristics and cellular uptake process. we found that pei-dna complexes, obtained by the k pei at n/p ratio > , were more stable in the presence of tissue culture medium & serum, and did not dissociate in nacl easily. key words: polyethylenimine, polyplex, polycations, dna, transfection, gene delivery. effect of nanopatterned substrate on neuronal growth cones activity in the last years an increasing interest has been address to explore, at the level of the single cell, the physical interaction between neurons and micro-and nano-patterned surfaces which mimic the biological environment and can induce specific biological behavior. a consistent number of substrates have been tested (nano-grooves, pillars, gap nanowires) but the effect of nano-topographical features at subcellular level e.g. branching and pathfinding of growth cones(gc)is still unexplored. using nanoimprinting lithography we fabricated gratings on glass with grooves of variable pitch, depth and width all in the nm range. embryonic stem cell derived neurons were seeded on nanostructured and on flat control glasses. we investigate gc morphology with nm resolution by afm. a significant effect on sub-cellular architecture was observed.on nanopatterned substrates % of gc were branched with a large number of long and thin filopodia(average length and height . µm and nm)while on control only %of gc were branched with an higher percentage of long and thick filopodia (average length and height , µm and nm). on the contrary we did not observe a significant influence of the nanopatterning on the alignment and elongation of neurites. in both cases the distribution of angles between axon and filopodia showed a preferential direction at • . in conclusion, the tested nanopatterns do not influence the neurite directions but do enhance the gc morphology and explorative activities. growth enhancement and adhesion control of pc on micropatterned ns-tio thin film c. lenardi , a. v. singh , p. milani c.i.ma.i.na., dip. di scienze molecolari applicate ai biosistemi, università di milano, italy, c.i.ma.i.na., semm, european school of molecular medicine, fondazione ifom, milano, italy, c.i.ma.i.na., dip. di fisica, università di milano, italy cluster assembled nanostructured titanium dioxide (ns-tio ) has been explored as novel substrate for in vitro cell culture. in this work, we report micropatterned ns-tio thin film as putative microdevice for neuron culture and growth. in additions, we show a simple scheme of molecular patterning of bovine serum albumin (bsa) as cell anti-adherent substrate complementary to ns-tio micropattern which favors a selective spatially confined adhesion of neurons. bsa was drop coated and physisorbed over glass coverslip covered with a thin conducting layer of indium tin oxide (ito) and then pmma was spin coated over it using standard protocols. later, using combinations of e-beam lithography and pulsed microplasma cluster source (pmcs), a thin layer of ns-tio was deposited over micropatterned pmma. further, lift off process enabled us to generate complementary micropatterns of hydrophobic bsa (cell repellent) and hydrophilic ns-tio (cell adhesive). cell culture studies have confirmed that pc cells like to grow on ns-tio substrate and not on bsa layer. the technique offers a novel approach for neuronal cell assay applications. gene delivery with chitosan: influence of chain length on intracellular trafficking and dissociation s. lélu , c. d. l. davies , n. reitan , s. p. strand department of physics, ntnu, trondheim, norway, department of biotechnology, ntnu, trondheim, norway chitosan, a cationic polysaccharide presenting low cytotoxicity, is a promising nonviral gene delivery vector. chitosan complexes dna into nanoparticles, and the complexation and cell transfection efficacy are function of the chitosan/dna ratio and of the intrinsic properties of chitosan, i.e. degree of polymerization dpn, charge density, and molecular structure. nanoparticles formed by shorter chitosan (dpn - ) mediated higher transgene expression than nanoparticles based on longer chitosans. the purpose of this work is to relate the dpn of linear chitosan with the cellular uptake, intracellular trafficking and dissociation of chitosan-pdna complexes, measured by confocal laser scanning microscopy and fluorescence correlation spectroscopy (fcs). cells were incubated with oligomers (dpn and ) either free or complexed with plasmid-dna. both chitosan oligomers seem to penetrate cell nucleus in association with or free from pdna. hours after incubation, accumulation of oligomers in cell nucleus is similar for free and complexed dpn , whereas it increases for complexed dpn compared to free, indicating a possible delayed dissociation of the complexes based on dpn in the nucleus and suggesting a dissociation of pdna-dpn in cytoplasm. these data are consistent with previous studies which suggested that longer chitosan chains led to tighter complexes, and hence to a delayed dissociation process and lower transfection efficiencies compared to shorter chitosans. material surface properties greatly influence dna purification and pcr yield in microsystems c. potrich , l. lunelli , l. marocchi , l. pasquardini , g. guella , d. vozzi , l. vanzetti , p. gasparini , c. pederzolli fbk, trento, italy, univ.trento, italy, univ.trieste, italy modern microchip platforms integrate dna purification, target amplification by pcr and dna detection in a single device. combination of these processes minimizes sample loss and contamination problems as well as reduces analysis time and costs. different strategies are available to perform dna extraction on a chip. here we exploited amino-coated materials as a tool for specific binding of dna through the electrostatic interaction between amine groups and nucleic acids. we analyzed the ability of different treated substrates to selectively absorb/desorb the genomic dna with the aim to purify dna from unwanted components. amino-coated substrates were characterized by afm, xps, fluorescence microscopy and absorption spectroscopy to define the surface chemical and morphological properties. the distribution of the dna adsorbed on materials was homogeneous and the eluted dna was tested for pcr. same materials were analyzed for their compatibility with pcr and the use of different enzymes and reagents or proper surface treatments was employed. we established the best conditions for dna amplification in silicon/pyrex microdevices depending on the type and the fabrication method used and on the quality of reagents more than on the passivation treatment or increment in standard taq polymerase concentration. nanoporous alumina fabricated using unconventional acids for enhanced biomolecular physisorption n. patra, m. salerno, r. losso, r. cingolani italian institute of technology, genova, italy in the last decades the available porecell sizes of porous alumina have been extended, but operating conditions to obtain some pore diameters still have to be optimized, particularly below the nm limit. in this range the use of biocompatible porous alumina thin films could find applications in biosensors, after functionalization by appropriate physisorbed biomolecules. while pore ordering and film growth rate are mainly influenced by the electrical parameters of voltage and current, respectively, the typical pore size primarily depends on the electrolyte. in the search for alternative anodization conditions, we have investigated several acids that have never been used before, namely gallic, lactic, propionic, and glycolic acid. the anodizations were carried out in galvanostatic conditions at fixed concentration and durations, by varying the current. atomic force microscopy was used to test the oxidized surface morphology. in particular, lactic and propionic acids demonstrated feasible. lactic acid gave best results for ∼ ma/cm current density, corresponding to roughly constant ∼ v potential, with resulting pore diameter in the nm range, whereas propionic acid performed best for ∼ ma/cm , corresponding to ∼ v, resulting in nm pore diameter. both kind of films looked lightgray, different from the yellowish oxalic acid porous alumina, which can be a hint of lower ion contamination. chitosan-arabic gum nanoparticles as potential vehicles for peptide delivery l. moutinho, s. rocha, m. coelho, m. c. pereira lepae, chemical engineering department, faculty of engineering, university of porto, portugal chitosan (cs) -arabic gum (ga) nanoparticles were produced by an ion-ion interaction process, using different weight ratios of polysaccharides. according to zeta potential (zp) measurements, particles are ionically stable. zp values ranged from to mv for cs:ga ratios of : . and : . , respectively. cs:ga : yielded uncharged nanoparticles and aggregates, showing that, at this ratio, the negative charges of ga neutralize the positive charges of cs. the particle diameters ranged from to nm, as measured by dynamic light scattering (dls), and displayed a slight tendency for decreasing as the ga concentration increased. transmission electron microscopy (tem) images showed numerous spherical nanoparticles. peptides were encapsulated within the nanoparticles, by mixing them with chitosan solution prior to adding ga. this system can protect short peptides from rapid metabolization, prolonging their blood half-life. physical characterization of nanocarriers for drug delivery s. motta , y. gerelli , g. sandri , e. ricci , p. brocca dept. of chem, biochem and biotech for medicine -university of milan, italy, dept. of physics -university of parma, italy, dept. of pharmaceutical chem -university of pavia, italy nanoparticles used as nanocarriers for pharmaceuticals can improve solubility and bioavailability of problematic drugs and protect labile or toxic molecules. fundamental parameters like drug encapsulation efficiency and release kinetics are tuned by the physico-chemical features of the drug/carrier complex. a challenging aspect of the pharmaceuticallyoriented issues resides in the relatively low number of molecules allowed to build up nanosystems with different properties and morphology, according to the specific drug and to the intended therapy. we have studied: ) soybeanlecithine/chitosan nanoparticles for progesterone and tamoxifen delivery; ) solid-lipid nanoparticles (compritol ato, poloxamer , tween and chitosan) for cyclosporine-a delivery via ophthalmic formulation. both void carriers and drug-loaded nanoparticles have been studied, to understand how the structure of the carriers can be modified by the active molecules. several non-invasive physical techniques have been used to achieve a detailed knowledge of the systems: a) dynamic light scattering for the dimensional distribution of the nanocarriers, b) zeta potential to determine surface charge, c) cryo-tem for morphological analysis d) x-ray scattering (in small angle configuration) and dsc to access information on the inner structure. . a. schollier , a. halperin , m. sferrazza , g. fragneto institut laue-langevin, grenoble, france, universite libre de bruxelles, belgium, cnrs-ujf grenoble, france protein adsorption on surfaces is responsible of several unwanted effects in technological and pharmaceutical applications: fouling of contact lenses, clotting in blood containing devices, inflammation around artificial organs for instance. those phenomena can be repressed with certain types of polymer brushes, in particular peg (polyethylene glycol) brushes, and a better understanding of the mechanism of adsorption could lead to improvements in the design of biomaterials. we have developed a theory describing this mechanism and carried out measurements to confirm the theoretical predictions [langmuir , , ] . three cases are predicted: primary adsorption at the grafting surface, secondary adsorption at the outer edge of the brush, and ternary adsorption within the brush itself. we prepared samples with different grafting densities and different degrees of polymerization and used different protein size and concentrations. neutron reflectivity experiments, able to determine the structure and composition of material interfaces with a fraction of nanometer resolution, were carried out and, by using deuterated proteins (anti-freeze protein, dihydrofolate reductase and myoglobin) on different peg compositions and grafting densities, it was possible to locate the proteins in the brush and to distinguish between the different kinds of adsorption : primary adsorption is dominant for short brushes and ternary adsorption for long brushes. effect of powder air polishing on nanocomposite dental materials measured by atomic force microscopy m. salerno , g. derchi , a. m. genovesi , l. giacomelli italian institute of technology, genova, italy, istituto stomatologico tirreno, lido di camaiore, italy in dentistry, airpolishing with glycine and bicarbonate powders is widely used to remove accumulated plaque. however, the resulting teeth and gingival surface roughness, which is a risk factor for further plaque accumulation, has to be considered. in this in vitro study the effect of the above mentioned technique on the surface of a novel nanocomposite dental material is evaluated by means of atomic force microscopy (afm). square specimens ( . × . cm size) were airpolished either with glycine or bicarbonate, using different application times ( , , sec) and distances ( , , mm) . four specimens were evaluated for each timedistance combination, and checked versus untreated specimens (controls). the specimens were imaged with tappingmode afm at two different scan sizes ( × and × µm ) in two different regions for each sample. the surface roughness was measured as the rms of the feature heights. for the sec mm group airpolishing resulted in an increased surface roughness as compared to the controls; however, glycine was associated with a lower roughness than bicarbonate (glycine: ± nm; bicarbonate: ± nm; controls: ± nm; p< . for treated specimens vs controls, p< . for glycine vs bicarbonate, anova test). similar results were obtained for all the other timedistance combinations. the work comprises the design of polysaccharide based nanoparticles for drug delivery. chitosan/arabic gum and arabic gum/maltodextrin nanoparticles were prepared by polyelectrolyte complexation and spray drying [ ] , respectively. dynamic light scattering characterization established that arabic gum/maltodextrin nanoparticles are more polydisperse (diameters ranging from to nm) than chitosan/arabic gum particles (diameters of approximately nm). scanning electron microscopy measurements demonstrated that the particles are spherical and have a smooth surface. the systems are highly stable to forces up to nn as observed by atomic force microscopy. due to their nature they are hydrophilic and biodegradable. the nanoparticles were used to entrap short peptide sequences and antioxidants and were proved to be efficient in maintaining the biological activity of the molecules. kyotorphin (ktp) was found in in bovine brain. despite revealing remarkable analgesic properties, analgesia was only induced after central delivery. this limited ability to cross the blood-brain barrier (bbb) combined with the unknown mechanism of action largely confines its pharmacological use. to surpass these problems, we designed new ktp derivatives: ktp-rc and ktp-rc-lipogen. biophysical studies were carried out using fluorescence methodologies to characterize the peptides' interaction with biomembrane model systems. partition coefficient quantification showed a clear preference of the derivatives towards fluid zwitterionic and anionic membranes. moreover, a relationship between anionic lipid percentage and in-depth insertion in membrane was established. additionally, studies with fluorescent probe di- -anepps revealed different membrane-interaction profiles of morphine and ktp-derivatives, suggesting distinct actions between them. the analgesic efficacy of the compounds was studied in vivo after systemic administration in models of acute and tonic pain. unlike ktp, both ktp-rc and ktp-rc-lipogen displayed high efficacy from doses as low as . and . mg/ g, respectively, indicating bbb crossing. the observed correlation between higher partition/insertion in the membrane and enhanced analgesic action proved the biophysical rationale to be a powerful strategy for early screening in cns drug development. metabolic syndrome (ms) is now regarded as a major risk factor for cardiovascular disease. as prooxidant/antioxidant balance is disturbed in the course of this disorder, it is possible that melatonin may exhibit protective effect against oxidative damage of blood cells. ms was diagnosed according to international diabetes federation definition ( ) . the investigated group consisted of patients before and after the melatonin supplementation ( mg per day for two months) and was compared to control group of healthy individuals on normal diet. our experiments show that erythrocytes from patient group exhibit significantly higher tbars and total cholesterol levels whereas the protein thiol concentration, na + k + atpase and glutathione peroxidase activities were decreased in comparison to those of healthy volunteers. after two month melatonin supplementation, tbars and cholesterol concentrations significantly decreased, whereas the na + k + atpase, catalase and glutathione peroxidase activities increased. the glutathione concentration was also higher. these results show that melatonin supplementation has a protective effect on erythrocytes of ms patients. action of ferritin, a nanoparticle model, on ros formation and glutamate uptake in synaptosomes t. waseem, a. alekseenko, s. fedorovich institute of biophysics and cell engineering, minsk, republic of belarus nanoparticles are currently used in medicine as agents for targeted drug delivery and imaging. however it has been demonstrated that nanoparticles induce neurodegeneration in vivo and kill neurons in vitro. the cellular and molecular bases of this phenomenon are still unclear. we have used the protein ferritin as a nanoparticle model. ferritin contains iron particles (fe + ) with size nm and a protein shell. we investigated how ferritin influences uptake and release of [ c]glutamate and free radical formation as monitored by fluorescent dye dcfda in rat brain synaptosomes. we found that even a high concentration of ferritin ( µg/ml) did not induce spontaneous [ c]glutamate release. in contrast the same concentration of this protein inhibited [ c]glutamate uptake two fold. furthermore ferritin induced intrasynaptosomal ros (reactive oxygen species) formation in a dose-dependent manner. this process was insensitive to µm dpi, an inhibitor of nadph oxidase and to µm cccp, a mitochondrial uncoupler. these results indicate that iron-based nanoparticles can cause ros synthesis and decrease glutamate uptake, potentially leading to neurodegeneration. functionalized carbon nanotubes (cf-cnts) have a promising future as vectors for drug delivery and for neuronal cell growth and communications . it has been demonstrated that cnts can be employed in drug delivery systems. the toxicological properties of cnts result strictly correlated with nanotube solubility . in this study we focused our attention on multi-walled cnts (mwcnts) because they are easy to manipulate and we covalently functionalize them in different ways to increase their solubility.. we grew different derivatives of a dendrimer on mwcnts surface with positive charges at the periphery, in order to study the different solubilisation properties and correlate them to the biological activity in gene therapy . slowdown of - β-peptide aggregation by addition of two synthetic biocompatible polymers p. sciortino , r. carrotta , g. cavallaro , d. bulone , p. l. san biagio ibf-cnr, palermo, italy, ctf dept., university of palermo, palermo, italy fibril deposit formation of amyloid β-protein (aβ) in the brain is a hallmark of alzheimer disease (ad). fibrils formation is triggered by molecular conformational changes and protein-protein interactions involving partially unfolded regions of different aβ peptide molecules. increasing evidence suggests that toxicity is linked to diffusible aβ oligomers, which have been found in soluble brain extracts of ad patients, rather than to the insoluble fibres. new therapeutical approach, based on searching molecules capable of regulating the peptide aggregation, is currently developing. here, we study the effects on the aggregation of aβ - peptide of two different synthetic polymers with structure similar to that of a protein (α,β-polyasparthylhydrazide -pahy and α,β-polyasparthylhydrazide-polyethyleneglycol -pahy-peg). static and dynamic light scattering measurements showed that the aggregation kinetic is slowed down by the presence of both polymers. optical microscopy revealed the presence of aggregates of different dimension in all samples. transmission electron microscopy allowed to establish that all aggregates are made of fibers, as confirmed by fluorescence spectroscopy measurements on thioflavine binding. a. smorodchenko , d. sittner , a. rupprecht , j. goyn , a. seiler , a. u. bräuer , e. e. pohl institute of cell biology and neurobiology, charité-universitaetsmedizin, berlin, germany, german federal institute for risk assessment, zebet, berlin, germany ucp is a member of the mitochondrial anion transporter family and one of the three ucps (ucp , ucp and ucp ) associated with the nervous system. in our previous work we have shown that ucp appears in brain at embryonic day (e ). we hypothesized that the ucp expression can be related to neuronal differentiation. to prove this idea we now investigated protein and mrna levels in two different systems: (i) mouse embryonic stem cells of line d (mesc) and (ii) brains from pre-and postnatal mice. ucp was not present in undifferentiated mesc. during differentiation of mesc in neurons the expression of neuronal marker map and ucp started at the same time -as early as on day in culture -and were increasing simultaneously. (ii) the comparative analysis of gene transcripts prepared from whole embryos, brains and different brain regions (neocortex, hippocampus, cerebellum) demonstrated that levels of ucp mrna were increasing from e till e , reached an expression peak between e and postnatal day (p ) and remained constant in adult animals. in contrast, expression of ucp was increasing permanently until birth, whereas ucp expression was invariant in time. our results suggest that ucp contributes to specific neuronal functions. the plant hormone abscisic acid stimulates the proliferation of human hemopoietic progenitors through the second messenger cyclic adp-ribose s. scarfì , c. fresia , c. ferraris , s. bruzzone , f. fruscione , c. usai , m. magnone , m. podestà , l. sturla , l. guida , g. damonte , a. salis , a. de flora , e. zocchi advanced biotechnology center, genova, italy, dept of experimental medicine, biochemistry section, and cebr, university of genova, italy;, institute of biophysics, cnr, genova, italy, stem cell center, s. martino hospital, genova, italy abscisic acid (aba) is a hormone involved in pivotal physiological functions in higher plants. recently, aba was demonstrated to be produced by human granulocytes, β pancreatic cells and mesenchymal stem cells (msc) and to stimulate cell-specific functions. here we show that aba expands human hemopoietic progenitors in vitro, through a cadpr-mediated increase of the intracellular calcium concentration. incubation of cd + cells with micromolar aba also induces transcriptional effects, which include nf-κb nuclear translocation and transcription of cytokines encoding genes. stimulated human msc produce and release aba at concentrations sufficient to exert growth-stimulatory effects on co-cultured cd + cells, as demonstrated by the inhibition of colony growth in the presence of an anti-aba monoclonal antibody. these results provide a remarkable example of conservation of a stress-hormone from plants to humans and identify aba as a new hemopoietic growth factor involved in the cross-talk between hp and msc. t. golovanova, g. b. belostotskaya sechenov institute of evolutionary physiology and biochemistry, russian academy of sciences, saint petersburg, russia a subpopulation of small cells (volume ± µm ) have been found in the neonatal cardiac myocytes culture whereas the same parameter of the remaining cells was ± µm on the st day. in contrast to main part of hypetrofied cardiomyocytes, the small cells were able to proliferate, form colonies and differentiate spontaneously into cardiac myocytes in the culture. on the th day there were slight, slow ( - beats/min), arrhythmic contractions in the centre of colonies. pulsing cells were not united by common contraction and had individual beating profile. on the - th days, the colonies displayed comprehensive contractile activity with the pulsation rate - beats/min and reached beats/min on the d - th day and beats/min on the - th days in the culture. it has been shown that receptors of the surface membrane and sarcoplasmic reticulum of colony cells gradually mature. by estimating the ca + transition under the specific agonists action: acetylcholine, kcl and caffeine, we have detected the activity of basic structural and functional elements of excitation-contraction coupling in contractile cells inside colonies. the small cells ability to proliferate and differentiate under the influence of near mature cardiomyocytes allows us to put forward a hypothesis, that they belong to a category of resident stem cells. changes in the gating properties of na + channels were studied in es-derived neural stem (ns) cells during in vitro neuronal differentiation with the use of the whole-cell and cell-attached variants of patch-clamp technique. ns cells represent a novel stem cell population remaining stable and highly neurogenic over multiple passages.voltage-clamp recordings during neuronal differentiation of ns cells indicated significant changes in the key properties of na + currents. a voltage-gated and tetrodotoxine-sensitive na + current,absent under self-renewal conditions, was first recorded following application of differentiative agents. current density increased with time of exposure to differentiating conditions. whole-cell and single channel analysis revealed that the observed increase in current density was due at least in part to changes in steady-state activation and inactivation properties. namely, half activation potential shifted from - mv to - mv, while half-inactivation potential shifted from - mv to - mv. furthermore, a contribution to the increase in na + current density could also be given by an enhancement in channel expression, as suggested by an augmentation in the number of single channels per patch area, with increasing neuronal differentiation. interestingly, those changes in na + channel activity well correlate with the capability of ns cells to generate action potentials during in vitro neuronal differentiation. a. viale, g. bonizzi, a. cicalese, f. de franco, c. pasi, s. pece, a. orleth, p. di fiore, p. pelicci european institute of oncology, milan, italy we are characterizing the biological differences between normal and transformed scs. scs are defined by their ability to generate more scs, termed self-renewal, and to produce cells that differentiate (asymmetric cell division). scs, however, possess the ability to expand in number (i.e. during development, in adulthood after injury/disease); this increase is not accounted by asymmetric divisions, in which only one daughter cell maintains sc identity. recent findings in invertebrates indicate that scs can also generate daughter cells destined to acquire the same fate (symmetric cell division). on the other hand, sc quiescence is critical to maintain tissue homeostasis after injury. here we show increased symmetric divisions of cscs in breast tumors (due to inactivation of the p tumor suppressor) and dependency of leukemia development on quiescent leukemia scs (due to transcriptional up-regulation of the cell cycle inhibitor p by leukemia-associated fusion proteins). we suggest that sc asymmetric divisions function as a mechanism of tumor suppression, that sc quiescence is critical to the maintenance of the transformed clone and that symmetric divisions of scs permit their geometric expansion. a. uccelli department of neurosciences ophthalmology and genetics, genoa, italy stem cells are considered as a possible source of cells for tissue repair. in this scenario damaged tissues will be reconstructed by newly formed cells with the result of a recovery of lost functions. thus, the rationale for utilizing stem cells for the treatment of neurological diseases such as multiple sclerosis (ms) stemmed from the idea that they differentiate in neural cells regenerating the damaged tissues at the basis of irreversibile disability. however, while multipotential embryonic stem cells may provide in the future an optimal source of cells competent for myelin and axons repair in ms, their use is still far from being exploitable in a clinical setting. moreover, it is widely accepted that adult stem cells may in vitro transdifferentiate in neural cells but recent experimental data have challenged the biological importance of this event in vivo. nevertheless, several experimental studies have provided new evidences supporting the use of adult stem cells derived form different human tissues for the treatment of ms. in the cases of mesenchymal stem cells and neural stem cells current experimental data support an unexpected therapeutic plasticity mediated by diverse paracrine mechanisms including neuroprotection, induction of local neurogenesis and modulation of the immune response. therefore, current experimental and clinical studies support the use of stem cells for the treatment of neurological diseases such as ms. unravelling the structure of the water splitting site of photosynthesis and implications for mechanism of catalysis j. barber imperial college london, u.k. photosystem ii (psii) is a multi-subunit membrane protein complex which catalyses the oxidation of water to molecular oxygen and reducing equivalents. the reaction occurs at a catalytic centre composed of mn ions and a ca ion, is thermodynamically demanding and generates highly oxidised species. unavoidable side reactions cause detrimental effects on the protein environment leading to the rapid turnover of the reaction centre d protein. to understand the mechanisms of water oxidation and d turnover structural information is required. initially the positioning of various protein subunits and their transmembrane helices were determined by electron microscopy. more recently a refined structure of the cyanobacterial psii unit has been elucidated by x-ray crystallography giving details of specific environments of the redox active cofactors. the implications of these structural studies will be discussed in relation to the unique facets of psii function, particularly the water splitting reaction. importantly this new knowledge is providing a blue print for the design of photochemical catalysts which can mimic the photosynthetic water splitting reaction and thus give hope that new technologies will emerge to provide humankind with a sustainable energy supply. photochemically controlled molecular devices and machines v. balzani department of chemistry "g. ciamician", university of bologna, italy the macroscopic concepts of a device and a machine can be extended to the molecular level. molecular-level devices and machines operate via electronic and/or nuclear rearrangements and, like macroscopic devices and machines, they need energy to operate and signals to communicate with the operator. the extension of the concepts of a device and a machine to the molecular level is of interest not only for basic research, but also for the growth of nanoscience and the development of nanotechnology. if a molecular device or machine has to work by inputs of chemical energy, it will need addition of fresh reactants ("fuel") at any step of its working cycle, with the concomitant formation of waste products that compromise the operation. currently there is an increasing interest in the use of light to power molecular devices and machines. the lecture will illustrate examples of recent achievements [ , , ] , which include molecular wires, switches, plug-socket systems, extension cables, antennas, and light powered nanomotors. biophysics laboratory, institute of botany, azerbaijan national academy of sciences, baku, azerbaijan it is well known that fast component of induction curves of millisecond delayed chlorophyll fluorescence (ms-df) originates via radiative recombination of reaction center with product on the donor side of psii. in view of our previous data (j. photochemistry and photobiology b: biology , ) it was shown that partners for radiative recombination of p q a − localized as a hole on the donor side of psii. depending on ph this hole might be on the camn -cluster or on y z and their recombination with p q a − can be monitored by the ms-df fast component. for analysis of site of damages, photoinhibition of psii particles from spinach at different ph was monitored by ms-df. during photoinhibition of psii ( µmol photons m − s − ) the fast component of ms-df was shown to be more stable at ph . and ratio of the fast component to the steady-state level of ms-df was approximately constant, while at ph . the fast component essentially decreased, the steady-state level increased and ratio of these components rapidly went down. it is possible concluded that strong light damaged of recombination reaction with in the presence ppbq -artificial electron acceptor -the protection of psii from photoinhibition has been observed only at acidic condition. stress factors such as heavy metals, strong light and low temperature were investigated by measurement of transient pictures of ms-df in intact plants, isolated chloroplasts and pure psii particles. the heavy metals action on psii was investigated on wheat seedlings. the targets for toxic action of al, mn and co ions were found to be a q a -q b acceptor side of psii. the damage site for cd + may be partners for recombination with p + -depend on of medium ph -either y z or camn -cluster. investigated ions (mn + , al + , cd + and co + ) have lead to reduction of chlorophyllprotein complexes pigment fund and cd + and co + destroyed also an apoproteins, especially of psii. the photoinhibition of psii was investigated in intact leaves of barley and maize seedlings at low and normal temperature. a very sharp reduction of intensity of ms-df second fast component, possibly y z * p ÿq a − radiative recombination of maize seedlings was observed even after short illumination by strong light at • c. ph dependence photoinhibition of chloroplasts and pure psii particles have shown that strong light damaged of camn -cluster in great degree than y z . hydrogenases are considered as potential energy sources. in particular, the ability of the green alga c . reinhardtii to reduce protons to hydrogen gas upon illumination by means of a [fefe]-hydrogenase is a phenomenon of great scientific interest, as it would need only light and water to generate energy. however, the catalytic activity is strongly inhibited by the o produced during photosynthesis; furthermore, the protein is expressed at very low levels, only in conditions of strict anaerobiosis. this mutually exclusive nature of o and h photoproduction represents a crucial problem in the development of h bio-production. the study of the structurefunction relationship of [fefe] hydrogenases, which would help to clarify the molecular mechanisms underlying both h production and o sensitivity, requires the characterization of purified native and modified proteins, which can be obtained by site-directed mutagenesis. we expressed the algal hydrogenase in the cyanobacterium synechocystis sp. pcc , which holds a bidirectional [nife]-hydrogenase with a well known maturation system ( ) . we obtained two constructs to stably transform synechocystis, enabling it to express the c. reinhardtii hydrogenase in an active form. this suggests that the [nife]-hydrogenase maturation pathway is able to drive the biosynthesis of functional [fefe] enzymes. these data open new perspectives about the indispensable presence of hyde, hydf and hydg auxiliary proteins ( , ) to obtain a correctly folded [fefe]-hydrogenase. the a low energy monomeric chlorophyll of cp to investigate the role of low energy spectral forms in energy transfer among chlorophyll protein complexes, we characterized one of the redmost chlorophylls of psii antenna complexes, the a of cp . pigment stoichiometry of the wild type (cp wt) and mutant lacking the a binding residue (cp a ) reconstituted complexes confirmed the loss of a chlorophyll a. to exclude the possibility of a marked excitonic interaction of the a with other chlorophylls, we quantified the decrease in dipole strength after mutagenesis as a function of the frequency and performed a second derivative analysis of the difference absorption spectrum cp wt minus cp a . these data, along with circular dichroism spectra of both complexes, support the idea that the a is a monomer in cp , in disagreement with recent suggestion of involvement in an excitonic dimer (mozzo et al., bba ) . chla a reorganization energy and inhomogeneous broadening were then determined through a thermal broadening analysis in the - k temperature range. these parameters allowed us to calculate the förster overlap integral of the homogeneously broadened a bandshape, a fundamental factor for energy transfer rate estimations between chromophores. a comparison with foi of chlorophyll in solution suggests that chla a may connect cp complex to psii core favoring energy transfer from cp towards other inner antenna low energy chlorophylls. in oxygenic photosynthetic organisms, reaction centre complexes catalyze electron transport from water to co using light energy absorbed by tetrapyrrole pigments bound to so called "antenna proteins". a major challenge in performing this type of photosynthesis consists on the difficulty of operating "one electron" transport through a multi-step pathway in the chloroplast environment which is the source of oxygen of biosphere. in fact, synthesis of ros, mainly singlet oxygen and superoxide anion and consequent photoinhibition is an intrinsically unavoidable consequence of photosynthesis that must be prevented and/or controlled in order to avoid photodestruction and death. mechanisms involved include chlorophyll (chl) triplet quenching, ros scavenging and controlled heat dissipation of excess chl singlet excited states. the latter process has been studied for over years with little success in elucidating its mechanism. reverse genomics and ultrafast-spectroscopy led to the proposal that the transient formation of carotenoid radical cations, followed by charge recombination might be the underlying mechanism of energy dissipation while three proteins belonging to the lhc superfamily could be the site hosting the reaction. probing the dark cycle to reveal regulatory networks controlling photosynthetic efficiency the food, fibre and fuel needs of an ever-increasing population are one of the challenges facing current society. higher plant photosynthetic efficiency is ∼ % whereas the theoretical maximum is thought to be ∼ - % giving potential for great improvements. it is not however clear where in the photosynthetic process the additional losses are occurring. we are adopting a systems biology approach to reveal the regulatory networks that control photosynthetic efficiency, the challenge being to make quantitative measurements of the input parameters to models of the network of photosynthetic reactions, and also to identify missing physical parameters and processes. the aim being to understand how they interact with other key physiological pathways and what impact they hold over photosynthetic efficiency. we report initial results in this poster from experiments using conventional and novel proteomic methods. e.g. an optical technique based on a non-linear dir method is being developed for proteomic and metabolomic analysis. unlike many conventional proteomic methods, which provide information on the relative levels of various proteins and metabolites, the viability of the dir method as a quantitative, sensitive and high throughput proteomics platform has recently been demonstrated. in collaboration with klug this project will employ dir as a both a proteomic and metabolomic tool. t. morosinotto , p. arnoux , g. saga , g. m. giacometti , r. bassi , d. pignol dip. di biologia, padova, italy, cea, cadarache, france, dip. di biotecnologie, verona, italy violaxanthin de-epoxidase (vde) is the enzyme responsible for zeaxanthin (z) production. the synthesis of this carotenoid in plants exposed to high light conditions is an important photoprotection mechanism, enhancing excess energy dissipation and reactive oxygen species scavenging. the inactive enzyme is normally soluble but, upon activation by low ph, it binds to the thylakoids membrane, where its substrate is found. we present here the first structural data on this enzyme obtained at both acidic and neutral ph. at neutral ph, vde is monomeric with its active site occluded in the lipocalin barrel. upon acidification, the barrel opens up resulting in a functional dimerization of the enzyme. the channel linking the two active sites of the dimer can harbour the entire carotenoid substrate and thus allow the parallel de-epoxidation of the two violaxanthin β-ionone rings, making vde an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate. structural data opened the possibility to investigate deeper this enzyme and further work allowed the identification of its active site, the protein domains responsible of its membrane association as well as key residues involved in the ph dependent conformational change. hydrogen is considered the fuel of the future if produced from sun light-driven water splitting. a hydrogen economy based on genetically modified organisms, immobilized enzymes or biomimetic synthetic catalysts requires a profound knowledge of the structure and function of the respective enzymes in nature. light-induced water splitting is performed by a tetranuclear manganese cluster located in photosystem (ps) ii of oxygenic photosynthesis. the protons generated by ps ii can be converted to molecular hydrogen by the enzyme hydrogenase, which is for example found in green algae and cyanobacteria. [nife]-and [fefe]-hydrogenases are the two main classes of this enzyme. they contain bridged binuclear transition metal cores, which are tuned by a special ligand environment to efficiently convert protons to hydrogen -or vice versa -via a heterolytic mechanism. rhodobacter sphaeroides strain a- isolated from mineral springs in armenia produces h with high rate in anaerobic conditions under light. in our previous work the inhibition of h production by n,n' -dicyclohexylcarbodiimide (dccd), the f f -atpase inhibitor, was shown, and dccd-inhibited atpase activity was determined. therefore it is possible to admit a role of this atpase in h production by r. sphaeroides; otherwise dccd inhibition of hydrogenase is not ruled out. in order to examine the mediatory role of proton-motive force (pmf) or proton atpase in this process transmembrane electrical potential (∆Ψ) and ∆ph are determined and the atpase activity is studied in r. sphaeroides grown under light. pmf was determined under anaerobic conditions in the dark. at ph . the ∆Ψ was of - mv and the reversed ∆ph was + mv, resulting in the pmf of - mv. but ∆Ψ was not affected by dccd. moreover, adenine nucleotide phosphates (anp) content is essential for cell functioning and may result with atpase activity. the percentage of atp was calculated from the total quantity of anp in whole cells, it was . %. a relatively high concentration of adp (∼ %) and amp (∼ %) and accordingly low energetic charge were noted; these might be indicative for the atpase activity, a further study is required. relationship between h production and atpase activity by rh. sphaeroides is suggested; possible mechanisms are discussed. identification of the sites of chlorophyll triplet quenching in relation to the structure of lhc-ii from higher plants. evidence the chl a molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric lhc-ii are identified by time-resolved and pulse epr techniques. the concept of spin conservation during triplet-triplet energy transfer is exploited. the sites with the highest probability to form triplet states, which are quenched by the central luteins, are chl and chl . unquenched chl triplet states are also produced by photo-excitation in the lhc-ii complex. putative sites of these triplet states are chl , chl , chla and chl , since they do not contribute to the formation of the observed carotenoid triplet states. fluorescence lifetime spectrum of the plant photosystem ii core complex the photosystem ii kinetic model (diffusion or trap-limited) is still much debated. there is discussion about whether energy transfer from the core antenna (cp and cp ) to the reaction center complex (d -d -cyt b ) is rate-limiting (transfer to trap-limited). this study investigates this problem in isolated core particles by exploiting the different optical properties of the core antenna and the reaction center complex near nm, due to p and an isoenergetic pheophytin. this was used as a marker feature for the reaction center complex. if the transfer to the trap-limited model were correct, assuming excited-state thermalization, the specific reaction center fluorescence decay lifetime should be shorter near nm, where there is reaction center complex specificity, than at the other emission wavelengths. such a selective reaction center feature was not observed in fluorescence decay measurements. at the experimental resolution used here, we conclude that the traplimited energy transfer to the reaction center could, at the most, be % limiting. thus, the transfer to the trap-limited model is not supported. photosynthetic apparatus response under heat stress n. pshybytko , i. divak , l. kabashnikova , e. lysenko institute of biophysics and cell engineering, national academy of sciences of belarus, belarus, institute of plant physiology, russian academy of sciences, russia the mechanisms of psii thermoinactivation and adaptation under high temperature impact and participation of hydrogen peroxide at these processes were studied. the twice suppression of oxygen evolving activity of thylakoids with simultaneous decrease in d protein content and the release of extrinsic kda polypeptide from woc after - min heating at o c were registered. using inhibitor analysis it was shown that thermoinduced degradation of d protein after min heating occurred by proteases. the participation of ftsh protease in thermoinduced d protein degradation was observed. level of transcription of psba gene in chloroplast was raised after min heating and was decreased through h. the content of hydrogen peroxide was increased three times after min of heating and was decreased to normal level through h and was raised after . h again. it is interesting that level of peroxidation lipids products was increased after . h heating only. received data indicated that hydrogen peroxide is signal molecular at the photosynthetic apparatus under heat stress. during heating the inactivation of woc and d protein is occurred. as result the h o is generated. hydrogen peroxide as signal molecule activates transcription of psba. turnover of psii is occurred. more long heating induces degradation of proteins and lipids and h o represents as the destructive agent. a. nurisso , m. a. morando , f. j. cañada , j. j. barbero , a. imberty cermav-cnrs, grenoble, france, centro de investigaciones biológicas, madrid, spain the mechanism of symbiosis between legume plants and rhizobial bacteria is a relevant topic of interest since it is at the basis of the nitrogen fixation process. this mechanism is strictly related to the production of lipochitooligosaccharides, nod factors (nf), which allow the bacterial invasion into the legume host roots. in medicago truncatula, the recognition of nf from the symbiont sinorhizobium meliloti requires the nfp gene, able to encode a lysm-motif receptor-like kinase. the extracellular part of this protein is characterized by three lysm motifs: only one of them seems to be involved in the nf recognition. herein, we report an in silico investigation of different structural aspects of this process. first of all, the conformational behaviors of a new generation of nf analogues were elucidated by mds simulation. then, a homology model of the lysm domain from m. truncatula was proposed and compared with a model of lysm domain from pisum sativum: docking calculations of natural nf identified a common binding site in which the carbohydrate portion is the main responsible of the binding. both studies provide support for the idea that the carbohydrate part of nf plays a key role in the interaction with lysm domains, while the lipid moiety modulates ligand specificity probably interacting with a potential second receptor. interaction between frutalin with biomembrane models and its correlation with cell adhesion property frutalin is a homotetrameric lectin d-galactose (d-gal) binding that activates natural killer cells in vitro and leukocyte migration in vivo, being a potent lymphocyte stimulator. in this study we investigated the interaction of frutalin with different phospho/glyco-lipids using langmuir monolayers as biomembrane mimetic system. the results attest the specificity of the protein for the carbohydrate d-gal when attached to the biomembrane model. the adsorption kinetics for frutalin to the mixed monolayers containing glycolipids showed that the interaction depends on the presence of charged groups and on the position of the d-gal on the polar head of the glycolipid. using brewster angle microscopy (bam), we investigated the morphology of the interface for the binary mixtures containing galcer, where small domains were formed at high lipid packing, suggesting that frutalin can induce the formation of likelipid rafts domains in vitro. the results obtained with the membrane models were associated with those from fibroblast adhesion induction. the cell adhesion promoted by frutalin is in accordance with the results observed in langmuir monolayers, which probes the specificity of the interaction between the lectin and d-gal on cell-membrane surfaces. based on these results, frutalin can be considered as a promise biotechnological tool to actuate in tissue engineering regeneration. supported by fapesp, cnpq and capes aggregation and glycation processes of proteins are of peculiar interest for several scientific fields. serum albumins are widely studied proteins for their ability to self-assemble in aggregates and also to undergo to non enzymatic glycosylation in cases of diabetes. in this work we report a study on thermal aggregation of glycated bovine serum albumin (bsa) prepared with different concentrations of glucose at ph . . increasing concentration of sugar modulates the effect of different glycation levels on the protein aggregation. fluorescence spectroscopy, ftir absorption, static and dynamic light scattering are used to follow the time evolution of the aggregation process and of protein conformational changes. conformational changes of secondary and tertiary structures are measured by ftir absorption; the kinetics of amide i, amide ii and amide ii' bands are monitored. the kinetics of tryptophans fluorescence give complementary information on the tertiary structure changes and on the polarity modification of the fluorophores environment. the aggregates growth is studied by dynamic light scattering measurements and rayleigh scattering peak. the results show that the partial unfolding of the protein is not affected by the glycation, while the presence of increasing amounts of glycated molecules progressively inhibits the aggregates formation. solvent occupancy analysis in ligand structure prediction: the case of galectin- s. di lella, m. a. martí, d. a. estrin inquimae-conicet, universidad de buenos aires, argentina formation of protein ligand complexes is a fundamental phenomenum in biochemistry. during the process, significant solvent reorganization is produced along the contact surface. using md simulations in explicit solvent combined with statistical mechanics analysis, thermodynamic properties of water molecules around proteins can be computed and analyzed in a comparative view. based on this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin- (hgal- ). specifically, we defined water sites (ws) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. we then succesfully extended the analysis of the role of the surface associated water molecules in the ligand binding and recognition process to many other carbohydrate binding proteins. our results show that the probability of finding water molecules inside the ws, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an oxhydril group of the ligand in the protein-ligand complex. this information can be used to predict possible complex structures when unavailable, and suggest addition of ohcontaining functional groups to displace water from high p(v) ws to enhance drug, specially glycomimetic-drugs, protein affinity and/or specificity. glyconanoparticles: nano-biomaterials for application in biotechnology and biomedicine s. penadés laboratory of glyconanotecnology, cicbiomagune -ciber-bbn, san sebastian, spain to study and intervene in carbohydrate interactions our laboratory has developed a chemical strategy (glyconanotechnology) to produce sugar functionalized gold nanoclusters with multivalent carbohydrate display (glyconanoparticles, gnps). by combining these tools with biophysical and analytic surface techniques (itc, afm, tem, spr) we have demonstrated and evaluated ca + -mediated carbohydratecarbohydrate interactions involved in cell adhesion processes. the gnp system complements other currently available multivalent systems incorporating carbohydrates and presents some advantages as: ) exceptionally small core size; ) high stability in water and physiological buffers; and ) multivalency and multifunctionality with control over ligands number. specific gnps have been applied to the inhibition of melanoma metastasis in mice and as inhibitors of hiv-trans infection. magnetic gnps have also been used in cellular labelling and imaging as probes for mri. in this lecture, the glyconanotechnology strategy will be presented and some application will be highlighted. -glycobiophysics - expanded ataxin- induces membrane mechanical instability: evidences from model systems and cells c. canale , s. averaimo , d. pesci , d. paulis , v. fortunati , a. gliozzi , m. mazzanti , c. jodice , a. relini iit, genoa, italy, university of milan, italy, university of tor vergata, rome, italy, university of genoa, italy spinocerebellar ataxia type is a neurodegenerative disorder involving the expansion of a polyglutamine stretch beyond a threshold in the protein ataxin- , with intracellular deposition of amyloid aggregates. ataxin- variants with polyglutamine stretches of pathological length are not associated to disease when the stretch is interrupted by histidines. mounting evidence suggests that ataxin- aggregates interact with the nuclear membrane. to get insight into the mechanisms leading to neurological disorders, we studied model lipid membranes containing an expanded pathological form of ataxin- or expanded normal forms interrupted by histidines. electrical measurements on planar bilayers showed the occurrence of current steps, much larger for the pathological form, indicating the formation of pore-like structures. atomic force microscopy measurements showed that the longer the polyglutamine tract, the smaller was the force required to penetrate the bilayer with the afm tip. the smallest penetration force, and then the strongest membrane destabilization, was observed for membranes containing the expanded pathological form. experiments on cos cells provided evidence that pathological protein aggregates damage the nuclear membrane eventually causing cell autophagy. modification by dopamine adducts links αsynuclein to oxidative stress in parkinson disease m. bisaglia , e. greggio , l. tosatto , f. munari , i. tessari , p. polverino de laureto , s. mammi , m. r. cookson , l. bubacco university of padova, italy, nih, bethesda, md, usa oxidative stress has been proposed to be involved in the pathogenesis of parkinson disease (pd). a plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (da) and produce various toxic molecules, i.e., free radicals and quinone species. α-synuclein (αsyn), a small protein found in lewy bodies characteristic of pd, is also thought to be involved in the pathogenesis of pd. to investigate the possibility of a synergistic role of oxidative stress and αsyn in pd, we analyzed the modulation of da toxicity by αsyn overexpression in dopaminergic human neuroblastoma cells. our results indicate that the increased expression of αsyn enhances the cellular toxicity induced by the accumulation of intracellular da. we then correlated our results with the structural modifications induced by the oxidation products of da on αsyn by studying two potential pathways for the oxidative chemistry associated with da. the first one is the auto-oxidation reaction which leads to the concomitant formation of radical and quinone species. the second is the enzymatic oxidation, mediated by tyrosinase, which leads only to the accumulation of quinones. our data suggest a link between da and αsyn in the progression of pd and provide novel insights on both the mechanisms involved in the oxidative chemistry and the aggregation properties of αsyn. dynamic light scattering aggregation studies of aβ ( - ) peptide s. bertini, r. beretta, d. gaudesi, a. naggi, g. torri ronzoni institute, milan, italy heparan sulfate (hs) proteoglycans play a role in formation of amyloid plaques by facilitating formation of amyloid aggregates. heparins and low-molecular weight heparins which mimic hs sequences could interact with amyloid peptides putatively involved in neurodegenerative processes. the aim of this work is to study the influence of chain length and structure of heparin oligosaccharides on the aggregation of amyloid. to this purpose, the interaction with amyloid peptide aβ − with a number of heparin/heparin oligosaccharides had been investigated using dynamic light scattering (dls) which permits to determine the size and the stability of molecular aggregates in solution. as indexes of aggregation in solution, two parameters were chosen, i.e., the area under the autocorrelation function (af) and the average hydrodynamic ratio (rh). we evaluated the aggregation kinetic of different preparations of aβ and the ph of solubilization. a clear indication was obtained for the trend of aggregation of the peptide aβ − alone and in the presence of oligosaccharides. the size of the aggregate in the presence of the tetrasaccharide is definitely lower than for the peptide alone. the size of the aggregates increases with increasing size of the oligosaccharides. in fact, the octasaccharide promotes further aggregation. it is crucial for pharmaceutical protein formulations to have low aggregate content. using the monoclonal igg antibody rituximab as a model system, we have studied the mechanisms by which antibodies aggregate at physiological ph when incubated at - • c. light scattering showed two coupled stages: an initial fast stage followed by several hours of exponential growth of the scattered intensity. data analysis showed the fast formation of a large species, which subsequently increased in size. the aggregate number density had a maximum suggesting that aggregates increase in size by coagulation. the analysis also predicted the actual underlying increase in aggregate mass to be linear and reach saturation. this was confirmed by size-exclusion chromatography of incubated samples. in an arrhenius plot the activation energy of the first stage was similar to the unfolding energy of the ch domain, suggesting a pivotal role of this domain in the aggregation process. cd and fluorescence showed only minor structural changes in the temperature interval studied. we conclude that coagulation is the main mechanism driving rituximab aggregation and that aggregation is due to small structural changes. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils -abstracts solid-state nmr structural studies of alzheimer's disease amyloid β( - ) in lipid bilayers j. d. gehman , a. k. mehta , f. separovic school of chemistry, bio institute, university of melbourne, vic, australia, department of chemistry, emory university, dickey dr., atlanta, ga, usa amyloid-β peptides are believed to cause loss of nerve cell function in individuals suffering from alzheimer's disease, where evidence suggests that interaction with the cell membrane correlates strongly with cytotoxicity. previous studies report a range of different but plausible structures, which depend on the molecular environment of the peptide. this sensitivity to sample preparation suggests that the structure relevant to disease is found in lipid bilayer membranes. while model membrane vesicles are too large to be studied by conventional solution nmr, solid-state nmr is one of the few technologies available to study such systems. we present recent measurements which suggest a novel peptide structure in a lipid bilayer environment: (i) rotational-echo double-resonance (redor) distance measurements between selectively enriched c-carbonyl and n-amide positions constrain dihedral angles of intervening residues and suggest that at least part of the aβ( - ) peptide folds into a β-sheet like conformation, in contrast to the helical and coiled structures in previous reports; and (ii) double quantum filtered draws measurements indicate that the extended strand does not assemble into an in-register parallel sheet as reported for amyloid fibrils. a mutation in app gene with dominant-negative effect on amyloidogenesis: a light scattering study e. del favero , g. di fede , f. tagliavini , m. salmona , l. cantù university of milan, segrate, italy, "carlo besta" national neurological institute, milan, italy, istituto di ricerche farmacologiche "mario negri", milan, italy we studied the effect of a single-point mutation (a v) on the aggregative properties of alzheimer's peptides aβ − . by laser light scattering it was possible to follow the early stages of aggregation of aβ − wt and aβ − mut (within hours after sample preparation), when intermediates in the aggregation pathway, rather than the mature insoluble fibrils, are formed. results show that both the kinetics and the extent of aggregation are strongly enhanced by the mutation. on the reverse, coincubation of mutated and wild-type peptides slows down the aggregation process and results in aβ aggregates that are much more unstable against dilution. it is evident that the interaction between mutant and wild-type aβ − interferes with nucleation or nucleation-dependent polymerization, hindering amyloidogenesis. these results account for the highly amyloidogenic effect of the mutation in vitro, if alone, reversely reduced by the mutated-wild type mix. also a clinical case exists that constitutes the in-vivo evidence of these two opposing effects. this finding may offer grounds for the development of therapeutic strategies, based on modified aβ peptides or peptido-mimetic compounds, for the potential treatment of alzheimer's disease. the collagen i is the major structural protein of the body and it is found organized in a hierarchical manner in the extra-cellular matrix of several tissues (bone, tendon, cornea and sclera, etc..). it is responsible for their specific architecture. it is already known that collagen i is capable of forming cholesteric liquid crystalline phases that once stabilized by a ph increase, lead to collagen matrices that mimic the organization of the organic matrix found in bone [ ] . in the present work, we analyze physico-chemical ways to modulate long range organizations obtained in this liquid state. we study the effect of collagen concentration, ph ( . / . ) and acids (hydrochloride acid / acetic acid) over the liquid crystalline order. in a original approach, correlation of collagen endofluorescence and shg signals has enabled us to quantify cholesteric pitch and phase transition as a function of collagen concentration within a gradient. cholesteric pitch have proved to be very responsive to physico-chemical conditions. indeed, the results allowed us to quantify a i / cholesteric transition as a subsequent transition from cholesteric to a phase hexagonal or colummar. prion diseases are deadly neurodegenerative diseases affecting human and mammalian species. according to the 'protein-only' hypothesis, the key event in the pathogenesis is the conversion of the α-helix-rich monomer (prp c ) into a polymeric β-sheet-rich pathogenic conformer (prp sc ). we have used a combination of biophysical techniques with molecular dynamics simulations (md) to elucidate the molecular mechanisms of prp c unfolding and polymerization. under well established conditions, three β-sheet-rich soluble oligomers were generated from the partial unfolding of the monomer, which were found to form in parallel. to obtain a deeper insight into the molecular events, doublecystein mutants were designed, thus 'locking' different regions of prp. single mutations were also performed, which affected dramatically and selectively the prp oligomerization pathway. furthermore, we have now identified the minimal region that leads to the same oligomerization profile as the full-length prp, namely, h h . the existence of at least three distinct oligomerization pathways and the effect of single mutations reveal the conformational diversity of prp and a possible relationship with prion strain phenomena. the identification of domains involved in the conversion process may lead to a better understanding of the effect of mutations or gene polymorphism on the evolution of prion pathology. investigating toxic protein nanoclusters and their interactions with living cells s. nag, b. sahoo, a. bandyopadhyay, c. muralidharan, r. abhyankar, s. gurav, s. maiti tata institute of fundamental research, homi bhabha road, colaba, mumbai , india amyloid protein aggregation is responsible for many neurodegenerative diseases, such as alzheimer's and parkinson's. it appears that quasi-stable small soluble aggregates are the key to amyloid toxicity. though amyloid aggregation appears to be a nucleation mediated process, simple nucleation theory is unable to explain the stability of these intermediate species. we investigate this issue with fluorescence correlation spectroscopy in solution and on cell membranes. our initial data, varying the ph and the ionic strength of the solution, show that a charged colloid model can explain the overall stability of these species. in addition, this understanding suggests possible ways of modulating the stability of these aggregates in solution. some of these strategies successfully decimate the stable aggregate population, and also reduce the toxicity of amyloid beta to cultured neurons. afm studies of artificial amyloid fibrils and filaments p. mesquida , a. kurtossy , c. macphee department of mechanical engineering, king's college london, london, u.k., school of physics, university of edinburgh, edinburgh, u.k. amyloid fibrils are beta-sheet-rich superstructures of peptides or proteins. although these aggregates have first been found in connection with protein-misfolding diseases, such as alzheimer's or parkinson's disease, there is evidence that the ability to form fibrils is a generic property of any polypeptide rather than a result of specific, disease-related amino-acid sequences. fibrils can easily be formed in vitro from nondisease-related proteins and even from synthetic peptides. these artificial fibrils can thus serve as model systems to investigate the biophysical properties of amyloid fibrils. the system presented here is built from the artificial peptide ttr − , which contains amino-acids and which forms well-defined nanorods of ca nm diameter. we present atomic force microscopy (afm) results of their inner morphology by chemically dissecting the rods into their constituent filaments without completely breaking up the aggregates. in contrast to the stiff rods, their constituent filaments of ca - nm diameter were much more flexible. this shows that the particular way filaments are arranged around each other can massively change the mechanical rigidity of the resulting structures in amyloid fibrils, which could be one cause of the different strains in amyloid diseases. the formation of insulin fibrils is characterized by an initial apparent lag-phase, related to the formation of oligomers, protofibrils and aggregation nuclei. afterwards, the aggregation proceeds via fibril elongation, thickening and/or flocculation, and eventual gelation. here, we focus on the formation of such a gel, made of insulin amyloid fibrils, upon incubation at high temperature and low ph. by light scattering and rheological techniques, we monitor the development of the structural, dynamical and mechanical properties of fibrillar aggregates, up to the dynamic arrest of the sample and to the appearance of a non-ergodic behaviour, which marks the onset of gelation. our experiments were able to reveal the structural details hidden in the apparent lag-phase, displaying the slow fibril nucleation and elongation. this initial stage is followed by the known exponential growth of structures of different sizes. these two kinetic stages of structural growth are mirrored by the kinetics of the viscoelastic properties and, in particular, by the growth of the elastic modulus. our results show that the appearance of a noteworthy elastic network, is associated with the initial fibril nucleation and elongation more than with the formation of large structures, which causes the eventual gelation. capturing the initial events of in-cubo crystallization of membrane proteins c. v. kulkarni , o. ces , s. iwata , r. h. templer chemical biology centre and department of chemistry, imperial college london, united kingdom, division of molecular biosciences, imperial college london, united kingdom, institute für chemie, heinrichstrasse- , university of graz, austria lipid based methods for the crystallization of membrane proteins including in-cubo crystallization are becoming popular in recent times. however, a complete understanding of the basic principles behind these methodologies is still elusive. the crystallization of membrane proteins in the lipid cubic phases involves following major steps: removal of membrane proteins from the native membrane using detergentlike molecules, followed by their insertion and equilibration into a lipid bilayer. the crystallization itself commences with precipitant induced osmotic dehydration which in turn stimulates the nucleation that subsequently leads to a crystal growth. using time-resolved x-ray scattering (saxs) and ultraviolet spectroscopy we have been able to gain new insights into the mechanism behind protein insertion into these complex three dimensional lipid structures including information on the timescales of protein folding relative to crystal growth and the effect of protein insertion on the morphology of the surrounding lipid matrix. several proteins can form amyloid fibrils under given environmental or thermodynamic conditions that affect their native conformation. lysozyme forms amyloid fibrils upon incubation of solutions at acid ph and at about • c for a few days. differential scanning calorimetry experiments confirm that lysozyme is mainly unfolded above • c at ph . we monitored the growth of aggregate size by dynamic light scattering for some days at different temperatures. our results show that the fibrillogenesis is characterized by an initial apparent lag phase and a subsequent growth with quadratic dependence upon time of the scattering intensity. this behaviour recalls a simple kinetic model of nucleation and elongation, with nuclei in equilibrium with monomers. at the end of the incubation at high temperature, we collected atomic force microscopy images which show fibrils with a diameter of a few tens of nanometers and a length of a few microns, characterized by a periodicity along the elongation axis. interestingly, the fibrils morphology exhibits no branching or thickening. this is consistent with the non-exponential growth observed in light scattering experiments. in order to elicit the role of repulsive electrostatic interaction in protein unfolding and self-assembly we extended our study at different ph. in this work we used afm to follow the amyloidogenesis pathway of transthyretin (ttr) to form protofilaments. single-molecule force spectroscopy (smfs) of native ttr and protofilaments were also compared in order to evaluate dynamic and structural differences. we observed that this pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers. although implicated in cytoxicity, the role of such oligomers within the amyloidogenesis pathway is poorly understood. we show that the annular species display a tendency to stack, forming tubular-like structures that precede the formation of protofilaments. the protofilament height and pitch resemble those of ttr amyloid reported in previous structural studies. upon solvent exchange, we also observed protofilament disassembly that revealed structures reminiscent of the initial ttr annular oligomers. smfs of protofilaments showed a time-dependent increase in the length of the manipulated structures, suggesting that associations between monomers stabilize with time. force spectra of native ttr and protofilaments contained transitions spaced by ∼ nm, indicative of sequential unfolding of individual β-strands. based on these results a model of ttr protofilament assembly is proposed. ability to undergo amyloid aggregation is affected by protein size c. parrini , h. ramshini , f. chiti , a. relini physics department, university of genoa, italy, biochemical sciences department, university of florence, italy short polypeptide chains, typically between and residues, are generally involved with the conversion from the native state into insoluble fibrillar aggregates. the low prevalence of large proteins is disproportionate with their high occurrence in the human proteome. in order to explore the propensity of large proteins to form amyloid-like fibrils, the -residue hexokinase-b from saccharomyces cerevisiae (yhkb) has been induced to aggregate under two separate conditions, at low ph in the presence of salts and at ph . in the presence of trifluoroethanol. such conditions are among the most promising to form amyloid-like fibrils by normally globular proteins. under both conditions yhkb aggregates very rapidly into species with significant β-sheet structure, as detected by circular dichroism, and a weak thioflavin t and congo red binding. atomic force microscopy revealed globular aggregates eventually clustering into large amorphous aggregates at low ph, while in the presence of trifluoroethanol ribbon-like structures with distinct morphology from typical amyloid fibrils were observed. they had irregular width, no twist, and were connected by thinner fibril segments perpendicular to them. in general, yhkb aggregates displayed an unusual softness, as they were very easily perturbed by the afm tip. these results suggest that inability to form amyloid fibrils may prevent large proteins from being associated with protein deposition diseases. the initial stage of proteins aggregation leading to amyloid fibrils: a saxs study m. g. ortore , f. spinozzi , f. carsughi , t. narayanan , g. irace , s. vilasi , p. mariani dip. saifet, univ. pol., ancona, italy, esrf, grenoble, france, dip. biochim. biofis., ii univ., napoli, italy under some conditions, a protein converts from its soluble form into highly ordered aggregates called amyloid fibrils, which are associated with many human diseases. in order to tackle the prevention and treatment of these diseases, we need to understand the mechanism of the pathological aggregation of proteins. in vitro amyloid fibril formation is preceded by the formation of metastable non fibrillar forms, which are responsible for cytotoxicity underlying neurodegeneration. the molecular mechanisms leading proteins into prefibrillar aggregates are still unclear. we present a saxs study performed at id beamline of esrf on the apomyoglobin mutant w fw f, which at physiological ph, firstly aggregates in prefibrillar forms that are cytotoxic and then forms amyloid fibrils. the first stages of w fw f oligomerization are induced by a ph jump. data show that big changes in w fw f in solution happen in less than ms. singular value decomposition (svd) of the data yields a set of functions, from which all the scattering curves can be reproduced. the major result of our study is the determination of the presence of different oligomers in each step of the process. hence, time-resolved saxs experiments together with the estimation of different oligomers via svd method, can be a new and useful approach to investigate the first stages of amyloidogenesis. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils - study of structure/toxicity relationship of amyloids by infrared spectroscopy h. p. ta amyloid diseases (alzheimer, parkinson, type ii diabetes, prion) correlate with protein aggregation. all the proteins associated with these pathologies aggregate into amyloid fibrils with a common ß-cross structure. according to many in vitro studies, the toxicity of various amyloids seems to be linked to some intermediates formed during the aggregation pathway and to their interaction with membranes. our aim is to understand the link between structure and toxicity of amyloids by studying their interaction with model membranes. for this, we use as an amyloid model, the prion-forming domain pfd - (wild type wt) of het-s, a prion protein of the fungal p. anserina. when expressed in yeast, wt is not toxic whereas one of these mutants, m is toxic. in vitro, m forms very unusual short amyloid fibers contrary to wt which polymerizes as long fibers. furthermore, atr spectra have shown that m is essentially assembled into mixed parallel and anti-parallel ß-sheets whereas wt displays a predominant parallel organization. we are currently studying the interaction between wt or m with lipid langmuir film by polarized modulation-infrared reflexion absorption spectroscopy (pm-irras). this technique allows determining the secondary structure of proteins and their orientation in the membrane. the study of interactions with different charged phospholipids is following. a. stirpe, m. pantusa, r. bartucci, l. sportelli, r. guzzi dipartimento di fisica, laboratorio di biofisica molecolare & udr cnism, università della calabria, rende (cs), italy an increasing number of experimental studies is demonstrating that the propensity of forming amyloid fibrils is not limited to proteins related to neurodegenerative diseases but it is a more general phenomenon. we present data on human serum albumin (hsa) aggregation induced by a combination of thermal and metal ions effects investigated by optical density, fluorescence and electron paramagnetic resonance (epr). the turbidity experiments as a function of temperature show that the hsa aggregation starts at about • c which is higher than the denaturation temperature (∼ • c). in the presence of copper and zinc metal ions the onset temperature for protein aggregation is markedly reduced as the metal:protein molar ratio is increased from : to : . moreover, the copper is more effective in inducing protein aggregation compared to zinc ion. the hsa aggregation analyzed by tht fluorescence at different incubation temperatures lacks a lag phase and the kinetic traces can be fitted by double exponential functions. the tht fluorescence increase evidences the formation of protein aggregates with fibrillar features. epr experiments for the cu(ii)-hsa complex show the binding of cu(ii) in the protein native state in a square planar coordination with equatorial n atoms, and is not influenced by the heat treatment of the protein. the overall results suggest that hsa aggregation is compatible with a downhill process that does not require the formation of an aggregation nucleus. protein condensation diseases -a colloid physicists viewpoint p. schurtenberger adolphe merkle institute, university of fribourg, ch- marly , switzerland a broad class of diseases, such as cataract, alzheimer's disease and sickle-cell disease, involve protein association phenomena as an essential aspect. the basic element common to all members of this class of molecular condensation diseases is the subtle interplay between protein interactions that produces condensation into dense, frequently insoluble mesoscopic phases. among this class of diseases, cataract is particularly important as the world's leading cause of blindness. this disease is most often the consequence of an uncontrolled aggregation (or phase separation) of the proteins in the eye lens that results in a loss of its transparency. the high concentration protein mixtures present in the eye lens are normally stable and produce a high refractive index that aids the eye in adaptive focusing of light. moreover, the proteins themselves exhibit a rich variety of repulsive interactions, attractive interactions, sizes, phase transitions and self-association. these mixtures also exhibit the pathological aggregation and opacification of the cataract disease that has inspired their study. in my presentation i will illustrate how we can use a combination of small-angle neutron and xray scattering experiments combined with molecular dynamics computer simulations to identify, measure and model the molecular interactions and emergent optical and viscoelastic properties and the phase behavior of the relevant, complex cytoplasmic mixtures. fluorescence microscopy studies of iapp fibrillation at model and cellular membranes d. c. radovan , n. opitz , r. winter department of physical chemistry i -biophysical chemistry, tu dortmund university, dortmund, germany, max-planck institute for molecular physiology, dortmund, germany type diabetes mellitus (t dm) is characterized by islet amyloid deposition and beta cell death, the main culprit being a small a.a. peptide hormone, islet amyloid polypeptide (iapp), which forms fibrils under pathological conditions. we studied the interaction of iapp with giant (guvs) and large (luvs) unilamellar vesicles as well as with ins- e cells. by using confocal / two-photon excitation fluorescence microscopy and guvs, we tested the influence of charge (dopc:dopg : ) and model raft systems, displaying liquid-ordered (l o ) / liquid-disordered (l d ) phase coexistence (such as dopc:dppc:cholesterol : : ), respectively, on the kinetics of iapp fibril formation. a preferential partitioning into the l d phase was observed and fibrils grew along with lipid uptake. fluorescence spectroscopy leakage experiments with carboxyfluorescein-filled luvs and the corresponding tht kinetics of iapp fibril formation were carried out as well. moreover, using the wst- reduction assay and fluorescence microscopy, we could show that the red wine compound resveratrol is a potent inhibitor of iapp fibrillation and its cellular toxicity on ins- e cells, these findings highlighting the potential role of resveratrol in future clinical applications, i.e., in the treatment of t dm. a remarkably high viscosity has been induced in aqueous solutions of lysozyme by the addition of certain structurally related organic solvents, such as tetramethylurea (tmu), dimethylsulfoxide (dmso), dimethylformamide (dmf), and hexamethylphosphortriamide. dmso-induced gelation is observed in samples fulfilling the two following requirements: ( .) lysozyme concentration in excess of mm, and ( .) volume fractions of dmso exceeding . . based on spectroscopic data, the whole process was characterized as consisting of two mutually independent stages. the first involves an extensive transition of the polypeptide backbone, from a predominantly helical to increased random coiled and beta-sheet structures, with the occurrence of non-orthodox protein secondary structures at regions above the solvent critical point. the second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. here we present a set of scattering and microrheology experiments that investigate both the structural and dynamic properties of the gels under various experimental conditions. studying alpha-synuclein aggregation by fluorescence polarization based kinetics l. tosatto, f. munari, i. tessari, g. de franceschi, m. pivato, p. polverino de laureto, m. bisaglia, l. bubacco università degli studi di padova, padova, italy alpha-synuclein (syn) is linked to parkinson's disease (pd) by two evidences: the accumulation of amyloid fibrils of the protein and the autosomal dominant forms of the disease (a t, a p and e k mutants). protein oligomers seem to be the most toxic species, causing dopaminergic neuronal death, probably disrupting cell membrane (volles & lansbury, ) . the aggregation of syn follows a nucleation dependent mechanism, i. e., aggregation is favoured only after the formation of an oligomer composed of a critical number of monomers (wood et al., ) . as the constitution of the nucleus is a rare event, early aggregation stages are difficult to study. to explore the early stages of the aggregation process a fluorescence polarization (fp) based method (luk et al., ) was applied to study syn oligomerization. fp depends on the size of the fluorescent molecule, therefore it is suitable for the detection of oligomers formation. we measured aggregation kinetic properties under several different conditions. a to mixture of syn and oregon green labelled syn was used to analyze the aggregation behaviour of wild type (wt) and pd mutants. the wt protein shows the fastest aggregation rate. this aggregation process is slowed down by the presence of the chaperone - - eta in wt syn samples. finally, dimers formed by a disulfide bond at the n-terminus or c-terminus of syn, when tested for their aggregation behaviour, show a different propensity to aggregate. a. pardini , r. bizzarri , a. diaspro , p. bianchini , c. usai , p. ramoino , g. checcucci , g. colombetti istituto di biofisica, cnr, italy, a) nest, sns, iit udr, pisa, italy; b) nest, cnr-infm, pisa, italy, univiversity of genova, genova, italy the colored ciliates blepharisma japonicum and fabrea salina show photomotile responses triggered by endogenous pigments of the hypericin family. b. japonicum exists in two forms: the wild one contains red blepharismin; the other one, generated by irradiating the cells with dim visible light, blue blepharismin. b. japonicum shows step-up photophobic response, whereas f. salina, that contains fabrein, shows step-down photophobic responses and positive phototaxis. we showed by confocal microscopy that the pigments are localized not only in pigment granules, but also in the cilia. this fact implies that the structure of the pigment in the cilia differs from that in the cell body, (pigment granules are too big to fit in the axoneme) and suggests that ciliary pigments might play a decisive role in photoreception. fluorescence lifetime imaging microscopy (flim) shows that there is a spatial distribution of lifetimes, which are shorter for the pigment in the cilia. this might indicate a functional role for these pigments. furthermore, lifetimes for f. salina are always longer than those of blepharisma. in order to further characterize the structure of ciliary pigments by means of spectroscopic methods, we have also performed spatially resolved static and time-resolved fluorescence anisotropy measurements. biosensing applications of micro/nano structured silicon several topics related to porous silicon (ps) biosensing properties were carefully considered in this study. ps allows the increasing of the immobilised biomolecule number on its surface and the creation of stable covalent bonds due to its controllable chemistry. beside this, ps is suitable for electrical (conductance, impedance), electrochemical and optical amplification of the detected signal. the experimental results on the fabrication of the ps microstructures such as: (i) protein immobilization and detection using microarray technique; (ii) dna biomolecule detection by impedance or by fluorescence spectroscopy; (iii) very sensitive sers biosensors (raman signal of -mercaptoundecanoic acid); (iv) sensitive element for neurons in nutmix culture are in detail presented. various characterisation techniques have been used, optical and scanning electron microscopy (sem), x-ray diffraction, raman, laser fluorescence and impedance spectroscopy for investigation molecule attachment on the au/ps structures. we have demonstrated that different morphologies of ps as-prepared or coated with gold nanoparticles have an important role in biomolecule/cell detection, due to its large internal surface combined with specific optical properties, being in the same time sensing element/support for immobilization of sensing biomolecules as well as transducer for biochemical interactions. heterotrimeric g-proteins interact with their g-protein coupled receptors (gpcrs) via key binding elements comprising the c-terminal segment of the α-subunit and the two lipid anchors at the α− and γ−subunit. direct information about diffusion and interaction of gpcrs and their g-proteins is mandatory, as these properties will affect the timing of events in the complex signal transduction cascade. in the case of the photoreceptor rhodopsin, receptor packing in the membrane and the related diffusion coefficients are discussed controversially ( , ). by using single particle tracking we show that the encounters of rhodopsin with the fluorescently labeled cterminus of the α-subunit as well as with the holo-g-protein transducin change upon rhodopsin light-activation. our results indicate confined areas of interaction for the c-terminal segment of the α-subunit with inactive rhodopsin disk membranes and less restricted diffusion of the receptor-bound cterminal segment after light-activation. this suggests dynamic short-range order in rhodopsin packing and specific structures for efficient interaction ( ) in vivo, skeletal muscle actively shortens and also resists lengthening, for example when landing after a jump. we study the energy during shortening and during lengthening by measuring the rate of pi release which results from atp hydrolysis. we show here the rate of pi releases in an in vitro protocol that involves muscle stretched followed by muscle shortening. it is hypothesised that the magnitude of deceleration of pi release in the stretch phase is less than the acceleration during the release due the energy input of the motor that is used to apply the length changes. therefore the end point of pi release is similar to isometric conditions. pi release responses to a ramped stretch ( %) followed ms later by a symmetrical release at low velocities ( . l /s − ) were measured in permeabilised fibre bundles of rabbit psoas at • c. laser diffraction and high speed video were also recorded to confirm length change. we show that the rate of pi release drops during the stretch phase, returns to isometric levels during the length hold phase, and finally accelerates during the ramped release. tracking of sarcomere length change using video analysis demonstrated that laser diffraction is unreliable at times during stretches due to lack of uniformity of the sarcomere spacing. microbial rhodopsins: receptors, channels, and pumps from a single design j. l. spudich, e. negri-spudich center for membrane biology, university of texas, houston, texas, u.s.a. the microbial rhodopsin family is comprised of ∼ homologous proteins containing transmembrane helices forming a pocket for the chromophore retinal. most are lightdriven ion pumps ("transport rhodopsins") and others are photosensory receptors ("sensory rhodopsins"). phylogenetic analysis indicates frequent lateral gene transfer of proton pumps among prokaryotic and unicellular eukaryotic species, followed by coupling of the pump's mechanism to the cell's existing signal transduction machinery to create photosensors. this evolutionary path is strongly supported by our studies of sensory rhodopsins in various organisms which demonstrate remarkably diverse signaling mechanisms with diverse transducer partners. the best studied are the phototaxis receptors in haloarchaeal prokaryotes (sri and srii) and in eukaryotic algae (channelrhodopsins). sri and srii transmit signals by protein-protein interaction to control a phosphorylation cascade that modulates motility. channelrhodopsins are light-gated cation channels that depolarize the membrane mediating calcium ion influx into the flagellar axoneme. crystallography and molecular biophysics have begun to clarify how modifications of the same architecture enable the rhodopsins to carry out their distinctly different molecular functions. interconversions of their functions by mutation reveal the elegant simplicity by which evolution uses existing genes to create proteins with novel functions. gold colloids-fluorophore complexes for protein detection assay l. sironi , s. freddi , l. d'alfonso , m. collini , m. caccia , g. tallarida , s. caprioli , g. chirico dipartimento di fisica, università di milano-bicocca, italy, laboratorio nazionale mdm, agrate brianza (mi), italy noble metal nanoparticles (np) are endowed with peculiar optical properties related to the surface plasmon resonances (spr). the interaction of surface plasmons of gold nanoparticles with fluorophores a few nanometers away from the surface modifies their brightness and excited-state lifetime, and this effect can be exploited to obtain nanodevices for proteinprotein recognition. we studied different types of constructs based on gold nps on which derivatives of fluorescein were bound. the interaction of this fluorophore with the gold surface plasmon resonances, mainly occurring through quenching, affects its excited-state lifetime, that is measured by fluorescence burst analysis in standard solutions. the binding of proteins to the gold nps through antigen-antibody recognition further modifies the dye excited-state lifetime. this change can therefore be used to measure the protein concentration. streptavidin-functionalized gold nps of size - nm are used to bind biotin-fluorescein and biotin-antibodies for specific proteins. we have first tested the constructs for bovine serum albumine (bsa) detection. the data reported here indicate that one can measure the concentration of bsa in solution with an apparent limit of detection of ± pm. we have then extended the study to the antitumor protein p -p antibody interaction in standard solution and directly in cellular extracts. calcium transport and phototransduction in isolated rod outer segments g. rispoli dip. biologia ed evoluzione, università di ferrara, italy ca + concentration in photoreceptor rod outer segment (os) strongly affects the generator potential kinetics and light adaptation. light stimuli may produce voltage changes exceeding mv: since the os ca + extrusion is entirely controlled by the na + :ca + ,k + exchanger (nckx), it is important to assess how the nckx ion transport is affected by voltage and intracellular factors. the nkcx regulation was investigated in whole-cell recorded os, using ionic conditions that activated maximally forward and reverse exchange. in all species examined of amphibia and reptilia, the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. since hyperpolarization increases ca + extrusion rate, the recovery of the dark level of ca + (and of the generator potential) after light stimuli results accelerated. mg-atp doubled the size of forward and reverse exchange current without modifying their voltage dependence, indicating that mg-atp regulates the number of active exchanger sites and/or the nckx turnover number. ca + jumps achieved via photolysis of caged-ca + produced current transients, possibly originating from electrogenic partial reactions. no monovalent cation substituted for na + at the nckx binding sites, but rb + substituted for k + , while sr + , ba + , mg + substituted for ca + with an apparent permeability ratio of . , . , < . : , respectively. a. pfeifer , k. zikihara , s. tokutomi , j. heberle , t. kottke bielefeld university, bielefeld, germany, osaka prefecture university, sakai, japan the blue light receptor phototropin regulates the growth of plants towards the light. it contains two light-, oxygen-, or voltage-sensitive (lov) domains and a kinase domain. lov domains bind noncovalently flavin mononucleotide (fmn) as chromophore. upon illumination, the triplet excited state of flavin reacts within few microseconds with a nearby cysteine under formation of a photoadduct, which represents the signaling state. in response to adduct formation, a jα helix adjacent to the lov domain dissociates and allows for autophosphorylation by the kinase domain. the mechanism of the photoreaction and the signaling pathway from fmn to the jα helix are still unclear. we have investigated the lov domain of arabidopsis phototropin by microsecond ft-infrared spectroscopy. the difference spectrum recorded at µs provides evidence that the flavin is unprotonated in the triplet excited state. therefore, a previously proposed ionic mechanism of bond formation is disfavored. changes in secondary structure were detected concomitant with adduct formation that relax with a time constant of µs. this early adduct intermediate has not been previously characterized. the final adduct state is formed in milliseconds by further alterations in secondary structure. these findings raise the question of whether the early or late adduct intermediate propagate the signal to the jα helix. -photosensory biophysics - the psychostimulant amphetamine increased no generation measured by epr as well as amino acid release in the rat brain nitric oxide (no) is a novel messenger that modulates many functions of the nervous system. the involvement of no in brain damage was shown mainly by indirect evidence and the data are controversial. the short half-life of no makes its direct detection difficult. we measured no generation using epr spectroscopy based on determination of the amount of paramagnetic mononitrosyl-iron complexes. the aim was to elucidate whether psychostimulant drug amphetamine (amph) modulates formation of no and lipid peroxidation (lpo) products as well as the neurotransmitter release in rat brain. the output of glutamate, aspartate, gaba and acetylcholine (ach) was monitored in striatum by microdialysis with hplc detection. amph produced fold elevation of no generation and lpo formation in brain areas. while amph increased the aspartate, gaba and ach release, the glutamate output was not affected. pretreatment with the neuronal nos inhibitor was highly effective in abating the rise of no and neurotransmitter levels but failed to influence the lpo intensity elicited by amph. the findings suggest that activation of no synthesis is a potent factor in the amph-induced neurotransmitter release. light scattering study of dna over a wide chain length range: comparison with wormlike model p. baeri, m. zimbone dipartimento di fisica e astronomia, università di catania, italy this work reports light scattering measurements on dna in aqueous solutions ( mm nacl , mm edta and mm tris-hcl buffer, ph . ) over a wide range of molecular weights ( - base pairs) and shows that, in the above standard solvent, shorter chains ( < base pairs) behave as a "wormlike chain" and their diffusion coefficient as obtained by dynamic light scattering measurements, confirm the prediction of standard wormlike model, whilst longer chains ( > base pairs) behave in a different manner. dynamic and static light scattering and sem analysis indicate that dna molecules base pairs long, condense into compact structures in our solvent condition. calculations done using a wormlike model are also presented and discussed in comparison both to our experimental data and to other data reported in the literature. a. asandei, t. luchian 'alexandru i. cuza'university, faculty of physics, laboratory of biophysics and medical physics, blvd. carol i no. , iasi, romania amphotericin b (amb) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. in this study we investigated the influence of ph upon the selectivity and the transport properties of amb channels inserted in reconstituted, ergosterolcontaining zwitterionic lipid membranes. our electrophysiology experiments carried out on single and multiple amb channels prove that at ph= . these channels are anion selective, whereas at neutral and alkaline ph's (ph= and ph= ) they become cation selective. we attribute this to the ph-dependent ionization state of the carboxyl and amino groups present at the mouth of amb molecules. surprisingly, our data reveal that the single-molecule ionic conductance of amb channels varies in a non-monotonic fashion with ph changes, which we attribute to the ph-dependent variation of the surface and dipole membrane potential. we demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low ph values, amb channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition. we report a detection method for the redox state of proteins which combines fret-based fluorescence/confocal microscopy on dye-labeled protein with cyclic voltammetry. by using this combined method, electron transfer properties can be revealed from protein to electrode or from redox enzyme to substrate. we applied the fluorescent detection to azurin, a blue copper protein from the bacterium ps. aeruginosa, fluorescently labeled on the n-terminus for monitoring the redox state of the protein. the dye fluorescence is quenched by energy transfer to the copper in oxidized, but not in reduced azurin. fluorescence results demonstrated that cy -labeled wtazurin switched in fluorescence intensity by up to % by varying the applied potential. labeled zinc-azurin was used as a control sample and did not show any fluorescence switching. for single molecule studies wt-azurin was labeled with atto dye and a mixed sam was used, with -hydroxy- octanethiol as a blocking agent to prevent non-specific binding of protein on the surface, and , decanedithiol for the specific covalent binding to the protein. preliminary data show that single-molecule fluorescence switching with the potential is indeed possible. -single molecule biophysics - is there a specific erythrocyte membrane receptor for fibrinogen? an atomic force microscopy approach f. a. carvalho , s. connell , r. a. ariëns , n. c. santos instituto de medicina molecular, univ. lisbon, portugal, university of leeds, u.k. fibrinogen (fg) contributes to erythrocyte (rbc) hyperaggregation by an increase in. rbc-fb protein binding considered to be non-specific. glycoprotein α iib β is a specific integrin receptor for fibrinogen on platelets. we showed that there is a single molecule interaction between fg and an unknown receptor on rbc membrane, with a lower affinity when compared with platelet binding. we evaluated if rbc-fg binding is through an integrin-like receptor or not. interactions between fg and platelet/rbc receptors were studied by force spectroscopy. force curves were performed between fg-functionalized atomic force microscope tips and rbc or platelets. to evaluate if the fg-rbc binding is calciumdependent, similar studies were performed in the presence of ca + or edta. we also carried out studies in the presence of a α iib β inhibitor and of methyl-ß-cyclodextrin (to disrupt lipid rafts by cholesterol depletion). rbc-fg single forces were of - pn and of - pn for platelet-fg binding in presence of calcium. a significant decrease of the platelets-fg force-rupture was obtained in the presence of edta or α iib β inhibitor. significant lower fg-rbc force value was obtained in the presence of mβcd, but not in the presence of edta. conclusion: fg-rbc binding seems not to be calcium-dependent but the existence of cholesterol on rbc membrane is important. nanoscopic and spectroscopic investigation of p -based complexes at single molecule level a. r. bizzarri biophysics and nanoscience centre, cnism, facoltà di scienze, università della tuscia, largo dell'università - viterbo, italy p is a transcription factor that plays a widely recognized role in preventing cancer development in response to dna damage. the tumor suppressor activity of p involves the formation of several complexes whose detection and study, at single molecule level, could be extremely relevant to understand, in detail, the mechanisms governing the cancer defense processes, as well as to develop ultrasensitive biosensors. p -based complexes, are investigated at molecular level, by combining atomic force microscopy (afm), atomic force spectroscopy (afs) and surface enhanced raman spectroscopy (sers) with the support of computational docking. our attention is mainly devoted to study complexes between p and the electron transfer azurin which has been demonstrated to interact with p , by promoting its stabilization. a possible competition between azurin and the cellular oncogene mdm is also investigated. unwinding the dna helix in force clamp condition p. bianco , l. bongini , m. dolfi , l. vincenzo physiolab, dbe, university of florence, italy, university of florence and centro interdipartimentale studio dinamiche complesse, firenze, italy we use a dual-laser optical-tweezers (dlot, smith et al., science, ) to define the highly cooperative conformational transition in the molecule of dna, where the natural b-dna has converted into a new overstretched conformation called s-dna (bensimon et al., phys. rev. lett., ; cluzel et al., science, ) . single molecules of double stranded λ-phage dna (in a solution with mm naci, mm tris-hcl, mm edta, ph . . and • c) are stretched either in length clamp or in force clamp mode. when the dna molecule is stretched in length clamp mode with a ramp lengthening, it shows the previously described highly cooperative overstretching transition at ∼ pn, attributed to unwinding from the b-form to the . times longer s-form. stretching the molecule in force clamp mode with a staircase of force steps at s intervals (step size - pn, rise time - ms) shows, for any given clamped force f in the region of the overstretching transition, different amounts of dna elongation (∆l) with exponential time courses. the analysis of the elongation rates allows to recover all the necessary parameters for an effective two-state model able to reproduce the out-of-equilibrium properties of the system. the results imply an unwinding cooperativity of bps. this value is significantly lower than that obtained assuming force independent rate constants. supported by miur, ente cassa di risparmio di firenze and itb-cnr (milano). c. m. becker , a. benedix , b. l. de groot , a. cafisch , r. a. böckmann saarland university, saarbrücken, germany, mpi for biophysical chemistry, göttingen, germany, university of zürich, zürich, switzerland modifying the stability or the binding behavior of the involved proteins by mutation can influence the activity of cellular processes. for an efficient identification of possible mutation-sites a fast calculation of the free energy of proteins is crucial. here we developed a fast and reliable method (cc/pbsa) [ ] for the prediction of the change in stability of proteins and binding affinity of protein-protein complexes upon mutation. the energy function of cc/pbsa is based on gas phase energies, solvation free energies and entropic contributions. the protein flexibility is taken into account by generating random conformations based on geometrical constraints only applying the concoord [ ] program. we applied cc/pbsa on the tem -blip complex, which is important in bacterial antibiotic resistance. the results of single-and double-point alanine scanning are used to detect hot spots, cooperative effects, and the corresponding energy distribution. cc/pbsa is freely accessible on our web-server: http://ccpbsa.bioinformatik.uni-saarland.de the human recombinase hrad is a key protein for the maintenance of genome integrity and for cancer development. this protein plays a central role is the dna strand exchange occurring during homologous recombination. here we report the polymerization and depolymerization of hrad on duplex dna observed with a new generation of magnetic tweezers, allowing the measurement of dna twist with a resolution of • in real time. at odds with earlier claims, we show that, after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition of single proteins, involving dna twisting steps of ± • . simple numerical simulations support that this mechanism is an efficient way to minimize nucleoprotein filament defects. this behavior, consisting of different stoichiometry for nucleation and growth phases, may be instrumental in vivo. fast growth would permit efficient continuation of strand exchange by rad alone while the limited nucleation would require additional proteins such as rad , thus keeping this initiation step under the strict control of regulatory pathways. besides, our results combined with earlier structural information, suggest that dna is somewhat less extended ( . versus . Å per bp) and more untwisted ( . versus • per bp) by hrad than by reca, and confirm a stoichiometry of - bp per protein in the hrad -dsdna nucleoprotein filament. biofunctional micropatterned surfaces to study the spatio-temporal organisation of lfa- r. diez-ahedo , d. normanno , c. g. figdor , a. cambi , m. f. garcia-parajo bionanophotonics, ciber-bbn and ibec, barcelona, spain, tumor immunology, nijmegen center for molecular life sciences, the netherlands lymphocyte function associated antigen- (lfa- ) adhesion depends on receptor occupancy and lateral organization on the cell membrane. however, the signals and mechanisms which dynamically reorganize lfa- into high avidity clusters are still a subject of many studies. to obtain deeper insight on the mechanisms that control and regulate lfa- clustering, patterned surfaces of immobilized lfa- ligand areas were fabricated using microcontact printing. the diffusion of lfa- expressed by monocytes stretched over patterned surfaces was followed in time using single molecule tirf microscopy. single lfa- nanocluster trajectories on individual cells showed an increase of immobile lfa- fraction and a slow-down of diffusing lfa- on the ligand areas compared to the non-ligand areas. moreover, single-cluster intensity analysis indicated a reorganization of lfa- nanoclusters in microclusters upon ligand binding. finally, single particle motion analysis of lfa- trajectories in close neighborhood to the ligand areas showed no assisted diffusion of lfa- towards the adhesive regions, consistent with random ligand-encountering and binding. we are currently investigating the effect of cell membrane organizers to regulate the spatio-temporal organization of lfa- . r. diez-ahedo et al, small, in press. optical and electrophysiological detection of single phages across a lipid membrane n. chiaruttini , p. boulanger , m. de frutos , l. letellier , u. bockelmann , v. viasnoff nanobiophysique, espci paristech, cnrs, paris, france, ibbmc, université paris xi, cnrs, orsay, france, lps, université paris xi, cnrs, orsay, france we present an investigation study of the ejection of single t bacteriophages. in vivo studies of dna ejections from the bacteriophage capsid show that the t genome is introduced in the bacterial host in two steps. first % of the genome is ejected then after a pause of a few minutes the rest is internalized. bulk in vitro studies showed that various mutants of t eject their genome in solution following a single or a multistep process. by immobilizing single bacteriophages on a surface and following their ejection by fluorescence microscopy we showed that in all cases the ejection occurs in one step, but some mutants seem to have a subpopulation for which the triggering signal of the ejection is transmitted more slowly to the capsid entrance. we then reconstituted the phage receptor fhua into giant liposomes and followed the ejection of the dna into the liposome by fluorescence. finally we incorporated fhua in a suspended bilayer and followed the infection of the phages through the bilayer both by fluorescence labeling and electrophysiological measurements. we will discuss the influence of the cross membrane potential on the ejection speed of the dna. helixlike pili is a prerequisite of uropathogenic e. coli to adhere to host and withstand urine flow the gram-negative uropathogenic escherichia coli (upec) bacteria, invades the urinary tract region and cause in some cases severe infections, pyelonephritis, if they can withstand the rinsing action of urine and ascend to the kidney, via the bladder and ureters. to mediate adhesion, upec express quaternary surface organelles that are assembled from ∼ identical subunits into a helix-like coil, with a single adhesin located at the tip. it is believed that the single adhesin mediate attachment to host cells while the helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow. to unravel the biomechanical properties of such quaternary structures, in particular in terms of their force-elongation and kinetic behavior, force-measuring optical tweezers (fmot) have been used. a plethora of different types of pili have been identified in the literature and we show, using fmot, that those dissimilarities might reflect the host environment. for example, we have found differences among pili expressed at diverse environment inside the urinary tract, which imply that pili presumably have evolved to resist specific forces under in vivo conditions. it is thus worth striving for understanding bacterial adhesion in order to figure out alternative to the over-abundance of antibiotics worldwide. single molecules studies have probed protein conformational fluctuations and monitored the oscillating activity of enzymes that remained masked in ensemble measurements. various statistical models have attempted to connect the conformational motions to the fluctuating activity of enzymes and suggested that they adopt inter-converting conformations each one of them exhibiting different catalytic activity. the main drawback of these studies is the non specific interactions that may bias the observed catalytic behavior. we have developed new innovating tools to study the behavior of single enzymes. as a first step we utilized liposomes as scaffold to confine enzymes while keeping them under physiological conditions. keeping them in their native conformation allowed us to monitor the inherent properties of their behavior. as a second step we developed a new model that accurately connects enzyme activity behavior to conformational motions. using this approach we accurately predicted the behavior of single lipases in a highly controlled environment. we modulated the enzyme's conformational mobility and activity by systematically varying its accessibility to liposomes. as we anticipated, that resulted in altering the time the enzyme spends on active conformations.our results provide new insights on interpreting the behavior of enzymes. simulations of single-molecule fret experiments on ribozymes m. hajdziona, a. molski adam mickiewicz university laboratory for dynamics of physicochemical processes, poznan, poland rnazymes are important biological molecules and that can catalyze the cleavage of their own nucleotide chains. this precess is called self splicing [ ] . there are several methods to to study kinetic properties of ribozymes at the singlemolecule level. in this work we focus on fret (förster resonance energy transfer) of single, immobilized molecules. single-molecule fluorescence spectroscopy gives an insight into the behavior of individual molecules rather than the average behavior of an ensemble of molecules, which makes it possible to observe the kinetic heterogeneity. we simulated fret trajectories for single immobilized ribozymes. in our computer simulations we consider two-and three-state kinetic models motivated by actual experiments, published in [ ] and [ ] . we fitted the histograms of on and off times to recover the kinetic parameters of the models. we investigated the bias and standard deviation of the recovered parameters when one or two thresholds are applied to fret trajectories between adjacent states. we found that two thresholds give better parameter estimates than one-threshold analysis. references frap probes the average dynamical properties of a population of fluorescently labeled molecules whereas spt obtains these properties from observations of the trajectories of individual molecules tagged with a colloidal particle (the "bead"). when results of frap are compared with those of spt, frap yields significantly higher diffusion constants than spt. to understand the origin of this difference, we have developed and tested a model system to evaluate the influence of the bead on the dynamics of a diffusing molecule. we use a dsdna tether to attach a bead to a supported lipid bilayer (slb). the dna tether is modified at one end by cholesterol for anchoring to and diffusion in the membrane, and at the other end by biotin for tagging with a bead. with this system, we can vary several parameters: distance between slb and bead, size and nature of the bead, lipid composition of the slb, external force. we can also model the brownian motion of the bead and the hydrodynamic flow around it. we will present results using φ = nm neutravidin-coated latex particles attached to an eggpc slb via a bp to bp dna tether. elucidation of the mechanism of the lambda bacteriophage epigenetic switch l. finzi physics department, emory university, dowman dr, atlanta, ga , usa the lambda bacteriophage epigenetic switch determines the growth lifestyle of the virus after infection of its host (e. coli ). it is now clear that the switch consists of a ∼ . kbplong dna loop mediated by the lambda repressor protein. using tethered particle microscopy (tpm), magnetic tweezers and afm, our laboratory has novel, direct evidence of loop formation and breakdown by the repressor, the first characterization of the thermodynamics and kinetics of the looping reaction and its dependence on repressor non-specific binding and dna supercoiling. these in vitro data provide insight into the different possible nucleoprotein complexes and into the lambda repressor-mediated looping mechanism which leads to predictions for that in vivo. the significance of this work consists not only of the new insight into a paradigmatic epigenetic switch that governs lysogeny vs. lysis, but also the detailed mechanics of regulatory dna loops mediated by proteins bound to multipartite operators and capable of different levels of oligomerization. mechanical force at the molecular level is involved in the action of many enzymes. for example, the phi dna polymerase mechanically unwinds the dna helix as it moves processively along the dna replicating one strand of the dna molecule. using optical tweezers we have developed a single molecule mechanical assay to elucidate the physical mechanism of dna unwinding by the phi dna polymerase as the protein replicates the dna. a single dna hairpin is hold between an optical trap and a mobile surface. as a single polymerase works on the dna hairpin, its replication and unwinding reactions can be measured in real time by measuring the change in extension in the dna polymer, revealing the fluctuations of its rate in response to the dna sequence. moreover, by gradually pulling on the opposite strands of the dna hairpin we can promote the controlled mechanical unwinding of the dna helix and determine the effect of increasing unwinding forces on the polymerization and unwinding rates. the effect of force and dna sequence on the activities of the wild type and an unwinding-deficient polymerase mutant will allow us to determine the inner workings of this molecular motor. single centrosome manipulation reveals its electric charge and associated dynamic structure we demonstrate laser manipulation of individual early drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively-charged organelle with a very low isoelectric region ( . ± . ). from this single-organelle electrophoresis, we infer an effective charge smaller or of the order of electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. by investigating the centrosome hydrodynamic behavior, we show that its charge has a remarkable influence over its own structure. specifically, we find that the electric field which drains to the centrosome expands its structure to a physiological size a % larger than previous electron microscopy determinations, a self-effect which modulates its structural behavior via environmental ph. this methodology further proves useful to study the action of different environmental conditions, such as the presence of ca + , over the thermally-induced dynamic structure of the centrosome. magnetic contrast neutron reflectivity delivers significant improvements in resolution for membrane structure analysis neutron reflection (nr), with its high resolution and use of contrast variation, is a unique tool to gain information on the orientation and structure of lipid bilayers in the z-axis perpendicular to the surface. to improve the use of nr we have used a highly oriented and stable layer of membrane proteins and lipids made possible by self-assembly upon a gold surface. this also provides a model of the bacterial outer membrane. as the dimensions of the layer can be predicted with accuracy, the system provides a molecular ruler for improvements in methodology and the complexity of the layer structure adds significantly to the modelling challenge. early improvements in resolution were obtained through sample preparation and gold smoothness. further improvement however required clearer discrimination between similar models. this was achieved using the new approach of magnetic contrast variation which uses a magnetic layer to provide two different scattering length densities for oppositely polarised neutrons. during this data collection the sample is unchanged. we show that this approach delivers significant improvements in data analysis and resolution of the protein, lipid and solvent structures. the method will provide a new level of understanding of membrane structure and dynamics. single molecule force spectroscopy and recognition imaging p. hinterdorfer institute for biophysics, johannes kepler university of linz, altenbergerstr. , a- linz, austria in single molecule force spectroscopy, interaction forces of ligand-containing tips with receptors on probe surfaces are quantified. here he attachment of human rhino virus (hrv ) to the cell surface, the first step in infection, was characterized. sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra. this suggests that multiple receptors bound to the virus in a timely manner. unbinding forces required to detach the virus from the cell membrane increased within a time frame of several ms. the number of receptors involved in virus binding was determined and estimates for on-rate, off-rate, and equilibrium binding constant were obtained. furthermore, we show that accurate free energy values of membrane protein unfolding can be obtained from single molecule force measurements. by applying a statistical theorem developed by jarzynski, we derived equilibrium unfolding free energies of from unfolding force data acquired at different force loading-rates and temperatures. finally, we present a method for the localization of specific binding sites and epitopes with nm positional accuracy. a magnetically driven afm tip containing a ligand covalently bound via a tether molecule is oscillated at a few nm amplitude, during scanning along the surface. in this way, topography and recognition images on membranes and cell surfaces were obtained simultaneously. mapping of the forces acting on biomolecules in cell membranes has spurred the development of effective labels, e.g. organic fluorophores and nanoparticles, to track trajectories of single biomolecules [ ] . standard methods use particular statistical observables, namely the mean square displacement (msd), to extract cues on the underlying dynamics. yet, msd is not an appropriate tool to access force fields and becomes easily a biased estimator in the presence of positioning noise. here, we introduce general inference methods [ ] to fully exploit information hidden in the experimental trajectories, providing sharp estimates for the forces and the diffusion coefficients within membrane microdomains. rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. the inference method is then applied to infer forces and potentials acting on the receptor of the ε-toxin labelled by lanthanide-ion nanoparticles. results show a constant diffusivity inside a complex force field confining the receptor inside a specific domain. our scheme is applicable to any labelled biomolecule, and results presented here show its general relevance to the issue of membrane compartmentation and protein motion. large scale domain motions are structural rearrangements often adopted by enzymes to achieve their full functionality. understanding the mechanism responsible for such movements by means of computational approaches is of great interest especially in rational drug design, since induced fit or population shift effects are closely related aspects of the problem. unfortunately, the simulation time required to sample these events by conventional techniques such as plain molecular dynamics, is unfeasible. here, the domain motion required by adenosine kinase (ak) to achieve its (pre-)catalytic conformation was studied using well-tempered metadynamics [barducci, a. et al. phys rev lett. ( ) , : ] with path collective variables (pcvs) [branduardi, d. et al. j chem phys. ( ) , : ]. first, a low energy path for the apo form of the enzyme was obtained, and the potential of mean force along the pcvs was reconstructed. then, the large scale movement was simulated in the presence of two inhibitors known to bind to the enzyme in different conformational states. the adopted approach was proven to be successful both to understand the mechanistic features of the ak domain motion and to provide a picture of the population shift effects upon ligand binding. force measurement study of the interaction between s rrna and the ribosomal protein l single molecule force measurements enable to probe the complex structure and folding dynamics of rna molecules and furthermore to investigate the numerous interactions with proteins that affect rna folding in vivo. we use a double optical tweezers setup to study a region of the ribosomal rna s from e.coli and its interaction with the essential ribosomal protein l . the apparatus permits us to probe the dynamics of the rna structure with milisecond time-resolution. first, we pull the rna and show that it mechanically unfolds in several reproducible steps. we use these results in combination with structure prediction tools to evaluate the possible structures of the rna fragment in vitro. the most probable structure exhibits a unique binding site for l . then, force measurements are done on the rna in presence of l . we show that the protein specifically binds the rna and stabilizes it. the binding site is recognized with a resolution of a few bases. finally, two bases are mutated on the rna fragment. in presence of these mutations, in vivo and in vitro binding of l to s rna is abolished. the single molecule approach gives an explanation of this result: the force measurements show that the mutated rna folds differently from the natural one and that it does not bind l . control of the translocation of single dna molecules through alpha-hemolysin nanopores g. maglia, h. bayley department of chemistry, university of oxford, ox ta, oxford, uk the analysis of single nucleic acid molecules by electrophoretic threading through nanopores is under intense investigation as a rapid, low cost platform for dna sequencing. biological nanopores such as staphylococcal alpha-hemolysin (hl) have an added advantage over solid-state nanopores because they can be modified by genetic engineering with atomistic precision. although we showed that all four dna bases can be identified in an immobilized ssdna molecule ( ), the translocation of free dna is too quick to observe single bases. here we show that by a small increase of the net internal charge of the hl nanopore (e.g. by introducing a ring of arginine residues), we have augmented the frequency of dna translocation events through the pore and dramatically lowered the voltage threshold required for dna translocation ( ) . by further increasing the net positive charge of the transmembrane barrel region of the pore (e.g. by introducing extra positive charges) we have also reduced the speed at which dna translocate the pore by more than two orders of magnitude. these experiments provide a means of controlling dna translocation with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. the atomic force microscope (afm) is a tool for imaging, measuring and manipulating matter at the nanoscale. single-molecule pulling experiments give information on the thermodynamics and kinetics of biomolecules. the purpose if this work was to develop software to simulate single-molecule pulling experiments and to analyze singlemolecule pulling data. the long term objective is to asses the accuracy and precision of the parameters recovered from single-molecule pulling data.we carried out simulations for two different models [ , , ] , using a wide range of loading rates. from the simulated force-extension curves we extracted the kinetic parameters and compared them with the values used for simulations. the kinetic parameters were the intrinsic rate coefficient (k kramers rate), the location of transition state (x ) and the free energy of activation (∆g). we have found that the loading rates have a small effect on the recovery of the free energy of activation, but have a significant effect on the recovery of the kramers rate. we report the tracking of single myosin v molecules in their natural environment, the cell. myosin v molecules, labeled with quantum dots, are introduced into the cytoplasm of living hela cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. we perform intracellular measurements of key parameters of this molecular transporter: velocity, processivity, step size and dwell time. our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells. a mathematical model of neurotransmission at the input stage of the cerebellum t. nieus , s. solinas , l. mapelli , e. d'angelo dept. neuroscience and brain technologies, iit, italian institute of technology, genova, italy, dept. of biomolecular sciences and biotechnology, milan, italy, dept. physiological and pharmacological sciences and cnism, university of pavia, italy the granule cell (gc) of the cerebellum has some peculiar properties compared to other cells of the vertebrate brain. the gc has a small soma (diam= microm) and just a few ( to ) excitatory and inhibitory inputs (e&i). the e&i gc synaptic inputs are formed inside the cerebellar glomerulus, which favors neurotransmitter diffusion between neighboring sites ( ) protracting postsynaptic receptor activation and ( ) causing cross-tall between e&i synapses through presynaptic receptors. neurotransmitter release probability (p) can be regulated by long-term plasticity (ltp and ltd at e synapses) and by gaba b and mglu presynaptic receptors (both at e&i synapses). the p change in turn modifies shortterm plasticity, affecting the first response in a train much more than the followings. to gain insight into the role that p changes might have in computations performed at the cerebellum input stage, we have built detailed biophysical models of the e&i synapses. the model has allowed to investigate glomerular processing of high frequency inputs reaching the cerebellum. optimal synaptic transmission through the mfs inputs resulted when gos inhibited synchronously the gcs. the role of feed-forward inhibition onto synaptic transmission is under investigation. unzipping dna with a nanopore j. muzard, n. chiaruttini, u. bockelmann, v. viasnoff cnrs/espci nanobiophysique, rue vauquelin, paris, france the use of proteinaceous or artificial nanometer size pores has become a promising approach for sensing biomolecules at the single molecule level. it was shown that alphahemolysin, a toxin from staphylococcus aureus, can be employed to sense the translocation of dna strands through a lipid bilayer. the pore dimension allows the electrophoretically driven passage of single stranded dna whereas double stranded structures need to open prior their translocation. we study the unzipping process of the double stranded part of dna both experimentally and theoretically. we show that in a first approximation the duplex opens progressively in a sequence dependent manner. the experimental results can be accounted for by modeling the unzipping process as a free diffusion of the unzipping fork in the energy landscape defined by the sequence of the basepaires. we then discuss the effect of the pore/dna interaction on the translocation process. we further characterize the effects of the pore geometry, showing that the pre-confinement of the dna in the pore vestibule is essential to the unzipping process. we eventually discuss the possibility to use this nanopore approach to sequentially probe rna secondary structures. we report on the interaction forces in the range -- pn determined between pairs of the lectin soybean agglutinin (sba) and a modified porcine submaxillary mucin (tn-psm) using a single-molecule approach. lectins are carbohydrate-binding proteins with biological activities related to i.e. cellular recognition, adhesion, growth and metastasis. here, dynamic force spectroscopy is used to investigate pairs of sba and tn-psm with the aim to understand the mechanisms of binding and cross-linking of multivalent lectins. the unbinding force increased from pn to pn with increasing force loading rate and the lifetime of the complex in the absence of applied force was . - . s. published kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range . x − - . x − s. the long lifetime of the sba -tnpsm complex is compatible with a previously proposed "bind and jump" mechanism. this mechanism has also been suggested for lectins binding to multivalent carbohydrates and globular glycoprotein. the bind and jump mechanism is also similar to that observed for binding of proteins to dna, and suggest a common conserved binding mechanism of ligands to the two biopolymers and possibly between ligands and all biopolymers, as recently suggested. the rate of topoisomerase ii activity correlates with persistence length of dna q. shao , l. finzi , d. dunlap dept. of physics, emory university, atlanta, georgia, u.s.a., dept. of cell biology, emory univ. med. school, atlanta, georgia, u.s.a. type ii topoisomerases catalyze the transection of one double helical segment by another to modify the topological state of dna. reversible ' linkages to the phosphate-sugar backbones are established on each strand of the "gate" segment to create an opening through which the enzyme drives the "transfer" segment. a recent crystal structure shows that the "gate" segment bends approximately • upon binding to the enzyme. bending a stiff polymer like dna requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. using modified deoxyribonucleotides in pcr reactions, more rigid dna fragments have been produced and used as substrates for topoisomerase ii-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. before relaxation the persistence lengths were measured by fitting force extension data with the worm-like chain model. topoisomerase ii was found to release mechanically introduced supercoils more slowly in stiffer dna suggesting that dna bending might be a rate limiting step in topoisomerase ii activity. h. seidel , t. klose , h. lilie , c. g. hübner institute of physics, ratzeburger allee , lübeck, germany, institute of biochemistry and biotechnology, kurt-mothes-str. , halle, germany one essential element of a virus is its protein shell, the viral capsid, which encloses the viral genome. the murine polyomavirus is a non-enveloped dna tumor virus with an icosahedral t= d structure. besides the knowledge of the structure, it is of utter importance to understand the process of viral assembly [ ] . the assembly reaction of polyoma vp does not show the typical sigmoidal kinetics in light scattering experiments. the apparent kinetics is of fourth order, which appears rather unrealistic. in order to gain knowledge on capsid composition during assembly beyond ensemble average, we apply methods of single molecule fluorescence, namely fluorescence correlation spectroscopy (fcs), fluorescence-intensity-distribution-analysis (fida), and single-particle-imaging (spi). the molecular brightness and the diffusion time reported by fcs are in agreement with the results from light scattering. fida as well as spi, moreover, point to a polymerization of subunits to complete capsids along the assembly pathway without pronounced intermediates. mixing experiments show that already early in the course of the assembly reaction no exchange of pentamers between capsids occurs, and that the effect of breathing [ ] can be excluded for polyoma vp . genetic information coded in dna provides complete instructions to cells regarding metabolism and proper functioning. a number of endogenous and exogenous sources can damage the genomic dna. unrepaired dna damage can give rise to mutations and may cause cell death. cells have evolved different mechanisms to repair damaged dna and to protect genetic integrity. uracil in dna occurs as a result of incorporation of dump in the place of dtmp during replication and deamination of cytocine, resulting in u:a and u:g base pairs, respectively. uracil-dna glycosylase (ung) is a dna repair protein that searches and removes uracil from dna. ung removes mismatched base by flipping it out from the base stack into the active site. the exact mechanism by which ung searches dna for uracil is unknown. therefore, in our study we use atomic force microscopy (afm) to investigate the interaction between single ung molecules with dna carrying the u:a or u:g mismatches. furthermore, we study the complexation of dna and ung protein. afm images of ung bound to dna reveal the structural changes at the level of single complex, e.g. dna kinking that may occur upon binding of ung to dna. imaging of dnaprotein complexes can provide a a new level of understanding of ung-dna interaction. atomic force microscopy (afm) has been a useful device to visualize cellular and molecular structures at a single-molecule resolution. especially, afm imaging under the trec t m (topography and recognition) mode (recognition imaging) can map a specific protein of interest within an afm image. in this study, we employed an antibody-coupled afm cantilever in recognition imaging, and dissected the structural/functional domains of α actinin- , an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction. a use of monoclonal anti-α actinin- antibody enabled us to map its epitope in the amino-terminal actin-binding domain within a single molecule afm image of α actinin- . counting fluorescent molecules by photonantibunching h. ta, m. schwering, d. p. herten bioquant, heidelberg university, germany information on the stoichiometry of labelled biomolecules is highly desirable. a method called photo-antibunching has successfully been used to determine the number of fluorescence emitters in multichromophoric systems. a statistical model to estimate the number of fluorescent molecules in a confocal observation volume is established based on photonantibunching in time-correlated single-photon counting (tcspc) scheme with avalanche photon diodes (apds) for detection of individual photons. numerical simulations based on realistic experimental conditions show that the model is able to estimate the number of molecules with moderate errors. experiments on immobilized double-strand dna oligonucleotides with photon stabilizing agents show promising results even when the number of molecules is ∼ . the proposed method allows us to monitor labelled single molecules and in the near future we plan to implement it in complex biological systems (cell extracts/live cells). single molecule afm force spectroscopy and steered molecular dynamics of contactin- protein j. w. strzelecki, k. mikulska, a. balter, w. nowak institute of physics, nicolaus copernicus university, grudziadzka , - torun, poland contactin- (cntn ) is an axonal cell adhesion protein that contains six igc and four fniii domains and is responsible for creating neural outgrowths. recent research shows that mutation of cntn gene may be responsible for autism, while its absence in transgenic mice results in lack of smell sense. we use a homemade afm single molecule puller and steered molecular dynamics simulation to test its elastic properties through force spectroscopy. stretching experiments show unfolding of fniii domains while igc domains stay coiled as they are stabilized by disulfide bonds. unfolding of contactin- molecule appears to play role of buffer that helps to protect neural contacts from damage when neural cells are subjected to shock or tumor. single molecule studies of the thermophilic bacillus ps f -atpase have revealed kinetic and structural information that cannot be discerned using other methods, including the presence of and degree physical substeps (yasuda et al. ) and the order and kinetics of chemical substeps. we are interested in using single molecule techniques to observe the effects of mgi mutations on enzyme kinetics and torque production in f from the yeast saccharomyces cerevisiae. using a high speed imaging camera, we have captured the rotation of wild-type and mutant forms of yeast f -atpase. rotation data for the wild-type and preliminary data for some mgi strains will be presented. we show for the first time that at saturating atp, wild-type yeast f rotates approximately four times faster than the thermophilic f . kinetic and substepping behaviour in yeast appears to be similar to that observed in bacterial f . -single molecule biophysics - biophysical characterization of a dna gquadruplex formed in the promoter of human mef d gene w. zhou , l. ying molecular medicine, national heart and lung institute, imperial college london, london, sw az, uk, chemical biology center, imperial college london, sw az, uk g-quadruplex is believed to be involved in many crucial biological processes, such as the gene regulation and the maintenance of human telomere. mef d, a member of mef (myocyte enhancer factor- ) family of transcription factors, regulates the response of heart to cardiac stress signals. we found that a g-rich sequence starting at - bp of mef d promoter can form a very stable intramolecular g-quadruplex. here we present a detailed biophysical characterization of this quadruplex. we also found that this quadruplex is more stable than its duplex form under physiological conditions by cd melting and single molecular fret. we observed subpopulations in smfret measurements possibly due to different conformations of the quadruplex. we characterized its unfolding process by monitoring the change in fret when it hybridizes to its c-rich complimentary strand. finally, we proposed several possible structures of this quadruplex based on the smfret and fluorescence dms footprinting results. this research is supported by national heart and lung institute foundation. by stretching a polymer in solution using single molecule techniques it is possible to infer about its physical properties. afm stretching experiments allow for a full characterization of the elasto-mechanical properties of the sample under study. in the presented work, single molecule afm force spectroscopy experiments were used to determine mechanical properties of a peptide obtained from exon (ex ) of the human elastin gene. elastin is a protein with important mechanical properties and, in particular, it shows quasi ideal elastic behavior associated to the presence of many hydrophobic unstructured domains (such as ex ) into the protein structure. ex coded polymer was used as a starting point to obtain bio-materials with specialized elastomechanical functions. in particular, a mutated polypeptide based on the ex sequence was synthesisized with the aim of obtaining a new polymer with the same mechanical and physical properties of the native molecule but with increased aggregation properties, induced by a cross-linking reaction. afm stretching experiments were used to verify the mechanical properties of the engineered proteins at a single molecule level. the obtained results allowed not only to address this question, but also to give some insight into the first aggregation steps of the polymer towards the formation of reticulated structures. luminescent lanthanide-ion doped nanoparticles (nps) are attractive single-biomolecule labels. they are synthesized directly in water, are highly photostable, and display narrow emission without intermittency. we coupled y . eu . vo nps to ε-toxins produced by clostridium perfringens, which bind to a specific receptor on mdck cells. single-molecule tracking experiments using these labels produce long ( min) uninterrupted trajectories with temporal resolution down to ms or localization precision down to nm. we found that the toxin receptor exhibits confined motion in cell membrane microdomains. to analyze the receptor trajectories, we used a novel approach based on an inference method [ ] . this method fully exploits the information of the ensemble of the trajectory, in contrast to the usual mean square displacement analysis. applying both techniques to recorded trajectories, we highlight the difference in extracted parameters. from the shape of the confining potential, obtained by mapping the forces inside the domain, we can deduce information about the mechanism of confinement. in combination with experiments on cholesterol depletion and cytoskeleton destruction, this technique will shed light into the nature of the membrane micropatterning. background: implantable cardioverter defibrillators (icds) are save-life device for patients at risk of sudden cardiac death, and help cardiac patients to avoid slow beat-rate by means of anti-bradichardia pacing (abp) feature. however, recent literature evidenced the presence of ventricular tachyarrhythmias (vts) immediately following abp, leading to the hypothesis that icd devices might be pro-arrhythmic. aim: study differences between pacing-associated tachyarrhythmias (pat) and other spontaneous vts. signals and methods: spontaneous vt episodes are retrieved from patients with icd. vts are examined and pat episodes, classified. characterization of vts is based on the analysis of seconds immediately preceding vt-onset quantified by: heart cycle (hc-prevt), prevalence of premature ventricular contraction (pvcprev) and prevalence of paced beats (pprev). significant differences are evaluated by student-t or chi-square tests with p< . . results: vt episodes from patients are pat episodes ( %). cardiac activity preceding pats vs. non-pats differs: pat episodes have higher hc-prevt (p< . ), greater pprev (p< . ), and greater pvcprev (p< . ). analysis also shows that pat episode often occur when the paced beat immediately follows a premature contraction. conclusion: the study indicates that new icd generations should avoid abp after premature ventricular contraction. towards mechanistical understanding of adsorption: combining technologies in situ and in real time p. h. bjöörn q-sense ab, västra frolunda, sweden a growing number of researchers in different surface related fields present evidence from more than one analytical technique when detailing their findings. thus a logic and useful development is to combine technologies for simultaneous measurements on a single sensor surface. to develop a biosensor platform such as an assay fast and accurately, mechanistical understanding of why the platform works is essential. quartz crystal microbalance-with dissipation (qcm-d) technology and instrumentation provides an open platform and enable easy and precise quantification of mass, thickness and viscoelastic properties of surface bound molecules. these parameters provide both a reference tool in assay development, but also detecting and identifying your target molecules as the combination of mass and material property info in many cases provide a unique response for a specific molecule. by combining qcm-d with other technologies, a range of useful info is put within easy reach of the researcher. recent advances will be presented where simultaneous real time and in situ measurements using qcm-d together with electrochemistry, ellipsometry and fluorescence microscopy enables manipulation of films as well as quantification of the variation of water content as a result of conformational changes in immobilized molecules. examples will include new data from the formation of protein films, polyelectrolyte multilayers and polymer brushes. intrinsic gravity versus metabolically inert infrastructure and basal metabolic rate in living mass i. r. bhattacharjee , r. kashyap , b. shaptadvipa assam agricultural university, jorhat- , india, international institute of intrinsic gravitation biology (i gb), jorhat- , india 'self gravitation bio' is an emerging concept in biophysics. intrinsic or 'self' gravitational force might exert when mass increases beyond critical level in biological particle pyramid. (http://en.wikipedia.org/wiki/biomechanics of intrinsic gravity) 'metabolically inert infrastructure' (mii) consists of total body mass (body water, dissolved substances, mineral and organic deposits) and serves as storage of nutrients, transport and distribution of these materials. to act independently as living body, mii is suggested also to provide structural support to the organism with density-gradient buoyant force against intrinsic and extrinsic gravitational attraction for the biological mass. it is shown that 'amniotic fluid', 'isotonic saline to ailing patient', 'growth factors', 'cultural medium' and other 'medium matrices' act as counter-gravity mechanism for micro to macro living organisms under center-of-biomass reference frame. controversy over relationship between bmr (basal metabolic rate), rmr (resting metabolic rate), pal (physical activity level), on one hand, and mass of the living organism, on the other, (as described in rubner's / law, kleiber's / law that continued to be contested by many) can be favorably resolved substituting the concept of self gravitation bio, considering 'mass' as synonym of gravitational force under center-of-biomass reference frame. bioenergetics would be an unequal but opposite entity of self-gravity reinforced by extrinsic gravity. platelet arrest on von willebrand factor (vwf) occurs transiently via the platelet receptor gpib. depending on the combination of shear stress and surface density of vwf binding sites the platelets either only adhere, pull tethers or generate microparticles. these processes are influenced by the properties of the platelet membrane and the cytoskeleton. under shear flow conditions these platelet characteristics were examined with reflection interference contrast microscopy and a viscosimeter. in addition we quantified platelet adhesion energy and tether pulling forces. disrupting platelet f-actin had several effects. the tethers were shorter, the membrane contact area was larger, the receptor αiibβ density of the microparticles was depleted and no platelet spreading occurred. breaking up the microtubular system had different implications. the number of severed tethers tripled, the contact area was smaller, microparticle formation was increased and the area of spread platelets was reduced. however changes in the membrane elasticity had no effect on the platelets. our results suggest that platelet attachment and adhesion is not only determined by the platelet adhesion receptors and their cytoplasmic linker proteins, but that cytoskeletal structures have also a crucial influence on how platelets interact with thrombogenic surfaces. gene expression is orchestrated by a host of regulatory proteins that coordinate the transcription of dna to rna. regulatory proteins function by locating specific binding sequences of dna and binding to these sequences to form the transcription initiation complex. in many instances, these regulatory proteins only have several hundred copies that must efficiently locate target sequences on the genome-length dna strand. the non-specific binding of regulatory proteins to random sequences of dna is believed to permit the protein to slide along the dna in a stochastic manner. periodically, a thermal kick or an interaction with another bound protein will disengage the regulatory protein from the dna surface, leading to three-dimensional diffusion. eventually, the protein will reattach to the dna at some new location that is dictated by both the diffusivity of the protein and the dna configuration. cycling through these random events constitutes a search strategy for the target site. we build a reaction-diffusion theory of this search process in order to predict the optimal strategy for target site localization. the statistical behavior of the dna strand acts as a necessary input into the theory, and we consider several governing behaviors for the dna strand. we explore the impact of dna configuration and protein occlusion on target site localization in order to predict how protein expression will vary under different experimental conditions. a. di garbo , s. alloisio , s. chillemi , m. nobile istituto di biofisica cnr, via g. moruzzi , pisa, italy, istituto di biofisica cnr, via de marini , genova, italy in the nervous system of vertebrates there are more glial cells than neurons: from to times, depending on the animal type. glial cells are not able to generate action potentials but, nevertheless they play an important role for the functioning of the different brain's area. the astrocytes are the more abundant cells of the macroglia and through their processes they modulate synaptic activity. in this contribution a biophysical neural network model consisting of a pyramidal neuron, an interneuron and the astrocyte is studied. the corresponding dynamical properties are mainly investigated by using numerical simulations. the results show that the presence of the atp and of the interneuron strongly impacts the neural activity. moreover, it is shown that the fluxes of calcium through the cellular membrane strongly affect the modulation of the neural activity arising from the astrocyte. microscopic origin of the very-low energy vibrational dynamics in proteins g. d'angelo , v. conti nibali , c. crupi , a. paciaroni , u. wanderlingh department of physics, faculty of science, messina university, italy, department of physics, faculty of science, perugia university, italy important functions of biological processes require large atomic rearrangements and conformational fluctuations. for proteins, atoms are mostly displaced along the soft directions identified by the delocalized, low frequency vibrations. the study of very low energy vibrational spectrum of proteins is therefore of particular interest. as a step toward understanding their functional role, we have investigated the low frequency vibrational motions ( . ÷ mev) in different proteins by performing inelastic neutron scattering and low temperature ( . ÷ k) specific heat measurements. for the first time for biological systems, the well-known boson peak found in neutron scattering spectra at ∼ mev is put in relationship with a clear anomaly of the measured specific heat located at around k. this departure from the debye-like behavior further expands the analogy of proteins with glassy systems. quite interestingly, in the very low sub-mev energy range and below ∼ k, we observe an additional anomalous behaviour, which could be ascribed to the existence of twolevel-systems states. increasing the hydration degree, the low energy vibrational region is drastically altered, revealing that the addition or removal of the hydrogen bond network around the protein deeply modifies the interatomic forces, affecting the character of the vibrational modes. a. cupane , m. levantino , m. cammarata , g. schirò department of physical and astronomical sciences, university of palermo, via archirafi , i palermo, italy, european synchrotron radiation facility, grenoble, france our efforts in recent years have been to study in great detail the way hemoglobin works in confined geometries [ ] [ ] [ ] . to this aim we have contributed to the development of a new experimental technique, time-resolved wide-angle x-ray scattering (tr-waxs), that enables one to watch proteins at work in solution [ , ] . a very recent and challenging application of this technique is the study of hemoglobin functioning inside intact red blood cells (rbc). our preliminary results show that, by using laser pulses of about ns, it is possible to photolyse hemoglobin inside rbcs obtaining about % - % photolysis. good structural signals from hemoglobin are obtained, with limited radiation damage to the cells: this opens the possibility of studying the conformational transitions of hemoglobin in its "natural" environment. preliminary results concerning the timing of the r->t quaternary transition inside the rbc and comparison with the behaviour in dilute solution will be discussed. physical review letters and physical review e invite submission of your best work in biological physics. submissions must present significant new results in physics; topics may range from the microscopic to the macroscopic. we will provide information on the different types of articles published in the journals, on authors and referees, and on the review process. for publication in physical review letters, the work should be important and of broad interest. for rapid communications in physical review e, the work should be important for the field. biological physics papers published in physical review are indexed in medline. in an effort to bring important research to the attention of a wider community, the physical review journals have recently begun highlighting important work with viewpoints in the online publication physics. all physical review journals may be accessed through the website http://publish.aps.org/. modeling of fibrin gels using confocal microscopy, light scattering and turbidimetry f. ferri , d. magatti , b. cardinali , a. profumo , m. rocco dipartimento di fisica e matematica, università dell'insubria, como, italy, istituto nazionale per la ricerca sul cancro (ist), genova, italy fibrin gels are biological networks playing a fundamental role in blood coagulation. confocal microscopy of fibrin gels shows a collection of straight fibers, not uniformly distributed in space, connected together at low-order ( ) ( ) branching points. although each fiber is quite straight (mass fractal dimension d m = ), for the overall system d m > . based on the confocal images, we generated threedimensional ( d) synthetic gels made of cylindrical sticks of diameter d, joined together at randomly distributed nodal points. the resulting d network is no more random but ordered on length scales of the order the average fiber length, and exhibits a fractal morphology with d m ∼ . - . . the gel structure is analyzed by means of its d correlation function g d (r)=<n(x) n(x+r)> x , where n(x) is the gel local density. since the gel is isotropic, g d depends on the modulus r=|r| and can be obtained by averaging d correlation functions evaluated at different heights of the gel volume. from this analysis we recover the fiber diameter d (fwhm of g d ), the fractal dimension d m (power-law decay of g d ) and the gel mesh-size ξ (minimum in g d ). furthermore, the d-fourier transform of g d (r) gives the gel power spectrum i(q), which compares quite well with elastic light and multi-wavelength turbidimetry data taken on real gels. a model coupling vibrational and rotational motion for the dna molecule e. drigo filho, j. r. ruggiero, r. a. d. s. silva unesp -sao paulo state university, brazil a largely used mechanical model for vibrational motion of dna has been proposed by peyrard and bishop (pb). in this model, dna is represented by a pair of harmonic chains coupled by a nonlinear potential. the most frequently used nonlinear potential for this purpose is the morse potential. some extensions of the original model are proposed considering, for example, the helicoidal structure of dna. an important objective for this kind of model is to understand the phenomenon of thermal denaturation and, through this, get some knowledge about other processes such as the genetic transcription and drug intercalation. it is possible to obtain from this type of model interesting properties, such as the average stretching between base pairs as a function of the temperature using the transfer integral operator. dynamical properties of this model were also explored and several phenomenological quantities are studied, such as energy localization. in this work, we extend the original pb model by introducing rotational motions for the nucleotides. in this way, both the vibrational and rotational motion for each nucleotide are considered. the stretch of the base pairs is given by the morse potential in the same way as in the original pb model. however, the coupling between the two kinds of motion, rotation and vibration, is obtained through a nonlinear combination of them in the morse potential. a. dobovišek , m. brumen , p.Županović , d. juretić university of maribor, faculty of natural sciences and mathematics, maribor, slovenia, jožef stefan institute, ljubljana, slovenia, faculty of science, university of split, split, croatia mepp is a physical principle, widely used for quantitative explanation of non equilibrium phenomena in physics, chemistry and biology [ ] [ ] [ ] . here, we applied mepp to study two and three state reversible michaelis-menten kinetic schemes of enzymatic reactions. by applying constraints as a diffusional limit for kinetic constants, constant free energy differences between enzymatic states and constant thermodynamic force, we calculated shannon information entropy and entropy production of the entire reaction system as a function of forward rate constants. we found maxima in the net steady-state metabolic flux, total entropy production and shannon entropy for equal values of forward rate constants. moreover, for these values an analytical expression derived gives a relation between substrate and product concentrations at which enzymes operate in the optimal way. in conclusion, we demonstrated that mepp is an appropriate selection principle for evolutionary optimization of enzymes. attractive interaction between like-charged lipid surfaces mediated by spherical nanoparticles with spatially distributed charge is theoretically described by using functional density theory and mc simulations. the spatial distribution of charge within a single nanoparticle is considered by two effective charges at a finite distance. the minimization of the free energy is performed to obtain the equilibrium configuration of the system. both, the rigorous solution of the variational problem and the mc simulations show that orientational ordering of nanoparticles subject to the gradient of the electric field gives rise to an attractive interaction between charged lipid surfaces for high enough charge densities of the interacting surfaces and large enough separations between charges within the nanoparticle. the attraction is explained by orientational ordering of dimeric charges in the electric field which lowers free energy. viral genomes encode for a series of membrane proteins which are embedded or attached to the lipid membrane of the host, or penetrate them during the very first step of viral invasion. we are focussing especially to those of the former which are known to assemble and from channels or pores for small ions or substrates. with these channel forming proteins the virus manipulates the host cell interior for the benefit of its replication. strategies are developed to answer the question about the assembly process of these proteins based on the 'two stage model' and the substrate flux. only few experimental data are available to answer these questions, consequently we stress computational methods to derive the adequate answers. the methods applied are ab inito molecular dynamics (md) simulations based on density functional theory (dft) and conventional md simulations. potential routes for assembly of the three transmembrane domains of a protein from sars-cov will be outlined and compared with computational data from other viral membrane proteins. with a novel pore lining motif suggested for a, the dynamics of ions at selected positions within the putative pore will be assessed. probing the molecular mechanism of antibiotics diffusion through the ompf channel e. hajjar , a. kumar , m. winterhalter , p. ruggerone , m. ceccarelli department of physics, university of cagliari, italy, jacobs university, bremen, germany in gram-negative bacteria, the outer membrane porin-f (ompf) is the preferred entry point of antibiotics. bacteria can resist to antibiotics by altering the expression and the structures of ompf. a key feature in the structure of ompf is the presence of a constriction zone, characterized by both spatial and electrostatics restrictions. to study the process of antibiotics translocation at a molecular scale, we performed molecular dynamic simulations accelerated with the metadynamics algorithm. we studied the diffusion of antibiotics with different structural and chemical properties through ompf wild-type and variants. the free energy surface suggests faster translocation for the cephalosporins compared to the penicillins antibiotics, and also for ompf mutants compared to the wild type. further, the conservation of favored orientation and affinity sites of antibiotics inside the ompf channel reveal which specific interactions govern translocation. the calculated energy barriers and rate determining interactions for translocations compared well with the electrophysiology measurements and liposome swelling assays from our collaborators. this study demonstrates how theory and experiments can be combined to reveal the structural determinants and mechanism of ompf permeation. this will benefit to the design of antibiotics with improved transport properties. m. g. gauthier, j. bechhoefer dept. of physics, simon fraser univ., burnaby, bc, canada dna replication requires two distinct processes: the initiation of pre-licensed replication origins and the propagation of replication forks away from the fired origins. experiments indicate that these origins are triggered over the whole genome at a rate i(t) (the number of initiations per unreplicated length per time) that increases throughout most of the synthesis (s) phase, before rapidly decreasing to zero at the end of the replication process. we propose a simple model for the control of dna replication in which the rate of initiation of replication origins is controlled by the interaction with a population of ratelimiting proteins. we find the time set by reaction-diffusion kinetics for such proteins to find, bind to, and trigger a potential origin. the replication itself is modeled using a formalism resembling that used to study the kinetics of first-order phase transitions. analyzing data from xenopus frog embryos, we find that the initiation rate is reaction limited until nearly the end of replication, when it becomes diffusion limited. initiation of origins and hence i(t) is suppressed when the diffusionlimited search time dominates. we find that, in order to fit the experimental data, the interaction between dna and the rate-limiting protein must be subdiffusive. we also find that using a constant nuclear import of the limiting proteins leads to a more accurate description of the experimental data. the microsoft research -university of trento centre for computational and sisytems biology, trento, italy we propose a new method for inferring the structure of a biochemical network from the time-series of the reactant species. it consists of two parts: the first is the quantification of the correlation between the time-series profiles. correlation in time series data can be used to reveal dependencies between variables and to infer the graph of connectivity among species. the second part consists in the elimination from the connectivity graph of the relationships that have a non-null correlation coefficient, but that are not biochemically plausible. the cutting of false correlations from the graph is performed through the estimation of the parameters ("calibration") of the network. to calibrate the network for detecting null dynamics correlations, we developed the software tool kinfer (knowledge inference). based on a new probabilistic model of the variations in reaction concentrations, kinfer infers the values of the kinetic rate constants, their confidence regions and the level of noise in the input data by maximizing the likelihood to obtain the observed variations given the network model. the a priori knowledge required as input is minimal, as it consists only in the time-series of reactant concentrations. the minimal a priori knowledge and the probabilistic formulation of the calibration method make the accuracy of the predictions strong against experimental, biological and stochastic noise, and allows to use it to cut the null-dynamics edges of the connectivity graph. a. kuzmanic, b. zagrovic mediterranean institute for life sciences, split, croatia root-mean-square deviation (rmsd) is a measure used to give information on the global structure of macromolecules. for example, pairwise rmsd (prmsd) is used to assess similarity of the lowest energy nmr structures or for clustering large ensembles of structures. on the other hand, to obtain information on the local structure of a macromolecule and its dynamics, root-mean-square fluctuation (rmsf) is often used. rmsf can be calculated from md simulations, but also from experimental x-ray b-factors. since prmsd and rmsf report on different features, it is interesting to ask what the relationship between them is. first, we provide a mathematical derivation showing that, given a set of conservative assumptions, the <prmsd> rms is directly related to the <rmsf> rms and, consequently, experimental b-factors. second, we demonstrate this on structures taken from distributed-computing md simulations of the native and unfolded state of villin headpiece. both our analytical and computational results suggest a strong correlation: <rmsf> rms = [(s- )/ s] / <prmsd> rms , where s is the number of compared structures. furthermore, if <prmsd> rms is defined as a generalized radius of gyration in the space of d structures and using rmsd as a measure of distance, the following identity holds: <rmsf> rms = <prmsd> rms. our results provide a basis for determining the level of structural diversity of molecular ensembles, as captured by <prmsd> rms , directly from experiment. charge migration along dna helices may be biologically important because extended electronic states could play a role in the processes of sensing of dna damage and/or dna repair via long-range charge transfer. we measured conductivity and other physical characteristics of several models of natural and diversely damaged molecules of dna. dna polymers were mimicked by various sequences of nucleotide-long double helices with fully watson-crick (wc) paired bases, with several central bases mismatched, and also with chemical modifications that included removal of bases from a few central nucleotides (abasic dna), and neutralization of phosphate charges by their derivatization. the model dnas were investigated by scanning tunneling microscopy, time-resolved thz spectroscopy, raman spectroscopy, circular dichroism, and modeled by molecular dynamics simulations. dna has the highest conductivity in its biologically most relevant double helical form with wc base pairs and negatively charged phosphates equilibrated with counterions. mismatches and all chemical modifications always lower the conductivity. the mechanism of charge transfer is consistent with electron or hole hopping between parallel stacked bases. these observations and data showing that the natural dna has also the most regular double helical form suggest that the continuous base stacking is critical for charge transfer. to control the passage across the bacterial cell wall nature created a large number of "nano"-channels which may act as selective gates for water soluble molecules. here we focus on porins from e. coli which control permeation through interactions with the channel surface. comparing single channel temperature dependent conductance measurements with all atom modeling allow conclusions on the mode of permeation. for example, surprisingly modeling ompf-conductance revealed not only a good agreement with the experiment over a broad range of temperature but also the selectivity for ions. the primary task of porins is to provide facilitated diffusion. we investigated facilitated diffusion of maltooligosaccharide or antibiotics. time resolved conductance measurements allows conclusion on the flux and molecular modeling identifies the limiting interactions with the surface, reveal potential barriers and pathways. exploiting the selectivity of natural or bioengineered channels has promising applications for detecting molecules, characterizing molecular interaction, sequencing dna, protein folding etc. traditionally, studies of diffusion-controlled reaction of biological macromolecules have been made in diluted solutions. however, the high concentration of macromolecules in intracellular environments results into non-specific interactions (macromolecular crowding), which have a great importance on the kinetics and thermodynamics of possible reactions that occur in these systems. in the literature there are studies concerning monte carlo (mc) simulations, giving results that are satisfactory agreement with experimental data, showing, for example, that the protein diffusion in cell cytoplasm is reduced considerably. in addition, there are mc studies about enzymatic reaction, which predict a temporal dependency of the velocity constant in macromolecular crowding. in this work, we try to compare the predicted behavior by mc simulation with the results obtained from the study of the diffusion and reaction of a model protein (alpha-chymotrypsin) using spectroscopic techniques in highly confined media in order to study experimentally the temporal dependence of its diffusion and reaction coefficients. a. paciaroni , a. orecchini , c. petrillo , a. de francesco , f. sacchetti university of perugia, italy, cnr-infm, genova, italy the single-particle and collective dynamical properties of protein hydration water have been studied by neutron scattering experiments in a wide temperature and hydration range. an unprecedented accuracy has been achieved thanks to the availability of a large amount of fully deuterated protein powder and the use of the high-flux spectrometers in and brisp. the protein under investigation was the maltose binding protein (mbp), which is a well-known and widely studied model of biosensor systems. we found that the low-temperature single particle dynamics of mbp hydration water shows clear features that can be traced back to amorphous systems. more in detail, its vibrational density of states is simply described as the superposition of the contributions of low-density and high-density amorphous ice. the quasielastic signal, which appears at the higher temperatures, can be excellently described with a fractional power law which may put in relationship with the peculiarities of fractal systems. quite strikingly, there is a strong similarity, on both the qualitative and quantitative point of view, with the behaviour of hydrated proteins. the collective dynamics of protein hydration water is characterised by the presence of two modes, whose dispersion curves are reminiscent of those of bulk water. however, the relevant damping factors suggest a strong similarity with glassy systems. m. malferrari , f. francia , s. sacquin-mora , g. venturoli università di bologna, bologna, italy, cnrs upr , paris, france the coupling between electron transfer and protein dynamics has been compared in reaction centers (rc) from the wild type (wt) and the carotenoid-less mutant r , by combining brownian dynamics simulations and the kinetic analysis of charge recombination. upon incorporation of the rc into a progressively dehydrated trehalose matrix the electron transfer between the primary photoreduced quinone and the photoxidized donor accelerates progressively and becomes broadly distributed. this behaviour reflects the hindrance of protein relaxation following charge separation and the inhibition of interconversion between conformational substates. in extensively dehydrated matrices the recombination kinetics is two-times faster and three-times more distributed in the wt rc, indicating a larger inhibition of the internal protein dynamics. in line with this findings brownian dynamics simulations reveal a larger rigidity of the carotenoid-containing structure, in which a cluster of residues close to the quinone acceptors is stiffened as compared to the r rc. the in silico and experimental results indicate that the introduction of an internal void in the rc structure has long-range effects on the protein dynamics and that the coupling between the glassy matrix and the rc interior depends markedly on the local mechanical properties of the protein. n. maghelli , v. krstic , n. pavin , f. julicher , i. tolic-norrelykke mpi-cbg, dresden, germany, mpi-pks, dresden, germany in the fission yeast schizosaccharomyces pombe, the nucleus is positioned at the cell center. since the nucleus determines the cell division site, keeping the nucleus at the center is crucial for ensuring symmetrical cell division ( ) . microtubules push against the cell ends and exert force on the nucleus ( ), but how the cell regulates these forces in order to center the nucleus remains unknown. here we tackle this problem by using a combination of live cell imaging, cell manipulations by optical tweezers, and a theoretical model. we show that microtubule pushing forces can center the nucleus because of a larger number of contacts between the microtubules and the proximal cell end than the distal one. moreover, kinesin- motors (klp / ) increase the rate of microtubule catastrophe (transition from growth to shrinkage) in a microtubule length-and contact-dependent manner. thus, the motor behavior results in a longer contact between a microtubule and the proximal than the distal cell end. taken together, our experimental and theoretical results provide a novel centering mechanism, where kinesin- motors increase the efficiency of nuclear centering. electropermeabilization is a commonly used physical method which can induce a transient permeabilization of the cell membrane allowing the entry of therapeutic molecules into the cell and is thus of great interest in the fields of cancer treatment and gene therapy [ ] . however, very little is known about the mechanisms occurring at molecular level. there is clearly some microscopic reorganization of the membrane which is responsible for this change in its transverse transport properties. rather than studying the change of these transport properties, we adopt a simple strategy based on the use of giant unilamellar vesicles and videomicroscopy, as described below. we apply a series of permeabilizing electric pulses to the liposomes, and we observe a size decrease down to a critical radius beyond which their size no longer changes. this decrease in size points to the fact that during the physical processes leading to electropermeabilization, lipids are lost from the vesicles. our results published in [ ] suggest different possible modes for lipid loss, which can be small vesicles, pores, or tubules formation. imaging of brain activation using core techniques as fmri, pet and synchrotron radiation in parallel c. poitry-yamate, g. margaritondo, r. gruetter ecole polytechnique fédérale de lausanne, switzerland functional magnetic resonance imaging (fmri), magnetic resonance spectroscopy (mrs), positron emission tomography (pet) and synchrotron x-ray emission imaging form a highly complementary set of imaging core technologies for studying brain function and energy metabolism. owing to differences in the information each conveys and the temporal and spatial scales on which they measure labeled substances, a single working hypothesis can be tested from different angles to provide a cross-validated, consistent and coherent explanation. the cibm is a unique research facility in europe for advancing our understanding of biomedical processes in health and disease. the housing of a -tesla human magnet, . and . -tesla animal magnets, an animal pet imaging facility and fully-equipped neurochemistry and rf laboratories has enabled us to develop and perform well-targeted experiments around one research theme. in parallel, a longterm collaborative project using synchrotron x-ray transmission and emission imaging at elettra enables us to combine these core technologies towards understanding brain function in vitro and in vivo, from tissue to cells. a brief presentation of these methods will be followed by their application in studying the visual system from man to mouse. p. picone , r. carrotta , d. giacomazza , m. di carlo dip. chimica e tecnologie farmaceutiche, università di palermo, italia, ibf -cnr, palermo, italia, ibim -cnr, palermo, italia diabetes and alzheimer's disease are connected in a way that still is not completely known. diabetes has been implicated as a risk factor for developing alzheimer's disease. some diabetes drugs appear to decrease the cognitive decline associated with alzheimer's disease. it has been recently demonstrated that extracellular injection of insulin is able to protect neurons against a-beta amyloid cell death. one of the proposed theories to explain such an effect is that the hematic glucose levels affect the metabolism of the hippocampus, a part of the brain (associated with memory, emotion and motor skills), which is strongly damaged in alzheimer's patients. the aim of this study is to investigate the effect of insulin on the a-beta induced degeneration and oxidative stress on the neuroblastoma lan cell line. in particular, the present study looks into the role of insulin in the inhibition of abeta specific degenerative apoptotic pathways. preliminary results indicate that insulin dissolved in culture medium in its hexameric form (as tested by absolute scale light scattering) is able to reduce neurodegeneration induced by a-beta amyloid in a dose dependent manner. the link between diabetes and alzheimer's disease may provide new targets for future alzheimer's treatments. moreover, due to the increased incidence of diabetes in western countries, a deeper understanding of such a link is relevant in order to control the escalation in the number of people dealing with dementia. a. pelizzola dipartimento di fisica, politecnico di torino, torino, italy many features of protein folding have been shown to be described by an ising-like model (one-dimensional, with longrange, multispin interactions) whose equilibrium thermodynamics is exactly solvable [ ] [ ] [ ] . we have generalized such a model to the problem of mechanical unfolding. the equilibrium thermodynamics is still exactly solvable, and the characteristic kinetic responses found in force ramp and force clamp experiments are well reproduced [ , ] . unfolding and refolding pathways and intermediates can also be studied, again with good agreement with experiments [ ]. applications to various proteins and rna fragments will be discussed. [ the key role of water in living systems has been widely studied in literature, along with its anomalies, consequence of the extensive three-dimensional hydrogen bonding of water molecules. moreover protein-water interactions take place at protein surface where cell water has been recognized to behave differently from bulky water. the two-states theory of water assumes that water is a mixture of microdomains of different structure and density, the low-density water (ldw) and the high-density water (hdw) domains, and ions partition selectively into ldw or hdw domains. the idea developed in this work was to explore the ordered water structure by measuring delayed luminescence (dl) from salt aqueous solutions in which water structuring is anticipated. it appeared that dl signal from salt solutions is significantly relevant when prevalence of ldw domains is foreseen, with a decay time probability distribution function characterized by a broad maximum in the microsecond range. the obtained results support the ability of dl to reveal the different properties of ldw and hdw domains induced by salt molecules. moreover, the results reveal the existence of clusters, whose characteristics strongly depend on the specific ion effects, of surprisingly long lifetimes not observed till now. this could give new insight into biological water properties. self assembly of patchy particles and dnafunctionalized dendrimers f. sciortino dipartimento di fisica e infm-cnr-soft università 'la sapienza', roma, italy i will report numerical results on the phase behavior of very simple models of patchy particles with the aim of understanding the interplay between phase separation and selfassembly and how the fraction of surface allowing for attractive interactions controls the collective behavior of the system. the case of janus particles, particles characterized by a surface divided evenly into two areas of different chemical composition, will be discussed. i will also discuss the self-assembly of a simple model for four single strands of dna tethered to a central core, and show that the model exhibits a rich phase diagram that includes at least four thermodynamically distinct amorphous phases (polyamorphism) in a one-component system. the dense phases consist of a hierarchy of interpenetrating networks, reminiscent of a woven cloth. peptide dimer motifs in the phospholipid environment -structure, interaction and molecular design p. e. schneggenburger , a. beerlink , t. salditt , u. diederichsen iobc, universität göttingen, germany., irp, universität göttingen, germany. based on recently reported homodimeric peptide pores with a d,l-alternating configuration a novel double helical hairpin-motif of a membrane active gramicidin a analog was designed. [ , ] the cd spectroscopic analyses of the peptide-lipid complexes revealed the structural preservation and elucidated the importance of a zwitterionic interaction of the peptide termini. [ ] the peptide design was enhanced regarding the versatile functionalization with analytical probes as well as molecular recognition moieties like peptide nucleic acids (pnas) to observe the effects of aggregation and specific organization within model lipid membranes even at high peptide-to-lipid ratios. [ ] x-ray reflectivity on lipid bilayer stacks in combination with heavy atom labeling and spectroscopic studies of vesicle systems provides information about the peptide structure and interaction in the native fluid state of the membrane system. [ , ] for this, the fmoc-diiodo-allylglycine building block was created to serve as a novel iodine label pinpointing at a certain position with respect to the membrane normal. we study thermal undulations of giant unilamellar vesicles (guvs) of lipids by flickering spectroscopy. getting values for the mechanical parameters of lipid bilayers requires the experimental fluctuation spectra to be scrutinized in view of the classical helfrich's theory. pure bending modes are revealed unable in predicting the large fluctuations systematically found at high wavevectors. hybrid curvature-dilational modes have been invoked as a more efficient mode of motion in producing high curvatures. a bimodal spectrum of the thermal undulations has been theoretically developed for the shell-like topology. from this new description, two important consequences emerge a priori, the dependence of the fluctuation dynamics on either vesicle size and on bending/compression parameters. for popc and dopc vesicles containing cholesterol the experimental fluctuation spectra are well described by the new spectrum. reconciliation between experiments and theory is achieved when this bimodal spectrum is considered. the new theory opens enormous possibilities for better exploring membrane mechanics in guv models. under normal conditions, platelets circulate in the vascular system having very low interaction with each other and with other cells. the platelets become activated when the biological system is disturbed, for instance by vascular damage in which blood gets in contact with collagen. upon activation, different types of receptors/molecules are exposed on the cell membrane to support adhesion, spreading and aggregation of the platelets onto the damaged vessel. the measurement of altered platelet function is particularly important in cardiovascular diseases such as thrombosis. we are investigating biosensor technologies for the detection of functional properties of platelets. an important study is the specific and non-specific stimulation of platelets in a biosensor cartridge. we will present experimental results on biosensor platelet activation using the platelet-specific membrane markers p-selectin and gp b. multi-joint analysis of locomotion in the first neonatal rats flown in space d. sulica, j. vinersan "carol davila" university of medicine and pharmacy, bucharest, romania the first mammalian neonatal animals in space were the rats flown on the space shuttle endeavor during a -day mission, sts- . the development of locomotion in weightlessness was evaluated using two litters of neonatal rats, launched at postnatal days and . age-and cage-matched animals were used as ground controls. free walking was videotaped from the landing day. although preliminary analysis of walking showed differences in both hindlimb and forelimb joint angles and a hyperextension of the hindlimbs was apparent, the numerical values reached the significance level only for the ankle angle measured at specific moments of the step cycle: foot contact, maximum loading with weight, foot lift and maximum flexion during swing. we report here on the behavior of all the joints during the whole step cycle, by computing the integral of these angles over the step cycle. the results were affected by the differences in the walking speed (the young animals walked faster than the controls), so we scaled the integrals by the step cycle duration. we found that, besides the ankle, the knee was also more extended throughout the whole step cycle in both groups of animals. moreover, all the joints (including the toe and the hip) were affected in the same way (hyperextended), since the differences were still significant when we added together these angles. the animals recovered slowly, with significant differences remaining after days of readaptation. the effect of dextran concentration on red blood cell deposit formation j. strzelecka, b. grzegorzewski department of biophysics, collegium medicum in bydgoszcz, nicolaus copernicus university - bydgoszcz, poland red blood cell (rbc) deposit formation was examined by means of an optical method. blood was obtained from healthy donors and measurements were performed at initial hematocrit %. the intensity of scattered light was measured during sedimentation of rbcs suspended in saline -dextran solutions at different polymer concentrations ( - g/dl). the changes in the intensity of the scattered light manifest rbc aggregate formation, their sedimentation and the process of deposit formation. the deposit formation curve was determined. it is shown that the concentration of dextran affects the deposit formation. an empirical model has been used to describe the experimental data. the parameters of the deposit formation curve as a function of dextran concentration are analyzed. water is essential to life and a major scientific interest lies in a detailed understanding of how it interacts with biological macromolecules in cells. we studied water dynamics in whole cells with neutron scattering [ , , ] . the cellular environment is extremely crowded with macromolecules and water molecules are permanently in close contact to biological interfaces. we measured water dynamics in e. coli and human red blood cells with neutron scattering [ , ] . the data revealed two populations of water in the cells: a major fraction which has dynamical properties similar to those of bulk water (relaxation times ∼ps) and a minor fraction in the order of ∼ % which is interpreted as bound hydration water with significantly slower dynamics (relaxation times > ps). in this contribution we report on investigation of model membrane dynamics by means of quasi elastic and inelastic incoherent neutron scattering and on the effect of membrane inserted pore forming peptide gramicidin. model membrane are realized by highly oriented, hydrated phospholipid bilayer stacks of dmpc ( , -dimyristoyl-snglycero- -phoshatidylcholine) hydrated with d o in excess of solvent condition. the bilayer were supported on mica substrates and prepared at different concentrations of gramicidin, a -residue oligopeptide showing antimicrobial activity by forming pores on the membrane surfaces which allow water and small ions to permeate across the membrane. incoherent qens and ins spectra, measured on in and in spectrometer at ill, allows to obtaining information on the mean dynamics of the hydrogen atoms in the system. moreover, by proper orientations of the membrane plane respect to the scattering wave vector q, we were able to derive information on in plane and out of plane motions of the phospholipids. the using of media products for the creating attracted bioenergetic brain rhythmus advertisement v. i. vlastopulo, v. g. nikolajev str. gen. petrova , app. , odessa, ukraine the technical efficiency of bioenergetical influence of advertisement is present with assistance of consciousness of memory at revision and hearing of advertisement on tv, radio stations, mobile phones, at supermarkets and other places. in a basis of useful model it is put a task to improve the method of creating the attracted advertisement, in which the creation of bioenergetical influence by the oscillation of not less electromagnetic fields or video-images is introduced with their creating as the base on the spatial or flat structure formative macro matrix or matrixes with repeatable structure with the brain α-rhythm frequency of extreme attention and δ-rhythm frequency of meditation. the point of the patent on the device is in the bioenergetical influence increases in addition to the information influence by advertisement of pictures and audio oscillations the bioenergetical influence increases at the consciousness contribution of human memory at the moment of watching or hearing of television, radio stations, working in the internet, music in the supermarkets, banks, clubs, metro and other places. cell adhesion and motility are processes involved in fundamental biological phenomena. they imply multimolecular scaffolds as anchorage points and actin cytoskeleton filaments to build internal stress and eventually crawl onto the substrate. these processes, very dynamic by nature are out of equilibrium. we study cell adhesion on micro-patterned substrates where the introduction of a finite distance between the possible anchorage points of the cell modifies drastically the organization of the cytoskeleton and the anchorage point's distribution. because statistical quantification shows that some shapes are more likely than other, we believe they represent particular organizations of the system which should minimize the energy dissipation. we checked this hypothesis by using the cellular potts model. shapes obtained by simulation are in excellent qualitative agreement with experimental shapes. they depend on phenomenological parameters such as interaction between cells and the extra cellular matrix, a line tension and an elastic modulus. the aim of this work is to link model parameters to physico-chemical properties of cells and to establish phenomenological relations between relevant biochemical regulators controlling the cytoskeleton organization. c. canale, d. ferrera, f. benfenati, l. gasparini the italian institute of technology, genova, italy alzheimer disease (ad) is characterized by cerebral extracellular deposits of β-amyloid (aβ) fibrils. aβ aggregation is a multi-step process involving the formation of various conformational species including soluble intermediate species (i.e. aβ oligomers), protofibrils and fibrils. such aggregates may have various effects on neuronal and glial function and differentially contribute to ad neurodegeneration. aim of this study was to investigate the structural properties of distinct aβ aggregated species and dissect out their effects on neuronal viability. recombinant aβ and aβ peptides were aggregated in vitro in conditions differing by ionic strength, temperature and ph and were analyzed by gel electrophoresis, thioflavin t binding assay and atomic force microscopy (afm). afm analysis was performed using both hydrophilic and hydrophobic substrates, to analyze the full spectrum of structural species. stable low molecular weight oligomers were obtained when aβ was incubated at • c for days in low salt concentration buffer. doughnut-shaped conformational species were detected by afm alongside globular aggregates ( . - . nm height range). acidic ph promoted aggregation of aβ into thioflavin-positive fibrils and protofibrils. protofibrils appeared as beaded chains having a mean height of . ± . nm. effects of aβ on viability of mouse hypppocampal neurons were assessed and correlated with their conformational features. a. bellova , e. bystrenova , m. koneracka , p. kopcansky , f. valle , j. bagelova , f. biscarini , z. gazova institute of experimental physics, slovak academy of sciences, kosice, slovakia, ismn cnr, bologna, italy peptide amyloid aggregation is a hallmark of amyloid diseases including azheimer's disease or type ii diabetes. recent works have addressed the potential of nanoparticles to affect amyloid aggregation. the experimental data are very controversial suggesting that particle characteristics markedly influence the final effect of nanoparticles on the amyloid aggregation (initiation, acceleration or inhibition of amyloid aggregation). we investigate the ability of electrostatically stabilized magnetic nanoparticles of fe o to affect the amyloid aggregation of lysozyme, as a prototype amyloidogenic protein. we have used a combination of spectroscopic (tht fluorescence) and local microscopic techniques (afm). we found, that the ability of magnetic nanoparticles to inhibit formation of amyloid aggregates or destroy pre-formed amyloids exhibit concentrationdependence. the values of inhibition ic and depolymerization dc were determined suggesting that nanoparticles interfere with lysozyme aggregation at stoichiometric concentrations. the observed features make magnetic nanoparticles of potential interest as a therapeutical agent against amyloid diseases. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid, vega grants , and and eu-strp biodot.). a. bellova , l. balogova , b. chelli , e. bystrenova , f. valle , j. imrich , p. kristian , l. drajna , j. bagelova , f. biscarini , z. gazova institute of experimental physics, sas, kosice, slovakia, faculty of sciences, p. j. safarik university, kosice, slovakia, ismn cnr, bologna, italy numerous diseases have been linked to a common pathogenic process called amyloidosis, whereby proteins or peptide clump together to form amyloid aggregates in the body. an attractive strategy to develop therapies for these diseases seems to be reduction of polypeptide aggregation. we have tested several acridine derivatives characterized by various glycosyl groups for their potential to affect the lysozyme amyloid aggregation in vitro. the ability of glycosyl acridines to interfere with lysozyme aggregation was investigated by tht assay. we found that structure of acridine side chain is factor affecting their anti-aggregation activity significantly. for the most effective compounds the values of ic and dc were obtained. the reduction of protein aggregation was confirmed by afm. to investigate influence of the glycosyl acridines on the cell processes we examined effect of compounds on cell viability. we performed glycosyl acridines characterization by high anti-aggregation activity and low toxicity suggesting their possible application for therapeutical purpose. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid and vega grants , and and eu-strp biodot.). a. j. beevers, a. m. dixon department of chemistry, university of warwick, uk due to the immense medical importance of proteins which span the membrane of cells, detailed molecular structural information of these systems is essential. practical difficulties in employing high-resolution structural elucidation techniques have resulted in a relative paucity of fully resolved membrane protein structures. therefore a variety of lower-resolution techniques are used to determine structural information of the transmembrane (tm) domains of proteins. one example of such a membrane protein is erbb- , a receptor tyrosine kinase responsible for triggering cell division and which is prone to a mutation in its transmembrane domain resulting in permanent activation and oncogenic effects. we have predicted an interface for the mutated tm domain dimer using site-specific infrared spectroscopy containing a repeating sequence of ile, val and leu . applying the in vivo toxcat assay to the tm domain sequence and to specific mutants of it, confirms this proposed interface whilst another proposed interface is discounted. current studies are focussing on the effect of the tm mutation to the activation of the erbb- receptor and to any possible change in this interface. bacteriophages are complex molecular assemblies which multiplication relies on bacteria infection. the process starts with the binding of the phage on its specific host receptor and the injection of its genome into the host cytoplasm. our work aims to determine the physical mechanisms and forces driving the dna transfer from the phage capsid. the in vitro dna ejection has been analyzed by using light scattering and gel electrophoresis measurements for three phages (t , spp and lambda) belonging to the same family (syphoviridae). our results reveal two forces contributing to drive the dna transfer: the first one is originated from the pressurization due to the strong confinement of dna into the capsid; the second one comes from a pulling mechanism originated by the presence of condensed dna outside the capsid. these two contributions were characterized in in vitro conditions but they likely play a role in the in vivo transfer. the ejection kinetics was also analysed and the characteristic time of the mechanism was studied as a function of the temperature. it appears to follow an arrhenius law, allowing the determination of the activation energy that governs the transfer. the energy values are close for the different phages, suggesting that the mechanism regulating the ejection is common for a given phage family. below these general features, our studies also reveal differences between the three phages. the effect of &beta-amyloid peptide on polymer cushioned membranes s. dante , r. steitz , t. hauss , c. canale , n. a. dencher istituto italiano di tecnologia, genova, italy, bensc, helmholtz center berlin, germany, tu-darmstadt germany beta-amyloid (aβ) is a peptide implicated in the neurodegenerative process characteristic of the alzheimer's disease (ad). to clarify its mechanism of action it is crucial to elucidate the interaction of aβ with the neural membrane. in previous work we demonstrated the capability of aβ to penetrate and perturb stacked lipid bilayers. in this study we considered polymer cushioned lipid bilayers as a model for neural membranes. the polymer cushion is aimed to preserve the membrane natural fluidity; it is obtained depositing charged polyelectrolites layer-by-layer; the lipid membrane is built on the top of the polymer film by fusion of unilamellar vesicles. the floating membranes were kept always in contact to the subphase. the kinetics of adsorption of the lipid double layer at the polymer/water interface was monitored by neutron reflectivity; different experimental conditions to obtain the best surface coverage were exploited. after administration of aβ to the subphase the lipid membrane still adhered to the polymer cushion, but its structure was modified by the interaction with aβ. neutron reflectivity showed a change of the scattering density profile in the direction perpendicular to the membrane plane, suggesting a penetration of aβ inside the double layer. a change in the surface morphology was detected by afm imaging; afm film-rupture experiments showed that aβ weakens the lipid packing. x. cheng , r. pacheco-gomez , a. rodger , h. matthew , d. i. roper university of warwick, u.k., university of birmingham, u.k. ftsz, the ancestral homolog of eukaryotic tubulins, is a gt-pase that assembles into a cytokinetic ring structure (z ring) essential for cell division in prokaryotic cells. the z ring also recruits other proteins (e.g. zapa, ygfe, zipa) to the division site, where they participate in formation of the septum that separates the two daughter cells. we have studied ftsz polymerization and its dynamic behaviour in real time by right angle light scattering. similar to tubulin, ftsz polymerizes into dynamic protofilaments in the presence of gtp; polymer assembly is accompanied by gtp hydrolysis. the kinetics of inorganic phosphate (p i ) released from the gtp hydrolysis have been studied as well, employing a fast and sensitive colourimetric assay. at ph . , approximate % of the p i was released into the media within minutes of gtp addition. the effects of gtp, ph, k + , and mg + were studied in both cases, and the results were used to build up a model for the mechanism of fibre assembly and disassembly. ygfe, a ftsz accessory protein, is identified as a functional zapa orthologue. finally, we have studied the ygfe bundling to ftsz polymers. it strongly promotes ftsz bundling and is an inhibitor of the gtpase activity. many genomes of viruses encode small membrane spanning proteins which are proposed to modify membrane permeability for ions and small molecules. these channel or pore forming proteins are getting into the focus for antiviral therapy since they are essential for some of the viruses. one of the general themes of the mechanism of function of the proteins is to self-assemble to form the functional form. we present a study on the open reading frame (orf) a membrane protein encoded in structural region of human severe acute respiratory syndrome coronavirus (sars-covs). the full length orf a protein is residues long and contains a single transmembrane (tm) domain. full length protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers forms cation-selective ion channels. the bilayer recordings show cation selection channel activity with a major conductance level of around . ps also at elevated temperatures ( . • c). in silico studies with a amino acid tm domain are done to assess conformational space of the monomeric orf a helix. with this monomeric helix homooligomeric helical bundle models are built and embedded in a fully hydrated -palmitoyl- -oleoyl-sn-glycerol- phosphatidylcholine (popc) bilayer. results of both experimental channel recordings and computational modeling show sars orf a to act as a channel forming protein. -biomolecular self-assembly - microtubules are involved in many vital processes. their rigid structure can resist high forces while their intrinsic ability to switch stochastically between growth and shrinkage phases allows them dynamically to reorganise. in cells, a sizeable network of microtubule binding proteins control and regulate microtubule dynamics. alp and dis are members of the dis / xmap family that are major players in s.pombe. the deletion phenotypes of alp and dis are similar, but nonetheless distinct. both are involved in the formation of spindles but alp is also involved in the maintenance of cytosolic microtubules in interphase. the restrictive temperatures of alp -deletion and dis -deletion mutants are different. alp interacts with alp , a potential member of the tacc protein family. i am working to reconstitute alp /dis -dependent microtubule dynamics in vitro, using purified s. pombe tubulin. both alp and dis express well in insect cells and can be readily purified. preliminary data show that both proteins bind tubulin at low salt concentrations and that both influence the dynamics of pig brain microtubules. my goal is a complete functional analysis of alp and dis , individually and in combination, to test candidate molecular mechanisms for alp /dis -catalysis of s. pombe microtubule dynamics. the syphoviridae coliphage t is a well-suited model to study the assembly of large viral capsids. biochemical and biophysical approaches were used to reconstitute in vitro the assembly pathway of its capsid. the t structure was recently solved from cryo-em and image reconstruction. its icosahedral capsid (t = ) is built from the major head protein (pb , copies) forming both the pentons and hexons and from the portal protein (pb , copies) located at one vertex. its assembly proceeds by steps. pb and pb first assemble into a precursor structure called prohead i, which is converted to prohead ii by proteolysis of pb and pb by a head maturation protease. packaging of the kbp dsdna is then driven through the portal pore by a molecular motor, the terminase. this promotes expansion of prohead ii leading to the mature capsid. the different assembly steps and the conformational changes accompanying capsid maturation were characterized using proheads i either self-assembled from the overproduced and purified capsid proteins or isolated from a phage mutant. these precursor capsid structures were analysed by small angle x-ray scattering. the d structure of prohead ii and of the expanded capsid were solved from cryo-em. our data show that the assembly process of a large icosahedral capsid can be efficiently reconstituted in vitro. amyloid beta peptide fibril formation modulated by phospholipid membranes e. hellstrand , e. sparr , s. linse lund univ., department of biophysical chemistry, sweden, lund univ., department of physical chemistry, sweden disease-causing amyloid fibril formation can be modulated by many factors including interactions with biological lipid membranes. an increasing amount of evidence suggests that the process of fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. alzheimer's is probably the most well-known amyloid disease and the associated amyloid beta peptide originates from the membrane incorporated amyloid precursor protein (app). we use recombinant abeta m - and abeta m - produced in escherichia coli, which allows us to perform large scale kinetics assays with good statistics where the amyloid formation process is followed in means of thioflavin t fluorescence. the lipid membranes are introduced in the system as large unilamellar vesicles composed of dopc, dppc and sphingomyelin, with and without incorporation of cholesterol. we find that the phase behanviour of the membrane in the vesicles has a large effect on the lag time of the amyloid formation process for both abeta m - and m - . all membranes increase the lagtime to some degree but dppc has the largest effect. by comparing different phases we can conclude that the translational diffusion in the membrane seems to be more important than the acyl chain ordering. furthermore, electrostatics, concentration dependence and membrane addition at different time points in the amyloid formation process have been investigated. equilibrium/non-equilibrium transitions in macromolecule interactions p. dumas , g. gibrat , s. bernacchi , e. ennifar ibmc-cnrs, strasbourg, france, llb (cea/cnrs), saclay, france usually, so called 'relaxation phenomena' occur on a fast time-scale and 'p-jump' or 't-jump' techniques are required to follow such events lasting (much) less than ms. we report that, during stability studies of proteins or nucleic acids, such relaxation events can be observed on astonishing long time-scales. we first performed 'melting studies' with nucleic-acid duplexes by using linear variations of temperature (t) with time (t). we observed that even very low rates dt/dt could lead to a frozen state for temperature values below a sharp temperature range, and relaxation to equilibrium beyond that range. this allows defining a 'relaxation temperature' t r separating the two regimes. numerical simulations very accurately described the related hysteresis phenomenon observed upon a heating-cooling cycle, which is the hallmark of departure from equilibrium. analogous observations were made with protein oligomers submitted to either a variable pressure, or variable concentration in denaturant. importantly, a single theoretical frame predicts that the critical relaxation value x r (x standing indifferently for temperature, pressure or denaturant concentration) depends on ln(dx/dt). one may ask whether some thermosensor rnas known for switching on or off genetic expression by 'feeling' a temperature variation, might also 'feel' dt/dt. if true, the exact switching temperature would depend on dt/dt and faster temperature changes would increase t r . -biomolecular self-assembly - the major component of amyloid plaques in the gerstmann-sträussler-scheinker disease is a prion peptide fragment from - to - residues. here, we present a structural study of prp - in form of oligomers and fibrils by fourier transform infrared spectroscopy (ftir) and atomic force microscopy (afm). after incubation at • c, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands and by a wide distribution of oligomer volumes. after a lag phase, a conformational rearrangement of oligomers into fibrils, with a parallel orientation of the cross β-sheet structures, was observed. by afm, different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly. in addition, we also studied thermal and random aggregation. the prp - peptide was found to undergo several aggregation pathways, whose end products display different structural properties and intermolecular interactions. these findings underline the high plasticity of the prion peptide, a peculiar feature of prion proteins to overcome species barriers (natalello et al. j.mol.biol. ; : - ). the role of proline isomerisation in the aggregation process and fibril formation of alpha-synuclein j. meuvis , m. gerard , v. baekelandt , y. engelborghs lab.of biomolecular dynamics, ku leuven, belgium, lab.of biochemistry, campus kortrijk, belgium, lab.for neurobiology & gene therapy, ku leuven, belgium alpha-synuclein (α-syn) plays a central role in parkinson's disease. the aggregation of this protein, which contains five proline residues (p ,p ,p ,p ,p ), is accelerated in vitro by fk binding proteins (fkbps), a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (ppiase). fkbps catalyze the cis-trans conformational change of proline, often a rate limiting step in protein folding. to elucidate the role of the proline residues in aggregation, we constructed a mutant p( , , , , )a α-syn . the kinetics of the aggregation of the mutant were studied with turbidity and thioflavin t fluorescence (tht). turbidity measurements show the formation of early, tht negative aggregates which is as fast for both wt and mutant. fibril formation however is faster for the proline-deficient mutant. we also studied the effect of fkbp on the aggregation of the mutant. although wt α-syn early aggregate formation is accelerated by the addition of µm fkbp , this effect disappears in the mutant. addition of ( pm- µm) fkbp accelerates the fiber formation of wt α-syn, which is abolished in the mutant. we can conclude that α-syn fiber formation is accelerated for the proline-deficient mutant, which suggests a role for the proline residues in fiber formation. furthermore all accelerating effects of fkbp are abolished in the mutant which suggests that the ppiase activity of fkbp is responsible for the accelerating effect on the aggregation of wt α-syn. materials used as gene delivery vehicles must be able to condense dna into small sizes to facilitate transport and crossing various barriers. one of the polycations investigated for dna compaction is chitosan, which has the advantage of being safe and biodegradable. as a step towards reducing the aggregation behaviour of dna-chitosan complexes, chitosans were modified by grafting peg-chains on the backbone. it is known that the transfection efficacy depends on the chitosan chain length. additionally, the degree of pegylation might influence the condensation process. here a systematic biophysical study of pegylated chitosans and how the interplay between chitosan chain length and degree of pegylation affect the compaction of dna in terms of particle size and structure, stability in pbs and when exposed to serum, and transfection efficacy is presented. three different chain lengths of chitosans are employed, and for each sample three pegylation degrees are investigated and the properties of the dna-pegchitosan complexes compared to complexes formed when employing the original, chitosan for dna compaction. it is found pegylation of chitosans can be used to increase both the stability of the dna-chitosan complexes when exposed to serum as well as increase their transfection efficacy in hek cells. max-planck institute for polymer research, mainz, germany model membranes mimic the essential function of a natural membrane. however, the complexity is reduced in order to allow the study of fundamental processes. tethered membranes consist in principle of a lipid bilayer that is covalently linked to a solid support through a spacer group. this architecture allows the characterization of the membrane itself as well as of incorporated membrane proteins using surface analytical techniques. we have established a versatile system of various anchor lipids, which allow membrane formation on different surfaces. the architectures have been characterized by surface plasmon techniques, neutron reflectivity and electrochemical methods. the membranes are electrically insulating and allow for the functional incorporation of ion channel proteins. polymerizable lipids allow to pattern the membrane and to study lateral diffusion processes. furthermore, the membranes can be used as a sensing platform, where embedded membrane proteins act as actual sensing units. a. perico, s. pietronave, l. arcesi, c. d'arrigo consiglio nazionale delle ricerche (cnr), institute for macromolecular studies (ismac) the electrostatic free energy (fe) of two parallel rigid likecharged polyelectrolytes (pes) is given as a function of the separation distance. for high linear charge density, z , the fe shows a minimum due to the increasing of the counterion condensation and condensation volume as the two pes approach. the interaction fe is governed by a critical linear charge density, z c , inversely proportional to the counterion valence. for highly charged pes (z > z c , like dna), the pes attract the stronger the smaller is the counterion valence, because the fe is dominated by the entropic term due to condensation of counterions in a volume displaying a maximum at short distances. for weakly charged pes (z < z c / ) the pes remain undercritical in the whole separation range and therefore repel. for moderately charged pes (z c / < z < z c ), the infinitely separated pes are undercritical but become supercritical as they approach a critical distance and charge condensation and condensation volume expansion start: in these circumstances the pes may attract if the counterion valence is sufficiently large. in the case of many dna rods, hexagonal clusters may be formed. upon interaction with hydrophobic surfaces, proteins show a tendency to expose regions that are normally buried in the hydrophobic core. unfolding is generally perceived as an undesired process in studies aimed to anchor functional proteins at surfaces. upon an upset of perspective the fine control of the unfolding/re-assembly process could be regarded as a strategy to build up molecular nanostructures for the development of organic-inorganic assemblies. we show that molecular layers patterned at the nanoscale, with longrange order properties extending over the microscopic scale, can be obtained upon adsorption of proteins onto the hydrophobic and ordered surface of pyrolytic graphite. upon adsorption, proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. the first layer, in contact with the substrate, and the second molecular layer show corduroy-like nanopatterns of different periodicity, with a relative orientation between the first and second layer patterns of • . surface-induced protein unfolding and polypeptide chain reassembly according to a layered ordered structure is a rather general phenomenon since it is observed for different proteins irrespectively of their specific structural properties. the possibility of using these ordered molecular structures as templates for the subsequent patterned deposition of supramolecular aggregates will be discussed. understanding protein-protein interactions and assemblies to control the hierarchical building of well-ordered supramolecular structures is highly relevant to new tailormade biomaterials. we previously evidenced that contrary to native calcium-loaded α-lactalbumin (holo α-la), calcium-depleted form (apo α-la) has the ability to selfassemble with lysozyme (lys) to form different supramolecular structures in temperature-dependent manner. in the present work, the events occurring at molecular scale were explored using fluorescence techniques. fluorescence anisotropy and fluorescence lifetime measurements provide a powerful and sensitive mean to measure intermolecular interactions. we showed that lys interacts with both apo α-la and holo α-la to form oligomers, assumed to be heterodimers, at • c and • c. the dissociation constants for dimerization, found to be in the µm range, were sensitive to the ionic strength. correlation time calculations suggest that formed heterodimers holo α-la/lys and apo α−la/lys differed in their shape and/or conformation. such conformation differences could explain why holo α-la/lys complexes are trapped as heterodimers while the apo α-la/lys complexes have the ability to further self-assemble into previously reported various supramolecular structures. polyglutamine aggregation and neurodegeneration g. nicastro, l. masino, a. pastore national institute for medical research, the ridgeway, nw aa london, u.k. polyglutamine (polyq) diseases are rare but dominant misfolding diseases linked to neurodegeneration. they are caused by the expansion of cag codon repeats, which encode polyq tracts in the corresponding gene products. aggregation of polyq proteins is thought to be triggered by polyq expansion but be strongly modulated by the protein context. in the attempt of understanding the molecular bases of polyq diseases, we are studying the structures, interactomes and aggregation properties of selected polyq proteins. here, we present recent work on ataxin- , taken as a representative example of the whole family. ataxin- is a ubiquitin specific cysteine protease, involved in the ubiquitinproteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. the enzymatic site resides in the n-terminal josephin domain of ataxin- . we have characterized, using different biophysical techniques, the structure in solution and the aggregation properties of josephin both in isolation and in a ubiquitin complex. we demonstrate that interaction with ubiquitin strongly modulates the aggregation properties of ataxin- and suggest the importance of protein-protein interactions in preventing aggregation. our study also provides new insights into the molecular mechanisms which determine ataxin- specificity for poly-ubiquitin chains of the correct length and cross-linking. förster resonant energy transfer (fret) from an optically excited to a non-excited molecule has been widely used to probe molecular interactions in living cells. changes in the molecular makeup of a cellular region occurring during the acquisition of fluorescence images place tight constraints on the fret technology and data analysis, which could not be addressed satisfactorily until recently. we will describe a method for imaging protein complex distributions in living cells with sub-cellular spatial resolution, which relies on a spectrally resolved two-photon microscope (raicu et al, , nature photon. : - ) and a simple theory of fret in oligomeric complexes (v. raicu, , j. biol. phys. : - ) . then, we will overview recent results on the determination of the supra-molecular structure and distributions in living cells of oligomeric complexes of some g protein-coupled receptors. observing protein aggregates on surfaces m. rabe, d. verdes, s. seeger institute of physical chemistry, university of zurich, switzerland protein aggregation is an important topic of current protein research as it is associated with several human diseases including alzheimer's disease, parkinson's disease, and type ii diabetes. although protein aggregation mechanisms and conditions have been comprehensively investigated, studies on the formation and the fate of protein aggregates in contact with solid interfaces are scarce. we have comprehensively investigated the structure of protein assemblies that form spontaneously upon protein adsorption on solid interfaces using a surface sensitive fluorescence imaging technique based on super critical angle fluorescence (saf) detection. combining this technique with fret we not only succeeded to detect protein aggregates deposited on surfaces but also to characterize their behavior in real time, i.e., their emergence, growth, or spreading. the model protein bsa, for instance, was found to exhibit a certain tendency for aggregation in the buffer solution. these protein clusters can deposit onto solid surfaces and spread resulting in a large, flat structure after some time. a different possibility how protein aggregates emerge on surfaces consists of a direct deposition of protein monomers to pre existing aggregates. such a growth of protein aggregates on the surface has been observed in a model system using the protein α-synuclein, which is tightly associated with the parkinson's syndrome. we present the results of a spectroscopic ellipsometry (se) study of the adsorption process of yeast cytochrome c (ycc) on gold and graphite substrates, according to methods already applied to study the growth dynamics of organosulphur sams on gold [ ] . se investigation was carried out both in situ, at room temperature during protein deposition, and ex situ. on gold, se data demonstrate the formation of an about - nm thick layer, consistent with the formation of a dense monolayer of ycc molecules, confirmed by afm inspection. both in situ and ex situ measurements were characterized by well defined spectral features related to the soret band. analysis of the fine position of this feature allowed to obtain information on the oxidation state of the iron ion of the heme group. se data suggest that proteins have preserved their native structure. a completely different adsorption mechanism was observed on highly oriented pyrolytic graphite (hopg) [ ] . ex-situ se data on ycc/hopg, supported by afm observations, indicate the formation of an ultrathin molecular layer (∼ . nm) related to complete protein unfolding at the hydrophobic surface. the role of phospholipid anisotropy in the stability of inverted hexagonal phase was considered. the equilibrium configuration of the system was determined by the minimum of the free energy involving the contribution of the isotropic and deviatoric bending and the interstitial energy of phospholipid monolayers. the shape and local interactions of a single lipid molecule were taken into account. the minimization with respect to the configuration of the lipid layers was performed by the monte carlo simulated annealing method. at high enough temperature the lipid molecules attain a shape exhibiting higher intrinsic mean and deviatoric curvatures which fits better into the inverted hexagonal phase than into the lamellar phase. for the mathematical model the advanced geometry with non-spherical cross-section of inverted hexagonal phase was calculated, resulting in lower energy in non-spherical cross-section. theoretical results are in a good agreement with the small angle x-ray scattering experimental data. for a long while the conventional view has been that alzheimer disease is brought about by the beta-amyloid fibrils found in the senile plaques, but more recently it has been suggested that the main neurotoxic species would be the soluble oligomeric species, apparently prone to interact with cell structures and macromolecules potentially inducing neuronal dysfunction. peptide-peptide interactions resulting in self-assembly phenomena of beta-amyloid yielding fibrils can be modulated and influenced by small organic molecules that might also be effective therapeutic tools to ideally target both oligomeric and fibrillar species. in this perspective, polycyclic aromatic molecules are of special interest because they might disrupt the molecular architectures precursors of beta-amyloid fibrils by means of weak, non-covalent aromatic interactions, like stacking interactions. we have performed an in vitro spectroscopic study (light scattering, circular dichroism, ftir and fluorescence) of the effects on beta-amyloid fibrillogenesis of the natural pigment hypericin extracted from hypericum perforatum. our results show that, thanks to its structural characteristics and peculiar spectroscopic features, hypericin can be easily used to in vitro monitor the appearance of initial aggregation states of beta-amyloid peptides and, more importantly, that hypericin can interfere with the early stages of polymerization process, playing the role of an aggregation inhibitor. peptaibols are peculiar peptides produced by fungi associated to plants. they are composed by to amminoacidic residues and exhibit antibiotic and antifungal properties. due to their amphypatic nature, they can form ion channels in biological membranes. by making use of experimental models of biological membranes (biomimetic membranes) currently employed in the laboratory of bioelectrochemistry, and models of plant membranes (corn seed root), that are used in the international laboratory of plant neurobiology (linv), we characterized synthetic peptides such as trichogin gaiv and its shorter homologues ( and residues). we studied these peptaibols in a dioleoylphosphatidylcholine monolayer supported by hg using different electrochemical techniques (ac,vc,eis). the experimental technique employed in the linv (clark microelectrode coupled to mife system) allows to measure oxygen flux in the solution contacting plant cell membranes, after treatment with different peptide concentrations. preliminary results might indicate that short peptides can influence the whole metabolism of the plant and can therefore be used as "elicitors" in order to induce an acquired systemic resistance. supported biomimetic membranes (sbm) were developed for protein-membrane interactions studies. phospholipid vesicles were chemically linked onto amine grafted gold or glass surfaces; after an osmotic choc and liposomes fusion a continuous membrane bilayer was formed. the anchoring phospholipid molecule (dspe-peg-nhs) incorporated into the vesicles allowed the formation of a water-filled compartment between the surface and the bilayer. this first sbm model was used to monitor the membranes binding properties (dependent of calcium) of the adenylate cyclase toxin (cyaa) from bordetella pertussis. the sbm model was improved in order to study the translocation of the catalytic domain of cyaa across the bilayer. naturally, the cyaa catalytic domain, when it reaches the target cell cytosol, associates with intracellular calmodulin (cam) an activator of the adenylate cyclase activity of cyaa. to mimic this biological phenomenon, cam was first immobilized on the surface (gold or glass) and in a second step membrane construction was performed over the cam layer. the formation of the biomimetic membrane onto the cam layer was monitored by spr while membrane fluidity and continuity were analysed by fluorescence. our results demonstrated the potentialities of sbm for the study of protein insertion into and translocation across biological membranes. a multi-resolution approach to the structure and function of integrin αiibβ m. rocco , c. rosano , j. w. weisel , d. horita , r. r. hantgan istituto nazionale per la ricerca sul cancro (ist), genova, italy, university of pennsylvania, philadelphia, pa, usa, wake forest university, winston-salem, nc, usa. integrins are heterodimeric transmembrane receptors involved in mechanical anchoring and two-way signaling. each α and β subunit has a modular structure with a large extracellular portion, a single transmembrane region, and a cytoplasmic domain. integrins activation mechanism is regulated by controversial conformational changes: while crystallography revealed similar bent shapes for resting and primed extracellular region constructs, ligand binding-induced large structural rearrangements in smaller constructs suggested extension, "opening" and tails separation. in a multiresolution approach, we used experimental and computed hydrodynamics to discriminate among αiibβ integrin models built on x-ray, nmr, and em data. in contrast with xray data and d em maps, an extension is needed to match the hydrodynamics of full-length, solubilized αiibβ ; an electron tomography-based model fares better. consistent with that, and with our averaged d em images, a conformational change in the head region (β hybrid domain swingout) coupled to a simple transmembrane helices shift matches priming agents-induced frictional changes in full-length αiibβ . our multi-resolution study thus suggests that in integrins extension and immediate tail separation are uncoupled from head domain rearrangements following activation. -biomolecular self-assembly - stabilizing effects of α s -casein, a natively unfolded protein, on the aggregation of biomolecules a. trapani, r. carrotta, p. l. san biagio, d. bulone ibf-cnr palermo, italy α s -casein is one of the four types of caseins, a group of calcium phosphate-binding proteins that, in the form of micellar aggregates, makes up the largest protein component of bovine milk. the structure of α s -casein is that of a triblock polymer with a hydrophilic tract interposed between two hydrophobic blocks. due to the lack of a compact folded conformation, this protein can be classified as one of the intrinsically disordered (or natively unfolded) proteins. this class of proteins is known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces that partially unfolded molecules may expose to the solvent. here we present results on α s -casein effects on the thermally induced aggregation of gluthathione stransferase, a ligand-binding anzyme, and - β-amyloid peptide involved in alzheimer's disease. by means of light scattering and circular dichroism experiments, we attempt to reveal the molecular details of α s -biomolecules interaction. bacterial protein self-assembly on surfaces of well-defined chemistry s-layers are one of the most common cell envelope components of prokaryotic organisms and represent the simplest biological membrane developed during evolution. these (glyco)proteins, which can self-assemble into -d crystalline nanostructures on lipid films, liposomes, and polymers, play already an important role in nanobiotechnology. in this work, we present new findings concerning the recrystallization of bacterial proteins on substrates with defined chemistry. manipulation of the protein-sample interaction was carried out by changing the relative height of oh and ch terminated moieties in self-assembled monolayers (sams). we have found that differences in chain length lead to: i) protein bilayer-protein monolayer transition, ii) preferential protein side adsorption, and iii) increase of the crystal lattice parameters. further manipulation of the protein-sample interaction was achieved by using silane chemistry. we will show that sample hidrophobicity speeds up recrystallization kinetics and reduces the crystalline domain size (and layer compliance). the protein-adsorbed mass per unit area on these substrates is reported for the first time. self-assembly of phenylalanine oligopeptides: insights from experimental and computational studies p. tamamis , l. adler-abramovich , m. reches , k. marshall , p. sikorski , l. serpell , e. gazit , g. archontis dpt. of physics, univ. of cyprus, cyprus, dpt. of molecular microbiology and biotechnology, tel aviv univ., israel, dpt. of biochemistry, univ. of sussex, u.k. the diphenylalanine peptide (ff) forms well ordered nanotubes and its derivatives form nano-assemblies of various morphologies with promising material applications. we demonstrate for the first time by electron and laser microscopy and ftir spectroscopy that the related, triphenylalanine peptide (fff) assembles into rather planar nanostructures, rich in β-sheet. in addition, we conduct . -µs replica exchange m.d. simulations of aqueous ff and fff solutions in implicit solvent. the peptides coalesce into aggregates and participate frequently in open or ring-like linear networks, as well as elementary and network-containing structures with β-sheet characteristics. polar and nonpolar interactions, as well as the surrounding aggregate medium contribute to the network stabilities. within a network, consecutive peptides are linked by head-to-tail interactions; the aromatic sidechains of neighbor peptides assume approximately "t-shaped" orientations. these features are observed in ff crystals and could characterize early formations, or stabilize the mature nanostructures. the fff aggregates acquire higher stability and peptide-network propensity compared to the ff aggregates due to energetic contributions , . chlorophyll biosynthesis is light-dependent in angiosperms because the reduction of protochlorophyllide (pchlide) into chlorophyllide is driven by a photoenzyme, nadph:pchlide oxidoreductase (por). the unique properties of por are due to its ability to assemble into dimers and oligomers within the prolamellar body (plb) membranes of etioplasts studied mainly in leaves of dark-grown seedlings under laboratory conditions. we extended these studies to plant organs developed in the nature: cabbage heads, leaf primordia inside buds, pericarp-covered regions of sunflower cotyledons, potato tubers and seedlings germinating under the soil. in electron microscopic and fluorescence spectroscopic studies we found in many of these organs poorly developed plbs in which por was mainly in monomer state. as a consequence, the chlorophyll accumulation was slow and photo-oxidation processes occurred at illumination. in vitro we artificially induced the aggregation of por monomers into oligomers in glycerol and sucrose containing buffers. this resulted in the increase of the photoreduction rate at the expense of photo-oxidation. these results underline the importance of the self-assembly of por and the plbs in chloroplast development and chlorophyll synthesis in nature. v. vetri , g. ossato , v. militello , m. a. digman , m. leone , e. gratton dsfa, university of palermo, palermo, italy, lfd, university of california, irvine, ca, usa we report an experimental study on concanavalina (cona) aggregation in live cells. in vitro, close to physiological temperature, cona readily forms fibrils involving secondary structure changes leading to β-aggregate structures. the effect of cona on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. in particular, we monitored protein aggregation in live cells by means n&b analysis, cross-n&b and rics. n&b showed the aggregation kinetic and the progressive formation of cona oligomers at cell surface. this suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. in parallel, the morphology of the cells changes indicating the progressive cell compaction and death. aggregation and binding of small aggregates to the cell surface were assessed by rics: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. oligomers formation may stimulate non-specific cellular responses due to the exposure of reactive regions of protein structure and of progressive formation of cross−β structures. moreover, aggregates stoichiometry was measured during the kinetic by cross-variance n&b. the two conductive pathways of p x purinergic receptor: different modulation and selectivity r. barbieri , s. alloisio , a. di garbo , m. nobile institute of biophysics, cnr, via de marini , genoa, italy, institute of biophysics, cnr, via g. moruzzi , pisa, italy the p x purinoceptor (p x r) is an atp-gated cation channel that is able to activate a cell permeabilizing pore. p x r cytosolic c-terminal tail is thought to modulate this function. this study was aimed to characterise the biophysical properties of p x r compared to those of the variant lacking the entire c-terminus tail (trp x r) by measuring whole-cell currents and intracellular ca + variations. a mathematical model is used to describe the experimental results. in p x r expressing hek- cells, the potent agonist '-o-( -benzoyl)benzoyl adenosine '-triphosphate (bzatp) -evoked ionic currents depending on concentration and frequency of agonist applications. the currents were strongly inhibited by extracellular mg + in a noncompetitive way. by contrast, in trp x r cells, only high bzatp concentrations elicited small currents not affected by mg + . interestingly, bzatp-induced ca + influx was present both in p x r and in trp x r cells, albeit in the latter the intracellular ca + elevation was smaller. importantly, in trp x r the intracellular ca + rise maintained a competitive mechanism of mg + inhibition similar to that observed in p x r. the experimental data and the modelling findings support the tenet of a functional structure of p x r possessing two distinct conductive pathways. the review of our data on the effects of physical and chemical weak signals on physicochemical properties of water, cell volume, activity and the number of membrane proteins (receptors, ionic channels and enzymes, na + /k + pump and na + /ca + exchanger), intracellular signal systems in norm and pathology (cancer and nerve disorders) would be presented. light microscopic, cell voltage-and patch-clamp, isotope, standard biochemical and genetic engineering methods were used. weak signal-induced effects on cell functional activity (intracellular enzymes activity, the number of functionally active membrane proteins) are realized by changing the physicochemical properties of extra-and intracellular aqua medium. the latter induces the modulation of na + /k + pump-induced cell hydration, which serves as a primary mechanism through which the autoregulation of pump and regulation of membrane excitability and chemosensitivity are realized. by genetic engineering method in oocytes it was shown that the correlation between na + /k + pump and na + /ca + exchanger, which is realized through intracellular messenger systems, plays a crucial role in weak signals transduction in cells and determination of aging-induced increase of cell pathology. listeriolysin pore forming ability in planar lipid membranes at different ph listeriolysin o (llo) is a cholesterol-dependent cytolysin secreted by the intracellular pathogen listeria monocytogenes. its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. llo exhibits optimal cytolytic activity at low ph but it is still able to bind membranes at physiological or even slightly basic ph values in a cholesterol-dependent fashion. high cholesterol concentrations in the membrane restore the low activity of llo at high ph values. based on this broad ph activity we investigated the electrophysiological properties of pores formed by llo at room temperature and at different phs using planar lipid bilayer technique. llo is able to form pores both at ph . and . with a similar permeabilizing ability and similar heterogeneous conductances in the range of picosiemes to nanosiemens. cholesterol content directly correlates with llo activity but it does not change the pore characteristics. collectively, our results demonstrate that llo activity at physiological ph cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for listeria pathogenesis. s. aimon, g. toombes, p. bassereau institut curie, paris, france the physics of membrane/channel and channel/channel interactions is difficult to investigate in cells where it is nearly impossible to modify relevant parameters to deduce physics laws. to overcome these difficulties we built a model system in which voltage gated ion channels were reconstituted in giant unilamellar vesicles (guvs) for the first time. as a first step, we successfully expressed kvap (a voltage gated potassium channel) in e-coli. the channel was purified, fluorescently labelled and reconstituted in small liposomes. its functionality was checked with electrophysiology via fusion of these liposomes into black lipid membranes (blm). as a second step, guvs were formed from these small proteoliposomes using electroformation in a buffer containing mm kcl salt. the proper incorporation of proteins into guvs was controlled using confocal microscopy. functional proteins were detected using the patch clamp technique. with our protocol, we are thus able to prepare guvs containing functional voltage-gated ion channels. one goal is now to study the effect of channel activity on its spatial distribution in these guvs. ryr activation in cultured shr cardiomyocytes at the end of the prehypertensive period the rate of [ca + ] i elevation after the ryanodine receptor (ryr) activation by -chloro-m-cresol ( -cmc) and l-type ca + channels (dhpr) activation by bay k was studied in cultured ( days) cardiomyocytes of spontaneously hypertensive (shr) and normotensive rats (wky, wistar) during weeks of postnatal development. the differences in dh-prs and ryrs activities began to be evident after weeks age when cicr formation has finished and became more expressed at the end of prehypertensive period ( weeks). in response to -cmc ( mm), a drastic increase in the rate of [ca + ] i accumulation ( . ± . times) in shr myocytes was registered after days age versus a decrease in the rates of ca + efflux from the sarcoplasmic reticulum of wistar and wky rat cardiomyocytes. bayk ( µm) also induced more sharp [ca + ] i elevation in shr myocytes ( . ± . times) as compared with wistar ( . ± . times) and wky ( . ± . times) ones of the same age. our results argue that in shr and wky cardiomyocytes, as opposed to normotensive wistar rats, gradual growth of dhpr activity is observed, which follows in parallel with cicr formation in the excitation-contraction coupling during early postnatal ontogenesis, and drastic activation of ryr in shr myocytes after the process termination. cytochrome ba from thermus thermophilus belongs to the large family of structurally related heme-copper oxidases. it accepts electrons from cytochrome c at the p-side of the membrane and uses them to reduce oxygen to water. the energy released in this reaction is used for proton pumping across the membrane to form of an electrochemical proton gradient, used by the cell for formation of atp. in this work we followed the kinetics of single-electron injection into the oxidized nonrelaxed state (o h →e h ) of cytochrome ba by time-resolved optical spectroscopy. two main phases of electron transfer were resolved. the first (τ∼ µs) includes oxidation of cu a and simultaneous reduction of both low and high spin hemes. the second (τ∼ µs) reflects reoxidation of both hemes by cu b . this is in significant contrast to the o h →e h transition of aa -type oxidases, where the fastest phase is due to transient reduction of the low-spin heme a only. on the other hand, the single-electron reduction of the relaxed o state in ba oxidase consisted of only rapid electron transfer from cu a to heme b, which is similar to that in aa oxidase. this indicates a functional difference between the relaxed o and the pulsed o h states of cytochrome ba . as opposed to the phospholamban pentamer, sarcolipin forms anion-selective channels in biomembranes l. becucci , c. b. karim , d. d. thomas , g. veglia , r. guidelli chemistry department, florence university, florence,italy, chemistry department, university of minnesota, minneapolis, mn , department of biochemistry, molecular biology, and biophysics, university of minnesota, minneapolis, mn sarcolipin (sln) and the phospholamban pentamer (pln) are two membrane proteins that inhibit ca-atpase of the sarcoplasmic reticulum at low concentrations. in contrast to pln, sln stimulates maximal ca + uptake rates. sln and pln were incorporated in a bilayer lipid membrane (tblm) tethered to a mercury electrode through a hydrophilic spacer. electrochemical impedance spectroscopy measurements show that sln forms channels permeable to chloride ion, weakly permeable to phosphate ion and impermeable to inorganic cations such as na + and k + . a relationship between this property of sln and its regulatory function on ca-atpase of sarcoplasmic reticulum is proposed. atp increases the permeability of a tblm incorporating sln to phosphate ion by associating to sln with an association constant of . µm. an explanation for this behavior is provided. sln can be identified with the " p i transporter" described by a.g. lee et al. conversely, both electrochemical impedance spectroscopy measurements and molecular dynamics simulations provide strong evidence that the pore of the pentameric form of pln does not act as a chloride channel. "social" domain organization and dynamics of nicotinic acetylcholine receptor at the cell membrane f. j. barrantes unesco chair biophys. & mol. neurobiology. univ. nac. sur, b. blanca, argentina a combination of experimental techniques (patch-clamp, confocal frap and fcs, single-particle tracking, highresolution fluorescence microscopy) has been used to analyze the supramolecular organization of the acetylcholine receptor (achr), the dynamics of the receptor at the cell surface, and the kinetics of receptor internalization. changes in cholesterol (chol) content affected muscle and neuronal-type achr organization and dynamics at the cell surface. chol depletion produced gain-of-function of single-channel dwell time. submicron-sized (∼ nm) domains, stable over a period of hours at the cell membrane, could be resolved into achr "nano-clusters" with a peak size distribution of ∼ nm by sted microscopy. chol depletion reduced the number of nanoclusters, increasing their size, and changed their supramolecular "social" organization on larger scales ( . - . microns). frap, fcs and spt experiments provided information on the dynamics of achr nanoclusters, disclosing the dependence of their mobility on chol content and cortical cytoskeleton. chol content at the plasmalemma may thus modulate cell-surface organization and dynamics of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute achr losses from the cell surface. m. czaplinska, k. gwozdzinski, a. koceva-chyla division of research of structure of biopolymers university of lodz, lodz, poland doxorubicin (dox) and paclitaxel (ptx) are anticancer drugs commonly used in chemotherapy of breast cancer therapy, however, the use of these drugs is limited by the risk of developing heart failure. generation of reactive oxygen species contributes to the cardiotoxicity of doxorubicin. nitroxides are low molecular weight, stable free radicals reacting with ros and they present antioxidant properties. the aim of this study was to analyze the effects of pirolid (pd) on the oxidative stress induced by dox and taxane in mcf- breast cancer cell line. results from mtt test revealed that ptx is more cytotoxic towards mcf- cells than dox. the ic was . µm and µm, respectively. pd alone does not influence cell viability. pd in combination with both drugs did not change viability of cells. both drugs increased the level of carbonyl groups in cells. the highest level of peroxide was observed in cells incubated with dox (approx. -fold). nitroxide alone did not influence the level of peroxide in the whole range of concentrations. combination of pd with dox and ptx reduced the level of carbonyls depending on its concentration. pd did not affected on the level of peroxide in cells suspension. dox and ptx increased ( -fold) the level of carbonyls. pd decreased the level of peroxides in cells treated with dox and ptx. the lowest concentration of peroxide was observed at µm of pd. these results show that pd protect mcf- cells against oxidative stress induced by drugs. open channel structure of mscl from fret microscopy and simulation mechanosensitive channels open in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing and osmoregulation. here, we have determined the likely structure of the open state of the mechanosensitive channel of large conductance from e. coli (mscl) in a natural environment using a combination of patch-clamp studies, fret spectroscopy, epr data, molecular and brownian dynamics simulation. structural rearrangements of the protein are measured while controlling the state of the pore by modifying lipid bilayer morphology. fret efficiency changes can be related to distance changes using a monte carlo analysis program in conjunction with detailed orientational analysis. these measurements are used as restraints in all atom molecular simulations in order to determine the likely structure of the open state, whose probable conductance is derived from brownian dynamics simulations. transition to the open state occurs via large rearrangements throughout the protein that create a wide pore nearly a in diameter. both transmembrane helices are found to line part of the pore. the n terminal helix is found to lie along the face of the membrane where it can act to sense membrane tension and directly transfer this to the pore lining helices. the method of coupling spectroscopic data with simulations is likely to be of great value for studying conformational changes in a range of membrane proteins. putative potassium channels in synechocystis sp. pcc v. checchetto, m. zanetti, g. m. giacometti, i. szabò, e. bergantino department of biology, university of padova, italy we are interested in the identification and characterization of potassium channels in the cyanobacterium synechocystis sp. pcc , an organism which is considered the ancestor of plant chloroplasts. a bioinformatic screening of synechocystis proteome identified, among others, two proteins on which we focused our attention. the first one (syncak) displays sequence homology to mthk, a ca + -dependent potassium channel from m . thermoautotrophicum. the second one (synk) is predicted to contain six transmembrane regions and the typical selectivity filter of all potassium channels. our goal is to understand their roles in the physiology of cyanobacteria. we cloned their coding sequences in fusion with gfp and the hybrid proteins were expressed in chinese hamster ovary cells. we evaluated the presence of both proteins in plasma membrane by fluorescence microscopy and then we proceeded to their functional characterization using patch clamp technique.this analysis will allow us to gain information about channel activity, regulation and pharmacology. we also plan to evaluate the importance of syncak and synk channels in photosynthesis. to test the hypothesis that they could be involved in regulating this process, we will produce deletion and site-specific mutants in synechocystis. finally we would also identify the homologous of these channels in the higher plant a. thaliana and obtain some information about their localization and function. j. braunagel max-planck institute for polymer research, ackermannweg , mainz, germany the cyclododecadepsipeptide valinomycin is composed of two amino acids (l-valine and dvaline) and two hydroxyl acids (d-α-hydroxy-isovaleric acid and l-lactic acid). they form a membered ring of alternating amino and hydroxyl acids. in the cyclic structure, the polar groups are oriented towards the central cavity, whereas the rest of the molecule is relatively nonpolar. this enables the complexation of ions and valinomycin acts as a selective ion transporter (k + ) in lipid membranes. when one of the amino acids is exchanged, e.g one l-valine by an l-lysine, selected functionality can be engineered into the depsipeptide while maintaining its ion conducting properties. we induced several modifications into valinomycin, i.e. a biotin binding unit or a ferrocene group to induce an electrochemical active center. the ion conducting properties of the modified ion carriers have been probed in planar lipid bilayers as well as in solid supported membranes. the role of the membrane dipole potential (ϕ d ) is of a particular interest due to a powerful impact of this potential on the membrane permeability and lipid-protein interactions. channel forming activity of gramicidin a, alamethicin, syringomycin e, hpa peptide, and ompf porin are influenced by ϕ d . we have studied the effect of the membrane dipole modifier, phloretin, on the properties of single channels formed by a wild-type alpha-hemolysin in planar lipid bilayers. the single channel of a ∼ ps conductance exhibits transitions into a number of low-conductance states as the transmembrane voltage exceeds ∼ mv (regardless of the voltage polarity). the phloretin addition to the bathing solutions ( µm) (after the hemolysin channel was formed in the membrane) shifts dramatically the channel voltagedependence. transitions to the low-conductance states are observed at ∼ mv. the effect of the phloretin addition was not observed in the case when it was introduced into the bathing solution before alpha-toxin. since phloretin reduces ϕ d , the data may report on the influence of the electric potential profile on the energy of the low-conductance state of the alpha-hemolysin channel. the alternative explanation of this effect consists in a specific interaction between the phloretin and toxin channel. the work is supported in part by rfbr (# - - ), ss (# . . ) , and the program of the ras «molecular and cell biology». atypical mechanism of conduction in potassium channels c. domene , s. furini physical and theoretical chemistry laboratory, department of chemistry, university of oxford, oxford, u.k., department of electronics, computer science and systems, university of bologna, bologna, italy potassium channels can conduct k + ions with rates of up to ∼ ions per second at physiological conditions, and they are selective to these species by a factor of over na + ions. ion conduction has been proposed to involve transitions between two main states, with two or three k + ions occupying the selectivity filter separated by an intervening water molecule. the largest free energy barrier of such a process was reported to be of the order of - kcal mol − . here, we present an alternative mechanism for conduction of k + in k + channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of k + -water-k + . we exploited the au-deposited self-assembled monolayers of the type: [−s−(ch ) n −ch ] (where n = , and ) with hydrophobically adsorbed redox protein -azurin to verify intrinsic electron transfer mechanisms according to the charge-transfer theory. the enthalpies and volumes of activation were determined through the variation of temperature ( - o c) and pressure ( - mpa) and experimental values were compared with those expected on the theoretical grounds. for the case of n = the activation enthalpy definitely contains a large contribution originated from frictional-like dynamics of protein, and the activation volume has a small positive value. for n = the value of activation enthalpy directly matches / of that for the reorganization energy, and the activation volume attains substantially negative value. for n = we observed the intermediary performance. the whole kinetic pattern is consistent with the smooth changeover between adiabatic and nonadiabatic mechanisms of electron transfer. two gating modalities in the pore of the miniature k + channel kcv kcv is a viral protein that forms functional k + channel in heterologous systems. because of its miniature size ( amino acids) we use kcv as a model system to study and manipulate basic properties of the k + channel pore. by analysing single-channel recordings we highlighted two voltage-dependent modalities of gating in kcv: a slow and a fast gating. the presence of a slow gating is revealed by the very low (in the order of - %) mean open probability. slow gating is not related to the presence of a bundle crossing, as shown by accessibility of the cavity to mts reagents. channel opening might involve the transient formation of salt bridges between residues at the n and c termini of the channel, as suggested by mutational experiments inspired by molecular dynamics simulations of kcv. fast gating, analyzed by beta distributions, is responsible for the negative slope conductance in the single-channel i/v curve at extreme potentials and can be explained by depletion-aggravated instability of the filter region. aca is a type b caatpase with a regulatory n-terminus whose autoinhibitory action can be suppressed by binding of calmodulin (cam). aca n-terminus is able to bind a region of the small cytoplasmic loop connecting transmembrane domains and . to define the role of this interaction in autoinhibition we have analysed a number of single point mutants produced by mutagenesis of aca e -n sequence. mutation to ala of any of acidic residues (e , d , d , d , e , d ) originates an enzyme with normal activity in the presence of cam, but less camstimulated. these results highlight the relevance of a negative charge of the surface area of the small cytoplasmic loop in aca autoinhibition. the most deregulated mutant is d a aca , which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the d a mutant is stronger than that of d n aca suggesting a more direct involvement of this residue in autoinhibition. of the other mutants (i a, n a, p a, p a, v a, n a), only p a aca has a basal activity higher than that of the wt. these results provide the first evidence that the small cytoplasmic loop of a type b caatpase plays a role in the attainment of the autoinhibited state. complex i, the first member of the respiratory chain, serves as a proton pump catalyzing transfer of two electrons from nadh to ubiquinone coupled with the translocation of four protons across the membrane. so far the mechanism of energy transduction by complex i is unknown. the nadh-binding cavity of complex i has a very prominent feature -the presence of two invariant amino acid residues, glutamate and tyrosine, that are exposed to the solvent and located in the vicinity of the fmn, the primary electron acceptor in the enzyme. it was suggested that they might be involved in the binding of nadh through interaction with its nicotinamide moiety. in this work we assessed the function of corresponding glu from the nuof subunit of e. coli complex i by mutation for glutamine. we showed that the negative charge of glutamate in the catalytic site is needed for the electrostatic repulsion of negatively charged phosphates of nucleotides. this process facilitates release of the product nad + and, as a result, accelerates turnover of complex i. we also found that glutamate, as one of the four negatively charged amino acid residues surrounding the isoalloxazine ring of the fmn at a distance of - Å, has a share of mv of the overall mv depression of the midpoint potential of this redox cofactor. l. erokhova , p. kügler , p. pohl institute of biophysics, johannes kepler university, linz, austria, ricam, austrian academy of sciences, linz, austria the mechanism of water transport through epithelia is still under debate. in the present work we tested the hypothesis of isosmolal water transport using mdck cells stably expressing the human sodium-glucose cotransporter (hsglt ). tagging hsglt with egfp enabled determination of its abundance in the plasma membrane by fluorescence correlation spectroscopy (fcs). by monitoring tiny shifts in the concentration of water-soluble dyes in the vicinity of epithelia, fcs also allowed assessment of the water fluxes through confluent cell monolayers grown on permeable supports. fitting the set of differential equations for the osmotic drift and for the back diffusion to the experimentally determined dye distribution permitted calculation of water flow both in the presence and in the absence of an osmotic gradient. from the calorimetric measurements of glucose transported across the cell monolayer, the water:glucose stoichiometry was derived. dividing the increment in osmotic water flux due to hsglt expression by the number of hsglt copies in the plasma membrane resulted in a single transporter water permeability p f of . x − cm /sec. thus, p f is close to the single channel water permeability of aquaporin- . consequently, even small osmolyte concentration differences between the cytoplasm and the basolateral buffer solution are sufficient to drive a substantial water flux. m. emre, s. kavak cukurova university school of medicine, department of biophysics, balcali-adana, turkey experimental studies have shown that the at -receptor antagonists telmisartan (tel) has a ppar-activating property, but there does not appear to be a class effect. to test telmisartan's importance, we investigated its effect on electrical activities (ea) in diabetic (d) rats. the purpose of this study was to investigate the effects of the tel on ea of diabetic papillary muscle (dpm) with stz-induced. in this study, we used four groups: ( ) nondiabetic control (ndc) group (c), ( ) tel-treated ndc group (c+tel), ( ) diabetic group (d), and ( ) tel-treated d group (d+tel). diabetes was induced by a single i.v injection of stz. in the study, membrane potential (mp) and action potential (ap) recorded after the establishment of diabetes. ) mp was decreased significantly in both tel-treated c and d rats (from - , ± , to - , ± , mv and from - , ± , to - , ± , mv). ) ap unchanged in d group, whereas c+tel and d+tel groups showed increase in ap compared with c and d groups. ) repolarization time was prolonged in diabetic rats. ) in c+tel and d+tel groups depolarization rate values increased significantly. on the other hand, in d group repolarization rate values descreased increased significantly compared to baseline values in tel solution. as a result, our data suggest that the beneficial effects of tel-treatment on the ea of the dpm appear to be due to the diminished k + currents. cytochrome c oxidase is a terminal complex (cco, complex iv) of a respiratory chain that is located in an internal membrane of mitochondria or plasma membrane of bacteria. cco is an electron transfer enzyme that reduces o and uses the redox energy of the o reduction for the proton translocation across the membrane. the electron-and proton-transfer generate a transmembrane electrochemical gradient (∆µh + ) that is used for atp synthesis and all other kinds of work for the cell needs. the proton translocation mechanism of cco requires 'channels' for the h + uptake and expulsion within the enzyme. the proton transfer occurs on a time-scale of micro-to-milliseconds. in order to study the proton transfer in cco, a flow-flash approach based on a time-resolved ftir spectroscopy was developed and applied. our ftir flow-flash approach (the measurement of the reaction of cco with o ) allows to reach a time resolution up to tens milliseconds. with this approach and site-specific mutants of cco where the catalysis is slowed down, separate steps of the proton transfer were studied. the results showed that a unique cross-linked tyr- (in a combination with a time-resolved visible spectroscopy and electrometry) serves as a proton donor for the dioxygen bond cleavage during the o reduction by cco. furthermore, the protolytic transitions of glu- -a key amino acid in the proton transfer mechanism in cco -were shown for the first time. role of calcium ions in nickel potentiation of nmda currents p. gavazzo, i. zanardi, p. guida, c. marchetti biophysics institute ,cnr, genova, italy nmda receptors are glutamate-gated channels distributed throughout the brain in the excitatory synapses and are critical for the nervous system function. they are assembled from two types of subunits, the essential nr and at least one nr (a,b,c,d). nickel (ni) modulates the current flowing through nmda receptors in a different way depending on the nr subunit present. we have recently identified several domains of the channel involved in ni interaction, but many aspects of this modulation remain elusive. in this work we intended to determine the role of calcium (ca) ions in the potentiation induced by ni on the current through nr /nr b recombinant nmda receptors. when ni was applied in the presence of the physiological concentration of ca ( . mm) , a voltage-independent potentiation of the current was observed with a kp of . µm. this effect was progressively reduced by decreasing ca concentrations and it was no more detectable with . mm ca or in the presence of barium (ba, . mm). in this last case the effect of ni on nr /nr b receptors was mainly inhibitory (ki(- mv)= µm). therefore a physiological concentration of ca is necessary to induce ni amplification of the current. many data in the literature indicate a correlation between ca ion entrance through the channel, nmda current facilitation and cytoskeleton; however, in our experiments, the application of the actin perturbing agent cytochalasin-d did not produce major modifications in ni effect. heteromerization properties of voltage dependent potassium channels f. gambale , l. pedemonte , a. naso , i. testa , c. usai , a. diaspro , c. picco istituto di biofisica, cnr, genova, italy, lambs-microscobio, department of physics, genova, italy voltage-gated potassium channels are either homomeric or heteromeric tetramers composed of four α-subunits. in order to bring a contribution to the comprehension of channel heteromerization we have been investigating the properties of two plant voltage-gated k + -channels by using electrophysiological and fluorescence techniques. experiments were focussed on kdc and kdc , coexpressed in xenopus laevis oocytes. kdc , the first potassium channel cloned from daucus carota, belongs to the subfamily of α-modulatory silent channels as it doesn't form functional homomeric channels by itself. on the contrary kdc forms functional heteromeric channels when coexpressed with homologous subunits. kdc , the last k + channel cloned from d. carota, belongs to the kat family and shares an overall identity of % with kat . to correlate kdc functional properties with its localization in oocytes, kdc and/or kdc subunits were labelled with gfp and their properties investigated by confocal microscopy and voltage-clamp. we found that the kdc -egfp fusion protein is not targeted to the plasma membrane unless it is coexpressed with kdc . moreover electrophysiological experiments demonstrated that the heteromeric kdc -kdc channel has altered selectivity and activation properties with respect to homomeric kdc channel. circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome (ards) [ ] . we present a microfluidic investigation of the roles of actin organization and myosin ii activity during the different stages of leukocyte trafficking through narrow capillaries using specific drugs. the deformation rate during entry reveals that cell stiffness depends strongly on f-actin organization and hardly on myosin ii activity, supporting microfilament role in leukocyte sequestration. in the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. conversely, membrane unfolding was independent of leukocyte stiffness. the surface area of sequestered leukocytes increased by up to % in absence of myosin ii activity, showing the major role of molecular motors on microvilli wrinkling and zipping. finally, cell shape relaxation was largely independent of both actin organization and myosin ii activity, whereas a deformed state was required for normal trafficking through capillary segments [ ] . [ ] g.s. worthen et al., science, , - ( ) excitation-contraction coupling in skeletal and cardiac muscle is tightly regulated by the calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor (ryr). we could previously show that suramin is a potent activator of the ryr via the calmodulin binding site. calmodulin shows dualistic action, i.e. activation or inhibition of the ryanodine receptor, depending on the absence or presence of ca + . screening of suramin analogues identified nf as a use-dependent inhibitor of the skeletal muscle ryr (ryr ). here we show that nf inhibits high affinity [ h]ryanodine binding and single channel recordings of the purified ryr . nf induced a reduction of open probabilities in a concentration dependent manner, with no effect on current amplitude and unitary conductance. importantly, nf triggers flickering episodes of channel openings and closings before the ryr is frozen in a complete non-conducting state, which is fully reactivated by the ryr agonist atp. moreover, zwitterionic behaviour of nf facilitates plasma membrane permeation, which prevented caffeine induced ca + transients in skeletal muscle cells and cardiomyocytes. conversely, ip mediated ca + signals were not altered by nf . this work was supported by herzfelder'sche familienstiftung and fwf . beyond steady-state protein dynamics t. hauß , j. pieper , a. buchsteiner , r. e. lechner , n. a. dencher helmholtz-zentrum berlin für materialien und energie, berlin, germany, technische universität darmstadt, germany, technische universität berlin, germany to study protein dynamics beyond steady-state experiments we have developed a novel laser-pump:neutron-probe experiment which allows us to monitor temporal changes in protein dynamics during a working cycle of a protein. protein dynamics has been extensively studied, but so far, the correlation of internal protein dynamics with the function of proteins was investigated only indirectly in steady-state experiments by variation of external parameters by variation of external parameters like temperature or hydration. the method comprises of an in-situ optical activation of a protein and a time-dependent sampling of the dymamic response using quasi-elastic neutron scattering. with the membrane protein bacteriorhodopsin, a light driven proton pump, we can demonstrate for the first time temporary alterations in the protein dynamics after triggering the working cycle. this observation is a direct proof for the functional significance of protein structural flexibility, in connection with the largescale conformational changes in the protein structure occurring during the operation of a "molecular machine". the slow vacuolar (sv) channels are ubiquitous in all tissues of higher plants. the sv channel is a non-selective cation channel permeable to both monovalent and divalent cations. sv currents recorded in a typical patch-clamp experiment require unphysiologically high cytosolic and low vacuolar calcium concentrations for full activation. we aim at looking for endogenous plant substances which might be able to modify or shift the voltage activation threshold of this channel towards more physiological conditions. flavonoid naringenin [nar] is present in all plant species where it plays a central role in the flavonoid biosynthetic pathway. nar is stored in the vacuoles in glycosylated form called naringin. when nar was added to cytosolic bath solution, we recorded a dose-dependent reversible decrease in sv channel activity. when we investigated the effect of nar on the voltage dependence of the channel, we observed that the activation threshold of the sv channel is shifted towards more positive voltages. our group has evidences that approximately % of the total sv current at high (e.g.> mv) positive voltages is mediated by calcium. therefore, in order to verify whether nar affects both potassium and calcium conductance, we performed experiments by combining the patch clamp technique with fluorescence measurements using the fluorophore fura- : both sv currents and calcium signals were abolished by mm [nar]. determination of calcium currents in cation channels using a novel fluorescence/patch-clamp approach p. v. k. gutla, a. gradogna, a. carpaneto istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy the patch-clamp technique combined with fura- fluorescence detection is suitable to investigate calcium fluxes. we used the excised patch configuration and focused the photomultiplier to the tip of the recording pipette where the fluorescent dye was present (fluoresence combined with excised patch = flep). this configuration has several advantages, i.e. absence of delay in loading the fluorophore, of interference by endogenous calcium buffers and of photobleaching. here we present an application for the determination of fractional calcium currents (pf) in a plant non-selective cation channel, showing that pf can be modulated by cytosolic calcium and potassium. flep is very efficient for measuring small calcium currents (< pa) of sufficiently long duration; fluorescence signals are amplified by integration in time, as the calcium/fura- complex accumulates at the tip of the recording pipette and diffuses slowly. we propose this technique not only for the study of calcium transport pathways, but also for other transporters of divalent cations as nickel and manganese known to quench fluorescence thus reducing both and nm components. moreover, using the appropriate fluorophore the technique may be extended to further ion species, e.g. bcecf for investigating proton transport pathways. ref: we introduced an original method for the monitoring of the changes in the electrostatic surface potential, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. this approach opens new way to understanding the dynamic processes within the proteins. our experiments revealed that the conformation of the na + /k + -atpase large cytoplasmic loop (c ) in the presence of the atp (without magnesium) substantially differed from the conformation in the presence of mg + or mgatp or in the absence of any ligand not only in the sense of geometry but also in the sense of the electrostatic surface potential. moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. this property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain. influx of antibiotics into the periplasm of gram-negative bacteria is facilitated by porins that form channels in the outer membrane. we propose that certain natural antibiotics have been optimized by co-evolution to take advantage of the charge distribution in non-specific porins to achieve binding and thereby facilitating their uptake in bacteria. we investigate the permeation pathways of antibiotics into bacteria by reconstitution of a single porin into an artificial lipid bilayer and measuring the binding of antibiotic molecules through the time-resolved modulation of a small ion current. we have been able to characterize facilitated translocation of several antibiotics through escherichia coli and enterobacter aerogenes porins. noise analysis of ion currents through a porin in the presence of effective antibiotics revealed binding kinetics at a single molecule level. we report for the first time temperature dependent antibiotic translocation that revealed complete energy profile. combining these results with microbiological assays and molecular dynamics simulations, we conclude the molecular mechanism of antibiotic permeation. our approach may contribute to the rational design of new antibiotics against clinical bacterial strains for the most efficient delivery to target sites. mitochondria regulate ca + influx and determine patterns of er ca + refilling in acinar cel o. kopach, i. kruglikov, t. pivneva, n. voitenko, n. fedirko bogomoletz institute of physiology, kiev, ukraine store-operated ca + entry (soce) is mediated by activation of soc-channels of plasma membrane following the emptying of endoplasmic reticulum (er) ca + stores. the soce is required for calcium signaling, secretion of neurotransmitters and proteins, but the mechanisms of natural soce regulation are not well understood. we utilized several imaging methods to measure ca + signals in cytoplasm ( ] mit transients, observed both in egta-and bapta-buffering inside solutions. these data suggest that ca + sequestration by mit is associated with the formation of microdomains and prevents ca + -dependent socc inactivation. we also found that inhibition of mit under prolonged cell stimulation resulted in complete inhibition of soce as well as decrease and deceleration of er refilling. thus, mit regulate calcium recycling and maintain the soce controlling dynamic interplay between soce and sustained er refilling under prolonged stimulation. bioelectrochemical devices composed of au electrodes coated by self-assembled monolayers (sams) of different composition and thickness are ideal systems to probe et patterns and mechanisms for redox proteins. representative proteins, cytochrome c and azurin were studied by using the combinations of four different strategies including the variation of sam thickness ([−s−(ch ) n −ω], with n running over the range to , throughout), solution viscosity (varied by adding of the viscose additive -glucose), temperature ( to o c) and hydrostatic pressure (up to mpa), aiming the identification of different intrinsic et patterns and interplay between them in the framework of generalized charge-transfer theory. we demonstrated the full adiabatic (frictional) control for the case of thinner sams, the intermediate (mixed) regime, and the complete changeover to the nonadiabatic mechanism (long-range tunneling) for the case of thick sams owing to the variation of electronic coupling, in a nice agreement with theoretical predictions. h. nury , f. manon , b. arnou , m. le maire , e. pebay-peyroula , c. ebel institut de biologie structurale (ibs) cea cnrs ujf grenoble france, cea ibitec cnrs ura univ. paris-sud gif-sur-yvette france adp/atp carriers (aacs) are major and essential constituents of the inner mitochondrial membrane. they drive the import of adp and the export of newly synthesized atp. they were described as functional dimers from the s until the structures of the aac shed doubt on this consensus. we aimed to ascertain the published biophysical data claiming that aacs are dimers and to characterize the oligomeric state of the protein before crystallization. analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine aac is a monomer in -laurylamido-n,n dimethylpropylaminoxide (lapao), whereas in triton x- and reduced triton x- , higher molecular mass species can also be identified. neutron scattering data for monomeric bovine aac in lapao does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. we discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of aacs within the mitochondrial membrane. local anaesthetic binding to shaker channels: role of aromatic residues j. nilsson, h. ullman, k. sahlholm, p. arhem the nobel institute for neurophysiology, department of neuroscience, karolinska institutet, se- stockholm, sweden local anaesthetic, antiepileptic and antiarrhythmic drugs acting on nav and herg channels have been assumed to bind to aromatic residues in the internal vestibule; to f and y in nav (ragsdale et al., and to y and f in herg (mitcheson et al., ) . despite a lack of such residues in kv channels, local anaesthetics, antiepileptic and antiarrhythmics bind to kv channels with a considerable affinity. to explore the role of aromatic residues for the binding we investigated the effect of bupivacaine, benzocaine, phenytoin and quinidine on shaker channels mutated to residues corresponding to the most c-terminal of the two aromatic residues in the s segment of the nav and the herg channels, (v y and p f respectively). the channels were expressed in xenopus oocytes and the currents measured with the two-electrode voltage-clamp technique. the results suggest that aromatic residues do not increase the binding affinity of the studied compounds to kv channels. rather, the affinity decreases (as reflected in typical k d values for bupivacaine on v f, p f and wildtype channels, being , and µm, respectively). thus, aromatic residues seem not to be necessary for high-affinity binding of the studied compounds to kv channels. how this relates to their suggested roles in the nav and herg channels, remains to be evaluated. (molina et al, ) . the occurrence of a similar behavior in other channels points out to clustering and coupled gating as a potentially important drug target to modulate channel activity. we have identified molecular determinants involved in single and coupled channel gating in kcsa. first, we detected that clustering and coupled gating of kcsa is modulated by anionic lipid. also, a model for the interaction between two kcsa open channels was built. the docking predicts intermolecular sites which includes the non-annular lipid binding site. this explains how an excess of anionic lipid disrupts interactions between channels, destabilizing clustering and coupled gating in kcsa. in addition, the docking model reveals molecular determinants involved in single and coupled channel gating. this interaction involves w , which affects the neighbouring channel through specific interactions in the extracellular mouth stabilizing the selectivity filter in an open conformation. the coupled gating is also explained since this mechanism affects the opposite channel in a mutual manner. finally, mutants kcsa e a and kcsa w a disrupt the coupled gating of kcsa, thus, supporting the model. we think that this coupled gating phenomenon could correspond to the second gate previously detected by fluorescence methods (blunck, et al, ) . supported by grants from the spanish bfu - /bmc and csd - . a. kumar, e. hajjar, p. ruggerone, m. ceccarelli university of cagliari, monserrato, italy the striking presence of outer membrane (om) in gramnegative bacteria of e.coli represents a strong barrier for any molecule to penetrate inside bacteria. in particular for β-lactam antibiotics, which have their target located inside the bacteria; the first step towards reaching inner part of bacterium is the cellular uptake. ubiquitous presence of porins (such as for instance ompf, ompc) in the om, function as a channel facilitating the transport of molecules (such as, for instance antibiotics) across the om. bacteria can exhibit resistant towards antibiotics by: (i) decreasing their uptake by under-expressing the porins or/and (ii) production of inactivating enzymes such as ß-lactamases. to combat the latter mechanism ß-lactamase inhibitors (such as, sulbactam for instance) are prescribed together with the antibiotics. like the antibiotics, the inhibitors must penetrate the om, the main path being through porins. it is thus evident the biological relevance of investigating the mechanisms by which porins can regulate entry/exit across the om. to achieve this goal, molecular dynamics simulations were performed to explore the structure and dynamics of pores formed by ompf and ompc porin. from the analysis of data obtained from our simulations, we identified the key residues buried behind the l loop, which may play be crucial for porins to exert their biological role. as a case study, we report results about the diffusion of sulbactam through the two porins. the pentadecapeptide gramicidin forms a cation-specific ion channel in membrane environment. the two main conformations are the head-to-head helical dimer (hd) known as the channel conformation and the intertwined double helical form (dh) often referred to as non-channel conformation. in this comparative study [ ] , the energetics of single potassium ion permeation by means of the potential of mean force (pmf) for both gramicidin conformations embedded in a dmpc bilayer has been addressed by molecular dynamics simulations. a significantly decreased free energy barrier by ∼ kj/mol for potassium ion passage through dh as compared to hd is reported. favorable electrostatic side chain-cation interactions in hd are overcompensated by phospholipid-cation interactions in dh. the latter are coupled to an increased accessibility of the channel entrance in dh due to distributed tryptophans along the channel axis. this result underscores the importance of the lipid environment of this channel not only for the equilibrium between the different conformations but also for their function as cation channels. y. sawada , m. murase , m. sokabe dept. physiol. nagoya univ. grad. sch. med., nagoya, japan, icorp/sorst cell mechanosensing, jst, nagoya, japan the bacterial mechanosensitive channel of large conductance mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer α-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. although several models for the opening process have been proposed with molecular dynamics (md) simulations, as they do not include mscllipid interactions, it remains unclear which amino acids sense membrane tension and how the sensed force induces channel opening. we performed md simulations for the mechano-gating of mscl embedded in the lipid bilayer. upon tension in the bilayer, phe in the outer helix was dragged by lipids, leading to a tilting of the helices. among amino acids in the outer helix facing the bilayer, phe at the water-lipid interface showed the strongest interaction with lipids, thus may work as a major tension sensor. neighboring inner helices cross each other in the inner leaflet, forming the most constricted part of the pore. as tension increases, the crossings move toward the cytoplasm associated with an expansion of the constricted part. during the movement, a hydrophobic water block environment around the constricted part was broken followed by water penetration and permeation. the k + channel kcsa is an integral membrane protein from s.lividans, used as a model system for studies on ion channels and oligomeric membrane proteins. its atomic structure has been solved by x-ray diffraction, which shows an assembly of four identical subunits around a central aqueous pore, including the so-called selectivity filter. this channel is able to permeate k + at high flux rate and it is blocked by na + (physiological blocker). fluorescence, circular dichroism and fourier transform infrared experiments carried out in our laboratory demonstrated that k + and na + are able to bind to kcsa in a competitive manner. this binding is indeed associated with channel conformational changes, which seem to be related to the permeation and blockade processes. to further investigate this phenomenon we carried out a detailed study of chemical and thermal denaturation of wild-type and mutant kcsa channels. these two types of experiments were in agreement and indicate that both cations are able to stabilize the channel through conformational changes, being k + the more efficient one, even more when lipids are present. particularly, mutant channels with a structurally altered selectivity filter show that ion and lipid-induced global conformational changes are intimately associated to the conformation of this selectivity filter (conductive and non-conductive forms). modulation of the voltage-gated sodium channel nav . by rcssii, a toxin from the scorpion centruroides suffusus c. picco , g. corzo , l. d. possani , g. prestipino institute of biophysics, cnr, genova, italy, instituto de biotecnologia, unam, cuernavaca, mexico the main cardiac voltage-gating sodium channel, na v . , generates the fast depolarization of the cardiac action potential and plays a key role in cardiac conduction. its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene scn a, which encodes na v . , that are linked to various cardiac deseases. subsequently, studies of this channel localization have led to its identification in immature and denervated skeletal muscle and in the brain neurons. in our effort to identify high affinity ligands for this channel, we have investigated the effects of the recombinant cssii (rc-ssii), a four disulfide-bridged scorpion toxin isolated from the venom of the scorpion centruroides suffusus. human cardiac sodium channel α subunit scn a was expressed in cho cells and macroscopic na + currents were recorded with patchclamp technique in whole cell configuration. the electrophysiological experiments have highlighted a strong affinity for the channel at low nanomolar concentration. compared with control conditions, rcssii toxin affects in reversible way the kinetics of activation and inactivation and marked decrease the peak na + influx. the extrusion mechanism of substrates in rnd family efflux pumps: a molecular dynamics study a. v the rnd transporters of the acrab-tolc (e.coli) and mexab-oprm (p.aeruginosa) systems are able to export structurally and chemically different substrates outside bacteria through the membrane, being responsible of multidrug resistance. on the basis of crystallographic information, an extrusion process conceived as a three-cyclic peristaltic pumping has been proposed, but further microscopically well-funded investigations are needed to understand the mechanism. using different computational methods like adaptive bias force (abf) and targeted molecular dynamics (tmd), we have investigated the mechanism of substrate uptake and pumping at a molecular level. with the first method we have investigated the passage of antibiotics from the periplasm into the internal pore of the pump, while tmd has been used to assess the effect of conformational changes on the extrusion of drugs (which have been located into one of the proposed binding pockets). comparison between the active pumps acrb and mexb (which show different resistance patterns despite their homology) provide insights into the microscopic details of their functioning. in arabidopsis thaliana there are twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iglrs). each protein displays a pore-forming loop, flanked by two conserved helices (plus a third c-terminal helix), a glutamate-binding domain and an n-terminal region. through pharmacological and/or genetic approaches, many physiological functions have been attributed to plant glurs, such as the regulation of cytosolic calcium, photomorphogenesis, water balance and carbon/nitrogen sensing and assimilation. according to the endosymbiotic theory, the cyanobacteria are considered to represent the precursors of the present chloroplasts. the first prokaryotic glutamate receptor (glur ) was identified in the cyanobacterium synechocystis. the putative products of the atglr . and atglr . genes display a possible targeting sequence for chloroplast location and show a high degree of homology with glur . using specific antibodies and confocal microscopy, we have localized the two members of the atglr subgroup , glr . and glr . (splicing variant) , to the chloroplast in arabidopsis and to the inner envelope membrane in spinach. electrophysiological experiments indicate the presence of an activity which is compatible with that of glutamate receptors. furthermore, oxygen evolution measurements suggest that chloroplast-located glutamate receptors may play a role in the regulation of photosynthesis. under extreme conditions many cells control their volume responding to osmotic challenges by unloading or loading solutes to recover their original volume. a faster volume regulatory role triggered by membrane tension has been disclosed for aquaporins in kidney proximal tubule cells, where aqp is the main water channel, using isolated brush border membranes. in conventional osmotic studies in animal cells it is common to disregard internal hydrostatic pressures because they are insignificant compared to osmotic forces. however, by using low osmolarity buffers in small radii vesicles, we detected a rise on the internal pressure that creates surface tension and causes membrane stress, with a negative outcome on aquaporin water permeability. these findings suggested a mechanism for volume regulation in kidney proximal tubule epithelia where massive solute and fluid transport occurs. to further explore aquaporin regulation by membrane tension, yeast cells were used as a model that could bare surface tension some orders of magnitude higher than animal cells due to the existence of a cell wall. the effect of increasing levels of membrane tension on yeast water channel activity was evaluated. an impairment of aquaporin activity correlated with the increase of membrane tension corroborates the volume regulatory role of aquaporin in different cells. deuterium isotope effects on fast gating of the chloride channel clc- g. zifarelli, a. r. murgia, p. soliani, p. michael istituto di biofisica, cnr, via de marini, , i- genova, italy gating of the torpedo cl − channel clc- is modulated by intracellular and extracellular ph, but the mechanism responsible for this regulation has remained so far elusive. using inside-out patch clamp measurements we studied the dependence of the fast gate on ph int and [cl − ] int . only the closing rate, but not the opening rate showed a strong dependence on these intracellular factors. using mutagenesis we excluded several candidate residues as mediators of the ph int dependence. we propose a model in which a proton generated by the dissociation of an intrapore water molecule protonates e leading to channel opening. deuterium isotope effects confirm that proton transfer is rate limiting for gate opening and that channel closure depends mostly on [oh − ]. the model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on ph int and [cl − ] int . deletion of the c-terminus destabilizes phosphorylated na/k pump state containing na ions n. vedovato, d. c. gadsby the rockefeller university, new york, u.s.a. the na/k pump's extended c-terminus (compared to the serca ca pump's) links transmembrane helices, and its truncation lowers cytoplasmic na affinity for forming the occluded e p(na ) state. here we test the effects of c-terminal truncations on interactions with external na. we deleted the last (yy) or (kesyy) residues in xenopus α β pumps made ouabain resistant by mutations q r-n d (rd) or c y (c-y), and then used two-microelectrode voltageclamp recording in xenopus oocytes to measure pump currents as mm ouabain-sensitive currents while endogenous na/k pumps were silenced with µm ouabain. inhibition by external na of steady outward pump current ([k] o = mm) at large negative voltages was somewhat weaker in both rd and c-y pumps than in wt pumps, but was severely impaired in all c-terminal truncated pumps. consistent with this, the voltage dependence of transient charge movements under na/na exchange conditions ([k] o = mm) was strongly shifted to more negative potentials in the truncated pumps relative to the parent rd or c-y pumps, shifts comparable to those seen in wt pumps on decreasing [na] o several-fold. together, the results suggest that these c-terminal deletions lower the apparent affinity for external na ions to bind and become occluded in the na/k pump. the c-terminus therefore provides contacts important for stabilizing the occluded e p(na ) conformation, regardless of the route of na ion entry into the binding pocket. muscle contraction is driven by molecular motors that adapt their energy utilization according to the demands made on them. we test the hypothesis that rate constants controlling the biochemical steps involved in atp hydrolysis by myosin atpase are affected by the force of the muscle. here we use fluorescence lifetime imaging microscopy (flim) of a fluorescently labelled atp analogue to investigate changes in the environment of the myosin atpase, caused by different loads applied to skeletal muscle. single muscle fibres were subjected to cycles of stretches and releases in the presence of rigor solution and µm of coumarin-labelled atp. flim acquisition was synchronised with stretch/release cycles and force measurements, which allow us to investigate the effect of strain on the lifetime of the labelled atp bound to the actomyosin complex. characterization of the fluorescence decay by a bi-exponential function resolved the time constant of two populations, namely, free fluorophore (τ = . ± . ns; mean ± s.d.) and fluorescent nucleotide bound to the actomyosin complex (τ = . ± . ns at low strain). these experiments showed that while the time constant of the free fluorophore did not change with force, the time constant of the fluorescent nucleotide bound to actomyosin showed a linear dependence with the force applied to the muscle of . ± . ps/kpa. neck linker docking coordinates the kinetics of kinesin´s heads i. derenyi, a. czovek, g. j. szollosi department of biological physics, eotvos university, pazmany p. stny. a, h- budapest, hungary conventional kinesin is a two-headed motor protein, which is able to walk along microtubules processively by hydrolyzing atp. its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. considering the two neck linkers as entropic springs that are allowed to dock to their head domains and incorporating only the few most relevant kinetic and structural properties of the individual heads, we have developed the first detailed, thermodynamically consistent model of kinesin that can (i) explain the cooperation of the heads during walking and (ii) reproduce much of the available experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck linker length and composition, etc.). besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion. the positive role of noise on the transport efficiency of na, k atpase c.-h. chang , t. y. tsong institute of physics, national chiao tung university & physics division, national center for theoretical sciences, hsinchu, taiwan, institute of physics, academy of sciences, taipei , taiwan na, k atpase is a molecular motor which is able to transport ions through cell membranes, even against the transmembrane ion concentration gradient. while in vivo this nanoscale soft machine consumes atp, it may be driven by external fluctuating electric fields, no matter they are periodic or random. theoretically, the motor conformations can be described by a conformation vector v(t) governed by a multi-dimensional kinetic equation. given an oscillating electric field with a slight fluctuation, the boltzmann distributions of these conformations will change with time. the instantaneous transported ion flux is a functional of the quasi-cyclic trajectory v(t) of this non-autonomous dynamical system. various interesting dynamical properties of this ion pump, including stochastic resonance, can be studied theoretically, some of which have good agreement with recent experimental findings. in situ measurements of the molecular motor of muscle with nanometer-microsecond resolution in a contracting muscle, arrays of the dimeric motor protein myosin ii pull the actin filament towards the centre of the sarcomere during cyclical atp driven interactions. when the external load is smaller than the array force, the sarcomere works as a motor, converting metabolic energy into mechanical work; when the external load is larger than the array force, the sarcomere acts as a brake resisting the load with reduced metabolic cost. to investigate the molecular basis of the work production and the braking action of muscle, we use sarcomere-level mechanics and x-ray interferometry in intact single cells from frog skeletal muscle. during isometric contraction, each motor bears a force of about pn. during shortening against high and moderate loads, the number of myosin motors attached to actin reduces in proportion to the external load while the force per attached motor is maintained similar to the isometric value (piazzesi et al., cell , - , ) . rapid stretches of - nm between each overlapping set of myosin and actin filaments in a muscle sarcomere cause the stiffness of the array of myosin motors to increase up to twice the isometric value within ms (brunello et al., pnas usa , - , ) , indicating that the high resistance of active muscle to stretch is due to recruitment of the second motor domain of the myosin molecules with the first domain already attached to actin. supported by miur and ente crf (italy), nih (usa), mrc (uk), embl, esrf. kinesin- is a molecular motor that moves cellular cargo along microtubules. its functional mechanism is well understood for individual motors. however, the way that many kinesin- motor proteins bound to the same cargo move together is not. we addressed the structural basis for this phenomenon using video microscopy of single microtubulebound full-length motors and various spectroscopy methods were employed to study synthetic peptides derived from hinge- region. these peptides show an unexpected profile of secondary structure forming propensities. video microscopy of single microtubule-bound full-length motors reveal the sporadic occurrence of high compliance states alternating with longer-lived, low compliance states. the deletion of hinge- abolishes transitions to the high compliance state. from the results we hypothesize that strain accumulated during multiple kinesin motility populates the high compliance state by unfolding helical secondary structure in the central hinge domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple motor situations. titin is a giant protein of vertebrate skeletal and cardiac muscles. cardiac titin is expressed in two main isoforms: short n b (∼ kda) and long n ba (∼ kda). we have studied changes of titin isoform composition in myocardium of hibernating ground squirrels and spontaneously hypertensive rats (shr). using electrophoresis we have revealed considerable decrease (by - times) in the content of titin relative to myosin heavy chains in shr heart as compared with that for normotensive rats. surprisingly that the data of qrt-pcr showed the increase in mrna content of n ba and n b-isoforms in hypertrophic heart more than times in comparison with norm. we suppose that such a result is an effect of depressed translation of mrnatitin in pathology. we have observed the decrease (by , times) of total titin amount in heart of hibernating animals in comparison with that for summer active animals. however n ba/n b ratio in the heart upon hibernation was increased by times. similar trend was not revealed for the mrna level of corresponding isoforms, although we have showed the decrease of mrna of both titin isoforms in heart of hibernating ground squirrels as compared to their content of summer animals. the decrease in total mrna level may be explained by repressed transcription or mrna degradation in the cell during hibernation. these discrepancies in protein and mrna levels may be considered as the posttranscriptional regulation of titin isoforms expression. actomyosin cross-bridges formed when the globular heads of myosins bind to actin filaments are the molecular engines that drive muscle contraction, fuelled by atp hydrolysis. critical to this process is the change in shape of the cross-bridge and the change in the interactions with actin, in response to force applied to the muscle, and to the status of the nucleotide in the binding pocket. although molecular detail is known from x-ray crystallography and biochemistry, understanding of the interplay between cross-bridge shape and chemical state requires studies in muscle fibres generating force. we use fluorescence life time imaging microscopy (flim) as a probe of the cross-bridge environment.with a fluorescent analogue of atp , fluorescence life-time (flt) changes when the crossbridge binds to actin. now, we show preliminary experiments on the effect of force on flt. the essential light chain of myosin (elc) is a ∼ kda peptide that wraps around a nmlong α-helix of the myosin cross-bridge known as the lever arm which tilts during force generation. using a recombinant elc, labelled with a fluorophore at a strategic cys, we replace the native elc and introduce the fluorescent elc in muscle fibres. preliminary experiments demonstrate that the elc fluorophore also is sensitive to force applied to the muscle fibre. in addition, förster resonance energy transfer occurs between the nucleotide and elc fluorophores, opening the way for studying structural changes in cross-bridges during force generation by fret. muscle contraction: pitfalls in the determination of the contractile response e. grazi dipartimento di biochimica e biologia molecolare, università di ferrara, ferrara, italy the contractile response of an active muscle depends on the load. the load is a force /cross-section. there are three fundamental dimensions: the mass, m; the space, l; and the time, t. from these three dimensions are built up all the physical dimensions. as an example the acceleration, a, is given by, a=l.t − . once the direction and versus are settled the modulus fully defines the physical effect of the acceleration. what about the force? the force, f, is given by, f=m.a. at variance with the acceleration, once the direction and versus are settled, the modulus does not define the physical and the biological effects of the force: the same force is generated by an infinite number of mass-acceleration couples that display different physical and biological effects. the same occurs with the load. thus defining the load that opposes the contractile force does not define the contractile system. in the studies on muscle contraction the acceleration of the load is not considered nor it is provided a way to extract this information. thus these systems are poorly defined from the physical as well as from the biological point of view. models of muscle contraction that consider explicitly both the mass and the acceleration of the load show that, at the same load, the decrease of the acceleration of the load significantly delays the pre-steady state of the contraction and decreases the stiffness of the active fibre. r. shahapure , f. difato , a. laio , d. cojoc , e. ferrari , j. laishram , g. bisson , v. torre int. school for advanced studies, trieste, italy, iit-sissa unit, trieste, italy, lab. nazionale tasc, trieste, italy polymerization of actin filaments is the main source of motility in lamellipodia and is controlled by many regulatory proteins. the underlying molecular mechanisms are only partially understood and now a determination of the dynamical properties of force generation is needed. using optical tweezers we measured with millisecond temporal resolution and pn sensitivity the force-velocity (fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. when force and velocity are averaged over - s, fv relationships can be flat. on a finer time scale, random occurrence of fast growths and sub-second retractions become predominant. maximal power dissipated by lamellipodia over a silica bead with a diameter of µm is − w. due to the presence of adhesion forces, beads in close contact with a lamellipodium can seal on its membrane reducing the amplitude of brownian fluctuations often by more than times. under these conditions, when lamellipodia grow and push the beads, discrete jumps varying from about to nm are detected. when lamellipodia retract, pulling the beads, no discrete events are observed. our results on the dynamical properties of force generation are: a) force generation is a probabilistic process; b) underlying biological events have a bandwidth up to at least hz; c) fast growths of lamellipodia leading edge alternate with local retractions; d) force generation is produced in discrete steps with varying amplitude up to . pn. pushing on microtubules: dominant spindle centering mechanism in c. elegans embryo? j. pecreaux, s. redemann, a. a. hyman, j. howard mpi-cbg, pfotenhauerstr , dresden, germany asymmetric cell division, where the content of the two daughter cells -as well as their sizes -differ, is found in many organisms. strikingly, the spindle, first centered, starts to be displaced out of the center only in late metaphase. in c elegans embryo, the spindle rocks and is posteriorly displaced during anaphase by force generators asymmetrically localized on cell cortex. prior to anaphase onset, the spindle is usually assumed to be centered by the same pulling force. it thus requires the force generators to be carefully repressed to distribute forces symmetrically. on live embryos, we measured positional fluctuations of centrosomes during metaphase with nm accuracy. fourier analysis shows an extremely accurate centering respect to the number of force generators and microtubules. furthermore, spectrum is close to a lorentzian, modeled by a spring and a dashpot, suggesting a spindle centering more likely by pushing on microtubules than pulling. deviation at high frequencies indicates a subdominant pulling force. rnai of gpr- / , known to control force generation, increases slightly centering accuracy; this result supports the hypothesis of an independent centering mechanism. conversely, zyg- (rnai), a microtubule growing factor, decreases centering accuracy, modeled spring stiffness and damping modulus. conclusion: first, the spindle centering mechanism is independent of cortical pulling force generator. second, microtubules pushing is likely to center the spindle. mechanical forces are important in the regulation of cellular adhesion and migration. the focal adhesion kinase (fak) has been suggested to transduce cellular forces and govern cell migration. to obtain more insight in the functioning of fak, a fret-based optical biosensor for fak was designed to relate integrin-mediated conformational changes in its ferm domain to focal adhesion behavior during cell spreading and migration in living cells. imaging of the kinetics of ferm-based fak conformational changes in spreading cells revealed two consecutive stages of focal adhesion activation. heterogeneous ferm conformational responses were observed in individual focal adhesions of adherent motile cells, with the active ferm conformation being enriched in growing and sliding fas, but not in stable and shrinking focal adhesions. inhibition of the cellular actomyosin system revealed the involvement of rho-rock rather than mlckinduced tension signaling in the modulation of the ferm response. our results place the ferm conformational change of fak at the interface between integrin and force sensing. the time course of inorganic phosphate release in permeabilized cardiac trabeculae of the rat c. mansfield, t. west, m. a. ferenczi imperial college london, u.k. the rate of p i release was determined in permeabilized rat trabeculae. contraction was elicited at • c by laser-flash photolysis of npe-caged atp, and time-resolved p i release was monitored using mdcc-pbp, a coumarin-labelled phosphate binding protein, which increases its fluorescence intensity five-fold upon p i binding. the atpase rate during the first turnover of the total crossbridges (assuming µm myosin heads) was s − . the rate decreased to a steady state of s − after the eighth turnover ( . - . s after activation). this steady state rate is comparable to published values of - s − , made ∼ s after activation using an nadh-linked enzyme assay of adp release. the advantage of using mdcc-pbp is that the control of mechanochemical coupling can be examined from the onset of force production and as it progresses toward the steady state. force production and p i release were simulated using a seven step scheme. force was attributed to the states in the sequence a.m.adp.p i ↔ a.m.adp ↔ a.m.adp, with strain sensitivity incorporated into the isomerisation of a.m.adp. the a.m.adp.p i and a.m.adp states populated rapidly as force was increasing. in contrast, the a.m.adp state accumulated slowly after the force plateau was reached and became the dominant force bearing state at the time of the eighth crossbridge turnover. experiments are on-going to examine how the distribution of a.m states changes in response to rapid length-changes. pulling as a factor in forming the heterophasic structure of immunoglobulin proteins structure of proteins of immunoglobulin superfamily: human igg kuc and muscle protein titin, has been investigated by methods of electron microscopy and diffraction with the use synchrotron radiation. super elasticity of titin, the protein of immunoglobulin superfamily, is a key parameter that determines the mechanical properties of muscle. however, the structural-physical mechanism of titin elasticity under tension remains poorly understood. here both tension transduction and high elasticity of titin are explained in terms of crystalline polymer physics. x-ray data suggest a model of titin as a nanoscale, morphological, aperiodical array of rigid ig-and fn -type domains covalently-connected by conformationally variable short loops. the line group symmetry of the model can be defined as s m with axial translation τ ∞ . homologous domains would have similar stability, but the structure of different domains on stretching is subject to different forces because they have different orientations relative to the axis of the molecule. under the force influence the structure of any domain can become either rigid or flexible depending on its orientation in the titin strand. pulling geometry forms an active axial structure from latent isotropic random coil structure of titin strand. we are suddenly faced with nanophase-separated morphology of igg kuc. study was supported by rfbr grant - - . strain response of myosin essential light chain in permeabilized skeletal muscle fibres d. s. ushakov, d. ibanez-garcia, t. g. west, p. m. w. french, m. a. ferenczi imperial college london, uk we applied fluorescence lifetime imaging microscopy (flim) to investigate the relation between conformation of myosin head and mechanical force in skeletal muscle fibres. recombinant myosin essential light chain (elc) was expressed in e.coli and labelled at cys- with coumarin. the labelled elc was exchanged with native elc in single permeabilized rabbit m.psoas fibres. fluorescence lifetime was measured using leica sp upright confocal microscope equipped with becker & hickl time-correlated single photon counting module and x . na leica planapo dipping objective, with the two-photon fluorescence excitation at nm by sapphire pulsed laser. after acquiring flim images of muscle fibres in relaxed state, the solution was changed to ca-free rigor. further images were acquired in rigor with or without . - % stretch applied by a motor. both single and double exponential fluorescence decay analysis showed that the lifetime in rigor was lower compared to relaxed (about ps difference for single exponential fit) and to rigor fibres under strain (about ps). these data suggest a change in the microenvironment of coumarin induced by nucleotide binding and strain. this change is likely to be due to interaction between c-terminal domain of elc and the n-terminal domain of myosin heavy chain related to the lever arm re-orientation process. supported by bbsrc. alpha-synuclein and its a p mutant affects the actin cytoskeleton structure and dynamics v. sousa , s. bellani , g. ronzitti , f. valtorta , j. meldolesi , e. chieregatti department of neuroscience, hsr, milano, italy, department of neuroscience, iit, genova, italy alpha-synuclein (syn) is a soluble protein abundant in the brain, primarily enriched at pre-synapses. syn overexpression and the expression of its a p mutant participate in the pathogenesis of parkinson's disease. many roles have been proposed for syn, including the regulation of synaptic vesicle pools and of neurotransmitter release. the actin cytoskeleton regulates many aspects of synaptic function and its dysregulation may be a cause of neurodegeneration. working both in cell-free and in vivo conditions we demonstrate that syn and the a p mutant have different effects on the actin cytoskeleton dynamics. our results show that syn binds actin, and decreases actin polymerization rate probably by monomer sequestration. on the contrary, a p accelerates actin polymerization in vitro and disrupts the cytoskeleton of intact cells. in particular, during dynamic cytoskeleton remodeling, a p induces the assembly of discrete actin-rich foci. actin trapping and the impairment of filaments reassembly lead to inhibition of cell movement and of the re-establishment of cell-cell contacts. in a p expressing cells cytoskeleton-based processes, such as cell migration and the exo/endocytic traffic are inhibited. elucidating the dynamics of syn interaction with actin may contribute to the understanding of its role in neuronal physiology as well as in neurodegeneration. on the physics of muscle contraction m. l. shur ural state university, yekaterinburg, russian federation whichever energy source is chosen as an engine, its force will decrease with increasing velocity. this is connected with a limited power of any engine. thus, we state that hill's formula is a mere sequence of the law of energy conservation. to derive a mathematical dependence "force-velocity", all the means of consumption of fuel energy should be determined -in our case, the energy of the atp hydrolyze. moreover, the conformation energy of the crossbridges attached serves as the force source as well. we state that part of the energy release transforms into the energy of oscillations of myosin proteins; the other part goes into thermal energy of the sarcoplasmic solution. interaction of the oscillating myosin system with the sarcoplasmic solution controls the process of force generation by a muscle. it is just this interaction that leads to the temperature dependence of force. the presentation is devoted to constructing a theory based on these simple considerations. a discussion and constructive critic is especially wanted. -biological motility and molecular motors - meiotic nuclear oscillations in the fission yeast schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. we report a mechanism of these oscillations based on collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. by combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. the redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, due to the intrinsic motor properties, generate regular large-scale movements in the cell ( ) m. versaevel, s. gabriele, p. damman university mons-hainaut, mons, belgium the remodeling of blood vessels in response to changes in blood flow is mainly realized by endothelial cells (ecs) that convert mechanical stimuli from flowing blood into changes in cell signaling through a process called mechanotransduction. many of the biological responses to external forces originate at two types of microscale structures: focal adhesions linking cells to their extracellular matrix and adherens junctions that link adjacent cells. this study aims to elucidate the role of the cytoskeleton, cell-matrix and cell-cell junctions in transducing fluid shear stress into intracellular signals in ecs. by using microcontact printing of proteins, we design substrates with defined adhesive islands in order to control shapes of living cells. this confinement of ecs allows to study the organization and the contractile activity of the cytoskeleton in order to redistribute their intracellular forces in response to externally applied forces. we design microfluidic channels with sizes and geometries close to small blood vessels to apply a physiological range of shear stresses on ecs. our results indicate that cells deposited on a precisely defined adhesive area inside microchannels and subjected to shear stress reorganize their cytoskeleton, their focal adhesions and adherens junctions in response to blood flow. drugs interfering with the cytoskeleton are used to underline the role of its different components in the cellular adaptation to the mechanical environment. s. mahdavi , b. ranjbar , s. gharibzadeh , m. toosi , m. javan tarbiat modares university, tehran, iran, amirkabir university of technology, tehran, iran multiple sclerosis (ms) is the main known pathology of myelinating cells. an autoimmune reaction occurs against myelin sheets of neurons, so action potential (ap) propagation along the affected nerve fibers has been destroyed and it causes various disorders. here, we propose a novel strategy for ms symptoms treatment. we modeled neuron by orcad software and simulated the action potential propagation along the axon in normal condition. our model simulated normal neuronal behavior. then we destroyed the myelin sheet as it occurs in ms and observed destroyed ap propagation as it was reported in ms disease. we investigated the effect of changes in the voltage-gated sodium channel (vgsc) threshold on the efficiency of ap propagation. the results demonstrated that reduction of vgsc threshold improves the propagation of ap by increasing the amount of sodium flux during ap propagation. although, some researches have proposed vgsc blocker as ms symptom treatment, our result suggests that the increase of sodium current produced by reduction of vgsc threshold, improves ap propagation and probably cure some ms symptoms. so, we suggest that vgsc gating modifiers can be considered as novel strategy for ms treatment. surely, this results needs to be confirmed by experimental studies. a. gradogna, e. babini, a. picollo, m. pusch istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy clc-ka and clc-kb are highly homologous cl − channels expressed in the kidney and the inner ear where they mediate transepithelial chloride transport. both channels heteromerize with the beta subunit barttin. mutations in clc-kb and barttin genes lead to bartter's syndrome. we analyzed the modulatory effect of extracellular ca + and h + on clc-k channels using the xenopus oocyte expression system. clc-ka currents increased with increasing [ca + ] ext without full saturation for [ca + ] ext up to mm. however, in the virtual absence of ca + , clc-ka currents are about % of currents measured in mm [ca + ] ext , demonstrating that ca + is not strictly essential for opening. vice versa, clc-ka was blocked by increasing the [h] + ext with an almost complete block at ph . among various reaction models tested, the model that best fitted all state-steady data predicts an allosteric regulation of channel opening by separate binding sites for ca + and h + . moreover, the best fit suggests that one ca + and two h + bind to the channel. kinetic analysis of current responses upon [ca + ] ext and ph jumps confirmed the allosteric character of modulation. in support of the presence of two separate binding sites we identified several mutations that selectively altered ca + or h + sensitivity. our data represent a first step towards a molecular picture of ca + and proton regulation of clc-k channels and suggest that it is of physiological relevance. the extracellular matrix molecule hyaluronic acid modulates l-type voltage-dependent ca + channels e. dvoretskova , g. kochlamazashvili , o. bukalo , c. henneberger , d. rusakov , m. schachner , a. dityatev italian institute of technology, genova, italy, centre for molecular neurobiology, hamburg, germany, institute of neurology, university college london, london, uk we studied the effects of hyaluronic acid (ha), a major extracellular matrix molecule, on activity of l-vdccs in a heterologous expression system and in hippocampal slices. we recorded currents mediated by a major neuronal subtype of l-vdccs (ca v . c, β b, and α δ ) expressed in cho cells. a five-minute application of . mg/ml ha potentiated l-vdcc currents at - , - , and + mv by approximately %. analysis of boltzmann curves showed that ha increased maximal conductance rather than other parameters (v . or k ). treatment with hyaluronidase removed endogenous ha in murine hippocampal slices and specifically impaired long-term potentiation (ltp) induced at ca -ca synapses by repetitive theta-burst stimulation. blockade of l-vdccs reduced ltp in control slices to the levels seen after hyaluronidase treatment. a potentiation of l-vdccs with bay k fully restored ltp after hyaluronidase treatment. removal of ha reduced ca + transients elicited by backpropagating action potentials in individual dendritic shafts and spines of ca pyramidal cells, whereas pretreatment with nifedipine fully occluded this effect. thus, ha potentiates postsynaptic l-vdccs and by this way influences use-dependent synaptic plasticity. whole-cell patch clamp recordings from a variety of human cancer cells showed that functional voltage-gated sodium channel (vgsc) expression occurred specifically with strongly metastatic cells. in addition, where studied, this was accompanied by down-regulation of outward (mainly potassium) currents. this has led to the celex ("cellular excitability") hypothesis of cancer according to which metastatic cell membranes are excitable and this promotes their hyperactivity. importantly, the vgsc genes expressed are embryonic splice variants, which are normally developmentally regulated, hence the phenomenon is 'oncofetal'. in breast cancer, where the predominant vgsc is nav . the neonatal and adult forms are significantly different and this is reflected in channel activity whereby the neonatal vgsc has much slower inactivation kinetics. the double-charge change at position is critical for this difference. the slow kinetics results in much greater influx of na + into cells and one consequence of this is activation of protein kinase a. this is a tonic effect and, under steady-state resting conditions, it results in a positive feedback effect promoting post-translational trafficking of vgsc protein to plasma membrane. the unique amino acid sequence of the spliced region has enabled the production of a polyclonal blocking antibody specific to neonatal nav . . it is concluded that vgscs represent novel biophysical targets for clinical management of metastatic disease. m. pusch cnr, istituto di biofisica, genoa, italy clc proteins form an evolutionary conserved gene-family that comprises members in mammals. four of the human clcs are passive plasma membrane cl − ion channels. the other clcs are expressed in intracellular organelles. clc- and clc- , mutations of which lead to dent's disease, are secondary active cl − /h + antiporters, similar to the bacterial clc-ec , and with identical cl − : h + stoichiometry. cl − and h + transport activity of the exchanger clcs depends on two glut residues. mutating a 'gating glutamate' (e in clc- ) converts the exchanger into anion conductances. neutralizing the 'proton glutamate' (e ), but not its replacement by some other titratable groups, abolishes cl − and h + transport. noise analysis indicated that clc- switches between silent and transporting states with an apparent unitary conductance of . ps, indicating a very large transport turnover. no − uncouples h + transport but mutating the highly conserved s to p, as found in the plant no − / h + antiporter atclca, led to coupled no − : h + exchange. clc proteins are a fascinating example of how a very similar protein architecture can be used to provide either a passive electrodiffusive permeation pathway or a strictly coupled secondary active ion transporter. (supported by telethon italy -grant ggp ). the role of ion dynamics in zebrafish fin regeneration the specific and directional ion transport across cell membranes or tissue layers results in differential accumulation of ions and endogenous electric currents. these phenomena have been shown to be important for vertebrate organs regeneration. however, the specific ion nature of such electric currents remains unknown, as well as the role of cellular ion dynamics during regeneration and the molecular signalling pathway that transduces electric cues into cellular responses. we use zebrafish caudal fin as an adult regeneration model to unveil the specific ion composition of the currents associated with wound healing and regeneration, using a non-invasive ion-specific scanning microprobe setup. our data suggests a role for potassium (k + ), calcium (ca + ) and protons (h + ) at different stages of the regeneration process. k + and ca + extracellular effluxes have both been detected during the wound healing stage. h + efflux is triggered during wound healing and is maintained throughout regeneration. we are validating these data with genetic and pharmacological approaches, as well as advanced ion imaging. overall, our results suggest ion-driven mechanisms underlie adult tissue regeneration and its comprehension may open way for new therapeutic strategies, both in regenerative and developmental medicine and in cancer therapy. cardiac effects of anabolic steroids: an electrophysiological approach anabolic androgenic steroids (aas) have been used by athletes and non athletes for almost five decades in order to improve performance. however, the illicit abuse of high-doses of aas has been attributed as a main cause of several cardiovascular disorders such as arterial hypertension, lipid profile abnormalities, heart failure, hypertrophic cardiomyopathy, arrhythmia and sudden death. the aim of this study was to investigate qt interval and transient outward potassium current (i to ) changes in rats treated with nandrolone decanoate (deca). male wistar rats received weekly mg/kg of deca (n= ) or vehicle (control, n= ). electrocardiogram was recorded weekly, and qt interval was measured. after weeks hearts were excised and single myocytes were isolated from the ventricles of animals. i to was recorded by means of the whole cell patch clamp technique. qt interval was larger in deca group from th to th week (p < . ). analysis of i to showed a decreased current density (p < . ) in ventricular cardiomyocytes of deca group, compared to control group. in conclusion, our results show that alterations on ventricular repolarizaton may constitute an early consequence of the chronic administration of high doses of anabolic steroids in rats, and demonstrated qt prolongation and i to density reduction, which may constitute an important marker of arrhythmia vulnerability and sudden death. -ion channels in channelopathies and cancer - denitrifying bacteria control no and no cytosolic levels by regulating the expression of denitrification gene clusters via redox signalling of specific transcriptional factors that may act as no sensors in vivo. a protein belonging to the subclass dnr (dissimilative nitrate respiration regulator) from pseudomonas aeruginosa has been recently suggested to be a heme containing protein. very recently the three dimensional structure of the apo-form of dnr (in the absence of heme) has been determined by x-ray crystallography, whereas the holo-form (in the presence of heme) has not yet been crystallized. we have investigated the heme local structure in solution of ferric, ferrous, co bound and no bound holo-dnr by x-ray aborption spectroscopy (xas) and we added a kinetic study of the co bound form by means of a flash photolysis setup using uv-visible absorption as a spectroscopic probe. the combination of fe k-edge xanes fingerprints and kinetic study reveal a heme pocket able to bind exogenous ligands like no and co with increased plasticity, thus supporting its role as the cofactor involved in no sensing activity. molecular examination of motifs that lead to the formation of s-nitrosylated proteins i. alicea, e. r. schreiter university of puerto rico rio piedras, san juan, puerto rico physiologically, a wide range of proteins experience structural and functional modifications after the addition of a nitric oxide (no) moiety to cysteine thiol. this posttranslational modification, known as s-nitrosylation, regulates a large number of cellular processes like vasodilatation, cell signaling and others, and the products of s-nitrosylation can be involved during the development of different human diseases. however, little is known about the mechanism by which different proteins specifically bind the no moiety to their cysteine. here we show a bio-statistical analysis of some properties of cysteine that are s-nitrosylated in different proteins, including the pk, electrostatic environment, solvent accessibility of the target cysteine and identity of surrounding amino acids. we also chose model proteins (human thioredoxin and s protein) to make specific targeted amino acid substitutions around selected cysteines to alter the properties described above. the reactivity and stability of these mutant proteins towards s-nitrosylation will be examined. sampling the flexibility of ppar-γ s. aci-sèche , n. garnier , d. genest , s. bourg , c. marot , l. morin-allory , m. genest upr cnrs , orléans, france, fr pcv cnrs , orléans, france, umr cnrs , université d´orléans, france a promising approach to consider the flexibility of proteins in docking studies consists in performing multiple rigid docking on a representative set of the receptor conformations. molecular dynamic (md) simulation is one of the best adapted methods for structural sampling, but exploring the conformational diversity of a protein is computationally expensive. we present a protocol for generating a wide range of conformational states of a receptor using restrained md and a partitioning protocol to select a few representative conformations of the binding site from this md. a way to speed up efficiently md calculation is using an implicit model to represent the solute-solvent interactions. we explore a protocol using a distance-dependant permittivity function to represent solvent effect and an ensemble of controlled restraints applied on a subset of specific atoms in order to prevent artefactual structural distortions, but preserving receptor's flexibility. ten ns simulations have been performed using different sets of parameters and compared to a reference ns simulation with explicit solvent. to select a representative set of conformations, partitioning (k -means algorithm) was applied on the ensemble of simulated conformations. this methodology was applied to the ligand binding domain of peroxysome proliferator-activated receptor-γ. department of biomedical sciences, university of antwerp, antwerp, belgium the complex system of cavities identified in human neuroglobin (ngb) has been postulated to be of functional significance to the putative no dioxygenase activity of the protein. the interconnected hydrophobic cavities may support this catalytic activity by acting as reservoir for reactants and providing preferential pathways assisting product removal from the active site. we thus decided to investigate co rebinding kinetics to ngb embedded in silica gels to expose ligand migration processes in the geminate phase. encapsulation of the co complexes of reduced neuroglobin, leads to a slight increase in geminate recombination after nanosecond laser photolysis. increasing the viscosity of the medium, by soaking the gels in glycerol, completely inhibits escape of the photodissociated ligand to the solvent, and highlights a complex, multiphasic kinetic pattern. this finding can be rationalized by assuming the existence of a discrete set of temporary docking sites, capable of trapping the photodissociated ligand for very long times, up to a few ms after photolysis. g. bartolommei, f. tadini-buoninsegni, m. r. moncelli bioelectrolab -department of chemistry, university of florence, italy ion pumps are integral membrane proteins devoted to ion transport through a lipid membrane phase. the ion pumps ca-atpase and na,k-atpase are prominent members of the p-type atpases family. due to fundamental physiological roles of these proteins, they are very promising drug targets. bioelectrolab has a wide expertise in the study of ion transport by these proteins [ ] . our attention has been recently focused on the interaction of these enzymes with molecules of potential pharmacological interest. frequently, drugs exert an inhibitory action on the transport activity of an ion pump, usually confining it in an inactive conformation. molecules like thapsigargin and cyclopiazonic acid belong to high (nanom) affinity inhibitors of the ca-atpase, whereas clotrimazole and curcumin are medium (microm) affinity inhibitors of both ca-atpase and na,k-atpase. for each of these compounds a mechanism of action is proposed. moreover, recent results concerning ca-atpase inhibition by clotrimazole analogues will be shown: the relevance of this type of molecules is due to their potential employment as an alternative to traditional drugs against malaria parasite. financial support of ente cassa di risparmio di firenze and of miur (prin project) is gratefully acknowledged. [ ] tadini-buoninsegni f., bartolommei g., moncelli m.r., fendler k. . arch. biochem. biophys. : - (review). s. asthana , s. shukla , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy studies of protein-inhibitors interactions are helpful to elucidate the mode of action of ligands and thereby providing clues for rational drug design. bvdv, is an important target of drug discovery activities largely because it is essential for viral replication. in bvdv rdrp no specific nni binding site has been reported till now. experimental results have shown that different class of inhibitors (benzimidazole, imidazoquinolines and pyridoxyquinolines), have resistant mutations located in the finger domain of the rdrp. all the reported mutations are spatially very close to each other. thereby, indicating that binding sites of nni's may lie in the finger domain for these different class of inhibitors.herein, we have utilized docking procedure to investigate binding sites, binding modes as well as binding affinity of different class of inhibitors.we then used all atom molecular dynamics (md) simulations to investigate the stabilizing interaction between inhibitor-receptor pairs. our md results are in good agreement with experimental data and provide deep insights into the dynamical features of the high affinity inhibitorreceptor binding. thus identifying the binding modes of our inhibitors and mechanism leading to inactivity of the enzyme can help us to build a microscopically well-funded picture of the functioning of these enzymes. we present an investigation of the molecular basis of ligand binding and reactivity of heme proteins using computer simulation. a combination of classical molecular dynamics and hybrid quantum-classical (qm-mm) calculations are applied to explore distal and proximal effects on diatomic ligand binding to the heme. trends in binding energies and in the kinetic constants are illustrated through a number of selected examples. an investigation of the interplay between ligand migration and protein dynamics obtained through classical molecular dynamics techniques in combination with advanced sampling tools is also presented to yield information about free energy profiles and possible secondary docking sites. results for truncated n hemoglobin of mycobacterium tuberculosis, presented as an illustrative example, suggest that the truncated hemoglobin n has evolved a dual-path mechanism for selective/distinct migration of o and no to the heme, to achieve efficient no detoxification. finally, we present also an analysis of the molecular basis of hexacoordination in human neuroglobin, which suggest that the flexibility of the cd plays a key role in determining the ligand binding properties. thermodynamic bases of nucleoplasmin-histone complexes recognition by the nuclear transport machinery i. arregi, j. falces, s. bañuelos, m. a. urbaneja, s. g. taneva unidad de biofísica (csic/upv-ehu), departamento de bioquímica y biología molecular, universidad del país vasco, spain the nuclear transport of the chromatin remodeling (nucleoplasmin) and chromatin building (histones) proteins is mediated by importins. nucleoplasmin (np) contains a classical bipartite nuclear localization signal (nls) that is recognized by importin α, while histones present multiple sequence elements (nls-like motifs) for nuclear targeting. besides, ternary importin/np/histone complexes might represent a putative coimport pathway for nuclear import of linker (h ), nucleosomal core (h ah b) histones and their chaperone protein np, enhancing the histone import efficiency. to better understand np and histone recognition by the transport machinery we studied the thermodynamics of complex formation of importin α (a truncated form) and importin β with histones and np, and with np/histone binary complexes by means of isothermal titration calorimetry. data show that importins interact with the two histone types and np, and that importin and histones can simultaneously bind to np. analysis of the binding energetics reveals an enthalpy driven formation of high affinity binary and ternary complexes. we demonstrate that different amount of importin molecules can be loaded on np/binary complexes dependent on the histone type, linker or core, and the amount of the bound histones. g. breuzard, i. di maïo, p. barbier, d. allegro, c. brault, v. peyrot cro inserm u , ufr de pharmacie, marseille, france since a decade, our laboratory has already studied the interaction of tau variant with tubulin (tub) or microtubules (mts) and phosphorylation process on this interaction. indeed, fret assay was a powerful tool to achieve binding parameters between tau and tub in vitro: / with mts stabilized by fluorescein-coupled taxol (flutax- ) as donor and rhodamine-labelled tau (rho-tau) as acceptor, and / in living cells by confocal laser scanning microscopy (clsm) with tau/α-tub fused to egfp/mcherry, respectively. results revealed ± % energy transfer efficiency from flutax- to rho-tau and a donor-to-acceptor distance of ± Å. by titration, the dissociation constant of tau was determined to . ± . µm. a cleavage procedure of αβ-tub was performed to determine the influence of the c-term tails of αβ-tub on the tau-mt interaction. no difference in distances and binding parameters was observed. clsm images displayed a heightened concentration of fluorescent tau in patches along mts. fret experiments revealed in particular higher efficiencies between gfp-tau to mcherry-α tub proteins in these locations. overall, our results suggested no involvement of the hypervariable and highly acidic c-term tails of tub in mt/tau binding. a molecular model is proposed in which flutax- is directly accessible to tau molecules. besides, the modified distribution of fluorescent tau could be implicated in local change of mechanical properties of mts. a. boreham , k. winkler , c. gebhard , k. rueck-braun , p. henklein , e. michalsky , r. preissner , r. misselwitz , a. ziegler , u. alexiev freie universität berlin, berlin, germany, technische universität berlin, berlin, germany, charité-universitätsmedizin berlin, berlin, germany peptide presentation by major histocompatibility complex (mhc) molecules is crucial for immune responses. photocontrol of peptide dynamics by means of photo-switchable peptide analogs will provide insights in peptide dynamics and its dependence on mhc polymorphism ( ) ( ) ( ) . we have designed a hemithioindigo (mhti) photo-switch bearing peptide using the viral epitope rrrwrrltv (plmp ) as a template. incorporation of mhti in the peptide backbone should result only in a minor change of the overall peptide structure given the relaxed conformation of the zisomer. indeed, the human mhc molecule hla-b* can tolerate nonpeptidic elements in plmp , as evidenced by normal peptide binding. using the autofluorescent properties of mhti we determined the stability of the hla-b* /mhti-plmp complex. photoswitching from z →e results in a decrease of hla-complex stability. based on computer modeling this decrease is due to a reduced interaction of the peptide c-terminus with hla-b* . truncated hemoglobins (trhbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. three phylogenetic groups (n, o, and p) have been identified in trhbs. the crystal structure of truncated hemoglobin o of b.subtilis, does not show an evident tunnel/cavity system connecting the protein active site with the solvent, a fact that cannot be easily rationalized considering the very high oxygen association rate. moreover, resonant raman results of the co bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. for these reasons we performed classical molecular dynamics simulations of the oxy, carboxy and deoxy protein, and computed the free energy profiles associated with ligand migration to the active site. our results suggest that there is a key residue, glne , that may present an alternate conformation in which a wide ligand migration tunnel is formed, consistently with the kinetic data. the results for the co and o bound protein show also that glne is directly involved in the stabilization of the coordinated ligand, playing a similar role as tyrb , and trpg in other trhbs. our results not only reconcile the structural data with the kinetic information, but also provide additional insight about the general behaviour of trhbs. patterned functionalization of surfaces for guided transport on molecular motors tracks m. bhagawati , s. ghosh , t. surrey , j. piehler department of biophysics, university of osnabrueck, osnabrueck, germany, european molecular biology laboratory, cell biology and biophysics unit, heidelberg, germany chelator head groups with multiple nitrilotriacetic acid (nta) moieties have been very well characterized and successfully utilized as high affinity adapters for functional immobilization of oligohistidine tagged proteins to surfaces. we have recently established a generic method for patterning nta functionalized surfaces by selective photodestruction via a light induced fenton reaction. efficiency of different transition metal ions for catalyzing this reaction was tested. functionality of the patterned protein was confirmed using the interaction between interferonα and its receptor. implementation of this technique in a confocal laser scanning microscope allowed us to control surface density of binding sites, providing the possibility to vary the surface concentration of immobilized proteins in a spatially resolved manner. we also applied this approach for exploring guided transport of microtubules by kinesin selectively immobilized onto trisnta patterns. rheumatoid arthritis (ra) is an autoimmune disorder, leading to pathological damage at the level of joints, associated with the hla class ii allele hla-dr . although etiology of ra is unknown, type ii collagen (cii) is a potential antigen candidate and it is believed that t cell responses in collagendependent ra are directed towards the immunodominant pathogenic epitope cii( - ). despite recent advances in characterization of class ii major histocompatibility complex (mhc) and t-cell receptor (tcr) contacts in this epitope, the atomic details of tcr-cii( - )-mhc complex are not known. here, homology modeling and molecular docking studies have been used to derive a three-dimensional model of tcr β chains, obtained from a dr + subject, in complex with cii( - )/hla-dr . the best complex from docking was further refined using molecular dynamics simulations for ns. the proposed model represents a reasonable structural basis for understanding cii( - )-mhcii complex recognition by tcr and for rational design of inhibitors targeting tcr-pmhc interface. probing bio-molecular bonds with magnetic force for biosensor applications a. m. de jong , a. jacob , x. j. janssen , j. m. van noorloos , l. j. van ijzendoorn , m. w. prins eindhoven university of technology, eindhoven, the netherlands, philips research laboratories, eindhoven, the netherlands we investigate new technologies to be applied in next generation biosensors, which not only measure biomarker concentrations but also probe bio-molecular interactions. the concept is based on the response of ligand-receptor pairs to an applied force or torque [ , ] . we use functionalized magnetic beads and magnetic fields to apply translational and rotational forces on the molecular bonds. in a model experiment, polystyrene surfaces were coated with anti-biotin and beads were coated with biotin. after incubation, a constant magnetic force was applied to the beads and the number of bound beads was measured as a function of time. this was repeated for a range of forces. the dissociation rate (k o f f ) is determined for each force and k o f f at zero force is extracted from these data. rotational forces were exerted on protein-g coated beads bound to igg antibodies immobilized on a surface. rotating fields revealed an oscillating behavior, which can be understood from the balance between the applied magnetic torque and the torque due to the deformation of the biological bond. studying interactions between cop regulatory protein and hy transcription factor d. s. dalafave the college of new jersey, ewing, nj , usa this work addresses important questions of protein-ligand interactions and selective protein recognition. proteinligand bindings are crucial for many cellular processes. dependable methods for predicting binding sites would lead to a better understanding of proteins' selective recognition and would, in turn, help research on fighting diseases. presented here is a study of interactions between the wd domain found in a regulatory protein cop and the motif v-p-e/d-Φ-g (Φ=hydrophobic residue) found in a transcription factor hy . cop and hy proteins have opposing roles in developmental regulation. cop can repress hy by directly binding with it. the repression involves specific interactions between the wd domain and the motif v-p-e/d-Φ-g. previous experimental research showed that mutations in the motif's v-p pair resulted in a large decrease in hy repression. to study effects of similar mutations, residues in the motif v-p-e/d-Φ-g were systematically substituted with other residues. interactions between the mutated motif and the wd domain were studied. distributions of binding sites, bond lengths, local shape complementarity, and interaction potentials were modeled for each residue substitution. the study identified binding sites critical for the cop -hy binding. some hy residues in the vicinity of the motif were also found to be important in the binding. the significance of the results for understanding selective protein recognition is discussed. determination of protein-ligand binding thermodynamics by thermal shift assay p. cimmperman, a. zubriene, l. baranauskiene, e. kazlauskas, j. matuliene, d. matulis laboratory of biothermodynamics and drug design, institute of biotechnology, vilnius, lithuania thermal shift assay determines the effect of ligand binding on the protein thermal denaturation equilibrium. several models have been derived to describe the general cases of protein-ligand binding. first, the model describing protein stabilization and destabilization by ligand binding to the native and unfolded states. second, the split of protein melting transitions by tightly binding ligands is presented when the transition of free protein and ligand-bound protein occur separately. mathematical equations describing the protein unfolding and ligand binding thermodynamic parameters at various temperatures are presented. the protein melting temperature shift can be determined by various techniques such as differential scanning calorimetry, circular dichroism, and intrinsic or extrinsic fluorescence. the advantages of fluorescent techniques are presented. the melting temperature shift caused by ligand binding is dependent on the thermodynamic parameters of protein unfolding and ligand binding, including enthalpy, entropy, and heat capacity, thus allowing determination of binding thermodynamics. application of the models in the design of hsp chaperone and carbonic anhydrase inhibitors is discussed. comparison with isothermal titration calorimetry data is presented. recent reports have shown that the bacterial redox protein azurin can enter into cancer cells and induce apoptosis by stabilizing p . the formation of a complex between the two proteins has been demonstrated, but little is known about binding features. for the first time, we show here that azurin binds to the n-terminal region of p with a dissociation constant in the - µm range. trp phosphorescence lifetime measurements revealed conformational changes of azurin induced by the interaction with p ( - ). acrylamide quenching of trp phosphorescence also indicated a significant increase of the overall flexibility of azurin upon binding to p . no change of the fluorescence emission of p ( - ) was detected in the presence of azurin. the latter finding suggests that w of p is not directly involved in domain binding to azurin, indicating that the binding site is distinct from that of mdm . the present results may assist the design of novel cancer treatments based on p stabilization by azurin. a. eleta , r. georgieva , h. bäumler , j. l. toca-herrera cic biomagune, donostia-san sebastián, spain, charité-universitätsmedizin berlin, berlin, germany human serum albumin (hsa) is the most abundant nonglycosilated plasma protein in the human body. this multifunctional protein has ligand-binding and transport properties, antioxidant functions and enzymatic activity [ ] . bilirubin interacts with albumin in order to be transported from the blood to the liver where it is secreted. the interaction occurs specifically in hsa i-domain of its three domains [ ] . in our work, we investigate the interaction between hsa and bilirubin by quartz crystal microbalance with dissipation (qcm-d) and atomic force microscopy (afm) [ , ] . albumin was adsorbed on negatively charged silicon oxide. however, hsa was removed after rinsing with pbs. hsa adsorbed on positively charged polyelectrolyte multilayers leads to a stable layer of surface mass density of ng/cm . cross linked albumin with glutaraldehyde after its adsorption on nh -terminated thiols is also stable reaching surface mass density of ng/cm . a preliminary bilirubin adsorption results on cross linked hsa substrate show that ng/cm is immobilized. taking into account that hsa-bilirubin stoichiometry is : , the outcome demonstrates that the % of hsa i-domains remain active. antimicrobial peptides (amps) are short positively charged polypeptides. they are important due to their potential to provide an alternative to conventional therapy against bacterial infections. rbpi is a kda peptide based on the nterminal region of the neutrophil bactericidal/permeabilityincreasing protein (bpi). it was shown that this amp possesses bactericidal effects on gram-negative bacteria and higher affinity for lipopolysaccharide (lps), neutralizing its effect. the peptide use against meningitis, is in phase iii clinical trials. here, we demonstrate that rbpi promotes aggregation of negatively charged large unilamellar vesicles (luv) and lps aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (popc) luv the size remains unchanged. the aggregation increases with peptide concentration until peptide promotes massive aggregation followed by sample flocculation/precipitation. with the rbpi -lipid interaction there is a progressive change in the zeta-potential of the luv systems and lps aggregates. luv systems composed of phosphatidylglycerol (popg) and popc:popg mixtures have higher zeta-potential variations than popc luv. for lps aggregates, rbpi neutralizes the surface charge and at higher peptide concentrations overcompensates it. results demonstrate that the interaction of the peptide rbpi with lps aggregates and luv systems has electrostatic and hydrophobic contributions. serratia marcescens heme acquisition system: heme transport and protein:protein interactions m. delepierre unité de rmn des biomolécules cnrs ura , institut pasteur, paris, france heme transport systems in bacteria are required and might be potential target for antibacterial drugs. the heme acquisition system, has, exists in pathogenic as well as in opportunistic bacteria but only for the latter one extensive studies have been conducted, constituting as such a model system. the outer membrane receptor hasr, the central component of this system, functions in synergy with a secreted high affinity heme binding protein, the hemophore hasa. hasa extracts heme from host hemoproteins and returns it to hasr. then, the energy given by a protein complex of the inner membrane is used to allow heme entrance across the bacterial membrane and to eject the empty hemophore from the receptor. reconstitution of this heme acquisition system in e coli, overexpression and purification of its various components have allowed us to obtain sufficient amount of protein to perform nmr and biophysical studies to analyse at the molecular level the different steps of heme acquisition by hasr. protein-protein interactions (ppi) are the central pillar supporting most of biological functional activity on the molecular level. a binding event between two proteins typically consists of two stages: ) diffusional search of the binding partners for each other, and ) specific recognition of the compatible binding surfaces followed by the formation of the complex. we focus here on the non-specific component of ppi, which refers to all physico-chemical properties of the binding partners (such as size, charge, isoelectric point, hydrophilicity etc.) that are independent of the exact details of their binding sites, but which could in turn affect their localization or diffusional search for one another. it is known that proteins co-localize due to segregation into different cellular compartments, sequestration via anchor and scaffold proteins or even chemical modifications. we suggest that the non-specific component of ppi determines in part the co-localization and clustering of the binding partners, which then directly in a non-specific fashion influences their interactions. we examine the possibility that such signature might be encoded within the experimental d structures of a large set of known mutually interacting proteins. we provide preliminary evidence that this indeed may be the case, and corroborate our findings by using different statistical tests to compare those features of the known interacting partners, and ascertain correlations and commonalities between them. a link between hinge-bending domain motions and the temperature dependence of catalysis in ipmdh i. hajdú, a. szilágyi, j. kardos, p. závodszky institute of enzymology, hung acad sci, budapest, hungary enzyme function depends on specific conformational motions. since conformational flexibility strongly depends on temperature, temperature dependent enzyme kinetic studies with measurements related to dynamics can give us some insight at atomic level into these functionally relevant motions. the catalytic efficiency (k cat /k m ) of -isopropylmalate dehydrogenase for its substrate (ipm) has unusual temperature dependence, showing a local minimum at • c. in search of an explanation, we measured the individual constants k cat and k m,ipm as a function of temperature, and found that the van't hoff plot of k m,ipm shows a sigmoid-like transition in the - • c temperature range. by means of various measurements including h-d exchange and fret, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperature above • c. the thermodynamic parameters of ligand binding determined by itc as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. because the binding of ipm is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with ipm binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency. a simulation approach to multiple sclerosis: study of a peptide with a pharmaceutical potential c. guardiani , s. marsili , p. procacci , r. livi centro dinamiche complesse, università di firenze, italy, dipartimento di chimica, università di firenze, italy, dipartimento di fisica, università di firenze, italy multiple sclerosis (ms) is an autoimmune disease of the central nervous system, leading to premature death. one of the potential targets of the autoimmune reaction is the myelin protein mog that has been crystallized in complex with the - c ms-autoantibody. the analysis of contacts and buried surface area combined with an alanine scanning computation reveals the key role of mog fragment - for the interaction with - c . a docking simulation shows that the - fragment, excised from mog, and kept in crystal-like conformation, is still capable of fitting into the binding pocket of the antibody. we then studied, through replica exchange molecular dynamics simulations, the structural equilibrium distribution of the free peptide and of a number of analogs stabilized by a disulfide bond. we found that the free peptide yields a significant fraction of crystal-like conformations and the proportion of native-like structures is further increased by the disulfide bridge. when we tried to dock the centroids of the most populated clusters to - c , we discovered the existence of a docking funnel whose bottom is populated by stable complexes where the peptide occupies the same spatial region as in the crystal. we therefore conclude that the mog - fragment may be used to develop a diagnostic assay or a drug for ms. the escherichia coli membrane insertase yidc reversibly binds its substrate pf coat protein u. gerken, s. winterfeld, a. kuhn institute of microbiology and molecular biology, university of hohenheim, germany the membrane insertase yidc of e. coli belongs to the oxa family of mitochondria and plays an essential role in facilitating the insertion and assembly of membrane proteins. we have previously shown with detergent-solubilized (c pc) yidc, labelled with ans, and pf coat that the initial step of the membrane insertion process, the binding of the substrate pf coat to yidc, is reversible [biochemistry , - ( ) ]. the dissociation constant k d for that particular system is about µm. in order to obtain data for the native system we used in this study membrane-reconstituted (dopc and dope/dopg) yidc. the effect of the initial binding was examined in vitro by fluorescence quenching of the tryptophan (trp) residues of yidc which are highly sensitive fluorescent probes for changes of the tertiary structure. quenching of the trp fluorescence after titration with a trp-free pf mutant indicates a change in the yidcs tertiary structure upon binding to its substrate. the binding data show a k d value in the range of . - . µm. the influence of different environments (lipid membranes, ddm micelles) on the secondary structure of yidc as well as on the yidc large periplasmic domain p was investigated by circular dichroism (cd). the cd data show that the secondary structure of yidc changes upon reconstitution into a membranes when compared to the detergent solubilized state. particularly, the p domain is considerably affected by the detergent c pc. b. karasulu, b. erman, o. keskin koc university, istanbul, turkey histone proteins are fundamental to the cells since they are involved in cell regulatory processes, such as chromatin regulation, gene silencing and transcription, cell cycle control, and epigenetics, which are controlled via post-transcriptional modifications of the histone protein tails. these modifications are categorized under four main groups: methylation, acetylation, ubiquitination, and phosphorylation. among them methylation has been very recently proven to be reversible with the discovery of histone demethylase proteins and this modification type has been extensively studied, because the abnormal methylation rates cause the excess proliferation of the cell, which, in turn, triggers the cancer. therefore, understanding of the details of reaction mechanisms of histone methylation/demethylation dynamics provide the required knowledge basis for preventing the abnormal methylation rates by designing proper inhibitor (drug) molecules. in this study, we display possible reaction mechanisms (such as amine oxidation via lsd ) for the demethylation of specific histone tail proteins. we try to explain the role/importance of residues that take place in or near to the reaction pocket for the demethylation reaction. we also carry out md simulations and reaction path (free energy profile) analysis using free energy perturbation (fep) method for qm/mm hybrid systems in order to compare different possible reaction pathways. the sonic hedgehog (shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors patched (ptc) and smoothened (smo) , which lead to birth defects, cancer or neurodegenerative diseases. however, little is known about ptc, the receptor of the shh protein, and the way ptc regulates smo, the receptor responsible for the transduction of the signal. to develop structure-function studies of these receptors, we expressed human ptc (hptc) in the yeast saccharomyces cerevisiae. we demonstrated that hptc expressed in a yeast membrane fraction is able to interact with its purified ligand shh, indicating that hptc is produced in yeast in its native conformational state. using surface plasmon resonance technology, we showed that fluorinated surfactants preserve the ability of hptc to interact with its ligand after purification. this is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor ptc. this work will allow the scale-up of hptc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by shh signalling dysfunction. dissecting the colicin translocon c. l. johnson , a. solovyova , p. callow , s. a. holt , l. a. clifton , k. weiss , j. h. lakey inst. for cell and molecular biosciences, newcastle univ., uk, inst. laue langevin, grenoble, france, isis, rutherford appleton lab., didcot, uk, oak ridge national lab., centre for structural molecular biology, oak ridge, usa pore-forming colicin n hijacks e. coli outer membrane protein ompf and exploits it as both a receptor and translocator to cross the outer membrane [ ] . it is currently a matter of debate if the translocation route is through the ompf lumen or the interface between ompf and the lipid bilayer. recent electron microscopy data from our laboratory suggests the latter route for translocation [ ] . the colicin n/ompf complex in detergent has been studied by sans to examine the translocation pathway undertaken by colicin n. by using a combination of deuterated ompf and hydrogenated colicin we have been able to derive a low resolution structure of individual proteins in the binary complex. low resolution structural studies supplemented by targeted mutagenesis and screening techniques including itc, potassium efflux assays and auc have allowed us further our understanding of colicin n translocation. dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined. here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. effect of osmolytes on the dhfr activity, structure and dynamics b. legrand , s. renaud , m. collen , c. tascon , s. bonnassie , e. gautier , j. mellet , c. blanco , e. le rumeur , j.-f. hubert , a. bondon rmn-ilp, duals, cbp, umr cnrs , univ. de rennes , france osmolytes are small molecules accumulated by a wide variety of organisms in response to hyperosmotic stress. they contribute to save the cellular integrity and to stabilize the macromolecules from environmental stress. dihydrofolate reductase activity is inhibited by several osmolytes. we studied the impact of osmolytes on the dhfr structure and dynamics by various techniques. we observe that substrate (dhf) and cofactor (nadph) diffusions are quite different in glycerol and betaine despite similar viscosities. we demonstrate that the overall structure is maintained at high osmolyte concentrations while no direct interactions can be detected with the enzyme. the k o f f of substrate analogues decreases with increasing the osmolyte concentrations. the enzyme dynamics, in various media, has been compared with the dhfr behaviour in water described in the literature. the osmolyte impact appears only partly conditioned by its viscogenic properties which reduce the molecules diffusion and the k o f f of the product controlled by the m loop of the dhfr. we suggest that the osmolytes decrease m loop mobility. comparing the results obtained with different osmolytes, we offer a better understanding of the osmolyte nature dependence of the dhfr inhibition. estrogen receptor (er) is a well characterized member of the nuclear receptor superfamily that modulates the expression of estrogen-responsive target genes in response to estradiol and other natural and synthetic chemicals mimicking the estradiol structure. in human, two ers, erα and erβ, lied on two distinct chromosomes, are known. er exhibits several functional domains: two conserved domains, a short domain c involved in dna-binding and a large domain e/f responsible for ligand-binding and hormone-dependent transcription activation, are linked by a hinge domain d; a poorly conserved a/b domain, at the n terminus, mediating interactions with the general transcription machinery, is involved in hormone-independent transcription activation. upon estrogen binding, ers can specifically bind to a dna fragment, called estrogen response element ere, and activate the transcription. the optimal ere sequence consists of two six base-pair half-sites, aggtca, organized as inverted repeats with a three base-pair spacing. in this study, we have investigated, by fluorescence methods, the effect of kcl concentrations, on the protein conformational flexibility and the thermodynamic stability of hers -eres complexes. we show, here, that electrostatic interactions, inside hers, contribute to its conformational flexibility and its thermal stability. moreover, the specific interaction between hers and eres is poorly sensitive to changes in ionic strength, in opposite to unspecific complexes. kinetic and structural explanation for the low enantioselectivity of human -phosphoglycerate kinase p. lallemand , j. rouhana , l. chaloin , b. roy , s. arold , t. barman , c. lionne cpbs umr , bd henri iv cs , montpellier cedex , france, ibmm umr , cbs umr l-nucleosides comprise a new class of antiviral and anticancer agents that are converted to pharmacologically active nucleoside triphosphates in vivo. the last step of the cascade may be catalyzed by -phosphoglycerate kinase (pgk), an enzyme that has low specificity for nucleoside diphosphate: ndp + , -bisphosphoglycerate ↔ ntp + -phosphoglycerate. here we compare the kinetics of formation of the complexes of human pgk with different d-and their mirror images, l-nucleoside diphosphates, and the effect of -phosphoglycerate thereon. two types of experiment were carried out: equilibrium experiments allow the estimation of dissociation constants, and stopped-flow experiments the transient kinetics of the interactions. in addition, by the rapid-quench-flow technique, we compare the kinetics of the phospho-transfer and product release steps with each d-or l-nucleotide. crystallographic and molecular modelling studies allow defining the structural reasons for the low enantioselectivity of pgk. the aim of this basic work on the mechanism of action of human pgk with non-natural nucleotides is to obtain information for the optimization of therapeutic nucleoside analogues that are poorly phosphorylated by pgk. anrs is gratefully acknowledged for financial support. a new itc assay for measuring high-and lowaffinity protein-ligand interactions g. krainer , j. fanghänel , s. keller leibniz institute of molecular pharmacology (fmp), berlin, germany, bayer schering pharma ag, berlin, germany isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions [ ] . however, reliable determination of the dissociation constant (kd) is typically limited to the range µm > kd > nm. nevertheless, interactions characterised by a higher or lower kd can be assessed indirectly, provided that a suitable competitive ligand is available whose kd falls within the directly accessible window [ ] . unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of high-and low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-affinity (kd < nm) and low-affinity (kd > µm) protein-ligand interactions. a molecular dynamics study of the cftr nucleotide binding domains interaction v. martorana , r. noto , o. moran cnr-istituto di biofisica, palermo, italy, cnr-istituto di biofisica, genova, italy the cystic fibrosis transmembrane conductance regulator (cftr), the dysfunctional protein in cystic fibrosis, contains two transmembrane domains, two nucleotide-binding domains (nbds), and a regulatory domain. opening of the pore have been linked to the atp-driven tight dimerisation of nbd. we have studied the nbd -nbd interactions on wild type and cystic fibrosis-related mutations by steered molecular dynamics simulations (smd). a fully solvated dimer, including the two bound atps, was separated by pulling one monomer with an external, increasing force. interestingly, the force needed to break the mutated (g d) dimer is significantly smaller than in the wild type case. the effect of a cftr potentiator, the genistein, has also been tested by repeating the smd simulations with the small molecule docked at the interface between the two nbd domains. to test the validity of our results we have repeated the separation process for different simulation lengths and force strengths. the amount of distortion on the pulled nbd domain has also been studied. this work is partially supported by the italian cystic fibrosis foundation (prog ffc # / ), with the contribution of "mille bambini a via margutta"onlus"blunotte", "lega italiana fc toscana" stability and aggregation studies of human septin (sept ) j. n. a. macêdo, r. c. garratt, a. p. u. araújo instituto de física de são carlos, usp, são carlos, brazil the septins are a conserved family of nucleotide binding proteins firstly identified in saccharomyces cerevisae as proteins required for the completion of the cell cycle. septins are implicated in several cellular functions such as cytokinesis, vesicle trafficking, exocytosis, cytoeskeletal dynamics, cell polarity and sperm motility. furthermore, they are associated with alzheimer's and parkinson's disease. sept was identified in rat brain being highly enriched in presynaptic nerve terminals. it colocalizes with synaptophysin and dynamin i and is associated with synaptic vesicle. in this work, human sept without its n-terminal domain (sept gc) was expressed in e. coli and purified by affinity and size exclusion cromatographies. structural stability studies were performed with recombinant sept gc using circular dichroism spectroscopy (cd), right-angle light scattering, and fluorescence spectroscopy. the sept gc cd spectrum showed a conformational transition from a predominantly α-helical starting structure to one dominated by β-sheet, just above physiological temperatures. the formation of irreversible aggregates, detected by light scattering, and their ability to bind thioflavin-t suggested that sept forms amyloidlike structures, as has been previously observed for human septins and . our data suggest that amyloid formation by isolated septins in vitro may be a general phenomenon whose physiological relevance needs to be further investigated. pln is an antimicrobial peptide produced from lactobacillus plantarum nric . analog pln (pln a) and a ser-derived (pln s, tyr to ser replacement) were synthesized on solid phase and their interactions with biomembrane model systems and inhibitory property on s.aureus and p.aeruginosa were investigated by cd, leakage assays, fluorescence spectroscopy, and differential scanning calorimetry. both peptides share the same unordered structure-like cd spectrum in aqueous solution, but a helicoidal induction in the presence of negative vesicles were observed, however pln s showed lower helical content than pln a. the ser-derived peptide presented % decrease of its leakage activity in different liposome compositions, a threefold increase in the dissociation constant from the liposomes than pln a, and close to % reduction for the inhibitory activity against p. aeruginosa growth. we can conclude that besides the electrostatic contacts between the amphipathic &alpha-helix, formed by the cationic residues from pln and negatively charged phospholipids, the pln -membrane binding is a process driven for the hydrophobic interactions from non-polar residues. in this case, there was a significant contribution of the tyr residue, which must be allocated in a lipid interface, described as the preferential position to this residue in membrane proteins. supported: fapesp label free detection using deep-uv laser-based fluorescence lifetime imaging microscopy q. li, s. seeger physikalisch-chemisches institut, universität zürich, winterthurerstrasse , ch- zürich, switzerland label free detection based on native fluorescence excited at uv region shows great potential for the life sciences. it offers simple, low-cost and fast method for sensitive detection of important biological analytes without modification. in this contribution we present a deep uv fluorescence lifetime imaging microscopy system (duv-flim) based on a picosecond deep uv laser using time-correlated single-photon counting method. the described setup is well-suited for biological applications for ultrasensitive detection. we have showed single uv dye (bmq) and single protein (ecβ gal ) molecules detection using duv-flim. further, the label free detection of protein interaction between ecβ gal and monoclonal anti-ecβ gal has been demonstrated by means of steady-state and time-resolved fluorescence spectroscopy. we achieved detection sensitivity for the ecβ gal/ anti-ecβ gal pair down to the nanomolar concentration range. we also extended this method to study the interaction of therapeutic drug porphyrin with bsa protein. fluorescence resonance energy transfer between protein and alexa fluor has been investigated using duv-flim. the intrinsic fluorescence and fluorescence lifetime changes of donor biotin β-galactosidase have been measured. energy transfer efficiency and donor acceptor distance have been obtained. fluorescence images of acceptor af due to fret have been observed when excited at nm. the monomeric heme-containing indoleamine , dioxygenase (ido) catalyses the oxidative cleavage of the indole moiety of l-tryptophan (l-trp). enhanced l-trp degradation contributes to various physiological disorders including depression or failure of the immuno-regulating system. the regulation of ido by inhibitors is extensively studied. however, the catalytic mechanism of ido on the molecular level is still unknown. in addition to l-trp, a wide range of substrates are degraded including d-tryptophan, melatonine or tryptamine. in contrast, indole or histidine do not function as substrates for ido. to understand the determinants for substrate specificity, we investigate the interaction of the heme iron, the heme-bound ligand and the substrate. we use (time-resolved) uv/visible and fourier transform infrared (ftir) spectroscopy over a wide temperature range ( - k) to monitor the binding of diatomic ligands to the heme iron and the binding of different substrates to coligated ido. changes in the spectra upon addition of l-trp are analyzed and compared to those induced by other substrates or inhibitors. archaeal protoglobin d-structure: novel ligand diffusion paths and heme-reactivity modulation protoglobin (pgb) from methanosarcina acetivorans c a, a strictly anaerobic methanogenic archaea, is the latest entry in the hemoglobin superfamily. our previous crystallographic studies on pgb have shown that protoglobinspecific loops and a n-terminal extension completely bury the heme within the protein matrix ( ) . access of o , co, and no to the heme is granted by protoglobin-specific apolar tunnels reaching the heme distal site from locations at the b/g and b/e helix interfaces. here we report structural and kinetic data on pgb mutants engineered to probe the protein structural and kinetic properties. six crystal structures (pgb mutants: ∆ (missing nter residues), y(b ) →a, y(b ) →w, f(b ) →w, f(g ) →w, i(g ) →f) show that the mutations engineered essentially restrict access to ligand tunnel . an accurate molecular level description of the signaling mechanism in ca + transducers necessitates the knowledge of the kinetics and energetics of conformational changes associated with ca + binding to various calcium binding proteins. with this in mind, we have developed an approach that combines the laser-induced photolysis of photolabile "caged" ca + compound, dm-nitrophen, with the photothermal beam deflection (pbd) technique to determine thermodynamic profiles associated with the ligand binding to calcium chelator, edta, and ca + sensor, calmodulin. this approach allows us to monitor time profiles of volume and enthalpy changes on the microsecond to millisecond timescale. the initial pbd study of ca + photo-relase from ca + loaded dm-nitrophen reveals the presence of two phases. the first step takes place within first µs upon and is associated with a volume decrease of - ml mol − and enthalpy change of kcal mol − . on the longer timescale (τ = µs), the second event with a positive volume change of ml mol − and enthalpy change of kcal mol − was detected. on the other hand, ca + photorelease in the presence of calmodulin is accompanied with an additional phase with a distinct lifetime and volume and enthalpy changes that reflects the metal binding to calmodulin and concomitant structural changes. antimicrobial peptides: linking partition, activity and high membrane-bound concentrations antimicrobial peptides (amps) have been intensively studied at micro and macroscopic levels for over twenty years. knowledge from these two domains has, however, contributed little to a comprehensive understanding of amp action; rather, in vivo amp performance has been only remotely correlated to biophysical properties. we focus on the assessment of peptide accumulation on bacterial membranes as an example of this separation: amp-bilayer interactions have been subject to extensive biophysical characterization, but conversion of that information into educated estimates of in vivo membrane-bound amp concentrations is lacking. this has led to the overlooking of important factors for activity. using simple partition models we were able to analyze available information on amp activity and interaction with membranes to show that unexpectedly high membranebound peptide concentrations are likely in vivo and may, in some cases, be required for triggering bacterial death. e. e. schäfer , s. schuy , a. janshoff georg august-university göttingen, germany, johannes-gutenberg-university mainz, germany retrovirus entry into cells occurs through fusion of the lipid bilayers that surround the virus and the lipid bilayer of the host cell. fusion proteins, present on the surface of the virus membrane play an essential role in the early stage of virus entry. understanding of the molecular mechanism is important for the design and function of modern fusion inhibitors. in this project we analyze the fusion of active conformation of the envelope gp from the human and simian immunodeficiency viruses (hiv and siv). during the infection process gp undergoes a sequence of conformational changes where the n -terminal nhr develop a trimer pre-hairpin intermediate. afterwards the three chr fold back towards the central nhr and a six helix bundle is formed. this rearrangement forces the viral and the host membrane into close contact and fusion pores may induce membrane fusion. this decisive molecular step in retroviral fusion has been modeled by rational design of lipopeptide assemblies that mimic a coiled-coil structure serving as a receptor for potential antagonists. for this purpose, sslbs were functionalized in an in situ coupling reaction with peptides originating from the nhr (n-peptides) of siv and hiv to monitor the interactions with the specific chr peptides (c and t ). binding of antagonists to surface confined coiled-coil structures has been quantified by ellipsometry, quartz crystal microbalance and was visualized by atomic force microscopy. a. rupprecht , e. sokolenko , e. e. pohl institute of cell biology and neurobiology, charité -universitätsmedizin, berlin, germany, frumkin institute of physical chemistry and electrochemistry russian academy of sciences, moscow, russia the production of reactive oxygen species (ros) in mitochondria is very sensitive to the proton motive force and can be decreased by mild uncoupling, mediated e.g. by uncoupling proteins (ucp) . in contrast, the activation of uncoupling proteins by ros as a negative feedback loop is a highly controversial hypothesis. ucp activation in mitochondria by -hydroxy- -nonenal (hne, aldehydic product of lipid peroxidation) was first demonstrated by echtay et al. . here we investigate the ability of hne to activate and/or to regulate the expression of ucp in two different systems: (i) in lipid membranes reconstituted with recombinant ucp and (ii) in primary neuronal cells. total bilayer conductance was enhanced in the presence of hne, but this effect was independent on ucp and ucp . the results concerning the hne-mediated ucp expression after induction of ros-production and/or after exogenous addition of hne are discussed for three brain-associated proteins (ucp , ucp , ucp ) in view of their possible functions. . beck, v et al. . faseb j. : - . . echtay, k. s. et al. . t. rudack, j. schlitter, k. gerwert ruhr university, department of biophysics, nd north, bochum, germany the gtpase ras p which is linked to the membrane via a lipid anchor, is a crucial switch in the cellular signal transduction processes that control cell growth and proliferation. docking simulations can be seriously hampered by the great difficulty to accurately estimate the ligand-protein binding affinity constant k, which is usually derived via the computation of the binding free energy ∆g. unfortunately, due to the involved logarithmic relationship, errors of less than . kcal/mol in the computation of ∆g result in about one order of magnitude inaccuracy on k. this can hinder computational methods from discriminating micromolar from nanomolar compounds. to improve the reliability of docking prediction, we make use of enhanced sampling methods, ranging from steered molecular dynamics to metadynamics . we also test several descriptors, such as the recently developed path collective variables , to identify the most suitable reaction coordinates accounting for binding and unbinding processes. using these approaches we investigate ligand docking and undocking and we attempt to describe at an atomistic level the kinetics of binding, which we intend to exploit for drug design purposes. here, an example of application in drug design is reported. . isralewitz, b. et al.; j. mol. graph. model. , , - . . laio, a. and parrinello, m.; pnas , , - . . branduardi, d.et al.; j. chem. phys. , , . prediction of protein-protein complex structures using wang-landau simulations a. solernou, barcelona supercomputing center, jordi girona , barcelona, spain protein-protein interactions are essential in the majority of life processes, so they have keen interest in several knowledge areas. however, experimental data on protein complexes is being produced at a quite low pace in comparison to that of the individual components. thus, in recent years attention has focused into computational approaches to the proteinprotein docking problem. a variety of docking protocols have been recently reported, sharing usually the following strategy: fast rigid-body search of the interacting subunits, followed by scoring and refinement of the interfaces. although this kind of strategy has proven some good results in the capri blind test it has two main limitations. on one hand one should include full flexibility on the protein structures. on the other, the evaluation should be made with free energy calculations instead of using a scoring function. in this work we propose to search the native complex structure in the minimum of the free energy landscape. we use the coarse grained potential unres to get the potential energy of the possible conformations. they are generated changing the dihedral angles, the side chain rotamers, and lastly the mutual orientation using a new sampling protocol we have developed (rotation-based uniform sampling; rotbus). finally, the free energy calculations are performed using an omp parallelization of the wang-landau algorithm. s. shukla , s. asthana , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy the virus encoded rdrp has emerged as a prime target in the search for specific hcv and other flaviviridae antivirals. recently, the determination of the hcv rdrp structure in complex with certain benzimidazoles has been reported, these nni's bind to the surface of the thumb domain, thereby disrupting its interaction with the finger domain, which is necessary for catalytic activity. on the other hand, we have found that, in bvdv, the mutations conferring resistance to same class of inhibitors lie in the finger domain of rdrp, indicating that the mode of inhibition of benzimidazole class of compounds is different in both hcv and bvdv rdrp. herein, we have applied docking approaches (binding orientation), molecular dynamics (md) simulations combined with metadynamics to elucidate the microscopic mechanism of the interactions between the ligand and the receptor in order to identify features barely seen in experiment. the recently designed algorithm overcomes the time scale problem by accelerating properly defined reaction coordinates. it mimics the real dynamics of a ligand staying or leaving the receptor and in doing so reconstructs the free energy surface, which in turn gives an idea of the residence time of the inhibitor in the cavity.finally we identified the binding modes and different mechanism of inhibition of benzimidazole class of compounds in rdrp of two closely related rna viruses. . c. seifert , w. stacklies , f. graeter protein mechanics and evolution, bioquant, inf bq , heidelberg, germany, ag graeter, picb, yueyang lu, shanghai , china we use a new method that detects force distribution in proteins. based on molecular dynamic simulations, changes in inter-atomic forces are calculated, here caused by different ligands. these changes will then be analyzed to detect a signal transfer through a protein initiated by the binding of a ligand to the protein. chaperones are ubiquitous proteins, which help other proteins to fold into a native conformation. they are able to refold misfolded proteins with a great variety of mechanisms. in this project, our group focuses on molecular dynamic simulations of htpg, an e. coli homolog of the human hsp (heat shock protein kda). the full structure of htpg was published by shiau et al. in , but the mechanism of how htpg performs its function is still not understood. the folding mechanism is an atp driven reaction cycle, in which the functional entity is a homodimer of htpg. we separate the cycle in three main states: apo, adp-and atpbound state. conventional molecular dynamics simulations are used to build a stable simulation system and provide structure trajectories and primary information about the behavior of htpg in its different states of the folding process. experiments indicate that the molecular movement is atp driven. we use force distribution analysis to elucidate how atp effects htpg conformation and dynamics. the small size of myoglobin makes it the preferred candidate to investigate the structure-function paradigm. in its interior five docking sites have been identified and for long time these xenon cavities have been classified as packing defects. recently, it was shown that they might be involved in ligands migration path. however, some questions regarding its role as oxygen carrier no scavenger remain yet open as well as the microscopic mechanism regulating these biological functions. in this work we made use of standard md simulations of solvated myoglobin to characterize internal cavities. our principal results is that we have found several secondary cavities showing volume and occurrence at least comparable to that of xenon cavities. in order to analyze and rationalize internal cavities we applied special cluster-analysis: we classified all cavities with respect to the position, size and occurrence ascribing them to different clusters. this analysis highlights possible intrinsic migration paths for small ligands within the protein matrix controlled by spontaneous fluctuations of the protein itself. moreover, we identified some key residues playing a fundamental role in controlling internal pathways. our suggestion that the secondary cavities constitute the preferred path for ligand escape is also supported by explicit metadynamics simulations of ligand escape. a. varga , p. lallemand , j. szabó , p. závodszky , m. vas , t. barman , l. chaloin , c. lionne institute of enzymology, brc, hungarian academy of sciences, budapest, hungary, cnrs-université montpellier -université montpellier , institut de biologie, umr , montpellier, france -phosphoglycerate kinase (pgk) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. pgk is a key enzyme in glycolysis; it catalyses the reversible reaction , -bisphosphoglycerate + adp ↔ -phosphoglycerate + atp. here we explored the catalytic role in human pgk of the highly conserved lys that has been proposed to be essential for pgk function, by a transient and equilibrium kinetic study with the active site mutant k a. by the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type pgk and mutant k a. by the use of a chemical sampling method (rapid-quench-flow) under multi and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild type pgk follows product formation, whereas with the mutant it is the phospho-transfer step itself that is rate limiting. these data are supported by saxs measurements which showed no direct role of lys in the domain closure of the enzyme i.e. the isomerisation step of the reaction. the results are explained by the structural data of the enzyme. characterization of different recombinant nrp proteins and interactions with heparin k. a. uniewicz, y. ahmed, d. g. fernig school of biological sciences and centre for glycobiology, biosciences building university of liverpool, l zb liverpool, u.k. neuropilin- (nrp ) is a mammalian membrane glycoprotein involved in tip sprouting processes like angiogenesis and neurogenesis. it has been shown that the interaction of nrp with heparin/heparan sulfate is implicated in its enhancement of growth factor signalling, however the mechanism is not yet known. commercially available extracellular domain of nrp is available either as a truncated his-tagged human protein (hnrp ) or as the rat protein fused to histagged human fc and expressed as a dimer (fcrnrp ). biochemical properties such as kinetics of heparin binding and structural requirements for sugar binding together with biochemical tests of protein properties were performed in order to characterise these commercial proteins. fc rnrp shown high affinity to heparin (kd= . nm) and required a minimum of dp to effectively compete with fc rnrp binding to immobilised heparin. competition experiments with various modified sugars show that interaction of fc rnrp with heparin is highly ionic and dependent on the position of sulfate groups along the heparin chain. the hnrp did not bind to heparin immobilised via nhs-biotin, though it did bind to some heparin affinity chromatography matrices. organic compounds of tin are among wide spread environmental pollutants. due to physical and/or chemical actions, poly-substituted alkyltins speciate into less substituted, more toxic species. tetra-or tri-alkyltins show a marked delay in their toxic action with respect to the corresponding di-and mono-alkylated analogs. it has been hypothesized that the delayed toxicity may results from the progressive dealkylation of alkyltins in more toxic di-and mono-derivatives, which bind and inhibit essential enzymes. it has been proposed that alkyltins preferentially target enzyme sulphydryl groups. previously, we showed that a nine amino acid linear peptide (i lgcwcylr ) containing a cxc motif is able to bind and dealkylate tri-substituted alkyltin compounds into the corresponding dialkyl derivatives. here, we investigated the time dependence of the degradation of the most common alkyltin derivatives by this peptide. we monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position of the peptide. we found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudo-enzymatic reaction mechanism. the end-point of the reactions was the formation of a covalent complex between the disubstituted alkyltin and the peptide. these data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds. parkinson's disease (pd) is a multifactorial neurodegenerative condition characterized by the progressive loss of dopaminergic neurons in the substantia nigra and by the presence of intracellular inclusions, composed predominantly of fibrillar alpha-synuclein (as). post-mortem studies indicate the presence of oxidative damage in the nigral neurons. dopamine oxidation, which leads to the formation of highly reactive quinones (daq), may account for the specificity neurodegeneration observed in pd. daq have many potential protein targets for chemical modifications. among them, we focused our attention on dj- , of which mutated forms have been found in familial cases of pd. a possible function of dj- is its redox-dependent chaperone activity that could prevent as aggregation and fibril formation. in the present work, we analyzed the structural and functional modification induced on dj- by daq. n-hsqc spectra of dj- were recorded in the presence of different amounts of daq and chemical shift perturbations were used to identify the dj- residues target of daq and their relative reactivity toward daq. aggregation assays were also performed to evaluate the functional effects of the daq modifications on the chaperone activity of dj- . rabies virus (rabv) infects neurons exclusively and causes lethal encephalitis. pathogenic rabv strains favor neuronal survival, whereas non-pathogen strains lead to neuronal apoptosis. the use of recombinant rabv showed: / the g protein determined the induction of the survival or death phenotypes; / the last cooh amino acids of the g protein cytoplasmic domain (cytog) are critical. these residues form a binding site for pdz domain (pdz-bs). one of the amino acid differences between survival and death gproteins are located in this pdz-bs. results of two-hybrid experiments showed that cytog survival interacted pdz domain of ser/thr kinases (mast), while cytog death interacted also with additional pdz containing host proteins. to understand the fine structural basis for the specificity of the pdz-cytog complexes, we determined the d structure and the dynamics of the mast-cytog complexes by nmr. the structures, as well as the affinities and constant kinetics, of the mast pdz complexes with both cytog are similar. we conclude that difference by one aa in the pdz-bs of the two strains cannot drastically modify the interaction with mast -pdz, in agreement with the two-hybrid data. preliminary results suggest that the interaction of cytog death with one additional cellular partner blurs the pro-survival signals engaging the infected cells through apoptotic trails. functional protein immobilization on glass-type surfaces s. waichman, m. bhagawati, y. podoplelova, j. piehler institute of biology, department of biophysics,university of osnabrück , barbara st. osnabrück, germany the immobilization of membrane proteins onto solid supports enables protein interactions and conformational changes to be probed by spectroscopic techniques under highly defined conditions. here, we present a novel method for covalent protein immobilization on glass-type surfaces using a bottom-up approach. in this approach the 'phosphopantetheinyl group of coenzyme a (coa) was transferred to the acyl carrier protein-derived ybbr tag of the target protein by means of the phosphopantetheinyl transferase sfp. the glass-support was rendered biocompatible by coating it with an ultrathin layer of peg (polyethylene glycol), followed by functionalization with coa through maleimide chemistry. immobilization of ybbr-tagged proteins in presence of sfp was followed in real time by label-free detection using reflectance interference spectroscopy. the immobilization procedure was thus systematically optimized by means of binding specificity, enzyme activity and functionality of the immobilized protein. this approach was employed for immobilizing the type i interferon ifnα in order to probe ligand recognition by ifnar and ifnar and ligand-induced ternary complex formation. the versatility of this technique was further enhanced by its combination with photopatterning methods. this immobilization technique can provide a beneficial tool for bioanalytical and biophysical applications at the single molecule level. r. vijayan, p. c. biggin department of biochemistry, university of oxford, south parks road, oxford, ox qu, uk ionotropic glutamate receptors mediate excitatory synaptic transmission in the brain and are heavily implicated in memory and learning as well as in numerous neuropathological conditions. one family of iglurs, the kainate receptors, show unusual sensitivity to changes in external ion species resulting in an apparent requirement for both sodium and chloride ions for activation. our recent work revealed the location and selectivity of the cation binding sites. despite this progress, it is still unclear how the cation binding sites confer sodium selectivity and how apparent affinity for chloride is influenced by the presence of cations. we have attempted to address these questions by performing extensive free energy calculations using all-atom molecular dynamics simulations. the rank order of cation binding obtained from relative binding free energy calculations is in agreement with experimental measurements of apparent affinity. these calculations also reveal that the pair of cation binding sites in the dimer interface act independently. binding free energy calculations performed using a reduced model of the binding site show that cation selectivity can been attributed to both the rigidity and high charge density of the binding sites. finally, a potential of mean force derived from umbrella sampling simulations indicate that the presence of cations stabilize the anion binding site considerably. the effect of toxins on the inorganic phosphate release during the actin filament formation a. vig, t. kupi, g. hild, m. nyitrai university of pécs, faculty of medicine, department of biophysics, szigeti str. , pécs, hungary actin can be found in monomeric (g-actin) and filamentous (f-actin) form in eukaryote cells under physiological circumstances. the first step of actin polymerisation is the formation of actin nuclei by atp-binding actin monomers. the next step in is the elongation when monomers are associated to the previously formed nuclei or to the ends of the growing filaments. after the association of monomers the bound atp is hydrolysed to adp.p i , and with first order kinetics the release of inorganic phosphate occures. the rate constant of the release is , s − , which is a slower process than the hydrolysis itself ( , s − ).phalloidin, a cyclic peptide from amanita phalloides can bind to the filaments and stabilizes their structure. jasplakinolide is another cyclic peptide from marine sponge (jaspis johnstoni) which binds actin filaments. the aim of this study was to investigate whether the binding of toxins to the newly created filaments has an effect on the kinetics of the inorganic phosphate release or not. we used absorption photometry measurements to measure the rate of phosphate release. phalloidin decreased the rate of the release substantially. although the effect of jasplakinolide was weaker, the results showed that the binding of these toxins to the actin can modify the rate of the release of inorganic phosphate from the filaments. these observations are in agreement with the molecular mechanisms by which these toxins stabilise the actin filamens. fluorescent proteins (fps) are invaluable fluorescent markers in cell biology. however, their use is often limited by photobleaching of the chromophore, notably in single-molecule, time-resolved or super-resolution imaging studies. we will present the crystallographic studies at near atomic resolution of a photo-activatable fluorescent protein irisfp that has been observed in a transient radical state en route to photobleaching. we took advantage of x-rays to populate the radical, which, under illumination with visible light, presumably forms with low probability from the triplet state. the combined x-ray diffraction and in crystallo spectroscopic data (from uv-vis and raman spectroscopies) reveal that radical formation in irisfp involves strong but reversible distortion of the chromophore, suggesting a transient loss of pi-conjugation. these results will help unravel the mechanisms of blinking and photobleaching in fps, which is of importance to rationally design variants of higher photostability. optimizing photoactivatable fluorescent proteins for live-cell imaging recently, novel fluorescent proteins (fps) have been reported which perform spectral changes in response to irradiation with light of a particular wavelength. reversibly photoactivatable proteins switch between a bright and a dim fluorescent state. this process is accompanied by photoisomerization of the protein chromophore. irreversible photoactivation results from photochemical processes within the protein, e.g., photolysis of an amino acid side chain, or an extension of the alternating π-electron system of the chromophore by a β-elimination reaction. fps have became valuable tools in live-cell imaging because they allow intracellular protein labeling by using them as fusion tag, and photoactivatable fps are powerful tools for application in novel subdiffraction imaging techniques. however, the available fps still offer potential for improvement in various ways. they frequently show a tendency to aggregate or oligomerize, incomplete chromophore maturation, fluctuating emission and low photostability. here, we will present our recent progress towards engineering the 'perfect' fp. simultaneous intracellular chloride and ph measurements using gfp-based sensor in ldcv d. arosio , f. ricci , l. marchetti , l. albertazzi , f. beltram italian institute of technology, udr pisa, italy, nest, scuola normale superiore, pisa, italy, nest, infm cnr, pisa, italy chloride ion participates in many physiological functions including control of neuronal resting potential, charge balance during endosome acidification, and regulation of cell volume. as a consequence dysfunctions in regulating membrane chloride permeability lead to severe diseases including motor disorders, cystic fibrosis and epilepsy. at present processes regulating intracellular chloride ion concentrations are still widely unexplored mainly as a consequence of limiting methods to quantify chloride fluxes in living cells. in the present work a highly specific, genetically encoded sensor is developed for detecting simultaneously intracellular ph and chloride concentration. the sensor is obtained by fusion of a red fluorescent protein (dsred-monomer), insensitive to chloride and ph, to a gfp variant containing a specific chloride-binding site (gfp-chl). dsred-gfp-chl binds the chloride ion following a fluorescence static quenching mechanism, which allows measurements of intracellular ph in a chlorideindependent manner. the sensor has been successfully tested in different living cells, in a ph range - and chloride concentration up to mm. for the first time, to the best of our knowledge, it allowed to measure the chloride concentration of dense core vesicles in the secretory pathway. applicability to high-throughput screening, range of validity and accuracy of time-lapse maps will be discussed. we aim to understand the basis of the photophysical changes in fluorescent proteins (fp) induced by reactive oxygen species (ros). indeed, ros might be involved in photochemistry of fp, leading to their photobleaching or photoconversion. in addition, fp may be used to investigate cellular events like phagocytosis or mitochondrial activity, where ros are naturally produced. in the latter cases, an accurate analysis of fp's fluorescence signals requires the full knowledge of reactions between ros and fp. in the future, this work may help in developing either photoresistant or photoswitchable fp and improving their use for imaging under oxidative stress conditions. using γ-radiolysis as a quantitative source of ros, we investigated the reactions of oh and o − radicals on the cyan fluorescent protein (cfp) and the modifications of the cfp's photophysical properties by oh radicals were explored in detail (submitted). in order to address the corresponding chemical changes in the protein, we devised a mild proteolysis protocol that for the first time offers a peptide mass fingerprint almost covering the cfp sequence (alvarez et al. biochemistry ). then, we achieved the meticulous characterization of the cfp oxidation products by mass spectrometry and proposed a mechanism to account for their formation by pulse radiolysis. since the cloning of the green fluorescent protein from aequoria victoria, numerous screens have been performed to improve the brightness of this protein, its spectral variants and fluorescent proteins from other species. the improvement is evaluated by comparing fluorescence intensity of individual bacteria or colonies. in this way also expression level, folding and maturation efficiency, and thickness of the bacterial colony contribute. here we report a screening method that, in addition to fluorescence intensity, quantifies the excited state lifetime of a fluorescent protein, which is independent of intensity or expression level, and provides a direct measure for the quantum efficiency of the fluorescent protein. the novel approach was used to screen a library of cyan fluorescent protein (cfp) variants randomly mutated at position , and , yielding an improved bright cyan fluorescent protein named, mposeidon, with a markedly increased fluorescence lifetime and quantum yield, increased photostability and improved fluorescence intensity in vivo. it is shown that mposeidon is the brightest cyan fluorescent protein in mammalian cells. in addition, several lifetime variants were identified that can be used for lifetime unmixing. it is demonstrated that three cfp variants can be separated and their distribution quantified in a single detection channel. a. r. faro , p. carpentier , d. bourgeois , e. rosny irtsv, cea, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cnrs, ufj, grenoble, france enhanced yellow fluorescent protein (eyfp) is extensively used as a fluorescent marker. like other photo activatable fluorescence proteins (pafp), it exhibits photo-switching properties. however, the mechanism by which fluorescence can be swiched on or off upon light irradiation is not fully understood at the molecular level. the bright to dark conversion involves a protonation step and structural rearrangements of the chromophore, but it is not clear which of these two steps is the triggering event. to answer this question, we carried out photo-switching experiments at cryotemperatures. our data suggest that a photo-induced protonation step (probably in the triplet state) is the primary event in the bright to dark conversion. our results may bear relevance to other pafps, such as iris, eos, dronpa, padron or kaede. how misfolding and aggregation of proteins constitutes a toxic insult to neurons remains largely unknown. in order to obtain insight into the molecular biology of neurodegenerative disease, we have developed a number of gfp-based biosensors for the detection and quantification of cellular clearing mechanisms for aggregated proteins. the high load on these protein quality control mechanisms, and their failure to meet normal physiological demand ultimately results in a "de-compensation" condition from which the nerve cell cannot recover. our fret/flim-based bioassays visualize protein ubiquitination and degradation; proteasomal activity; foldase activity using a folding-impaired gfp mutant which gains fluorescence conditional on the upregulation of chaperone activity; chaperone binding to unfolded proteins; and autophagosome formation/lysosomal integrity via the targeted and sensitive fret-based measurement of ph changes. these sensors are employed in cellular model systems for parkinson's and alzheimer's disease, and amyotrophic lateral sclerosis (als) to delineate the molecular pathway of cellular demise, and to gain a mechanistic understanding of the toxicity of protein aggregates and the basis for the vulnerability of neurons. millisecond photo-switching dynamics of e q gfp mutants for sensor and imaging applications m. collini , v. quercioli , l. d'alfonso , g. baldini , b. campanini , s. bettati , g. chirico dipartimento di fisica, università di milano bicocca, italy, dipartimento di biochimica e biologia molecolare, università di parma, parma, italy e q mutants of the green fluorescent protein are known to possess photo-chromic properties: the anionic emission, primed by a pump nm laser beam, can be switched between two levels of different brightness by irradiation with a blue, ∼ = nm, probe laser light. we have studied here the amplitude and the dynamics of the brightness enhancement of the e q mutant of gfpmut . the fluorescence emission increases almost threefold, under saturating probe laser excitation, for pump excitation intensity in the linear regime. two characteristic activation times, estimated by means of modulated two colour fluorescence correlation spectroscopy, are detected in the - ms range, independent of solution temperature and viscosity. the brightness enhancement factor and the characteristic activation times depend markedly on the solution ph. these results indicate that this mutant can be used as a high sensitive intracellular marker for local proton concentration and for modulated excitation imaging. the advent of fluorescent proteins (fp) gave researchers the opportunity to study proteins in situ. fluorescence resonance energy transfer (fret) benefited from this. cell fixation is a commonly used approach when working with microscopy. however, we have found that fret efficiency (e) in cells transfected with cerulean and venus chimeras could not be reproducibly measured after fixation. to evaluate this problem in detail, we measured e of cerulean-venus standard constructs by acceptor photobleaching fret, intensity-based ratiometric fret and flim-fret. the constructs were produced as standards (biophys j, , , ) with , and % e values, comprising donor-acceptor separations of , and amino acids, respectively. transient transfection of the fusion plasmids was performed into hela cells and e was measured in live and pfa or methanol fixed cells. literature e values were reproduced when measuring live cells. conversely, cell fixation caused a deviation of e values. methanol fixed cells showed e between - % for all the constructs. the effect of pfa fixation on both fluorescence intensity and fret varied vastly among independent experiments regardless of the measurement modality. thus, fixation should be avoided due to the effects it has on fp's fluorescence and consequently on fret efficiency. to explain the improvement in the fluorescence properties of cerulean when compared to ecfp, we have determined the x-ray crystallographic structures of these two proteins at physiological ph, and performed molecular dynamics simulations. both proteins exhibit a structural heterogeneity in the nterminal half of their seventh strand, which forms a specific set of van der waals interactions with the chromophore. the critical h d mutation present in cerulean induces a modification of these interactions, and allows the chromophore to be more planar and better packed, albeit only intermittently. as a consequence, the probability of non-radiative decay is significantly decreased. our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins, and provide insights which should allow the design of mutants with enhanced optical properties. the fluorescence lifetime of green fluorescent protein g. jung biophysical chemistry, saarland university, saarbruecken, germany biotechnological design of the green fluorescent protein (gfp) and the discovery of other proteins boosted the development of the life sciences in the past decade. tracking protein movements and high resolution microscopy are only a few recent applications which were realized by fluorescent protein technology. among these examples, the switching between two chromophore state is exploited. our aim is to establish fluorescent proteins for bioanalytical fluorescence lifetime measurements. despite the progress in other fields, quantification with gfp still imposes practical problems [ ] . in the past, we observed that the uv-light driven decarboxylation of the nearby aminoacids glu distorts the fluorescence lifetime of gfp [ ] . we found out that this chemical reaction also occurs under excitation of the anionic chromophore state with a high quantum yield [ ] . recently, we could show by time-resolved spectroscopy that, indeed, the more susceptible state for this kind of photoconversion is the anionic chromophore state [ ] . suppression of this reaction therefore enables the design of autofluorescent proteins which can be used e.g. for the quantification of ions and which are beneficial as donors in fret applications. the fluorescent properties of tryptophan residues (w) in proteins are highly dependent on their immediate protein environment. however, the multi exponential decay of single w proteins is not completely understood. the most cited hypothesis contributes a multi exponential decay to the existence of several micro conformations (rotamers) of the w residue within the protein matrix. to determine rotamers we apply a method based on dead-end elimination (dee) and molecular dynamics simulations (md). low energy rotamers are calculated by dee while dynamics and further refinement is accomplished using md. the method was applied on several test cases including the protein mutant bc-csp l e from bacillus caldolyticus, which contains a solvent exposed w residue. as resolved by x-ray crystallography, this w residue occupies two conformations. using dee and md we were able to retrieve the w conformations found in the x-ray structure. the results demonstrate the ability of the method to obtain valuable w rotamers, both for solvent shielded as exposed residues. the determined conformations were compatible with the findings based on a method using replica exchange simulations. k. nienhaus , h. nar , r. heilker , j. wiedenmann , g. u. nienhaus inst. of biophysics, universität ulm, ulm, germany, dept. of lead discovery, boehringer ingelheim, biberach/riss, germany, national oceanography center, university of southampton, southampton, uk, inst. of applied physics, universität karlsruhe (th), karlsruhe, germany eqfp is a red fluorescent protein (rfp) with the chromophore in a co-planar trans orientation, whereas the cis isomer is preferred by most other rfps. by using x-ray crystallography, we determined the structures of the dimeric variants d eqfp and d eqfp at high resolution (up to . Å). for d eqfp , we had previously seen a redshifted species upon irradiation with -nm light. concomitant changes in the raman spectrum were interpreted as evidence of a trans-cis isomerization of the chromophore. here we have combined x-ray crystallography and site-directed mutagenesis to assess whether we can create a stable fluorescent, red-shifted eqfp variant with a cis chromophore. in a first step, we introduced the n s substitution. this variant, d rfp , is highly fluorescent, with the absorption (emission) maximum red-shifted by ( ) nm. with an additional s c mutation, the chromophore is found completely in the cis form. the variant, rfp , is highly fluorescent, with excitation and emission maxima at and nm. still further red shifts appear to be in reach. k. nienhaus , v. adam , d. bourgeois , g. u. nienhaus of biophysics, universität ulm, ulm, germany, esrf, grenoble, france, institute of applied physics, universität karlsruhe (th), karlsruhe, germany dendra is an engineered, monomeric gfp-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green-to a red-emitting state upon exposure to purple-blue light. this process occurs in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized π-electron system. we have measured the x-ray structure of green dendra and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. the structure, which is very similar to those reported for the closely related proteins eosfp and kaede, revealed a local structural change next to the chromophore, involving mainly arg and a water molecule. we propose that this structural change explains the blue shift of the absorption and emission bands, as well as the markedly higher pks of the hydroxyphenyl moiety of the chromophore. the -fold enhancement of the neutral species in dendra at physiological ph accounts for the observed higher photoconversion yield of this protein in comparison to eosfp. photochromic green fluorescent protein mutants: chromophore states unveiled by raman spectroscopy s. luin, v. voliani, g. lanza, r. bizzarri, r. nifosì, p. amat, v. tozzini, m. serresi, f. beltram nest, scuola normale superiore, cnr-infm and italian institute of technology, pisa, italy the most widespread genetically-encodable fluorescent markers used for studies in living cells and tissues belong to the green fluorescent protein (gfp) family. reversibly switchable fluorescent proteins (rsfps) were developed that can undergo repeated transitions between different states, e.g. bright and dark forms. this property makes rsfps particularly attractive as active labels in biological systems for selective photolabeling applications or subdiffraction imaging. we shall present pre-resonant raman results unveiling the photophysical mechanism underlying the observed photochromic behavior. the variation of spectral properties before and after photoconversion of chemically-synthesized isolated chromophores under different protonation and/or isomerization have been analyzed, and compared to results obtained for the case of complete folded proteins comprising the same chromophores. experimental results have been analyzed within a time-dependent density functional theory, allowing us to assign all relevant vibrational modes. these results make it possible to discriminate between the effect of cis-trans isomerization and of diverse protonation states of the chromophore in the photoproducts of these proteins. s. luin et al, j. am. chem. soc. , - ( ). r. bizzarri et al., anal. bioanal. chem. , - . a combined study of the interaction of outer membrane proteins with cephalosporin antibiotics m. lovelle, i. barroso, m. j. feio, p. gameiro requimte , fac. ciências, universidade do porto, portugal gram-negative bacteria characteristically are protected by an outer membrane that serves as a selective permeation barrier. most of the β-lactam antibiotics appear to penetrate the outer membrane through these non-specific channels, and it becomes important to understand the possible interactions between β-lactams and the porin. fluorescence techniques have been largely used to characterize both the conformation and the dynamic behavior of large biological structures such as membranes and proteins. the fluorescence of the tryptophan residues is quenched in the presence of the different cephalosporin antibiotics. this reaction between the excited state of the fluorophores and the drug can be described as a formation of a non-fluorescent complex. the dependence of the fluorescence intensity upon quencher concentration for static quenching is proportional to the binding constant for complex formation. since β-lactam susceptibility is closely related to the presence of these non-specific porins, minimum inhibitory concentration (mic) by micro-broth dilution in microplate were used to assess the bactericidal activity of cephalosporin antibiotics upon on escherichia coli strain bl (de ) and a series of bl (de ) mutated in different outer membrane proteins. this combined study of the interactions at single molecular level and at in vivo level provides new insights for a better understanding of the antibiotic translocation. when used in combination with e.g. a cyan fluorescent protein, keima offers the unique opportunity to perform dual color fluorescence cross-correlation spectroscopy using a single laser line to excite both fluorophores. the molecular determinants of the large stokes shift of mkeima have been characterized structurally by combining x-ray crystallography with in crystallo uv-visible absorption, fluorescence and raman spectroscopy. our results reveal a ph-dependant "reverse chromophore protonation" of mkeima, driven by the key residue asp . moreover, the chromophore protonation state is shown to be coupled with different chromophore conformations, cis conformation at ph . , and mostly trans conformation at ph . . these results will help unravel the mechanisms of fluorescence in fps, which is of importance to rationally design and develop new fluorescent markers. recently reversibly switchable fluorescent proteins (rsfps) have become a new branch of the green fluorescent protein (gfp) like family. the rsfps may be reversibly switched between a fluorescent and a non-fluorescent state by irradiation with light of distinct wavelengths. the key process of this switching behaviour is a light induced change of the chromophore between a cis-and a trans-isomeric state. the proteins are becoming increasingly important for diverse applications like protein tracking, sub-diffraction resolution microscopy and data storage. based on the switching mechanism, we created novel rsfps with unique and improved characteristics. padron and bs-dronpa are two of these new switchable proteins. padron features a reversed switching mechanism as compared to all other green rsfps known to date while bsdronpa exhibits a very broad absorption spectrum extending into the uv. utilizing the characteristics of both proteins, we performed multi label single detection colour microscopy as well as dual colour sub-diffraction microscopy, the latter with a resolution of nm. further, we recently introduced the first monomeric rsfp exhibiting red fluorescence: using a semi rational mutagenesis approach on the non-switchable mcherry, we generated the switchable monomeric protein rscherryrev. the use of this protein in single molecule switching microscopy allowed us to record time-lapse live-cell images of the endoplasmic reticulum with sub-diffraction resolution. a spectroscopic approach to the study of chromophoric dissolved organic matter (cdom) in the sea c. santinelli, r. lavezza, l. nannicini, a. seritti cnr-ibf, via moruzzi , pisa, italy dissolved organic matter (dom) in the ocean represents the largest reservoir of reactive carbon on the earth. it is produce at each level of the marine food web and it represents the food for heterotrophic bacteria, which can use the different pools of dom (labile, semi-labile, refractory) with a large range of turn-over times (from minutes to millennia). dom plays a central role in the global carbon cycle and it has a high ecological significance. chromophoric dom (cdom) is the fraction of dom capable of adsorbing light at uv and visible spectral regions. it determines the underwater light availability in open and coastal regions with important implication on primary production and biological activity. it is photodegraded to co , co, with a significant impact on the role of the ocean as source and/or sink of atmospheric co and to highly reactive compounds, dangerous for marine organisms. in its pool "humic-like" and "protein-like" fluorophores have been individuated. cdom optical properties (absorption and fluorescence) together with doc data collected in some key regions of the mediterranean sea will be presented and discussed, in order to (i) investigate the powerful of cdom optical properties to get information on cdom "quality" (i) asses the role of dom in carbon export at depth, (iii) underline the main unresolved question on dom and cdom dynamics in the ocean, with particular emphasis to their biological lability. mechanism and applications of photo-and redox-switchable fluorescent proteins s. j. remington, j. n. henderson, x. shu, j. lohman institute of molecular biology, university of oregon, u.s.a. photoswitchable fluorescent proteins have significant advantages over conventional fluorescent labels, and in a revolutionary application, now allow cell biologists to exceed the diffraction limit in light microscopy by a factor of ten. atomic resolution crystal structures and time resolved spectroscopy of both reversible (mtfp . ) and irreversible (pa-gfp) photoswitchable fluorescent proteins in the light and dark states explain the long term stability of either state, as well as how illumination at the appropriate wavelength causes the molecules to switch between states. the photoswitching mechanisms will be discussed in terms of photochemistry, light induced chromophore isomerization and excited state proton transfer (espt). mutagenesis of espt pathways provide insight into the nature of the ratedetermining steps in proton transfer and lead to practical applications, such as redox-sensitive gfp biosensors. k. i. willig, b. hein, s. w. hell max-planck-institute for biophysical chemistry, goettingen, germany stimulated emission depletion (sted) nanoscopy is a light microscopic technique offering a resolution far beyond the diffraction limit: the excitation beam is overlapped with a doughnut-shaped, red-shifted sted beam, which switches off the ability of the molecules to fluoresce in the outer region of the excitation spot. scanning the nanosized focal spot through the sample renders sub-diffraction images with a sub-second frame rate. we used the yellow fluorescent protein citrine to image individual structural elements of living mammalian cells: vimentin and the tubular network, structures of the cytoskeleton, were recorded with a lateral (x,y) resolution < nm inside the living cell, corresponding to a -fold improvement over that of a confocal microscope. also, time lapse sted imaging of dendritic spines of yfppositive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. as an alternative to fluorescent proteins we used a genetically encoded protein tag which can be labelled with modified organic dyes within living samples. thus nanoscale imaging of structures in the interior of living cells greatly expands the scope of light microscopy in cell biology. pulling membrane tubes from solid-supported lipid bilayers with atomic force microscopy j. w. armond , j. v. macpherson , m. s. turner department of physics, university of warwick, u.k., department of chemistry, university of warwick, u.k. information on the elastic and dynamic properties of membranes is essential for a thorough understanding of biological processes such as exocytosis, endocytosis, cell division, and pore formation. we use an atomic force microscope to pull on solid-supported lipid bilayers and observe that an energy barrier must be overcome prior to the formation of membrane tubes. we have modified elastic models of lipid bilayer vesicles to describe the free energy of a planar lipid bilayer in adhesive contact with a surface. from this model we are able to extract force-distance curves for the formation of a membrane tether, including the associated energy barrier. the experimental data can thus be understood in a quantitative fashion. this work enables the atomic force microscope as a quantitative instrument for measuring membrane pulling mechanics, and in future work will allow two-dimensional mapping of elastic properties under tension. from pores to micelles -a peptide-membrane interaction study t. l. andresen, j. r. henriksen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark. email: thomas.andresen@nanotech.dtu.dk. research in the partitioning of peptides into lipid membranes has been intense for several years. along with scattering techniques and nmr, isothermal titration calorimetry (itc) has proven to be a powerful tool for thermodynamic characterization of peptide-membrane interactions. we have studied two antimicrobial peptides, mastoparan-x and melittin, and found that these peptides induce a range of structural transitions of popc and popc/popg membrane systems at different peptide-lipid ratios. we have found that itc can be used to elucidate the threshold where transitions occur, including the threshold for pore formation and micellation. this has been achieved using a thermodynamic model based on gouy-chapman theory, which provides the partitioning constant of the peptide-membrane interaction and thereby the concentration of peptide on the membrane surface. the structural changes have furthermore been visualized by cryo-tem. we have further investigated the pore formation in detail and found that the thermodynamic parameters of pore formation can be fully characterized using a system specific temperature where the enthalpy of peptide partitioning becomes zero. this allows for an exclusive study of the pore formation process. lactoferrin is a glycoprotein with two globular lobes, with of two domains each. since its discovery, the research on antimicrobial properties has been extended to peptides derived from this protein. the largest family studied so far is known as lactoferricin b, obtained from the protein by digestion with pepsin. more recently, a new family of antimicrobial peptides derived from lactoferrin was discovered by bolcher et al and named lactoferrampin (lfampin). the original sequence of lfampin contained residues - from the n domain of lactoferrin. we studied peptides derived from lfampin obtained by extension and/or truncation at the c-or n-terminal sides, keeping the essential characteristics, in order to unravel the main structural features responsible for antimicrobial action. the peptides were tested against model membranes. the ability to adopt helical conformation was followed by cd, the perturbation of the membrane phase transition by dsc, the energetics of interaction by isothermal titration calorimetry (itc), the partition of the peptide to the membrane by trfs and the importance of charge effects assessed by zeta potential measurements. the results are discussed and compared to the antimicrobial and hemolytic activities obtained by microbiology techniques. defensins are small cysteine-rich cationic proteins or peptides found in both vertebrates and invertebrates. they can be active against bacteria, fungi and many enveloped and non-enveloped viruses; thus, being also classified as antimicrobial peptides (amps). in the present work a comparative study between psd (a plant defensin with antifungic properties obtained from the garden pea pisum sativum) and hnp (a human neutrophils defensin) was conducted, in order to shed some light on the mechanism of action at the molecular level of these defensins. large unillamelar vesicles with different lipid compositions were used for this purpose as biomembranes model systems; namely, popc/cholesterol (characteristic of the outer leaflet of vertebrates cell membranes) and popc/ergosterol (fungal) mixtures. changes on the intrinsic fluorescence of the tryptophan residues present in these peptides upon membrane binding/insertion were followed by fluorescence spectroscopy. experimental results show the affinity of both defensins for mammalian and fungal sterols. the partitioning behavior of psd showed a high selectivity for ergosterol rich membranes, while hnp has preference for cholesterol rich membranes. preliminary characterization of atomistic computer models of galactolipid bilayers k. baczynski, m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university, krakow, poland the main lipid component of thylakoid membranes are galactolipids, which constitute more than % of its lipid composition. the most common types of galactolipids found in thylakoid are monogalactosyldiacylglycerol(mgdg), whose headgroup consists of a single molecule of beta-d-galactose and digalactosyldiacylglycerol(dgdg), whose headgroup consists of two galactose molecules: alpha-d-galactose and beta-d-galactose linked by o-glycosidic bond majorty of thylakoid galactolipid have gamma-linolenic acid both in the sn- and sn- position. atomistic computer models of mgdg and dgdg molecules have been constructed and parametrized in the opls-aa force field. the molecules were used to construct three bilayer systems for molecular dynamics (md) simulations:( ) composed entirely of dgdg molecules,( ) composed entirely of mgdg molecules,( ) composed of a mixture of dgdg and mgdg molecules the ratio : . the systems have been md simulated using the gromacs . . package. the generated trajectories were analysed to determine a number of structural parameters among them: membrane thickness, average area per lipid, electron density profile accross the bilayer, order parameters, organisation of the bilayer/water interface as well as several dynamic parametrers like diffusion coefficients, lifetimes of conformational states, lifetimes od lipid lipid interactions. single mixed-lipid guv method reveals interaction of viper venom with lipid membranes n. m. ayvazyan, n. a. zaqaryan, n. a. ghazaryan dpt. biophysics, yerevan state university, armenia studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including bilayer lipid membranes (blms), small and large unilamellar vesicles. giant unilamellar vesicles (guvs) with a mean diameter of µm have a minimum curvature and mimic cell membranes in this respect. guvs are ideal for studying lipid/lipid and lipid/protein interactions using microscopy techniques with membrane fluorescence probes. guvs were formed from the total lipid fraction from bovine brain by the electroformation method, developed by angelova and dimitrov ( ) . vipera lebetina obtusa venom was added to the sample chamber before the vesicles were formed. the membrane fluorescence probes, ans and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. ans and pyrene allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. the presence of viper venom in guvs media reveals a noticable decreasing of membrane fluidity compare the control, while the binding of fluorophores with guvs modified by venom lead to appearance of channel activity. it was recognized early that the vipers' venom components preferred an organized lipid substrate near the lipid's phase transition and were particularly active against micellar lipids. these studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom. the attenuated total reflection fourier transform infrared (atr-ftir) spectroscopy is ideal for highly absorbing samples such as water suspensions or even bulk water due to the small light penetration depth. it is also suited for experiments on lipid membranes in excess water. for example, we have studied the hydrogen bonding (h-bonding) between cholesterol (ch) and phosphatidylcholine (pc) or sphingomyelin (sm), which could be important for the stabilization of the cholesterol-rich lipid domains. the atr-ftir method enabled comparison of the carbonyl band shape for pc/ch samples in excess h o or d o, and has confirmed similar behavior for both [ ] . secondly, we were able to analyze the amide ii band for sm/ch samples in excess h o. the observations confirm the presence of h-bonds between ch and n-h group in sm [ ] . another example is the study of the interlamellar water structure, which could influence the water-mediated phenomena in membranes. h-bonds in interlamellar water in partially hydrated lipid multibilayers are stronger with respect to bulk water. in contrast, we show by atr-ftir spectroscopy that the h-bonds are weaker in multibilayers in excess water [ ] . the bactericidal activity of antimicrobial peptides is linked to their ability to perturb the permeability of bacterial cells. they often show α-helical conformation, and many peptides have a kink in the middle of this structure, caused by pro or gly. in order to understand the role of the kink we designed various analogues of p , in which the central pro residue was moved from its central position, or removed altogether (in analogue p f). displacement of the pro residue caused a decrease of the antimicrobial activity, and an increase in the toxicity against erythrocytes, with the less active and more toxic peptide being p f. fluorescence studies suggest that both p and p f bind on the membrane surface. fluorescence experiments show that the activities of the two analogues correlate with their affinity for different kinds of lipid bilayers: the kinked p peptide has a dramatically higher affinity for negatively charged vesicles (mimicking the composition of bacterial membranes) than for neutral liposomes (similar to mammalian cells), while analogue p f exhibits comparable affinities for both membranes. therefore, our data suggest that the central pro-induced kink is involved in selectivity, inhibiting peptide binding to the membranes of eukaryotic cells. d. behn , h. hoffmeister , r. witzgall , c. steinem institute for organic and biomolecular chemistry, university of göttingen, germany, institute for molecular and cellular anatomy, university of regensburg, germany polycystin- , encoded by pkd , is an integral membrane protein with a size of kda and amino acids. the protein, which is known to be a ca + permeable, non selective cation channel, interacts with several proteins such as polycystin- , pigea or pacs- /- etc. the interaction takes place through a proposed coiled-coil domain of polycystin- located at the c-terminus of the protein.if mutation of either pkd or pkd (gene product of polycystin- ) occurs, the interaction between the proteins is disturbed leading to increased formation of renal cysts. this autosomal polycystic kidney disease (adpkd) is one of the most common genetic diseases causing renal failure due to the enormous cyst formation. in this study, the interaction of the c-terminus of polycystin- with its postulated specific interaction partners has been investigated in terms of its biological relevance. binding affinities as well as kinetic parameters of the interaction were determined. in particular, the interaction of c-polycystin- and pigea immobilized on solid supported membranes with c-polycystin- has been quantified by means of the quartz crystal microbalance (qcm) method. a dissociation constant of about nm was obtained. the results are compared with those obtained by surface plasmon resonance (spr) technique using a different immobilization strategy. correlation of the lateral structure of lipid bilayers and monolayers using two photon excitation fluorescence microscopy l. a. bagatolli membrane biophysics and biophotonics group/memphys -center for biomembrane physics, bmb, university of southern denmark most of the reported fluorescence microscopy applications on langmuir lipid films are focused in obtaining fluorescence intensity images using particular fluorescence probes. in this type of experiments the probes are generally utilized to obtain "contrast" between membrane regions (lipid domains) displaying dissimilar physical properties. the obtained information largely depends on the partition of the fluorescent probes for the lipid domains and the obtained fluorescence intensity pictures are not providing any information about the local physical properties of the lipid film. local physical properties of these distinct regions can be determined by exploring fluorescent probe related parameters such lifetime, emission shift, polarization. however these fluorescent probe's properties are almost unexplored in this type of experiments. this presentation will focus in describing a new experimental setup that includes a specially designed film balance on top of a custom built multiphoton excitation fluorescence microscope. this setup allows obtaining laur-dan gp images ( ) many studies on phase separations of lipids in bilayers and their leaflet asymmetry make use of fluorescence techniques. we used a dithionite assay, steady-state fluorescence anisotropy and fluorescence resonance energy transfer (fret) for characterization. dithionite assay was performed to measure the fluorophore distribution in the inner and outer leaflets of symmetric membranes. for low concentrations, the fluorophore is distributed almost homogeneously, whereas for concentrations > . mol % it accumulates in the outer leaflet. we discuss these effects taking into account the presence of multilamellar liposomes and dynamic effects produced by higher local bending elasticities. the results point out possible artifacts in the use of fluorophores to characterize bilayers under the assumption of their homogeneous distribution. dithionite relative bilayer permeability is discussed. the results won by fret and steady-state fluorescence anisotropy regarding lipid phase transitions are in good coincidence to each other and to results reported in the literature. the photosensitizing properties of three chlorins are compared in solution and when incorporated in dioleoylphosphatidylcholine vesicules. in solution, they possess a similar efficacy to generate singlet oxygen and a similar ability to induce the peroxidation. however, the role of the photosensitizer localization within the lipidic bilayer is strongly highlighted, when chlorins are incorporated in liposomes, both by the changes in order of peroxidation efficacy but by the measurements of the photoinduced permeation of the liposomes. the results are discussed in relation with the technology of photochemical internalization, pci. then, using giant unilamellar vesicles, we asymmetrically oxidize the membranes. we observed different shape transitions, such as budding, typical of membrane curvature modifications. the asymmetry of the shape transitions are in accordance to a lowered effective spontaneous curvature of the leaflet targeted. this effect is interpreted as a decreased preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation. permeabilization of guv by photooxidation is interpret as the opening of a pore above a critical membrane tension due to the budding of vesicles. the effective spontaneous curvature of photosensitized vesicle at lysis was estimated. additionally photooxidation was shown to be fusogenic. influence of polyphenol extracts from fruits on biological and model membranes d. bonarska-kujawa, h. pruchnik, j. sarapuk, j. oszmiański, h. kleszczyńska univ. of environmental and life sciences, wroc law, poland biological activity of polyphenol extracts from apple, strawberry and chokeberry was studied. polyphenols were shown to be good antioxidants and to act as antyhemolytic agents. both the activities are the result of polyphenols incorporation into biological membranes. to check potential changes they induce in membranes some experiments were performed with the use of erythrocytes, and lipid membranes. it was found that the extracts studied induced shape transitions of erythrocytes. they were classified according to the bessis-brecher scale. strawberry extract induced mainly discocytes and discoechinocytes. populations of discocytes, echinocytes and some discoechinocytes were found on applying apple extract, while chokeberry induced mainly the formation of echinocytes and spheroechinocytes. the results evidence that the polyphenols incorporate into the external monolayer of lipid bilayer of the erythrocyte membrane. the results of the fluorimetric experiments showed that all the extracts changed fluidity in the hydrophilic part of rbc membrane. the changes observed were biggest for chokeberry extract and smallest for strawberry one. incorporation was also followed by changes in electrical resistance of black lipid membranes and in shifting the temperatures of main transition (t m ) and pretransition (t p ) in liposomes. this work was sponsored by ministry of science and education, scientific project no. n n . a. boll , n. czudnochowski , m. geyer , c. steinem institue of organic and biomolecular chemistry, university of göttingen, germany, mpi for molecular physiology, dortmund, germany hiv- tat belongs to the accessory proteins of hiv and has regulatory functions. tat is concentrated in the nucleus and nucleolus of infected cells. the protein is composed of amino acids with a molecular weight of kda. tat is a transcriptional activator protein, which stimulates rna polymerase ii-mediated transcription elongation. therefore, tat interacts with cyclin t and binds to the tar rna element. tat has different domains. with respect to the interaction with lipid membranes, the most important structural motif is its basic region, including arginine and lysine residues. the peptide derived from this basic region belongs to the cell-penetrating peptides and is able to translocate through lipid membranes. the major aim of this study is to investigate the influence of full length hiv- tat on artificial lipid membranes. as a starting point solid-supported membranes composed of -palmitoyl- -oleoyl-sn-glycero- phosphocholine (popc) immobilized on silicon dioxide were used. the influence of the lipid head groups on the interaction with tat was analysed by using membranes composed of the negatively charged lipid -palmitoyl- -oleoyl-sn-glycero- -[phospho-l-serine] (pops) in a mixture with popc. fluorescently labelled tat was used to localise the protein in the membrane. the impact of tat on lipid membranes was investigated by fluorescence and atomic force microscopy, showing that it is capable of perturbing lipid membranes. g. bocchinfuso , a. palleschi , b. orioni , g. grande , f. formaggio , c. toniolo , y. park , k. s. hahm , l. stella univ. of rome tor vergata, dept. of chemistry, italy, dept. of chemistry, univ. of padova and cnr, italy, chosun univ., rcpm, south korea. most antimicrobial peptides exert their activity by perturbing the permeability of bacterial membranes, but the molecular details of this process are still debated. here, we compare fluorescence experiments and molecular dynamics simulations regarding the interaction of two antimicrobial peptides (pmap- and trichogin ga iv) with lipid membranes, showing that their mechanisms of bilayer perturbation are significantly different. pmap- , a cationic peptide member of the cathelicidin family, associates to the membrane close to its surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. on the other hand, trichogin ga iv, a neutral peptide member of the peptaibol family, undergoes a transition from a surface-bound state to an inserted orientation. in the first arrangement it does not cause any strong membrane perturbation, while in the second orientation it may span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. lipopolysaccharide (lps) is the main component of the outer membrane of gram negative bacteria. lps is also known as endotoxin because of its potency to induce sepsis, a serious source of mortality in many clinical cases. among lps-neutralizing agents, polymyxin b (pxb) is considered as the "gold standard" due to its high efficiency of binding and detoxification of endotoxin. in this work, we have further explored the interaction of pxb to lps from the rough mutant of salmonella minnesota (re-lps) both in the gel and in the liquid crystalline phase, using isothermal titration calorimetry (itc) and fluorescence based techniques. the effect of pxb binding on lps-membrane integrity was determined with a fluorescence quenching assay treating vesicles of lps labeled with nbd-pe with dithionite. thermodynamic parameters associated with the binding process as well as binding stoichiometry were obtained from itc experiments. finally, itc was conducted with enterococcus faecalis and salmonella typhimurium, as representative examples of a gram negative and a gram positive bacterium respectively. pressure jumps -an accessible trigger for biomolecular transformations high pressure can be used to induce many structural changes in biological systems: from triggering phase changes in model membranes to causing protein unfolding, in fact any change involving a volume reduction is promoted by pressure. as well as being broadly applicable, pressure changes can be applied very quickly both up and down in contrast to other structure change triggers such as temperature jumps. fast pressure jumps allow the trigger to be decoupled from structural changes, so with fast structure probe techniques such as time resolved x-ray diffraction, the out-of-equilibrium evolution of these systems can be monitored. despite great advantages, high pressure remains under-utilised primarily due to its technical difficulty. in response to this technology vacuum a high pressure user facility based around a pressure jump cell for small and wide angle x-ray diffraction has been commissioned at beamline i , diamond light source, uk and will be freely available to the user community. the cell is highly robust requiring virtually no user intervention during an experiment and the pressure system is computer controlled with a graphical user interface and is integrated with the beamline. pressures between and . gpa are accessible and jumps can be carried out in approximately ms at temperatures from - to • c. sample changing has been made simple and fast with a dedicated sample loading port and modular sample holders allow optimisation for a broad range of samples. the transformation of vesicle and lateral distribution of mobile membrane inclusions b. bozic institute of biophysics, faculty of medicine, university of ljubljana, lipičeva , si- ljubljana, slovenia membrane inclusions such as membrane embedded peptides or proteins exhibit curvature dependent interaction with the surrounding lipid matrix due to the mismatch between their intrinsic curvature and the local membrane curvature. this interaction causes an inhomogeneous lateral distribution of the inclusions and a corresponding adjustment of the vesicle shape. by taking into consideration that the membrane free energy includes elastic energy of the lipid bilayer and a contribution due to an inclusion-membrane interaction, the configurations of lipid vesicles with mobile inclusions have been studied theoretically. the variational problem to calculate equilibrium vesicle shapes is solved by applying a ritz method based on an expansion in spherical harmonics. in general, vesicle shapes adjust to the presence of inclusions by increasing regions with favorable curvature and decreasing regions of unfavorable curvature in such a way that the lateral distribution of inclusions becomes inhomogeneous. if the number of inclusions or the inclusion-membrane interaction exceeds a certain value, the prolate shapes become globally stable. investigating the structure of pores formed by antimicrobial peptides using epr spectroscopy m. bortolus , k.-s. hahm , a. l. maniero universita' di padova, padova, italy, chosun university, kwangju, south korea spin label electron paramagnetic resonance (epr) is a spectroscopical technique effective to study the molecular mobility of membrane components and the membranepeptide interactions, as the timescale of epr is optimally matched to the rotational motions of lipids in membranes. we applied epr to study the pore-forming mechanism of two antimicrobial peptides (amp) that create pores of different dimensions when interacting with liposomes. hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) is derived from the n-terminus of helicobacter pylori ribosomal protein l , and hpa is an hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) analogue where gln and asp at positions and were substituted by trp. we studied the interaction of the two amp with zwitterionic and negatively charged vesicles, doped with phospholipids spin labelled in the lipid head or at the c or c positions; the different phospholipids allow us to study the peptide-membrane interaction at different depths relative to the membrane surface. we studied the interaction of the amp with vesicles following the influence of amp on label mobility as a function of temperature and membrane depth. we also prepared spin-labelled dmpc/dhpc bicelles, doped with lanthanide ions (dy + /tm + ) that allow us to macroscopically orient the system using the magnetic field of the epr spectrometer. we studied the interaction of amp with the oriented bicellar system monitoring the effect of amp on the order parameter of the phase. a. chattopadhyay centre for cellular and molecular biology, hyderabad, india we addressed the organization and dynamics of the human serotonin a receptor fused to enhanced yellow fluorescent protein (serotonin a r-eyfp) expressed in cho cells. serotonin a receptors are prototypical members of the gprotein coupled receptor superfamily and represent a prime target for therapeutic actions of several anxiolytic and antidepressant drugs. interestingly, we observed significant retention in fluorescence of serotonin a receptors upon triton x- treatment of intact cells at low temperature demonstrating their detergent insolubility. we analyzed the role of cholesterol in the plasma membrane organization of the serotonin a receptor by fluorescence recovery after photobleaching (frap) measurements with varying bleach spot sizes. our results show that lateral diffusion parameters of serotonin a receptors are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin a receptors in the plasma membrane. interestingly, results from frap measurements performed under conditions of mild cytoskeletal destabilization suggest that receptor signaling is correlated with receptor mobility, in agreement with the 'mobile receptor hypothesis'. our current work is focused on exploring the oligomerization of the receptor using photobleaching anisotropy measurements and indicates the presence of constitutive oligomers of the serotonin a receptor in live cells. expression and reconstitution of connexin in pore-suspending membranes c. carnarius , s. kaufmann , m. tanaka , c. steinem institute of organic and biomolecular chemistry, university of göttingen, tammannstrasse , göttingen, germany, institute of physical chemistry, university of heidelberg, im neuenheimer feld , heidelberg, germany the intercellular communication and electronic coupling between adjacent cells of vertebrates are mediated by gap junctions. these proteins are composed of two connexon hemichannels, whereas each connexon consists of six connexin subunits. each subunit is characterized by two conserved motives: two extracellular loops and four transmembrane α-helices. in this study, we focused on cx . to obtain large amounts of this protein, we expressed cx and cx +gfp in a rather new expression system: dictyostelium discoideum. in contrast to human tissue cultures, the system allows for high cell densities up to million cells per ml and the cells can be cultivated by fermentation. cx +gfp was successfully visualized in d. discoideum by confocal laser scanning microscopy, where it was preferentially found in the plasma membrane.after the cells were harvested, plasma membranes were prepared and both proteins (cx and cx +gfp) were verified by western blot analysis. the proteins were solubilized by addition of % n-octyl-β-d-glucopyranoside and purified by ion metal chelate affinity chromatography. the activities of both proteins were confirmed by a cytochrome c assay. after the purification of both proteins, they were reconstituted in µm-sized pore-suspending membranes. in the near future, we plan to determine the mobility of cx and cx +gfp in these membranes by fluorescence recovery after photobleaching. o. cañadas, c. casals dpt. biochemistry and molecular biology i, and ciber enfermedades respiratorias, complutense university of madrid, madrid, spain inhaled bacterial lipopolysaccharide (lps) may incorporate into the lung surfactant monolayer. in this study, the effect of smooth lps (s-lps) on the surface activity of lung surfactant was evaluated. to that end we investigated the behavior of dppc films containing s-lps, with and without surfactant protein a (sp-a) in the subphase, using epifluorescence microscopy combined with a surface balance. our data show that s-lps injected into the subphase incorporated into dppc films forming mixed dppc/s-lps monolayers. cospread s-lps fluidized the dppc monolayer as demonstrated by epifluorescence images and changes in the compressibility modulus of the monolayer as a function of s-lps molar fraction (x s−lps ). the presence of low amounts of s-lps in the monolayer promoted early collapse, preventing high surface pressures to be reached. moreover, s-lps hampered the re-spreading of dppc molecules during dynamic compression at s-lps concentrations as small as x s−lps = . . such inhibitory effects could not be relieved by repeated compression-expansion cycles or by adding surfactant protein a. however, sp-a facilitated the squeeze-out of s-lps from dppc/s-lps mixed monolayers, suggesting that sp-a is an s-lps scavenger. cholesterol displaces ceramide from its tight packing with sphingomyelin in the absence of ld phase j. v. busto, j. sot, j. requejo-isidro, f. m. goñi, a. alonso unidad de biofísica (csic-upv/ehu) and departamento de bioquímica, u. país vasco (upv/ehu), spain a set of biophysical approaches have been applied to study the phase behaviour of palmitoylsphingomyelin (psm)/cholesterol (chol) model membranes upon palmitoylceramide (pcer) addition. fluorescence spectroscopy of di- -aneppdhq-stained psm/chol vesicles reveals no segregation of large liquid-ordered (l o ) microdomains. in contrast, the formation of disperse, compositionally homogeneous l o psm/chol ( : ) nanodomains over a psm gel (l β ) phase is proposed. dsc measurements show that pcer addition to vesicles with coexisting l o and l β phases (low [chol] ) induces the formation of large gel-like psm/pcer microdomains, coexisting with a l o psm/chol phase. the ∆h for the psm/pcer phase at high psm/(chol+pcer) ratio is close to that of the binary mixture in the absence of chol, supporting immiscibility, but no displacement, between chol and pcer-rich phases. on the contrary, both confocal microscopy of guvs and the dsc data coincide in showing that a rise in pcer increases the gel-like phase to a lower extent than in the pure psm/pcer mixture, revealing some cholinduced restriction. in the presence of a pure l o psm/chol phase (high [chol] ), pcer addition is unable to induce the formation of large psm/pcer microdomains. the present data support the role of chol as the key determinant in controlling its own displacement from l o domains by ceramide. hydroquinones modifying lipid membrane morphology c. di vitta , l. marzorati , v. rebbin , s. s. funari iqusp, university of são paulo, são paulo, brasil, hasylab, hamburg, germany quinones are important molecules in cells. flavina, ubiquinone and coenzyme q, coq, are associated with electron transfer. coq, having a hydrophobic tail, is soluble in the internal lipid membrane of mitochondria. their reduction leads to the equivalent hydroquinones. we synthesized novel alkylthioquinones aths, to investigate their interaction with phospholipids. one or more long hydrophobic chains attached to the quinone ring alter their hydrophobicity and electron availability. we aimed at their influence on the structure of membranes. different phs highlights the charge/polarity effect on the interaction and on the morphology of the bilayer. for that, pope and popc, lipids with different charges, but identical chains were selected. x-rays diffraction on pope/ , bath, / at different temperatures and ph, showed cubic phases coexisting with the usual phases formed by simply hydrated pope, at different phs. for investigation of the charge/polarity, we turned to the popc/ , bath system (smaller charge/polarity). despite decrease in temperature of phase transition and dimensions of the lattice, the structures were the same as in hydrated lipid, illustrating a less significant effect than on the similar lipid pope. another additive, , ath, mixed with pope showed the effect of multiple thioalky chains. no morphology change was seen, compared to pure lipid. interestingly, despite both additives differ by one thioalkyl chain only, their influence on the pope matrix is so different. cross-linking of phospholipid membranes by calcium-sensitive synaptotagmins synaptotagmins are vesicular proteins implicated in many membrane trafficking events. they are highly conserved in evolution and the mammalian family contains isoforms. we now show that the tandem c domains of several calcium-sensitive synaptotagmin isoforms tested, including drosophila synaptotagmin, rapidly cross-link phospholipid membranes. in contrast to the tandem structure, individual c domains failed to trigger membrane cross-linking in several novel assays. large-scale liposomal aggregation driven by tandem c domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic lightscattering and both confocal and negative stain electron microscopy. high-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem c domains results in a constant distance of approximately nm between the apposed membranes. our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem c domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. membrane proteins can be challenging samples to work with and as such often require the use of multiple techniques. here we present an insight into the structure of the antimicrobial peptide melittin in lipid membranes using various techniques. we explore the conformational changes of membrane-bound melittin and its interaction with model membrane lipid systems with a series of spectroscopic methods that can be used in parallel. the techniques used include linear dichroism and ft-ir for orientation information and circular dichroism for conformation information. we use dynamic light scattering for molecular sizing, fluorescence emission for information on peptide environment, thin-layer chromatography for lipid identification, analytical ultracentrifugation to identify oligomerisation state and calorimetry to investigate the thermodynamics. we observe how the physical properties of both the peptide and the membranes affect the insertion kinetics of the peptide in the membrane. phospholipid membranes dynamics: molecular dynamics vs neutron scattering v. conti nibali , m. tarek , u. wanderlingh , g. d'angelo department of physics, faculty of science, university of messina, italy, unité mixte de recherche cnrs/uhp , université henri poincaré, nancy, france collective dynamics and single-particle dynamics of hydrated multilamellar phospholipid bilayers ( , -dimyristoylsn-glycero- -phosphatidylcholine, dmpc) have been studied by means of all-atom molecular dynamics simulations. here we report results of a gel phase bilayer at k and of a liquid crystal phase bilayer at k. coherent and incoherent dynamic structure factors and meansquare displacements have been calculated from the trajectories for both the inplane and out-of-plane lipid dynamics. moreover, the results have been compared to recent quasi-elastic and inelastic neutron scattering data. optical tweezers allow trapping of particles of different types in a wide range of sizes [ ] . among these, unilamellar vesicles are of interest as they are known to be effective vectors in drug delivery and they are also studied as models for cell membranes. this work focuses on the interactions between optically confined unilamellar vesicles and their cross-linking by proteins [ ] . synaptotagmins are vesicular proteins implicated in many membrane trafficking events having an accelerating effect on the membrane fusion. calcium-sensitive synaptotagmins are thought to confer calcium sensitivity to the fusion of secretory vesicles with target membranes [ ] . the novel optical assay reported here allowed us to visualize the cross-linking of nm liposomes mediated by synaptotagmin and calcium. the use of the optical tweezers approach to investigate the function of other fusogenic proteins related to exocytosis (i.e., snare proteins) is discussed as well. antimicrobial peptides (amps) have received increasing interest as the search for new potential antibiotics has become imperative due to increasing bacterial multiresistance. acylating the natural occurring polymyxin b (pmb) significantly enhances antibacterial activity towards two representative gram-negative bacteria e. coli and k. pneumonia compared to the nonacylated pmb. the aim of the presented work was to study various biophysical parameters, such as partitioning coefficients, zeta potentials and effective charges of a selected amp, mastoparan-x (mpx) and a propanoylated (pampx) and octanoylated (oampx) analogue, respectively. fluorescence spectroscopy, isothermal titration calorimetry and ζ-potential measurements were the main techniques used for the measurements. we employed different luv model systems; a partially charged (popg/popc : ) and a neutral system (popc). for the neutral luvs there was an increase in partitioning with increasing length of the acyl chain, whereas partioning into the partially charged luvs was governed by a balance of hydrophobic and electrostatic contributions. the selectivity for the partially charged luvs over the neutral luvs was in the order pampx, mpx and oampx. the modeled effective charge for the peptide followed the same trend as the partitioning coefficient for the three peptides for the respective luv systems. does the lysosomal membrane need triglycerides? a spectroscopic study of a simple model membrane l. duelund , k. pakkanen , m. vuento , j. h. ipsen memphys -center for biomembrane physics, university of southern denmark, odense, denmark, nanoscience center, university of jyväskylä, jyväskylä, finland lysosomes are intracellular organelles in which proteins and other macromolecules are degraded. morphological and functional changes in different compartments of the endocytic pathway are connected to several diseases. a crucial step in understanding biogenesis of lysosomes and their role in disease conditions, is to characterise the properties of the lysosomal membrane. by the use of tlc we have found that lysosomes contain non-neglible amounts of triglycerides (tg). to investigate how the presence of tgs could influence the lysosomal membrane, we have investigated the properties of a mixture of popc and triolein, as a simple model for the lysosomal membrane. we found the system to form two types of popc-rich membranes. these were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. by using epr and fluorescence spectroscopy the physical properties, including order, fluidity and water penetration, of these phases were found to differ markedly despite of their almost identical composition. the results suggest that presence of tgs on lysosomal membranes could have a crucial effect in the barrier functions and thus, the integrity of the organelle. model membranes with the capacity to align in magnetic fields such as bicelles and magnetically sensitive vesicles are of high interest, since their macroscopically alignment allows for the determination of structure, dynamics and topology of molecules within the membrane [ ] . we performed p solid state nmr on a magnetically sensitive lipid possessing a large positive magnetic anisotropy introduced in form of a biphenyl unit during lipid synthesis in one of its acyl chains ( -tetradecanoyl- -( -( biphenyl) butanoyl)-snglycero- -phosphocholine (tbbpc)). the phosphorus lineshapes of tbbpc mlvs studied at various magnetic fields ( . t, . t, . t, . t), revealed a drastic change in shape upon exposure to fields > . tesla: resonances resulting from phospholipid molecules oriented perpendicular to the magnetic field decrease whereas resonances resulting from parallel oriented molecules increase. this is a sign for magnetically induced vesicle deformation from vesicle to oblate ellipsoid shape. factors influencing this drastic deformation, such as magnetic anisotropy, membrane elasticity, lipid chain length and field dependency are discussed based on existing theories [ ] . hepatitis g virus (gbv-c/hgv) is an enveloped viruses belonging to the flaviviridae family. clinical findings have suggested that in people co-infected with gbv-c/hgv and human immunodeficiency virus (hiv), delayed progression of aids has been observed ( ). the mechanism by which this virus may inhibit the progression of aids remain to be elucidated. enveloped viruses acquire their lipid membranes by budding through host cellular membranes ( ) , in this process; fusion peptides play an important role. study of the interaction of hgv-c peptides with lipid membranes could lead us useful information about the mechanism that takes place. in this work we present a study on the effects of e peptides on the fluidity of and polarizability of model membranes (dmpc and dppc liposomes) by dsc and fluorescence polarization. both techniques showed that the presence of the studied sequences in phospholipid mixtures already affected the thermotropic properties of the gel to liquid-crystalline phase transition. systems were thermodynamically charac- small angle neutron scattering (sans) studies have been performed to study the structural changes induced in membranes of vesicles prepared from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations of the anti-fungal drug, amphotericin b (amb).the vesicles, sonicated to a mean size∼ nm, were prepared using dimyristoylphosphatidylcholine(dmpc) or dmpc-cholesterol or dmpc-ergosterol mixtures -with both of the mixed systems involving mol% sterol. analysis of the sans data show that when the concentration of amb added is just above the drug's cmc (∼ µm) there is an increase in the membrane thickness of both the dmpc-chol and dmpc-erg vesicles (both cases + Å), but the thickness of the pure dmpc vesicle membranes remains the same as in the absence of amb. when amb is added at a concentration in excess of its cmc (∼ µm), the mixed-sterol vesicles show the same changes in membrane thickness as observed with the lower amb concentration, and the pure dmpc vesicles again remain unaffected. on the basis of these studies, therefore, there appears to be no difference in the structural changes induced by the insertion of amb into the model fungal cell membranes (mimicked by dmpc-erg vesicles and those resulting from its insertion into the model mammalian cell membranes (mimicked by dmpc-chol vesicles). the third transmembrane helix of bacteriorhodopsin, also known as phlip, is a unique model system for studying the interactions of a natural transmembrane domain with lipid membranes: depending on ph, the water-soluble peptide either adsorbs superficially or inserts as a transmembrane helix on addition of lipid vesicles [ ] . published values for the free energies of these processes were based on a stoichiometric model invoking two distinct sets of binding sites [ ] . however, discrepancies between data obtained from different experimental techniques and inconsistencies between experimental and expected temperature dependencies suggest that these values should be taken with caution. we therefore reassessed membrane interactions of phlip using titration calorimetry and fluorescence spectroscopy. if electrostatic effects at the membrane surface are taken into account, the data can be described quantitatively by a partition equilibrium, but not by a stoichiometric binding model. the thermodynamics of membrane partitioning differ substantially from those determined previously [ ] and draw a different picture of peptide-lipid interactions. beyond deepening our insights into the first step of the two-stage model of membrane protein folding, this also sheds light on the ability of phlip to drag cargo molecules across lipid membranes. adsorption of monolysine and polylysines at the surface of lipid membranes of varied composition was studied by two methods. both methods -the electrokinetic one, measured the surface potential of liposomes, and the intramembranouse field compensation sensitive to boundary potential of planar bilayer lipid membranes -detected the positive changes of the potentials for membranes with negatively charged component (cardiolipin, cl). neither monolysine, nor polylysines adsorbed at neutral membranes (phosphatidylcholine, pc). pentalysine show the difference between these potentials for the membranes composed from cl/pc mixtures. this difference is attributed to dipole part of boundary potential and indicates the changes in lipid packing. polylysines show high affinity to the membrane and saturation with plateau. these saturation levels correspond to surface charge densities . and . c/m for oligomers with and units, and . c/m for polymers with and units. these values do not depend on the ionic strength of background electrolyte but proportional to the content of negatively charged components in the lipid bilayer. the polylysine layer at the mica surface was studied by atomic-force microscope (afm) technique. it was shown that pentalysine molecules cover the surface by the layer of . nm thickness and polylysines of high molecular weight by the layer up to nm. effects of lysolipids on the mechanical stability of lipid membranes j. r. henriksen , l. feldborg , j. h. ipsen , t. l. andresen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark., university of southern denmark, department of physics and chemistry, memphys center, campusvej , odense m, denmark lysolipids (lpcs) and fatty acids (fas) are the natural products formed by the hydrolysis of phospholipids. lpcs and fas form micelles in solution and thus act as detergents in the presence of lipid membranes. in the present study we investigate the detergent strength of a homologous series of lysolipids (lpcs) on popc lipid membranes by use of isothermal titration calorimetry (itc) and vesicle fluctuation analysis (vfa). the membrane partition coefficient (k) and critical micelle concentration (cmc) are determined and found to obey an inverse proportionality relation (cmc x k ∼ constant). the partition coefficient and critical micelle concentration are used for the analysis of lpc's effect on the membrane bending rigidity. the dependence of the bending rigidity on the lpc membrane coverage has been analyzed in terms a phenomenological model based on continuum elastic theory which yield information about the curvature inducing properties of the lpc molecule. the results reveal: (i) an increase in the partition coefficient with lpc acyl-chain length and (ii) the degree in acyl chain mismatch between lpc and popc determines the magnitude of the membrane mechanical perturbation per lpc molecule on the membrane. patch clamp electrophysiology remains the gold-standard for ion channel research because of the information richness of the data produced. automated planar patch clamp devices, with their higher throughput and high data information content, have made the technique accessible to a wider audience. nanion technologies offers two planar patch clamp workstations combining higher throughput with high data quality. the port-a-patch records from a single cell at a time and the patchliner from up to cells simultaneously with high success rates (typically - %). both, the port-a-patch and the patchliner are bench top patch clamp rigs which uses a planar borosilicate glass chip for obtaining a giga-seal for electrophysiological recordings. suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. both workstations have been successfully used for whole cell, perforated patch, and cell attached recordings as well as for guv-bilayer recordings. due to the versatility of nanion`s products it is possible to study a wide variety of ion channels including nav, kv, cav, ligandgated, herg or reconstituted proteins such as ksca, cx , ompc. special features unique to nanion products, including but not limited to internal solution exchange and temperature control, expand the experimental possibilities. waves on lipid monolayers j. griesbauer, s. boessinger, a. wixforth, s. f. matthias univiversity of augsburg, experimental physics i, biological physics group, augsburg, germany " for the sake of illustration we shall try to provide a physical basis for the equations, but must emphasize that the interpretation given is unlikely to provide a correct picture of the membrane." [ hodgkin&huxley, ] to explain the occurrence of reversible heat production during nerve pulse propagation it has been suggested by multiple authors [wilke,kaufmann,heimburg] that travelling sound waves and not ion channels may offer a better explanation than the hodgkin&huxley theory. therefore, if sound waves are an essential feature of the nerve membrane they should also appear on lipid monolayers where ion conductivity is evidently absent. here we demonstrate that sound waves can be excited on a lipid monolayer by using a set of planar electrodes incorporated into the monolayer and driven by an alternating voltage. not only do our results indicate propagating waves on lipid monolayers in accordance with their thermodynamic predictions, but importantly, no significant attenuation is detected proposing an adiabatic phenomenon. in order to provide evidence that our physical explanation provides a correct picture of the membrane, direct detection of the waves was done, whereas a clear transduction of signals was shown. finally, the impact of toxins, neuropharmaca and anaesthetics needs to be integrated in our physical picture of the nerve membranes, what can easily be done now and could deliver a new approach for understanding their physical mechanism. our earlier studies showed that organometallic compounds (otc) in the presence of uvc radiation enhanced the degree of phosphatidylcholine (pc) liposome oxidation, whereas quercetin effectively protected the membrane. the present investigation is concerned with the effect of uvb and otc (chlorides of diphenyl-and dibutyltin, triphenyl-and tributyltin), both in combined action and separately, on oxidation of erythrocyte proteins, pc liposome and albumin. the degree of oxidation of proteins and liposomes induced by otc and radiation, also in the presence of selected antioxidants (trolox, quercetin) was determined on the basis of changes in the number of sulfhydryl groups, carbonyl groups and malone dialdehyde, respectively. the studies indicate that uvb induces pc liposome and erythrocyte oxidation, whereas in the case of albumin it causes both an increase in the number of c=o groups and free sh groups (most probably, braking sulphonic bridges). otc compounds interact with membrane biomolecules both as weak pro-and antioxidants, also in combined action (uvb plus otc). the prooxidative effects are markedly diminished by application of antioxidants. the action of quercetin results from its ability to incorporate into membranes and formation of complexes with otc (liposome>erythrocyte>albumine). this work was supported by grant n n . a phospholipase c/ sphingomyelinase from pseudomonas aeruginosa has been assayed on giant unilamellar vesicles (guv) consisting of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol, at equimolar ratios. the enzyme activity modifies the chemical composition, thus the physical properties of the bilayer, and conversely the latter influence the enzyme activity. biochemical assays of enzyme activity, together with confocal fluorescence microscopy examination of guv provide novel information about the system. the original lipid composition in the absence of enzyme gives rise to lateral phase separation of liquid-ordered and liquid-disordered domains in the guv. the two enzyme end-products, diacylglycerol and ceramide, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase. morphological examination of individual guv shows that the enzyme binds preferentially the more fluid (or more disordered) domains, and that, in most cases, it causes the fluidification (disordering) of the other domains.. cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. to this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures ( - mol% cholesterol) have been measured and compared with the original mixture before lipid hydration. there is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the bilayers is much lower than expected. the maximum solubility of chol in bilayers containing saturated pc or pc with less than four unsaturations is - mol%, while in polyunsaturated pc, e. g. dic : , and in sphingomyelin the maximum chol contents is mol%. a quantitative analysis of the vesicles is thus required before any experimental study is undertaken. membrane fusion assay based on poresuspending lipid bilayers i. höfer, c. steinem institute of organic and biomolecular chemistry, university of göttingen, germany membrane fusion has attracted significant interest due to its biological relevance. thus in recent years a great variety of fusion assays has been developed. since the process of membrane fusion is not yet fully understood, our aim is to develop a new fusion assay based on pore-spanning membranes, which have been proven mechanically stable, highly insulating and rather tension free. the application of these so-called micro-blms should allow simultaneous monitoring of lipid mixing, content release as well as electrical readouts. furthermore both membrane sides can be addressed individually to apply transmembrane potential or fusion modulating compounds. first results show that the micro-blms provide the opportunity to investigate lipid bilayer fusion by means of fluorescence microscopy. the formation of porespanning membranes is achieved by the painting technique of dphpc doped with oregon green dhpe dissolved in ndecane. the addition of large unilamellar vesicles doped with texas red dhpe allows direct observation of single fusion events. lipid mixing during fusion leads to a decrease of the donor fluorescence (oregon green) and an increase in acceptor fluorescence intensity (texas red) in the plane of the planar bilayer due to fret. in future work simultaneous monitoring of lipid-mixing and content release combined with electrical measurements are planned to gain further insight into different intermediate steps of membrane fusion. the kinetics of membrane-peptide folding and orientation m. r. hicks department of chemistry, university of warwick, uk. understanding interactions of peptides with lipid membranes is essential if we are to be able to design new and better antibiotic peptides. there are different steps in these interactions and following the kinetics of these processes requires that we combine the information from different biophysical techniques. here we present data on the kinetics of peptidemembrane interactions using circular dichroism to report on conformation and linear dichroism to report on orientation of the peptides in/on membranes. these are combined with other techniques such as dynamic light scattering and fluorescence spectroscopy to elucidate the mechanisms of action of the peptides. one of the most important aspects of membrane-active peptide design is that of specificity. we investigate specificity for different types of membranes by using libraries of lipids with different properties of charge, chain saturation and curvature stress. in this way one can test for effects of e.g. negatively charged head groups found in bacterial membranes or cholesterol found in animal and human membranes. using these approaches it is proposed that we will be able to modify peptide sequences, test what part of the kinetic processes are affected and subsequently use this information to design new and better antibiotics. k. kubica, h. misiak wroclaw university of technology, wroclaw, poland reversible membrane electroporation, that can be observed as a effect of electric field influence on biological membranes, finds application in cell delivery of biologically active compounds. the membrane susceptibility for creation of stable pores, depends on electric field parameters applied to the membrane as well as on the membrane environment (ionic strength, ph, temperature) and membrane molecular composition. we have already shown that the changes of van der waals lipid chain interaction effect the process of pore formation. there are known factors which increase or decrease the membrane respond to electric field. because pores appear in lipid membrane matrix it make sense to study this phenomenon using model planar lipid membranes. as lipids are very receptive on oxidizing factors we decided to study the relation between lipid oxidation and membrane reaction on electric field. with the use of four electrode galvanostat we are examining the influence of factors initiating lipid oxidation (uv, fe + ) and the efficiency of some antioxidant compounds of lipid protection on electric properties of lipid membrane. the product of lipid oxidation is determined by characteristic reaction with tiobarbituric acid. we want to determine the role of polyunsaturated fatty acids on membrane properties also. results of our work will be useful in optimization of medicine distribution, aided by electric field, into cells. budding of giant phospholipid vesicles induced by β -glycoprotein i j. kovacic, b. bozic, s. svetina institute of biophysics, faculty of medicine, university of ljubljana, ljubljana, slovenia β -glycoprotein i (β gpi) is classified among amphitropic proteins: in its inactive form it is dissolved in plasma and in its active form it is bound to membrane. in comparison to other amphitropic proteins it exhibits a distinct membrane interaction behavior. a patch of positively charged amino acid residues contacts anionic phospholipids via electrostatic interactions, and a hydrophobic loop anchores the protein into the outer leaflet of the membrane via hydrophobic interactions. the binding constant depends on physical properties of the membrane such as membrane lipid composition, surface potential, lipid packing density and curvature. the binding of an amphitropic protein alters the membrane's spontaneous curvature as well as the difference between the equilibrium areas of membrane leaflets. theoretical model has been developed to predict vesicles shape transformations realized by the formation of buds. it was shown that the effects are stronger for flaccid vesicles which were therefore chosen for our experimental investigations. vesicles were composed of % pops and % popc, and flaccidity was achieved by an osmotic adjustment. the solution containing β gpi was subsequently injected to a chamber with flaccid vesicles by a syringe pump. observation took place under a phase-contrast microscope. experiments showed a concentration dependent occurrence of buds. their number and size were related to the degree of vesicle flaccidness. the interplay between detergent cohesion and peptide adsorption on the structure and dynamics of a glycophorin a tm-detergent complex j. khao, j.-p. duneau, j. n. sturgis lism, cnrs -aix marseille university, marseille, france in spite of the major interest of membrane proteins at functional, genomic and therapeutic scales, their biochemical and structural study remain challenging as they require, solubilization in detergent micelles. the complexity of this task arises from the structural dependence of membrane proteins on their anisotropic environment and in particular by a delicate balance between different physico-chemical properties. in order to study these properties in a small protein detergent complex, we have used molecular dynamics simulations on the transmembrane part of glycophorin a (gpatm) solubilized in micelles of the detergent di-hexanoyl-phosphatidylcholine(d pc). we show that the molecular aggregates organizes to give distinct populations of detergent molecules. those molecules which loosely interact with the peptide are preferentially involved in highly cohesive inter-detergent interactions that impose a global bilayer structure to the micelle. interaction profiles of other detergents with gpatm depend upon the nature of residues along the surface of the peptide. this topology dependence leads to different modes and strength of interactions that ultimately constrain the orientation of the micelles around the peptide. this simple model illustrates how differential detergent selectivity for faces and strong constraints coming from purely environmental features could influence transmembrane helix packing, membrane protein structure and assembly. in this paper we present a study of a new family of bolaamphiphiles. these amphiphiles are unsymmetrical bolalipids having one sugar polar head and the second glycine betaine polar head. they are potentially useful for pharmaceutical or cosmetics applications as vectors for drugs. therefore it is important to investigate their self-assembled properties. the chemical variations that we introduced in this new family concern the length of the main chain that connects two polar heads as well as the length of the side chain placed on the position of the sugar moiety. another variation concerns the introduction of a diacetylenic unit into the main chain in order to rigidify it. we have performed the saxs (small angle x-ray scattering) measurements on the dehydrated compounds as a function of temperature and observed the lamellar liquid crystalline structures. we also measured the saxs spectra of aqueous solutions of these compounds that have shown lamellar l α structure in all cases. these measurements are compared with polarised optical microscopy measurements that confirmed our interpretation. formation of membrane tubular structures induced by phase separation in giant vesicles y. li, r. lipowsky, r. dimova max planck institute of colloids and interfaces, science park golm, potsdam, germany tubular membrane structures (also known as tethers) exist widely in eukaryotes. abundant work has been done on tube extrusion from cells and model membranes under the application of external forces. we present a novel system allowing tube formation in the absence of external forces. aqueous solutions of two chemically dissimilar polymers, polyethylene glycol (peg) and dextran are encapsulated in giant vesicles, a cell-size model system. the exposure of vesicles to hypertonic solutions induces phase separation of the internal aqueous polymer solution. the excess membrane area created by this vesicle deflation, engages in the formation of tubular structures. membrane tube formation and phase separation are coupled processes. hydrodynamic flows and changes in the membrane spontaneous curvature during phase separation might be the driving forces for tube formation. the tubes are rather stable: without external perturbation, they can exist for several days. they prefer to be located in the peg-rich phase at low polymer concentration. at high concentration, they are absorbed at the interface of the liquid phases to lower the surface energy of the system by decreasing the contact area between the liquid phases. the membrane tubes can be retracted back to the vesicle surface by increasing the membrane tension via vesicle aspiration. membrane tubes, which can form and be retracted easily, might be relevant to lipid storage in cells. insight into the antimicrobial mechanism of a de novo auto-assembling peptide b. legrand , m. laurencin , e. duval , c. zatylny , j. henry , m. baudy-floc´h , a. bondon rmn-ilp, umr cnrs -univ. de rennes , france, icmv, umr cnrs -univ. de rennes , france, pemm, umr ifremer -univ. de caen, france a short ( residues) de novo antimicrobial peptide (k ) composed of a cationic polar head and a hydrophobic tail was studied. it exhibits a broad spectrum of antimicrobial activity on bacteria. no haemolytic activity or cytotoxicity on eukaryotic cells are observed at mic. when bacteria are lysed by the k , spherical objects are observed on the sem micrograph. k was structurally studied using various membrane mimetic media such as different micelles and small unilamellar vesicles (suv) of different composition. cd revealed that k adopt various structures (random, β-turn, α-helix) in the presence or the absence of detergents or phospholipids. nmr structures confirmed the α-helical structure of k hydrophobic tail in presence of sds. k self-assembles at high concentration as observed by sem and dls. we suggest that k may act as a surfactant building mixed microsomes composed of peptide and lipids. this destabilization mechanism of the bacterial membrane support the "detergent like model" previously described in the literature. oxidative stress and the membrane dipole potential; modulation with tocopherol s. le-nen-davey , b. m. davis , j. l. richens , k. a. vere , m. w. tilley , p. g. petrov , c. p. winlove , p. o'shea biomedical physics group, university of exeter, uk, cell biophysics group, university of nottingham, uk tocopherol, a component of vitamin e well know for its antioxidant properties, has recently been thought also to influence the structure of cellular membranes. tocopherol treatment reduces hyperglycaemia induced oxidative stress and the associated endothelial dysfunction which is a precursor to the vascular complications of diabetes. tocopherol and insulin interactions are also modified by hyperglycaemia. it is unclear whether these clinically important effects arise from the anti-oxidant and/or structural properties of tocopherol. we have therefore measured the dipole and surface potentials of phosphatidylcholine vesicles containing different amounts of cholesterol, ketocholesterol and tocopherol. we have also investigated the effects of hyperglycaemia, ketocholesterol and tocopherol, alone and in combination, on microdomain formation and interactions of insulin within the membranes of cultured endothelial cells. both sets of experiments indicate that tocopherol causes significant modifications of the membrane dipole and surface potentials. the physiological significance of these changes will be discussed. t. d. lazzara , a. janshoff , c. steinem georg-august university göttingen, institute for organic and biomolecular chemistry, tammannstr. , , germany, georg-august university göttingen, institute for physical chemistry, tammannstr. , , germany cell penetrating peptides (cpp) have been shown to penetrate cellular membranes. they have been of interest for their ability to translocate not only themselves through cellular membranes, but also carry along with them, cargo as large as iron nanoparticles. the exact entry mechanism remains unclear, but has been shown to vary with peptide sequence. their translocation properties have been demonstrated through different experiments involving vesicles, cells and living animals. we plan on using nano-black lipid membranes (nano-blm), which span the pores of nanoporous materials as a model system to study the interaction between cpp and lipid membranes. the nanopores can be used as cellular containers whose interior surface can be functionalized with receptors for biotin-streptavidin recognition. we hope that this system will provide greater control over experimental variables, such as the type of cpp and lipid used, as well as provide kinetic data that can be used to evaluate cpp activity and the kinetics of cargo transport. ethanol induces phospholipid acyl chain interdigitation. while much is known, important issues remain unclear, such as the role of lipid domains. the main purpose of this study was to follow, in real time, the changes induced by ethanol on supported lipid bilayers with nano to microdomains by in situ atomic force microscopy. to this goal, a pure lipid in the fluid phase (dopc), a pure lipid in the gel phase (dppc), binary phospholipid mixtures with gel/fluid phase coexistence, and cholesterol-containing, raft -forming mixtures (dopc/dppc/cholesterol and dopc/sphingomyelin/cholesterol) were investigated. from the height differences observed upon ethanol addition to pure lipids (dppc and dopc), and to dopc/dppc mixtures, it is shown that in the binary system the interdigitation of the fluid phase occurs prior to the gel phase. however, for the lipid rafts mixtures the simultaneous interdigitation of both raft and non raft portions of the membrane is observed both in mica and silicon substrates. for all compositions studied, domain formation or rearrangement accompanied by lipid bilayer expansion occurs as a consequence of interdigitation. these results show the ability of ethanol to influence the bilayer properties in different ways according to membrane composition. ethanol may exert its biological effects by reducing bilayer thickness, and also by changing membrane proteins conformation and lateral distribution, as a consequence of the altered properties of the lipid bilayer. interactions between non-steroidal anti-inflammatory drugs and a pc/cholesterol bilayer m. markiewicz , t. librowski , p. serda , m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university " department of pharmacodynamics, medical college, jagiellonian university " regional laboratory, jagiellonian university, krakow, poland. the non-steroidal anti-inflammatory drugs (nsaids) are among the most frequently prescribed and used drugs [ ] . there are several side effects connected with frequent use of nsaid, mainly gastrointestinal ulcers and bleedings. a plausible molecular mechanism of these effects are direct interactions of nsaids with gastric phospholipids [ , ] . the influence of three well-known non-steroidal antiinflammatory drugs with a diverse gastric toxicity (aspirin, ketoprofen and piroxicam) and three newly-synthesized xanthone derivatives (belonging to the nsaids) on the structure and dynamics of lipid bilayers was studied using small angle x-ray scattering and molecular dynamics simulations. the results showed some correlation between nsaid toxicity and its binding to the lipid polar groups. this binding increases membrane fluidity by reducing its density due to an increased membrane surface area. a reduced lipid packing in the membrane most likely increases gastric mucosa permeability, which can result in a decreased resistance of the gastric mucosa to luminal acid. we studied the partition of the anionic amphiphile pyrenesulfonate (psa) into mlv and luv (produced by extrusion), composed by zwitterionic popc and zwitterionic/anionic mixtures with pops (until mol %). psa is an anionic amphiphile that mimics several xenobiotics (e.g. pharmaceuticals, pesticides) and endogenous substrates that interact with biological membranes. we found increasing b. lorenz, s. schuy, a. janshoff georg august university, göttingen, germany cell-cell and cell-virus interactions are ubiquitous in living organisms. the analysis of the forces acting between two cells or a cell and a virus gives insight into details of these interaction processes such as their stochastics, cooperativity, reversibility, and energy landscape. using colloidal probe microscopy in conjunction with solid-supported lipid bilayer techniques, we mimic the contact between two membranous compounds displaying sponge glycolipids or viral peptides on their surfaces. after spreading functionalized lipid bilayers on both -a colloidal probe and a silicon wafer surface -the molecular interactions are quantified by means of force-distance curves. by probing the dynamic interaction strength between the viral peptides n and c we aim for a deeper understanding of the role of these peptides in the complex process of the formation of the prehairpin intermediate as the key step in retroviral fusion. as far as the cellular interaction of marine sponge cells is concerned, we intend to investigate the strength, specificity, and the ca + dependency of the self-recognition between sponge glycans. the systems advantages are the flexibility of the membrane composition and the control over the distribution of receptor molecules in the membranes. since adhesion in biological systems relies heavily on cluster formation within the biomembrane, we plan to mimic the clustering by "printing" interaction domains and comparing the results to homogeneous samples. development of video-rate imaging microscope using laurdan and its applications to lipid raft t. ohba, k.-i. muto, t. kiuchi, k. ohki department of physics, graduate school of science, aobaku, sendai, japan - , ohki@bio.phys.tohoku.ac.jp various biological functions are located on cell membranes, and the biomembranes maintain heterogeneity as microdomain even in its dynamic structure. existence of such domain was reported as phase-separated 'rafts' in a cell. and the bio-functions of membrane protein are affected by physical property of its surrounding lipid bilayers. laurdan is a useful fluorescence dye to monitor membrane fluidity, and we have developed an instrument to image spatial and temporal change in membrane fluidity by use of laurdan. in order to examine role of 'micro-domain' in biomembrane, we applied this imaging instrument to a giant liposome, cho cells and pc d cells. fluorescence microscope was equipped with a home-build dual-view optical unit. microscopic image of membranes stained with laurdan is separated into an image at nm and an image at nm by the combination of monochromatic filters and dichroic mirrors. the each image is focused on an image plane of a ccd camera side by side. and generalized polarization (g.p.) image is calculated from those images by personal computer according to the definition of g.p. the g.p. imaging at video rate was applied to a giant liposome of composed of dmpc and dmpe in order to observe phase separation. it was also examined that specific interaction of sphingomyelin and cholesterol in living cho cells and neuritis protrusion from raft region of pc d cells stimulated by neuron growth factor. current fluctuations in biological lipid membranes from human cell lines s. nuschele, a. wixforth, m. f. schneider university of augsburg, experimental physics , univer-sitätsstrasse , d - augsburg, germany lipid membranes can undergo phase transitions at physiological temperatures. not only do single lipid component membranes show melting behaviour but also biological lipid membranes from eukaryotes as well as from prokaryotes. performing calorimetric and monolayer studies we are able to detect phase transitions in extracted membranes from different human cell lines (e.g. keratinocytes and melanoma cells). close to and in the melting transition regime we measured distinct channel like current fluctuations. the opening times of these current fluctuations can be predicted based on the lateral compressibility measured from the monolayer isotherm and agree well with the experimental data [*]. the applied method of extraction excludes the presence of functional proteins in the membrane rendering the lipid bilayer as the source of the observed current fluctuations. the molecules are composed of single hydrophobic tail and two hydrophilic aldonamide-type groupings (gluconyl c -dga or lactobionyl c -dla) linked by the propylene chain at the nitrogen atom. the micellization processes of c -dga and c -dla were studied by means of itc. the critical micelle concentrations, the enthalpies (∆h m ) and the entropies (∆s m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (∆g m (hy)) were calculated. qspr analysis was also used to predict cmc of studied compounds. the interactions of c -dga and c -dla with model membranes (dppc and dppc/chol. bilayers) were studied by means of dsc. using quantum computations some basic molecular properties were calculated. the conformational space was explored using molecular mechanics. obtained results were compared with those for analogical compounds with single head groups, e.g. with also synthesised by us n-alkanoyl-nmethyllactitolamines (c n mela) and common sugar-based surfactants c gluc and mega- . enfuvirtide (t- ) was the first hiv- fusion inhibitor peptide approved for clinical use. t- is an inhibitor still under evaluation. previous studies, based on tryptophan intrinsic fluorescence, showed that the peptides interact differentially with membrane model systems (luv) with different lipid compositions. studies with human blood cells were necessary to further establish the role of membranes on these peptides mode of action. an experimental strategy was applied based on the membrane dipole potential, as measured by the fluorescent probe di- -anepps. human erythrocytes and peripheral blood mononuclear cells (pbmc) were successfully labeled. for both systems, a fusion inhibitor concentration-dependent decrease on di- -anepps fluorescence excitation ratio (a measure of the spectral shift and dipole potential) was observed. the quantitative analysis of these variations indicated that t- has an approximately ten-fold higher affinity towards erythrocyte and pbmc, when compared with enfuvirtide. this is in agreement with the previously known adsorption of t- on cholesterol-rich membrane domains and with its higher partition constants. hiv associates with erythrocytes in vivo, which can constitute a route to deliver peptide to the viral membranes (also rich in cholesterol). lymphocyte membranes can concentrate and accelerate the drug interactions with its molecular target, gp in its exposed conformation. oxidation of low density lipoprotein (ldl) is known to be a key step in atherogenesis, leading to inflammation, proliferation and apoptosis of cells of the arterial wall. these effects are largely exerted by oxidatively fragmented phospholipids, which are highly exchangeable between cells, tissues and lipoproteins. in particular, pgpc has been identified in minimally modified ldl and has been reported to elicit a wide range of pathophysiological responses in vascular cells, e.g. the activation of apoptotic signaling pathways. we investigated here the behavior of the fluorescent oxidized pgpe-alexa compared to dhpe-bodipy in artificial supported lipid bilayers with different cholesterol contents. the two labeled lipids differ in the type of membrane insertion: while dhpe-bodipy is anchored to the membrane via two fatty acids, pgpe is incorporated with only one fatty acid. the second chain is an acyl fragment in sn- position, which represents the oxidation product of an unsaturated acyl chain. with increasing cholesterol content we observed a decrease in the diffusion coefficient for both lipids. interestingly, the diffusion of the oxidized lipid was reduced in a higher degree compared to that of the non-oxidized lipid. the calculated ratios of the diffusion constants of pgpe-alexa and dhpe-bodipy suggest a different type of interaction with cholesterol. membrane proteins account for a third of all proteins encoded for by the human genome, and play a vital role in a number of cellular processes. few membrane protein structures have been determined to date in comparison to soluble proteins. this discrepancy is due to experimental difficulties in preparing membrane protein samples for structural analysis. traditional techniques for studying membrane proteins by x-ray crystallography or solution nmr use detergent solubilised proteins which can differ from their native confirmations. solid-state nmr allows the study of transmembrane proteins in lipid bilayers, representing a more native like environment in which to obtain biologically relevant structural information. we have been working on the development of reliable methods for reconstitution of transmembrane peptide into liposomes using glycophorin a as a model tm protein. using reconstitution methods based on the removal of detergent by bio-beads, we have used electron microscopy to screen the ideal conditions for insertion of gpa into liposomes in preparation for mas nmr experiments. em has allowed us to identify conditions favourable for insertion of peptide into lipid vesicles and those that result in aggregation. in order to confirm the secondary structure and insertion of gpa into lipid vesicles, techniques such as cd, ocd, ftir and dls have been used to provide quantitative information in addition to the visual results from em. nonesterified fatty acids regulate a broad spectrum of metabolic activities and are involved in many physiological, pathological and/or pharmacological processes in living cells. they spontaneously transfer between donors and acceptors such as fatty acid binding proteins and lipid membranes. we focus on protein-lipid dispersions of human serum albumin (hsa) and sterically stabilized liposomes (ssl) composed of dppc and appropriate amount of peg: -dppe in which stearic acids (sa) are inserted either in the protein or in the ssl. exploiting the fact that hsa has a single tryptophan residue and that the intrinsic trp-fluorescence emission signal is quenced by the presence of sa, the kinetics of sa transport between hsa and peg: -grafted dppc membranes is studied by means of fluorescence. it is found that the transfer of sa between hsa and ssl is a first-order process and the kinetics of transfer depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers), and on the grafting density of the peg-lipids at the protein/lipid interface. indeed, in the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer and it is faster for the passage from dppc bilayers to hsa. the presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer-chains, especially for lipid membranes in the fluid phase. a. orth , w. römer , l. johannes , c. steinem institute for organic and biomolecular chemistry, university of goettingen, germany, laboratoire trafic et signalisation, institut curie, paris, france shiga toxin (stx) from shigella dysenteriae is an ab -class bacterial toxin. infections with stx lead to the haemolyticuraemic syndrome, which is known to be a major cause for renal failure at an early age. the interaction of the homopentameric b-subunit (stxb), which is responsible for binding and intracellular transport of the holotoxin, with its cellular receptor, the glycosphingolipid gb , is the first step for endocytosis of the toxin. one b-subunit can bind up to gb -molecules to form stxb-gb -clusters causing negative curvature of a membrane. the binding of stxb to giant unilamellar vesicles, composed of dopc, cholesterol and gb induces tubular membrane invaginations, which were also found in experiments with energy-depleted hela-cells. in recent studies, protein and lipid reorganization processes after attaching stxb to solid supported membranes, composed of dopc/sphingomyelin/cholesterol/gb have been investigated. the compaction of gb -molecules led to an additional stxb-gb -enriched phase, which was also observed in lipid monolayers at the air/water interface. in this study, the influence of stxb on model membranes will be investigated combining the advantages of free-standing lipid membranes with those of ssms. the impact of stxb on pore-suspending membranes will be followed by confocal laser scanning mi croscopy and atomic force microscopy. a. robaszkiewicz , c. spickett , p. sicinska , g. bartosz , m. soszynski department of molecular biophysics, university of lodz, lodz, poland, strathclyde institute of pharmacy and biomedical sciences, glasgow, u.k. hypochlorite generated in vivo under pathological conditions is a known oxidant, able to initiate lipid peroxidation process, which affects the stability of biological membranes. the aim of this study was the analysis of the products formed during the reaction of hypochlorite with phosphatidylcholines containing unsuturated fatty acid residues ( -stearoyl- -oleoyl-, -stearoyl- -linoleoyl-, stearoyl- -arachidonylphosphatidylcholine) and their effects on the human erythrocytes. using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in the decrease of the rotational correlation time and rotational motion freedom of liposomes estimated by epr using spin probes ( -and doxylstearic acid). unilamellar chlorohydrin liposomes had lower diffusion coefficient for calcein than liposomes made of parent lipids. flow cytometry demonstrated fast incorporation of uni-and multilamellar chlorohydrin liposomes labeled with nbd-pe into erythrocytes. this effect was accompanied by the formation of the erythrocyte subpopulations of higher volume, decrease of the rate of fluorescein diacetate hydrolysis, estimated by flow cytometry, and increase of affinity and maximal velocity of the membrane enzyme acetylcholinesterase. extensive bilayer perforation coupled with the phase transition region of an anionic phospholipid k. a. riske , l. q. amaral , m. t. lamy depto. biofisica, universidade federal de sao paulo, sao paulo, brazil, instituto de fisica, universidade de sao paulo, sao paulo, brazil at low ionic strength dimyristoylphosphatidylglycerol (dmpg) exhibits a broad phase transition region characterized by several superimposed calorimetric peaks. peculiar properties, such as sample transparency, are observed only in the transition region. we use differential scanning calorimetry, turbidity and optical microscopy to study the narrowing of the transition region with the increase of ionic strength. upon addition of salt, the temperature extension of the transition region is reduced and the number of calorimetric peaks decreases until a single cooperative event is observed in the presence of mm nacl. the transition region is always coupled with a decrease in turbidity, but a transparent region is detected within the melting process only in the presence of up to mm nacl. optical microscopy of giant vesicles shows that bilayers first rupture when the transition region is reached and subsequently lose optical contrast. fluorescence microscopy reveals a blurry image in the transparent region, suggesting a different lipid self-assembly. overall sample turbidity can be related to the bilayer optical contrast. our observations are discussed in terms of the bilayer being perforated along the transition region. in the transparent region the perforation is extensive and the bilayer completely loses the optical contrast. financial support: fapesp. label-free imaging of biological membranes using surface imaging techniques j. l. richens, k.-a. vere, p. o'shea cell biophysics group, university of nottingham, nottingham, uk surface plasmon resonance (spr) is a detection technique traditionally used for specific protein detection, which is now exploited routinely as a generic label-free sensor. spr responds to changes on a metal surface conditioned to sense the binding of analytes. thus, the composition of the external medium and metal surface will be fundamental to the signal output. here we use a model phospholipid membrane system to investigate the effect of altered buffer and surface compositions on spr signals. comparisons are undertaken between continuous gold surfaces and gold nanoparticles. we demonstrate that surface electrostatics and the salt composition and molarity of a buffer all have significant impacts on spr output. the association of respiratory syncytial virus matrix protein with membrane microdomains the association of the matrix protein from respiratory syncytical virus with membranes has been characterised by tensiometry, brewster angle microscopy, and atomic force microscopy following deposition of langmuir monolayers onto modified silica substrates. association of the protein with monolayers containing phosphocholines and cholesterol leads to the formation of materials with new properties that differ from those of either of the pure components. the behaviour of the protein in monolayers rich in cholesterol and sphingomyelin exhibits a significant concentration dependence. at low concentrations, the protein exhibits a simple partioning behaviour. at higher concentrations, lipids are extruded from the monolayer below a critical surface area. these findings are discussed in relation to the recently published structure of the protein (pnas, , , - ) , the documented formation of viral filaments during key stages of the infection cycle and the isolation of the protein from detergent-resistant membrane fractions. structure and dynamics of closed melting membranes v. m. rondelli , s. santorsola , p. brocca , e. del favero , l. cantù , m. zimbone dep. of chemistry, biochemistry and biotechnologies for medicine, university of milan, segrate, italy., dep. of physics, university of catania, catania, italy. we studied the dynamical and structural properties of large unilamellar vesicles (≈ nm luvs) of phospholipids (dmpc, dc pc and dmpc : dc pc = : molar mixture) in the temperature range around the chain-melting transition, ≈ • c wide, with . • c resolution and . • c accuracy. small-angle (saxs) and wide-angle x-ray scattering (waxs) measurements show that across the transition the vesicle behaves as an 'evolving membrane', passing through several different states, each of them being characterized by different proportions of coexisting fluid-and gelchains molecules. noteworthy, no kinetics has been detected. on the same samples, a unique and very sensitive laser light scattering technique allows to determine the characteristic times of thermally induced shape fluctuations, connected to the elastic properties of the membranes. results indicate a clear softening of the membranes in correspondence to the chain-melting transition, as indicated by a manifold increase of the corresponding fluctuation characteristic time. meanwhile the overall size of the vesicle is not sensibly changed. this softening is likely to be due to the presence of structural defects, eventually driving to local morphological modifications. thermodynamics characterization of isolated lung surfactant assembled as lamellar bodies e. x. rodriguez , r. alvarez , r. barrio , c. irles , a. ortega biochemistry depto., school of medicine, inst. of physics unam, nat. inst. of perinatology, mexico city, mexico lungs are a large extension of ∼ m of one layer cells, which is structured as compacted sacs called alveoli, where lung function takes place: the gas exchange. alveolar endotelium is formed by two kinds of cells: neumocyte type i (nti), which covers ∼ % of the alveolar surface where gas exchange occurs and, ntii where lung surfactant (ls) is produced. ls are a mixture that lay over a water film in the luminal surface of the alveoli and it is thought to decrease the surface tension to avoid alveolar collapse during expiration. ls are made of phospholipids and proteins, which are determinant for the structure of ls under particular conditions of pressure and temperature. ls inside the cell is packed in organelles called lamellar bodies (lb), and is released to the water/air interphase in other conformation. in the present study we isolate lb from pig's lung and study lb thermodynamic characteristics by microcalorimetry in the presence and in the absence of structural proteins. phase transition profile of lb with and without proteins is basically the same; while ls in the alveolar lumen have a higher transition temperature (tm). mayor changes in tm are observed between lb and ls from alveolar lumen. although ls lipid composition in and outside the cell is assumed to be the same, the ground for the differences in tm under these two conditions is unknown. m. rodrigues , g. rádis-baptista , b. g. de la torre , m. castanho , d. andreu , n. c. santos instituto de medicina molecular, portugal, pompeu fabra university, spain, federal university of cearà, brasil nucleolar-targeting peptides (nrtps) have been recently designed by structural dissection of crotamine, a toxin from the venom of a south america rattlesnake (radis-baptista et al. . j med chem : ) . at µm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work is to decipher the molecular mechanism for the translocation of nrtps into cells. quantification of partition into membranes was carried out, based on intrinsic tyrosine fluorescence. the role of the bilayer phase, anionic lipids, reducing agents and peptide concentration on the extent and kinetics of partition were studied. both nrtp and nrtp exhibited high partition to popc (neutral) lipid vesicles (k p ≈ × ), which was enhanced by the anionic lipid popg for nrtp , but not nrtp . the peptides showed a decrease in partition for popc:cholesterol (liquid ordered state) or dppc (gel) membranes. depending on the lipid composition, the peptides either increased or decreased their quantum yield upon membrane insertion. quenching experiments with acrylamide showed no peptide aggregation in solution. once the translocation mechanism is fully understood we will test nrtps as carriers of relevant cellular cargos, evaluating their potential clinical application in drug delivery or gene therapy among other applications. the ingestion of trans fatty acids (tfa) formed during the partial hydrogenation of vegetable oils has been linked to a detrimental impact on health by an, as yet, unknown mechanism. we synthesized deuterated analogs of -elaidoyl- -stearoylphosphatidylcholine (t : - : pc) that contains a single "unnatural" trans double bond and -oleoyl- -stearoylphosphatidylcholine (c : - : pc) that contains a single "natural" cis double bond. solid state h nmr, complemented by molecular dynamics (md) simulations, was then employed to compare molecular organization in model membranes prepared from these isomeric molecules. analysis of spectra recorded as a function of temperature reveals a higher chain melting temperature for the trans isomer, indicating tighter molecular packing in the gel state. in the liquid crystalline, however, the difference between the trans and cis isomers is subtle. order as probed by the perdeuterated [ h ] : sn- chain, and corroborated by computer simulation, coincides within < %. only in the conformation of the double bond is an appreciable difference implied. thus, our results contradict the conventional view that tfa resemble saturated fatty acids, which is > % more ordered. (supported by acs, prf -ac .) photooxidation and lateral membrane diffusion of dipole molecules v. s. sokolov , e. a. sokolenko , a. a. lents , p. pohl a.n frumkin institute of physical chemistry and electrochemistry, russian academy of science, moscow, russia, institut fuer biophysik, johannes kepler universitaet linz, austria the photodynamic oxidation of phloridzin, of the styryl dye di- -anepps and of unsaturated lipids was monitored "online" by measuring the collapse of the dipole potential which has been introduced to the membrane by these molecules. their photodamage occurred at different rates when the target molecules and the singlet oxygen generating photosensitizer (phthalocyanin) were adsorbed to the same or to opposite sides of the planar lipid bilayer. the difference in the oxidation rates were attribute to singlet oxygen transport through lipid bilayer and therefore we were able to estimate the permeability of lipid bilayers to singlet oxygen. however, the apparent permeabilities derived from experiments with different targets were differ from each other. therefore, we tested the hypothesis that the lateral membrane diffusion of target molecules and oxidation product may have biased our analysis. in line with this anticipation we found that the apparent permeability is dependent on the size of the planar bilayer. the development of a new mathematical model, which takes the mobility of all reacting species in the aqueous and lipid environments into account, allowed estimation of the real membrane permeability to singlet oxygen. it appeared to be very close to that of oxygen in the ground state. a number of complex three-dimensional lyotropic liquid crystal phases are already known, such as the bicontinuous cubic phases, but so far only a single example has been found -a cubic phase of spacegroup fd m -of a structure based upon a complex close packing of inverse micelles ( ) . we now report the discovery ( ) of a novel lyotropic liquid crystal phase, of space group, p /mmc, whose structure is based upon a hexagonal close packing of identical quasi-spherical inverse micelles. the model membrane system consists of a hydrated mixture of dioleoylphosphatidylcholine, dioleoylglycerol, and cholesterol. this novel phase has a number of unique features which may render it useful for a range of applications. firstly, it is the only known self-assembled lyotropic phase whose structure consists of a periodic close packing of identical inverse micelles. secondly, it is stable in excess aqueous solution, which is very important for potential biological or biomedical applications. references ( ) v. luzzati, v., r. vargas, a. gulik, p. mariani, j.m. seddon, and e. rivas, biochemistry , - ( ) . dystrophin is a rod-shaped muscular subsarcolemal protein. its deficiency is one of the root causes of duchenne muscular dystrophy. dystrophin rod domain contains homologous repeats, where the sub-domain constituted by the repeats to (r - ) was reported to bind actin and membrane lipids. we analyzed the interaction of r - with lipid monolayers. to better understand the assembly mechanism of this protein with lipids, we studied its adsorption behavior at the air-liquid and lipid/liquid interface using ellipsometry, surface pressure, and atomic force microscopy (afm). using two different mixtures of phospholipids, ellipsometry and pressure surface data show that r - interacts with the lipid monolayers, but is inserted into the monolayer formed by dopc-dops while it lies below the monolayer of dopc-dope. this indicates that r - interacts more strongly with dopc-dops than with dopc-dope. afm images show that the pressure of lipid monolayer influences the organization of r - . when the lipid surface pressure is mn/m, r - forms a striking network, indicating a protein-protein interaction in addition to the protein-lipid interaction. this unique behavior of one part of the central domain of dystrophin may explain its key role in muscle cell. studies on polyphenol extracts from helichrysum l., fagopyrum mill., crataegus l. and hypericum l. were performed in order to find if they may be applied as a natural free radical scavengers protecting biological membranes against peroxidation. ghosts erythrocytes were used in the experiments. they were suspended in tris-edta solution, ph , , then.irradiated with a bactericidal lamp without (control) or with a proper amount of the extracts studied. the product of lipid peroxidation was malonic dialdehyde (ma). the colour reaction of ma with thiobarbituric acid (tba) enables to determine the concentration of ma spectrophotometrically. it was found that peroxidation increased with the irradiation time. however, it significantly decreased when concentrations of poliphenols increased. the best antioxidtive property was found for hypericum l. the antioxidative efficiency sequence of the plant extracts studied was the following: hypericum l. > crataegus l. > fagopyrum mill. > helichrysum l. the results obtained indicate that polyphenol extracts exhibit excelent antioxidative properties that make them good free radical scavengers for efficient protection of biological membranes. this work was sponsored by ministry of science and education, scientific project no. n n . interaction of cationic porphyrins with neutral and negatively charged liposomes i. voszka , g. corradi , p. maillard , h.-j. steinhoff , g. csík institute of biophysics and radiation biology, semmelweis university budapest, hungary, research institute for solid state physics and optics, hungarian academy of sciences, budapest, hungary, institute curie, section de biologie, orsay, france, fachbereich physik, universitat osnabrück, germany porphyrin derivatives are used in photodynamic therapy of tumors. the knowledge of the photosensitizer location within the cell is important. the effect of porphyrin derivatives containing cationic side groups was examined on neutral and negatively charged liposomes by esr. the esr signal was first examined as a function of temperature and porphyrin concentration in the dark. a significant change related to the appearance of quasi free spin labels was obtained for spin probes at the th carbon atom and was more expressed for the asymmetrical derivative. illuminating the samples the esr amplitude decreased for all positions of the spin probe but to different extent. the effect was more expressed in case of the symmetrical derivative, especially for label positions and . for the asymmetrical derivative the effect changed from moderate to weak from the th to the th position. this indicates that the asymmetrical derivative is incorporated nearer to the lipid head groups while the symmetrical one may be located deeper in the membrane. under oxygen-free conditions both derivatives showed weaker but still pronounced effects.. single channel recording of α-hemolysin in nanopore-spanning tethered bilayer lipid membranes n. t. thet , i. pfeiffer , w. knoll , i. köper max planck institute for polymer research (mpi-p), ackermannweg , mainz, austrian research centers gmbh -arc, tech gate vienna, vienna, austria artificial lipid bilayer membranes mimic biological cell membranes in many aspects and can be used to study functional processes such as ion channeling, signal transducing, transport of nutrients etc. as most of the functions of a cell are accomplished by membrane proteins, research has been ongoing in studying and characterization of membrane proteins embedded in model lipid bilayer membranes. black lipid membranes (blms) were studied for decades but their potential for practical applications is hindered, mainly due to their lack of stability and limited lifespan. recently, tethered bilayer lipid membranes (tblms) proved to have long life span of up to several months without significant changes in membrane resistance and capacitance. in order to combine advantages of blms and tblms, we have designed a membrane system which is freely spanned across a single nanopore in a silicon nitride membrane. this system not only mimics cell membranes, but also it allows control over the chemical composition of buffer with unlimited ionic reservoirs on both sides of the membrane. this freestanding tblm maintains structural stability and lifetime of up to hours without significant decrease in its structural integrity and electrical sealing. for the validation of the tblm formation, we have inserted well known α-hl pores. we are able to control the amount of α-hl pores insertion and measure single channel ion transport across the tblm by α-hl pores using a capacitor feedback amplifier. the language of shape: biological reactions are dramatically affected by the shape of lipid membrane d. stamou university of copenhagen, copenhagen, denmark a plethora of biological process are taking place on the surface of lipid membranes. as a rule membranes in vivo are curved in a variety of complex geometries. here i will present a quantitave study on the influence of membrane curvature on protein-membrane and membrane-membrane interactions. to gain systematic access to a continuum of membrane curvatures we immobilized liposomes on a surface at dilute densities. using confocal fluorescence microscopy we imaged single liposomes of different size, and therefore different curvature, and monitored their interaction with a binding partner (proteins or other liposomes). i will discuss unpublished data on two important classes of biomolecular interactions that exhibited dramatic curvature dependence: a) snare-mediated docking and fusion b) anchoring of peripheral proteins. the following references provide partial information on the single-liposome assay: a. zettergren, c. gudmundsson, t. nylander, e. sparr physical chemistry , lund university, lund, sweden there is accumulating evidence of substantial amounts of phospholipids in the cell nuclei , although the function of these lipids is still not fully understood. it has been shown that the chromatin complex composed of dna, rna and proteins also includes phospholipids, and that rna colocalize with these . although the rna-phospholipid interactions may have important implications to biological function, in gene therapy and in medicine, very little work has been dedicated to the characterization of rna interaction with phospholipids. the objective of this work is to investigate the adsorption behavior of short single stranded bases long rna (ssrna ) molecules (similar to mirna) to lipid monolayers at the air-water interface as well as to study how the presence of rna affect the domain formation in the monolayers using fluorescence microscopy. monolayer studies have shown adsorption of ssrna to monolayers consisting of zwitterionic dppc as well as to monolayers consisting of cationic dodab. the adsorption behavior of these very short nucleic acids differ significantly from the adsorption process for longer nucleic acids as for example a base pairs long ds dna (dsdna ) which has been used as a reference system . the presence of ssrna significantly changes the compression isotherm of both dppc and dodab monolayers. plant defensins are cysteine-rich antimicrobial peptides found in various plant species. they share a common threedimensional structure, stabilized by eight disulphide-linked cysteines consisting of three antiparallel β-strands and one α-helix. most plant defensins show antifungal activity with no effect on mammalian and plant cells. expression of these peptides in plant tissue is induced by pathogen infection. the mechanism of defensin action is based on membrane permeabilization. this occurs through an interaction with high affinity binding sites on fungal membranes, resulting in alteration of membrane potential. the genes encoding for a peach ppdfn and a grape vvamp defensins were expressed in e. coli and purified to homogeneity. they were tested for antimicrobial activity against some fungi and showed to have an inhibitory effect on the spore germination. biophysical analysis showed that defensins were able to interact with artificial membranes. binding of defensins to membranes was dependent on lipid composition, increasing with the sphingolipids content. interaction between peptides and sphingolipids could lead to insertion of the defensins into the membrane resulting in its destabilization. extracts of the plant perilla frutescens have many uses in the asian kitchen, e.g. as a popular garnish used in sushi. the plant is also employed in eastern traditional medicine to treat a variety of ailments including colds, food allergy and depression. two of the main constituents of the plant are limonene and perilla aldehyde. by bio-oxidation, these molecules can be converted to perillic alcohol and perillic acid. these cyclic terpenes possess antibacterial and anticarcinogenic properties. the modes of action for these compounds are at present not understood, but their remarkably diverse pharmacological properties suggest that they might target the phospholipid matrix of the cellular lipid membrane. indeed, the cyclic terpenes can bind to, and alter the physiochemical properties of the lipid bilayer of the membrane, the effects of which can cascade down to several essential cellular processes. here, we use molecular dynamics (md) simulations to investigate the effect of limonene and its derivatives on the properties of lipid bilayers including the changes in acyl chain order parameters, bilayer thickness and the area per lipid. md can afford molecular-scale dynamic information, often not easily accessible from experimental measurements. this information can be used to interpret existing experimental data obtained by e.g. isothermal titration calorimetry, electron paramagnetic spectroscopy and differential scanning calorimetry. catalysis in the membrane: interfacial mechanism of phospholipase a h. p. wacklin , r. k. thomas institut laue langevin, grenoble, france, physical and theoretical chemistry laboratory, oxford university, u.k. phospholipase a cleaves the sn- acyl chains of membrane phospholipids and performs a range of physiological functions. one of the least well understood aspects of the its mechanism is how its activity is regulated by the interaction with the substrate membrane. we have used neutron reflection to monitor changes in membrane structure during lipid hydrolysis [ , ] . the penetration depth of pla depends on lipid packing and increases during the lag phase of porcine pancreatic pla . by using a selectively deuterated lipid substrate, d -popc, we determined the relative membrane-water partitioning of the lipid products in-situ. the lyso-lipid product partitions into the solution phase, while fatty acid accumulates in the membrane and increases the affinity of pla [ ] . pla is inhibited at ph , which is consistent with protonation of the catalytic histidine. however, irrespective of ph, pla is fully activated by me-betacyclodextrin, which facilitates the release of the lyso-lipid from the enzyme-substrate complex. me-beta-cyclodextrin does not interact directly with the membrane surface or the substrate lipids, indicating that product release occurs outside the immediate membrane-water interface. diffraction limits resolution in optical microscopy, but many interesting biological problems occur on shorter (molecular) length scales. recently, methods to circumvent the diffraction limit have been presented. fluorescence photoactivation localization microscopy (fpalm) uses activation of many small subsets of photoactivatable or photoswitchable fluorescent probes (pafps) to generate images with effective resolution in the tens of nanometers. pafp molecules are photoactivated, imaged, localized, and photobleached in small numbers. the process is repeated for many subsets to build up data on thousands to many hundreds of thousands of molecules. the positions of all localized molecules are used to construct an image of the sample with resolution limited not by diffraction, but by the localization precision and molecular density. results will be shown from a variety of biological systems, including live and fixed cells expressing a variety of pafp-tagged proteins. bi-plane fpalm can image in three dimensions with demonstrated resolution of nm x nm x nm. polarization fpalm can image both molecular positions and anisotropies simultaneously with lateral resolution of ∼ - nm. using these powerful capabilities, many potentially interesting biological problems can be addressed. binding of the hiv- ncp on oligonucleotides at the single-molecule level j. godet, p. didier, a. jouonang, y. arntz, y. mély laboratory of biophotonics and pharmacology, umr cnrs , university of strasbourg, france the nucleocapsid protein (ncp ) of the hiv- is a small basic protein which plays key functions in the viral life cycle. the activity of ncp mainly rely on its potent rna-and dna-chaperone activities that direct the rearrangement of numerous nucleic acid molecules into their most stable conformation. two main features of nc's chaperone activity are its abilities to aggregate and destabilize nucleic acids. in addition, the rapid kinetics of ncp interaction with nucleic acids was recently proposed as another major component of nc's chaperone function based on the apparent correlation between an indirect measurement of the nucleic acid dissociation kinetics of ncp and its overall chaperone activity. but so far, no direct measurement of the on/off rates of ncp binding to oligonucleotides was performed. in the present work, we realized single molecule fluorescence resonance energy transfer (smfret) measurements to probe the transient interactions of a tmr-labelled ncp with a short cy -labelled dna oligonucleotide confined into nanovesicles. after confirming the efficiency of the ncp /odn complex encapsulation into the nanovesicles by fcs, the vesicles were tethered to the surface for immobilization. integrity of the entrapping vesicles attached on the surface was confirmed by afm. finally, the association and dissociation constants retrieved from these smfret measurements were discussed in the context of the dna-chaperoning activity of the protein. high-resolution spatiotemporal organization of the integrin lfa- m. f. garcia-parajo , t. s. van zanten , g.-j. bakker , r. diez-ahedo , a. cambi , c. g. figdor bionanophotonics group; ibec, barcelona, spain, tumour immunology dept; ncmls, nijmegen, the netherlands lfa- is a leukocyte-specific integrin involved in different steps of the immune response. on monocytes, lfa- plays a key role in the regulation of monocyte-endothelial interaction during rolling, arrest and extravasation into the underlying tissue. i n-vivo experiments showed that blood borne lymphocytes can 'switch' within seconds from rolling to arrest. furthermore, tem observations of pro-active, ligandindependent nanoclusters confirmed that affinity and clustering are complementary processes required in adhesion. yet, the mechanisms leading to fast-switching remain obscure. in our group, we used a combination of single molecule fluorescence techniques to study the spatiotemporal organization of lfa- on monocytes. we performed optical nanoimaging of lfa- nanoclusters in relation to membrane rafts with a resolution of nm and accuracy of ∼ nm. in quiescent cells, lfa- nanoclusters do not associate with membrane rafts and diffuse freely on the membrane. binding of the integrin to its ligand icam- induces the formation of microclusters that further associate with rafts and exhibit reduced mobility, consistent with cytoskeleton interactions. our work highlights the markedly different spatiotemporal organization of lfa- that might explain its concerted action to form larger and stable platforms on the cell surface required for rapid and effective cell adhesion. c. ciobanasu, u. kubitscheck rheinische friedrich-wilhelms-universität bonn, germany cell penetrating peptides like the hiv tat peptide have the property to rapidly translocate the cell membranes and the capability to deliver a wide range of cargoes. the mechanism of the membrane translocation is still under investigation and object of considerable controversy. we applied and single-molecule and confocal laser scanning microscopy (lsm) to study peptide-membrane interactions. electron-multiplying ccd cameras yield images of single fluorescent molecules with a time resolution in the range of a few milliseconds only, which allows the tracking of fluorescently labelled peptides and lipids at bio-interfaces in realtime with a localization precision of a few nanometers. we formed giant unilamellar vesicles (guvs) from different lipid mixtures and examined their interaction with fluorescently labeled tat peptides. we found that the passive peptide internalization process depends on lipid composition, charge of the lipid bilayer, and the ionic properties of the medium. a translocation of cationic tat peptides was observed in membranes containing at least mol% of lipids with a phosphatidyl ethanolamine or a high mol fraction of the phosphatidyl serine head group. in salt-free solution tat efficiently bound to guvs, however, in a physiological nacl solution tat binding was completely abrogated, but the peptides efficiently equilibrated across the guv membrane. new approaches to measure interactions in the live cell plasma membrane g. j. schütz biophysics institute, johannes kepler university linz, austria in my lecture, i will show examples how to obtain insights into the organization of the cellular nanocosm by single molecule experiments. our primary goal is an understanding of the role of such structures for immune recognition. brightness and single molecule colocalization analysis allows us to study stable or transient molecular associations in vivo. in particular, i will present results on the interaction between antigen-loaded mhc and the t cell receptor directly in the interface region of a t cell with a surrogate antigenpresenting cell. in addition, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions. the method allows identifying and characterizing interactions between an arbitrary fluorescence labeled protein ("prey") and a membrane protein ("bait") directly in living cells. cells transfected with a fluorescent fusion protein of the prey are plated on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of the bait; the fluorescence copatterning is used as readout for the interaction. we applied this tool for the study of the interaction between cd -the major coreceptor for t cell activation -and lck, an important tyrosine kinase in early t cell signaling. in addition to the well-known zinc-clasp structure, we found strong contributions of lck membrane anchorage for the binding of the two proteins. developing a fluorescent redox sensor for monitoring metal-ion mediated catalysis in biosystems a. rybina , a. kiel , b. thaler , a. sprödefeld , r. krämer , d. p. herten bioquant and, department of inorganic chemistry, heidelberg university, germany a fluorescent redox sensor is an electron photo-switching device that can be used for the characterization of the redox state of a given environment. it combines a fluorescent fragment with a redox-active unit that senses the media by a redox reaction and controls the light emitting properties of the fluorophore. such reversible sensor can help to examine the electrochemical state and changes in biological systems during biochemical processes in real time. recently a new fluorescent molecular sensor with a redox-active hydroquinoneunit covalently linked to fluorophore rhodamine b was developed. (kierat r.m. et al., bioorg. med. chem. lett., -accepted) . the reduced hydroquinone-form of the sensor is fluorescent while its oxidation to benzoquinone-derivative leads to a significant decrease of the fluorescence. although the above method shows great promise for applications in biological systems, the exact mechanism of this process is not fully understood yet. we use fluorescence spectroscopy to investigate oxidation reactions on the ensemble and single-molecule level and study kinetic rates. the proposed strategy is to use cu (ii) complex as oxidation mediator immobilized on surface via dna linker to examine the oxidation mechanism. fluorescently labeled atp as a probe of the outer mitochondrial membrane barrier: role of vdac fluorescence correlation spectroscopy (fcs) was applied for studying the distribution of fluorescently labeled atp (bodipy-atp) in isolated mitochondria. the setup and peak intensity analysis (pia) was described in our recent paper (perevoshchikova et al. biochim.biophys.acta : . the binding of bodipy-atp to mitochondria was maximal in the non-energized state, whereas the addition of succinate (respiratoty substrate) or atractyloside (adenine nucleotide translocase inhibitor) led to a decrease in the binding. nadh reduced the fcs signal from bodipy-atp added to non-energized mitochondria more than nad+ did under the same conditions suggesting the control of nucleotide transport through voltage-dependent anion channel (vdac) residing in the outer membrane. konig's polyanion also decreased the bodipy-atp binding to mitochondria with the effect being reduced by alamethicin or digitonin. control experiments showed that bodipy-atp did not bind to liposomes showing minor role of unspecific binding. it was suggested that bodipy-atp in combination with fcs can be used to monitor the functional state of mitochondrial vdac which is considered to be a principal regulator of mitochondrial function. fluorescence correlation spectroscopy studies of lysozyme partition to phospholipid vesicles a. m. melo , a. coutinho , m. prieto cqfm and in, ist, - lisboa, portugal, dqb, fcul, - , lisboa, portugal binding to membrane lipids has been increasingly recognized as an important step in the aggregation and cytotoxicity of several amyloidogenic proteins [ ] . in addition, it has been recently reported that membranes containing negatively-charged phospholipids can also trigger rapid amyloid-like fiber formation by a variety of several nonamyloidogenic proteins, such as cytochrome c and lysozyme [ ] . our study aims to elucidate the factors that govern the formation of these lipid-protein complexes. given the importance of electrostatic interactions between the proteins and the acidic phospholipids in the putative membrane-induced protein misfolding step, it is essential to first characterize quantitatively the protein partition behavior towards liposomes prepared with variable anionic lipid content. in this study, lysozyme was chosen as a model protein and fluorescence correlation spectroscopy (fcs) was used to monitor its binding to liposomes after its conjugation to alexa fluor . most organic dyes labelling techniques produce a mixture of populations of molecules labelled with a different number of fluorophores. the influence of this poli-dispersity of labelled molecules on the protein partition behaviour will be explored, namely the ability of the fcs technique to detect the production of non-competent membrane-binding species. t. wohland , p. liu , x. shi , y. h. foo , t. sudhaharan , s.-w. chong , v. korzh , s. ahmed chemistry dept., singapore nat. univ., medical biology inst., singapore, molecular & cell biology inst., singapore biomolecular interactions have been measured mostly under in vitro conditions because of higher accuracy and ease of measurement. however, it has become clear that the cellular environment has an important influence on these interactions. it was therefore necessary to develop new tools to allow the measurement of interactions in cells and organisms. recently, we have developed a modality of fluorescence cross-correlation spectroscopy (fccs) called single wavelength fccs (sw-fccs), which uses one-photon excitation to excite two fluorophores with overlapping absorption but separable emission spectra. sw-fccs has been used to determine e.g. dimer fractions of proteins in live cells. here we aim to extend the use of sw-fccs to cells and organisms. in the first part we determine the dissociation constants of a small rho-gtpase (cdc ) with an effector domain (crib) and two effectors (n-wasp or irsp ). in the second part we measure the binding between cdc and a scaffolding protein (iqgap ) in dependence of their expression levels in cho cells and in live zebrafish embryos. by using gfp/mrfp fusion proteins we can excite both fluorophores at nm and detect them separately in two different wavelength channels. we quantitatively determine the dissociation constants and compare their differences in vitro, in cells, and in embryos. these experiments demonstrate that sw-fccs is a powerful biophysical tool for the quantitation of biomolecular interactions in cells and organisms. addressing plasma membrane structure at the nanoscopic length-scale s. wieser, m. axmann, g. j. schütz biophysics institute, johannes kepler university linz, altenbergerstr. , a- linz, austria there is increasing interest in a detailed understanding of the structure and dynamics of the cellular plasma membrane, primarily based on recognizing its essential role for controlling cellular signaling processes. we employed single molecule fluorescence microscopy to study diffusion of cd , a gpi-anchored protein, in the plasma membrane of living t cells at sub-wavelength resolution, both on the cell body and on tunneling nanotubules connecting cells. the lateral motion of this single fluorescence labeled molecule was imaged on a millisecond time scale. within the experimental errors, no indications for confined diffusion for cd on the cell body in t cells have been found. furthermore by separating longitudinal and transversal mobility, we found isotropic diffusion behavior on the surface of tunneling nanotubules. in both studies we analyzed the mean square displacement as a function of the time-lag and the distribution of displacement steps. however, a closed analytical theory for these analysis is only available for the simplest models. to address a suspected diffusion process we reasoned that a full analytical description may not be required; it may well be sufficient to compare the experimental data with monte carlo simulations of the process. we demonstrated the working principle for this simulation based analysis for free diffusion, hop diffusion and transient binding of the tracer molecule to slowly moving receptors. n. chakroun , f. fraternali , m. malfois , h. rezaei , c. a. dreiss king's college london, u.k., diamond light source, didcot, oxfordshire, u.k., inra, jouy-en-josas, france prion(prp) diseases are fatal neurodegenerative diseases affecting mammals including human and sheep.they are characterized by the accumulation of extracellular βrich fibrillar deposits of a structurally modified form (prp sc ) of the cellular prp c .despite the increasing interest for prp diseases,the mechanism of prp c /prp sc conversion is still unknown.studies on prp diseases suggest that neurotoxicity arises from small pre-fibrillar oligomers.we have used a range of biophysical techniques combined with molecular dynamics simulations (md) to resolve the oligomerization pathways of sheep prp (sprp).we have shown that under well established conditions, sprp oligomerizes into three oligomers, which form in parallel.in addition, we have now identified the minimal region of sprp leading to the same oligomerization profile of the entire sprp,namely h h .low resolution shapes of sprp, h h and resulting oligomers have been determined by small-angle x-ray scattering.time-resolved studies have been used to follow the oligomerization of sprp and h h monomers into the oligomers.the conversion of sprp sc at the molecular scale was studied by md.simulations of the h h region recreating experimental conditions revealed a complete unfolding of h helix followed by h helix.these crucial steps are followed by the formation of β-sheet structures leading to a stable βrich double hairpin structure. single-molecule force spectroscopy investigation of the conformational equilibria of alphasynuclein m. brucale , a. rampioni , m. sandal , i. tessari , l. tosatto , l. bubacco , b. samorì istituto di biochimica g.moruzzi, università di bologna (italy), dipartimento di biologia, università di padova (italy) alpha-synuclein (asyn) is an abundant intrinsically disordered protein (idp) primarily located at presynaptic terminals. mutations in the gene encoding asyn have been linked to early-onset parkinson's disease (pd). by means of single molecule force spectroscopy (smfs) experiment, we show how the conformational equilibrium of monomeric wild type (wt) asyn shifts toward more compact structures in several unrelated conditions linked to pd pathogenicity [ ] . the conformational heterogeneity of pathological alpha-syn mutants a p, a t and e k has also been characterized, revealing marked differences in the conformational behaviors of the mutants with respect to wt asyn [ ] . all the mutants show a distinctively higher propensity, with respect to wt, to acquire a monomeric compact conformation that is compatible with the acquiring of beta structure. the same smfs experimental methodology is then used to characterize the conformational behavior of wt and mutant asyn in a variety of conditions, in an attempt to gain insight about the multiple and contrasting parameters controlling the equilibrium. in vitro protein folding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble proteins. this is not the case for integral membrane proteins, which constitute about one third of all eukaryotic proteins and more than half of all validated and potential drug targets. fully reversible denaturant-induced unfolding remains limited to a few β-barrel porins, whereas the much larger and more relevant class of α-helical membrane proteins has thus far evaded this approach. we report here the first example of an α-helical membrane protein that can be unfolded completely and reversibly by a chemical denaturant: mistic, a -residue protein from bacillus subtilis [ ] , dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. using spectroscopic and microcalorimetric techniques, we exploited this unique property to provide (i) a quantitative comparison of membrane protein stability in different membrane-mimetic systems; (ii) an experimental test of controversial predictions [ ] regarding the folding core of mistic; and (iii) a convenient setup to study the spontaneous, translocon-independent membrane insertion of this unusual membrane protein. the mechanical functioning of biological tissues is important from many viewpoints such as diseases, clothing and even food. the protein collagen makes up the greater part of these tissues, and is remarkable for its many uses in the body, however there are at least two other major components. one of the most interesting properties of these tissues is their non-linear behavior under stress. this behavior is essential to prevent a catastrophic failure such as an aneurysm. at least three major theories have been proposed within the past few years to explain this behavior, but have been impossible to verify. in order to determine a correct description of the mechanical structure of the tissue we have been using cutting edge technological solutions to address the single molecules within the extra cellular matrix. this technique combines optical methods with single molecule force spectroscopy, allowing stiffness measurements over the nanoscale as well as determining the individual protein tensions within the extra cellular matrix. the results show that this method can be used to determine the network properties even in the complicated aortic wall enabling better understanding of disease states, which in this case include marfan's syndrome and ascending aortic aneurysms. beta amyloid peptide abetapy - shows a faster aggregation kinetics than abeta - c. d'arrigo , m. tabaton , a. perico institute for macromolecular studies, national research council, genova, italy, department of neurosciences, university of genova, genova, italy we test directly the differences in the aggregation kinetics of three important beta amyloid peptides, the full-length abeta - and the two n-terminal truncated and pyroglutamil modified abetapy - and abetapy - , found in different relative concentrations in the brains in normal aging and in alzheimer disease. we find by following the cd signal and the tht fluorescence of the solution in phosphate buffer, a substantial faster aggregation kinetics for abetapy - . this behavior is due to the particular sequence of this peptide which is also responsible of the specific oligomeric aggregation states, found by tem, during the fibrillization process which are very different from those of abeta - , more prone to fibril formation. in addition abetapy - is found here to have an inhibitory effect on abeta - fibrillogenesis, coherently with its known greater infective power. this is an indication of the important role of this peptide in the aggregation process of beta-peptides in alzheimer disease. the puzzle of the anomalously long denaturation kinetics of green fluorescent protein (gfp) mutants still is largely unveiled. in this study we have followed the effect of mutation h g on the stability of gfpmut (mut g) in the presence of guanidinium hydrochloride (gdnhcl). different techniques of fluorescence spectroscopy have been employed in order to obtain information concerning the unfolding event: time resolved fluorescence, fluorescence correlation spectroscopy (fcs), and fluorescence anisotropy. the substitution of the histidine with glycine affects protein stability versus ph: in particular mut g kinetics is not ph dependent and at basic ph values the protein is less stable. the fluorescence properties (quantum yield, lifetime) and the rotational correlation time are unchanged during the unfolding dynamics, while the number of fluorescent proteins decreases exponentially. an extrinsic probe, bound to cysteine , has been employed in order to gain more insights on the unfolding process, monitoring the stability of a different region of the protein. in particular, it has been found that a softer region is present around cysteine in both gfp variants, showing that the unfolding process does not follow a simple two step mechanism. recently, negatively-charged membranes were reported to catalyze amyloid fiber formation by amyloidogenic peptides/proteins and also to induce formation of "amyloidlike" fibrils by nonamyloidogenic proteins. here, we used an approach which combines steady-state and time-resolved fluorescence measurements to obtain structural information about these supramolecular assemblies and to gain insights about the factors that control their formation. by exploring a wide range of lipid concentrations, the interaction of alexa -lysozyme with phosphatidylserine-containing membranes was found to be a complex multi-step process, critically dependent upon the protein-to-lipid molar ratio (p/l) used. upon increasing the total lipid concentration in solution, there was a balance between an increased overall protein binding to the lipid vesicles and a progressive protein dilution on the membrane surface. as the surface potential of the vesicles decreased upon increasing the protein interfacial coverage of the liposomes, the protein binding mode was found to switch from a peripheral binding of lysozyme to the anionic headgroups (at low to intermediate p/l) to a partial insertion of the basic protein into the hydrophobic core of the membrane (at a high p/l). it is hypothesized that disruption of the protein tertiary structure might be a stepwise process beginning with loosening of the structure caused by its deeper insertion in the membrane bilayer. unexpected scaling laws in the mechanical unfolding of single protein molecules m. clusel, e. i. corwin, h. lannon, j. brujic center for soft matter research, physics department, new york university, new york, ny, usa it is a question of fundamental importance to understand the response of proteins to a stretching force, particularly in the case of mechanically active proteins, such as those in muscle fibers. we aim to understand how the structure and topology of a protein affect its resilience to external forces and presumably its function. owing to recent advances in single molecule force-clamp spectroscopy using the atomic force microscope (afm), we are now able to probe the structure and dynamics of single proteins under a constant stretching force by measuring their end-to-end length over time. the probability distribution of unfolding times at a given force allows us to estimate the strength of the protein in terms of a characteristic energy barrier, while the shape of the distribution provides a window into the microscopic mechanism by which the protein breaks apart. here we show a novel scaling of the unfolding kinetics with the stretching force, which deviates significantly from the currently accepted bell model. instead, we propose a physical picture for forced unfolding that is analogous to the mechanics of fracture. v. garcía-gonzález, j. mas-oliva instituto de fisiología celular. universidad nacional autónoma de méxico. méxico, d.f. méxico. studies focused on the thermodynamic and kinetic analysis have demonstrated that transfer of neutral lipids such as cholesterol esters through an aqueous phase is a highly costly biophysical event. therefore, nature has developed a series of lipid transfer proteins such as the cholesterylester transfer protein (cetp) designed to efficiently lower the energy barrier for transfer of cholesterol-esters between lipoproteins through an aqueous environment. employing circular dichroism we evaluated the secondary structure stability of a small peptide derived from the carboxy-end of cetp (helix y ) in a wide range of ph's. the percentage of α-helix is diminished only at extreme temperatures and acidic ph's in a reversible way. we report that while a mixture of phosphatidylcholine/cholesteryl-ester forms large aggregated particles independently of ph, inclusion of helix y to the mixtures close to neutral ph's allows the formation of small micellar-like structures confirmed by dynamic light scattering and electron microscopy. these results suggest that helix y when close to physiological ph values presents the ability to organize a micellar structure around itself. this type of organization allows the process to dramatically lower the energetic barrier for lipid transfer through aqueous media, phenomenon directly related with the facilitation of lipid transfer between lipoproteins. mimicking metastasis by a novel microfluidic approach there is increasing evidence that cancer metastasis shares commonalities with thrombosis. the von-willebrand-factor (vwf), a mechanical active blood clotting protein appears to be a particular potent candidate to bridge the gap between clotting and cancer extravasation. modeling the crucial physiological conditions of the blood circulatory system, for an in situ study of blood clotting-metastasis connections is not only, absolutely necessary, but also a challenging task. here, we present acoustically driven flow as a novel microfluidics method for mimicking the blood flow. this method enjoys very beneficial advantages of possibility of handling very little volumes of fluids, together with freedom to model most of the geometries present in our microcirculatory system. one technologically challenging, yet physiologically important factor, is the hydrodynamic condition in a bifurcated vessel, where complex shear profiles arise. we present a model to mimic these conditions and discuss the impact of hydrodynamics on vwf mediated cancer cell adhesion in bifurcated vessels of our microcirculatory system. protein structural changes occurring in flows stresses inherent to viscous fluid flow have previously been associated with protein unfolding, although structural changes have not been well documented as a function of relevant hydrodynamic parameters. we have used raman spectroscopy to monitor the structure of various protein solutions in situ for multiple flow scenarios within a concentric cylinder fluidic device ( ) . the flows, which ranged from circular couette to wavy taylor-couette flow, were characterised experimentally using particle image velocimetry. several proteins were observed to change conformation when exposed to these flows, although the nature of these changes was protein specific. shearing hen egg white lysozyme in water altered the protein backbone structure, while similar shear rates in a % glycerol, % water solution affected the solvent exposure of the side chain residues near the exterior of the α-domain. the solventdependent response may be due to the flow topology, viscous stress, or the surface hydration properties. comparison of spectra acquired at different time points, including before and after flow, confirmed that the observed changes are reversible and independent of fluid stress exposure time. the scripps research institute, la jolla, ca, usa. intrinsically disordered proteins are increasingly found to play major roles in cell biology and disease. we are utilizing single-molecule fluorescence methods to probe these complex and highly dynamic molecules, allowing more direct measurements of structural distributions and dynamics, while avoiding loss of information due to ensemble averaging. in one example, we investigated the structural dynamics of sup -nm, whose regulatable amyloid formation is believed to have functional significance in yeast. using a combination of single-molecule fret as a molecular ruler, coincidence to interrogate intermolecular interactions, and correlation analysis to probe conformational dynamics, we showed that the monomeric protein populates an ensemble of compact and rapidly interconverting conformations. a particularly interesting feature of intrinsically disordered proteins is that they are relatively unstructured in isolation, but can gain stable structure by interaction with binding partners. in this context, we used single-molecule fluorescence to characterize the complex folding pathway for the parkinson's-related idp alpha-synuclein induced by binding to a lipid-mimic. this combined single-molecule fluorescence methodology provides a powerful approach for detailed studies of the coupling of folding and dynamics with interactions and biology of this important class of proteins. m. ito , j. johansson , r. stromberg , l. nilsson department of biosciences and nutrirtion, karolinska institutet, huddinge, sweden, department of anatomy, physiology and biochemistry, swedish university of agricultural sciences, the biomedical centre, uppsala, sweden amyloid β-peptide (aβ) is a - amino acid polypeptide and known to aggregate and form insoluble amyloid fibril, which is regarded as a primary cause of alzheimer's disease (ad). the discovery of practical and effective treatments and drugs for ad has been waited eagerly. in a recent experimental study, it was suggested that stabilization of the helical conformation of the aβ middle region, which strongly favors collapsed coil formation in the extracellular environment, would reduce the aβ fibril formation. based on the experimental evidence, inhibition of the unfolding of the aβ α-helix can be a forceful strategy to repress the aβ fibril formation resulting in prevention of ad. however, the detailed mechanism of the unfolding of the aβ α-helix has remained unclear, because the x-ray or the nmr structure of the aβ α-helix in aqueous solution has not been reported due to its instability. the aim of this study is to find effective ways to inhibit the unfolding of the aβ middle region, which is a prerequisite for the aβ fibril formation. in this study, we attempted to elucidate the molecular mechanism of the aβ unfolding by molecular modeling and molecular dynamics (md) simulations. the md simulations were performed for α-helical structures of a wild-type aβ( - ) model and a mutant aβ( - ) model. linker average hydrophilicity as a tool to discriminate between extended and non-extended calcium binding proteins a. isvoran , e. quiniou , c. craescu , l. mouawad west university of timisoara, department of chemistry, pestalozzi , timisoara, romania, inserm u , centre universitaire paris-sud, bâtiment , orsay, france the ef-hand calcium binding proteins (cabps) may exist either in an extended or a compact conformation, sometimes correlated with their functions. for the cabps with know structure and function, calcium sensors are usually extended and calcium buffers compact, hence the interest to predict the form of the protein starting from its sequence. in this study we used two different procedures, the sosuidumbbell algorithm and a novel procedure that is based on the linker average hydrophilicity (lah). the two procedures were tested on known-structure cabps and then applied to unknown-structure centrins. the so-suidumbbell algorithm yielded the right conformations for of the known-structure proteins and predicted that all centrins should are compact. the lah procedure discriminated well between the extended and non-extended forms of all the known-structure cabps and it reflected well the phylogenetic classification of centrins being a simple and powerful means to discriminate between extended and nonextended forms of cabps. only few residues that constitute the linker are responsible for the form of the cabp, showing that this form is mainly governed by short-range interactions. (http://u .curie.u-psud.fr/modelisation/lah) lipid and protein organization of hepatitis b antigen characterized by fluorescence spectroscopy v. greiner , c. egelé , s. oncul , f. ronzon , c. manin , a. klymchenko , y. mély laboratoire de biophotonique et pharmacologie, umr cnrs, faculté de pharmacie, université de strasbourg, france, sanofi pasteur, av. marcel mérieux, marcy l'étoile, france. hepatitis b surface antigen (hbsag) particles are nm lipoprotein particles, mainly composed of the major s surface viral protein containing trp residues and yeast-derived lipids. since the structure of these particles is still missing, we further characterized them by fluorescence techniques. fcs indicated that the particles diffuse mainly as monomers and contain about proteins per particle. fluorescence quenching and time-resolved fluorescence experiments showed that the fluorescence signal is largely dominated by the trp residues at the protein surface. moreover, time-resolved anisotropy measurements indicate that the protein motion is restricted and that the surface trp residues exhibit both local and segmental motions. the lipid part of the particles has been studied by environment sensitive -hydroxyflavone probes and viscosity-sensitive dphbased probes, and compared to lipid bilayers and low density lipoproteins (ldls), taken as models. the results suggest that the lipid part of hbsag is closer to ldls than to model lipid bilayers. we present an extensive calorimetric study of bovine alphalactalbumin for various ca++ content. equilibrium dsc raw data are analyzed and the melting temperature tm, the specific heat jump deltacp, the heat of unfolding deltahm are directly extracted. the binding of calcium on the native (n) state greatly stabilizes the protein, essentially by the enthalpic difference between the unfolded (u) and n states. we show that subsequent addition of calcium in the mm range stabilizes further the n state. the equilibrium calorimetric measurements are completed with out of equilibrium stopped flow refolding kinetics by cd spectroscopy performed at different temperature and ca++ concentrations. we discuss the possible stabilization mechanisms compatible with our measurements. protein unfolding/refolding in cellular compartments: application of luciferase constructs our studies show that a reporter enzyme, firefly luciferase, can be used for evaluation of the stress-induced proteotoxicity within different cellular compartments such as the nucleus, cytoplasm or mitochondria. in transfected mammalian cells, firefly luciferase is localized in microsomes. we engineered plasmid constructs encoding luciferase with inserted specific sequences that ensure its cytoplasmic or intranuclear, or intramitochondrial localization. in addition, we fused luciferase to the green fluorescent protein (gfp) that enables to visualize patterns of the compartment-targeted product. using such gfp-labeled constructs we had a possibility to monitor protein unfolding, aggregation and refolding in the cytoplasm, nucleus and mitochondria of transfectants exposed to either stressful conditions. gfp-luciferase expressed in mammalian cells behaves as a relatively labile protein which can undergo reversible unfolding and aggregation in response to heat shock, atp depletion or action of toxic agents. in the case of cell recovery, refolding of denatured luciferase is carried out at the chaperone machine. we explored unfolding/refolding of gfp-luciferase in the cytoplasm, nucleus and mitochondria of ischemia-stressed rat cardiac cells and in several cancer cell lines treated with hyperthermia or some chemotherapeutic drugs. the results obtained have revealed intriguing correlations between the proteotoxic impact within either compartment and the viability of treated cells. amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid bilayers s. jha , d. sellin , r. seidel , r. winter biophysical chemistry, department of chemistry, tu dortmund university, otto-hahn str. , d- , dortmund, max-planck-institute for molecular physiology, otto-hahn str. , d- , dortmund, germany the islet amyloid polypeptide (iapp), which is considered as the primary culprit for β-cell loss in type diabetes mellitus patients, is synthesized in the β-cells of the pancreas from its precursor, the pro-islet amyloid polypeptide (proiapp). proiapp is co-processed in the secretory granules and co-secreted to the extracellular matrix together with insulin as iapp. here, we compare the amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. the two peptides show an enhanced amyloidogenic propensity in the presence of negatively charged membranes and similar secondary structural properties. however, proiapp shows a much less amyloidogenic propensity, probably due to the increased net charge on proiapp, compared to iapp. unlike iapp, proiapp forms small oligomeric structures at the lipid interface, having heights of ∼ . nm. this morphological distinction can be attributed to the presence of the pro-region, flanking the amyloidogenic iapp. the addition of proiapp to iapp marginally delays iapp fibrillation, probably by interfering with the interaction between amyloidogenic iapp cores of distinct iapp molecules. thus, it appears reasonable to speculate that the pro-region of the proiapp could serve to delay the fibrillogenesis of iapp at negatively charged lipid bilayers. the role of transmembrane domain interactions in the kinetics and folding of cpt z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon chemistry dept., univ. of warwick, coventry, uk, clinicalȃsciencesȃres.ȃinst., warwick univ., coventry, uk carnitine palmitoyltransferase i (cpt ) enzymes are polytopic integral membrane proteins in the outer membrane of mitochondria (omm). cpt controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for βoxidation. the two catalytically active isoforms, cpt a and cpt b, are different in their inhibitor binding kinetics and structure (interaction between n-and c-segments, interactions of transmembrane domains (tmd)). it has been suggested that inter-and intramolecular tmds interactions are important for cpt a, but not for cpt b function. cpt a has also been implicated in formation of oligomeric complexes through its tm segments. the study of tm helix-helix interactions in cpt isoforms could lead to a better understanding of their function and inhibitor binding kinetics, and will contribute towards the design of pharmacological strategies aimed at modulating the activities of cpt enzymes in conditions such as diabetes. to investigate the ability of the tmd in cpt to self-associate and the order of any oligomers formed, several biochemical and biophysical techniques have been used. we found the self-association of rcpt a tmds (tm , tm ) to be different as measured using the in vivo tox-cat assay. chemical cross-linking and analytical ultracentrifugation studies demonstrated formation of both trimers and hexamers by the rcpt a tm peptide. these results provide further evidence that tm plays role in formation of a channel in the omm. self-assembly of transmembrane domains in cpt : role of gxxxg(a) motif in possible channel formation z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon department of chemistry and, warwick medical school, university of warwick, coventry, uk carnitine palmitoyltransferase a (cpt a), a membrane protein that controls the rate of oxidation of long-chain fatty acids, is of key importance in diabetes and has recently been reported to exist as an oligomer in vivo. we have investigated full-length cpt a and find that the protein exists as a hexamer in liver mitochondria. using mutants of cpt a expressed in yeast mitochondria, we have localised key protein interactions in the hexamer to the transmembrane (tm) domains of the protein. detailed study of the tm domains in isolation, in both e.coli membranes and detergent micelles, demonstrated that while tm shows little self-assembly, tm displayed significant self-association. biophysical analyses of a tm -derived synthetic peptide revealed oligomerization behaviour identical to native cpt a in mitochondria, providing a strong link between tm helixhelix interactions and cpt a hexamer formation. this is significant in light of a recent suggestion that cpt a oligomerization may lead to formation of a channel in the mitochondrial outer membrane through which acylcarnitine gains access to the inter-membrane space. our data supports this new theory, and we go on to demonstrate experimentally the structural determinants of hexamer (channel) formation, specifically gxxxg(a) motifs in the tm domain which pack favourably in the hexamer and stabilize the oligomer. investigation of flexible loop role in structure and thermodynamic stability of firefly luciferase p. maghami, b. ranjbar, s. hosseinkhani department of biochemistry and biophysics, faculty of basic sciences, tarbiat modares university, tehran, iran protein folding, like any chemical process, consists of two fundamental components, thermodynamics and kinetics, which determine the stability of the folded state and the pathway of folding, respectively. these processes are currently too difficult to be solved de novo by purely computational methods. experimental evidence is required to simplify the problem via protein engineering .in this research, the wild type firefly luciferase (photinus pyralis) and some of its mutants were over expressed and purified. then their unfolding thermodynamics were examined, using circular dichroism and conventional fluorescence measurements. the unfolding equilibrium constant were measured over a complete rang of denaturant conditions. the measurements were shown structural and physico-chemical changes between wild-type and mutant proteins. exploring intrinsic disorder of unstructured membrane proteins by surface polymer physics intrinsically unstructured proteins (iups) are considered as a separate class within the protein world because they lack a well-defined folded structure. because iup's function is indeed directly linked to structural disorder, they are assumed to be natively unfolded. several physicochemical techniques are available to discriminate the degree of disorder. however a clear structural classification is still lacking. in this context, polymer physics emerges as a powerful tool for getting inside on the conformational abilities directly related to structural disordered of iups. in the present contribution, surface pressure and ellipsometry experiments in conjunction with polymer physics have been used to infer structural data in terms of molecular conformation and flexibility of a membrane protein essential for bacterial division, zipa. this protein has been pointed to posses a high molecular flexibility and to adopt a random coil conformation. folding dynamics of peptides studied by timeresolved infrared spectroscopy c. krejtschi , o. ridderbusch , r. huang , l. wu , t. a. keiderling , k. hauser institute of biophysics, university of frankfurt, germany, department of chemistry, university of illinois at chicago, usa peptides are ideal model systems to study protein folding mechanisms. ir techniques provide both the necessary time resolution as well as the structural sensitivity. we initiate rapid heating by laser-excited ns temperature jumps (∼ • c) and study fast ns-to-µs relaxation dynamics [ ] . the dynamics of the α-helix to random coil transition of polyglutamic acid was analyzed under reversible folding/refolding ph-conditions. the observed relaxation kinetics allowed separation of the folding and unfolding process with additional use of ftir measurements in thermal equilibrium. sitespecific dynamics were monitored by use of isotopic labeling for a β-hairpin peptide whose conformation is stabilized by a hydrophobic core. various single and cross-strand isotopically labeled variants were analyzed. the isotope-edited kinetics show variations in local structural stability of the hairpin backbone. our data support a multistate dynamic behavior, and the site-specific kinetics are consistent with a hydrophobic collapse hypothesis for hairpin folding [ ] . small heat shock protein hsp was predicted to belong to the family of intrinsically disordered proteins. one of the features of these proteins is that they do not demonstrate cooperative thermal transitions on heating. we applied different methods (dsc, ftir and intrinsic tryptophan fluorescence) to investigate the thermal unfolding of hsp . dsc results have shown that thermal denaturation of hsp begins from o c and occurs, with very low cooperativity, within a broad temperature region (up to o c and above). the thermal unfolding of hsp is fully reversible. the ftir data show that heating of hsp from to o c results in complete disappearance of α-helices (from - % to ) and the decrease in β-sheets content from to %. studies on the temperature dependences of tryptophan fluorescence have shown a significant red shift of the spectrum. these changes occurred within temperature region from to o c with midpoint at ∼ o c. probably, this transition can be explained by destruction of β-sheets around trp , the only trp residue of the α-crystallin domain of hsp (other trp residues of hsp are localized in the n-terminal domain). the data obtained confirm the suggestion that hsp is a protein, whose significant part is intrinsically disordered. we propose that, on heating, the α-crystallin domain containing β-sheets melts at higher temperature than the n-terminal domain containing the most of α-helices. t. rosenkranz , a. katranidis , d. atta , j. enderlein , i. gregor , m. grzelakowski , p. rigler , w. meier , j. fitter isb- : molecular biophysics, research centre jülich, germany, institute of physics, biophysics/complex systems, georg august university, göttingen, germany, institut für physikalische chemie; universität basel, basel, swizerland the protein folding mechanism is the missing link in the biological flow of information from the dna to its specific function. since most of proteins within a cell consist of more than one domain studies on this protein class are of major importance. it is a common feature of multidomain proteins to aggregate under refolding conditions, which hinders a refolding. molecular encapsulation of single molecules prevents aggregation. by immobilizing the nanocapsule the observation period in a wide field microscope will be extended, so that slow or rare folding events can be detected. a major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies [ ] . polymer vesicles maintain their structural integrity even under harsh unfolding conditions. furthermore the nanocontainer proved to be permeable to guanidinium hydrochloride. using encapsulated phoshoglycerate kinase, labeled with atto- , a dye which experiences fluorescence quenching by photo-induced electron transfer (pet) with tryptophans, we demonstrate the remarkable properties of polymeric nanocontainers. applying pet as a folding probe we detected multiple unfolding/refolding transitions of single proteins. proteins frequently become irreversibly modified by carbonylation, a process of introducing the carbonyl group (carbon monoxide) in a reaction with reactive oxygen species (ros) such as superoxide, peroxide or ozone. the main targets for carbonylation in proteins are amino-acid side chains of lysine, arginine and proline. products of carbonylation are aminoadipic semialdehyde from lysine (asa) and glutamic semialdehyde (gsa) from arginine and proline. importantly, carbonylated proteins are marked for proteolysis by the proteasome, but can escape degradation and form aggregates that can be cytotoxic. carbonylation increases with the age of cells and it is associated with ageing and age related disorders such as alzheimer's disease, parkinson's disease and cancer. we have used the molecular dynamics method to study the stability of carbonylated proteins villin headpiece and ubiquitin. simulations were run after mutations of arginine, proline and lysine into gsa and asa had been performed. in addition, we have used thermodynamic integration on lysine, arginine, proline, asa and gsa residues in order to estimate their solvation free energy (related to relative hydrophobicity and hydrophilicity). our results suggest that carbonylation markedly decreases the overall stability of proteins, and that one potential reason for that may be a disruption of the balance between hydrophilic and hydrophobic regions in the protein. a. martino, d. crane, i. m. feavers, b. bolgiano division of bacteriology, national institute for biological standards and control, potters bar, uk the sensitivity to protein's secondary structure and progress in computational calculations have made circular dichroism (cd) an attractive technique to explore the optical properties of three promising vaccine candidates to neisseria meningitidis. clinical batches of a c-term deleted form of nada (genome-derived neisseria antigen -gna ) and the fusion proteins gna - (fp- ) and gna - (fp- ) were therefore studied. increases in temperature and denaturant concentration on secondary structures and folding/unfolding profile were monitored by cd in the far and near uv regions and complemented by trp fluorescence spectroscopy data. furthermore, epitope conformational changes on binding activities to immune-sera were investigated. the calculated secondary structure content was in broad agreement with the available predicted or solved protein structures. upon temperature incubation, a structural transition from a highly α-helical nada to a more unordered conformation, with a mid point at ∼ • c, was observed. fp- and fp- maintained their conformation up to • c or m guhcl in the case of fp- . unfolding was not always reversible. reductions in binding to monoclonal ab titrated along with increasing unfolding. folding/unfolding studies have proven useful in better understanding the solution behaviour and extent of folding of proteins. cold denaturation of yfh offers the clue to understand the effect of alcohols on protein stability s. r. martin , v. esposito , p. de los rios , a. pastore , p. a. temussi national institute for medical research, the ridgeway, nw aa london, u.k., laboratoire de biophysique statistique, sb/itp, ecole polytechnique fédérale de lausanne (epfl), ch- , lausanne, switzerland, dipartimento di chimica, università di napoli federico ii, via cinthia, i- napoli, italy although alcohols are well known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. here we present a study of the effects of alcohols on protein stability using yfh . exploiting the unusual property of this protein of undergoing cold denaturation around • c without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol,ethanol methanol. in all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. we suggest that alcohols, at low concentration and physiological ph, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation. biomolecular sciences and biotechnology tor vergata moro , rome, italy cholesterol plays an important role in regulating the structural properties of phospholipid and non-phospholipid membranes. in this study we have applied in situ energy dispersive x-ray diffraction (edxd) to investigate the effect of cholesterol on the structure of different phospholipid and non-phospholipid oriented membranes. in detail, phosphatidylcholine (pc) bilayers and niosomal membranes, made of a non-ionic surfactant centre for bioactive chemistry, department of chemistry department of chemistry this process is initiated at nuclear envelope remnants (ners) in the presence of atp and gtp. the mvs can be divided in two main populations: mv and mv . mv has a classical lipid composition while mv is enriched in phosphoinositides (pips: pi, pip, pip and pip ). ners have an unusual lipid composition, enriched both in cholesterol and pips. physicochemical properties of the pips were investigated as a function of ph and temperature (t) using nmr, saxs and dls to map out their phase state. pips-water dispersions are observed in lamellar, hexagonal or isotropic phases depending on t and ph. in parallel, model membranes mimicking mv and ners lipid composition were studied by h and p nmr. mv modelling shows a complex behaviour of pips on pc membranes: they order or disorder membranes, whereas the order of pc/pi/pip/pip membrane is lower than that of pc or pc/pi membranes c. manzo, t. s. van zanten, m. f. garcia-parajo bionanophotonics group, ibec-institut de bioenginyeria de catalunya, barcelona, spain membrane proteins play a fundamental role in intra-and inter-cellular functions. in particular, the proteins lateral mobility in the fluid membrane environment is crucial for the regulation of several mechanisms, as receptor-mediated signal transduction and establishment of immunological synapses. these mechanisms are controlled through protein crowding and reduced lateral diffusion, which induce macromolecular associations and limit the application of conventional single molecule fluorescence techniques. to measure proteins mobility on living cells membrane, we developed a fluorescent correlation spectroscopy (fcs) setup in which the sample illumination is obtained through near-field scanning optical microscopy (nsom) probes. the use of nsom probes is particularly suited for the observation of dynamics on the cell membrane and overcomes the drawbacks of other techniques. in fact, through a shear-force-based position control, the probe is kept at a fixed distance from the membrane and its sub-wavelength aperture (∼ nm) reduces the illumination area, allowing the observation of highly crowded regions of the membrane. on the basis of preliminary results, the nsom-fcs is expected to provide an additional insight on the proteins trafficking at the membrane level. the technique also presents several potential developments, as the further reduction of the illumination area and two-colors correlation. single molecule fluorescence microscopy of the store-operated calcium channel subunit orai j. madl, j. weghuber, d. bergmair, m. fahrner, m. muik, c. romanin, g. j. schütz johannes kepler university, institute for biophysics, linz, austria store-operated calcium entry (soce) is essential for many cellular signalling processes. the essential pore forming subunit of soce channels in the plasma membrane is orai . here we present single molecule fluorescence microscopy of orai which was performed in order to directly visualize the stoichiometry of mobile orai pores. the protein was fluorescently labeled with monomeric gfp. a novel single molecule fluorescence approach, toccsl (thinning out clusters while conserving the stoichiometry of labeling), was used for the determination of the stoichiometry. this technique allows reducing the density of fluorescently labeled molecules without affecting the stoichiometry of labeling. density reduction is achieved by completely photobleaching a defined area within the plasma membrane; nonbleached gfp-orai aggregates enter the bleached region subsequently by diffusion. our data indicate that there are different populations of orai present in the cell: most of orai is located in the plasma membrane. a second population of orai -mgfp was found to be localized in intracellular vesicles. a significant fraction of the plasma membrane orai exhibits a diffusive movement. we found by analyzing the bleaching characteristics of single orai -mgfp aggregates that in resting cells mobile orai is predominantly dimeric. a. kobitski , a. nierth , m. helm , a. jäschke , g. u. nienhaus university of ulm, germany, university of heidelberg, germany, university of karlsruhe, germany rna molecules have attracted enormous attention in recent years, and various novel roles were revealed for rna in biological processes. ribozymes are a class of rna molecules capable of catalyzing chemical reactions. we have studied a diels-alderase (dase) ribozyme, a small artificial -mer ribozyme, which is capable of catalyzing carbon-carbon bond formation between an anthracene diene and a maleimide dienophile in multiple turnovers. single-molecule fluorescence resonance energy transfer was employed to investigate the intramolecular dynamics of this rna molecule as a function of mg + ion concentration. folding into a functional state occurs via an intermediate state, and continuous fluctuations between these two states were observed on the ms time-scale at the midpoint concentration of mg + ions. an effect of substrates binding on the folding and catalytic reaction of the dase ribozyme is in the focus of our recent research with the ultimate goal to obtain a detailed structural view of the single-molecule conformational changes that accompany the catalytic reaction. a. katranidis , r. schlesinger , k. nierhaus , i. gregor , m. gerrits , g. bueldt , j. numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins. here we present an approach employing a two color single molecule sensitive fluorescence wide-field microscope in order to visualize surface tethered fluorescently labeled ribosomes and de novo synthesized gfp molecules in real time [ ] . fluorescence of co-translational folded proteins was observed from mature fluorescent gfp molecules which carry additional amino acids at the c terminus remaining linked to the ribosome. thus it was possible to co-localize fluorescence from labeled ribosomes and from gfp molecules. we demonstrate that the green fluorescence protein mutant gfp emerald is produced with a characteristic time of five minutes. the fastest gfp molecules appeared already within one minute. processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. in fluorescence spectroscopy, photobleaching is a process which leads to irreversible loss of fluorescent properties of a dye molecule, usually due to photochemical reactions. it is especially important for fcs experiments on slow-diffusion systems since for high excitation intensities it can have a strong impact on fluorescence intensity correlation function. usually it is observed as apparent shortening of the mean diffusion time of the dye molecules. the behavior of tmr-labeled fd-virus rods in water solution under various excitation conditions was investigated. the experiments were conducted for low ( : ) and high ( : ) tmr:virus ratios and for increasing laser intensities. the correlation function was measured in the experiments. the results were fitted using origin software to estimate the influence of photobleaching, and compared with computer simulations. a strong effect of photobleaching was visible for rods labeled with a single dye molecule, while rods labeled with tmr molecules showed little to no bleaching. a prolongation of characteristic diffusion times for highly labeled virus rods in comparison to low-labeled ones was also observed. k. toth , a. gansen , a. valeri , v. böhm , c. a. seidel , j. langowski abt. biophysik der makromoleküle, deutsches krebsforschungszentrum, heidelberg, germany, lehrstuhl für molekulare physikalische chemie, heinrich heine universität, düsseldorf, germanythe nucleosome has a central role in the compaction of genomic dna and the control of dna accessibility for transcription and replication. we studied the effect of dna sequence and selective histone acetylation on the structure, stability and disassembly of the mononucleosomes. quantitative single molecule fret measurements between dyes attached to different parts of the nucleosome permitted us to detect the equilibrium between several subpopulations of reconstituted nucleosomes in solution. we obtained that the heterogeneity and stability of the samples are correlated with each other and influenced both by the dna sequence and the histone acetylation. the path of the linker dna is more sensitive to all studied effects than the dna on the core. intermediates of the disassembly pathway were identified and characterized. j. strömqvist , s. johansson , y. ohsugi , k. andersson , l. xu , m. kinjo , p. höglund , j. widengren experimental biomolecular physics, kth, stockholm, sweden, department of microbiology and cell biology, karolinska institutet, stockholm, sweden, laboratory of molecular cell dynamics, hokkaido university, sapporo, japan dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined.here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. dynamic multiple-target tracing probes spatiotemporal cartography of cell membranes in order to decipher the non random and non homogeneity of the plasma membrane organization, we had performed fluorescence correlation spectroscopy measurements on live cells. this allowed us to establish the presence of nanoscale confining structures and to demonstrate their implication in signaling process . complementing these studies, we present here a new analytical method, namely multiple-target tracing (mtt) which takes advantage of the high resolution provided by singlemolecule sensitivity to generate dynamic maps at high densities of tracked particles. introducing deflation by subtracting detected peaks allows detecting peaks of lower intensity. we achieved an exhaustive detection of particles with performances reaching theoretical limits, and a reconnection of trajectories integrating the statistical information from past trajectories. we demonstrate the potential of this new method of analysis by applying it to the epidermal growth factor receptor labeled with quantum dots, in the plasma membrane of live cells. this has allowed us to build up a global representation of molecular dynamics in cell membranes. dual polarisation interferometry (dpi) is a surface analytical technique capable of dynamically measuring biophysical parameters of conformational change in biomolecular interactions. the technique measures three key parameters, namely layer thickness, layer density (ri) and mass, thereby enabling the resolution of conformational changes involved during binding. a number of different applications are presented. protein-protein interactions: understanding the biophysical nature of protein interactions can deliver insights into the mechanisms by which proteins interact, thereby elucidating protein function. dpi enables correlation between binding affinity and conformational change, greatly enhancing the study of structure-function relationships. lipid layers: the birefringence mode of dpi can be used to study the formation of lipid bilayers and biomolecular self-assembly. it is possible to use a combination of bilayer refringence and mass to study phase transitions associated with protein or peptide binding to the lipid bilayer. the individual stages of adsorption, absorption and micellisation can be distinguished. carbohydrate interactions: dpi uses a planar glass surface and a wide range of coupling chemistries. a carbohydratespecific surface can be used to study a wide range of biomolecular interactions, such as lectins, acidic proteins, extracellular matrix signaling and interactions with complex membranes. the experimental characterization of the elementary conformational steps constituting the protein folding pathway remains a big challenge. quenching of the triplet state of tryptophan by close contact with cysteine has been shown to provide a new tool for measuring the rate of intramolecular contact formation -one of the most elementary events in the folding process -in peptides and proteins using only natural probes. here we show an extensive study on a stabilized mutant of the second beta-hairpin of gb domain. steady state fluorescence and kinetics of contact formation between a natural tryptophan and a cysteine added to the c-terminal are measured for different temperatures and solvent conditions. we separately address the contributions of different structural elements to the overall stability of the hairpin. we extract kinetics parameter for contact formations in the unfolded state, formation of the hydrophobic core and tails pairing in the folded state. by means of a fragment peptide terminated with a tryptophan and a cysteine, we also estimate the structural propensity of the turn region. the data coherently combine with a simple model previously developed to describe the dynamics of unstructured chain [biophys. j. , ( )], here modified with the addition of attractive interactions between specific residues. catalytic power of partially denatured enzymes: implementation of molten-globule-like states m. shushanyan , d. e. khoshtariya , m. makharadze , t. tretyakova , r. van eldik institute of molecular biology and biophysics, gotua , tbilisi, georgia, department of physics, i. javakhishvili tbilisi state university, tbilisi, georgia, department of chemistry and pharmacy, university of erlangen-nürnberg, germany impact of nonspecific moderate denaturants, urea and dmso, on the kinetic (functional) and thermodynamic (stability) patterns of a hydrolytic enzyme, α-chymotrypsin (α-ct) has been investigated. furthermore, an impact of urea and guhcl in combination with of temperature on the kinetic pattern of carboxypeptidase a has been examined. for the case of α-ct, in particular, we have observed about tenfold increase of the apparent mickaelis constant when going from to m urea ( o c), whereas the value of catalytic constant remained almost unchanged, indicating that the protein is not unfolded even under those severe conditions. the matching microcalorimetric experiments revealed that both the temperature-induced melting temperature and transition enthalpy decrease gradually with the increase of the additive concentration. with m urea the peak-shaped calorimetric feature disappears totally. however, catalytic power of α-ct was preserved owing to its catalytic constant. for other studied enzyme/substrate/denaturant arrays diverse kinetic peculiarities due to mgls have been observed. rubredoxins are a class of iron-containing proteins whose biological role on electron transfer processes and metal binding is still unclear. the unfolding dynamics of the rubredoxin mutant rda c from the mesophile desulfovibrio vulgaris (dv) was studied on the temperature range from • c to • c and along time at • c. resonance energy transfer (ret) was used to determine the donor (d; trp ) to acceptor (a; , -iaedans) distance. from • c to • c the d-a distance increased Å. however the random coil expected d-a distance was only achieved after heating the protein solution during . hours at • c. from uv-vis absorption data it's clear that this protein is capable of maintaining its iron-sulfur center at • c. by melting the protein at the same temperature all iron-centers disintegrate and the protein unfold after . hour. the trp fluorescence also shifts nm to the red reflecting the partial exposition of the indole ring to the solvent. from fluorescence and anisotropy decay curves a breathing type movement of the protein structure was observed between • c to • c without lost of significant secondary structure. this structure flexibility should play an important role on the thermal stability of the dvrd the antimicrobial peptide novicidin (nc) -modified from the sheep self defense peptide smap- , for reduced mammalian cytotoxicity -is a cationic peptide (net charge +∼ . ) that adopts random coil structure in solution, but an α-helix in the presence of lipid vesicles. the conformation of nc in presence of the lipids dlpc, dlpg, dmpc, dmpg, dopc, dopg, dope, and dops in different combinations reveal the lipid selectivity, affinity, and phase dependent changes with varying l/p ratios and temperatures, observed from cd spectroscopy. it is understood that the conformational change is dependent on chain length and head group of the lipid. apart from the in vivo results on the nc activity, studies using qcm-d, dual polarisation interferometry, and calcein release assay reveal the kinetics and concentration dependent activity of nc in lipid bilayers and vesicles. preliminary studies on orientation of nc in various lipid environments using ssnmr, lsnmr, and molecular dynamics simulations apparently suggest that nc may form toroidal pores/detergent effects depending on the chain length of the lipids. further experiments on nc in presence of lipids using dsc, itc, ssnmr, oriented cd, ld, and confocal microscopy to determine the structure, thermal stability, orientation in lipid bilayers, and thereof the action of nc will help in proposing a comprehensive model for its mechanism of action in model membranes. dielectric method for measuring glass transition and denaturation temperatures of hydrated proteins g. e. thomas , s. bone , g. drago institute for bioelectronic and molecular microsystems, bangor university, gwynedd, uk., applied enzyme technology, pontypool, uk.the flexibility of protein structures is important in allowing the variety of motions, covering a wide range of magnitudes and frequencies, essential to biological activity. high frequency dielectric measurements can be used to study the flexibility of proteins by probing the relaxation of dipolar constituents of their structures. hydrated proteins exhibit a broad dielectric loss extending over the frequency range from mhz to ghz which can be decomposed into a number of constituent dispersions. one of these dispersions, with a relaxation time of ∼ ns, has been attributed to the relaxation of protein backbone peptide groups in the protein interior. in the work reported here, this dielectric dispersion was investigated as a function of temperature for the enzyme glucose oxidase. two critical temperatures were identified as the glass transition and denaturation temperatures, both of which were found to decrease with increasing protein water content. the results were consistent with a scheme in which the hydrated glassy protein undergoes a change in structural mobility at the glass transition temperature and experiences an irreversible change in conformation at a higher denaturation temperature. both glass transition and denaturation temperatures are key indicators of protein stability and are important in the production and storage of protein based pharmaceuticals. a. szymańska, k. kacprzyk, g.Ślósarek department of molecular biophysics, a. mickiewicz university, umultowska , - poznań, poland aggregation dynamics of proteins plays an important role in molecular biology and medicine as it permits explanation of several disease states. an interesting problem is to find out in which conditions the interactions of the protein molecules lead to formation of ordered structures and in which to disordered ones. in this study, dynamic light scattering, circular dichroism and also congo red dye experiments were performed to analyze various structural states of lysozyme induced under different concentration of ethanol solvent. at low ethanol concentration the attractive interaction between the protein macromolecules dominate. after addition of more ethanol solvent, the translational diffusion coefficient was much smaller than that for lysozyme solution at zero ethanol concentration. it can be explained by the structural transformation of the polypeptide chain leading to a partially folded conformation needed for oligomerization and fibrillation process. on the basis of the cd experiments we concluded that the ethanol solvent induces changes in secondary structure of lysozyme solution. on addition of % v/v ethanol solution the intramolecular hydrogen bonds were destabilized. above this ethanol concentration, β -sheet were the dominant secondary structure of lysozyme in solution. the phase diagram illustrating the formation of: monomers, oligomers at various structural states, protofilament formation state, protofilament and amyloid fibrils was constructed. key: cord- -ks g agf authors: prante, franz j.; bramucci, alessandro; truger, achim title: decades of tight fiscal policy have left the health care system in italy ill-prepared to fight the covid- outbreak date: - - journal: inter econ doi: . /s - - - sha: doc_id: cord_uid: ks g agf although austerity was particularly strong in the aftermath of the economic crisis of and its consequences in the euro area, italian fiscal policies have been characterised by tough consolidation periods ever since the s. what the government extracted from the national economy in terms of taxes has been larger than what people received in public services for almost three decades. figure also shows that periods with cuts in real health care expenditure tend to correspond with or follow periods of strong budget consolidation in the fi rst half of the s and in the euro crisis after . the development of italian health care expenditure is reported in figure together with data for selected european countries and the euro area average. three phases can be observed in the evolution of italian expenditure. in the s, unlike most other industrialised countries, italy experienced a decline in public and compulsory health care expenditure (measured in constant euros per capita). it was not until the end of the s that a slight upward trend began when spending increased in parallel to the other european countries until the late s. from onwards, a new phase of spending containment began, lasting until . in this period, public health care spending was similarly affected in portugal and spain and to a larger extent in greece, i.e. the countries hardest hit by the euro crisis and the subsequent austerity policies. by contrast, in this period a rapid increase in public and compulsory healthcare spending per capita took place in germany, france and belgium. ods ever since the s. over the years, the ssn has undergone a profound transformation aimed at containing costs and increasing effi ciency. the question now is whether the consequences of these measures have left the ssn unprepared to face the scourge of covid- . italy's ssn was founded in . based on the national constitution (article ), the state guarantees the universal right and largely free access to health care services. during the s, a fi rst series of far-reaching reforms was implemented in an attempt to contain costs in the face of the growing healthcare needs of an ageing population and rapidly improving technologies (pavolini and vicarelli, ) . these reforms were largely in line with the market-liberal 'new public management' approach and their primary objective was to limit italy's public defi cits and debt (pavolini and vicarelli, ) . cost containment was therefore motivated by the macroeconomic context of the time, characterised by italy's efforts to meet the maastricht criteria and the requirements of the stability and growth pact, which led to an overall tightening of public spending. more recently, the global fi nancial crisis and the policy response to the euro crisis put a further strain on the italian economy and signifi cant restrictions on health care spending returned to the national agenda (de belvis et al., ) . since the early s, the italian government has registered almost consecutive years of primary budget surpluses ( figure ). this signals that (right axis) forum research and development and other components) has suffered a drastic setback. from to , total public health care expenditure in nominal terms (i.e. including infl ation) increased by only . % in italy, while in germany it increased by . % ( figure a ). moreover, cofog data provides evidence of the extent of cuts in hospital services. unlike northern european countries, italy (together with portugal and even more so greece) has reduced public spending for hospital services. from to , cuts in public hospital services have contributed substantially to the negative dynamics of the percentage growth rate of the total public expenditure for health care (figure b ). although austerity policy placed a great burden on the health care system, the share of health care expenditures in total government spending figure shows the percentage change in government and compulsory health care per capita for these three different phases and for the entire period between and . from to , a fi rst phase of expenditure containment took place in italy, in which public expenditure increased by only . %. after a slightly expansive second phase from to , in which spending per capita in italy increased by . %, the growth of public health expenditure registered a reduction in the third time interval (as was the case in portugal, greece and spain). in this period, characterised by the most recent set of budget cuts, per capita spending in italy decreased by . % -less strongly than in greece but more than in spain and portugal. in contrast, the group of northern countries registered an increase. altogether, from to , public and compulsory health care expenditure per capita in italy increased by less than . %, which is by far the lowest value among the european countries reported in figure . in the last decade, the extent of cuts in the ssn was particularly dramatic. in the wake of the fi nancial and economic crisis of , total public healthcare expenditure in italy (including investments, intermediate consumption, i t a l y b e l g i u m s p a i n g e r m a n y n e t h e r l a n d s f r a n c e g r e e c e p o r t u g a l i t a l y b e l g i u m s p a i n g e r m a n y n e t h e r l a n d s f r a n c e g r e e c e p o r t u g a l i t a l y b e l g i u m s p a i n g e r m a n y n e t h e r l a n d s f r a n c e g r e e c e p o r t u g a l i t a l y b e l g i u m s p a i n g e r m a n y n e t h e r l a n d s f r a n c e g r e e c e p o r t u g a l forum of acute care hospitals in italy fell below the eu average. the trend continued to decline throughout the years of the euro crisis. the availability of acute care beds was reduced even more drastically than hospital capacity ( figure ). although a pronounced trend towards reducing acute care beds can be observed in many european countries, few european countries have reduced the number of available beds as much and to such a low level as italy. in , italy had seven beds per , inhabitants, a value close to germany and above the eu average. in , the number of acute care beds had dropped to . per , inhabitants, signifi cantly lower than in germany with six beds available per , persons and much closer to the historically low value of spain. thus within a rather short time period, italy found itself at the lower end of the spectrum in europe. there is also a considerable difference in the provision of intensive care beds, with italy again at the tail end in europe (rhodes et al., ; oecd, ) . although in recent years the number of intensive care beds in italy has remained relatively constant (figure ) , intensive care capacity has not been expanded (in contrast to e.g. germany) despite warnings of possible bottlenecks in accommodation capacity of intensive care patients (rhodes et al., ) . has increased from % in to . % in and remained above % after , according to cofog data. this may indicate that health care provision was important to the government despite general spending constraints. however, this did not prevent italian expenditures from falling below the international trend. in the eu, almost one-third of public health care expenditure is used to cover the running expenses of inpatient curative institutions (european hospital and healthcare federation, ) . over the years, hospitals have been subject to increasing pressure and have often been seen as a major potential source for cuts in public health systems (see mckee, ; popic, ) . cost containment strategies revised the use and provision of inpatient hospital care in favour of day hospital and outpatient services, thereby consistently sacrifi cing hospital capacity. data from the world health organization (who) show that since the beginning of the s, the number of hospitals has been drastically reduced throughout europe, but particularly in belgium and italy. acute care hospitals are currently a central element in the fi ght against cov-id- . a higher number of acute care hospitals could have also facilitated the isolation of infected patients, reducing the risk of contagion. figure shows that after starting from a level similar to germany in , italy has reduced per capita hospital capacity much more than many other countries within two decades. from on, the number health not elsewhere classified r&d health public health services hospital services outpatient services medical products, appliances and equipment total % forum is a matter of controversy among lung specialists and intensive care physicians (gattinoni et al., ) . the current research gap on covid- may therefore also require a comprehensive diagnosis of patients by lung specialists, which could lead to better treatment outcomes (see also begley, ) . in this context, the substantial reduction in the number of pneumological beds during the phase of intensifi ed austerity after in italy is particularly tragic. according to the data of the italian ministry of health, the number of pneumological beds has decreased from , in to , in -a reduction of %. the reduction of resources in the public health system and in particular in the operation of public hospitals in italy has been going on for almost years. the italian population is currently paying the price of prolonged tight budget policies in the ssn. the one-sided focus on fi scal constraints and debt reduction has deprived the italian health sector of an important part of its capacity to offer adequate protection to the population. the sizeable reduction of resources has caused severe diffi culty to the ssn in effectively tackling the consequences of covid- . the outbreak of the health crisis has sounded a wake-up call that cannot remain unheard. italy, as well as other european countries, would have been better prepared for adequate treatment of covid- patients in severe and critical condition if the capacity of acute and emergency care had not been reduced. oxygen-assisted beds are particularly relevant for the inpatient treatment of covid- . for some patients, breathing difficulties worsen in the course of the illness, making intensive medical care necessary. the public discussion therefore focuses primarily on the availability of intensive care capacities and mechanical ventilation equipment. however, the speed at which machine ventilation should be provided figure acute care hospitals per , inhabitants, eight largest emu countries and eu average sources: who; authors' calculations. acute care beds per , inhabitants, eight largest emu countries and eu average note: eu membership as of with country data available. sources: oecd; authors' calculations. spain eu average sources: italian ministry of health; destatis. intensive care beds in italy pneumological beds in italy intensive care beds in germany with ventilators running out, doctors say the machines are overused for covid- beyond containment: health systems responses to covid- in the oecd italy: a strange nhs with its paradoxes european health systems and covid- : some early lessons the variability of critical care bed numbers in europe the fi nancial crisis in italy: implications for the healthcare sector covid- does not lead to a "typical" acute respiratory distress syndrome country report italy : including an in-depth review on the prevention and correction of macroeconomic imbalances hospital in europe reducing hospital beds: what are the lessons to be learned?, european observatory on health systems and policies policy brief key: cord- -edc xr authors: bontempi, elza title: commercial exchanges instead of air pollution as possible origin of covid- initial diffusion phase in italy: more efforts are necessary to address interdisciplinary research date: - - journal: environ res doi: . /j.envres. . sha: doc_id: cord_uid: edc xr this communication aims to advocate a more coordinate activity mainly between medical and environmental scientists to clarify some confusing information related to airborne diffusion mechanisms of covid- . in this frame it is suggested that parameters other than environmental pollution (accounting for pollution-to human transmission mechanisms), as for example parameters involving commercial exchanges (accounting for human-to human transmission mechanisms), should be considered to better justify the difference in the initial diffusion of virus in italy. at the end of , covid- infections were found, for the first time, in wuhan (china). at the end of february , the first infection case was reported in northern italy (lombardy) , opening an unexpected sanitary crisis in europe. during the continuous diffusion of the virus in lombardy, the not complete knowledge of some aspects of infection spread (for example the virus transmission due to no-symptom patients), has alimented several debates and conflicting information about covid- airborne diffusion (zhiqiang zhai, ), making evident the need of more dialogue between different disciplines. in particular, due to several episodes of air pollution observed in the past in china (wang et al., ) and in northern italy, especially in the po valley (perrino c et al., ) , a correlation between airborne particulate matter (pm) concentration and the reported infection cases was proposed by a position paper (position paper). based on this work, several newspapers and some research articles (frontera et al., ) , (sterpetti, ) were published reporting the possibility that airborne pm acts as a carrier in covid- diffusion (pollution-to human transmission). however, the potential influence of pm in virus diffusion was proposed as exploratory evidence of potential risks, but never demonstrated so far (qin et al, ) . in particular, few knowledges are still available about interaction of virus and pm. on the contrary, it is well documented that the exposure to high pm concentrations can produce cardiovascular diseases, pulmonary oxidative stress and inflammation (hadei, et al. ). this situation can aggravate the health conditions of the infected subjects, due to the pneumonia often associated with covid- . concerning covid- diffusion in the air, some recent published papers support the idea that the airborne transmission is a mechanism of virus diffusion. moreover, these works concern the small respiratory droplets with viral content, that are generated by infected people (morawska et al., ) , and that can be transported by air current (human-to human transmission). indeed, this eventuality primarily concerns indoor situations, then different boundary conditions in comparison to transport that may be realized by outdoor pm. the lack of a clear definition of the covid- possibility to be transported by the air has alimented several discussions and different opinions, mainly depending on the experts' background. the aim of this letter is to highlight some limitations due to the deficiency in interdisciplinary activities focalized to study covid- diffusion. this work would demonstrate the need of more interdisciplinary collaborations in reporting and interpreting the available data, and the possibility that other parameters, different to those already proposed, should be considered. the next paragraph is devoted to expanding and discussing the examples cited in the introduction. the third part of this communication aims to propose a different point of view, considering also economic/commercial factors and suggesting some actions addressed to promote more efficacy in collaborations. theoretical framework: some limitations of available studies may be due to the lack of suitable interdisciplinary research the need to increase the collaboration concerning covid- infection, between different interdisciplinary areas, can be highlighted by the lack of a suitable and shared terminology. for example, the definition of airborne has a different meaning for medical and environmental scientists, with the consequence to make often confused the definition of airborne virus transmission. when public health officials discuss about airborne transmission, they specifically mean that virus is transported in aerosol respiratory droplets (generated indoor by infected people) smaller than micrometers in diameter (https://www.who.int/). compared to respiratory droplets of largest dimensions, bioaerosols are expected to be able to travel meters (morawska, et al. ). then, this concerns possible infection mechanisms mainly occurring indoor (human-to human transmission). on the other side, airborne has a different definition for scientist working in other scientific areas, such as atmospheric topics. indeed, in the air pollution studies, airborne generally refers to outdoor pollution, as for example, research concerning pm. in addition, bioaerosols consist of particles generated in biological processes, then they can consist of flowers, trees, carbohydrates, proteins, grass, lipids, dna, rna, fungi, bacteria, viruses, and so on (hyde et al., ) , that are only a limited part of pm, that usually contains large amount of inorganic components. the recent works suggesting virus diffusion in northern italy, concerning pm acting as a carrier, is an example of a possible mechanism concerning an outdoor virus diffusion possibility (pollution-to human transmission), that need further investigations. the possibility that the virus can attach for example to pm, which may modify the aerodynamic characteristics of these particles (prather et al, ), and increase the virus dispersion should be copuled with studies investigating the covid- infectivity, also depending on its dilution (the virus may be represent in insufficient amounts to produce infection) (national academic science, countries, china contribution is about % for export and about % for import in italy. then, these data can be considered a quite good picture of economical relationships occurring between different italian areas and china. figure reports trade data provided by istat, concerning import and export of italian it is very interesting to highlight that the image reported in figure is very similar to those shown in figure , strongly suggesting a possible correlation. in particular, i quantified the correlation between the infection cases (shown in figure ) and the trade import and export data (shown in figure ) methodological approach, that may start in clarifying the context and define a clear and recognized terminology specifically addressed to the covid- , select the fundamental parameters to investigate, and promote interdisciplinary international research activities. this goal can be obtained also with the support of political authorities, that often select their scientific committees specifically devoted to covid- , involving only medical and epidemiology specialists. this will allow not only to better address all the research efforts, but also to contribute to disseminate suitable messages and information to non-specialists, such as population and policymakers. figure : trade import (a) and export (b) data provided by istat, concerning italian regions with east asia in . geographical classification of italy, provided by istat is: ( ) north-west, ( ) north-east, ( ) centre, ( ) south, ( ) islands figure : global reported infection cases detected at the beginning of the sanitary crisis ( th march ), in all the italy, reported in accord with the geographical areas provided by istat for trade data (see figure ) highlights -some conflicting information can be found in literature about covid- transmission mechanism -the parameters used to evaluate the virus diffusion should be expanded -the commercial exchanges with china may be considered to account initial source of contagious in italy first data analysis about possible covid- virus airborne diffusion due to air particulate matter (pm): the case of lombardy (italy) environmental research ( ) , understanding covid- diffusion requires an interdisciplinary, multi-dimensional approach pm exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in factors determining the diffusion of covid- and suggested strategy to prevent future accelerated viral infectivity similar to covid regional air pollution persistence links to covid infection zoning the pollution particulate a platform to investigate the effects of particulate air pollutants on viral infectivity cardiovascular effects of airborne particulate matter: a review of rodent model studies contribution of bioaerosols to airborne particulate matter airborne transmission of sars-cov- : the world should face the reality seasonal variations in the chemical composition of particulate matter: a case study in the po valley part i: macro-components and mass closure long- term air pollution exposure and cardiorespiratory mortality: a review rapid expert consultation on the possibility of bioaerosol spread of sars-cov- for the covid- effect of outdoor air pollution on asthma exacerbations in children and adults: systematic review and multilevel meta- analysis plos one reducing transmission of sars-cov- genome biology longitudinal survey of microbiome associated with particulate matter in a megacity lessons learned during the covid- virus pandemic age-specific and sex-specific mortality in countries, - : a systematic analysis for the global burden of disease study facial mask: a necessity to beat covid- association between short-term exposure to air pollution and covid- infection: evidence from china this paper was conceived and written by only one author. the author declares that she has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -s fzex v authors: bayyurt, lutfi; bayyurt, burcu title: forecasting of covid- cases and deaths using arima models date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: s fzex v after the outbreak of severe acute respiratory syndrome (sars- / ) and middle east respiratory syndrome (mers- / ) in the world, new public health crisis, called new coronavirus disease (covid- ), started in china in december and has spread all over countries. covid- coronavirus has been global threat of the disease and infected humans rapidly. control of the pandemi is urgently essential, and science community have continued to research treatment agents. support therapy and intensive care units in hospitals are also efective to overcome of covid- . statistic forecasting models could aid to healthcare system in preventation of covid- . this study aimed to compose of forecasting model that could be practical to predict the spread of covid- in italy, spain and turkey. for this purpose, we performed auto regressive integrated moving average (arima) model on the european centre for disease prevention and control covid- data to predict the number of cases and deaths in covid- . according to the our results, while number of cases in italy and spain is expected to decrease as of july, in turkey is expected to decline as of september. the number of deaths in italy and spain is expected to be the lowest in july. in turkey, this number is expected to reach the highest in july. in addition, it is thought that if studies in which the sensitivity and validity of this method are tested with more cases, they will contribute to researchers working in this field. keywords: covid- , pandemi, arima, time series analysis most people from all of the world infected with the new coronavirus ( -ncov) at the present time. coronaviruses are enveloped, positive single-stranded rna viruses that infect humans and many animals. -ncov affects different people in different ways. the common symptoms of the disease are fever, fatigue and dry cough. coronaviruses have caused two major pandemics, such as sars and mers over the past two decades. , since the sars outbreak years ago, a large number of sars-related coronaviruses (sarsr cov) have been discovered in bats. zhou et al. ( ) reported that -ncov was identified in china (wuhan) that caused an acute respiratory syndrome epidemic in humans. the outbreak that started on december continues to spread worldwide and results in fatality. since there is no approved treatment for covid- currently prevention and preparation in healthcare services are crucial. this study include italy and spain that have high number of case and death from europe also turkey in which number of the case and death start to increase. from february , to march , ; means of the fatality rates of italy, spain and turkey are calculated as % , % and % , respectively. modeling and future forecast of daily number of confirmed cases and deaths can help the treatment system. aim of present study, the statistical prediction models could be meaningfull in forecasting and controlling this global pandemic threat. for this purpose, auto regressive integrated moving average (arima) model was used to predict the confirmed cases and deaths of covid- . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . the daily case and death data of covid- from february , to march , were collected from the official website of european centre for disease prevention and control (https://www.ecdc.europa.eu/en/publications-data/download-todays-data-geographic-distribution-covid- -cases-worldwide). excel was used to build a time-series database. spss and eviews statistical software was used to perform statistical analysis on the case and death datasets, and the statistical significance level was set at . . arima model consists of autoregressive (ar) model, moving average (ma) model and seasonal autoregressive integrated moving average (sarima) model. various methods are used to estimate whether a time series is stationary. one of these methods is the increased dickey-fuller (adf) unit root test. log transformation and differences are the preferred approaches to stationary the time series. seasonal and nonseasonal differences were used to stationary the term trend and periodicity. the non-seasonal arima model is usually denoted as arima ( , , ), in which is the order of the autoregression (ar) component, is the order of the differencing process to form a stationary times series, and is the order of the moving average (ma) process. in an arima model, the value of at time is estimated as equation ( ) where is the value at time , is the ar parameter, and is the ma parameter. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint before analyzing, time series must become station-based on mean and variance. the augmented dickey-fuller (adf) is used in recognizing stationary in the mean and ljung box test in recognizing whether the time series is stationary based on variance or not. the stationarity levels of the series are analyzed by dickey and fuller ( ) . after the extended dickey and fuller test (adf) expressed by equation ( ) spss and eviews statistical software were used to perform statistical analysis on the case and death datasets, and the statistical significance level was set at . . time series analysis were made for the number of cases and deaths in italy, spain and turkey due to covid- pandemic. when the time series graphs are examined, the trend is seen. autocorrelation (acf) and partial autocorrelation (pacf) graphics were used to see this more clearly and determine its stationary. in the acf graph, it is seen that the series is not stationary . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint since many delays exceed the confidence limits. in this case, the first order difference was applied to the series and it was ensured to become stationary acf and pacf graphs of the first order difference of the series for italy, turkey and spain are showed respectively ( fig. ) (fig. ) (fig. ) . after the first difference procedure, the series became stationary as seen from the autocorrelation graph. parameters of the arima model were estimated by autocorrelation function (acf) and partial autocorrelation (pacf) graph. to determine the number of case and death of covid- , the best arima models are showed (table i) . time series were created using the best arima models selected. goodness of fit criteria and coefficients for the time series models are given in (table ii) . according to the findings; it has been determined that the coefficients of the models used to predict the number of case and death in italy is statistically significant (p< . ). the constant coefficient for the number of cases and deaths was not included in the model since it was statistically insignificant (p˃ . ). it was determined that the explanatory power of the estimation equation for the number of case in italy was . % and the error terms were stationary as a result of ljung-box statistics. it has been determined that the model can be used for foresight due to the provision of necessary assumptions. the explanatory power of the model for the number of death in italy was calculated as . %, and it was determined that the error terms were stationary as a result of ljung-box statistics. models with a mape (mean absolute percentage error) value below % are very good, models with a range of - % are good, models with a range of - % are acceptable, and models above % are classified as incorrect and inaccurate. it is determined that models can . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint be used in making future predictions due to high determination coefficients (r ) and mape value is less than %. the coefficients of the models used to predict the number of case and death in turkey were determined to be statistically significant (p< . ). the constant coefficient for the number of case and death was not included in the model since it was statistically insignificant (p˃ . ). it was determined that the explanatory power of the estimation equation for the number of case in turkey was . % and the error terms were stationary as a result of ljung-box statistic. it has been determined that the model can be used for foresight due to the provision of necessary assumptions. the explanatory power of the model for the number of death in turkey was calculated as . % and it was determined that the error terms were stationary as a result of ljung-box statistics. it is determined that models can be used in making future predictions since mape value is less than %. the coefficients of the models used to predict the number of case and death in spain was statistically significant (p< . ). it was determined that the explanatory power of the estimation equation for the number of case in spain was . %, and the error terms were stationary as a result of the ljung-box statistic. it has been determined that the model can be used for foresight due to the provision of necessary assumptions. the explanatory power of the model for the number of death in spain was calculated as . %, and it was determined that the error terms was stationary as a result of ljung-box statistics. it is determined that models can be used in making future predictions since mape value is less than %. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . the confirmed and prediction graphs of the number of case and death of the countries are given in (fig. - ) . while number of cases in italy and spain is expected to decrease as of july, in turkey is expected to decline as of september. the number of deaths in italy and spain is expected to be the lowest in july. in turkey, this number is expected to reach the highest in july ( fig. - ) . pandemic in italy, china, south korea, iran and thailand. it is similar to our study in estimating the number of cases in italy. however, since the results of our study are obtained from more recent data, we think that we have obtained a more consistent forecast for the future. in conclusion, arima models have been created by considering the most appropriate aic and bic values for case and death numbers for each country. according to the results, while number of cases in italy and spain is expected to decrease as of july, in turkey is expected to decline as of september. the number of deaths in italy and spain is expected to be the lowest in july. in turkey, this number is expected to reach the highest in july. in addition, it is thought that studies in which the sensitivity and validity of these methods are tested with more cases will contribute to researchers working in this field. the authors have no conflicts of interest to declare in relation to this research article. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . daily confirmed cases and deaths according to first order difference acf and pacf plots in turkey. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . arima forecast case and death graph for the covid- in turkey . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . arima forecast case and death graph for the covid- in spain . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint the covid- epidemic identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a pneumonia outbreak associated with a new coronavirus of probable bat origin key: cord- -ow l li authors: qu, liang g.; perera, marlon; lawrentschuk, nathan; umbas, rainy; klotz, laurence title: scoping review: hotspots for covid- urological research: what is being published and from where? date: - - journal: world j urol doi: . /s - - - sha: doc_id: cord_uid: ow l li purpose: contemporary, original research should be utilised to inform guidelines in urology relating to the covid- pandemic. this comprehensive review aimed to: identify all up-to-date original publications relating to urology and covid- , characterise where publications were from, and outline what topics were investigated. methods: this review utilised a search strategy that assessed five electronic databases, additional grey literature, and global trial registries. all current published, in-press, and pre-print manuscripts were included. eligible studies were required to be original research articles of any study design, reporting on covid- or urology, in any of study population, intervention, comparison, or outcomes. included studies were reported in a narrative synthesis format. data were summarised according to primary reported outcome topic. a world heatmap was generated to represent where included studies originated from. results: of the search results, studies met final inclusion criteria, including pre-prints and ongoing studies from online registries. these studies originated from ten countries according to first author affiliation. most studies originated from china (n = ), followed by italy (n = ) and usa (n = ). topics of the study included pathophysiological, administrative, and clinical fields: translational (n = ), covid- -related outcomes (n = ), urology training (n = ), telemedicine (n = ), equipment and safety (n = ), urology in general (n = ), uro-oncology (n = ), urolithiasis (n = ), and kidney transplantation (n = ). conclusion: this review has outlined available original research relevant to covid- and urology from the international community. this summary may serve as a guide for future research priorities in this area. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the coronavirus disease , caused by severe acute respiratory syndrome coronavirus (sars-cov- , previously -ncov), has had a worldwide impact. health-care services in many nations have had to alter their operations due to the covid- surge [ ] . previously ongoing research trials have been suspended due to the sudden shift in resources towards managing this pandemic [ ] . in addition, surgical departments have had to prioritise and cancel surgeries, balance the safety of patients and staff, and assume the risk of operating where surgery is essential to life or limb [ , ] . there is ongoing research in many medical specialties in areas that relate to covid- , and urological research is one of these. studies have examined not only how covid- may affect bodily systems in relation to pathophysiology, but also how it may impact routine care in a speciality, from an administrative and clinical practice perspective [ ] . original studies are required to inform guidelines, provide country-specific recommendations, and advise how health-care services may operate effectively. current guidelines amidst this covid- pandemic frequently base recommendations on prior research [ ] [ ] [ ] . most of these publications consist of expert opinions, perspectives, or commentaries, with limited up-to-date research to guide these recommendations [ , ] . individual country-level recommendations seem warranted, given the vastly different impacts of covid- on nations, and the circumstances that surround them. thus far, it is not known where urology-related covid- research is published. this comprehensive review aimed to: identify all up-to-date original publications worldwide, reveal the origin of these publications, and summarise the studied topics in this field. this review has been reported according to the preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews-recommended checklist, with protocol available (supplementary file ) [ ] . this review aimed to capture all original urological research related to covid- . any manuscript was required to investigate covid- and urology in: the study population, intervention, comparison, or outcome. only original research observational or experimental studies were included. included articles could be published, in-press, or pre-print awaiting peer-review. case reports, editorials, reviews and guidelines were excluded. commentaries and letters were included if they reported original data. our review was not restricted by language. articles were restricted to / / onwards. our comprehensive search strategy was performed on may . our initial search was a combined search on medline and embase. the key search terms consisted of: 'covid- ', 'urology', 'kidney', 'ureter', 'bladder', 'laparoscopy' and 'robotic'. our primary search was adapted to: cinahl, global index medicus, and the cochrane library. grey literature was searched using google scholar. the top urology journals by impact factor were also searched. a reference search was conducted from included manuscripts. a search of trial registries identified currently ongoing studies [ ] . registries included: australian new zealand clinical trials registry, international standard randomised controlled trial number registry, international clinical trials registry platform, and clinicaltrials.gov. after extraction and removal of duplicates, two authors (lq and mp) contributed to the screening of initial titles and abstracts, and subsequent full texts for eligibility and final inclusion. any disagreements were resolved through discussion. data recorded included metadata and study content. data synthesis was presented in a narrative format. available evidence was summarised and presented according to broad topic categories. a world heatmap was generated, using arc-gis desktop release . (environmental systems research institute, redlands, ca, usa), demonstrating location of covid- -related urological publications by affiliated countries. this was overlaid with up-to-date covid- case data at the time of our search strategy, according to the world health organization [ ] . risk of bias and quality assessment was performed using the critical appraisal skills programme checklist [ ] . studies were categorised as either poor, average, or good quality. all included studies were analysed due to the scoping nature of this review. the search strategy returned a total of initial search results (fig. ) . after first-stage screening, there were titles and abstracts for full text review. the primary reason for exclusion was for research that was not relevant or not original. in total, there were studies included- manuscripts were pre-print manuscripts and were ongoing studies from trial registries. quality assessment of included manuscripts (excluding seven registered trials) demonstrated / 'good', / 'average', and / 'poor' studies. all studies were included in our subsequent narrative synthesis. of the included manuscripts and trials, countries were represented from first author affiliations (fig. a) . china, italy, and the usa were the major contributing countries. there were countries represented by all co-authors ( fig. b ). all currently registered trials were single-country trials: four from france; two from italy, and one from poland. the most commonly reported field of literature was translational (n = ). other commonly reported topics were kidney transplantation and telemedicine (fig. a) . current studies suggest sars-cov- may involve the urinary tract; however, the possibility of urinary transmission has not been conclusively answered. farkash et al. investigated the link between sars-cov- and renal failure [ ] . using autopsy specimens, the authors demonstrated the presence of direct renal infiltration of sars-cov- . light microscopic examination demonstrated mild autolysis and some vacuolisation, which correlated to viral forms within tubular epithelial cells using ultrastructural analysis. peng et al. examined the presence of viral rna in pathological specimens using quantitative real-time polymerase chain reaction [ ] . of the nine patients with confirmed covid- , only one patient had detectable sars-cov- in urine, without any urinary tract symptoms. pan et al. have reported / seminal samples from men with covid- for sars-cov- , with a median of days from symptom onset to sample collection [ ] . song et al. reported negative semen samples in all men ( / ), but only one patient had a positive pharyngeal swab at the time of semen collection [ ] . quan et al. have reported / patients in their pre-print, but the presence of ongoing acute infection or time from symptom onset was not reported [ ] . these findings suggest sexual transmission is unlikely, but further clarification is required relating to acutely infected individuals. several studies suggest a possible association between angiotensin-converting enzyme ii (ace ), its co-receptor tmprss , and covid- . chai et al. reported the patterns of ace expression and survival for patients with covid- and cancers of over types [ ] . although papillary renal cell carcinomas (prccs) had increased ace expression, this was not associated with survival outcomes [ ] . yang et al. confirmed an association of [ ] . another pre-print examining ace expression throughout the urinary tract reported higher expression in testicular gametocytes and in renal proximal tubules [ ] . finally, wu et al. report in their pre-print, the relationship between androgen receptor activation and ace and the usa (n = ). b total cumulative incidence of covid- cases by country, as of may , has been illustrated (shades of blue). affiliated countries of all co-authors of included publications were tallied and displayed for each country (red circles) tmprss expression, and how this may contribute to the higher mortality from covid- in males [ ] . currently, two ongoing studies from france are registered. one study is examining kidney involvement in covid- -infected patients (nct ). another study is investigating the predictive value of interleukin- and disease severity in kidney transplant patients (nct ). two studies investigated androgens in patients with covid- , and associations with disease severity. in a pre-print by ma et al., a case-control study was performed with covid- male patients and controls [ ] . serum luteinising hormone to testosterone ratio was negatively associated with disease severity (p = . ) on univariate regression, but was not associated on multivariate analysis. a pre-print by schroeder et al. from germany reported on covid- patients in intensive care [ ] . numerous male patients demonstrated low testosterone ( . %) and low dihydrotestosterone ( . %) levels, correlating to a greater mounted viral immune response, suggesting testosterone may be associated with a protective immune response. clinical studies have supported the concept of androgen deprivation and a protective effect against covid- severity. an observational study in italy with covid- patients consisted of ( . %) men with prostate cancer. montopoli et al. reported patients not on androgen-deprivation therapy (adt) had significantly higher severity of covid- compared to prostate cancer patients receiving adt (odds ratio [or]: . ; % ci . - . ) [ ] . a pre-print by duga et al. reported on α-reductase inhibitors and adt in a longitudinal study of patients with covid- (n = ). the proportion of their sample on α-reductase inhibitors was lower compared to national population statistics ( . % difference, % ci . - . ). this may suggest a protective effect from α-reductase inhibitors from developing covid- infection; however, further clarification is required [ ] . one retrospective study has investigated covid- incidence post-urological procedures, comparing a covid- referral hospital to a covid- -free hospital in italy [ ] . there were no cases of covid- detected among the patients in the referral hospital. in contrast, of the patients at the covid- -free hospital, there were three covid- patient cases and two treating urologists infected. these findings suggest benefits of stricter safety measures in reducing transmission of covid- . the impact of covid- on resident well-being was assessed in a study from france. abdessater et al. surveyed the impact of covid- on french urologists in training ( . % response rate) [ ] . most respondents felt an increase in stress ( . %). on multivariable regression, greater experience level (or = . , % ci . - . ) and a need to manage covid- patients in the urology department (or = . , % ci . - . ) adversely affected the quality of their work. amparore et al. reported the results of a survey of urology residents ( % response rate), assessing the change in clinical and surgical activities in italy. involvement in training was reduced or suppressed in up to % of clinical and % of surgical activities. compared to junior residents, final year residents experienced a significant reduction in involvement in open (p = . ) and minimally invasive (p = . ) surgical procedures [ ] . an ongoing study in italy with recruited participants has been registered, investigating covid- and training across all surgical subspecialities (nct ). another study from poland is investigating the impact on surgical wards and procedures, aiming to recruit surgeons and surgical residents (nct ). several studies have reported increasing rates of telemedicine use across urology departments. in italy, the implementation of a telemedicine outpatient clinic was examined over weeks. of the scheduled patients, % had cancelled their appointments, and the resultant weekly proportion of face-to-face consultations decreased from to %. of faceto-face visits, % were regarding new or recurrent malignancies or dangerous clinical conditions, reflecting appropriate triaging of patients [ ] . a pre-print study in the usa reported the effective uptake of telemedicine before and during the covid- pandemic. clinic video visits increased from mean per week ( - %) pre-covid- to per week ( - %) during the pandemic [ ] . the level of patient satisfaction of the telemedicine consult has also been evaluated. in a study from germany, up to % of patients preferentially opted for telemedicine consult over face to face, with no difference between oncological versus benign diseases ( % vs %, p = . ) [ ] . similarly, davis et al. surveyed preference for telemedicine in a uro-oncology clinic over a -week period. lower no-show rates were identified with audio-visual consults compared to face-to-face ( % vs %, p < . ), while % of patients reported a preference for telemedicine consults in the future [ ] . a registered study in france (nct ) is similarly assessing the efficiency and satisfaction of telemedicine consults, aiming to recruit patients from a neuro-urology clinic. a study from the usa described the results from a riskstratified triage strategy for inpatient urology consultations over weeks. up to % ( / ) of inpatient consultations were successfully performed via telemedicine, and % ( / ) of covid- -associated consultations were managed using telemedicine alone. there were no reported covid- infections in urology team members [ ] . telemedicine in diagnostic procedures has also been studied. lobo et al. compared the proficiency and diagnostic capability of telecystoscopy with traditional cystoscopy in the covid- era. the authors reported % practitioner proficiency with telecystoscopy, while confidence in identification of abnormalities was equivalent [ ] . in the usa, sobel et al. reported personal protective equipment (ppe) use in urology to determine impact on limited resources. of the procedures, utilisation rates of masks, hats, gowns and gloves were higher in robot-assisted cases (p < . ). robot-assisted laparoscopic cystectomies required the most hats and masks ( . per case) [ ] . urology staff were surveyed regarding recommendations for urological procedures during covid- in a single institution in the usa. in summary, the responders recommend maximising telemedicine, reducing in-room time, appropriately using ppe, and managing consumable supplies for bedside procedures [ ] . wang et al. reported the impacts of covid- on surgical volume and ongoing research trials in china. the authors reported an % decrease in the number of procedures. all patients in ongoing clinical trials experienced protocol deviations of days on average [ ] . mandanelo et al. assessed the rates of urological emergency presentations before and during the pandemic in a single institution in portugal. a retrospective analysis of presentations demonstrated a . % reduction in presentations, with an associated reduction in hospitalisation ( % vs %, p < . ) [ ] . similarly, mantica et al. reported on emergency urological presentations in italy. over days, consultations decreased from to patients; stone-related cases decreased from to patients; while haematuria cases decreased from to patients [ ] . a currently registered study in france is assessing patients undergoing elective and emergency urological procedures to identify variables that affect post-operative outcomes (nct ). the study aims to assess patients to assist with limiting access to surgical care. uro-oncological management has been examined in three studies from italy. campi et al. described the burden of high-priority uro-oncological surgeries from three italian high-volume referral centres. of major surgeries, % were classified as high priority, suggesting that up to two-thirds of major surgeries could be safely postponed [ ] . ficarra et al. further characterised the proportion of uro-oncological surgeries, including transurethral resection of bladder tumours (turbts). of non-deferrable oncological procedures, were major surgeries and were turbts. there were patients who preferred to opt for treatment postponement, of which / were planned for turbts [ ] . a survey of medical oncologists managing urological cancers was conducted to investigate management changes with covid- . the majority of oncologists would still pursue first-line therapy where indicated without delay, with the primary treatment consideration still being survival benefit rather than risk of covid- transmission [ ] . a cross-sectional survey assessed practice changes in stone management from expert european endourologists [ ] . the survey achieved a % response rate from primary authors of the eau section of urolithiasis working groups. most experts reported managing patients with covid- by planning elective readmissions for definitive stone management. up to % of respondents did not perform surgery, while up to % did not accept covid- -positive patients. covid- in patients with kidney transplantation has been described. there were six case series included [ ] [ ] [ ] [ ] [ ] [ ] and one case-control study comparing patients with and without kidney transplants (table ) [ ] . sample sizes ranged from to patients, with reported median ages of - years. a range of - % of patients required intensive care admission, while - % patients died. almost all studies reported immunosuppressant cessation or reduction for the patients studied-zhu et al. however described one patient with mild covid- symptoms, who did not withhold immunosuppressants, and was subsequently discharged [ ] . currently, one ongoing study from italy has been registered, investigating hospitalisation for covid- in patients with transplantations of kidney, pancreas or pancreatic islet (nct ). our review aimed to highlight the current landscape of urological original research during the covid- pandemic. studies were included, investigating pathophysiological, administrative, and clinical outcomes relating to covid- and urology. current pathophysiological research appears to focus on characterising ( ) the extent of detectable viral load in human pathological specimens, and ( ) the role of ace , tmprss , and androgens. translational studies suggest ace and tmprss act as susceptible entry points for sars-cov- [ ] . given tmprss is a previously described dysregulated gene in prostate cancer, studies have investigated its relation to sars-cov- [ ] . this concept has been explored clinically, where androgen deprivation may downregulate tmprss activity, which may in turn influence covid- severity [ ] . although current findings are preliminary, they suggest that androgen deprivation may exhibit protective effects for patients from covid- . covid- infection rates have also been examined between different hospital urology units. notably, a urology unit within a covid- referral centre demonstrated no in-hospital covid- infections, while a non-covid- hospital recorded five new infections. despite the expected greater risk of transmission at a covid- referral hospital, the study findings suggest stricter safety measures employed at a referral centre may actually pose lower risk of viral exposure [ ] . current administrative research areas have been examined. the reduction of surgical activities of up to % for residents as reported by amparore et al. should be addressed through careful restructuring of residency programmes. urology surgical caseload has been associated with confidence and training satisfaction, as previously described for urology residents across europe [ ] . telemedicine initiatives have also demonstrated efficacy in implementation and patient popularity. these programmes may lead other healthcare services to adopt urological telemedicine services in both the inpatient and outpatient settings. clinical fields of covid- -related urological research seem to focus on uro-oncology, urolithiasis, and kidney transplant recipients. in addition to studies reporting general changes in urological presentations, uro-oncology studies have examined prioritisation of non-deferrable surgeries and choice of therapies, while one study has reported on expert management opinions for urolithiasis. studies of kidney transplant recipients are currently focused on characterising the clinical progress of covid- . of note, this comprehensive search did not identify any original research that examined endourology, reconstructive urology, paediatric urology, or functional urology. because reviews were produced as results from the search pertaining to these topics, the search likely had adequate sensitivity [ , ] . rather, this finding may represent a deficiency in original research in these fields. additional studies investigating sars-cov- in urine were not captured in this review [ , ] . this was likely due to a study focus towards other pathological specimens given negative detection in urine. likewise, authors from only countries produced original publications. these findings do not reflect the total research output relating to covid- , and other countries may contribute in other fields. of note, the countries contributing the most manuscripts were china, italy, and the usa. these [ ] italy case series ( %) a ( %) ( %) ( %) abrishami [ ] iran case series ( %) a - ( %) ( %) zhang [ ] china case series ( %) a - ( %) ( %) zhu [ ] china case-control c . range . - years ( %) a ( %) - ( %) countries represent some of the most affected nations across the world [ ] . although this review has highlighted key areas of current urological research during the covid- pandemic, these must be interpreted with caution, especially when informing clinical practice. studies were reported as if they represented entire nations when most were conducted within single institutions. furthermore, eight pre-prints were included to increase the scope of our review-these must first undergo a peer review. nevertheless, our review is the first to provide a comprehensive country-level analysis of current original urological research related to covid- . this review summarised original research relating to covid- and urology, spanning nations, and covering pathophysiological, administrative, and clinical research topics. this comprehensive overview has provided a landscape of what research exists and where it has been published, reflecting areas of high covid- burden across the world. this review may assist clinicians and researchers to identify current and future areas of study relating to their subspeciality or nation. lg qu: project development, data collection/ analysis, manuscript writing/editing. m perera: data collection/analysis, manuscript writing/editing. n lawrentschuk: manuscript writing/ editing. r umbas: manuscript writing/editing. l klotz: manuscript writing/editing. the authors declare no funding was used to support this study. conflict of interest the authors declare that they have no conflict of interest. data sharing data will not be available online, but will be shared upon request. informed consent not applicable. hospital surge capacity in a tertiary emergency referral centre during the covid- outbreak in italy surgical volume, safety, drug administration, and clinical trials during covid- : single-center surgical safety surgical safety in radical cystectomy: the anesthetist's point of view-how to make a safe procedure safer the impact of covid- on european health care and urology trainees impact of the covid- pandemic on paediatric urology practice in europe: a reflection from the european association of urology young academic urologists risks from deferring treatment for genitourinary cancers: a collaborative review to aid triage and management during the covid- pandemic european association of urology guidelines office rapid reaction group: an organisation-wide collaborative effort to adapt the european association of urology guidelines recommendations to the coronavirus disease era impact of covid- on urologists: learning on the go stepping forward: urologists' efforts during the covid- outbreak in singapore prisma extension for scoping reviews (prisma-scr): checklist and explanation infrequent use of clinical trials registries in published systematic reviews in urology world health organization ( ) coronavirus disease (covid- ): situation report critical appraisal skills programme (casp) ultrastructural evidence for direct renal infection with sars-cov- sars-cov- can be detected in urine, blood, anal swabs, and oropharyngeal swabs specimens no evidence of sars-cov- in semen of males recovering from covid- absence of novel coronavirus in semen and testes of covid- patients no sars-cov- in expressed prostatic secretion of patients with coronavirus disease : a descriptive multicentre study in china genetic alteration, rna expression, and dna methylation profiling of coronavirus disease (covid- ) receptor ace in malignancies: a pan-cancer analysis ace correlated with immune infiltration serves as a prognostic biomarker in endometrial carcinoma and renal papillary cell carcinoma: implication for covid- expression of ace , the sars-cov- receptor, and tmprss in prostate epithelial cells ) scrna-seq profiling of human testes reveals the presence of the ace receptor, a target for sars-cov- infection in spermatogonia the ace expression in sertoli cells and germ cells may cause male reproductive disorder after sars-cov- infection multiple expression assessments of ace and tmprss sars-cov- entry molecules in the urinary tract and their associations with clinical manifestations of suppression of androgen receptor (ar)-ace / tmprss axis by ar antagonists may be therapeutically beneficial for male covid patients effect of sars-cov- infection upon male gonadal function: a single center-based study the majority of male patients with covid- present low testosterone levels on admission to intensive care in hamburg, germany: a retrospective cohort study androgen-deprivation therapies for prostate cancer 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telecystoscopy compared to traditional cystoscopy, a pilot study personal protective equipment for common urologic procedures before and during the united states covid- pandemic: a single institution study considerations for bedside urologic procedures in patients with severe acute respiratory syndrome coronavirus- el impacto de la pandemia del covid- en la utilización de los servicios urológicos de urgencias stepping forward: urologists' efforts during the covid- outbreak in singapore eur urol assessing the burden of nondeferrable major uro-oncologic surgery to guide prioritisation strategies during the covid- pandemic: insights from three italian high-volume referral centres re: riccardo campi, daniele amparore, umberto capitanio, et al. assessing the burden of urgent major uro-oncologic surgery to guide prioritisation strategies during the covid- pandemic: insights from three italian high-volume referral centres the shifting landscape of genitourinary oncology during the covid- pandemic and how italian oncologists reacted: results from a national survey urolithiasis practice patterns following the covid- pandemic: overview from the eulis collaborative research working group preliminary data on outcomes of sars-cov- infection in a spanish single centre cohort of kidney recipients molmenti ep ( ) covid- in kidney transplant recipients the columbia university kidney transplant program ( ) early description of coronavirus disease in kidney transplant recipients in new york a single center observational study of the clinical characteristics and short-term outcome of kidney transplant patients admitted for sars-cov pneumonia clinical course, imaging features, and outcomes of covid- in kidney transplant recipients identification of kidney transplant recipients with coronavirus disease coronavirus disease pneumonia in immunosuppressed renal transplant recipients: a summary of confirmed cases in wuhan, china sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor current status of urology surgical training in europe: an esru-esu-esut collaborative study paediatric urology practice during covid- pandemic detection of sars-cov- in different types of clinical specimens clinical and virological data of the first cases of covid- in europe: a case series key: cord- -syy okhi authors: dettori, marco; deiana, giovanna; balletto, ginevra; borruso, giuseppe; murgante, beniamino; arghittu, antonella; azara, antonio; castiglia, paolo title: air pollutants and risk of death due to covid- in italy date: - - journal: environ res doi: . /j.envres. . sha: doc_id: cord_uid: syy okhi the present work aims to study the role of air pollutants in relation to the number of deaths per each italian province affected by covid- . to do that, specific mortality from covid- has been standardised for each italian province and per age group ( groups) ranging from - years to > years, based on the national population figures. the link between air pollutants and covid- mortality among italian provinces was studied implementing a linear regression model, whereas the wide set of variables were examined by means of lisa (local indicators of spatial autocorrelation), relating the spatial component of covid- related data with a mix of environmental variables as explanatory variables. as results, in some provinces, namely the western po valley provinces, the smr (standardized mortality ratio) is much higher than expected, and the presence of pm( ) was independently associated with the case status. furthermore, the results for lisa on smr and pm( ) demonstrate clusters of high-high values in the wide metropolitan area of milan and the po valley area respectively, with a certain level of overlap of the two distributions in the area strictly considered milan. in conclusion, this research appears to find elements to confirm the existence of a link between pollution and the risk of death due to the disease, in particular, considering land take and air pollution, this latter referred to particulate (pm( )). for this reason, we can reiterate the need to act in favour of policies aimed at reducing pollutants in the atmosphere, by means of speeding up the already existing plans and policies, targeting all sources of atmospheric pollution: industries, home heating and traffic. air pollution is at present one of the main problems for public health. according to reports from the world health organization (who) [ ], it is responsible for million deaths worldwide every year. in europe, an estimated , people die prematurely each year from exposure to air pollutants [ ] . the main air pollutants taken into consideration are particulate matter (i.e. pm . and pm ), carbon monoxide (co) and carbon dioxide (co ) and nitrogen-based components (e.g. nox). these substances mainly derive from anthropic activities (combustion, traffic, industry), agriculture and cattle breeding which alter the atmosphere's composition [ ] . the damage caused to human health varies and depends both on the concentration of pollutants, and on individual subjectivity and the duration of exposure. several conditions can be attributed to exposure and they vary from mild and transitory to chronic forms. furthermore, it has been shown that a continuous exposure to levels of pollutants above the regulatory limits causes a chronic inflammatory and hyperergic state which can lead to a greater predisposition to infections and to the symptomatic development of disease [ ] , as well as other pre-existing immune alterations [ ] . thus, living in an area with high levels of pollutants could lead a person to be more prone to developing chronic respiratory conditions and consequently susceptible to infectious agents. in relation to the above, since who has drawn up guidelines setting exposure limits for both the short and long term. despite this, regulatory limits are not the same worldwide and vary from country to country [ ] . the recent spread of the sars-cov- virus, responsible for the covid- pandemic, has raised various questions in the academic community in relation to why some countries were primarily subject to a greater spread of the virus and suffered higher lethality [ ] . italy, in particular, was the country most affected by covid- immediately after the appearance of the virus in wuhan, china. the disease has assumed dramatic characteristics throughout the nation, with , infections and , deaths recorded to date, a higher lethality than china and other european countries and numerous differences also within the same national territory [ , ] . different hypotheses have been put forward among the possible explanations for the primary spread of the virus within the country, also in relation to the recent observations showing various similarities from the geographical and social point of view between hubei province and northern italy [ ] . notwithstanding the commercial links between italy and china, and the various opportunities for contact and importation of the virus into our country, understanding why the virus had such a sudden spread in some italian territories is one of the aspects that currently stimulates the scientific debate on an international level. moreover, some other factors may be involved in facilitating the spread of viruses into the community. actually, a previously-proven phenomenon regarding the spread of other viruses (i.e., measles) [ ] attests that the levels of atmospheric pollution and, above all, of particulates, could act as a vehicle for the spread of the virus throughout the territory. nonetheless, setti et al. recently demonstrated the presence of the sars-cov- rna on particulate matter [ ] . therefore, a combination of factors related to air quality, such as pollution and, in general, a wide range of environmental conditions, could be considered responsible for targeting the respiratory tract and weakening the population at risk, at the same time increasing their likelihood of being affected by respiratory diseases like covid- [ , ] . in italy, the areas most affected are found within the po valley, known for its high levels of air pollution, apparently sparing a large part of central italy and most of southern italy. on the basis of these premises, and of a series of recent researches, that show a higher case fatality rate in the regions of the po valley [ , ] , the present work aims to evaluate the relationship between air pollution and covid- at a provincial level. in particular, it has aimed to study the role of air pollutants and a set of environmental variables, selected from recent observations [ , ] , in relation to the number of deaths per each italian province affected by covid- . the present study did not require ethical approval for its observational design according to italian law (gazzetta ufficiale n. dated . . ). italy is located in the southern part of the european peninsula, in the mediterranean sea, and has coasts on the tyrrhenian, ionian and adriatic seas. it covers a surface area of , . km² and its population numbers , , inhabitants [ ] for an average population density of inhabitants per square kilometre. from an administrative point of view, italy is divided into in regions -one of which, trentino alto adige, is split into autonomous provinces with regional competencies. most of the population is concentrated within the geographical area of the po valley, surrounded by the alpine and apennine mountains, and to the east by the adriatic sea towards the po delta and the area represents italy's economic "core". this geographical area includes the provinces of: turin; venice; vercelli; novara; milan; bergamo; brescia; verona; vicenza; padova; asti; alessandria; piacenza; parma; reggio emilia; modena; bologna; forlì-cesena; rimini; pavia; lodi; cremona; mantova; rovigo; ferrara; and ravenna. almost million people live in this area of approximately , km , with a density ( inhabitants per km ) double that of the rest of the peninsula, with a higher concentration in the main urban areas of the greater milan metropolitan area. covid- data takes into account the number of total infected people and the number of deaths as at th june at a provincial level, as reported by the italian ministry of health, and as collected by the national institute of statistics (istat) [ ] . environmental data come from istat, ispra (higher institute for environmental protection and research), il sole ore (an economic and business newspaper, which provides constant reports on economical facts), aci (automobile club d'italia), and legambiente (non-profit association for environmental protection) [ , , [ ] [ ] [ ] . the data relating to the environmental pollutants investigated,(i.e., pm , pm . and no ), come from the official italy-wide monitoring and refer to the average pollution values obtained from detection by the control units. the sources of data acquisition relating to environmental variables are shown in table . the spatial units selected are italian provinces, the intermediate levels between municipalities and regions, still in use as statistical units also by istat. while many provinces lose or change their administrative role, those selected are spatial units provided by istat as of . specific mortality from covid- has been standardised for each italian province and per age group ( groups) ranging from - years to > years, based on the national population figures. the indirect standardisation process initially provided for the calculation of national specific mortality by age group, obtained by dividing the number of covid- deaths to june and confirmed by the national institute of health (iss) [ ] by the defined age groups. thus, the number of deaths expected in the italian provinces for the age groups previously identified and based on the provincial populations, was calculated according to the formula: where n i is the specific age group population in each observed area (province); r i is the national mortality rate for the specific age group. the standardized mortality ratio (smr) was obtained by comparing the number of events observed in each province with the respective number of expected events: where d is the number of observed deaths for covid- ; e the number of expected deaths. finally, the % confidence intervals ( % ci) were calculated as proposed by vandenbroucke [ ] . in order to evaluate the correlation between the environmental variables a pairwise correlation analysis was performed. the link between air pollutants and covid- mortality among italian provinces was studied implementing a linear regression model. the analysis included, as independent variables, important air pollutants such as pm , no and pm . and various factors specific to the urban environment such as cycle paths, pedestrian streets, trees, soil, public and urban green areas, motorcycles and cars (table ) . these variables, previously studied in a recent observation [ ] , and selected on the basis of the direct (vehicular traffic) and indirect (urban green, cycle paths, land use and pedestrianised areas) role in relation to the presence of the air pollutants investigated, were compared with the dependent variable (smr) outcome. in particular, linear regression was carried out using the periodic averages of the atmospheric pollutants in question, obtained from the detections carried out by the official monitoring systems (control units). the level of significance was established at p < . with a type i error of %, the confidence intervals were calculated at %. statistical analysis was performed using stata . (stata corp, college station, tx, us), and medcalc (medcalc software ltd., ostend, belgium). for comparison reasons, some analyses on spatial autocorrelation among variables have been taken into consideration [ , , , ] . the wide set of variables were examined by means of lisa (local indicators of spatial autocorrelation), relating the spatial component of covid- related data (i.e., cases and deaths per province) with a mix of environmental variables as explanatory variables, such as annual average of pm , and pm , no , numbers of trees per inhabitants and urban green areas, number of vehicles and cycle paths, as reported in table . lisa enables an evaluation of the similarity of various observations to their neighbouring provinces, for each location [ , , , ] . in particular, lisa produces results as clusters of areas characterised by varying levels of similarity. high-high values indicate the presence of both strong values of the phenomenon and high similarity with its neighbouring provinces. low-low values represent low values of the phenomenon and low similarity with its neighbors. high-low values represent high values of the phenomenon and low similarity; at the same time, low-high values indicate low values and high spatial similarity. non-significant values represent situations of low importance of attribute and spatial closeness. the standardized mortality ratio (smr) compared the covid- mortalities with that which was expected, basing on national official data. in provinces, namely the western po valley provinces, including the mountainous ones, and on the adriatic coast of the regions of emilia romagna and marche, the standardized mortality ratio is much higher than expected (table ) . table shows the average annual values of pm by reference province. the data relating to smr for the selected provinces are shown graphically in figure . the correlation coefficients between the environmental variables investigated are shown in table . table shows the results of the linear regression analysis for the association between environmental variables (pm , no , pm . , cycle paths, pedestrian areas, trees, soil, urban green spaces, motorcycles and cars) and smr. the presence of pm (p= . , % ci: . - . ) was independently associated with the case status. no significant association with case status was found with the other variables (p > . ). considering only the pm variable, the relationship with the smr is shown in figure . furthermore, figure shows the nationwide situation, divided by province, in relation to the smr, and at the same time highlights the geographical distribution in relation to the data on pm . the map of smr distribution in italy considers provinces grouped in classes. the two classes below unity present covid- related mortality values lower than those expected. the intermediate class presents the value around unity and slightly higher than unity, thus showing a mortality in line with the expectations, or slightly higher, while the other remaining two classes present the values where covid- related mortality is much higher than that expected. with reference to the pm map, the ranges are based on the who and italian limits for particulate, based on multiple of (i.e., μg/mc is the who limit; μg/mc is the italian limit, recently reduced from μg/mc, etc.). regression analysis has been compared also in terms of spatial autocorrelation, for comparison purposes. the results of the spatial autocorrelation analysis are summarised and shown in figure and refer only to the smr and pm . figure shows [ , ] the results for lisa on smr and pm . the results demonstrate clusters of high-high values in the wide metropolitan area of milan and the po valley area respectively, with a certain level of overlap of the two distributions in the area strictly considered milan. covid- mortality highly depends on the prevalence of the disease and transmission rate, and human contacts among the population represent a key factor. nevertheless, other factors could play a role in favouring the infection, such as genetic factors [ ] , or environmental factors [ , ] . the existence of relationships between air pollution and covid- mortality was first hypothesized, taking into consideration air pollution from the particulates (pm . and pm ) and nitrogen based components deriving from human activities. the idea was that the presence of air-related pollutants can put pressure on the health conditions of the populations at risk and offer preconditions for the development of respiratory related diseases and their complications, including some that are life-threatening, which may explain the high case fatality rate observed in the po valley area [ ] . table ranks the italian provinces where the smr as of th june was higher than , indicating a higher increase in mortality than expected. this is a set of provinces, where, by means of example in the po valley area, a city like lodi, with a value of . , presents an increase times more than expected. it can be seen that the most affected areas are found in the po valley and are particularly characterised by a high average yearly value of pm . a similarity in the density classes, which mainly include between and people per square kilometer is also noticeable. the analysis of covid- related mortality shows quite a clear divide between northern italy on one side and central and southern italy on the other, along the apennine mountain chain, with values higher (much higher than expected) in the north, and values in line with the expected mortality in other italian regions, particularly in the south. thus, based on observations recently published by the authors, a set of environmental variables was selected in order to evaluate the association with mortality from covid- in the italian provinces. bearing in mind that the main source of some air pollutants may be the same, we performed a pairwise correlation analysis between the air pollutants, and some of these variables showed a statistically significant correlation. in particular, the highest correlation index (coefficient = . ) was found between pm and pm . . this aspect is attributable to the fact that the particulate detection systems currently in place return the average concentration of pm including the fraction relating to pm . . therefore, the two variables are strongly correlated. nevertheless, as uncovered by the linear regression, only pm was found to have a strong relation to the number of deaths distributed for each italian province affected by covid- . it is important to consider this factor for risk stratification, as close monitoring of air emissions and appropriate measures implemented at a local level may help to reduce air pollution. moreover, although, as mentioned, pm is strongly correlated with pm . , multivariate linear regression analysis has enabled us to highlight how the variable pm has a dependence relationship with specific mortality for covid- such as to be the only statistically significant variable, to the point that it can be considered as an independent predictor of mortality for covid- , and an early indicator of epidemic recurrence as suggested by setti et al. [ ] . furthermore, some spatial autocorrelation can be found regarding pm and smr, showing a certain level of similarity between the most affected provinces in northern italy by covid- and the highest recorded values of pm . numerous studies hypothesise a correlation between the presence of air pollutants and the mortality. in particular, in addition to a recent systematic review of the literature [ ] , which highlights the important contribution of pm , pm . and no as triggers for the spread and lethality of covid- , one us study has shown that a small increase in long-term exposure to pm , leads to a large increase in the mortality rate for covid- [ ] while, currently, a study by the iss and the ispra called pulvirus aims to investigate the controversial link between air pollution and the spread of the pandemic and the physical-chemical-biological interactions between fine dust and viruses [ , ] . in any case, these are observational studies, it will therefore be necessary to carry out further studies for an evaluation especially from an etiological point of view [ , ] . what has emerged therefore is that a profound reflection on the monitoring of air emissions is required, in particular of pm , which did not substantially decrease during the lockdown. in fact, through monitoring it is possible to verify the effectiveness of the measures implemented at a local level to reduce air pollution. this precaution should be included in the agreement of the po valley for the improvement of air quality, signed in bologna during the g environment ministers' meeting of th june , by the minister of the environment and the presidents of lombardy, piedmont, veneto and emilia -romagna. moreover, the pandemic could represent an opportunity to contrast the community outrage due to the perception of the environmental risks [ , ] . as far as the environmental pollution and both the spread of the virus in generating outbreaks and case fatality rate are concerned, this research appears to find elements to confirm the existence of a link between air pollution, this latter referred to particulate (pm ) and covid- mortality. particular atmospheric conditions in the early weeks of may have aggravated the environmental situation in the po valley area. quite an evident divide between northern italy on one side and central and southern italy on the other is clear to see, with provinces north of the apennine mountains presenting values higher in the north, and lower values of mortality -also lower than those expected -in the south, in all the periods considered. a spatial distribution of covid- related deaths through smr presents similarities in the spatial patterns drawn especially with particulates, as represented by the regression analysis thus far presented. as regards to suggestions in terms of policies, we can reiterate the need to act in favour of policies aimed at reducing pollutants in the atmosphere, by means of speeding up the already existing plans and 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doi: . / . . . sha: doc_id: cord_uid: brurrmi on march , , the world health organization declared the covid- outbreak, originally started in china, a global pandemic. since then, the outbreak has indeed spread across all continents, threatening the public health of numerous countries. although the case fatality rate (cfr) of covid- is relatively low when optimal level of healthcare is granted to the patients, the high percentage of severe cases developing severe pneumonia and thus requiring respiratory support is worryingly high, and could lead to a rapid saturation of intensive care units (icus). to overcome this risk, most countries enacted covid- containment measures. in this study, we use a bayesian seir epidemiological model to perform a parametric regression over the covid- outbreaks data in china, italy, belgium, and spain, and estimate the effect of the containment measures on the basic reproduction ratio r_ . we find that the effect of these measures is detectable, but tends to be gradual, and that a progressive strengthening of these measures usually reduces the r_ below , granting a decay of the outbreak. we also discuss the biases and inconsistencies present in the publicly available data on covid- cases, providing an estimate for the actual number of cases in italy on march , . lastly, despite the data and model's limitations, we argue that the idea of "flattening the curve" to reach herd immunity is likely to be unfeasible. more than countries ( ) in the world are currently affected by the coronavirus disease (covid- ) pandemic ( ) . covid- is a respiratory infectious disease caused by the sars-cov- virus (previously known as -ncov), and it originated in december in wuhan (china), most probably following a zoonotic event ( , ) . covid- epidemics are now affecting many european countries, which are at different stages of contagion and containment measures ( ) . the virus can be found in the respiratory tract of patients - days before the onset of symptoms, where it shows active replication ( ), persisting - days ( ) . italy was the first to be seriously affected ( ) , with spain, france, belgium, and other countries being - days behind. although definitive data on the covid- case fatality rate (cfr) are still missing and the current ones are biased by the testing policies and the demographic structure of the population, the observed cfr may be as high as of . % in italy, . % in china, . % in spain, and . % worldwide. in italy, it has been observed that - % of the cases present acute respiratory distress syndrome (ards) caused by sars-cov- pneumonia, and thus require respiratory support in intensive care units (icus) ( , ) . european countries tend to have between . (portugal) and . (germany) icu beds per , inhabitants ( ) (eu average is . ). this indicates that an exponential-like growth of the covid- cases can rapidly reach oversaturation of the available icu beds, thereby decreasing the quality of the medical treatments provided to patients and worsening the case fatality rate ( , ) . to avoid this scenario, almost every country affected by the covid- pandemic has put in place measures to contain the epidemic, in an attempt to relieve the strain on the healthcare system. when it comes to epidemic modeling, these measures affect the basic reproduction ratio r , which is the expected number of cases directly generated by an infected individual in a population susceptible to infection ( ) . current estimates of this value range from and . in china ( ) ( ) ( ) ( ) and . in the first phase of the outbreak in italy ( ) . in seir (susceptible, exposed, infected, removed) ( ) modeling of epidemics, r = β/γ, with β representing the number of contacts from an infected individual per unit of time and γ − the period in which a patient is infectious. when r > , the number of cases is growing, else, the epidemic is receding. countries affected by the covid- pandemic deployed containment measures that acted on these two parameters. china, for example acted on r by quarantining or hospitalizing cases as soon as they were becoming symptomatic, with an average time elapsed between symptoms and hospitalization of . ( , ) or . days ( ) . similar measures have been adopted by the european coun-tries in which covid- started spreading ( ) . italy was the first country affected in europe, and the first covid- cluster prompted the lockdown of the town of codogno and later of the lodi province. the further spreading of the cases led to the lockdown of the most affected regions in northern italy and eventually of the entire country. the growth of cases in spain started with a delay with respect to italy, but the growth trend prompted the government to lock down first madrid and then the entire country. similar incremental actions have been adopted in belgium, on the march , and on march , . in this study, we collected the publicly available data regarding cases, recovered and deaths related to the covid- epidemics in china, italy, belgium and spain and we trained a bayesian seir model to perform a parametric regression on these time series. we modeled the outbreak progression in those countries inferring the change of basic reproduction ratio r due to the introduction of government-issued containment measures aimed at slowing the outbreak. approaches similar to ours could help governments nowcasting the behavior of the outbreaks and detecting flaws in the containment measures in place and thus act as rapidly as possible, ensuring a proper containment of the disease. we show that the parameters learned by the seir model suggest an gradual effectiveness of the containment, with the most drastic effect observed in china, with a % reduction of r after the measures introduced on february , . we also provide an estimation of the actual number of covid- cases in italy for march , , suggesting that ) this number could have been at that time around times higher than the official count and ) unreported people in the - -yrs age group might have played a crucial role in the virus diffusion. finally, we argue that the idea of "flattening the curve" (i.e., reducing the r of the epidemic to a level that would allow the gradual build up of natural immunity in the population) is likely to be unfeasible, since reaching herd immunity at a manageable pace is probably not possible in a reasonable time scale. we performed a parametric bayesian regression (see methods) on the mainland china covid- epidemic data by training a seir model on the cumulative cases time series, with the goal of inferring the change in r = β/γ produced by the increasingly stringent containment measures introduced by the chinese government, which mainly aim at reducing the frequency of the contacts β − between individuals. we thus used the β i before and after the introduction of each containment measures as sole trainable parameters in our seir model. we kept γ − fixed to . , which is the mean between the current estimates for the mean time elapsed between insurgence of symptoms and hospitalization in china, which correspond to . ( , ) and . days ( ) . in this study we used an average incubation time δ − = . days, as reported in ( ) . the implementation of the first containment measure in china happened on february , , when all public transportation was suspended in wuhan, corresponds to a % decrease in the inferred r , bringing it down from . (ci % [ . , . ]) to . (ci % [ . , . ]). the introduction of the more stringent measures on february , , including closing all non-essential companies and manufacturing plants in hubei province corresponded to a further reduction to the r identified by our seir model, down to . . italy is the first european country that has been severely hit by the covid- pandemic, and, at the time of writing, it is the second nation in the world in terms of cases, with , . the italian government reacted to the epidemic by closing all schools and universities on march , , putting the north of italy under lockdown on march , , and extending this lockdown to the entire country few days later, on march , . on march , , the government introduced even stricter measures, banning open-air sports and closing parks and public green. we used the data of the italian covid- outbreak provided by the protezione civile to infer the r before and after the containment measures were implemented. we fixed γ − to be equal to , which is the median time between insurgence of symptoms and hospitalization, as estimated by the istituto superiore di sanità (iss) ( ) . the only free parameters in our bayesian seir regression were thus the β i associate to each date in which containment measures have been implemented as only free parameters, as shown in fig. . from this analysis, it appears that the initially inferred r = . (ci % [ . , . ] ) is in line with the . estimate provided in ( ) . the r = . (ci % [ . , . ]) inferred after the nationwide lockdown in effect from march , suggests that these measure had a gradual effectiveness and that did not provide an immediate dramatic change in r . for example, the effects these containment measures were initially less evident than the ones implemented in china, even though the data clearly departed from a situation without containment measures in place (see suppl. fig. ). this suggests that the measures were effective but their actual implementation was more gradual. allowing a change in β also on march , nevertheless shows that r decreased to . (ci % [ . , . ]), initiating the decrease of the new cases. belgium is one of the european countries in which the covid- pandemic arrived later, with the first confirmed case reported on february , . the growth of the number of cases in belgium started from march , , and on march , , the belgian government issued containment measures involving the closure of schools, cafes and restaurants starting from march , . the government then extended these measures, enforcing stricter "physical distancing", starting from march , , at noon. we modeled the growth of the covid- epidemic in belgium using the bayesian seir model to infer the effect of the containment measures through a change in the β i parameters. we fixed γ − to be days, since no official data on the mean time between symptoms onset and hospitalization is available from official sources and we assumed china to be an upper bound in terms of hospitalization efficiency. from fig. we can see that the seir model infers a change in r that decreases from the original . (ci % [ . , . ]) to . (ci % [ . , . ]), suggesting a situation similar to italy after the introduction of the first containment measures. the high initial value obtained for r might be partly explained by under-reporting of cases (see discussion). although the first covid- case in spain dates to march , , the epidemic there did not show worrying numbers until the end of february, with a rapid growth starting from the beginning of march. this crisis was answered first with containment measures in the community of madrid, enforced from march , , followed by nationwide measures enforced from march , . we used the seir model to infer the change in β due to the implementation of the containment measures. similarly to the italian and belgian case, we observe a clear decrease in the r , but the model deems it not drastic enough to reverse the trend of the epidemic already. this might be because of under-reporting of the actual number of covid- cases. testing. the official counts of covid- cases are in manu cases severely under-estimated and affected by clear biases, due to ) the limited number of tests that can be run and ) their preferential usage on symptomatic cases or high-risk subjects. in an attempt to address this issue, we computed an estimate of the actual number of covid- cases in italy on march , . to do so we relied on the fatality rate (cfr) of the disease and the age distribution of the cases in south korea, which adopted an extensive testing strategy to face the covid- crisis, administering one test every citizens, with no evident biases. we thus considered this to be the most reliable data available: south korea shows indeed a pearson correlation coefficient between the number of cases detected among -yrs age bins and its demographic structure of r = . (p-value= . ), while italy has an r = . (p-value= . ), suggesting a much more skewed testing. our estimation is based on three other assumptions: ) that the disease propagated similarly in south korea and italy over the different age bins; ) that south korean and italian healthcare have similar standards, thus suggesting a comparable fatality rate once the testing bias is addressed; and ) that the healthcare system in italy (e.g., the availability of icu beds) has not reached saturation, and to satisfy this condition we indeed chose to perform this estimation for march , , as lockdown measures in italy appear to be the result of the healthcare system rapidly approaching saturation. we first adjusted the south korean number of cases by age group with respect to the demographic structure of the italian population. as reported on figure (green bars), the age of confirmed patients is heavily skewed towards older individuals in italy, while it is more consistent with the demographic structure in the south korean data. we argue that the skewness of the italian cases towards older age groups results from the fact that on the february , on the italian testing strategy changed from blanket testing to focusing on symptomatic and high-risk individuals ( ) , thus introducing a clear sampling bias. we then adjust the proportion of cases per age in italy (see suppl. material) by computing the proportion ... (orange bars in figure ), and we computed the expected number of cases in italy based on the number of deaths and use it to further adjust the age distribution. in our case, bins from to years old were undefined, but we addressed this issue by using the corrected age distribution of italian cases ... . using the data available on march , for italy and south korea, we find an estimated number of real cases of , , instead of the , officially reported, indicating a three fold increase. analysis of the healthcare system strain-level during the epidemic progression. the covid- pandemic has been putting immense pressure on the healthcare systems of many countries because it spreads widely in the population in an asymptomatic or mild form ( ), but a significant percentage of the symptomatic cases ( - % in italy ( , )) requires icu treatment, which is a limited resource in any country, including european countries ( ) . the availability of icu beds is crucial ( ) because so far there is no established curative treatment for covid- and the clinical best practice is to put patients suffering from acute respiratory distress syndrome under respiratory support, for a period that may last up to two weeks ( ) . saturation of icu capacity causes a dramatic decrease of the quality of the medical treatments provided to patients, thereby worsening the observed case fatality rate (cfr) ( , ) . to analyze the burden that the covid- epidemic brings to the national healthcare systems of the affected countries, we plot the evolution over time of the log-cfr (the log-ratio of deaths per confirmed covid- case). during the first days of the epidemics, we expect this rate to be noisy: due to the delay between infections and deaths, the number of deaths remains very low during the first days while the number of cases grows. after this transition period, first deaths occur and if icu units do not reach saturation, we expect this rate to be stay constant. however, if this rate increases, this suggests that the healthcare system is under strain as it tries to cope with the growing number of patients requiring ards treatment. towards the end of the epidemics, when the cumulative number of cases flattens out, the rate is expected to increase, again due to delay between the reporting of cases and the occurrence of deaths. fig. shows this log-ratio over time in china. after some expected initial oscillations (also possibly due to the change in the testing strategy), china showed a steep increase incidence of deaths with respect to the number of cases, indicating a significant strain over its healthcare system, and possibly the degradation of the quality of the care provided. towards, the end of the epidemics, when the number of cases flattens, we observe a slower steady increase. the same plot is shown for italy in figure . shortly after the first days of the epidemics, the mortality rate started growing quickly, suggesting an increasing strain on icus and the italian healthcare system, as reported also in ( ). effectiveness of containment measures. containment measures in china significantly reduced covid- spreading. the first lockdown resulted in a % decrease of the reproduction factor r and the second, stricter wave of measures eventually managed to bring it to close to . we do however observe that while the model was able to fit the italian, belgian, and spanish data relatively well, its fit of the data from china was rather mediocre. we are unsure about what could have caused this discrepancy. by contrast, containment measures in italy appear to have had a more gradual effect. the reason for this is not entirely clear. data from the lombardy region, based on anonymous cell phone tracking (not showed here), suggests that almost % of the population of lombardy were still commuting and moving around notwithstanding quarantine measures, although the trend from february , and march , clearly indicates a progressive reduction of displacements. such large percentage of the population moving across lombardy might in part be explained by the fact that factory closures were only partial until march , , when the italian government issued a decree mandating the immediate halt of all non-essential production, industries, and businesses across the country. data from the italian interior minister ( ) also indicates that during the lockdown . million police controls were carried out with infractions to the containment registered in % of controls. the analysis of the data from belgium suggested high values for r , despite the introduction of strict quarantine measures, can possibly be explained by some under-reporting in the data. because of limited testing of the population, the number of infectious patients is probably significantly underestimated. because the seir model assumes that the reported infectious patients are the ones infecting new patients, the inferred r may be over-estimated, although is in the same range of r estimations from other sources (r estimates range from and . in china ( ) ( ) ( ) ( ) and has been estimate to be . in the first phase of the outbreak in italy ( ) ). limit the ability to draw clear conclusions. the models fits presented in the results section are based on the officially available covid- cases counts from china, italy, belgium, and spain. even from a superficial analysis of this data, several biases that hinder the modeling of these outbreaks become clear. first, the number of tests that can be run each day is finite, because of the limited availability of supplies and personnel, making blanket testing currently impossible to perform in many countries. this results in a large number of unreported cases with respect to the available data. second, if tests are performed mainly on symptomatic patients for diagnostic purposes, because of the generally higher age of the hospitalized cases, the resulting official covid- cases data will show a striking proportion of patients over years old, regardless of the actual demographic structure of the population (see suppl. fig. ). another reason why the sheer number of tests performed is not a clear indication of the level of bias present in the data is that the number of test performed is just an upper bound for the actual number of individuals screened, because for example medical personnel with high risk of exposure may undergo periodic tests. moreover, the directives of the italian ministry of health indicates that a covid- patient must be negative to two consecutive tests performed with a h delay ( ) to be considered as having recovered from the disease. the cumulative number of cases we used to fit the seir model is therefore most probably both severely underestimated and skewed towards older age groups in the population. both in belgium and in italy, for instance, patients who are diagnosed as suspect covid- case over the phone by their gp, but who present no immediate risk of complication, are nor tested, nor reported as new cases. as the epidemic progresses and healthcare resources become mobilized, testing capacity increases and we observe a growing number of newly tested individuals. interestingly, the estimation of the actual (vs. reported) number of cases on march , in italy suggests that, although heavily under-represented in the official data because of testing bias, the - age group is the most affected by covid- . given that age group is particularly socially active, one might speculate that infections via this age group may have played a key role in the spread of covid- across italy, even though these cases ended up almost completely unreported. there are however some limitations to this analysis. while south korea's testing strategy has clearly been comprehensive, it is not clear that it has been completely unbiased. in particular, the low number of cases in the - years bin compared to the - years bin might be explained by a radical difference in the true proportion of cases between those two age groups, but also by lower testing among younger individuals because they might have been considered at very low risk of complications and/or unlikely to be infectious. the information available does not allow us to discriminate between those explanations. a key assumption of our model is that new infections are caused by contamination from currently reported infectious individuals, because our modeling is based on the observed cases, for which official data exists. however, in practice, many of the newly diagnosed patients have been infected by the majority of unreported infectious people. our model will thus infer an higher r to compensate for the underestimated pool of infectious patients. this might explain the seemingly high values of r estimates in spain for instance. moreover, every country adopted its own specific strategy for testing and reporting of cases, resulting in heterogeneity of the covid- data coming from different countries. for example, south korea opted for blanket testing of its population and selective quarantine of the positive cases, while italy focused on testing high-risk and symptomatic subjects and generalized lockdown of the country to reduce the r by acting on the frequency of social contacts. even within the same country, the reporting strategy changed over time in some cases, leaving a trace in the data. for example, the number of daily new cases in china presents an unlikely spike of , new cases in a single day (february , ) because of a change in the reporting strategy, since also clinically diagnosed covid- cases started to be included in the cases count, alongside laboratory tests. this measure was probably necessary to overcome the saturation of the maximum number of tests that could be performed every day, but caused the sudden inclusion of previous "suspect" cases in the official count. similarly, italy opted for testing only high-risk individuals and symptomatic cases from february , on ( ) . limitations of the model. in suppl. material we discuss some minor limitations of our model. one aspect of covid- which is still unclear is whether presymptomatic individuals (during at least - days of the incubation period) are likely to be contagious with a degree of infectivity that is not yet well characterized. the seir model does not account for these effects and the high value of β obtained might in part be caused by the need to account for those missing contagion events. moreover, the number of symptomatic individuals might also be underestimated because testing is in some cases being focused on most severe cases, which similarly will lead to the inflation of β and r . the parameter γ was set for each country based on prior knowledge obtained from the literature rather than estimated from the data. the rationale for this approach is that parameter estimation from a single time-series of the autonomous response of a time-varying system is inherently challenging. we thus chose to use the information to carry out bayesian inference for a piecewise stationary model with a single parameter β i . more sophisticated models (with more patient compartments) might better capture the different effects described before, but such models will have significantly more free parameters, which means that those parameters might simply be unidentifiable from the available data or that overfitting is likely. about "flattening the curve". despite their limitations, our models show that the idea of "flattening the curve" (i.e., reducing the r of the epidemic to a level that would allow the gradual build up of natural immunity in the population) is likely to be unfeasible. any significant reduction of r that would not bring it extremely close to would overwhelm the healthcare system because the icu capacity and the height of the epidemic peak in a immunologically naïve population are simply on different scales (in the sir, the proportion of the population infectious at the epidemic peak is given by − /r − ln(r )/r . for example, % of the population is infectious at the epidemic peak for r = , while the icu capacity in for example belgium is . beds per , inhabitants ( ) ). even if the epidemic could be controlled at a fixed level corresponding to a heavy but non-overloading load of the icu capacity, the time needed to build herd immunity would be measured in years. as an example, an estimation for belgium based on a permanent icu capacity of , beds for coronavirus patients (compared to the pre-existing capacity of , ( ) beds, which would mean a major continuing strain on the hospital system and thus the need to maintain supplementary capacity for several years), assuming an average icu stay of days ( , ) , and assuming that % of patients affected in the general population would eventually require icu care, would mean that patients would be ad-mitted at icu care per day and that , individuals in the general population would be infected by the disease each day. reaching a level where % of the population (of about million people) has achieved natural immunity would require , days or years. given that the immunity to the disease might be relatively short-lived (around years for sars ( ) ), it might simply be next to impossible to achieve herd immunity without overwhelming the healthcare system. moreover, such a strategy would require maintaining the number of cases in the population at a tightly controlled level with r being maintained on average at . whenever r would be above , the disease would flare up, which would quickly overload a healthcare system maintained at saturation. when r would be below , the disease would start vanishing, which would extend the time needed to build herd immunity. given that it is completely unclear what the precise impact of any containment measure is on r , a strategy based on lifting and reimposing measures to switch between r slightly below and r slightly above does not appear realistic. if a treatment became available that would greatly diminish the risk of complications (for example, by a factor ), or if it turns out that the proportion of the general population that develops severe complications when infected by sars-cov- is much lower than %, it might be possible to revisit strategies based on "flattening the curve". in the absence of such a silver bullet treatment, the only plausible option for the moment seems to be the immediate quashing of the epidemic together with the development of strategies to try to contain the disease at a minimal level driven by imported cases, while waiting for greatly improved treatments or a vaccine. in such strategies, as currently deployed by south korea, hong kong, and singapore for example, patients only arise from imported cases and small local clusters that are rapidly quashed. it is likely that such strategies will sometimes fail in insufficiently prepared populations leading to the reimposing of heavy quarantine measures during the time needed to quash the new epidemic flare. it seems advisable to reimpose strict quarantine measures as soon as uncontrolled local circulation of the disease is suspected. data collection. we collected covid- data from official sources and we list them in supplementary material. seir model. the seir (susceptible, exposed, infected, recovered) is a widely used mathematical model for the description of the behavior of infectious disease outbreaks. we provide the full details in suppl. material. the parameters of this model, which are responsible for tuning the dynamics of the epidemics are δ, β and γ. δ − can be interpreted as the average incubation period (i.e., the average time spent in pool e before becoming infectious i). β corresponds to the average number of infections an infectious individual will cause per unit of time and γ − corresponds to the average time necessary to recover from the disease (i.e., going from i to r). the average number of new infections arising from a single infectious person is then r = β γ . in this study we used δ − = . days ( ) and we adapted γ to the estimated value for the country under scrutiny (see the corresponding country section in results). we consider that those parameters are constant over time, before and during lockdowns. the effect of lockdown measures is then modelled by a change in β. in this work, we considered that a lockdown enforcement resulted in a discrete, instantaneous change in β. in practice, however, the β might have changed only progressively after the adoption of confinement measures, but modeling this will require more parameters, and thus also more and higher quality data will be recommended for the inference. we inferred the β parameters during each period by fitting the cumulative number of cases c(t) with mcmc (metropolis-hastings). we use a poisson likelihood and uniform priors for β. the details of the model are available in suppl. material. . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint time to use the p-word? coronavirus enter dangerous new phase who director-general's opening remarks at the media briefing on covid- - the proximal origin of sars-cov- european centre for disease prevention and control clinical presentation and virological assessment of hospitalized cases of coronavirus disease covid- and italy: what next? the lancet covid- in italy: momentous decisions and many uncertainties. the lancet global health the variability of critical care bed numbers in europe at the epicenter of the covid- pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation pandemic potential of a strain of influenza a (h n ): early findings. science estimation of the transmission risk of the -ncov and its implication for public health interventions early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study time-varying transmission dynamics of novel coronavirus pneumonia in china. biorxiv the mathematics of infectious diseases report sulle caratteristiche dei pazienti deceduti positivi a covid- in italia parere del consiglio superiore di sanità: definizione di paziente guarito da covid- e di paziente che ha eliminato il virus sars-cov- clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study. the lancet duration of antibody responses after severe acute respiratory syndrome key: cord- - mtn m authors: lenti, marco vincenzo; corazza, gino roberto; di sabatino, antonio title: carving out a place for internal medicine during covid‐ epidemic in italy date: - - journal: j intern med doi: . /joim. sha: doc_id: cord_uid: mtn m internal medicine has been immediately involved in the coronavirus disease (covid- ) epidemic in italy, which started in late february . the first few covid- cases were suspected and diagnosed in internal medicine wards, including, among others, a young adult male from codogno (referred to as "patient "), who was later transferred to the intensive care unit of our hospital. the rapid spread of the disease has put much pressure on the general population, patients, healthcare professionals and on the healthcare system as a whole, which was not prepared to face such an unpredictable event, especially in terms of number of patients needing admission to hospital all at once [ ] . the san matteo hospital foundation (pavia, italy), an academic, tertiary referral hospital, is located close to the first outbreak of codogno, and also relatively close to nearby cities in lombardy which were later hit by the epidemic, such as bergamo, brescia and cremona. the division of infectious disease, the molecular virology unit, the anaesthesia & intensive care unit, the emergency department, the pulmonology unit and the risk management unit dedicated all their efforts in order to overcome the growing epidemic, undergoing a rapid process of reorganization and expansion [ ] . internal medicine was also called to the front line and has proactively responded with great flexibility to the growing number of cases, transforming its divisions into departments dedicated to the care of covid- . paradoxically, a discipline that has chronicity as the main object of study found itself at the forefront of an acute epidemic. at our hospital, two entire internal medicine units, in a few days, were transformed into 'covid' wards. patients who were already admitted for reasons other than covid- were either transferred to 'non-covid' hospitals, or, when appropriate, discharged home within two-three days. during the first days since ward transformation, patients with covid- have been admitted, three quarters of them requiring noninvasive ventilation. this number is quite impressive, considering that the total number of beds in the two internal medicine units is and that the intensity of care markedly increased. one quarter of patients were discharged home after a mean time of nine days, whilst less than one in ten required to be transferred to the intensive care unit. from a clinical point of view, the internist was not floored by this 'viral' event, as the management of pneumonia and respiratory failure still pertains to her/his field of expertise. further, covid- , due to its heterogeneity, can be considered an internal medicine condition, as it is more than a singleorgan disease, being the gastrointestinal tract, the central nervous and cardiovascular systems all possible targets. this disease can onset with varied and nonspecific symptoms, such as cough, muscle pain, ageusia, anosmia and diarrhoea, and can be complicated by myocarditis, acute hepatitis and kidney failure [ ] . the internist is already prepared to treat this type of patients, as the management of complex diseases is the norm rather than the exception. important ethical and pragmatic principles are called into question, pointing at the need for transparency and inclusivity, as it has been recently stated [ ] . suffice it to say that italy has one of the world's most aged populations, and multimorbidity is the main clinical feature of the elderly population. ageing and multimorbidity are certainly the most important determinants of frailty, which is related to adverse health outcomes. in this scenario, internal medicine adaptability, spacing from a primary to a tertiary care setting, could also act as a link between hospital and territory medicine, especially for patients with multiple chronic conditions who cannot be left abandoned. the paucity of resources deriving from covid- , including medications, ventilators, available beds and physicians, forces us to carefully evaluate who to treat and how to treat. the elderly population is the most affected by covid- according to preliminary data reported daily by the italian ministry of health and the civil protection. the overall mortality rate is roughly %, and most deaths occurred in individuals aged more than years old. whether these patients died from, or died with, covid- will need to be ascertained, even if the bidirectional relationship between acute and chronic conditions cannot be dissolved. our first reaction to all these abrupt changes was that of frustration for the sense of helplessness deriving from the absence of specific therapies, the enormous workload under stressful conditions and for the fear of the risk of being infected. after the first moments of loss, the proactive implementation and the rapid reorganization of the department into 'dirty' and 'clean' pathways, and the availability of novel experimental therapies certainly raised doctors' morale. the use of monoclonal antibodiessuch as tocilizumab, which has the rational to counteract the cytokine storm underlying covid- is not uncommon in an internal medicine setting. also, given the relatively small 'window of opportunity' for the use of tocilizumab, a careful risk-benefit profile assessment should be made, taking into account patients' age, comorbidities and overall clinical presentation. despite the aforementioned corrective actions, the risk of burn-out amongst physicians still remains high. above all, incommunicability is the most relevant feature when coping with covid- . before entering the ward, physicians must wear protective clothing, including suit or gown, apron, head covering, gloves, mask and goggles. most patients also require oxygen therapy which constitutes another barrier to communication. in such a scenario, empathy can be transmitted only through eye contact or a quick touch of patient's hand, so to create a sort of 'healing bond', which will continue even after discharge. the role of the internist does not end with patient's discharge, as organ injuries induced by covid- might be irreversible thus predisposing to the development, or worsening, of chronic diseases such as pulmonary fibrosis and chronic kidney disease. hence, the internist could be in charge of the management of these patients since the onset of covid- , until the occurrence of late complications. in summary, internal medicine has responded promptly to covid- italian epidemic, due to its holistic attitudethe sick patient as an individual, rather than the disease, is the object of the studyand its methodological approach [ ] . bill gates, with regard to covid- , stated that 'leaders have two equally important responsibilities: solve the immediate problem and keep it from happening again' [ ] . paraphrasing this sentence, we may say that internal medicine could solve the immediate problem and could prevent it in the future. first, it has considered clinical reasoning prior over technical ability and technological instruments. secondly, it has proved capable of adapting in relation to the various clinical settings and to the changing burden of disease. finally, internal medicine has deemed humanism as an essential part of clinical practice [ ] . hence, this crisis may represent a great opportunity to rethink the healthcare system in a more rational and patient-centred way. none. from the department of internal medicine, san matteo hospital foundation, university of pavia, pavia, italy covid- and italy: what next? rapid response to covid- outbreak in northern italy: how to convert a classic infectious disease ward into a covid- response centre clinical characteristics of coronavirus disease in china facing covid- in italy -ethics, logistics, and therapeutics on the epidemic's front line issues for american internal medicine through the last century responding to covid- -a once-in-a-century pandemic? trusting internal medicine in hard times we thank all the internists who are currently facing the covid key: cord- -nx m authors: trisolino, giovanni; origo, carlo enrico; de sanctis, nando; dibello, daniela; farsetti, pasquale; gigante, cosimo; guida, pasquale; marengo, lorenza; panuccio, elena; toniolo, renato maria; verdoni, fabio; memeo, antonio title: recommendations from the italian society of pediatric orthopaedics and traumatology for the management of pediatric orthopaedic patients during the covid pandemic and post-pandemic period in italy date: - - journal: ital j pediatr doi: . /s - - - sha: doc_id: cord_uid: nx m the rapid spread of the covid- outbreak in italy has dramatically impacted the national healthcare system, causing the sudden congestion of hospitals, especially in northern italy, thus imposing drastic restriction of almost all routine medical care. this exceptional adaptation of the italian national healthcare system has also been felt by non-frontline settings such as pediatric orthopaedic units, where the limitation or temporary suspension of most routine care activities met with a need to maintain continuity of care and avoid secondary issues due to the delay or suspension of the routine clinical practice. the italian society of pediatric orthopaedics and traumatology formulated general and specific recommendations to face the covid- outbreak, aiming to provide essential care for children needing orthopaedic treatments during the pandemic and early post-peak period, ensure safety of children, caregivers and healthcare providers and limit the spread of contagion. the coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) was first identified in wuhan (hubei province, china) in late , rapidly spread worldwide, and, on march th, , the world health organization (who) officially declared covid- as a pandemic [ ] . in the same days, the government of italy imposed a national quarantine that locked down for about months all commercial and industrial activities (with some exceptions), school and universities, sports, cultural and leisure activities [ ] . as the contagion rate and death toll covid- related continued to decrease, on april th, the prime minister announced the so-called "phase " for the reopening and resumption of the activities scheduled for may th, . on may th, , the who reported , , confirmed cases of covid- ( , cases in italy), including , deaths ( , in italy) [ ] . so far, along with the hubei province, italy (and in particular northern italy) has had the longest period of lockdown during the covid- pandemic (almost months in northern italy). the rapid spread of the covid- outbreak has immediately caused the congestion of many hospitals, thus imposing the temporary interruption of all non-essential medical cares. this exceptional adaptation of the italian national health service was significantly evident also within non-frontline healthcare settings such as pediatric orthopaedic units, where limitation and temporary suspension of most routine care activities was necessary to reduce the risk of infection in patients, families, and healthcare providers and to reallocate healthcare personnel from routine tasks to emergency. on the other side, the limitation of healthcare services to the essential ones, along with the general reluctance among people to access care for fear of covid- exposure, led to a in increased risk and worsening of covid -unrelated diseases. in italy, an increase of death and worsening pediatric diseases due to delayed access or provision of care has been reported [ ] . therefore, a need for preventing the risk of delays in access to care is essential especially in children, in order to avoid complications due to the alteration or suspension of the typical patient care. given the profound uncertainty about the actions to be taken, on march , the advisory board of the italian society of pediatric orthopaedics and traumatology (sitop) launched an initiative to gather local experiences and epidemic risk management protocols from nine tertiary referral centers for pediatric orthopaedics and traumatology in italy concerning the covid- outbreak (fig. ) . this produced an initial emergency document that was approved on april th, , concerning the measures to be adopted during the pandemic period and a second document that was approved on may th, the present document provides general and specific recommendations for pediatric orthopaedic surgeons, who face the so-called "pandemic period" and "post-peak period". additional protocols and guidelines concerning the management of pre-pandemic and inter-pandemic periods are included into the who guidance document [ ] . the recommendations listed in this document have the following aims: . provide essential care to pediatric patients needing orthopaedic treatments during the covid- pandemic and early post-peak period; . ensure safety of children and caregivers in case of hospital admission; . ensure safety of medical staffs; . limit the spread of outbreak. these recommendations should be considered not as mandatory guidelines but rather as a support to reassure families and pediatric orthopaedic surgeons regarding the possibility to treat children safely during the pandemic and post-peak period. therefore, they can be adapted and modified depending on locally available resources and personal experience of the pediatric orthopaedic staff. we believe that they could be useful for future management of upcoming pandemics or during an eventual second wave of covid- , after that safety and effectiveness of these approaches will be analysed, using the data collected during this period. we are also confident that the experience gained across some of the main italian pediatric orthopaedic hospitals could be helpful also in other countries which are facing the pandemic at an earlier phase. separate access to triage areas, emergency, wards, outpatient clinics, operating theatres must be defined, based on suspected or confirmed infection (path a: sars-cov negative patients. path b: sars-cov positive or suspected patients). the paths must be physically separated, limiting asmuch as possible the possibility of communication between path a and path b [ ] . staff within path b must take level iii precautions and protective measures (see table ). the personnel within the path a and b should take precautions and protection measures, according to the current who guidelines [ ] (table ) . filter areas and covid- testing using nasopharyngeal swabs must be implemented for patients/caregivers scheduled for urgent/elective surgery before admission to hospital. cancel all deferrable outpatient appointments with clear notice to the patient through their phone numbers, in order to reduce the circulation of users and staff within hospital structures. regular follow-up visits should be temporarily suspended. appointments should be preserved for children requiring non postponable post-operative care (for example percutaneous k-wires removal, cast removal or renewal, medication of complicated wounds). children with recent onset and progressive exacerbation of pain or functional impairment, even in absence of trauma, should be also visited in person, in order to rule out severe diseases such as bone tumors, infections and acute rheumatic diseases (septic arthritis, osteomyelitis, juvenile arthritis) or severe developmental orthopaedic diseases (scfe, perthes disease, etc..), which may necessitate non deferrable treatments. newborns should be visited in person in agreement with neonatologist or pediatric recommendation. the interval between appointments must be prolonged, in order to avoid crowding the waiting rooms and allow disinfection of the outpatient facilities. in any case, telemedicine must be encouraged whenever possible. follow up imaging should be taken near the locality of the patient and e-mailed to the institute, only if likely to make a significant change to care [ , ] . carefully define and organize the management and priority of urgent and emergent interventions. children should be visited and treated by surgeons experienced in pediatric orthopaedics and nonoperative treatments should be encouraged and performed directly by the experienced senior surgeons. whenever feasible, use and teach parents to remove self-removable casts or splints, to reduce the follow-up requirement. when considering surgical management, the following priority protocol should be adopted: fractures or acute injuries with severe neurovascular involvement and potential risk for life, limb loss, or permanent damage. screen the patient using rapid response sars-cov tests (nasopharyngeal swab). if absent, or if surgery is required in less than - h, treat the patient as covid- -positive, until proven otherwise, in order to minimize infection spread. provide patient with ffp during transit from emergency to operation theatre. clean her/his in case of elective surgery, a strict priority must be maintained in the waiting list, especially if the duration of the pandemic period cannot be precisely estimated [ , ] . the advisory board of the sitop has provided a panel of priority levels in order to safely schedule deferrable surgical treatments, reducing the risk of missing children who require non postponable operations, during the pandemic and post peak period (see table ). this priority panel has considered several factors such as: . duration of the pandemic period and the local epidemic density; . availability and accessibility of hospitals and surgical rooms; . characteristics and severity of the pediatric orthopaedic disease; . range of age of patients, since the favourable outcomes and even the feasibility of some pediatric orthopaedic procedures (for example, closed or open reduction of severe cdh, ponseti method for severe ctev, growth modulation procedures,) are significantly impacted by age at treatment; . type of operation and surgical technique, since some procedures are at higher risk for dissemination of the infection. based on this priority panel elective surgical procedures should be categorized in four classes: table sars-cov- related personal protection management. adapted from the who guidance for rational use of personal protective equipment for covid- [ ] . ffp / = filtering facepiece particles. a the screening procedure refers to prompt identification of patients with signs and symptoms of covid- . b agp: tracheal intubation, non-invasive ventilation, tracheotomy, cardiopulmonary resuscitation, manual ventilation before intubation, bronchoscopy. c : this category includes the use of no-touch thermometers, thermal imaging cameras, and limited observation and questioning, all while maintaining a spatial distance of at least m. d : the number of visitors should be restricted. if visitors must enter a covid- patient's room, they should be provided with clear instructions about how to put on and remove ppe and about performing hand hygiene before putting on and after removing ppe; this should be supervised by a health care worker (continued) . priority a: intervention that should be performed within days from the start of the pandemic phase; . priority b intervention that could be performed within months from the start of the pandemic phase. cases belonging to this priority should carefully monitored both for age of the child and local epidemic density, in order to start treatments as much as possible close to the start of the postpeak phase . priority c: intervention that could be performed within the first months from the start of the postpeak period . priority d: intervention that can be safely performed at the end of the pandemic. healthcare providers must inform the family that only one parent/caregiver can assist the child during hospitalization. at hospital admission, both child and parent/caregiver must be mandatory screened for sars-cov . in patient care must be organized so that only one child and only one parent/caregiver per room are allowed. if the patient or parents are known or suspected to be covid -positive, the operation should be postponed until the covid tests are negative. as tests can occasionally become negative after more than month, if such delay can seriously threaten the health of the child due to the orthopaedic pathology, the surgeon must evaluate if the treatment must be anticipated and the patient can be safely managed through the path b. surgical treatments belonging to the priority class a or b should not be suspended during the pandemic period especially if the duration of this period lasts for more than two to months. the possibility to continue with high priority interventions in children should be preserved. wherever possible, depending on the local setting of healthcare services, the management of such pediatric orthopaedic conditions should be centralized in non-covid hospitals. strict surveillance of the infection should be maintained and all measures taken to ensure patient safety in the hospital (filter areas, separated paths, screening measures, protections, distancing measures) should be continued in anticipation of a possible second wave of infection. outpatient care can be progressively resumed, maintaining the distancing measures. wherever feasible, local health facilities should coordinate in order to redistribute outpatient visits, avoiding overcrowding of hospital facilities. patients should be contacted by phone and/or e-mail to confirm re-appointment. emergency and surgical care should maintain the same organization, recommendations and priority classes of the pandemic period. elective surgery can be gradually resumed, respecting the order of priority established during the pandemic phase. priority b-c surgical treatments could be prioritized close to the end of the pandemic period, depending on the local stage of outbreak, the accessibility and availability of operating rooms. when elective surgery must be scheduled in the immediate post-peak period consider to prioritize those operations, which: . require minimally invasive, arthroscopic or percutaneous techniques; . do not require post-operative intensive care unit recovery; . can be safely managed within day-surgery or with minimal in-patient care (possibly < days); . when possible, postponing at late stage of post-peak patients with comorbidities (especially cardiovascular or respiratory); . must be performed within a definite range of age (for example guided growth procedures). wherever possible we recommend using surgical techniques that significantly reduce the risk of aerosolgenerating procedures (see table ). local/regional anesthesia should be preferred to invasive airway management whenever possible for elective orthopaedic procedures of the upper and lower extremity [ ] . the covid- pandemic has dramatically impacted the health systems at a global level. the need for a rapid adaptation and response of the health providers to the pandemic has imposed the suspension of most routine healthcare services, potentially harming children who require covid unrelated care. the sitop working group has drawn up these recommendations with the aim of supporting the decisions of the pediatric orthopaedic surgeons for ensuring continuity of care in children requiring orthopaedic treatments during a pandemic. we are confident that the experience gained across the main italian pediatric orthopaedic hospitals could be helpful for other professionals involved in children's care, as well as for pediatric orthopaedic surgeons from other countries, which are facing the pandemic at an earlier phase. the sitop will also monitor, through multicenter data collection and analysis, the adherence to these recommendations and their safety and effectiveness, to estimate the impact of this coordinated initiative on the health of children with orthopaedic diseases. world health organization: rolling updates on coronavirus disease (covid ) covid- pandemic in italy delayed access or provision of care in italy resulting from fear of covid- world health organization. pandemic influenza preparedness and response. a who guidance document. geneva: world health organization surgery in covid- patients: operational directives rational use of personal protective equipment for coronavirus disease ( covid- ) and considerations during severe shortages: interim guidance recommendations for control and prevention of infections for pediatric orthopaedics during the epidemic period of covid- recommendations for the care of pediatric orthopaedic patients during the covid pandemic covid- -esska guidelines and recommendations for resuming elective surgery the sitop advisory board wishes to thank riccardo contessi for his work and support in organizing the web meetings which allowed the formulation of the recommendations contained in this article. authors' contributions gt, ceo and am conceived and wrote the manuscript. gt, ceo, nds, dd, pf, cg, pg, lm, ep, rmt, fv, am critically formulated the guidelines and recommendation included in the manuscript, edited drafts, read and approved the final manuscript. the authors did not receive funding for the present study.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -b vmnwv authors: cena, hellas; chieppa, marcello title: coronavirus disease (covid- –sars-cov- ) and nutrition: is infection in italy suggesting a connection? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: b vmnwv novel coronavirus disease (covid- ) was declared a global pandemic on march , . the outbreak first occurred in wuhan, hubei, china, in december and hit italy heavily in february . several countries are adopting complete or partial lockdown to contain the growth of covid- infection. these measures may affect people's mental health and well-being but are necessary to avoid spreading the pandemic. there has been a gradual increase in studies exploring prevention and control measures, and we recommend paying close attention to nutrition, which may contribute to modulating some important consequences of covid- infection, as such pro-inflammatory cytokine storm. novel coronavirus disease spread to all regions of italy on january , . the northern region of lombardy was identified as the center of the two main italian clusters of cases. on march , , the director-general of the who declared covid- a global pandemic as the virus spread rapidly from china to the rest of the world, particularly europe ( ) . currently, the scientific community is sharing potentially useful data to treat patients and protect the population, but the details of covid- infection are still largely unknown, and thus options for risk assessment and pharmacological intervention have only been partially developed. by march , more than , confirmed cases of covid- had been reported, of which , were registered in italy, so far the country with the highest mortality rate ( . ) ( ) . four regions in italy have reported , cases of covid- , including , confirmed infections solely in italy's epicentral lombardy region, corresponding to more than a third of the total number of infected people in the entire country (ministry of health). italy's mortality rate may be partially explained by the country's relatively higher proportion of older people. similarly to what has been observed in china, the most common symptoms were fever, cough, and fatigue ( ) . acute respiratory distress syndrome (ards) was the main cause of death ( ) . older adults and people with chronic illness are most vulnerable to the worst effects of the disease. knowledge about novel covid- is based on a few months of observation and some similarities to severe acute respiratory syndrome (sars) and to middle east respiratory syndrome coronavirus (mers-cov). despite the lower case fatality rate than mers and sars, covid- has so far proven extremely contagious. moreover, a significant percentage of those who are infected require hospitalization in an intensive care unit. here we speculate on a possible link between nutritional status and covid- mortality, based on data emerging from the italian national health system. furthermore, while waiting for clinical trials to shed light on the clinical efficacy and beneficial effects of antibodies and anti-inflammatory cytokines, we highlight nutritionally derived products that may inhibit the inflammatory cytokine secretion caused by covid- infection. subjects with diabetes are at risk of infections and have severe disease when infected with a respiratory virus, showing an increased risk of mortality ( ) . data from mers-cov, which broke out in in saudi arabia, showed that the severity and length of the pulmonary pathology observed were increased in those affected by type diabetes, which likely deregulates immune response ( ) . data on covid- in patients with diabetes is limited at present ( ). the chinese centre for disease control and prevention published a report of , cases of covid- , showing an increased mortality rate in subjects with diabetes ( ) . diabetes is the most common comorbidity observed in covid- -positive deceased patients in italy after hypertension ( ) . available data on pre-existing comorbidities updated on april , , and extracted from clinical charts showed hypertension and diabetes before hospitalization in . % (n = , ) and . % (n = ) of covid- -positive deceased patients, respectively ( ). the same data showed that among , covid- -positive deceased patients, . % (n = ) presented three or more comorbidities that had been diagnosed before covid- infection, . % (n = ) had two, . % (n = ) had one, and only . % (n = ) had no pre-existing pathology ( ). diabetes is prevalent in our population, and most patients with diabetes type ii are overweight or affected by obesity ( ) . however, obesity is not included within the who "fiveby-five" framework of non-communicable diseases (ncds) and risk factors, and data on bmi are not collected in a standardized manner . unfortunately, we do not yet have weight, height, and waist circumference data for all patients with laboratory-confirmed covid- , and, therefore, we cannot disentangle the effects of adiposity on lung function and immune response to viral infection. excess body weight and increased visceral adiposity are habitually associated with metabolic alterations such as insulin dysregulation, high fasting glucose levels, hyperlipidemia, or systemic hypertension, which cause dysregulation of the immune system through mediation in various immune, metabolic, and thrombogenic responses. however, the clinical impact of this immune dysregulation on susceptibility to and severity and outcome of viral infections and on lung function is not yet clearly understood ( , ) . nevertheless, preliminary data from giviti (https://giviti. marionegri.it/covid- /) presented on march , , showed a high prevalence of obesity ( %) and overweight ( %) in italian patients, median age years, from different italian icus, confirming evidence available so far in the literature. recent data on patients with laboratory-confirmed covid- treated at an academic health institution in new york city, the epicenter of the covid- outbreak in the united states, between march , , and april , , with follow up through april , ( , ) showed that obesity, after age, was linked to more severe coronavirus cases, with a substantially higher odds ratio than any cardiovascular or pulmonary disease. obese and obese-diabetic subjects undergo modifications of the innate and adaptive immune response at different phases, characterized by a state of chronic, and low-grade inflammation and a high basal concentration of several pro-inflammatory cytokines such as alpha-tnf, mcp- , and il- , leading to a defect in innate immunity ( ). recent evidence indicates that obesity not only increases the risk of infection and of complications for the individual but also increases the chance of appearance of a more virulent viral strain, prolonging virus shedding, and eventually increasing the overall mortality rate of an influenza pandemic ( ) . hence, both diabetes and obesity impair the immune response to viral infections like influenza and influenza vaccination through alterations of the cellular immune system ( ) . studies so far suggest that diabetics, as well as subjects with obesity, are at a greater risk of hospitalization and increased complications from influenza ( , ) . compared with vaccinated healthy-weight adults, vaccinated obese adults have twice the risk of influenza or influenza-like illness despite equal serological response to vaccination ( ) . this should be considered one of the challenges to be overcome in vaccine development and/or medications to combat this virulent respiratory virus and prevent future epidemics similar to covid- . obesity is also associated with chronic low-grade inflammation, dysbiosis, and increased secretion of inflammatory cytokines, including interleukin (il- ) ( ) . elevated plasma levels of pro-inflammatory cytokines are observed in covid- infected patients; in particular, il- and ferritin release have been identified as predictors of fatality ( ) . several studies focusing on previous outbreaks of severe influenza confirmed that the mortality caused by organ injury could be reduced by immunomodulatory agents ( , ) . currently, a study on the safety and efficacy of tocilizumab is being conducted (clinicaltrials.gov identifier: nct ) ( ) to assess its capacity to suppress the virally driven hyperinflammation and acute respiratory syndrome caused by covid- . as mentioned previously, covid- -related deaths are due to acute respiratory distress syndrome (ards); nonetheless, data indicate that the covid- virus is detectable in stool of infected patients, suggesting systemic manifestations ( ) . in line with this observation, reports indicate abundant expression of ace in absorptive enterocytes of the gi tract ( ) and diarrhea as among the most frequent infection symptoms ( ) . inflammation of intestinal mucosa may result in increased intestinal permeability with a consequent cascade of events that cause persistent inflammation, worsening the infectionrelated symptoms. immune homeostasis is a dynamic process maintained by a complex interplay between the gut microbiota and host mucosal immune system ( ) . dysbiosis, defined as imbalances in gut microbial species, is now a wellrecognized factor in the pathogenesis of age-associated frailty ( ) . it is possible that covid- mortality is increased in older patients with comorbidities associated with intestinal dysbiosis, as this could support systemic chronic inflammation in the host. nonetheless, only future studies will clarify this aspect. ace -expressing epithelial cells are the primary targets of covid- . similarly to other pulmonary viral infections, following primary exposure, the progeny proliferate in the host cells, which consequently die and release their contents. covid- is now able to infect other cells, including alveolar macrophages ( ) . activated or infected immune cells secrete excessive pro-inflammatory cytokines and chemokines, fuelling a vicious circle leading to pulmonary tissue damage. data accumulating from covid- patients indicate that these patients might have a cytokine storm syndrome, with markedly higher levels of ifn-γ, ccl- , ccl- , tnf, and the aforementioned il- ( - ). for optimal functioning of the immune system, an adequate nutritional status is required. this is well-acknowledged from evidence linking nutritional deficiencies to the functionality of the immune system ( ) . poor nutrition leads to poor immune defense, and it is frequently associated with figure | deleterious effects of the anti-covid- strategy (red) can impact the base of the mediterranean diet pyramid. an increase in polyphenol uptake and substitution of meat with legume-derived proteins can help prevent chronic inflammation, and reduction of caloric intake may compensate for the reduction in physical activity. alcohol consumption is discouraged for healthy people and is detrimental for patients, even in the absence of clinical symptoms. impaired immunity and increased susceptibility to infection. nevertheless, nutrient inadequacies and deficiencies in our habitual diet are common ( ) , and immune function may be improved by restoring nutrients to recommended levels, increasing resistance to infection, and hastening recovery once infected ( ) . furthermore, studies have revealed inadequate micronutrient levels in patients who were hospitalized in the infectious disease department, including thiamine, selenium, zinc, and vitamin b deficiencies ( ) , which were associated with adverse clinical outcomes. early detection, prevention, and treatment should aim at decreasing the inflammatory response and avoiding the excessive post-inflammatory immune suppression, defined as compensatory response ( ) , that is observed in many such patients ( ) . various micronutrients are essential for immunocompetence, particularly vitamins a, c, d, e, b , b , and b , folic acid, iron, selenium, and zinc, as well as macronutrients likely omega fatty acids ( ) and bioactive components as polyphenols ( ) . during recent years, our groups, together with several others worldwide, demonstrated the ability of nutritionally derived bioactive compounds to suppress inflammatory cytokine release ( ) ( ) ( ) ( ) . in particular, the administration of several plant-derived polyphenols to in vitro cultured immune cells suppressed the release of inflammatory cytokines ( ) . traditional chinese herbal medicines for influenza treatment demonstrated potential antiviral activity ( , ) . polyphenol bioavailability has been long discussed, as many have observed health benefits, but few have observed circulating traces of bioactive compounds ( , ) . nonetheless, exposure of the epithelial barrier to a polyphenol-rich environment can efficiently activate local immune suppression and tissue repair mechanisms ( ) , and systemic benefits may be related to the release of circulating microrna, defined as small non-coding rna molecules, with anti-inflammatory effects ( ) . indeed, even if orally introduced, bioactive dietary factors induce mirna synthesis, these are packaged into exosomes and released into the bloodstream to act systemically ( ) . dietary factors are emerging as anti-inflammatory mirna promoters able to regulate metabolic functions, inflammation, and oxidation systemically ( ) ( ) ( ) ( ) . furthermore, herb extracts may combine antiviral, anti-inflammatory, and antioxidant activity, and tissue-repair properties ( , ) (figure ) . altogether, these observations suggest that people may benefit from a correct nutritional intake, particularly during this period of uncertainty. choosing dietary regimes that may potentially work as adjuvants for preventing undesired hyper-inflammation might be particularly useful for patients with mild signs of infection (figure ). general recommendations for healthy adults over years of age observing a period of lockdown and thus with limited options for physical activity should focus on healthy dietary patterns. these can be generally described as those rich in plant-based foods, including fresh fruits and vegetables, soya, nuts, good sources of antioxidants ( ) , and omega- fatty acids ( ) and low in saturated fats and trans fats, animalderived proteins, and added/refined sugars ( ) . moreover, mild energy restriction is recommended for obese and obese-diabetic patients ( ) . most of these dietary targets can be met in our country by means of the well-known and traditionally familiar mediterranean diet ( , ) , which is rich in polyphenols with immune-protective and anti-inflammatory activities, playing an adjuvant role in both prophylaxis and therapy ( ) . the scientific community is already discussing how to manage future epidemic outbreaks by learning from the current experience ( ) . future studies should also focus on the effects of nutrition on immune function, identifying target population subgroups with the most vulnerable immune systems, such as the elderly and those with comorbidities. hc and mc wrote the manuscript. available online at clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china coronavirus covid- global cases by the center for systems science and engineering clinical 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compliance of the italian public with voluntary home quarantine has been very high, but little is known about the impact of psychological health on sociodemographic categories during the quarantine. the purpose of this study was to assess the prevalence of depressive symptoms in specific sociodemographic categories during the covid- quarantine lockdown and the potential factors that contribute to, or mitigate, these effects. in the very early stage of the nationwide lockdown, , quarantined italian adult residents ( % females, ranging from to years) participated in a web-based cross-sectional survey, including measures of depressive symptoms, which were measured by the teate depression inventory, and state anxiety levels. the overall prevalence was . % for moderate and . % for severe levels of depressive symptoms. a generalized logistic model was used to identify the factors associated with mental health problems. among these factors, sociodemographic variables (e.g., sex, age, employment status) and adherence to quarantine guidelines were analyzed. females, younger people, students, singles, residents in northern italy, people who were reluctant to adhere to quarantine guidelines, and people less worried about being infected with covid- were at high risk of developing depressive symptoms during the covid- epidemic, also after controlling for state anxiety. these findings showed that public levels of depressive symptoms did not increase the greater likelihood of being infected. our study suggested that the monitoring of psychological outcomes for outbreaks could identify groups at higher risk of psychological morbidities due to the current pandemic in order to target future psychological interventions for implementation. the pandemic of coronavirus disease , caused by the severe acute respiratory syndrome coronavirus (sars-cov- , previously known as -ncov) has affected the italian community since late january. according to the imperial college covid- response team (ferguson et al., ) , cumulatively, . ( . - . ) million people had been infected as of march , giving an infection rate of . % ( . - %) of the italian population. to contain the rapid spread of this pandemic, the italian government ordered nationwide lockdown by march : all public places were closed (included educational, religious, and public/cultural institutions, such as schools, universities, museums, and law courts), all public events and any form of congregation were banned, and a distance of at least m had to be maintained (government of italy, ). all italian people were in quarantine at home (#iorestoacasa) until may : people had to stay at home apart from essential tasks. the slowing growth in daily reported deaths in italy was consistent with the significant impact of these restrictions. the effective reproduction number, rt, dropped to close to around the start of the lockdown, with , ( , - , ) deaths averted. the widespread use of quarantine had the desired effect of controlling the epidemic, this was also due to the fact that the compliance of the italian public with quarantine guidelines had been very high (carlucci et al., ) . yet, the pandemic created a breeding ground for direct psychological consequences, suddenly throwing many individuals into daily lives filled with health threats, existential depression, and generalized stress (holmes et al., ) . a recent review of the psychological impact of quarantine, due to earlier outbreaks, suggested that there were high rates of negative psychological effects among the public, including post-traumatic stress symptoms, persistent depression, substantial anger, panic attacks, and suicidality (liu et al., ; maunder et al., ; brooks et al., ) . social distancing and isolation exacerbate the burden of stress, and often cause effects on immune, cardiovascular, and mental health because these measures frustrate the deep-seated human instinct to connect with others. on this point, social connection helps people to regulate negative emotions, remain resilient during difficult times, and cope with stress (rimé, ; hawkley and cacioppo, ; haslam et al., ; doré et al., ; jetten et al., ) . remarkably, the mental impact of quarantine can depend largely on the characteristics of participants and the quarantine variables selected. as documented by reynolds et al. ( ) , taylor et al. ( ) , and brooks et al. ( ) , while compliance with quarantine guidelines requirements are significant factors behind a higher level of post-traumatic stress disorder (ptsd), healthcare workers were more likely to be affected than the public. in italy, in the early phase of quarantine ( - days after the decree of nationwide lockdown), deleterious consequences on the population's psychological health were analyzed in a nationally representative survey of , participants (barari et al., ) . different demographic groups were struggling with different aspects of quarantine. older adults expressed worry or anxiety, while those who were likely working parents ( - years) cited consistent economic distress and struggles with home-schooling and smart-working, compared to other groups. younger people were struggling with increased boredom, perceived immobility, and conflicts within family, while vulnerable groups, like the elderly and health-compromised people, cited consistent loneliness relative to others. overall, the average level of anxiety surrounding the crisis in the italian population was high: no respondents reported being completely without anxiety. according to barari et al. ( ) , the negative psychological consequences of the quarantine were beginning to wear on people and seemed likely to become more serious over time. further findings derived from an online survey showed the prevalence of psychiatric symptoms in , participants drawn from the general population from march to . female gender, negative affect, and detachment were associated with higher levels of stress, anxiety, and depression. having an acquaintance infected was associated with increased levels of both depression and stress, whereas a history of stressful situations and medical problems was associated with higher levels of anxiety and depression. finally, those with a family member infected and young people who had to work outside their domicile presented higher levels of anxiety and stress, respectively. thus, it is important that the potential advantages of home quarantine are weighed against the possible mental costs (rubin and wessely, ; torales et al., ) . quarantine as an efficacious public health measure also needs to lower the psychological strain associated with it. research evidence aims of this study were to explore ( ) the likely effects of quarantine on mental health (anxiety and depressive symptoms), immediately after the nationwide lockdown issued by the italian government, and ( ) the factors that contribute to, or mitigate, these consequences. among these factors, sociodemographic variables (gender, age, employment status, marital status, education, geographic area, and income per year), worry about being affected by covid- , and adherence with quarantine guidelines were analyzed. depression was the principal outcome, while anxiety was used as a covariate, given its close association with depression (clark and watson, ; barlow and campbell, ) . respondents were italian quarantined adults aged and older with access to a networked computer. an online cross-sectional study was conducted from march to , immediately after the nationwide lockdown issued by the italian government on march (#iorestoacasa). a virtual snowball sample via social media was used within a wider web-based study including other psychological measures (carlucci et al., ) . this study has been approved by the department of psychological sciences, health and territory, university of chieti, italy review board. written informed consent was obtained from all the participants included in the study. of psychiatric illnesses and medical problems (e.g., hospitalizations) were collected. the -item teate depression inventory (tdi; balsamo et al., , b , developed via rasch analysis (rasch, ) , was employed to evaluate depressive symptoms in participants in the past weeks. respondent answers were measured on a point likert scale ranging from "never" to "always." cronbach's α coefficient in our study was . . the -item state scale of the state-trait inventory for cognitive and somatic anxiety (sticsa) (ree et al., ; balsamo et al., ; carlucci et al., ) was administered to evaluate cognitive (e.g., "i have trouble remembering things") and somatic (e.g., "my muscles are tense") symptoms of state anxiety. individuals rated how often a statement was true in the past weeks, from "not at all" to "very much so." cronbach's α coefficient was . . adherence to quarantine guidelines in response to the covid- outbreak was measured by a global index composed of items classified into preventive (i.e., handwashing with soapy water/alcohol-based solution) and avoidant (i.e., avoidance of gatherings in public or open to public places, handshaking) disease behaviors (carlucci et al., ) . respondents were asked about the frequency of which they had carried out quarantine restrictions on a -point likert scale from "never" to "always." cronbach's α coefficient was . . worry about being infected with covid- was assessed by a single item drawn from a multidimensional questionnaire of risk perception for the covid- infectious disease outbreak. responses were classified according to three levels of worry severity: none ("not worried at all"), moderate ("slightly worried"), and quite a lot ("really worried"). descriptive statistics were computed for sociodemographic characteristics, physical symptoms and health service utilization variables, knowledge and concern-related variables, precautionary measure variables, and additional health information variables. prevalence of depressive and anxiety symptoms during the covid- outbreak in the italian population were also computed for sex and age. in line with similar studies (e.g., giallonardo et al., ; wang et al., ) , the tdi outcome score was categorized into "minimal, " "mild, " "moderate, " and "severe" depression levels . a sticsa-s score of points or greater was indicated as the cut-off point for the presence of anxiety symptoms (van dam et al., ) . sensitivity analysis was conducted using the hmisc r package (harrell and dupont, ) in order to assess the power and sample size of ordinal outcomes under the proportional odds ordinal logistic model. next, generalized linear regression (glms) was applied to the explanatory model to analyze whether the severity of depression during the covid- quarantine could be predicted by high levels of adherence to quarantine guidelines and worry about being infected with covid- , and by sociodemographic variables (model ), that resulted as significant in our previous study (carlucci et al., ) . since depression was measured in terms of severity levels, we specified multinomial (ordinal) as the distribution and cumulative logit as the link function. predictors were selected according to a two-step process. firstly, a potential set of considered variables were correlated with the outcome variable. all the potential variables that correlated significantly with the outcome were selected as predictors in the glms model. hence, based on the test of model effects (wald chi-square statistic and p-values), the predictors were compared (guisan et al., ) . only the resulting significant predictors (p < . ) were retained in the model. a wald test (and its % confidence interval) based on robust estimates of the coefficients and covariance matrix were used to assess the models, and residual deviance as a goodnessof-fit statistic was applied to evaluate model overdispersion (mccullagh, ) . the model with the deviance/df ratio closest to the unit was retained as the most parsimonious model (mccullagh, ) . in addition, due to the high comorbidity between depression and anxiety symptomatology (clark and watson, ; barlow and campbell, ) , the model was re-estimated controlling for anxiety as the covariate (model ), in order to increase the ability to detect differences on dependent variables (depression severity levels) by an independent variable inserted as the covariate. differences between the two models were interpreted in terms of unique contribution of any independent variable on depression severity symptoms. the data were statistically analyzed with spss for windows . (ibm corp, ). statistical significance was set by p-values of less than . . the characteristics of the participants are shown in table . of the , respondents, , ( . %) were male and , ( . %) were female. the mean (±sd) age of the participants was . ± . years. a total of . % of them were located in the south of italy (n = ). among these, , ( . %) respondents held a high school diploma, while , ( . %) held a higher education qualification (bachelor/master/doctorate). in terms of occupational status and income per year, , ( . %) participants were students, ( . %) were healthcare workers, and , ( . %) were employed. concerning marital status, , ( . %) participants were unmarried/single, ( . %) were married, ( . %) were divorced/separated, ( %) were cohabiting, and ( . %) were widowed. most of the subjects had a high level of health: , ( . %) were found to show no physical disease, while ( . %) were detected as "fragile, " having more than three diseases, and with a long history of chronic medical illness. a total of ( . %) participants had previously carried out psychotherapeutic treatment. among these, . % of respondents had carried out at least one psychotherapy treatment (individual, family/couple, and/or group treatment), . % had undergone psycho-pharmacological treatment, and . % had participated in other psychological treatments. most ( . %) of the participants spent their quarantine period with family members. a total of , ( . %) were found to be highly adherent to quarantine guidelines, and , ( . %) reported that they were worried about being infected with covid- . a sample size of , was used for the statistical power analyses, and a : odds ratio was used as a baseline. the alpha level used for this analysis was p < . . the post-hoc analyses showed that the statistical power for this study was . . thus, there was an adequate power at the moderate to large effect size level. an n of approximately , would be needed to obtain statistical power at the recommended . level (cohen, ) . the overall prevalence was . % for severe, . % for moderate, . % for mild, and . % for minimal depressive symptoms. the overall prevalence of anxiety symptoms was . %, using the cut-off of > . taking together, the prevalence of depressive symptoms and state anxiety severity was significantly higher in female participants, and those younger than years compared to participants aged years or older (p < . , as shown in tables , ). in addition, those who received psychotherapeutic treatment in the past reported higher severity levels of depressive [χ = . ( ), p < . ] and anxiety [χ = . ( ), p < . ] symptoms relative to the general public. preliminarily, nonparametric correlations (spearman's rho) were performed in order to select independent variables as predictors in the glms. as expected, all the sociodemographic variables, as well as worry were found to correlate negatively with the outcome variable (depressive symptom severity levels) ranging from rho = . (age, p < . ) to rho = − . (education, p < . ), except for sex and worry (rho = . , p < . and . , p < . , respectively). parameter estimates of the generalized linear model and the exponentiated values of the coefficients [the "exp(b)" column] are displayed in table . the first model resulted in an underestimation of the data with no statistical association for education [wχ (df ) = . ( ), p = . ], and income per year [wχ (df ) = . ( ), p = . ] as predictors of depressive symptoms. to improve model fit, we discharged them and reestimated the model. the omnibus test [χ (df ) = . ( ); p < . ], and residual deviance/df ratio (deviance/df = . ) of the re-estimated models suggested that the refined model fit significantly better than the proposed model (mccullagh, as expected, a preliminary analysis showed that depression and anxiety symptoms shared approximately % of the common variance, as derived by the spearman rho coefficient (rho = . , p < . ). thus, state anxiety, as measured by the state sticsa, as the covariate was inserted in our model (model ). compared to the previous model, no statistical differences were found in sex, age, and adherence level to quarantine guidelines groups when predicting depression symptom severity, when controlling for anxiety (see appendix a). statistically significant differences were maintained in depression symptom severity for marital status, geographic area, and occupational status groups, after controlling for anxiety. in detail, divorced/separated participants were less likely to experience depressive symptoms compared to single people, with a significant decreased risk of depressive symptoms (odds ratio: . [ % ci, . - . ]). on the other hand, no statistical differences on depressive symptom severity was found in cohabiting participants compared to unmarried participants in the second model. concerning geographic area, the participants living in central italy were found less likely to experience severe depressive symptoms (odds ratio: . [ % ci, . - . ]) compared to those living in north-west italy, after removing anxiety effects. far from the previous model, the healthcare professionals' group were not found to differ from other occupational groups in predicting high levels of depressive symptoms compared to unemployed participants. interestingly, participants "moderately" worried about being infected with covid- were more prone to experience high levels of depressive symptoms (odds ratio: . [ % ci, . - . ]) compared to participants who were "quite a lot" worried, after controlling for the state anxiety effect. we fitted both the glms models (without and with anxiety as the covariate) in the subsample of participants with healthcare professionals (β = − . ; se = . ; p < . ) were less depressed compared to unemployed participants, with a decreased risk of depressive symptom severity (odds ratio: . [ % ci, . - . ]). next, those who reported to adhere to the quarantine guidelines had significantly lower levels of depressive symptoms compared to those who were not adherent (β = − . ; se = . ; p < . ), with a decreased risk of depressive symptoms (odds ratio: . [ % ci, . - . ]). no statistical association was found for worry about being infected with covid- and geographic area. surprisingly, in model , no statistically significant differences were maintained in depression symptoms for the sex, marital status, and occupational status groups, after controlling for anxiety. participants aged from to years reported significantly lower levels of depression scores compared to the younger participants (β = − . . ; se = . ; p < . ), with a decreased risk of depressive symptoms (odds ratio range: . [ % ci, . - . ]). concerning geographic area, the participants living in south italy were less likely to experience severe depressive symptoms (odds ratio: . [ % ci, . - . ]) compared to those living in north-west italy, after removing anxiety effects. as expected, participants "moderately" and "none" worried about being infected with covid- were more prone to experience high levels of depressive symptoms (odds ratio: . / . [ % ci, . - . / . - . ]) compared to participants who were "quite a lot" worried, after controlling for the state anxiety effect. quarantine has been used extensively in all countries of the world to lower the spread of the covid- infection and to protect individuals' health, at different times (sohrabi et al., ) . quarantine includes the separation and restriction of movement of people who have potentially been exposed to a contagious disease to ascertain if they become unwell, so reducing the risk of them infecting others. it is an unpleasant experience for those affected (hiremath et al., ) . imposed isolation and separation from loved ones, loss of mental health needs (freedom, social contacts, stimulation), uncertainty over disease status, family conflict, and boredom can, on occasion, contribute to the onset of psychological disorders (brooks et al., ) . due to the fact that the psychological impact of quarantine depends largely on the characteristics of participants and the quarantine variables selected, several sociodemographic characteristics have been selected here, with depressive symptoms measured by the tdi as the outcome. in the second model, anxiety, as measured by the sticsa state scale, was inserted as the covariate, given the close relationship with depression (brooks et al., ) . about gender, depressive symptoms were more likely to occur in female participants, with a risk of developing depressive symptoms higher . compared to male participants in our sample. this finding was in accordance with studies by qiu et al. ( ) and wang et al. ( ) among the chinese general population in the first weeks following the outbreak, as well as broche-pérez et al. ( ) among the cuban population. also, among the italian general population higher levels of psychological distress were reported in the female gender compared to their male counterparts rossi et al., ) . sex differences in depression were not caused by a higher prevalence of covid- infection in women because mortality and vulnerability to the covid- disease indicated that more men are dying from covid- (lancet, ) . thus, these differences seem to be caused by the fact that women carry a different kind of burden from this epidemiological emergency. gender inequities disproportionately affect the wellbeing and economic resilience during lockdown. households are under strain, but children and elderly care, as well as housework generally fall on women (cluver et al., ) . by increasing caregiving needs, covid- has intensified the pressure on women to uphold prescriptive feminine norms. women have to bear more of the burdens of providing additional support for children's distance learning, and alleviating children's emotional tedium, isolation, and anxiety of shelter-in-place (rosenfeld et al., ) . in addition, increased intimate partner violence has grown during the quarantine due to covid- because women are required to stay uninterruptedly with their partners and away from those people who can give help or at least validate their experiences and, particularly if these women live in small houses (bradbury-jones and isham, ; van gelder et al., ) . indeed, some studies suggest that sudden forced proximity with their immediate household members is a risk factor for domestic violence, and aggression (taylor et al., ; brooks et al., ) . in italy, since the beginning of the covid- quarantine, three domestic homicides and murder-suicides have been registered to date. furthermore, while covid- has coincided with greater rises in unemployment for women than men, the rise in unemployment for men remains substantial (bureau of labor statistics, ). about the age groups, depressive symptoms were most likely to occur in younger people (aged - years). with increasing age, depressive symptoms were less prevalent during the italian lockdown due to the covid- outbreak. our results were similar to those from previous studies, such as a study during the sars outbreak in taiwan (su et al., ) , a study of horse owners quarantined because of equine influenza (taylor et al., ) , and one recent study during the covid- epidemic in china (huang and zhao, ) . as well, like gender, for this sociodemographic variable, the prevalence of the depressive symptoms in different age groups and the probability of risk of developing depressive symptoms depending on age do not relate to the greater likelihood of being infected. being elderly has been reported to correlate with adverse clinical outcomes, including hospitalization and mortality (applegate and ouslander, ; zhou et al., ) . indeed, in italy the mean age of covid- patients who died was years (remuzzi and remuzzi, ) and the case fatality rate was % from to years, . % from to years, and % for years and older (livingston and bucher, ) . despite this, respondents older than years had the lowest risk for developing depressive symptoms compared to the younger age groups. in a population where loneliness and isolation have already been described as an epidemic (luo et al., ) , the impact of even short-term social distancing measures and the resulting distress did not influence the vulnerability to mental health issues (jeste et al., ; vahia et al., ) . this finding is in accordance with part of the literature. although mixed results derive across current and previous studies on the association between participants' age and depression as a psychological outcome of health-related emergency (hawryluck et al., ; qiu et al., ) , some authors reported that only young age was found to be associated with increased distress as a psychological outcome of the covid- quarantine (barari et al., ; and of the sars quarantine (hawryluck et al., ) . the higher psychological distress reported by the younger population could be due to their greater and uncontrolled access to the amount of information ("infodemic") through social media, which can easily trigger distress (cheng et al., ) . as regards marital status, unmarried/single people were the most depressed group with quarantine policies in the event of this outbreak. it is plausible that single people had greater difficulty in relying on or obtaining the assistance of others during the italian lockdown, thus are at risk of depressive symptoms and lower self-confidence more than cohabiting and married participants. this datum is in line with part of previous literature reporting that being married was protective for depression or associated with a lower risk of depressive symptoms (inaba et al., ; yan et al., ; bulloch et al., ) , although other studies conducted during the sars outbreak suggested that demographic factors such as marital status, as well as living with other adults, and having children were not associated with psychological outcomes (hawryluck et al., ; mihashi et al., ) . as to geographic area of residence, people living in the south of italy showed the lowest risk of developing depressive symptoms among all the groups, followed by participants from regions of central italy compared to residents from northern regions and the islands. as expected, residents in the most severely affected regions are at the highest risk of developing depressive symptoms. southern and central regions recorded a smaller number of deaths and diagnosed cases ( , and , deaths, respectively), compared to the north-east and north-west regions ( , and , deaths, respectively), where the disease spread first on a large scale. to explain this datum, it should be considered that the authorities introduced control measures in the northern regions (the "red zone"), before any other region and carried out extraordinary efforts to restrict the movement of people (carlucci et al., ) . in addition, residents from northern italy were found less adherent to restrictive measures compared to the those from the south of italy. people who have shown more adherence were found less at risk of depressive symptoms compared to people with less adherence (see under). as for occupational status, this study highlighted students as suffering from the highest level of psychological distress among all the other groups, including the unemployed group. also, in this case, the public's level of depressive symptoms did not increase with an increased probability of contracting the disease. since the physical spaces of universities were closed, students' mental well-being was affected by the sudden interruption of social interactions. however, the possibility of having online lessons and maintaining social contacts through social networks would not explain the onset of the depressive symptoms compared to other groups, for example employees who had been laid off or were retired. the reason for students' greater risk of depressive symptoms, reported also by wang et al. ( ) among the chinese population, could lie in a sense of uncertainty toward the future that this emergency, not only in health, but also in economic, social, and political areas, is eliciting all over the world (chong et al., ; wenzel et al., ; tan and enderwick, ) . compared with other professions and the general population, healthcare workers were associated with a lower risk of psychological outcomes compared to the unemployed in our sample. "learned helplessness" (seligman, ) could explain why health professionals were the least depressed group. after being exposed to inescapable difficult events, people become passive and stop trying after being exposed to events such as uncontrollable bursts of noise (alloy et al., ) and as a result show greater levels of anxiety and depression. on the contrary, health professionals, considered the real heroes of this emergency, were associated with a lower risk of psychological outcomes compared to unemployed participants. through the practice of their profession, they felt more useful to society, despite their increased risk for infection and transmission (al-rabiaah et al., ) . after students, the unemployed were at a higher risk of depressive symptoms compared to the other groups (stuckler et al., ; reeves et al., ) . this datum is inserted within the context of the covid- -related risk unemployment and economic losses and insecurity with the closure of community services and the collapse of industries negatively impacting the national economy. it should be a critical public health priority to prevent suicide. indeed, during the most recent economic recession, a % rise in unemployment was correlated with a rise in the suicide rate of . % in the united states ( % ci: . - . , p < . ) (reeves et al., ) . similarly, each percentage point increase in unemployment was accompanied by . % rise in suicide ( % ci: . - . , p = . ) in europeans aged years or less. as to adherence, people with low adherence were more likely to exhibited depressive symptoms relative to people with a great level of adherence to covid- preventive measures. as expected, adherence has been found to be a protective factor against mental health problems (hawryluck et al., ; koenig and schultz, ; brooks et al., ) . adopting the preventive behaviors contribute to lower the uncertainty of the epidemic progression which would cause higher psychological pressure on the public. as to worry about being infected with covid- , people with more worry were more depressed than people with a low level of worry. as for the whole sample, also in the subsample of participants with psychotherapeutic treatment history, the same sociodemographic factors and behaviors that contribute to, or mitigate mental effects of the quarantine in terms of depressive symptoms were reported. however, when anxiety symptoms or concomitant stressful events were present in comorbidity, these participants were found to experience higher levels of worry associated with increased depression symptomatology, compared to the whole sample. current evidence showed similar results. a history of stressful situations and medical problems was associated with a greater degree of depression and anxiety during the covid- quarantine in the italian population (hao et al., ) . again, psychiatric patients were significantly more likely to experience a higher degree of the negative mental impact of the outbreak, including stress, anxiety, and depression, compared to the general public (hao et al., ) . during the covid- quarantine outbreak in italy, female participants, younger people, single people, students, people living in northern regions, and who were less compliant with quarantine guidelines and less worried about being infected with covid- were at a high risk of displaying psychological issues. these findings suggest public levels of depressive symptoms did not increase with the greater likelihood of being infected. for example, although female and younger people reported a lower risk of covid- infection, they experienced higher levels of depressive symptoms during the covid- quarantine in italy. therefore, ongoing monitoring of the psychological strain associated with outbreaks of epidemic-potential, life-threatening diseases should become routine as part of preparedness efforts worldwide by establishing early targeted mental health interventions. in other words, more vulnerable groups, likes those cited above, should benefit from personalized "moraleboosting" interventions. or, intervention research could be valuable to combat amplifications of gender inequalities, particularly to address the added challenges women are likely to face. this research can provide timely insights for government agencies toward improving and safeguarding the psychological well-being of women, younger people, and categories of subjects at a higher risk of suffering from psychological distress on the occasion of subsequent waves of the spread of covid- or other epidemic diseases. this study has several limitations. firstly, the analyses presented here were derived from a crosssectional design, thus it is difficult to make causal inferences. secondly, given that the research was conducted in close temporal proximity to the period of the covid- quarantine, a webbased survey method was necessary to recruit a convenience sample by avoiding possible infections. this limited sampling in our study. as a consequence, a self-selection effect may have occurred and should be considered with those people who were experiencing the greatest or least levels of distress responding to the survey (saggino et al., ) . in addition, participants were required to access the internet and to be familiar with online devices to respond, which suggests that they might be more educated, younger or/and have a higher socioeconomic status than the overall surveyed quarantined population. thirdly, due to the uncontrolled occurrence of this health-related emergency, an accurate picture of the individual's psychological conditions before the covid- outbreak was not conducted. although it would been interesting to conduct pre-post analyses, these data could provide a baseline for future research on the psychological consequences of quarantine in the italian population throughout the rest of the current covid- pandemic. fourthly, depressive and state anxiety symptoms were measured by means self-report inventories that are notably biased by response set, such as social desirability (innamorati et al., ; carlucci et al., ; balsamo et al., a,b) . thus, future research should include methods, such as observational methods and psychophysiological or behavioral assessment, in order to objectively record the levels of these mood states (campbell and fiske, ) . fifthly, the assessment of state anxiety could be completed or replaced by adding the specific fear of covid- scale (ahorsu et al., ; broche-pérez et al., ; soraci et al., ) . in addition, it should be acknowledged that the study was carried out was not sufficiently heterogeneous for sex, with marked female preponderance, and age sample, with a prevalence of juveniles. hence, these findings may not translate accurately to the public at large. finally, it could not evaluate whether the outcomes considered in this study will be long-lasting after the covid- outbreak. however, follow-up with these participants will continue in order to facilitate our understanding about how long these outcomes will last. a deeper understanding of how the epidemic affects italians' psychological health by identifying which groups were at a high risk of psychological morbidities due to the current pandemic can help to guide and target future psychological intervention implementations. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. the studies involving human participants were reviewed and approved by the department of psychological sciences, health and territory, university of chieti, italy, review board. the patients/participants provided their written informed consent to participate in this study. both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. we thank maria pia pugliese, ines d'ambrosio, and gianluca balsamo for their assistance and support in data collection 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control marital status and risk for late life depression: a meta-analysis of the published literature clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © balsamo and carlucci. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -jp m ibe authors: naccarato, marcello; scali, ilario; olivo, sasha; ajčević, milos; stella, alex buoite; furlanis, giovanni; lugnan, carlo; caruso, paola; peratoner, alberto; cominotto, franco; manganotti, paolo title: has covid- played an unexpected “stroke” on the chain of survival? date: - - journal: j neurol sci doi: . /j.jns. . sha: doc_id: cord_uid: jp m ibe background: the covid- pandemics required several changes in stroke management and it may have influenced some clinical or functional characteristics. we aimed to evaluate the effects of the covid- pandemics on stroke management during the first month of italy lockdown. in addition, we described the emergency structured pathway adopted by an italian university hub stroke unit in the cross-border italy-slovenia area. methods: we analyzed admitted patients' clinical features and outcomes between th march and th april (first month of lockdown), and compared them with patients admitted during the same period in . results: total admissions experienced a reduction of % during the lockdown compared to the same period in ( vs , respectively), as well as a higher prevalence of severe stroke (nihss> ) at admission (n = , % vs n = , %). a dramatic prevalence of stroke of unknown symptom onset was observed in (n = , % vs n = , %). during lockdown, worse functional and independence outcomes were found, despite the similar proportion of reperfused patients. similar ‘symptoms alert-to-admission’ and ‘door-to-treatment’ times were observed. during lockdown hospitalization was shorter and fewer patients completed the stroke work-up. conclusion: in conclusion, the adopted strategies for stroke management during the covid- emergency have suggested being effective, while suffering a reduced and delayed reporting of symptoms. therefore, we recommend raising awareness among the population against possible stroke symptoms onset. thus, think f.a.s.t. and do not stay-at-home at all costs. as a result of the ongoing pandemic of covid- , caused by severe acute respiratory syndrome coronavirus (sars-cov- ), about one-third of the world population is currently living in a lockdown modality [ ] . the first case in italy was diagnosed on february th [ ] . the infectious disease sp read rapidly throughout northern italy regions and afterwards the whole country, reaching ' confirmed cases, with ' deaths as of th april, (http://www.salute.gov.it/portale/home.html). on the same date, friuli venezia giulia (fvg), a crossborder region between austria and slovenia, showed ' confirmed sars-cov- cases and deaths ( figure ). on th march the italian government imposed a national quarantine, restricting the movement of the population except for necessity, work, and health circumstances. in many italian regions, hospitals have been reorganized to properly manage covid- patients, creating new protected wards for sars-cov- positive patients both for intensive and sub-intensive care, including reorganizing many stroke units [ ] . the giuliano -isontina area of friuli-venezia giulia region represents a peculiar community determined by a high prevalence of j o u r n a l p r e -p r o o f elderly with polymorbidities [ ] , and by an international cooperation program for stroke management between italy and slovenia. to evaluate the effects of the covid- pandemics on stroke management, this report described the emergency structured pathway adopted by an italian university hub stroke unit in the cross -border italy-slovenia area (which serves ' people) (data from istituto nazionale di statistica-istat official report, th september , see http://dati.istat.it/), and compared clinical features and outcomes of admitted patients between th march (start of italy lockdown) and th april with stroke patients admitted during the same period in . figure . the study population was composed of consecutive patients of both sexes, above years of age, with acute focal neurological symptoms compatible with acute stroke. we excluded patients with acute and sub -acute stroke admitted to other departments. intravenous thrombolytic therapy (rtpa) ( . mg/kg) and endovascular thrombectomy (evt) were administered following the international guidelines (aha ), with the support of computed tomography perfusion (ctp) for tissue-based selection. nihss evaluation was carried out at the time of presentation at the stroke unit by a vascular neurologist trained in performing nihss examination [ ] . for this report, demographic characteristics, clinical and functional features, pre-hospital and intrahospital management characteristics were included in the analysis. for a complete description, see table . the study was conducted according to the principles of the declaration of helsinki. approval for the study was obtained from the local ethics committee (ceur fvg). j o u r n a l p r e -p r o o f subgroup analysis and data presentation was proposed for and patients, continuous variables were presented as medians ( th- th percentile) and non-continuous variables as percentages. differences between the two groups were tested with the appropriate nonparametric t ests (namely, mann-whitney u-test) and chi-square. a level of p < . was regarded as statistically significant. during the study period, patients were admitted to the stroke unit compared to who were admitted in the same period of (- %). all patients admitted to our stroke unit performed nasopharyngeal swab. none of the patients was positive to sars-cov- . among these, no differences were present in terms of demographic characteristics and stroke subtypes. in general, a lower absolute number of 'code stroke' activations ( vs ) and rtpa treatments ( vs ) was found in compared to . despite similar alert -to-admission and door-totreatment times, a higher prevalence of severe stroke (nihss> ) was found in (n= , %) compared to (n= , %), thus leading to worse functional outcomes. intrahospital management and complications highlighted a shorter hospitalization with a faster commencement of physiatric consultancy and a higher absolute number of respiratory infections in . a dramatic prevalence of stroke of unknown symptom onset (suso) was found in (n= , %) compared to (n= , %). a complete summary of these findings is reported in table . during the state of emergency, the attention of healthcare providers and health authorities is primarily focused on infected patients and the frontline responders. this had an impact also on other units dedicated to highly invalidant pathologies such as a stroke, which still is one of the leading causes of death and disability worldwide. in conclusion, the adopted strategies for stroke management during the covid- emergency have suggested being effective, while suffering a reduced and delayed reporting of symptoms. therefore, we recommend raising awareness among the population against possible stroke symptoms onset. were admitted to a specific protected 'dirty ed area' (separated from the 'clean ed area', for non -suspected covid- patients) where neurological examination and urgent hematological tests were performed. in "code stroke" patients, multimodal ct (including non-enhanced ct, ct angiography of the supra-aortic and intracranial arteries, and -in the cases of ischemic strokes -whole brain volume ct perfusion) was performed as usual. after neuroradiological examination in suspected covid- positive patients, the ct-room and equipment were properly sanitized. patients with diagnosis of definite or probable acute cerebrovascular disease were hospitalized in stroke unit where, similarly to the ed, 'dirty' and 'clean' areas were arranged. in both areas, patients were treated with the usual standardized protocols. all patients admitted to ed with stroke symptoms performed nasopharyngeal swab during the assessment process. the median time from swab collection to examination results was h. if covid- diagnosis was confirmed, the patient was transferred to a protected intensive care unit (icu) or other wards dedicated to covid- for sub-acute care. no lockdown for neurological diseases during covid pandemic infection critical care utilization for the covid- outbreak in lombardy, italy: early experience and forecast during an emergency response on being a neurologist in italy at the time of t he covid- outbreak ( %) ( %) ( %) ( %) ( %) ( %) ( %%) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( - ) ( %) ( - ) ( - ) ( % ) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( - ) ( - ) ( - ) ( - ) ( %) ( - ) ( % ) ( - ) ( - ) ( - ) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( %) ( - ) ( %) ( - ) ( - ) ( % ) ( %) ( %) ( %) ( %)) key: cord- -jxlrz ce authors: craxì, lucia; vergano, marco; savulescu, julian; wilkinson, dominic title: rationing in a pandemic: lessons from italy date: - - journal: asian bioeth rev doi: . /s - - - sha: doc_id: cord_uid: jxlrz ce in late february and early march , italy became the european epicenter of the covid- pandemic. despite increasingly stringent containment measures enforced by the government, the health system faced an enormous pressure, and extraordinary efforts were made in order to increase overall hospital beds’ availability and especially icu capacity. nevertheless, the hardest-hit hospitals in northern italy experienced a shortage of icu beds and resources that led to hard allocating choices. at the beginning of march , the italian society of anesthesia, analgesia, resuscitation, and intensive care (siaarti) issued recommendations aimed at supporting physicians in prioritizing patients when the number of critically ill patients overwhelm the capacity of icus. one motivating concern for the siaarti guidance was that, if no balanced and consistent allocation procedures were applied to prioritize patients, there would be a concrete risk for unfair choices, and that the prevalent “first come, first served” principle would lead to many avoidable deaths. among the drivers of decision for admission to icus, age, comorbidities, and preexisting functional status were included. the recommendations were criticized as ageist and potentially discriminatory against elderly patients. looking forward to the next steps, the italian experience can be relevant to other parts of the world that are yet to see a significant surge of covid- : the need for transparent triage criteria and commonly shared values give the italian recommendations even greater legitimacy. in late february and early march , while infection rates in china fell, italy became the european epicenter of the coronavirus (sars-cov- ) epidemic. at the beginning of may , italy entered the so called phase two, consisting in a progressive reopening of business and social activities while monitoring the eventual raise of the epidemic curve. "phase two" posed a number of ethical questions, such as finding a new balance between personal liberties and public health, privacy concerns from the use of movement tracking apps, and down the line access to vaccines or improved medications. we think that it is, however, important to look back and determine which lessons can be derived from the way italy confronted the scarcity of medical resources available to confront the epidemic, especially since this experience can prove invaluable for other countries that are still facing the epidemic peak. in the paper, we analyze the features of the italian recommendations, why the raised heated debate and why it is important to have ethical guidelines and balanced and consistent allocation procedures to prioritize patients. the recommendations issued by the italian society of anesthesia, analgesia, resuscitation, and intensive care in march and april , a huge effort was made by the national government to flatten the epidemic surge by enforcing increasingly stringent containment measures, in order to reduce the impact of the outbreak on the health system. despite facing a likely major social and economic crisis, the italian government imposed a nationwide lockdown on march: prohibition of all movements of people within the whole territory, and closure of all non-essential business activities. some think a systematic and strong response arrived too late ; such measures inevitably have a delayed impact, and hospitals were hit by what has been called a medical "tsunami," with high caseload punctuated by a stream of deaths. the enormous challenge for the health system-facing a dramatic shortage of icu beds and staff-has been how to meet the medical needs of patients affected by covid- . even though authorities state that no cases of people who failed to get into intensive care have been reported, many italian physicians working in icus in northern italy have stated otherwise, as has the mayor of bergamo, one of the hardesthit cities (ansa ; nacoti et al. ; rosenbaum ; guerzoni ) . professionals experienced uncertainty and distress about how to allocate the dramatically scarce resources available, as that situation was unprecedented for everyone: in some hospitals, at the peak of the surge, the rationing involved not only ventilators but oxygen as well. at the beginning of march, some hospitals around milan were already collapsing (some of them admitting more than patients with severe respiratory failure every day). as difficult allocation choices were already being made, clinicians (including one of the authors, mv) of the ethics section of the italian society of anesthesia, analgesia, resuscitation, and intensive care (siaarti) were asked to publish guidance on the allocation of limited resources. they worked between shifts to construct the recommendations (box ) that sparked a heated debate immediately after the release. box key elements of siaarti recommendations (vergano et al. a, b) . when the availability of resources is overwhelmed by their need, a decision to deny access to one or more life-sustaining therapies, solely based on the principle of distributive justice, may ultimately be justified . criteria for allocation should be flexible and adapted locally in response to available resources, the potential for patient transfer, and the ongoing or foreseen number of admissions . an age limit for admission to the icu may ultimately need to be set . together with age, the comorbidities and functional status of any critically ill patient should be carefully evaluated . every admission to the icu should be considered and communicated as an "icu trial." the appropriateness of life-sustaining treatments should be re-evaluated daily the recommendations issued by siaarti suggest that, if a choice to ration medical equipment and intervention is needed, the maximum individual benefit in terms of expected life years-likelihood of survival plus remaining likely years of a patient's life-should be prioritized. according to this principle, the recommendations suggested evaluating age, comorbidities, and functional status of any critically ill patient. this was subsequently criticized as ageist, as well as "unconstitutional" and discriminatory against elderly patients (fnomceo ; quotidiano sanità a, b; rodriquez ). it was perceived as not consistent with the values on which the healthcare system is grounded. according to the siaarti guidance, the drivers of decision for admission to icu should be the clinical picture taking into account "biological" (not mere chronological) age, comorbidities, and preexisting functional status. this kind of "soft" utilitarian approach is already applied in italy in specific fields of dramatically scarce resources, such as organ transplants (cillo et al. ) . this approach is justified by the need to maximize the achievable benefit in terms of life years gained, thus optimizing the use of available resources: not only the probability of survival and the "greatest life expectancy" are considered, but also the predicted length of the icu stay and hence the use of intensive care resources. nonetheless, this kind of approach is the opposite of the egalitarianism that pervades the wider italian healthcare system. sometimes in pursuing what is good, we run the risk of forgetting what is fair. in emergencies, the patient-centered "duty to care" needs to be balanced with publicfocused duties to promote equality of persons and equity in distribution of risks and benefits. also, individual allocating decisions must be supported by fair institutional processes that may include strategies such as preparing, conserving, substituting, adapting, re-using, and re-allocating resources (hick et al. ) . in every single country facing covid- emergency, if no ethical guidelines or balanced and consistent allocation procedures are applied to prioritize patients, there is a concrete risk for unfair choices (emanuel et al. ) . should the shortage arise with no rationing plan in action, decisions would be left to the ruling of local healthcare and hospital authorities or to the clinician's judgment in the heat of the moment, resulting in approaches both inconsistent and uncoordinated. one motivating concern for the siaarti guidance was the real chance that the prevalent criterion would be "first come, first served." such an approach appears to remove responsibility for choice over life and death, and can be thought as avoiding having to make ethically fraught choices between patients. we will only point out in passing that from an ethical standpoint, there is no actual difference between action and inaction when they both cause harm and injustice. choosing first come, first served is to be responsible for the foreseeable, avoidable deaths of many people in a pandemic. moreover, this approach would also carry the huge risk of social unrest and riots due to the rush to obtain hospital beds, or at the very least hospital overcrowding, with the ensuing risk of further spread of infection. major inequities would also arise from a "self-made" utilitarian approach by the clinicians left to their own individual intuitions. the risk of arbitrariness and disparities of judgment is only partially reduced by the recommendations that do not set specific thresholds and state that cutoffs "must remain flexible." the aim is to provide a guidance, not a substitution of the individual clinical judgment. last but not the least, individual physicians would be, as it happened in italy, under a tremendous moral distress facing such a terrible task of improvising decisions about whom to treat. they would literally be out on an ethical limb. in italy, as elsewhere in the world, we were dramatically unprepared for such a startling emergency, and not just because we had not enough icu beds, staffing, or funding. we were not prepared at all to face such dramatic choices. in retrospect, the siaarti recommendations might have been written differently. it would have been better to have had a chance to involve members of the wider community in reflection on how to make decisions in the event of such a health emergency, as was done in maryland in the usa in - (daugherty biddison et al. ) . in maryland, multiple forums were held with the general public and with healthcare workers and disaster professionals using a deliberative democracy approach. input from citizens was then fed into policy recommendations developed by an expert working group. this kind of approach would have given the recommendations greater legitimacy and might have provided additional support to clinicians. we do not know what the future will be like after the pandemic, but it is clear that we should try to rethink the whole issue of allocation of scarce resources in emergency conditions, finding out commonly shared and accepted values to construct a contingency plan with sound and consistent ethical guidelines and proper structures-such as triage committees-to help apply guidelines, relieving the individual front-line clinicians of that burden. the approach is similar to other settings, but this is not acknowledged by the general public, or better, by the critics of the triage recommendations. now more than ever, we are aware that a universal, functional, and proactive public healthcare system has the best chances of appropriately facing an unforeseen outbreak of a viral disease. its full functionality is important at the early stage, when no specific immunization or treatment interventions are available, since it guarantees proper infrastructures to mobilize a testing regime that provides the data that health-policy leaders need to make decisions, and it guarantees a fair and consistent access to supportive care. it will become crucial later, when drugs and vaccines will hopefully be available, to define their price and deliver them widely, effectively, and fairly. we do not know yet if this hard time will turn into a global catastrophe, but should learn from the history of past epidemics that one of the highest risks is to give wrong priorities (jones ) (box ). box key lessons from italy . in emergencies, the patient-centered "duty to care" needs to be balanced with public-focused duties to promote equality of persons and equity in distribution of risks and benefits. . in emergencies, when medical resources available are scarce, the first-come-first-served approach should be rejected. . as the development of rapid ethical guidance in emergency is difficult and politically fraught, an advance planning for intensive care-including decision-making in the event of overwhelming demand-is needed. . a political and public engagement/education in the ethics of resource allocation is needed to clarify priorities and values if they are to be reflected in allocation. author contributions lc conceived the paper and constructed a first draft. mv, js, and dw elaborated arguments and contributed to subsequent drafts of the paper. all authors revised the document for critical intellectual input, and all authors approved the final version. funding information js and dw received support from the uehiro foundation on ethics and education, and the wellcome trust via the wellcome centre for ethics and humanities (wt ) and js from the wellcome trust via the responsibility and healthcare project (wt ). through his involvement with the murdoch children's research institute, js was supported by the victorian government's operational infrastructure support program. conflict of interest dr savulescu reports grants from wellcome trust and grants from uehiro foundation on ethics and education, during the conduct of the study. dr vergano reports that he was the lead author on the siaarti covid- clinical ethics recommendations. there are no other declarations. mancano posti in terapia intensiva? non ci risulta on behalf of i-belt (italian board of experts in the field of liver transplantation). . a multistep, consensusbased approach to organ allocation in liver transplantation: toward a too many patients…a framework to guide statewide allocation of scarce mechanical ventilation during disasters fair allocation of scarce medical resources in the time of covid- nostra guida resta ilcodice deontologico coronavirus, gori su twitter: pazienti "lasciati morire duty to plan: health care, crisis standards of care, and novel coronavirus sarscov- . nam perspectives history in a crisis -lessons for covid- at the epicenter of the covid- pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation lessons from italy's response to coronavirus fnopi sul documento degli anestesisti-rianimatori no a rupe tarpea, la soluzione non è sacrificare gli anziani ci sono un ssn e un sistema paese in grado di dare risposte adeguate facing covid- in italy -ethics, logistics, and therapeutics on the epidemic's front line siaarti recommendations for the allocation of intensive care treatments in exceptional, resource-limited circumstances clinical ethics recommendations for the allocation of intensive care treatments in exceptional, resource-limited circumstances: the italian perspective during the covid- epidemic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -c y rtb authors: chiara, berardi; marcello, antonini; mesfin g, genie; giovanni, cotugno; alessandro, lanteri; adrian, melia; francesco, paolucci title: the covid- pandemic in italy: policy and technology impact on health and non-health outcomes date: - - journal: health policy technol doi: . /j.hlpt. . . sha: doc_id: cord_uid: c y rtb italy was the first western country to experience a major coronavirus outbreak and consequently faced large-scale health and socio-economic challenges. the italian government enforced a wide set of homogeneous interventions nationally, despite the differing incidences of the virus throughout the country. objective: the paper aims to analyse the policies implemented by the government and their impact on health and non-health outcomes considering both scaling-up and scaling-down interventions. methods: to categorise the policy interventions, we rely on the comparative and conceptual framework developed by moy et al. ( ). we investigate the impact of policies on the daily reported number of deaths, case fatality rate, confirmation rate, intensive care unit saturation, and financial and job market indicators across the three major geographical areas of italy (north, centre, and south). qualitative and quantitative data are gathered from mixed sources: italian national and regional institutions, national health research and international organisations. our analysis contributes to the literature on the covid- pandemic by comparing policy interventions and their outcomes. results: our findings suggest that the strictness and timing of containment and prevention measures played a prominent role in tackling the pandemic, both from a health and economic perspective. technological interventions played a marginal role due to the inadequacy of protocols and the delay of their implementation. conclusions: future government interventions should be informed by evidence-based decision making to balance, the benefits arising from the timing and stringency of the interventions against the adverse social and economic cost, both in the short and long term. the novel coronavirus (covid- ) has been declared a global pandemic by the who, with countries registering coronavirus outbreaks. governments have put in place various interventions to respond to the rapid growth of infection and death ( ) which have had considerable negative impacts on society. with no covid- vaccine available, countries have been relying on non-pharmaceutical public health interventions ( ) . mitigation and containment strategies were targeted to flatten the contagion curve and reduce the rate of transmission, and to ensure the sustainability of health care systems in dealing with limited icu capacity and equipment. the international experience suggests that technologies such as contact tracing, drones and robots have played a crucial role in the fight against the virus in some countries, however the experience is mixed and their overall effectiveness is still uncertain. ( ) . italy was the first western country to experience the covid- emergency with a spiral of infections and deaths placing the country at the top of the international rankings, overtaking china on th march . the covid- burden has challenged the cost and sustainability of regional healthcare systems and the concomitant safety of healthcare professionals, requiring a + . % gdp increase in public health expenditure compared to the previous year for hospital reorganisation, community infrastructure, health personnel recruiting and equipment supply ( ). the incidence of the virus has been particularly severe in northern regions, moderate in central regions and mild in the southern regions of italy ( ) . the italian government implemented a wide range of measures to balance the complex trade-offs between ethical, public health, legal and economic problems. in the early phase of the epidemic, the italian government applied targeted measures to the most affected areas. as of th march , the policy interventions were extended homogeneously to all the regions despite the varied severity of the spread. the national exit strategy plan announced at the end of april began on th may with the gradual relaxation of containment measures carried out in three different phases. this paper aims to investigate and assess policy interventions implemented in italy and the impact on health and non-health outcomes. the literature offers different measures providing a systematic cross-country tracking of covid- policies ( , ( ) ( ) ( ) . our analysis considers a set of interventions with targeted objectives in the escalation and de-escalation phases across italian regions from nd january to nd august . the remainder of this paper is organised as follows. in section , we present an overview of the health profile of the population and health care system of italy. section presents the analysis of covid- epidemiological trends at national and regional levels. in section , we describe and analyse scaling-up and scaling-down policies implemented in italy based on a comparative conceptual framework ( ) . this section considers various interventions such as measures to contain the spread of the virus, policies for prevention and cure, interventions for economic stimulus, and the introduction of new health technology. section gives an overview of the response of the health care system. in section , we discuss the long-term challenges and spillover effects arising from the pandemic and associated government interventions. the final section presents the implications of our results for policy and draws conclusions. it is important to understand italy's demographic and epidemiological features to recognise the factors associated with covid- . with a population of over million and a surface area of over , km , italy is one of the largest and most populous countries in europe. it is a highly developed country ( ) , with the eighth largest economy in the world. the government is a parliamentary republic, with a multi-level governance system across twenty administrative regions and provinces and metropolitan cities. the italian constitution recognises health as a fundamental individual and collective right and stipulates that care should be guaranteed to disadvantaged people ( , ) . this is reflected in the italian healthcare system (servizio sanitario nazionale, or ssn), established in to provide universal coverage to all citizens, eu nationals, and legal residents. further, emergency and basic services are provided for undocumented immigrants. since , health policies and constitutional reform have driven a decentralisation of the ssn ( ) . the decentralisation is reflected in the financing, provision, and governance of the twenty regional health systems (see in table in the appendix). regional models range from integrated model to a quasi-market in lombardy ( ) . italian healthcare expenditure amounts to . % of gdp, on par with oecd average ( ) . the general budget is pooled nationally and distributed to the regions. in , government compulsory healthcare expenditure per capita was usd , (ppp), below the oecd average of usd , ( ) . across regions the healthcare expenditure per capita is heterogeneous, ranging from usd , in campania to usd , in friuli venezia giulia ( ). national government financing accounted for . % of total health spending in , while out-of-pocket payments for . % and voluntary schemes for the remaining . % ( ). arguably, having one of the best healthcare systems worldwide ( , ) , italy has the second highest life expectancy at birth ( . years) among european countries, and the eighth highest in the world ( ) ( ). as a result, its population (median age . years, % over years) is the oldest in europe and the second oldest in the world ( , ). italy's longevity is associated with high morbidity rates, with % of the total population having a chronic condition, and nearly % being affected by multi-chronic conditions ( ) ( ) . behavioural risk factors such as diet, tobacco smoking, high body mass index, alcohol consumption and low physical activity levels contribute to the italian population burden of disease ( ). empirical data confirms that age and morbidity are factors associated with covid- mortality ( ) (see table in the appendix). among the deaths recorded in the sample period, .% are older than years, and % had at least one underlying comorbidity/condition ( ) (see figure in the appendix). in the early stages of the pandemic, the incidence was higher amongst men, however, in april, the distribution evened out (see figure in the appendix), limiting the role sex plays in the incidence of mortality arising from the diseases. as of st july , % of confirmed cases were female (see figure in the appendix in italy, covid- data is made available by different institutions at national and regional levels. the inconsistency of data between different administrative levels has been a major issue (see table in the appendix). the italian government started to publish data on th february , with a reasonable degree of transparency, but only a moderate level of accessibility. important data such as icu survival rates, hospitalised patients' outcomes, number and occupation of new beds introduced since the emergency are still missing (see table in the appendix). this section describes the epidemiological trend of covid- throughout the country. covid- appeared in italy in late january , when two chinese tourists tested positive. one month later, patient was detected in lombardy. in the following days, lombardy and veneto became the two initial clusters of infection, experiencing a rapid escalation of cases. increased surveillance, through contact tracing and testing of both symptomatic and asymptomatic persons exposed to positive cases, revealed that the virus had already been spreading in many municipalities of southern lombardy since january ( ). the contagious nature of covid- caused cases to rapidly spread throughout the country ( ). nationally, the peak of contagion to compare the epidemiological figures at the regional level, we used the day each region's th case was confirmed as the start of that region's outbreak. similarly, for the numbers of deaths, we used the day the th fatality was recorded. we arbitrary set these starting points to reduce potential bias of the testing strategy and deaths recording at the beginning of the outbreak. figure in the appendix shows the distribution of covid- cases and fatalities in italy. lombardy was the most affected region followed by veneto, emilia romagna and piemonte. to compensate for the likely underestimation of cases due to the classification method and the testing strategy, the italian bureau of statistics (istat) compared the excess deaths recorded in the first four months of with the average number of deaths across all causes in the first four months of - ( ). this empirical analysis covers a sample of italian municipalities ( % in march, % in april and . % in may). as a result, the integrated surveillance indicates that % of excess mortality registered in march, % in april and . % in may can be attributed to covid- . the unexplained number of deaths might be attributed to three main causes: i) the higher mortality associated with the cases that were not tested; ii) indirect mortality in untested patients who died from organ dysfunctions possibly caused by covid- ; and iii) indirect mortality due to strains on the healthcare system in the most affected areas ( ). the italian government declared a state of emergency on st january . as the first western country to experience a major outbreak of covid- , italy was faced with escalating crisis in a period of extreme uncertainty. in the absence of a covid- vaccine, the only measures to contain the spread of the virus are case isolation, contact tracing and lockdown measures. the decentralised nature of the italian health care system combined with the heterogeneous epidemiological incidence at the regional level created the need for a diverse set of policies responsive to emerging patterns, rather than a one-size fits all approach. the policy interventions ( ) are categorised as follows: . policy interventions to contain the spread of the virus (behaviour, containment, mitigation) (see roadmap and table in the appendix); policy interventions for prevention and cure (treatments, health monitoring) (see roadmap and table in the appendix); technological interventions for testing, tracing and treating (see roadmap and table in the appendix); each policy categorisation has its own spectrum of escalating and de-escalating measures. the escalation implies the implementation of stricter or more invasive policy intervention, while the de-escalation implies the opposite. scaling-up and scaling-down interventions are ranked on an ordinal scale gradient that ranges from to , where policies are classified as none ( ); minimum ( ); medium ( ); significant ( ); very significant ( ) based on their significance and invasiveness. therefore, the upper extreme of the gradient gathers the implementation of all the other measures that belong to the lower levels of the spectrum. for instance, referring to the policy interventions to contain the spread of the virus, the significance gradient ranges from no restriction to enforced lockdown ( table in the appendix). looking at the technology interventions, the gradient measures the invasiveness ranging from no interventions to centralised gps contact tracing ( table in the appendix). the comparative and conceptual framework ( ) uses a systematic approach to categorise policies targeted to specific objectives. it is a tool to assess the impact of policies on different sets of health and non-health related outcomes. we focus our evaluation on the following outcomes: daily reported number of deaths; case fatality rate; confirmation rate; icu saturation; ftse mib index value; and unemployment rate. despite death data might be biased as mentioned above, daily reported number of deaths and case fatality rate are selected to represent epidemiological outcomes. such outcomes are preferred to the confirmed cases indicators as the number of fatalities is less likely to be underestimated due to the country testing strategy ( ) . furthermore, the case fatality rate provides an estimate of the daily severity of the disease over time. icu saturation reflects the response capacity of the healthcare system. the confirmation rate shows the testing strategy variation over time, ceteris paribus . the financial and economic indicators reflect the investors' expectations of the italian economy. the unemployment rate provides a measure on how the containment measures impact the job market and the productivity nationally. we break down the analysis at regional level for all of the outcomes except for the ftse mib index value and unemployment rate, which are at national level. the analysis does not include other relevant health and non-health outcomes due to data availability or inconsistency during the period considered (i.e. length of stay in hospital, readmission rate, icu death rate and survival rate, public and private health care expenditure, public and private bed and personnel repurposing, public and private service provision, domestic violence, mental health demand). as a robustness check, the outcomes considered are compared with the oxford covid- government response tracker that includes the response, stringency, containment and health and economic support indices ( ). we report an overview of the policy categorisation considering their major impact on some outcomes of interest. among the four policy categories mentioned above, categories and are combined as there is an overlap between the preventive measures and the technology development and utilisation. in the absence of a covid- vaccine, the introduction of health resources and technology plays a fundamental role in preventing the spread of the virus. roadmap reports the principal interventions adopted by the italian government relative to the triple ts strategy: testing, tracing and treatment. technological solutions using geolocation tools have been used with success to control the spread of the virus in china, singapore and south korea ( ) . the effectiveness of such technologies relies on wide adoption, however, one possible barrier to this is the perceived invasiveness and potential breaches of privacy ( , ) . during the outbreak, the ministry of health issued national guidelines for testing. the testing criteria were updated at later stages (see table in the appendix), adopting who and european commission recommendations. the strictness of the criteria reflected the necessity to ration the supply of swabs, reagents and laboratory capacity. despite the national guidelines, a homogeneous testing strategy has not been consistently applied over time, across regions, neither in terms of number nor modality ( ) (see figure in the appendix). amongst the three most affected regions, lombardy ran fewer tests than veneto and emilia despite the huge expansion of the digital health sector in italy (with % increase, i.e. . billion euro), digital health strategies are decentralised, resulting in inconsistent utilisation across different regions ( ) . the covid- pandemic and the related lockdown measures have led to unprecedented economic costs around the world. the pandemic is a global shock that has affected the international economy, from financial markets where asset prices have decreased and volatility has increased characterising both the impact and future uncertainty involved with the pandemic ( ), to the impacts on the supply-chain ( ) . decision-making necessary to prevent an economic collapse in such a context involves a trade-off between public health and economic prosperity ( , ) . using some micro and macro indicators, this section shows that italy has suffered roadmap describes the principal economic interventions implemented by the substantial economic losses. italian government and the european central bank. to prevent the economic collapse of the country, italy has implemented several fiscal policies. the two most significant policies were the "cura italia" decree implemented on th march and the "decreto liquidità" implemented on the th april . the "cura italia" brought an immediate tax boost of billion euro to help most affected sectors, strengthen the healthcare system and provide unemployment benefits. the "cura italia" decree was reinforced when the council of ministers approved the "decreto liquidità" (decree-law of th april , n. ), allocating a total of billion euro to assist businesses by offering loan guarantees and a certain targeted tax relief. for instance, it provided billion euro as credit for small and medium enterprises, and injecting liquidity into the banking system. furthermore, the decree earmarks billion euro to support exporting enterprises located in italy to access liquidity. the state and the export credit agency cover respectively % and % of the guarantee to support enterprises financial obligations ( ). despite the substantial fiscal stimulus, the country has experienced the biggest quarterly economic contraction since the financial crisis. according to the latest data provided by the italian bureau of statistics (istat) ( ), the gdp in the first quarter of decreased by . % in comparison to the first quarter of . the overall gdp contraction estimated for is a contraction of . % ( ). the crisis also impacted international trade flows. figure in the appendix shows the monthly index of import and export in millions of euro. in the quarter march-may , despite the growth in may, the economic trend is conditioned by a sharp downturn of the previous months and is largely negative for both exports and imports (respectively - . % and - . % compared to the previous quarter december -february ) ( ). according to the latest data provided by the italian bureau of statistics (istat) ( ), in may , exports record a marked decline on an annual basis (- . %), but with improvements compared to april (- . %), for both the non-eu area (- . % ) and the eu (- . %). compared to exports, the contraction in imports (- . %) is wider and summarizes the drops in purchases from both markets (- . % from non-eu countries, - . % from the eu area). in may , the trade balance is estimated to increase by million euro (from + , million in may to + , million in may ). net of energy products, the balance is + , million euro (it was + , million in may ). considering the domestic market, retail sales recorded a collapse for non-alimentary goods, partly offset by a marked increase in e-commerce (see figure in the appendix). among the non-alimentary goods, the large negative variations correspond to the clothing and fur sector, followed by goods such as games, footwear and travel items. pharmaceutical products also recorded a negative variation ( ).the negative variation recorded in these sectors is likely to impact the economic fabric of the country, mainly composed by small and medium enterprises with limited investments in digitalisation directed toward to the online market. the perceived trade-off between public health benefits and the economic impact seemed to cover a central role in the exit phase as well. until the th april , the government imposed a homogenous exit strategy. as the number of cases decreased, regional governors put pressure on central government to relax some restrictions on economic activities. after th may , the government's policy changed, leaving the exit strategy to be decided by each region. this choice implied a heterogenous re-opening of economic activities, which helped small and medium scale companies to re-start their businesses. after the lockdown, with the de-escalation measures, the government faced a crucial phase in terms of economic recovery. to invert the negative economic trend, the italian government announced a massive fiscal and monetary stimulus on th may . the decree "rilancio" allocated around billion euro in five main areas with the aim of reorganising the hospital network to deal with covid- emergency. additionally, it guaranteed liquidity and support for italian companies, aiding their stability during the emergency period and encouraging their revival at the time of recovery. this section describes the policy implemented by the government to cope with the limited capacity of the health care system and the challenges of the covid- pandemic. the central government is responsible for public health interventions; however, the decentralisation of the italian healthcare system hindered the implementation of a homogeneous strategy. regional health care systems differ widely in terms of hospital organisation (public versus private), equipment (number of beds etc.) and medical workforce ( ). following a decree implemented on st february , the government facilitated the urgent increase of hospital beds in all regions by % in icu and % in pulmonology and infectious disease wards. the measure entailed the immediate redistribution of hospitalised patients to accredited private structures to ease the pressure on the public system. the national health system is composed of % public and % private beds, with substantial regional variations, ranging from . % of public beds in lombardy to . % in basilicata ( ). increasing the number of icu beds appears to have largely prevented saturation, except for lombardy, which experienced an overloading of the system from st april (see figure in the appendix). the available data does not give further information on patient outcomes. patients' length of stay, discharge, re-admission and mortality rate data are necessary to fully evaluate the healthcare system performance and the health policies implemented by the government ( ). the overall standard national health budget increase for amounts to . billion euro with a decree of th march ( ). as part of this budget increase, the government spent million euro to implement the "aid distribution system", distributing disposable and durable medical materials to each region ( ). the most common disposable materials distributed were masks ( %), gloves ( %), and diagnostic kits ( %). durables materials included glasses ( %) and thermometers ( %). veneto received the highest amount of materials and molise the least. between th march and th april , the government also distributed , ventilators, of which % went to lombardy and % to emilia romagna. following a decree implemented on th march , the government committed million euro to hire , medical personnel on six-month contracts. regions autonomously managed this hiring process, making it hard to access the relevant data ( ). on th may, "decreto rilancio" allocates , million euro to national emergency fund and , million euro to strengthen emergency departments and community care ( ). this section has two aims. firstly, we test whether the escalation and de-escalation stringency measures to contain the spread of the virus were justified by the underlying epidemiological trend for all the regions, using the t-test analysis. our goal is to test whether differences in means of scaling-up and scaling-down policies are statistically significant. the daily death trend is chosen as the indication of the epidemiological trend. secondly, we describe through a graphical analysis how the health and non-health outcomes were impacted by the policies presented in the conceptual framework. the graphical analysis aims at evaluating if differences in the levels of the policies gradient have had a detectable impact on given outcomes across different areas of the country and may be assessed over different lags of time . in the absence of a counterfactual scenario, we run a t-test analysis on the mean of the daily number of deaths for each region throughout the period of each single policy (see table ). the analysis defines whether the difference in the daily number of deaths between each containment policies implemented in the escalation and de-escalation phase is statistically significant to justify the implementation of a more or less stringent policy. despite the death trend might be influenced by other policy interventions (such as increase of the icu capacity and more effective preventive method), it still is more reliable compared to other epidemiological measures. the analysis covers the period th february to th august . a value between (no intervention) and (very significant intervention) is assigned to each policy to represent its strictness (see table in the appendix). the policy classifications of lombardy, emilia romagna and veneto are displayed in three separate columns since targeted lockdown measurers were implemented before the national lockdown (see table in the appendix). overall, the escalation measures were found to be justified by the underlying death trend. considering the very that the enhanced testing capacity corresponds to the flattening of the case fatality rate and to a reduced confirmation rate (see figure in the appendix). in early march , significant containment interventions were required to ensure the sustainability of the italian healthcare system, especially in northern regions. the lockdown implemented on the th of march , and the closure of business activities of the nd march coincides with a decreasing trend in daily mortality, especially in northern regions in late march (see figure in the appendix). faster policies escalation in the epicentre of the pandemic might have resulted in a lower peak of deaths, flattening the contagion curve (see figure in the appendix). despite the substantial distribution of equipment throughout the regions, the icu wards were close to full capacity in the northern regions. although northern and central regions faced a similar increase in the saturation rate until th march , the lockdown timing seemed to be effective in the central and southern regions where the severity of the contagion was mitigated, starting to flattener before than in northern regions (see figure in the appendix). the case fatality rate stabilisation coincides with the government's announcement of the exit strategy at the end of april (see figure in the appendix). during the period considered in the analysis, the government did not invest resources for the development of tracing technology, which was instead developed for free by a private company. on th may , at the start of the exit phase, the government launched a seroprevalence study on a sample of , individuals. however, significant technological interventions seemed to be far from having any impact on the outcomes considered (daily number of reported deaths and icu saturation) due to delayed implementation (see figure in the appendix). the case fatality rate flattened, and the confirmation rate decreased even though minimal technological interventions were in place (see figure in the appendix). the impact of significant technological interventions could be better assessed if a second wave of covid- (or similar diseases) were to occur in the future. the stringency of the measures is negatively correlated with socio-economic factors. figure in the appendix shows an inverse relationship between the stringency of the containment measures and the stock market index value. figure in the appendix shows the daily performance of ftse mib and the response to major fiscal stimulus packages in italy. the period from february to mid-march saw some of the most significant daily drops in the performance of ftse mib index. following the two major decrees, "cura italia" and "decreto liquidità", it recorded an increase. in particular, the week beginning th march showed an increase in ftse mib (see figure in the appendix). since th may , the ftse mib index has steadily increased, in response to the stimulus and improving expectations surrounding the recovery effort associated with covid- . the lockdown and the subsequent closure of most activities also affected the job market although the unemployment rate did not entirely reflect the lockdown effect due to the reduction in the labour force that decreased by % in april compared to january decreasing ( ) (see figure in the appendix). the results displayed in this analysis are consistent with the oxford covid- government response tracker indices (see figures , , and in the appendix) ( ). covid- also had a huge impact on patients' access to health care, essential services, and education facilities. the high saturation rate in icu due to a large number of severe covid- cases caused a . % decline in organ donation. as of th march , schools and universities closed their facilities and began offering online classes. although online schooling may represent an effective means of education provision, access is dependent on the availability of internet connection and electronic equipment (i.e. computer, laptop, tablets). with schools remaining closed during the exit strategy, and concerns for a potential second wave in autumn, the inequality in access may persist, with potential long-term consequences. the outbreak of covid- significantly affected italy with severe health, social and economic consequences. the production of future government policy; with the transparency and ready availability of data essential. evidence-based interventions is relevant for reducing uncertainty around the interventions, thereby maximising the resource and investment allocations. a detailed appraisal of the data management system between regions and central government is missing and represents a limitation for further studies. the threat of future pandemics should drive the government's investments and resources to prevent and promote public health, strengthening community and territorial services, which demonstrate to be particularly successful in some regions to respond to health services organisation and delivery challenges. as far as the sustainability of the healthcare system is concerned, policymakers should focus on the elaboration of the promotion, prevention and early intervention framework to prevent suicide and lower the long-term impact on people's mental health due to isolation, social distancing and high stress levels. mental health programs should be targeted for different population groups, prioritising those at higher risk. moving forward governments need to identify and implement plans to mitigate the negative effects of a pandemic on vulnerable groups across society which includes elderly in the home care facilities, students, families with children and the impacted workforce. ischemic heart disease ( %) atrial fibrillation ( . %) chronic renal failure ( %) chronic obstructive pulmonary disease ( . %) dementia ( . %) active cancer in the past years ( . %) hearth failure ( . %) source: data provided by iss ( ) ( ) prime minister signed "phase " decree-starting from the th may. it has three phases . from the th may: ) parks reopening; ) free movement in the same region; ) free movement in different regions has to be justified by heath, work reasons; ) relatives visiting with personal protections; ) sport activities is allowed at meters social distancing form others; ) athletes training will be allowed for individual sports; ) funeral ceremonies open air: people maximum; ) bar and restaurants take away; ). restart of activities: manufacturing, building companies, transportations respecting security and hygienic -new security guidelines . from the th may: ) reopening of commercial activities, museums, libraries; ) team sport activities allowed. from the  programs of the biomedical and telemedicine sector,  strengthening of the national system of production of medical devices and  services aimed at the prevention of health emergencies / / significant ( ) decree "rilancio"  billion financing workers, firms, healthcare system, touristic sector  billion to finance the firms' debt ( ) variation in government responses to covid- . version blavatnik school of government working paper impact of non-pharmaceutical interventions (npis) to reduce covid mortality and healthcare demand the covid- pandemic two waves of technological responses in the european union. hague centre for strategic studies; . . camera dei deputati. misure sanitarie per fronteggiare l'emergenza coronavirus covid- in italy: actual infected population, testing strategy and imperfect compliance. . . international monetary fund. policy responce to covid- -policy tracker key country policy tracker categorising policy & technology interventions for a pandemic: a comparative and conceptual framework united nations development programme italy: health system review the italian healthcare system. thomson s et al international profiles of healthcare systems la politica sanitaria in italia: dalla riforma legislativa alla riforma costituzionale. institute of public policy and public choice-polis quasi-market and cost-containment in beveridge systems: the lombardy model of italy. health policy world health organization. the wold health report : health systems: improving performance most efficient healthcare world bank -world development indicators. life expectancy at birth, total (years) population structure and ageing patologie croniche in costante aumento in italia con incremento della spesa sanitaria. la cronicità non colpisce tutti allo stesso modo: si confermano le diseguaglianze di genere, territoriali, culturali e socio economiche geographical tracking and mapping of coronavirus disease covid- /severe acute respiratory syndrome coronavirus (sars-cov- ) epidemic and associated events around the world: how st century gis technologies are supporting the global fight against outbreaks and epidemics quantifying sars-cov- transmission suggests epidemic control with digital contact tracing on the responsible use of digital data to tackle the covid- pandemic lessons from italy's response to coronavirus: harvard business review coronavirus, si estende in emilia-romagna il test drive-through: tamponi direttamente dall'auto covid- italia -monitoraggio della situazione survey nazionale sul contagio covid- nelle strutture residenziali e sociosanitarie the unprecedented stock market impact of covid- (no. w ) the covid- shock to supply chains. the university of melbourne pandemics depress the economy gianluca public health interventions and economic growth: revisiting the spanish flu evidence. ssrn comparison of the icu beds saturation rate with the capacity before and after the covid- note: the red line coincides with the total saturation of the icu capacity ( %) in the region considered. the x-axis reports the saturation rate = %; = %; = %. source: personal elaboration of data provided by minsitero della salute and protezione civile ( ) source: data provided by ministry of health technology intervention gradient and case fatality rate (panel a), confirmation rate (panel b), daily number of reported deaths (panel c) . patients become asymptomatic due to symptoms resolution . patient is negative to sars-cov- test clearance . rna and sars-cov- absence in body fluids for patient that presented symptoms and for those who did not present any symptoms . for asymptomatic patients that resulted positive, the test should not be repeated before days after the first positive diagnosis . two molecular tests need to be performed in hours. both of them have to be negative http://www.salute.gov.it/portale/nuovocoronaviru s/dettaglionotizienuovocoronavirus.jsp?lingua=i taliano&menu=notizie&p=dalministero&id= key: cord- -c be eii authors: martina, stefano; amato, alessandra; rongo, roberto; caggiano, mario; amato, massimo title: the perception of covid- among italian dentists: an orthodontic point of view date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: c be eii covid- has severely impacted dentists, who are at a great risk of infection. this study aimed to investigate if dentists are anxious about returning to their daily activities, and what the perception of the risk is for dentists and orthodontists regarding orthodontic procedures. an online questionnaire, including the patient health questionnaire- (phq- ), was sent to italian dentists during the final days of the lockdown with items about anxiety, fear, distress, perceived risk for operators, and concerns about orthodontic patients caused by working during the covid- outbreak. data were analyzed with a chi-square test and logistic regression analysis. the level of significance was set as p < . . a total of dentists completed the survey, including orthodontists. returning to their daily work activity was a source of anxiety for participants and this was associated with the level of distress (odds ratio (or) = . ; p < . ). most of the orthodontists ( . %) thought that they would increase the number of working hours during the week (or = . ; p = . ). italian dentists were mostly scared to return to their daily activities because they considered their jobs a high risk to them and their families. dentists with an exclusive/prevailing orthodontic activity were forced to increase their working day during the week. in december a novel coronavirus ( -ncov) was described in the chinese city of wuhan [ ] . the -ncov, afterwards renamed sars-cov- [ ], caused a highly contagious disease (covid- ) [ ] . covid- patients could have upper respiratory tract infection (rti), fever, dry cough, dyspnea and severe viral pneumonia with respiratory failure and even death, but there is a percentage of those infected that shows mild symptoms (pharyngodynia, nasal congestion, olfactory and gustative disorders) or no symptoms [ , ] . covid- could directly cause cardiovascular injuries such as pericarditis, myocarditis, myocardial infarction, heart failure, arrhythmias or thromboembolic events [ ] . in a few weeks, covid- had spread all over the world and on march , the world health organization (who) declared it a pandemic. the virus propagates between people through close contact and via respiratory droplets produced from coughs or sneezes [ ] , but several uncertainties currently remain, particularly regarding the contribution of asymptomatic versus symptomatic patients to the transmission of the virus [ ] . virus identification in human respiratory epithelial cells could be positive about h from exposure and - h before the onset of symptoms [ ] . the nasopharyngeal real-time polymerase chain reaction (rt-pcr) swab test was used to diagnose covid- . however, this test has high costs and complex execution methods and, even though one of the epidemic fight strategies is the identification and isolation of the positive subject, the nasopharyngeal swabs on the whole population is still not sustainable. the available diagnostic accuracy data suggest that the test has very high specificity, but a moderate sensitivity ( - %) [ ] . in the absence of a vaccine or an effective treatment, many countries including italy have adopted quarantine measures and lockdowns to control the spread of covid- , limiting mortality and maintaining healthcare demands within capacity. on may in italy , cases of covid- were diagnosed out of , , subjects tested, with , deaths [ ] . in italy, during the lockdown (also known as phase- ), the dental assistance in private practices was not suspended by law to reduce the burden on the hospitals dealing with the covid- patients. however, the national medical and dental committee and the category unions strongly recommended rescheduling dental activities, limiting these to emergencies, such as pulpitis, abscesses and phlegmons. it can be assumed that stopping dental procedures may be due to several reasons: to avoid the movement of people, to reduce the consumption of personal protective equipment (ppe) and to reduce the risk of infection. indeed, aerosols formed during dental procedures, contact spread, and contaminated surface spread are possible routes of transmission for sars-cov- [ ] . for these considerations, most of the treatments, including routine orthodontic appointments, were postponed. orthodontic patients were assisted virtually by means of photos, videos, or video calls, performing triage to select the real urgencies to be managed in person from remotely manageable situations [ ] . the lockdown ended in italy on may and the so-called phase- has started by imposing social distancing measures and the use of masks in everyday life. in a dental practice, it is obviously impossible to maintain these measures; therefore, dentists have a high risk of getting infected from patients and potentially spreading the virus. in light of this, it is not surprising for dentists to be afraid of being infected by their patients. on the other hand, orthodontics could be a less risky practice than other specialties of dentistry. orthodontic controls are usually quick, aerosol procedures are few and not very frequent during the same clinical day, and most patients are under years of age, a category that seems to be less affected by covid- [ ] . previous studies investigated the level of fear and psychological distress during the covid- pandemic [ , ] , finding that elevated psychological distress was present among dentists who have a background illness, those who feared contracting covid- from a patient, and those who had higher subjective overload. none of these studies were performed in italy. the purpose of this study was to investigate how the return to their daily practice during phase- influences dentists and what their perception of risk is regarding orthodontic procedures. the present cross-sectional study was conducted by sending an online survey questionnaire to italian dentists, from may to may . the survey was created on an online survey development cloud-based software called surveymonkey ® (svmk, san mateo, ca, usa). the participants were approached via facebook, whatsapp, and mailing lists. participants authorized the treatment of their data that were collected anonymously. the personal information of participants was protected and there were no inhumane questions or investigations. the questionnaire was comprised of multiple-choice questions: questions were about personal data (gender, age, region of residence); were on symptoms of covid- ; were about the perceived risk for operators during orthodontic procedures, anxiety and distress caused by working during the covid- outbreak; were about the fears for an infection and the concerns regarding orthodontic and temporomandibular disorders (tmd) patients whose treatment has been suspended due to the epidemic; were about emergencies and changes in clinical procedures and work organization as a result of the covid- . the last questions of the patient health questionnaire (phq- ) [ ] were included in the questionnaire to assess whether the general state of anxiety and depression could influence the other answers. the phq- was composed of four questions with a -item likert scale. scores between and indicated a slight distress, between and indicated a moderate psychological distress, and higher than , a severe distress. in the survey, most of the questions had a dichotomous answer (yes/no). dentists were asked about the number of emergencies they faced during the lockdown period (less than , more than ) and they had to classify what they feared most in the case of infection among the following: fear of dying, fear of the quarantine, fear of infecting family members, fear of economic loss, fear of interrupting treatments, or fear of discrimination. frequencies and percentages for categorical data were computed. a chi-square test was used to assess the association between gender (male vs. female), age (older than years old vs. - vs. - vs. - vs. - vs. - ), region of residence (south vs. centre-north), level of distress ( - vs. - ), orthodontic activity (general practice vs. main/exclusively orthodontic activity) and questions about risk, anxiety and distress caused by working during the covid- outbreak. furthermore, the chi-square test was used to evaluate the association between the level of distress and the orthodontic activity with the questions about emergencies and changes in clinical procedures and work organization as a result of the covid- . in the case of a statistically significant association, logistic regression analyses to calculate the odds ratio (or) were performed. a standard statistical software package (spss, version . ; spss ibm, armonk, new york, usa) was used. the level of significance was set at p < . . a total of ( m, f, not reported) dentists completed the survey. participants were asked to identify their age range and the most common were - years old ( ) and - years old ( ). most of the dentists lived with their families ( ) or alone ( ). a total of were from the south of italy and from the centre-north italy. among the participants, had a main ( ) or exclusive ( ) orthodontic practice, while had a more general practice. none of the participants were diagnosed as covid-positive but only were tested. the phq- reported different levels of distress: participants reported no distress, slight distress, moderate distress, and severe distress. these data are shown in the table . the level of distress was associated with questions about anxiety and distress; indeed, % ( / ) of the dentists with higher distress levels were more anxious about starting their daily work activities again (odds ratio (or) = . ; p < . ) compared with . % ( / ) of the dentists with less distress. furthermore, . % ( / ) of dentists with higher distress levels were more inclined to close their activities (or = . ; p < . ) compared with . % ( / ) of participants with less distress, for a total of subjects out of that were more inclined to stop their jobs (tables and ). only out of participants did not consider their work to be at a higher risk, and only did not consider their work to be of higher risk also for their families. among these , % ( / ) were younger than years old (or = . ; p = . ) and % ( / ) were from centre-north italy (or = . ; p = . ). returning to daily work activities was a source of anxiety for participants, mostly women; indeed, % of women ( ) replied yes against only . % of men ( ) (or = ; p = . ). similarly, this was a source of anxiety more for those participants who were younger than years old ( ) than for those older ( ), (or = . ; p = . ). regarding teamwork, out of dentists thought that their collaborators had less risk than dentists of being infected by covid- . most of the participants ( ) considered orthodontic procedures as a lower risk of contagion than general dental practices, even if adolescents and children were mostly considered a similar source of infection as adults (tables and ). the survey also asked about the number of emergencies faced during the lockdown period and dentists had more than patients during this lockdown period, but dentists were extremely scared and were moderately scared of dealing with emergencies. interestingly, there was an association between the number of dentists that dealt with fewer than emergencies during this period and the level of fear facing the emergencies with . % ( / ) of dentists that were strongly or moderately scared who faced less than emergencies compared with . % ( / ) of dentists that were slightly or not scared that faced more than emergencies (or = . ; p = . ). finally, there was an association between the level of distress (phq- ) and the fear during emergencies. indeed, among the dentists that had a high level of distress, % ( ) were strongly scared and only % ( ) were unafraid (or = . ; p < . ) ( tables and ) . furthermore, dentists thought that patients under fixed treatment who were not controlled for more than two months should be considered emergencies ( ), while only and dentists considered emergencies those patients with removable appliances or with temporomandibular disorders that had not been controlled for months (table ) . finally, most of the dentists ( ) thought that in this phase- they would reduce the number of patients during the day and thought that they would increase their number of working hours during the week. interestingly, among these , the greater percentage ( . %, / ) were dentists who had a main/exclusive orthodontic activity (or = . ; p = . ), and similarly among this group of dentists there was the greater percentage of participants that planned to postpone aerosol procedures ( . %, / , or = . ; p = . ). regarding the ranking of fears in case of contagion, most of the participants were afraid of infecting family members (indicated times as their first fear), dying (indicated times as their first fear), and being discriminated against (indicated times as their first fear, table ). this study was carried out during the final days of the lockdown in italy and just before dentists returned to their practices. the aim was to understand through a survey how dentists had approached phase- and how they were preparing for phase- and investigate whether these situations had created anxiety or distress. the sample was a good representation of different age ranges, different regions of residence, different levels of distress and different dental activities. none of the interviewed dentists were positive for covid- and almost % were tested. this percentage is higher than the total number of swabs done in italy (by may, % of the population). in italy there was a considerable difference in the incidence of covid- between central-northern and southern italy, with nearly % of total cases from the centre-north [ ] . therefore, the answers of dentists from the centre-north were compared with those from the south. despite the different number of cases, the perception of the problem by dentists between centre-north and south was similar. this could be due to the quarantine measures which were the same in all regions of italy and to the influence of the italian media which stressed the situation without any differences during phase- . the phq- showed that dentists out of ( . %) had moderate/severe psychological distress (score equal to or higher than ) in the last weeks. this percentage was greater compared with the data presented of the general population, where about % of the population presented these scores [ ] . it could be argued that the tough situation due to the pandemic crisis and the possible italian economic crisis might influence the psychological status of the participants. moreover, it was interesting that people with higher distress levels were those with more anxiety about returning to work, more inclined to close their activities, more afraid of facing emergencies, and more fearful of infecting their families. this critical situation has highlighted the difficulties that italian dentists had to face during their activities, and as a vicious circle in which more psychological distress led to more fear and anxiety, that led to a decrease in psychological wellness [ ] . most dentists interviewed considered themselves and their collaborators to be exposed to a high risk due to their jobs and they are afraid of infecting their relatives. these data are consistent with previous studies on the psychological impact in health care workers of similar infectious diseases, such as severe acute respiratory syndrome (sars), who were afraid of contracting an infection while treating an infected patient or infecting a family member [ , ] . however, some associations were found between dentists that did not think they were a higher source of infection risk for their relatives due to their jobs regarding age and region of residence. this is probably due to the fact that, although with different means of contagion, dentists with more experience are used to managing in their practice several infective diseases, such as hepatitis b, hepatitis c, human immunodeficiency virus infection, tuberculosis, following strict protocols for infection control [ ] . although many dentists treated less than patients in phase- , the management of these emergencies frightened them. the fear in dealing with emergencies was associated with the distress level of the dentists (the higher the distress, the higher the level of fear) but also with the number of emergencies seen during the lockdown period. indeed, the dentists that were more scared in facing the emergencies were those that treated fewer than emergencies. two hypotheses might explain this association: first, dentists who were more fearful tried as much as possible to solve any potential emergency by using telemedicine to avoid meeting patients. second, those dentists who performed more than emergencies understood that with adequate ppe the chance of contagion was low. however, due to the lack of ppe during phase in italy, the fear of performing any emergency procedure may have also been due to the lack of availability of proper ppe. fifty-five percent of respondents were worried about the return to their dental practices in phase- ; % contemplated definitively ceasing their activity. it is obvious considering that, since there is no vaccine or approved treatment, anxiety about being infected is very high in the population. dentists and health professionals in general are at higher risk of contracting infectious diseases, with an additional psychological distress component [ ] . moreover, dentists may be concerned about liability related to possible infection of workers or patients in their practices, and about the critical italian economic situation which could reduce the demand for dental treatment. women, dentists younger than years old, and participants with higher distress levels were more concerned about coming back to work compared with other categories. as already mentioned, psychological distress and the fear of coming back to work with an increased patient load strongly influenced each other due to the concern about the contagion, and probably women and young people were more worried about their families. indeed, when asked to rank the situations that would be most frightening in case of infection, most dentists ranked the fear of infecting their relatives and the fear of dying as the top two. few have attributed importance to the possibilities of being discriminated against, economic loss and the suspension of ongoing treatments. surprisingly, dentists from the south said they were more concerned with infecting family members. in addition, most dentists believed that children and adolescents represent the same risk of infection compared with adults. a part of the questionnaire was focused on orthodontics and about % of the participants were orthodontists. most dentists thought that, with orthodontics, the risk of being infected is lower. the explanation for this could be that, unlike other branches of dentistry, in orthodontics there are procedures that do not require aerosols. aerosol procedures were indicated as more infective procedures in several articles [ , ] . in fact, most orthodontists ( %) declared that they will postpone clinical procedures with aerosols as opposed to general practitioners, in which only % will postpone these procedures. sixty percent of participants believed that not examining a patient on fixed appliance treatment for more than months is a serious problem, whereas they believed it is less critical to postpone exams for patients who have removable devices. some orthodontists may be worried about orthodontic activations of fixed appliances given before the lockdown, which hardly can be monitored with telemedicine. other orthodontists may believe that not checking patients for more than months may have caused damage to the appliance, such as loose brackets and bands or worsened oral hygiene. these are all patient cooperation factors that could contribute significantly to increased treatment time [ ] . removable appliances may suffer less from these issues and generally cause less discomfort to patients as compared with fixed appliances [ ] . regarding rescheduling patients, compared with general practitioners a higher percentage of orthodontists declared that they would organize their weekly schedules to work more days during the week ( . % vs. . %). this is an important change in orthodontic practice because in italy most dentists that have a main/exclusive orthodontic activity work as consultants. due to the greater time needed to perform disinfection procedures, the chair time between two patients will increase and the number of patients that is possible to see in one day, obviously, will decrease forcing the dentists to work more days per week. regarding the management of tmd patients, most dentists thought that examining these patients is not an emergency; however, they believed that not controlling them for more than months may have worsened the patient's psychological distress. this study presents some limitations. it is a survey-based study; hence information is self-reported, and the collection time was only one week. however, it also presents some strengths, such as the high number of participants, the good distribution of the factors, and the focus on the orthodontic practice. this survey-based study investigated the perception of italian dentists of the return to their daily practices and of their risk during orthodontic procedures. italian dentists were mostly afraid to return to their daily activities, and women, younger dentists and dentists with higher distress levels were more frequently scared by this aspect. the worst fears in case of contagion was infecting family members and the fear of dying. dentists considered their job a high risk to them and their families; however, orthodontic procedures were considered a lower risk compared with general dental procedures. dentists with an exclusive/main orthodontic activity were forced to increase their working day during the week. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- novel coronavirus ( -ncov) situation report- clinical characteristics of covid- in china clinical presentation of covid- : a systematic review focusing on upper airway symptoms. ear nose throat j. , cardiovascular manifestation and treatment in covid- how covid- spreads estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship incubation period of novel coronavirus ( -ncov) infections among travellers from wuhan, china the appropriate use of testing for covid- dipartimento della protezione civile covid- , i casi in italia il • maggio ore transmission routes of -ncov and controls in dental practice management 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health care workers health hazard evaluation in private dental practices: a survey in a province of northen italy factors associated with mental health outcomes among health care workers exposed to coronavirus disease covid- outbreak: an overview on dentistry covid- ): emerging and future challenges for dental and oral medicine an evaluation of factors affecting duration of orthodontic treatment pain and discomfort associated with labial multibracket appliances vs. clear aligners funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -ylcv zvl authors: buonomo, b.; della marca, r. title: modelling information-dependent social behaviors in response to lockdowns: the case of covid- epidemic in italy date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ylcv zvl the covid- pandemic started in january has not only threatened world public health, but severely impacted almost every facet of lives including behavioral and psychological aspects. in this paper we focus on the 'human element' and propose a mathematical model to investigate the effects on the covid- epidemic of social behavioral changes in response to lockdowns. we consider a seir-like epidemic model where that contact and quarantine rates are assumed to depend on the available information and rumors about the disease status in the community. the model is applied to the case of covid- epidemic in italy. we consider the period that stretches between february , when the first bulletin by the italian civil protection was reported and may , when the lockdown restrictions have been mostly removed. the role played by the information-related parameters is determined by evaluating how they affect suitable outbreak-severity indicators. we estimated that citizens compliance with mitigation measures played a decisive role in curbing the epidemic curve by preventing a duplication of deaths and about % more contagions. in december , the municipal health commission of wuhan, china, reported to the world health organization a cluster of viral pneumonia of unknown aetiology in wuhan city, hubei province. on january , , the china cdc reported that the respiratory disease, later named covid , was caused by the novel coronavirus sarscov [ ] . the outbreak of covid rapidly expanded from hubei province to the rest of china and then to other countries. finally, it developed in a devastating pandemics aecting almost all the countries of the world [ ]. as of may , a total of , million of cases of covid and , related deaths have been reported worldwide [ ] . in absence of treatment and vaccine, the mitigation strategy enforced by many countries during the covid pandemic have been based on social distancing. each government enacted a series of restriction aecting billions of people including recommendation of restricted movements for some or all of their citizens, and localized or national lockdown with the partial or full closingo of nonessential companies and manufacturing plants [ ] . italy has been the rst european country aected by covid . the country has been strongly hit by the epidemic which has triggered progressively stricter restrictions aimed at minimizing the spread of the coronavirus. the actions enacted by the italian government began with reducing social interactions through quarantine and isolation till to reach the full lockdown [ , ] . on may , , the phase two began, marking a gradual reopening of the economy and restriction easing for residents. one week later, shops also reopened and the restrictions on mobility were essentially eliminated, with the only obligation in many regions to use protective masks [ ] . during the period that stretches between january , and may , , italy suered , ocial covid cases and , deaths [ ] . the scientic community has promptly reacted to the covid pandemic. since the early stage of the pandemic a number of mathematical models and methods has been used. among the main concerns raised were: predicting the evolution of the covid pandemic wave worldwide or in specic countries [ , , ] ; predicting epidemic peaks and icu accesses [ ] ; assessing the eects of containment measures [ , , , , , , ] and, more generally, assessing the impact on populations in terms of economics, societal needs, employment, health care, deaths toll, etc [ , ] . among the mathematical approaches used, many authors relied on deterministic compartmental models. this approach has been revealed successful for reproducing epidemic curves in the past sarscov outbreak in [ ] and has been employed also for covid . specic studies focused on the case of epidemic in italy: gatto et al. [ ] studied the transmission between a network of italian provinces by using a sepia model as core model. their sepia model discriminates between infectious individuals depending on presence and severity of their symptoms. they examined the eects of the intervention measures in terms of number of averted cases and hospitalizations in the period february march , . giordano et al. [ ] proposed an even more detailed model, named sidarthe, in which the distinction between diagnosed and nondiagnosed individuals plays an important role. they used the sidarthe model to predict the course of the epidemic and to show the need to use testing and contact tracing combined to social distancing measures. the mitigation measures like social distancing, quarantine and selfisolation may be encouraged or mandated [ ] . however, although the vast majority of people were following the rules, even in this last case there are many reports of people breaching restrictions [ , ] . local authorities needed to continuously verifying compliance with mitigation measures through monitoring by health ocials and police actions (checkpoints, use of drones, ne or jail threats, etc). this behavior might be related to costs that individuals aected by epidemic control measures pay in terms of health, including loss of social relationships, psychological pressure, increasing stress and health hazards resulting in a substantial damage to population wellbeing [ , , ] . as far as we know, the mathematically oriented papers on covid nowadays available in the literature do not explicitly take into account of the fraction of individuals that change their social behaviors solely in response to social alarm. from a mathematical point of view, the change in social behaviors may be described by employing the method of informationdependent models [ , ] which is based on the introduction of a suitable information index m (t) (see [ , ] ). this method has been applied to vaccine preventable childhood diseases [ , ] and is currently under development (see [ , , ] for very recent contributions). in this paper, the main goal is to assess the eects on the covid epidemic of human behavioral changes during the lockdowns. to this aim we build up an informationdependent seirlike model which is based on the key assumption that the choice to respect the lockdown restrictions, specically the social distance and the quarantine, is partially determined on fully voluntary basis and depends on the available information and rumors concerning the spread of the covid disease in the community. a second goal of this manuscript is to provide an application of the information index to a specic eldcase, where the model is parametrized and the solutions compared with ocial data. we focus on the case of covid epidemic in italy during the period that begins on february , , when the rst bulletin by the italian civil protection was reported [ ] , includes the partial and full lockdown restrictions, and ends on may , when the lockdown restrictions have been mostly removed. we stress the role played by circulating information by evaluating the absolute and relative variations of diseaseseverity indicators as functions of the informationrelated parameters. the rest of the paper is organized as follows: in section we introduce the model balance equations and informationdependent processes. two critical epidemiological thresholds are computed in section . section is devoted to model parametrization for numerical simulations, that are then shown and discussed in section . conclusions and future perspective are given in section . model formulation we assume that the total population is divided into seven disjoint compartments, susceptibles s, exposed e, presymptomatic i p , asymptomatic/mildly symptomatic i m , severely symptomatic (hospitalized) i s , quarantined q and recovered r. any individual of the population belongs to one (and only one) compartment. the size of each compartment at time t represents a state variable of a mathematical model. the state variables and the processes included in the model are illustrated in the ow chart in fig. . the model is given by the following system of nonlinear ordinary dierential equations, where each (balance equation) rules the rate of change of a state variable. the model formulation is described in detail in the next subsections. as mentioned in the introduction, the mitigation strategy enforced by many countries during the covid pandemic has been based on social distancing and quarantine. motivated by the discussion above, we assume that the nal choice to adhere or not to adhere lockdown restrictions is partially determined on fully voluntary basis and depends on the available information and rumors concerning the spread of the disease in the community. from a mathematical point of view, we describe the change in social behaviors by employing the method of informationdependent models [ , ] . the information is mathematically represented by an information figure : flow chart for the covid model ( )( ). the population is divided into seven disjoint compartments of individuals: susceptible s(t), exposed e(t), presymptomatic i p (t), asymptomatic/mildly symptomatic i m (t), severely symptomatic/hospitalized i s (t), quarantined q(t) and recovered r(t). blue colour indicates the informationdependent processes in model (see ( )( )( ), with m (t) ruled by ( )). index m (t) (see appendix a for the general denition), which summarizes the information about the current and past values of the disease [ , ] and is given by the following distributed delay this formulation may be interpreted as follows: the rst order erlang distribution erl ,a (x) represents an exponentially fading memory, where the parameter a is the inverse of the average time delay of the collected information on the status of the disease in the community (see appendix a). on the other hand, we assume that people react in response to information and rumors regarding the daily number of quarantined and hospitalized individuals. the information coverage k is assumed to be positive and k ≤ , which mimics the evidence that covid ocial data could be underreported in many cases [ , ] . with this choice, by applying the linear chain trick [ ] , we obtain the dierential equation ruling the the dynamics of the information index m :Ṁ in this section we derive in details each balance equation of model ( ). susceptibles are the individuals who are healthy but can contract the disease. demography is incorporated in the model so that a net inow rate bn due to births is considered, where b is the birth rate andn denotes the total population at the beginning of the epidemic. we also consider an inow term due to immigration, Λ . since global travel restrictions were implemented during the covid epidemic outbreak [ ] , we assume that Λ accounts only of repatriation of citizens to their countries of origin due to the covid pandemic [ ] . in all airports, train stations, ports and land borders travellers' health conditions were tested via thermal scanners. although the eectiveness of such screening method is largely debated [ ] , for the sake of simplicity, we assume that the inow enters only into the susceptibles compartment. in summary, we assume that the total inow rate Λ is given by: the susceptible population decreases by natural death, with death rate µ and following infection. it is believed that covid is primarily transmitted from symptomatic people (mildly or severely symptomatic). in particular, although severely symptomatic individuals are isolated from the general population by hospitalization, they are still able to infect hospitals and medical personnel [ , ] and, in turn, give rise to transmission from hospital to the community. the presymptomatic transmission (i.e. the transmission from infected people before they develop signicant symptoms) is also relevant: specic studies revealed an estimate of % of secondary cases during the presymptomatic stage from index cases [ ] . on the contrary, the asymptomatic transmission (i.e. the contagion from a person infected with covid who does not develop symptoms) seems to play a negligible role [ ] . we also assume that quarantined individuals are fully isolated and therefore unable to transmit the disease. the routes of transmission from covid patients as described above are included in the force of infection (foi) function, i.e. the per capita rate at which susceptibles contract the infection: the transmission coecients for these three classes of infectious individuals are informationdependent and given by ε p β(m ), ε m β(m ) and ε s β(m ), respectively, with ≤ ε p , ε m , ε s < . the function β(m ), which models how the information aects the transmission rate, is dened as follows: the baseline transmission rate β(·) is a piecewise continuous, dierentiable and decreasing function of the information index m , with β(max(m )) > . we assume that where π is the probability of getting infected during a persontoperson contact and c b is the baseline contact rate. in ( ) the reduction in social contacts is assumed to be the sum of a constant rate c , which represent the individuals' choice to selfisolate regardless of rumors and information about the status of the disease in the population, and an informationdependent rate c (m ), being c (·) increasing with m and c ( ) = . in order to guarantee the positiveness, we assume c b > c + max(c (m )). following [ ] , we nally set namely . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. presymptomatic individuals are infectious people that have not yet developed signicant symptoms. such individuals lie in a stage between the exposed and the expected symptomatic ones. they remain in this compartment, i p , during the postlatent incubation period and diminish due to natural death or progress to become asymptomatic or symptomatic infectious individuals (at a rate η). this compartment includes both the asymptomatic individuals, that is infected individuals who do not develop symptoms, and mildly symptomatic individuals [ ] . as mentioned above, the asymptomatic transmission seems to play a negligible role in covid transmission. however, asymptomatic individuals are infected people which results positive cases to screening (positive pharyngeal swabs) and therefore enter in the ocial data count of conrmed diagnosis. members of this class come from presymptomatic stage and get out due to quarantine (at an informationdependent rate γ(m )), worsening symptoms (at rate σ m ) and recovery (at rate ν m ). equation ( e): severely symptomatic individuals (hospitalized), i s (t) severely symptomatic individuals are isolated from the general population by hospitalization. they arise: (i) as consequence of the development of severe symptoms by mild illness (the infectious of the class i m or the quarantined q); (ii) directly from the fraction − p of presymptomatic individuals that rapidly develop in severe illness. this class diminishes by recovery (at rate ν s ), natural death and diseaseinduced death (at rate d). quarantined individuals q are those who are separated from the general population. we assume that quarantined are asymptomatic/mildly asymptomatic individuals. this population is diminished by nat- . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint ural deaths, aggravation of symptoms (at rate σ q , so that they move to i s ) and recovery (at a rate ν q ). for simplicity, we assume that the quarantine is % eective, i.e. with no possibility of contagion. quarantine may arise in two dierent ways. from one hand, individuals may be detected by health authorities and daily checked. such health active surveillance ensures also that the quarantine is, in some extent, respected. on the other hand, a fraction of quarantined individuals choose selfisolation since they are condent in the government handling of crisis or just believe the public health messaging and act in accordance [ ] . as mentioned in subsection . , we assume that the nal choice to respect or not respect the selfisolation depends on the awareness about the status of the disease in the community. therefore, we dene the informationdependent quarantine rate as follows. we assume that where the rate γ mimics the fraction of the asymptomatic/mildly symptomatic individuals i m that has been detected through screening tests and is`forced' to home isolation. the rate γ (m ) represents the undetected fraction of individuals that adopt quarantine by voluntary choice as result of the inuence of the circulating information m . the function γ (·) is required to be a continuous, dierentiable and increasing function w.r.t. m , with γ ( ) = . as in [ , ] , we set with d > , < ζ < − γ , potentially implying a roof of − ζ in quarantine rate under circumstances of high perceived risk. a representative trend of γ(m ) is displayed in fig. , bottom panel. after the infectious period, individuals from the compartments i m , i s and q recover at rates ν m , ν s and ν q , respectively. natural death rate is also considered. we assume that individuals who recover from covid acquire long lasting immunity against covid although this is a currently debated question (as of may, ) and there is still no evidence that covid antibodies may protect from reinfection [ ]. a frequently used indicator for measuring the potential spread of an infectious disease in a community is the basic reproduction number, r , namely the average number of secondary cases produced by one primary infection over the course of infectious period in a fully susceptible population. if the system incorporates control strategies, then the corresponding quantity is named control reproduction number and usually denoted by r c (obviously, r c < r ). the reproduction number can be calculated as the spectral radius of the next generation matrix f v − , where f and v are dened as jacobian matrices of the new infections appearance and the other rates of transfer, respectively, calculated for infected compartments at the diseasefree equilibrium [ , ] . in the specic case, if β(m ) = β b and γ(m ) = in ( )( ), namely when containment interventions are not enacted, we obtain the expression of r ; otherwise, the corresponding r c can be computed. simple algebra yields . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . ( )( ). and with (for more details see appendix b). the rst two terms in the r.h.s of ( ) describe the contributions of pre symptomatic infectious and asymptomatic/mildly symptomatic infectious, respectively, to the production of new infections close to the diseasefree equilibrium. the last three terms represent the contribution of infectious with severe symptoms, which could onset soon after the incubation phase or more gradually after a moderate symptomatic phase or even during the quarantine period. note that the latter term is missing in the basic reproduction number ( ), where the possibility for people to be quarantined is excluded. note also that r c = r when β = γ = . numerical simulations are performed in matlab environment [ ] with the use of platform integrated functions. a detailed model parametrization is given in the next subsections. the epidemiological parameters of the model as well as their baseline values are reported in table . the most recent data by the italian national institute of statistics [ ] refer to january , and . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . provide a countrylevel birth rate b = . / years − and a death rate µ = . / years − , as well as a resident population of aboutn ≈ . · ( ) inhabitants. fluctuations in a time window of just over a year are considered negligible. the immigration inow term Λ accounts for the repatriation of italians abroad. on the basis of data communicated by the italian ministry of foreign aairs and international cooperation [ ] , a reasonable value for Λ seems to be Λ = / days − , namely the average number of repatriated citizens is per week. from ( ), we nally obtain Λ ≈ . · days − . epidemiological data are based on the current estimates disseminated by national and international health organizations [ , , , , , ] or inferred by modelling studies [ , , ] . more precisely, the median incubation period is estimated to be from days, with a range from days, and identication of the virus in respiratory tract specimens occurred days before the onset of symptoms [ , ] . hence, we set the latency (ρ) and prelatency (η) rates to / . days − and / . days − , respectively. from [ ] , the specic informationindependent transmission rates for the presymptomatic (ε p β b ), asymptomatic/mildly symptomatic (ε m β b ) and severely symptomatic (ε s β b ) cases are such that ε m /ε p = . and ε s /ε m = . . they are in accordance with the observation of high viral load close to symptoms onset (suggesting that sarscov can be easily transmissible at an early stage of infection), and with the absence of reported signicant dierence in viral load in presymptomatic and symptomatic patients [ ] . we set β b = . days − , which, together with the other parameters, leads to the basic reproduction number r ≈ . , a value falling within the ranges estimated in [ , , , ] . as in [ ] , we consider that just % of infectious individuals show serious symptoms immediately after the incubation phase, yielding p = . . nonetheless, people with initial mild symptoms may become seriously ill and develop breathing diculties, requiring hospitalization. it is estimated that about in people with covid show a worsening of symptoms [ ] within days from onset [ ] , giving σ m = . / . ≈ . days − . instead, the possibility that the aggravation happens during the quarantine period is assumed to be more rare: σ q = . days − . governmental eorts in identifying and quarantining positive cases were implemented since the early stage of epidemics (at february , quarantined people were already registered [ ] ), hence we consider the daily mandatory quarantine rate of asymptomatic/mildly symptomatic individuals (γ ) for the whole time horizon. from current available data, it seems hard to catch an uniform value for γ because it largely depends on the sampling eort, namely the number of specimen collections (swabs) from persons under investigation, that varies considerably across italian regions and in the dierent phases of the outbreak [ , ] . since our model does not account for such territorial peculiarities and in order to reduce the number of parameters to be estimated, we assume that γ = . σ q , namely it is % higher than the daily rate at which members of the i m class hospitalize, yielding γ ≈ . days − . simulations with such a value provide a good approximation of the timeevolution of registered quarantined individuals at national level, as displayed in fig. , second panel. following the approach adopted in [ ] for a sars-cov epidemic model, we estimate the diseaseinduced death rate as where x is the case fatality and t is the expected time from hospitalization until death. from [ ] , we approximate x = % and t = days (it is days for patients that were transferred to intensive care and days for those were not), yielding d ≈ . days − . similarly, the recovery rates ν j with j ∈ {m, q, s} . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. ( ) versus mandatory quarantine and transmission reduction rates. intersection between dotted black and red (resp. blue) lines indicates the value after the rst (resp. second) step reduction. other parameters values are given in table . are estimated as where t j is the expected time until recovery or expected time in quarantine/hospitalization. preliminary data indicate that the virus can persist for up to eight days from the rst detection in moderate cases and for longer periods in more severe cases [ ] , suggesting t m = days is an appropriate value. as far as the time spent in hospitalization or quarantine, in the lack of exact data, we assume t s < t q , because hospitalized individuals are likely to receive a partly eective, experimental treatment: mainly antibiotics, antivirals and corticosteroids [ ] . moreover, shortages in hospital beds and intensive care units (icus) lead to as prompt as possible discharge [ ] . in particular, we set t s = and t q = days, by accounting also for prolonged quarantine time due to delays in test response (if any) and for who recommendations of an additional two weeks in home isolation even after symptoms resolve [ ] . crucially, we also estimate the initial exponential rate of case increase (say, g ), by computing the dominant eigenvalue of the system's jacobian matrix, evaluated at the diseasefree equilibrium. it provides g ≈ . days − , in accordance to that given in [ ] . we explicitly reproduce in our simulations the eects of the progressive restrictions posed to human mobility and humantohuman contacts in italy. their detailed sequence may be summarized as follows. after the rst ocially conrmed case (the socalled`patient one') on february , in lodi province, several suspected cases emerged in the south and southwest of lombardy region. a`red zone' encompassing municipalities was instituted on february and put on lockdown to contain the emerging . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . reactivity factor of voluntary quarantine · − ζ − ζ is the roof of overall quarantine rate . days − a /a is the average information delay / days − k degree of information coverage . . the next day, a decree evocatively entitled`i'm staying at home' was signed: the lockdown was declared for the whole country with severe limitations to mobility and other progressively stricter restrictions. soon after, on march , , the lockdown was extended to the entire country [ ] , with all commercial and retail businesses except those providing essential services closed down [ ] . finally, on march , , the phase one of restrictions was completed when a full lockdown was imposed by closing all non essential companies and industrial plants [ ] . on may , italy entered the phase two, representing the starting point of a gradual relaxation of the restriction measures. one week later, shops also reopened and the restrictions on mobility were essentially eliminated, with the only obligation in many regions to use protective masks [ ] . because data early in an epidemic are inevitably incomplete and inaccurate, our approach has been to try to focus on what we believe to be the essentials in formulating a simple model. keeping this in mind, we assume that the disease transmission rate incurs in just two step reductions (modelled by the reduction rate β in ( )), corresponding to • march (day ), when the lockdown decree came into force along with the preceding restrictions, cumulatively resulting in a sharp decrease of sarscov transmission; • march (day ), the starting date of the full lockdown that denitely impacted the disease incidence. in the wake of [ , ] , we account for a rst step reduction by % (that is β b − β | ≤t< = . β b ), which drops the control reproduction number ( ) close to (see fig. , dotted black and red lines). it is then strengthened by an additional % about, resulting in a global reduction by % ( β b − β | t≥ = . β b ) that denitely brings r c below (see fig. , dotted black and blue lines). the informationrelated parameter values are reported in table together with their baseline values. following [ , ] , we set ζ = . days − potentially implying an asymptotic quarantine rate of . days − if we could let m go to +∞. the positive constants α e d tune the informationdependent reactivity, respectively, of susceptible and infectious people in reducing mutual social contacts and of individuals with no/mild symptoms in selfisolating. in virtue of the order of magnitude of the information index m (ranging between and ), we set α = · − and d = · − , resulting in a receptive propensity to selfisolation for asymptomatics/mildly symptomatics and less evident degree of variability in contact . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . rate, being the latter already impacted and constrained by government laws (as shown later in fig. ) . values range for the information coverage k and the average time delay of information /a are mainly guessed or taken from papers where the information index m is used [ , , , ] . the former may be seen as a`summary' of two opposite phenomena: the disease underreporting and the level of media coverage of the status of the disease, which tends to amplify the social alarm. it is assumed to range from a minimum of . (i.e. the public awareness is of %) to . the latter ranges from the hypothetical case of prompt communication (a = days − ) to a delay of days. we tune these two parameters within their values range in order to reproduce the curves that best t with the number of hospitalized individuals (i s ) and the cumulative deaths as released every day at p.m. (utc+ h) since february , by the italian civil protection department and archived on github [ ] . we nd a good approximation by setting k = . and a = / days − , meaning a level of awareness about the daily number of quarantined and hospitalized of %, resulting from the balance between underestimates and media amplication and inevitably aected by rumors and misinformation spreading on the web (the socalled`infodemic' [ ] ). such awareness is not immediate, but information takes on average days to be publicly disseminated, being the communication slowed by a series of articulated procedures: timing for swab tests results, notication of cases, reporting delays between surveillance and public health authorities, and so on. of course, parameters setting is inuenced by the choice of curves to t. available data seem to provide an idea about the number of identied infectious people who have developed mild/moderate symptoms (the fraction that mandatorily stays in q) or more serious symptoms (the hospitalized, i s ) and the number of deaths, but much less about those asymptomatics or with very mild symptoms who are not always subjected to a screening test. in order to provide appropriate initial conditions, we consider the ocial national data at february , archived on [ ] . in particular, we take the number of mandatorily quarantined individuals (at that time, they coincide with q being the voluntary component negligible) and the hospitalized people (i s ). then, we simulate the temporal evolution of the epidemics prior to february , by imposing an initial condition of one exposed case ∆t days before in a population ofn individuals, withn given in ( ) . we assume β = and γ as in table (no social distance restrictions were initially implemented, but quarantine eorts were active since then) and disregard the eect of information on the human social . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . tables , and . behaviors in this phase (α = d = in ( )( )). the length of temporal interval ∆t is tuned in order to reproduce the ocial values released for q and i s at february and provide estimations for the other state variables, as reported in table . we obtain ∆t = . , indicating that the virus circulated since the end of january, as predicted also in [ , ] . let us consider the time frame [t , t], where t ≤ t ≤ t f . we consider two relevant quantities, the cumulative incidence ci(t), i.e. the total number of new cases in [t , t], and the cumulative deaths cd(t), i.e. the diseaseinduced deaths in [t , t]. for model ( )( ) we have, respectively: where β(m ) is given in ( ), and in fig. the time evolution in [t , t f ] of ci(t) and cd(t) is shown (rst and fourth panel from the left), along with that of quarantined individuals q(t) (second panel) and hospitalized i s (t) (third panel). the role played by information on the public compliance with mitigation measures is stressed by the comparison with the absolute unresponsive case (α = d = in ( )( )). corresponding dynamics are . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. other parameter values are given in tables , and . labelled by black solid and red dashed lines, respectively. in absence of reactivity to information, the cumulative incidence would have been much less impacted by lockdowns restrictions ( . · vs . · on may ) and the number of quarantined would have been reduced to those forced by surveillance authorities. as a consequence, the peak of hospitalized patients would have been about % higher and days timedelayed, with a corresponding increase in cumulative death of more than %. for all reported dynamics, the detachment between the responsive and unresponsive case starts to be clearly distinguishable after the rst step reduction of % in transmission rate (on march ). trends are also compared with ocially disseminated data [ ] (fig. , blue dots), which seem to conform accordingly for most of the time horizon, except for ci, that suers from an inevitable and probably high underestimation [ , , , ] . as of may , , we estimate about , contagions, whereas the ocial count of conrmed infections is , [ ] . we now investigate how the information parameters k and a may aect the epidemic course. more precisely, we assess how changing these parameters aects some relevant quantities: the peak of quarantined individuals, max(q) (i.e., the maximum value reached by the quarantined curve in [t , t f ]), the peak of hospitalized individuals, max(i s ), the cumulative incidence ci(t f ) evaluated at the last day of the considered time frame, i.e. t f = (corresponding to , and the cumulative deaths cd(t f ). the results are shown in the contour plots in fig. . as expected, ci(t f ), max(i s ) and cd(t f ) decrease proportionally to the information coverage k and inversely to the information delay a − : they reach the minimum for k = and a − = days. dierently, the quantity max(q) may not monotonically depend . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. tables , and . on k and a − as it happens for k ≥ . and a − ≤ days (see fig. , second panel, lower right corner). in such parameter region, for a given value of k (resp. a) there are two dierent values of a (resp. k) which correspond to the same value of max(q). the absolute maximum (max [k,a − ] (max(q))) is obtained for k = and a − ≈ days. note that the couple of values k = , a − = days corresponds to the less severe outbreak, but not with the highest peak of quarantined individuals. in the next, we compare the relative changes for these quantities w.r.t the case when circulating information does not aect disease dynamics. in other words, we introduce the index fig. . however, we report in table three exemplary cases, the baseline and two extremal ones: (i) the baseline scenario k = . , a − = days, representing a rather accurate and shortdelayed communication; (ii) the case of highest information coverage and lowest information delay, k = , a − = days; (iii) the case of lowest information coverage and highest information delay, k = . , a − = days. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint . · . · . · . · k = . . · - . . · . . · - . . · - . a − = days k = . · - . . · . . · - . . · - . a − = days k = . . · - . . · . . · - . . · - . a − = days table : exact and relative values of nal cumulative incidence, ci(t f ), quarantined peak, max(q), under circumstances of very quick and fully accurate communication (case (ii)), ci(t f ), max(i s ) and cd(t f ) may reduce till %, % and %, respectively (see table , third line). on the other hand, even in case of low coverage and high delay (case (iii)), the information still has a not negligible impact on disease dynamics: nal cumulative incidence and hospitalized peak reduce till %, nal cumulative deaths till % and quarantined peak increases of % about (table , fourth line). as mentioned above, information and rumors regarding the status of the disease in the community aect the transmission rate β(m ) (as given in ( )) and the quarantine rate γ(m ) (as given in ( )). in our last simulation we want to emphasize the role of the information coverage on the quarantine and transmission rates. in fig. a comparison with the case of low information coverage, k = . , is given assuming a xed information delay a − = days (blue dotted lines). it can be seen that more informed people react and quarantine: an increasing of the maximum quarantine rate from . to . days − (which is also reached a week earlier) can be observed when by increasing the value of k till k = (fig. , second panel) . the eect of social behavioral changes is less evident in the transmission rate where increasing the information coverage produces a slight reduction of the transmission rate mainly during the full lockdown phase (fig. , rst panel) . this reects the circumstance that the citizens compliance with social distancing is not enhanced by the informationinduced behavioral changes during the rst stages of the epidemic. on the other hand, a widespread panic reaction may lead people to`do it as long as you can' (see, for example, the case of stormed supermarkets at early stage of the epidemic [ ]). in this work we propose a mathematical approach to investigate the eects on the covid epidemic of social behavioral changes in response to lockdowns. starting from a seirlike model, we assumed that the transmission and quarantine rates are partially determined on voluntary basis and depend on the circulating information and rumors about the disease, modeled by a suitable timedependent information index. we focused on the case of covid epidemic in italy and explicitly incorporated the progressively stricter restrictions enacted by italian government, by considering two step reductions in contact rate (the partial and full lockdowns). the main results are the followings: • we estimated two fundamental informationrelated parameters: the information coverage regarding . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. tables , , . the daily number of quarantined and hospitalized individuals (i.e. the parameter k) and the information delay (the quantity a − ). the estimate is performed by tting the model's solution with ocial data. we found k = . , which means that the public was aware of % of real data and a − = days, the time lag necessary to information to reach the public; • social behavioral changes in response to lockdowns played a decisive role in curbing the epidemic curve: the combined action of voluntary compliance with social distance and quarantine resulted in preventing a duplication of deaths and about % more contagions (i.e. approximately , more infections and , more deaths compared with the total unresponsive case, as of may , ); • even under circumstances of low information coverage and high information delay (k = . , a = / days − ), there would have been a benecial impact of social behavioral response on disease containment: as of may , cumulative incidence would be reduced of % and deaths of % about. shaping the complex interaction between circulating information, human behavior and epidemic disease is challenging. in this manuscript we give a contribution in this direction. we provide an application of the information index to a specic eldcase, the covid epidemic in italy, where the information dependent model is parametrized and the solutions compared with ocial data. our study presents limitations that leave the possibility of future developments. in particular: (i) the model captures the epidemics at a country level but it does not account for regional or local dierences and for internal human mobility (the latter having been crucial in italy at early stage of covid epidemic); (ii) the model does not explicitly account of icu admissions. the limited number of icu beds constituted a main issue during the covid pandemics [ ] . this study did not focus on this aspect . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . but icu admissions could be certainly included in the model; (iii) the model could be extended to include age structure. age has been particularly relevant for covid lethality rate (in italy the lethality rate for people aged or over is more than double the average value for the whole population [ ] ). further developments may also concern the investigation of optimal intervention strategies during the covid epidemics and, to this regard, the assessment of the impact of vaccine arrival. in this case, the approach of informationdependent vaccination could be employed [ , , , ] . consider the scenario of an epidemic outbreak that can be addressed by the public health system through campaigns aimed at raising public awareness regarding the use of protective tools (for example, vaccination, social distancing, bednet in case of mosquitoborne diseases, etc). assume also that the protective actions are not mandatory for the individuals (or else, they are mandatory but local authorities are unable to ensure a fully respect of the rules). then, the nal choice to use or not use the protective tools is therefore partially or fully determined by the available information on the state of the disease in the community. the idea is that such information takes time to reach the population (due to timeconsuming procedures such as clinical tests, notication of cases, the collecting and propagation of information and/or rumors, etc) and the population keeps the memory of the past values of the infection (like prevalence or incidence). therefore, according to the idea of informationdependent epidemic models [ , ] , an information index m should be considered, which is dened in terms of a delay τ , a memory kernel k and a functiong which describes the information that is relevant to the public in determining its nal choice to adopt or not to adopt the protective measure (the message function). therefore, the information index is given by the following distributed delay: m (t) = t −∞g (x (τ ), x (τ ), . . . , x n (τ )) k(t − τ )dτ here, the message functiong depends generically on the state variables, say x , x , . . . , x n but it may specically depend only on prevalence [ , ] , incidence [ ] or other relevant quantities like the vaccine side eects [ ] . one may assume that: the delay kernel k(·) in ( ) is a positive function such that +∞ k(t)dt = . it represents the weight given to past history of the disease. the erlangian family erl n,a (t) is a good candidate for delay kernel since it may represent both an exponentially fading memory (when n = ) and a memory more focused in the past (when n > ). moreover, when an erlangian memory kernel is used, one can apply the so called linear chain trick [ ] to obtain a system ruled by ordinary dierential equations. for example, in the case of exponentially fading memory (or weak kernel erl ,a (t)), the dynamics of the information index is ruled byṀ = a (g (x , x , . . . , x n ) − m ) . for further details regarding the information index, see [ , ] . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint b the next generation matrix method following the procedure and the notations in [ , ] , we prove that the control reproduction number of model ( )( ), r c , is given by ( ) . similarly one can prove that the basic reproduction number is given by ( ). let us consider the r.h.s. of equations ( b)( c)( d)( e)( f), and distinguish the new infections appearance from the other rates of transfer, by dening the vectors as proved in [ , ] , the control reproduction number is given by the spectral radius of the next generation matrix f v − . it is easy to check that f v − has positive elements on the rst row, being the other ones null. thus, r c = (f v − ) , as given in ( ) . coronavirus: conte tightens lockdown, closes all nonessential businesses, after almost deaths in hours estimating the burden of united states workers exposed to infection or disease: a key factor in containing risk of covid infection evaluating covid public health messaging in italy bbc, british broadcasting corporation. coronavirus: more than british broadcasting corporation. coronavirus: the world in lockdown in maps and charts assessing the impact of the coronavirus lockdown on unhappiness, loneliness, and boredom using google trends. arxiv eects of informationdependent vaccination behavior on coronavirus outbreak: insights from a siri model oscillations and hysteresis in an epidemic model with information dependent imperfect vaccination modeling of pseudorational exemption to vaccination for seir diseases the early phase of the covid outbreak in lombardy, italy. arxiv expected impact of lockdown in Îledefrance and possible exit strategies. medrxiv on the denition and the computation of the basic reproduction ratio r in models for infectious diseases in heterogeneous populations heterogeneous social interactions and the covid lockdown outcome in a multigroup seir model. arxiv informationrelated changes in contact patterns may trigger oscillations in the endemic prevalence of infectious diseases vaccinating behaviour, information, and the dynamics of sir vaccine preventable diseases fatal sir diseases and rational exemption to vaccination vaccinating behaviour and the dynamics of vaccine preventable infections centre for disease prevention and control. disease background of covid contact rate epidemic control of covid : an equilibrium view. arxiv repatriation of eu citizens during the covid- crisis dynamics of icu patients and deaths in italy and lombardy due to covid analysis updated to march, day # evening preventing intrahospital infection and transmission of coronavirus disease in healthcare workers. safety and health at work spread and dynamics of the covid epidemic in italy: eects of emergency containment measures modelling the covid- epidemic and implementation of populationwide interventions in italy modelling strategies for controlling sars outbreaks temporal dynamics in viral shedding and transmissibility of covid sars-cov- latest available data italian civil protection department. chronology of main steps and legal acts taken by the italian government for the containment of the covid epidemiological emergency italian ministry of foreign aairs and international cooperation phase two: what's opening and what you can do italian ministry of health. faq -covid , questions and answers beyond just "attening the curve analysis of the mitigation strategies for covid : from mathematical modelling perspective. medrxiv nonlinear dynamics of infectious diseases via informationinduced vaccination and saturated treatment correcting underreported covid case numbers. medrxiv biological delay systems: linear stability theory modeling the interplay between human behavior and the spread of infectious diseases matlab. matlab release b. the mathworks mrc, centre for global infectious disease analysis. report -estimating the number of infections and the impact of nonpharmaceutical interventions on covid in european countries world tourism organization. covid related travel restrictions. a global review for tourism. second report as of reproduction numbers and subthreshold endemic equilibria for compartmental models of disease transmission the covid epidemic coronavirusscientic insights and societal aspects statistical physics of vaccination coronavirus disease (covid- ): situation report coronavirus disease (covid ) pandemic acknowledgements. the present work has been performed under the auspices of the italian national group for the mathematical physics (gnfm) of national institute for advanced mathematics (indam). [ ] b. j. quilty, s. cliord, cmmid ncov working group , s. flasche, and r. m. eggo. eectiveness of airport screening at detecting travellers infected with novel coronavirus ( ncov). eurosurveillance, ( ) : , .[ ] rijs, reuters institute for the study of journalism. types, sources, and claims of covid misinformation. fact sheet april , .[ ] g. sebastiani, m. massa, and e. riboli. covid epidemic in italy: evolution, projections and impact of government measures. european journal of epidemiology, : , .[ ] m. supino, a. d'onofrio, f. luongo, g. occhipinti, and a. dal co. world governments should protect their population from covid pandemic using italy and lombardy as precursor. medrxiv, .[ ] the guardian. italy charges more than , people with violating lockdown. https://www.theguardian.com/world/ /mar/ / italy-charges-more-than- -people-violating-lockdown-coronavirus.(accessed may , ).[ ] theguardian. italians struggle with`surreal' lockdown as coronavirus cases rise. https://www.theguardian.com/world/ /feb/ / italians-struggle-with-surreal-lockdown-as-coronavirus-cases-rise, .(accessed may , ).[ ] the guardian. leaked coronavirus plan to quarantine m sparks chaos in italy. https://www.theguardian.com/world/ /mar/ / leaked-coronavirus-plan-to-quarantine- m-sparks-chaos-in-italy, .(accessed may , ).[ ] who, world health organization.home care for patients with covid presenting with mild symptoms and management of their contacts. https://www.who.int/ publications-detail/home-care-for-patients-with-suspected-novel-coronavirus-(ncov) -infection-presenting-with-mild-symptoms-and-management-of-contacts, . (accessed may , ).[ ] who, world health organization. immunity passports in the context of covid . https://www.who.int/news-room/commentaries/detail/ immunity-passports-in-the-context-of-covid- , . (scientic brief april , , accessed may , ).[ ] l. zhu, x. zhou, y. li, and y. zhu. stability and bifurcation analysis on a delayed epidemic model with information-dependent vaccination. physica scripta, ( ): , . key: cord- -wl z l authors: putrino, alessandra; raso, mario; magazzino, cosimo; galluccio, gabriella title: coronavirus (covid- ) in italy: knowledge, management of patients and clinical experience of italian dentists during the spread of contagion date: - - journal: bmc oral health doi: . /s - - - sha: doc_id: cord_uid: wl z l background: the coronavirus infection that emerged in china in the last few months of has now spread globally. italy registered its first case in the second half of february, and in a short time period, it became the top country in europe in terms of the number of infected people and the first in the world in terms of deaths. the medical and scientific community has been called upon to manage the emergency and to take measures. dentists also need to take new precautions during their clinical activity to protect themselves, coworkers and patients from the risks of contagion and to avoid further spread of infection. methods: following the data published in the international literature as well as the guidelines and directives constantly updated by the who and by the national health authorities, a questionnaire to be completed anonymously was submitted online to italian dentists using social tools and online professional platforms. the collected data were processed statistically, providing descriptive data and analysis of correlations of the most significant parameters using the pearson’s χ , the likelihood-ratio χ , cramér’s v, fisher’s exact test, goodman and kruskal’s γ, and kendall’s τb (p < . ). results: a total of dentists from italy participated in the survey. a good level of scientific knowledge about coronavirus and the extra precautionary measures needed to limit the spread was related to the age of respondents and their sex. coming from areas with higher concentrations of cases affected knowledge, level of attention and perception of risk related to dental activity. conclusions: at the moment, there are no therapies or vaccines to contain the infection with the new coronavirus that is causing many infections, many of which are fatal, worldwide. dentists are one of the categories at highest risk of encountering diseases and infections because they work in close proximity with patients, and in their procedures, there is always contact with aerosols with high bacterial and viral potential. therefore, during this covid- emergency, it is important that dentists are properly informed and take the appropriate precautionary measures. human coronaviruses are a group of rna viruses able to cause respiratory, gastrointestinal and central nervous system diseases. the first human coronavirus (hcov) was detected in the mid- s [ ] . in december , the seventh coronavirus known to infect humans was found in china (wuhan city, hubei province) [ ] . on january , the world health organization (who) announced the temporarily named ncov- , now called sars-cov- , as the novel coronavirus pathogen responsible for the increasing number of new pneumonia cases [ , ] . since that day, the number of cases in china and progressively in many other parts of the world has increased (fig. ) , and with it, the number of people who died because of this infection as the primary or contributory cause of preexisting illness [ ] . on march , the who stated that the novel coronavirus outbreak was a pandemic (fig. ) . the scientific community is still making numerous efforts to clarify the etiology, pathogenicity, and characteristics of the virus to establish the mechanisms underlying human-to-human transmission and possible treatments [ ] [ ] [ ] [ ] [ ] . although there are many cases of recovery, the number of deceased subjects has increased with the spread, particularly affecting older subjects with previous severe pathologies [ , ] . the first detection of the virus in italy was in a case of two chinese tourists from wuhan who were later treated at the spallanzani hospital of rome. the entire tourist group of the chinese couple was then quarantined for two weeks in the same hospital and then released after negative results of the clinical and serologic controls [ ] . on february, the first locally transmitted case, a -year-old man who never travelled in china, emerged in northern italy, lombardy region, codogno town, and since then, the number of cases has increased (fig. ) in the whole area and progressively increased in many other northern regions until the virus has spread to affect the entire italian territory. as the number of swab tests for suspected cases has continued to increase, the italian authorities have started to adopt preventive measures to try to isolate the affected areas and block the spread of the infections and a nationwide lockdown went into effect on march. the death toll, at the (table ) . italian government, the ministry of health, civil protection and other competent bodies at the local level are constantly engaging in this emergency, providing instructions to citizens and health workers and updating the population on the evolution of the situation [ ] [ ] [ ] [ ] . even dentists have been involved in the management of this emergency through indications on prevention and safety measures to be observed in their clinical activity due to the high level of exposure for operators and dental patients. in the last weeks the number of health workers infected has risen: many nurses and doctors on the coronavirus front lines are working without adequate personal protective equipment (ppe), exposing themselves to great risk and some of them have been infected while on duty. already, doctors have died, and of them were dentists [ ] . a direct correlation between their death and coronavirus infection was not, however, ascertained but many of them were engaged in the management of infections. the novel coronavirus was recently identified in the saliva of infected patients. dental clinical procedures generate droplets and aerosols that can lead to viral transmission [ ] . contamination on surfaces and diffusion by contact, conceivable due to the characteristics of dental activity, require a great deal of attention from dentists, who should adopt simple but effective practical strategies to stop the possible spread of the virus. the suggested procedures include preappointment patient risk evaluation through a specific questionnaire; frequent hand hygiene; appropriate individual protective equipment; insulation of the oral cavity with a rubber dam after mouth rinses, which are suggested with peroxide %; the use of antiretraction handpieces; disinfection after every dental treatment; and appropriate waste management [ ] . in italy there are more than , dentists (about per inhabitants), differently distributed at the regional level ( table ). the management of dental activity can play an important role in limiting the infections. due to the increasing involvement of a large part of the population in the global epidemic situation in italy, the present study aimed to assess the knowledge about the new coronavirus, the perception of risk and the clinical management of the risk related to infection during the first month of the italian epidemic in an online survey of italian dentists. moreover, due to the rapid change in the number of infected individuals, a further analysis aimed to evaluate the progressive perception of the risks. this study used a questionnaire-based survey. the original version of the survey was piloted among a group of dentists to ensure suitability, validity, practicability and interpretation of answers. on the basis of the comments and suggestions obtained, the questionnaire was revised. the questionnaire was developed and forwarded to italian dentists in italian and translated into english for the presentation of this research (supplementary file ). scientific bases for the development of questions about the new coronavirus came from consultation of the scientific literature available on this subject, more specifically addressed to virologists, specialists, biologists, and general practitioners and epidemiological content that traced the data on coronavirus infection since its first appearance in china [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . additionally, as a source of scientific information and as an aid for the processing of a part of the questionnaire presented to italian dentists, circulars issued by the ministry of health containing protocols and guidelines aimed at health professionals during the outbreak and informative material disclosed by dental associations were also considered. the questionnaire was uploaded online to the free survey platform survio.com (© survio, hlinky , brno, czech republic) through a specially created user profile. the generated link was shared on professional group and contact networks on the main social channels (lin-kedin, facebook, and whatsapp), inviting italian dentists to share the link with other colleagues in further professional groups to widen the spread of the survey as much as possible. the assurance that those who would respond to the survey were regular dentists was given by the fact that all online professional groups and networks, of which none of the authors are an administrator, referred to (also for the dissemination of the questionnaire) by regulation are closed and approve registrations only when the registration number of the national professional register is originally provided. the responses were validated only for fully completed questionnaires, in fact the system automatically rejected incomplete questionnaires. the data collected were absolutely anonymous, and tracing the identity of the subjects was not possible. a total of open-and close-ended questions were developed (table ) . seven questions helped to obtain a profile of the practitioner (age group; sex; type of clinical activity-private, hospital or both; qualification-specialist or not; territorial provenance-specifying region; the number of citizens of their city; and the number of patients treated daily). six questions were intended to evaluate the direct influence of the coronavirus epidemic on the dentist's clinical activity (presence or absence of infected cases in their region; questions of patients about coronavirus; patients appearing to be worried or not about possible infections with coronavirus during dental procedures; effective decrease or not in patient appointment number since the coronavirus outbreak onset; adoption of special measures taken during professional activity since the coronavirus emergency started in italy; and which prevention methods are possibly used). the remaining eleven questions aimed to assess the level of scientific knowledge on coronavirus from a qualitative point of view and the dentist's perception of the problem related to this emergency in dental clinical practice. each respondent to the questionnaire corresponded to a form with all answers provided. the subjects were anonymous and were marked only with a number that reflected the chronological order of compilation. the form showed the day and time when the questionnaire was completed. the project did not need formal ethical approval since it collected general opinions that do not contain clinical data and neither personal data. according to the current regulation of the ethics committee of the higher institute of health (istituto superiore di sanità), the ethical aspects that need evaluation, approval and monitoring of trial protocols relate to epidemiological, evaluation and medical-social projects that require the collection of personal data. according to the national data protection authority (garante per la protezione dei dati personali), "personal data" are first and last name, images, tax code, ip address and license plate number. the compilation of the survey was anonymously carried out on a voluntary basis without the possibility to trace the identity of the subjects, as the system does not store even the ip addresses of the users accessing the link. before the start of the survey, informed consent was presented on the main page; the participant had to agree (by checking a box) that their anonymously provided answers could be used in this research for scientific purposes. for statistical examination of the data, the online platform automatically generated descriptive statistical analysis on the main page; the analysis could therefore be downloaded as an excel or spss spreadsheet for further statistical analysis. in this study, descriptive statistical analysis was carried out. several measures of association was performed including, the common pearson's χ , the likelihood-ratio χ , cramér's v, fisher's exact test, goodman and kruskal's γ, and kendall's τb. the level of statistical significance was set at . . the software used is stata . (statacorp llc, tx, usa). the pearson's and likelihood-ratio χ test for the independence of the rows and columns. the null hypothesis (h ) is that there is no relationship. to reject this we need a p < . (at % confidence). cramér's v is a measure of association between two nominal variables. it goes from to , where indicates strong association. γ and τb are measures of association between two ordinal variables (both have to be in the same direction, i.e. negative to positive, low to high). both go from − to . negative shows inverse relationship, closer to a strong relationship. γ is recommended when there are lots of ties in the data. τb is recommended for square tables. fisher's exact test is used when there are very few cases in the cells (usually less than , with an overall frequency of less than %). it tests the relationship between two variables. the null is that variables are independent [ ] [ ] [ ] [ ] [ ] . the survey was online for weeks from february to march . the link received visits, but only dentists responded to the survey by completing it. the results of the descriptive statistics were collected in table . most dentists carried out their professional activity in a private practice ( . %).the age group of up to years old (yo) and the group between and yo were the most represented (respectively, between . and . %). the distribution between the two sexes was equivalent ( . % males and . % females). over . % of dentists were general dentists, % are dentists without a recognized dental specialty (italian universities provide years of postgraduate programs in orthodontics, oral surgery and pediatric dentistry. they are the only recognized dental specialties) and . % are dental specialists ( . % were orthodontists, approximately % were oral surgeons, and just over % were specialists in pediatric dentistry). the answers related to the geographical location of the workplace mapped across the whole country, representing italy from north to south and including the larger islands (sicily and sardinia) (fig. ) . most of the respondents ( . %) were from moderately or highly populated cities. fourty % of the dentists who participated in the survey treat more than patients per day. almost % of dentists completed the questionnaire when there were positive cases in their region of sars-cov- infection. fifty percent of respondents did not notice a decrease in visits since the outbreak spread. more than % of patients asked questions about coronavirus to their dentist. according to the clinicians who participated in the study, the majority of patients (more than %) would not be worried about getting coronavirus infection during dental treatment. almost % of dentists said they were fairly informed about coronavirus. despite of the self-estimated knowledge about the infection, answers to the following questions assessing knowledge on the subject revealed a different reality. most respondents obtained scientific information about coronavirus through italian institutions ( . %); television, newspapers and social media ( . %); professional associations ( %); scientific literature (l . %); and other colleagues ( . %). only a very small percentage ( . %) specified other channels of information or that they were not fully informed ( . %). almost % correctly answered the questions about the definition of coronavirus, the . % correctly answered about ncov and . % about sars-cov- . most respondents, on the other hand, incorrectly answered the question on the definition of covid- (almost %). almost % of the subjects were very clear about the types of possible symptoms that accompany the infection, and in . % of cases, they correctly indicated how the new coronavirus is transmitted from person to person. however, the . % of dentists knew that the national federation of surgeons and dentists (federazione nazionale dei medici chirurghi ed. odontoiatri-fnomceo) has provided healthcare professionals with a free online course to disseminate useful information about the virus. sixty nine percent of dentists who completed the questionnaire had taken safety and prevention measures against workplace infection since coronavirus spread. almost % of them had taken all the recommended safety measures (telephone history collection, increased frequency of washing hands and environmental surfaces, and personal protective equipment such as gloves, disposable gowns and facemasks with adequate filters). fifty point % of respondents were concerned 'enough' about the spread of infection in italy. overall, almost % of dentists who took part in the survey considered the dental profession neither safe nor free from the risk of contagion for both patients and healthcare professionals. the measures of association results were collected in tables , and . in regards to the sex (table ) , both χ test statistics show a significance level < . for quality of information (question number ), level of information related to questions and and for risk perception related to question ; so we can safely assume that some differences exist between groups. therefore, we conclude that there is evidence of a statistically significant difference between male and female on these variables. we can confidently reject the null hypothesis that these two variables are statistically independent in that population. in other words, we can conclude that there is some relationship between sex and each of these four variables. in fact, for these variables the cramér's v values are > . , which indicates a non-negligible association. moreover, the goodman and kruskal's lambda for the relationship between sex and level of information related to question number , and sex and risk perception related to question is > . , in line with previous results. all these findings are confirmed by fisher's exact test results, since in these four cases the hypothesis of variables' independence is rejected, and we conclude that there is some kind of relationship between variables. concerning the age (table ) , the pearson and likelihood-ratio χ tests present a p-value < . only for the relationship with the variable quality of information (question number ). we reject the null hypothesis of no association at conventional level of statistical significance, because it emerges a dependence of the rows and columns. thus, in this case we can conclude that some differences emerge between groups. moreover, in this case cramér's v is > . : there is a small but statistically significant association between these variables. if we consider the region (table ) , the pearson and lr χ tests show a p-value < . for level of information related to questions number and , and correct risk management related to question number ; therefore, we conclude that some relationship exists between region and each of these three variables. here, the cramér's v are > . , which indicate a statistically significant association. since sars-cov- can be transmitted from person to person by droplets, contact and through saliva, dental patients and dentists and their coworkers can be easily exposed to novel coronavirus infections [ , , ] . in the period of contagion outbreak from the new coronavirus, information about the virus has become increasingly the subject of attention of the media, such as television, the internet, and social channels. however, it was only when the first cases began to register in italy that professional associations and dental professionals began to take a deeper interest in the problem. dental professionals had to refer only to the official communication of the ministry, whose law decrees lacked specific references to the dental profession. male dentists believed to be very well informed about coronavirus unlike female colleagues who had a more cautious opinion on their knowledge of the subject (pearson χ test . -p . ; lr χ test . -p . ). dentists between the ages of - believe they were well informed compared to younger colleagues who judged sufficient their knowledge (pearson χ test . -p . ; lr χ test, . -p . ). male dentists showed to have a significantly clearer idea of the taxonomic characteristics of the virus (pearson χ test . -p . ; lr χ test . -p . ). most were aware of the main features of coronaviruses but confused the term covid- with the virus itself ( . %). the definition of covid- was provided more correctly by the dentists of lazio, lombardy, emilia-romagna and sicily but the same regions, with the exception of sicily, reported the greatest number of incorrect answers (which overall exceeded the correct ones) the most informed dentists on the possibility to access a free online course on the new coronavirus promoted by the fnomceo (national federation of surgeons and dentists) were those from lazio, followed by those from lombardy, sicily and tuscany (lr χ test . p . ). quite important for the population and for the spread of epidemics is the preventive approach of dentists. for the possibilities of transmission from person to person, most are properly informed ( . %). female dentists were better informed on this aspect (pearson χ test . -p . ; lr χ test . -p . ). compared to age, younger dentists were significantly better informed about the transmission routes of the virus than other age groups (pearson χ test, . -p . ; lr χ test . -p . ). more than % of respondents to the survey were aware of the wide variety of symptoms with which the infection can occur, which is encouraging because it means that a diagnostic suspicion and a report to the authorities regulating the execution of swab tests of potentially infected individuals can also be appropriately carried out by a dentist. it is important to consider that transmission may occur through asymptomatic patients and that symptoms when covid- is present can also be mild and confused with a simple cold or flu [ ] . its manifestation does not always culminate with severe symptomatology accompanied by respiratory failure up to interstitial pneumonia. the asymptomatic incubation period takes approximately - days, and in these days, persons without symptoms can spread the virus. for this reason, it is important to add to the information required of the patient in the medical history, the report of a possible contact with infected or potentially infected people or of trips to the areas where the infection has spread. many dentists ( %) who responded to the survey chose to ask this question of their patients, judging it to be important. at the time of the virus's main spread, it was recommended to perform a telephone triage even before seeing the patient to assess whether to visit or to postpone the appointment. at the time of the survey, performed between february rd and march th, just over % of dentists did not notice a reduction in the number of visits despite the spread of the virus. it must be specified that after two weeks from the start of this research many work activities were suspended by the government by extraordinary decree, but the clinical dental activity was allowed only for the management of emergencies provided by dentists equipped with adequate personal protective equipment (ppe). the definition of "adequate ppe" for dentists is a matter of debate because above all the surgical masks used routinely by dentists would not have sufficient filters to protect from infection. the use of facemasks with ffp or ffp filters, highly protective than the surgical ones, does not seem to be considered necessary for routine dental activity, even if this has not been clearly said nor denied. the absence of a sample calculation and the methodology used in the dissemination of the survey represent limitations in this research. anyway the distribution of the respondents in the national territory was quite homogenous (proportionate to the extension of the individual regional territories) and the greatest proportion came from medium-large cities. although . % of dentists said that patients have asked questions about coronavirus, they agree that patients feeling worried about contracting the infection through dental care has not emerged ( . %). most of the dentists ( %) adopted additional preventive practical measures, a sign of a growing and widespread awareness ( . %) of the risk of contributing to the spread of contagion through dental activity. there were regions where the number of dentists who claimed to have taken additional contagion prevention measures during their clinical activity was significantly higher than others. this was especially true for regions such as lazio, lombardy, emilia romagna and campania. it is interesting to note that regions such as veneto which since the beginning of the spread of the virus in italy has been one of the first regions and among the most affected had not the same attitude (pearson χ test, . -p . ; lr χ test . -p . ). the female gender appeared significantly more concerned than the male gender about the spread of coronavirus infection (pearson χ test . -p . ; lr χ test . , p . ). dentists belonging to the younger age groups were found to be much more convinced than their older colleagues that this epidemic has future repercussions on the dental profession as it is not without risk (pearson χ test, . -p . ). dental treatment procedures always involve close contact with the patient, and this setup does not allow the maintenance of an adequate safe distance. it is extremely important that dentists equip themselves with appropriate individual safety devices (masks, gloves, protective goggles, hair caps and shirts). a recent article in the new york times, referring to the database "o'net" used by the department of labor to describe the various physical aspects of different professions, highlighted that the occupational categories in which you come into physical contact with others are those where the risk of covid- is highest. dentists are at the top of the ranking for work-related risk [ ] . in this survey, dentists affirmed the constant use of these safety devices as prescribed by the italian medical guidelines of safety in workplaces [ ] . when aerosol procedures are carried out, the presence of saliva and blood increases the spread of germs, bacteria and viruses. ensuring a change of air in the workplace and in the waiting room is a simple but important measure chosen by . % of dentists in this survey. this measure should always be adopted by dentists and not only in this situation. equally essential is to wash hands more frequently and disinfect them with alcohol-based solutions. this provision should also be encouraged for patients before entering the operating dental unit. these recommendations, together with those of not shaking hands with anyone, were accepted by . and . % of respondents, respectively. the data that emerged on the cleansing measures also include the cleaning of the clinical contact surfaces, such as buttons, handles and work surfaces. thorough cleaning has proven to be a mandatory and indispensable choice for prevention, as it is proven that the coronavirus family, including sars-cov- , can survive on plastic, metal and glass surfaces for up to days and can be efficiently deactivated through disinfection procedures with - % ethanol, . % hydrogen peroxide or . % sodium hypochlorite within min. the use of . - . % benzalkonium chloride or . % chlorhexidine digluconate does not have the same effectiveness [ ] . it must be said that the majority of dentists paid great attention to the measures to be taken; indeed, . % of them said they had adopted all the preventive measures listed so far. this is the most severe epidemic that has hit italy in the past years, and it will probably be one of the most severe viral pandemics of modern times. as no specific therapies are available at the moment for the new coronavirus, prevention and early containment of further spread can be crucial to control the pandemic. for this reason, dentists, similar to other medical practitioners, aware of the risk associated with carrying out their professional activity, at this moment limited to the management of dental emergencies only, have the responsibility in this situation to know the characteristics of the virus through precise and accurate information and to assume a careful and proactive attitude for the protection of their patients and of their entire community, working in the containment of this social emergency even if not directly involved in the treatment of affected patients. dentists at this time, however, should only work if they have the individual protective equipment recommended to high-risk healthcare workers [ , ] . after the pandemic emergency when people's professional activities and lives can slowly return to normal, the experience and the not-quite-finished risk of a recurrence of new cases of infection will require that dentists also follow new health safety protocols whose definition will be necessary. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file . "questionnaire" contains the english version of the questionnaire realized for the survey in this research. abbreviations who: world health organization; ppe: personal protection equipment connected to the survey. informed consent was presented on the main page of the online platform used to complete the survey. before the start of the survey the participant had to accept and agree that filling out the questions meant that his anonymously provided answers were used for the research presented in the introduction. not applicable. a new virus isolated from the human respiratory tract clinical features of patients infected with novel coronavirus in wuhan world health organization (who) novel coronavirus ( -ncov) severe respiratory disease associated with a novel infectious agent-letters to doctors genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a familial cluster of pneumonia associated with the novel coronavirus indicating personto-person transmission: a study of a family cluster a pneumonia outbreak associated with a new coronavirus of probable bat origin genome composition and divergence of the novel coronavirus (covid- ) originating in china genomic diversity of sars-cov- in coronavirus disease patients differential diagnosis in patients under investigation for the novel coronavirus (sars-cov- ) coronavirus disease (covid- ) -novel coronavirus severe adult respiratory distress syndrome in two cases in italy: an uncommon radiological presentation protezione civile italiana. emergenze coronavirus. elenco dei medici caduti nel corso dell'epidemia di covid- coronavirus covid- impacts to dentistry and potential salivary diagnosis transmission routes of -ncov and controls in dental practice mathematical methods of statistics the logic of inductive inference measures of association for cross classifications the treatment of ties in rank problems on the criterion that a given system of deviations from the probable in the case of a correlated system of variables is such that it can be reasonably supposed to have arisen from random sampling -ncov transmission through the ocular surface must not be ignored consistent detection of novel coronavirus in saliva. clin infect diseases transmission of -ncov infection from an asymptomatic contact in germany the workers who face the greatest coronavirus risk. the new york times, th testo coordinato con il d.lgs. agosto , n. in gazzetta ufficiale n. del aprile persistence of coronaviruses on inanimate surfaces and their inactivation with biological agents shortage of personal protective equipment endangering health workers worldwide world health organization. rational use of personal protective equipment for coronavirus disease publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions ap designed the study and was the major contributor to writing the manuscript. mr coordinated the development of the online questionnaire, data management and extrapolation. cm was responsible for statistical analysis of the results. gg researched the bibliography sources and reviewed the final manuscript. all authors read and approved the final manuscript. no funding needed. the datasets used and/or analysed during the current study available from the corresponding author on reasonable request. this research does not contain data require ethics approval. the current regulation of the ethics committee of the higher institute of health (istituto superiore di sanità, rome th may ) stipulates that projects with epidemiological, medico-social and evaluative contents need evaluation, approval and monitoring of trial protocols only if they contain personal data according to the legislative decrees on clinical trials and function of the ethics committees (decreto legislativo giugno , n. ; decreto ministeriale febbraio ). the official definition of "personal data" is given by the national data protection authority (garante per la protezione dei dati personali, https://www.garanteprivacy.it/home/diritti/cosa-intendiamo-per-dati-personali -regolamento (ue) / art. ). the term "personal data" includes information about first and last name, images, tax code, ip address and license plate number. the platform on which the anonymous questionnaire was completed does not allow to trace the ip address of the person who the authors declare that they have no competing interests. key: cord- -t b wpe authors: falcone, rino; colì, elisa; felletti, silvia; sapienza, alessandro; castelfranchi, cristiano; paglieri, fabio title: all we need is trust: how the covid- outbreak reconfigured trust in italian public institutions date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: t b wpe the central focus of this research is the fast and crucial impact of the covid- pandemic on a crucial psychological, relational, and political construct: trust. we investigate how the consequences of the pandemic, in terms of healthcare, state intervention and impositions, and daily life and habits, have affected trust in public institutions in italy, at the time when the contagion was rapidly spreading in the country (early march ). in this survey, addressed to italian citizens, we analyzed and measured such impact, focusing on various aspects of trust. this attention to multiple dimensions of trust constitutes the key conceptual advantage of this research, since trust is a complex and layered construct, with its own internal dynamics. in particular, the analysis focuses on how citizens attribute trust to public authorities, in relation to the management of the health crisis: with regard to the measures and guidelines adopted, the purposes pursued, the motivations that determine them, their capacity for involvement, and their effectiveness for the containment of the virus itself. a pandemic creates a bilateral need for trust, both in public authorities (they have to rely on citizens’ compliance and must try to promote and maintain their trust in order to be effective) and in citizens, since they need to feel that somebody can do something, can (has the power to) protect them, to act at the needed collective level. we are interested to explore how this need for trust affects the attributional process, regarding both attitudes and the corresponding decisions and actions. the most striking result of this survey is the very high level of institutional trust expressed by respondents: % of them trust italian public authorities to be able to deal with the covid- emergency. this is in sharp contrast with the relatively low levels of institutional trust characteristic of italy, both historically and in recent surveys. moreover, the survey allowed the discrimination of several potential predictors for trust, thus emphasizing factors that, during this crisis, are exhibiting an anomalous impact on trust. the great societal challenge presented by the covid- pandemic has prompted extraordinary efforts to meet such a challenge, from public authorities, civil society, and the scientific community. extreme policies for containment, mitigation, and co-existence with the virus have been implemented by the governments of most afflicted countries, as well as by relevant international institutions (e.g., the who and the eu). at the same time, scientific research worldwide has focused on tackling the many facets of this dramatic phenomenon, including its impact on social relationships and psychological well-being, as well as the key socio-cognitive factors in promoting effectiveness of the proposed countermeasures. several of these studies have highlighted the crucial and complex role of trust in dealing with the covid- pandemic. llewellyn ( ) puts it very succinctly and effectively: "in times of crisis, trust is the most important thing to consider if you want to communicate health advice." this blanket pronouncement is well-supported by previous evidence: in their systematic review on the importance of trust when preparing for and during a pandemic, siegrist and zingg ( ) found confirmation that "trust in health agencies positively influenced people's willingness to adopt recommended behavior." in addition, among the five recommendations for crisis communication highlighted by the authors' survey, two directly concern trust management: "the focus should be not only on trust but also on confidence, and establishing trust in health authorities before a pandemic occurs is important." this latter point is also stressed by lewnard and lo ( ) , with reference to the current pandemic: "the effectiveness and societal impact of quarantine and social distancing will depend on the credibility of public health authorities, political leaders, and institutions. it is important that policy makers maintain the public's trust through use of evidence-based interventions and fully transparent, factbased communication." it is worth noting that this emphasis on evidence and transparency, albeit crucial, describes only part of the relevant socio-cognitive dynamics that affect trust in public institutions: in particular, it collapses trust to confidence in information sources and their credibility, while a crucial problem is also trust in the institution's power to intervene, as well as trust in collective compliance with the proposed measures. finally, in specific circumstances, interesting inversions in cognitive causeeffect relationships can occur, as widely studied in cognitive sciences and social psychology (e.g., festinger, ; koller, ; kunda, ; epley and gilovich, ) . in fact, the relevance of trust for dealing with health emergencies is also linked to the limits of direct enforcement of the required behavioral change: without the active cooperation of the population, any drastic intervention is doomed to fail, because the desired behaviors (e.g., frequently sanitizing one's hands, wearing a facemask, and keeping a safe distance from others) cannot be effectively monitored on the required scale and with sufficient frequency. in a broad and comprehensive survey of social and behavioral results to support covid- pandemic response, van bavel et al. ( ) highlight how most measures needed to contain an epidemic are, by their very nature, difficult to enforce directly: this, in turn, makes trust in public authorities all the more relevant. based on scientific evidence gathered during previous outbreaks, van bavel et al. ( ) argue that "trust in institutions and governments (. . .) may play an important role." for example, trust in the liberian government was correlated with decisions to abide by mandated social distancing policies and utilizing clinics for care during the ebola outbreak. trust was also related to decisions to adopt preventive measures such as ebola vaccinations in the drc. conversely, a lack of trust in public health officials may lead to negative effects on utilization of health services. reliable information and public health messages are needed from national leaders and central health officials. however, local voices can amplify these messages and help build the trust that is needed to spur behavioral change (van bavel et al., ) . these expectations on the positive role of trust in promoting adherence and compliance with preventive regulations and guidance are finding ample confirmation also in recent studies on the ongoing crisis, both within and across various countries. in a nationally representative survey conducted in denmark during the covid- pandemic (n = ), olsen and hjorth ( ) measured the respondents' willingness to apply social distancing in order to reduce contagion: they found that both lower levels of political trust and lower generalized social trust are negatively associated with willingness to distance and that younger male respondents with the lowest levels of education and least political trust report lower willingness to distance. in a nationally representative survey of italian adults (n = ) conducted between the th and th of march , barari et al. ( ) observed high levels of understanding and self-reported compliance with containment measures, and noted that "even those who do not trust the government, or think the government has been untruthful about the crisis believe the messaging and claim to be acting in accordance." trust acts as a precious commodity both for institutions and for scientists, both of which are crucial actors in the public response to the covid- pandemic. in a large-scale background analysis of european social survey data on european countries (n = , ) focused on the covid- epidemic from january to april , , oksanen et al. ( ) found that institutional trust acts as a protective factor: countries with low levels of institutional trust prior to the outbreak (including italy) experienced significantly higher mortality rates during the crisis; moreover, their governments introduced restrictions against contagion later than countries with higher levels of institutional trust (calculated as the delta between the date when the restrictions came into effect and when the first confirmed covid- death was reported in that nation), which in turn contributed to the severity of their death toll. these results on the relevance of trust as a protective factor are in line with previous studies on other epidemics, e.g., ebola, showing how people with higher institutional trust are more likely to follow the advice and guidelines given by the health authorities (blair et al., ; vinck et al., ) , as well as investigating the interplay between scientific and non-scientific sources in modulating people's trust in healthcare information (falade and coultas, ) . as for trust in science, its role has been highlighted in a recent study by plohl and musil ( ) : using structural equation modeling (sem) on a sample of international, english-speaking respondents, the authors investigated whether and how risk perception and norm compliance for the covid- pandemic may be affected by several constructs, i.e., religious orthodoxy, conspiracy ideation, intellectual curiosity, and trust in science, all measured with validated scales. their results indicate that trust in science is by far the most important factor in producing appropriate risk assessment and high level of norm compliance. at the same time, trust in science, as opposed to the tendency to believe in alternative non-official sources, has been observed to be deeply affected by polarization and homophily (bessi et al., ) . looking at the specifics of the covid- pandemic, so far the most insidious threat posed by the virus has been the combination of the rapidity of its spread with the high number of patients requiring treatment in intensive care, resulting in unprecedented strain on the healthcare system of affected countries. this in turn has prompted an increasing number of national governments to adopt extreme measures to limit the spread of the virus, often imposing very demanding limitations on citizens' basic rights (e.g., social isolation, lockdown, and quarantine) and with dire socio-economic consequences (e.g., job insecurity, rising unemployment, loss of revenues, and inequalities). in such a unique scenario, the relevance of studying citizens' trust in public institutions is manifold: on the one hand, the effectiveness of these measures and the collective ability to overcome their costs is conditional on the compliance of the population, which in turn is affected by trust in institutions; for this same reason, institutions actively seek to promote citizens' trust, as a means to achieve their prevention goals; on the other hand, the very nature of the current crisis is likely to affect and shape how citizens conceptualize trust, and such socio-cognitive impact of the covid- pandemic needs to be understood. indeed, the current crisis acts as a magnifying glass in highlighting the essential role of trust in our societies (trust as "vinculum societatis, " the bond of society, to borrow john locke's famous expression), both for the psychological well-being of individuals and for the effective functioning of institutions. the study presented in this paper contributes to this fastgrowing body of knowledge on the interplay between trust in institutions and the covid- pandemic, by discussing the results of a large scale survey (n = ) conducted on italian citizens between march and march , . at that time, italy had the most active outbreak of the virus worldwide, and its death count was growing at alarming rates; at the same time, extreme prevention measures were still relatively recent and rapidly changing in nature, sometimes from day to day (e.g., on march new restrictions were introduced by the government, closing public places such as restaurants, pubs, and most shops). thus, our data offer insight into a time window in which the phenomenon was already in its acute phase in medical terms, yet still novel and unexpected for the population: this offers a privileged vantage point to observe how a pre-existing construct, trust in institutions, was affected by a sudden and profound change in the everyday functioning of the whole country, by a complete (albeit hopefully temporary) re-representation of one's role in society and in personal relationships, as well as in the relationship between citizens and institutions. the survey was theoretically inspired by the socio-cognitive model of trust developed by castelfranchi and falcone ( ) : we chose this theoretical framework because it provides a rich and nuanced description of various reasons for trust, thus allowing us to probe not only the degree by which italian citizens expressed trust toward the relevant public authorities engaged in the response to covid- but also on what grounds such attitude was based. our purpose, however, was not to look for direct validation of the theoretical model, but rather to collect as many detailed data as possible on the rapidly evolving italian response to the covid- emergency, from the standpoint of institutional trust: in this sense, this study was mostly intended as explorative. in particular, we wanted to compare our results with the welldocumented low levels of trust in institutions exhibited by italians before the onset of the crisis, which some have associated with tardiness in responding to the covid- emergency across various european countries (oksanen et al., ) : we intended to see whether such widespread distrust toward public institutions would be confirmed or subverted during the initial stages of the covid- outbreak in italy and to offer some insights and suggestions regarding the original and peculiar nature of any discontinuity in institutional trust that may be associated with the current pandemic. moreover, we intended to take a closer look at the cognitive and social factors responsible for trust toward public institutions in the face of pandemic threats: the survey was designed both to discriminate several potential predictors for trust, so that subsequent analysis would allow us to individuate the most relevant ones, and to facilitate comparison with the underlying theoretical model, thus emphasizing factors that, during this crisis, are exhibiting an anomalous impact on trust-either because they determine trust more intensely than usual (overcharged factors) or because their impact is minimal or non-existent (anesthetized factors). indeed, a key hypothesis that we wanted to test concerns the impact of covid- on the very nature of the institutional trust construct: not only the overall trust in public institutions is affected by the pandemic and how these institutions respond to it, but also the determinants of trust in institutions change and adapt to this crisis, in comparison with other situations. desperate times require desperate measures, and desperate measures induce a drastic reconfiguration of the cognitive underpinnings of trust in institutions. our survey was designed to collect data on such paradigm shift in how institutional trust was conceptualized by italian citizens during the early stages of the national response to the covid- pandemic. we used a snowball sampling method to determine the respondents: we collected a large sample (n = , % women, mean age = years, range = - years, sd = . ), relatively well-balanced in terms of geographical provenance ( % northern italy, % central italy, and % southern italy and main islands), with a significant portion of respondents ( %) residing in the regions most affected by covid- at that time (lombardy, veneto, emilia-romagna, marche, and piedmont) . the relatively uniform geographical distribution of the sample among the three macro-areas of italy, as well as the significant proportion of respondents from highly affected regions, allows interesting comparisons based on participants' residence. moreover, the introduction of more drastic restrictions by the italian government at the end of march , , invites considering also this temporal dimension in analyzing the data: in this respect, it is important that a fairly large set of participants (n = ) completed the survey after those new restrictions had been introduced. finally, it should be noted that the mean educational level of participants is very high: almost three quarters of respondents have a degree ( %) or postgraduate specialization ( %). the main characteristics of the sample are synthetized in table . data were collected with a -item questionnaire, using a fivepoint likert scale for most items: an english translation of the whole questionnaire is available in the supplementary materials. the questionnaire was based on the socio-cognitive model of trust developed by castelfranchi and falcone ( ) and explored the questionnaire was administered online using the google forms platform. the questionnaire fully complied with ethical guidelines for human subject research and participation was conditional on the preliminary approval of an informed consent by each subject; the compilation took an average time of min. data analysis was performed using the spss (version ) statistical software: the collected data were first analyzed through correlation analyses (given the asymmetric distribution of most variables, we considered spearman correlation values); secondly, given the high number of items in the questionnaire, we conducted a principal component analysis (pca) on each subsection of the questionnaire prior to running regression analyses on the aggregated data. full details on descriptive statistics for each item in the questionnaire are included in the supplementary materials, differentiating also based on geographical factors (northern, central, and southern italy; more affected vs. less affected regions) and temporal boundaries (before vs. after the march announcement of new restrictions by the national government). here, we report only the most relevant findings, prior to more in-depth analysis, and only in terms of aggregate data, since no significant differences emerged at this level between different areas and different dates (albeit some interesting patterns were detected via regression analysis, see section "regression analyses"). when asked to indicate which public authority is the most adequate to take decisions concerning the covid- emergency (item in the questionnaire), . % indicated the national government, . % indicated the civil protection, . % indicated the presidency of the republic, . % indicated the regional government, . % indicated the municipal authority, and . % indicated others. hence, the overwhelming majority ( . %) of respondents consider pandemics as a matter of national concern, which should be primarily addressed by national authorities. this should be taken into account while interpreting all other results, since most of the attitudes expressed by participants regarding features of public authorities (competence, intentionality, trust, etc.) should be understood with reference to national institutions, unless otherwise specified. moreover, it is remarkable that the presidency of the republic, which is mostly a moral authority, is seen as having a greater role than regional governments, in spite of their leading role in the healthcare system, which in italy is organized on a regional basis. equally significant is the fact that only . % of respondents (within the broader category "others") indicated any kind of international entity, including the european union, as having a primary role in facing a pandemic outbreak. in short, at this stage of the covid- emergency, italian citizens strongly believed that this pandemic was not to be prominently addressed by either regional or international authorities, but was rather mostly a matter of national concern. when asked to rank their overall trust in public authorities for the management of the covid- emergency (item in the questionnaire), % of respondents manifested either extreme ( . %) or high ( . %) levels of trust, . % were non-committal, and only . % expressed distrust (see figure , left panel). as we will see in the section "discussion and conclusions, " these numbers are in sharp contrast, to say the least, with the average institutional trust reported for italian citizens prior to the covid- crisis, especially considering that the main target of this newfound trust was national public authorities (see above). the competence of public authorities was assessed as their ability in planning both the right prescriptive measures (e.g., lockdown) and the appropriate behavioral guidelines (e.g., personal hygiene recommendations). on both counts, the majority of respondents expressed a positive belief in the public authorities' competence ( . % for measures, . % for guidelines), whereas only a relatively small minority was either undecided ( . % for measures, . % for guidelines) or skeptical ( . % for measures, . % for guidelines). moreover, correlational analysis indicates that competence scores for measures and guidelines are strongly and positively related (r = . , p < . ), suggesting that respondents did not really discriminate between prescriptive measures and behavioral guidelines, at least with respect to trust in public institutions: for this reason, in subsequent analyses, we collapsed these two items into a single competence value, calculated as the mean response for each subject to items (competence on measures) and (competence on guidelines) of the questionnaire (these are also the data reported in figure , central panel). other items in this section of the survey were designed to investigate the reasons behind participants' beliefs on the public authorities' competence: in summary, the overwhelming majority of the sample ( . %) believed that it was the public authorities' proper prerogative to take action and issue containment measures against the pandemic (item ), and most respondents ( . %) positively evaluated the use of experts' advice by the public authorities during the covid- crisis (item ); there was instead less confidence in the organizational capacity demonstrated by public authorities in the early stages of the emergency (item : . % expressed a positive evaluation, . % were undecided, and . % were critical), and the majority of the sample ( . %) agreed that institutional communication on the covid- presented some contradictions, either between different authorities or over time (item ). in spite of these partial concerns, a significant majority of the sample ( . %) did not express any skepticism on the competence of the public authorities in handling the emergency (item ). as for the competence, we inquired on the intentionality of public institutions separately for prescriptive measures and behavioral guidelines, asking participants whether they believed either type of intervention was both actively and honestly aimed at containing the covid- pandemic. again, respondents expressed an overwhelmingly positive belief in the good faith of public institutions, both in promulgating prescriptive measures ( . %) and in issuing behavioral guidelines ( . %): only a small minority was either undecided ( . % for measures, . % for guidelines) or skeptical ( . % for measures, . % for guidelines). correlational analysis reveals again that intentionality scores for measures and guidelines are strongly and positively related (r = . , p < . ), further confirming that respondents did not really discriminate between prescriptive measures and behavioral guidelines, when it comes to assessing the public authorities' trustworthiness in this emergency: hence, these two items on intentionality were collapsed into a unique intentionality value in subsequent analyses, using the mean response for each subject to items (intentionality on measures) and (intentionality on guidelines). other items in this section of the survey were designed to investigate the reasons behind participants' confidence, or lack thereof, in the nature of the public authorities' intentions: in summary, we found confirmation of the fact that most respondents ( . %) did not doubt that the intentions of the public authorities were consistent with their public statements (item ), whereas a smaller majority ( . %) considered the economic investment mobilized by the italian public authorities sufficient to fight the pandemic (item : notice that only . % considered it insufficient, with a significant portion of the sample, . %, remaining undecided). finally, asked whether other interests, e.g., political or economic, were at stake (item ), the larger part of the sample ( . %) answered in the negative, whereas . % acknowledged the presence of such ulterior motives and . % were unsure: as we will discuss further on, this question was probably easy to interpret in two markedly different senses-either negatively, as an accusation of having some hidden and problematic agenda, or positively, as the capacity to take into account all the key ramifications of the covid- crisis, including its political and socio-economic aftermath. overall, we registered strong confidence in the good faith of the intentions manifested by public institutions (figure , right panel): this parallels the belief in the public authorities' competence, and together, these attitudes support the high levels of institutional trust expressed by this sample. part of the survey was focused on the measures issued by public authorities as a response to the covid- pandemic, in order to estimate both their perceived usefulness and the goal attributed to these interventions by the participants. the vast majority of our sample ( %) perceived these measures as being either useful ( . %) or very useful ( . %) in fighting the pandemic, whereas only a tiny minority was skeptical ( . %), with the remaining . % being undecided (item ). when asked to assess the adequacy of the public authorities' intervention (item ), a more abstract notion involving a counterfactual comparison with alternative strategies, the majority rated current measures as adequate ( . %), . % were undecided, and only % considered them inadequate. in terms of the motivations associated with these measures, we asked participants to express agreement on three potential, non-mutually exclusive aims: reassuring the population (item ), curbing the spread of covid- (item ), and creating unmotivated alarm (item ). the vast majority ( %) agreed that the rationale of the public authorities' intervention is indeed to contain the pandemic, whereas only . % attributed to the public authorities the goal of reassuring citizens, and even fewer respondents ( %) regarded the proposed measures as a way of spreading unnecessary panic. when rating the personal burden of the proposed restrictions on their own lives (item ), % of participants expressed to feel a high level of impact, whereas . % indicated little discomfort for the current situation and the remaining . % reported medium levels of distress. however, regardless of the perceived impact on the public authorities' intervention, the overwhelming majority of respondents agreed that such sacrifices were crucially beneficial for themselves and their families (item , . % of agreement), for the society as a whole (item , . %), and for both (item , . %). moreover, when asked to assess the usefulness of one's personal contribution to these preventive measures, since they were intended for the whole population (item , a question aimed at implicitly measuring any "free-riding inclination" in our sample), as many as . % of the participants considered their personal role relevant for the collective effort. taken together, these data show that, albeit different people suffered more or less because of the containment measures, almost all agreed on their usefulness and on the necessity of personal sacrifice to deal with the pandemic: this suggests a mindset in which the shared goal of public safety trumps any individual concern, including personal discomfort, fear, and anxiety (an interpretation later confirmed by regression analysis, see section "regression analyses"). in terms of expectations on compliance with the sanitary restrictions by other fellow citizens (items - ), we observe a fairly varied pattern of response (see figure ): the most widespread belief ( . % of agreement) is that enough italian citizens, albeit not all, will comply with the regulations, thus making them effective (item ); in contrast, there is skepticism both on the most optimistic scenario, i.e., full compliance (item , . % of disagreement), and on the bleakest outcome, i.e., insufficient compliance (item , . % of disagreement), although it is worth noting that pessimism is rejected much more strongly than optimism. the possibility that only few people will comply, and yet their efforts will be useful (item ), is also rejected by the relative majority of the sample ( . % of disagreement), yet interpreting this result requires caution, since it could either express skepticism on figure | expectations on compliance by others. frontiers in psychology | www.frontiersin.org how many people will comply, or on the chances that limited compliance may indeed be useful. regarding the motivations useful to induce compliance, we asked participants to express agreement on four possible motivational triggers: the expectation that everybody else will follow the new regulations (item ), a personal concern for dangers (item ), a spirit of collaboration in the face of the emergency (item ), and trust in the fact that public authorities are doing everything in their power (item ). all four motivations engendered significant levels of agreement, with the highest being the feeling of a common cause against a shared threat ( %), followed by trust in maximum effort by the public authorities ( . %), concern for the associated risks ( . %), and expecting others to comply as well ( . %). it is interesting to note that a motivation tied to the collaborative dimension of trust in civil society, i.e., being united in pursuing a common goal, shows more than percentage points of distance from a motivation inspired instead by the sanctioning view of trust, i.e., being able to monitor compliance by others, possibly to punish free-riders, as well as from fear of personal harm: this suggests that emphasizing collaborative motives (a strategy employed quite consistently by the italian government in its public communications during the early stage of the covid- outbreak) may be more effective in promoting compliance than stressing individualistic goals. this section of the survey asked respondents to provide a metacognitive evaluation of the most relevant factors promoting their trust in how public institutions are handling the covid- crisis. of the eight factors explored, the type of measures adopted by the authorities was the most frequently cited as important (item , . %), followed by the information received on the crisis (item , . %), the capacity of public authorities to actually enforce protective measures (item , . %), the respondent's profession (item , . %) and his/her health condition (item , . %), the opinions expressed by social relations such as friends and relatives (item , . %) or colleagues (item , . %), and the political connotation of the relevant authorities (item , . %). later on, we will use regression analysis to investigate the extent by which these self-reported data correspond to the relative weight of the actual factors affecting participants' trust in institutions. for now, it appears that participants self-describe their theory of trust in fairly objective terms, giving priority to the factual nature of the proposed measures, the information they gathered (apparently with the exception of social channels; see below), and the extent by which public authority is able to enforce their recommendations; in contrast, relatively little weight is given to personal factors and social networks, and none at all to political partisanship. this last result suggests that the public response to the covid- crisis was initially perceived as a matter of shared concern of all political parties, which in turn prompted a temporary truce in the usual partisanship characteristic of italian politics; moreover, in their efforts to deal with the emergency, public authorities were regarded mostly for their institutional role, with little attention to their political affiliation (even when such authorities were the expression of certain political parties, as it was the case with the national government). this interpretation also helps to explain the extremely high level of trust in public institutions with respect to the covid- emergency during those few days, in a population well-known for its deep-seated distrust of politicians in general, and of political parties in particular: further analysis of this interesting anomaly will be presented in the section "discussion and conclusion." this section of the survey investigated both frequency of use (items - ) and perceived trustworthiness (items - and ) of various types of information sources in relation to the covid- pandemic, to get a better sense of what channels were most influential in affecting participants' opinions on this topic; in addition, we collected data on the trustworthiness directly assigned to public institutions as information sources (item ), which was high for . % of the sample, average for . %, and low only for . % of respondents. with respect to other information channels, the data summarized in table highlight four main findings: (i) official online channels, e.g., the website of the civil protection, and scientists are both frequently used and considered reliable as information sources; (ii) in contrast, traditional media, albeit often consulted, are regarded as reliable only by less than half of our sample; (iii) family physicians are in general considered trustworthy, yet they are rarely used as information sources; (iv), finally, both social relationships and unofficial online sources, e.g., social media, are neither frequently used, nor widely believed. the result on unofficial online channels is especially surprising: whereas the very low credibility associated to these sources is understandable and even commendable, the fact that only one respondent out of four admits to using them frequently is hard to swallow, especially at a time in which personal contact was severely limited in italy, thus making social media an even more attractive outlet for users. besides, recent national statistics on internet use in italy do not agree with the picture painted by these data: according to the global digital report , compiled annually by wearesocial and hootsuite, in , % of italian citizens were active social media users (with a growing trend with respect to ), and the average time spent on social media every day was a little less than h per person. besides social desirability effects (respondents may have been reluctant to admit gathering information via unofficial channels on such delicate topics), a possible explanation for this anomaly is in a common misperception of the role of social media as gatekeepers: someone who finds on facebook a link to an article on a traditional newspaper, or is made aware by a post on twitter of the latest press release on the official website of the civil protection, may be inclined to disregard the role of the social media in bringing these information to the user's attention. yet, this is how we use social media as information sources, often without even realizing it: we take advantage (or succumb, depending on the circumstances) of their agenda setting algorithms, which allow these platforms to act as powerful information brokers, rather than information producers. expectations on long-term impact on trust the final section of the survey intended to probe participants' expectations on the long-term impact of the covid- crisis on trust relationships between citizens and public institutions (item ), between citizens and the dominant economic model of development (item ), between citizens and the scientific community (item ), and among citizens as peers (item ). here, the big winner is expected to be science: . % of respondents believe that the current crisis will strengthen the trustworthiness of scientists as public figures. expectations on the impact of trust toward public institutions and among citizens are less triumphant, yet still positive: . % predict an increase in institutional trust after the covid- pandemic, whereas % make the same prediction with respect to social trust, i.e., trust among peers. finally, on future trust in the dominant model of economic development, our sample is evenly divided: % think that we will trust it more than before, . % are undecided, and . % expect an increase in distrust toward that model. as a preliminary step before running regression analyses, we used pca to identify strongly correlated items in the data set and simplify the variables' structure, in order to avoid multicollinearity issues in our regression models. since the survey was theoretically motivated by the socio-cognitive model of trust (castelfranchi and falcone, ) , we performed separate pca on subsets of items, to preserve relevant theory-based distinctions in the participants' responses. item , degree of trust toward public authorities in relation to the covid- pandemic, was not included in the pcas, since it was intended to act as the target of the regression models; we also excluded items (doubts on public authorities' competence) and (doubts on public authorities' intentions), since these were included in the survey merely as control questions for, respectively, items - and items - ; moreover, we kept separate from the pcas item (personal discomfort associated with public authorities' measures), item (usefulness of one's own personal contribution to the collective effort), and item (overall adequacy of public authorities' measures), since we wanted to test their role as individual predictors in the regression models; finally, item (expectation of very limited yet useful compliance by other citizens) was excluded for the pca and regression analysis, due to the ambiguity in its interpretation already mentioned in section "descriptive statistics." the remaining items led to the individuation of principal components, as summarized in table (full details on the pcas methods and results are provided in the supplementary materials). in order to be considered satisfactory, each pca had to explain at least % of the cumulative variance, and further components were added only if they improved by more than % the explained variance. in order to test our main hypotheses, we performed a multivariate regression model on raw data using ibm-spss software. the dependent variable to be predicted was the overall trust manifested by participants toward public authorities involved in the covid- response, i.e., item in the survey. after some explorative iterations and based on theoretical considerations, we decided to include independent variables in the final model: principal components identified via pcas (indicated with an asterisk in table ), individual items that were conceptually independent from the other sections of the survey (personal discomfort associated with public authorities' measures, usefulness of personal contribution to the collective effort, overall adequacy of public authorities' measures), and socio-demographic variables-age (coded as = - , = - , = - years of age), educational level (coded as = high school diploma or lower, = university degree or higher), region of residence (coded as = most affected regions, i.e., lombardy, emilia-romagna, veneto, marche, and piedmont, = all other regions), and time of data collection (coded as = before, = after the march new restrictions were announced). preliminary analyses indicated that the respondent's profession did not affect responses, so we excluded it from the model; as for gender, preliminary regressions showed no difference in the predictors of institutional trust between male and female respondents, so we excluded it from the final regression model and performed a separate set of analyses to assess its impact in our data (see section "gender effects"). we first run the regression analysis on the whole sample: the model had a good fit (r = . ) and explained % of the variance in the overall trust evaluation; out of independent variables were significantly correlated with trust (p < . ), and the most powerful predictors were positive indicators of competence of public authorities (β = . , p < . ), perceived adequacy of the adopted measures (β = . , p < . ), trustworthiness of official information sources (β = . , p < . ), public authorities' intention to ( ) the numbering used for items follows the order of presentation in the survey: the relevant items are from to , since item was the informed consent, whereas items - asked for demographic information. the asterisk (*) indicates principal components that were later used for regressions. contain the pandemic (β = . , p < . ), and perception that public authorities' efforts were focused on public safety, with no other agenda (β = . , p < . ). all other significant predictors had an absolute value of β equal to or lower than . . the non-significant predictors were personal discomfort due to the adopted measures, perceived usefulness of personal sacrifice, expectation of sufficient compliance (but notice that expectation of universal compliance was positively correlated with trust, whereas expectation of insufficient compliance was negatively correlated with it, both p < . , suggesting an "all or nothing" attitude toward compliance), individualistic reasons for compliance (while collectivist reasons for compliance were strongly and positively associated with trust, p < . ), educational level, time of data collection, and age (the last one showed a marginally significant negative correlation, β = − . , p = . ). we also applied the same regression model to subsets of participants, distinguishing first geographically (most afflicted regions vs. all other regions), then temporally (before and after the announcement of new restrictions by the italian government on march ), in order to detect differences in how trust was processed depending on the severity of the sanitary emergency in various areas, and the strictness of the measures implemented by public authorities while the pandemic was still progressing. we already knew from descriptive statistics that no overall change in trust toward public authorities was observed across these contexts, yet we wanted to probe for more subtle differences, e.g., different predictors of trust, or different contribution of the same predictors, depending on region of residence and time of data submission. all β and p-values for the various multiple regressions are reported in table ; in what follows, we will focus only on the most relevant results. applying the model only to participants from the most affected regions in italy at that time (lombardy, emilia-romagna, veneto, marche, and piedmont) revealed again a good fit (r = . ), explaining . % of variance in trust assessment; the same model also had a good fit when applied only to participants from all other italian regions (r = . , . % of explained variance). in both cases, the strongest predictors remained the same as in the whole sample, and also their order of importance was identical across regions, regardless of current outbreak severity (p < . for all the following predictors): positive indicators of competence (most affected: β = . ; other regions: β = . ), adequacy of the adopted measures (most affected: β = . ; other regions: β = . ), trustworthiness of official information sources (most affected: β = . ; other regions: β = . ), pa's intention to contain the pandemic (most affected: β = . ; other regions: β = . ), and perception that public authorities' efforts are focused on public safety, with no other agenda (most affected: β = . ; other regions: β = . ). in spite of the substantial similarity in how trust in public authorities was attributed by respondents in different areas of the country, some fine-grained distinctions emerge looking at those factors that were significant in one context but not in the other-and also exercising due caution, since a difference in significance does not necessarily imply a significant difference. in the most affected regions, we observed eight non-significant predictors, whereas there were only six in the other regions: four of these factors were irrelevant across both contexts (personal discomfort, perceived usefulness of the sacrifices, individualistic reasons for compliance, and time of data collection), whereas negative factors affecting competence of public authorities, intention to downplay the emergency, impact of personal effort, and trustworthiness of unofficial information sources were immaterial for respondents from the most affected areas, whereas they acted as significant predictors (albeit weak ones) for participants from other regions of italy; in contrast, an expectation of sufficient compliance from other people had a significant negative correlation with trust in the most affected regions (β = − . , p = . ), whereas it had a marginally significant positive correlation with it elsewhere (β = . , p = . ). taken together, these results suggest that participants living in areas that were currently experiencing very severe outbreaks of covid- had a more focused mindset when deciding whether to trust public authorities to deal with the emergency: less factors were considered relevant, and in particular, it was probably taken for granted that some inconsistency in public communication and intervention may occur, without necessarily jeopardizing trust (negative factors on competence), and that unofficial sources were not to be taken seriously when deciding whom to trust; at the same time, expecting that only a sufficient number of people would comply with the emergency measures had a negative impact on trust in public authorities, probably highlighting the fact that, in those regions, people believed that "enough is not enough"that is, either everybody cooperates in facing the crisis (universal compliance) or we will not be successful in overcoming it. this extreme mindset is confirmed by the fact that the relevance of one's own personal contribution did not affect trust attribution to public authorities in the most affected regions, whereas it did in other areas: this indicates again that collective compliance, not personal efforts, are perceived as the key to success by people currently facing the worst of the covid- pandemic. looking instead for short-term shifts in trust assessment over time, in relation to relevant public events (i.e., the introduction of new measures by the italian government on march ), we divided our sample based on time of data submission: before or after the public press release when the prime minister giuseppe conte announced the new restrictions to be implemented nationwide, to contain the covid- outbreak. the model performed well across both time windows (before: r = . , . % explained variance; after: r = . , . % explained variance) and the strongest predictors remained the same, as well as their relative order of importance (p < . for all the following predictors): positive factors affecting competence of public authorities (before: β = . ; after: β = . ), perceived adequacy of the adopted measures (before: β = . ; after: β = . ), trustworthiness of official information sources (before: β = . ; after: β = . ), attributing to public authorities the intention to contain the pandemic (before: β = . ; after: β = . ), and the perception that their efforts were focused on public safety, with no other agenda (before: β = . ; after: β = . ). again, we observed substantial stability over time in how trust in public authorities was attributed, with minor differences emerging only by comparing the significance and direction of some secondary variables. in general, the introduction of more severe restrictions had the effect of simplifying the metrics used to assess trust toward public authorities: before the march announcement, only four variables failed to correlate significantly with trust, whereas after it, the number of irrelevant predictors increased to , indicating a more narrowly focused mindset in assessing the trustworthiness of the institutions in charge of dealing with the emergency. in particular, intention to downplay the emergency, personal discomfort associated with the proposed measures, and trustworthiness of unofficial information sources became irrelevant for trust in public authorities; unfortunately, the expectation of universal compliance also became equally irrelevant (before: β = . , p = . ; after: β = − . , p = . ), while the negative correlation between expectation of insufficient compliance and trust was much stronger after the march announcement (before: β = − . , p = . ; after: β = − . , p = . ). this suggests a turn for the worst in people's expectations: before the new restrictions, trust was positively supported by expectation of universal compliance (the more i believe all others will behave responsibly, the more i trust the authorities), whereas after them, the influence of pessimistic fear became dominant (the more i doubt enough people will comply, the less i trust the authorities). as a possible reaction to this shift, it is worth noting that the positive correlation between impact of personal efforts in the covid- response and trust in public authorities became significant only after march (before: β = . , p = . ; after: β = . , p = . ), suggesting that the new measures strengthened in italian citizens a sense of personal responsibility for the collective reaction to the virus. finally, region of residence was a significant (albeit weak) predictor of trust before, but not after, the announcement of new restrictions by the italian government (before: β = − . , p < . ; after: β = − . , p = . ): this shows a stronger tendency to trust public authorities in the most affected regions before march , whereas this was no longer true after that date. since overall trust in public authorities did not decrease after march in the whole sample, this indicates a leveling in trust attribution across the country after the introduction of new measures, which in turn could be interpreted as a shift in the perception of the emergency: whereas in early march, a significant part of the italian population still believed the outbreak to be somehow contained to specific regions, and thus a local problem unlikely to affect everybody in the same way, the nationwide interventions announced on march made it crystal clear to all that covid- was indeed a national concern. overall, these regression analyses show that, in italy, trust in the capacity of public authorities to deal with the covid- emergency was attributed in a fairly consistent manner during the time window of this survey (march - , ) across different areas of the country, giving central prominence to positive indicators of competence in public institutions, assessing the adequacy of the proposed measures, verifying that proper intentions supported their application, and paying attention mostly to official information sources. all considered, this suggests a fairly reasonable and well-balanced judgment-making process for trust attribution, while the true anomaly remains the high levels of trust in public authorities recorded during the early stages of this emergency (see section "descriptive statistics"), which are in sharp contrast with both long-term trends and recent surveys on institutional trust in italy, prior to the covid- pandemic. at a more fine-grained level, region of residence and time of data completion did reveal some interesting shifts in trust assessment, yet these insights should be interpreted carefully, since they concern relatively minor changes in the significance of secondary predictors, within a regression model with a high number of independent variables. comparing male and female respondents, a χ test revealed a small but significant difference (p = . ) in institutional trust in relation to the covid- emergency: in particular, men were more likely to express high levels of trust toward public authorities involved in contrasting the outbreak ( . % men vs. . % women), whereas women were more often neutral ( . % women vs. . % men). running the regression model described in section "regression analyses" separately on male and female respondents showed that, although the main predictors remained the same (positive indicators of competence, adequacy of the measures, trustworthiness of official information sources, public intention to contain the pandemic, and institutional focus on public safety), age and region of residence were significant predictors only for women and not for men (age: women β = − . , p = . , men β = − . , p = . ; region: women β = − . , p < . , men β = . , p = . ). to further investigate this interaction between gender and other socio-demographic factors influencing institutional trust during the covid- emergency, we run a trivariate analysis on, respectively, gender × age × trust and gender × region × trust. the first analysis revealed that gender effects on institutional trust are significant (p = . ) only in the age range - years, which is also the most vulnerable to the virus: among respondents in this age range, the majority of those that expressed low levels of institutional trust were male ( %), whereas most of those neutral or highly trustful were female ( . and . %, respectively). it is also worth noting that, after performing a bivariate analysis on the impact of age on trust, we found a highly significant effect (p < . ), with . % of elderly respondents ( - years old) expressing high trust in public authorities, whereas this percentage drops to . % for participants in between and years of age: this further confirms the role of vulnerability to the covid- virus in eliciting higher attributions of trust, and it is consistent with previous findings on a negative correlation between age and willingness to comply with social distancing measures during the covid- pandemic (wirz et al., ) . the second analysis showed that the relationship between gender and institutional trust is significant (p = . ) only in those regions that were most affected by the covid- outbreak: in these areas, most of the respondents that manifested distrust in public authorities were men ( . %), while the majority of the neutral and trustful participants were women ( . and . %, respectively). taken together, these results suggest that, whenever the situation was most critical (i.e., for the most vulnerable age range and in the most affected regions), men were overrepresented in the (small) group of people expressing distrust toward public authorities, whereas women were overrepresented among those neutral or trustful. although this may suggest an interesting gender effect on resilience under extreme stress (women seem more likely than men to suspend judgment or look on the bright side, precisely when the situation is the most dire), it is worth noting that, regardless of gender, only a small minority of respondents were expressing distrust toward public authorities, even in the most affected age range (men . %, female . %) and in the most affected regions (men . %, female . %). thus, these gender effects invite further investigation, but on their own, they do not justify any hasty conclusion on how different genders may react against health emergencies. the most striking result of this survey is the very high level of institutional trust expressed by respondents: % of them trust italian public authorities to be able to deal with the covid- emergency. this is in sharp contrast with the relatively low levels of institutional trust characteristic of italy, both historically and in recent surveys: according to the demos & pi nd annual report on "the italians and the state" , based on a large representative sample (n = ) of italian citizens over years of age interviewed in december , only % respondents trusted the state, whereas both regional governments ( %), european union ( %), and municipal authorities ( %) fared better, while political parties were in the worst shape, with only italians out of willing to trust them; in fact, of the main national institutions, the only one with decent levels of trust was the presidency of the republic ( %, still in sharp decline with comparison to years before, in , when it was as high as %). also international estimates indicated relatively low levels of institutional trust: according to the eurofound report on eurofound ( ) , italians' trust in the national government has been declining in the last few decades and is now below %, while the more recent data of the eurispes report-italy , presented in february , indicated trust in institutions at . % ( . points lower than in ). institutional trust in italy in recent years is extremely weak not only in absolute terms but also in relation to other european countries: in their comparison of eu states, based on data from the european social survey, oksanen et al. ( ) reported very low levels of institutional trust in italy, measured by respondents' trust in five institutions (parliament, politicians, political parties, the police, and the legal system); in fact, only cyprus, poland, slovakia, and bulgaria expressed stronger institutional distrust than italy. moreover, this trend toward widespread distrust of public institutions is not a particularly recent feature of italian politics: while in recent decades, it developed mostly against the backdrop of increasing tensions between populist movements and traditional political parties (urbinati, ) , massive erosion of public confidence in political figures was already ongoing in italy well before the recent resurgence of populism worldwidein the last decade of the th century, following the corruption scandals of tangentopoli and its media resonance (giglioli, ; vannucci, ) , and with the largely failed shift toward bipolarism during the berlusconi age (viroli, ) . even before that, a longitudinal analysis reveals that the confidence gap between electors and political institutions, characteristic of many post-wwii democracies, appeared in italy much earlier than in other countries (segatti, )-so much so, that already in the s lapalombara ( ), a highly influential political scientist, described italians' attitudes toward politics with three emblematic words: alienation, fragmentation, and isolation. such a deeply rooted tradition of distrust in public institutions underscores the importance of the opposite trend registered in our survey, i.e., a sudden boost in institutional trust prompted by the covid- crisis-a significant result that is also supported by other data collected in this survey, as seen in the "results" section. moreover, insofar as this newfound trust is grounded on trust in the expertise of the scientific authorities involved, it is also at odds with the widespread anti-scientific sentiment considered to be on the rise at the global level, variously stigmatized as "the death of expertise" (nichols, ) and the crisis of epistemic deference (marconi, ) . surprising as it may be, there are several reasons to consider this finding on trust as reliable: (i) internal consistency: as discussed in section "results, " all other responses to the survey are consistent with a high attribution of trust to public authorities and indeed provide justification for such attribution. (ii) external validation: just a few days after data collection for this study was concluded, a survey on a representative sample of italian citizens (n = , - march ) was conducted by the independent research center demos & pi , providing substantial support to our main results: e.g., % trust both the italian government and the current prime minister, with % approval of the adopted measures, strong endorsement for the sanitary system ( %), the civil protection ( %), and the national government ( %), coupled with lower levels of confidence in political parties (none of them above % of approval) and a rising skepticism toward the european union ( % of respondents believe the italian response to the covid- emergency to be better than that of other eu countries, and only % consider the role played by the eu as positive in this crisis). (iii) low chances of social desirability effects: as demonstrated by the very low levels of institutional trust recorded in previous surveys, including recent ones, italians have no qualms expressing public distrust toward public authorities-quite the opposite, in fact. thus, there is no reason to assume that the current data on trust are inflated by social desirability effects. thus, there is a genuine phenomenon to be explained here: a veridical "trust boom" during the early stages of the covid- crisis in italy. the socio-cognitive theory of trust (castelfranchi and falcone, ) that inspired our survey provides the tools needed to craft a tentative interpretation of this remarkable fact, although the questionnaire itself was designed to record such a phenomenon, rather than explain it. thus, the speculative nature of our interpretation cannot be stressed enough: our study revealed a highly significant and surprising phenomenon, for which now we look for an explanation. the interpretation we favor is the one that, to the best of our knowledge, appears more adequate to account for the pattern of results obtained in this survey; later on, we will contrast it with other alternative explanations and argue in favor of its superiority. nonetheless, such interpretation remains tentative, and it is intended as a springboard and an inspiration for further studies that may either confirm or falsify it, rather than as something set in stone. with this in mind, let us focus on the fact that trust, at its cognitive core, entails the decision to delegate to someone else (the trustee) the realization of a goal that is important to the agent who is expressing trust (the trustor). as a result, being able to choose not to trust someone requires either having alternative means to achieve the desired goal (e.g., "i will do it myself " or "i will delegate it to someone else") or being ready to forsake that goal. however, neither of these options are available in the face of a pandemic: the relevant goal is personal and public safety, which is non-negotiable, i.e., it is not something we can decide to forget about, and the only course of action that offers reasonable chances of achieving it is to put our collective trust in public authorities, since there are no other available agencies we might appeal to (indeed, the only choice we have concerns the level of public authority we should confide in, and our sample clearly indicated the national level as the most pertinent one). in other words, a pandemic like covid- creates the preconditions for a collective case of necessary trust in public authorities, or institutional trust by force majeure: not in the sense that we are being manipulated by some hidden power, as some conspiracy theorists may be prone to believe, but because the very nature of the health crisis leaves us with no other option than to put our trust in public authorities (that is why we emphasize a need, a necessity for trust). it is worth noting that these pressures toward trust between citizens and public authorities in times of sanitary crisis are symmetrical: citizens have no alternatives to reliance in the relevant public institutions, yet these institutions themselves cannot help but trust in civic compliance to the proposed regulations, on pain of failure in containing the contagion, due to the limits of enforcement already emphasized in previous studies (siegrist and zingg, ; lewnard and lo, ; olsen and hjorth, ; van bavel et al., ) . necessary trust is a two-way street in health emergencies, for both citizens and public authorities. moreover, this two-way street is often cyclically traveled: in fact, the citizens themselves become fully aware (perceive the request and expectation) of the need for public authorities to receive the right degree of trust from citizens as a tool for achieving the common goal, and this awareness becomes one of the reasons for citizens to trust public authorities themselves. in other words, in the best-case scenario, this becomes a trust-based "alliance" toward a supreme common purpose. this civic alliance, or social pact, is grounded in a specific dynamic of trust: the trustor deliberately bestows trust on the trustee, even if partially skeptical of the trustee's qualities, in an attempt of motivating the trustee to "rise to the occasion" and become trustworthy. this is the sense in which trust breeds trust, as noted both by trust theorists (e.g., falcone and castelfranchi, b) and by political economists (e.g., feld and frey, ) . in the context of the early stages of the covid- pandemic in italy, we suggest that italian citizens put their trust in public authorities in charge of facing the crisis as a way of opening up a "trust credit line" and thus putting pressure on such authorities to prove themselves worthy of that credit. similarly, public authorities frequently manifested full trust in citizens' compliance with regulations (a topos often belabored on public occasions by all institutional actors, including the prime minister, the president of the republic, and representatives of the civil protection), precisely for the same reason: by declaring their trust in the common sense and civic responsibility of italian citizens, they were putting pressure on citizens to actually demonstrate such qualities. clearly, the objective need for trust created by a pandemic does not automatically evolve in greater trust toward public institutions. that need may find different outlets, so that other, bleaker outcomes may be equally possible: for instance, an already vulnerable trust relationship between citizens and public authorities may be shattered completely by a sudden crisis, especially if such crisis (or its poor management) are blamed on those authorities, possibly leading to a severe governmental crisis, and maybe even a takeover by authoritarian forces, or, in another scenario, public trust toward central authorities may dissolve, with citizens taking a turn toward tribalism and trying to face the crisis at the local level. while these options are certainly viable in general, our results suggest that neither of these paths was being seriously considered by most italian citizens in early march : our survey revealed a sudden increase of trust toward public institutions, rather than its collapse or further erosion, and that trust was directed toward national authorities, not toward specific charismatic leaders or local powers. according to our findings, faced with an unexpected need for public trust, the italian people in early march opted for putting their trust (at long last) in their elected representatives at the national level, rather than turning to authoritarian figures or local authorities for solutions. beyond the evidence of our data, how the management of the pandemic unfolded over those weeks provides further support to this interpretation. the italian government consistently acted as a mediator between all the social forces affected by the crisis, repeatedly demonstrating high reliance on the indications of the experts in crafting every containment measure: in short, the national authorities acted as the very antithesis of an authoritarian leader. at the same time, local authorities at all levels were relying on the guidance of the national government for facing the pandemic and, in some cases, were actively asking for its direct intervention to solve a crisis that they were not equipped to deal with; more generally, there was widespread consensus, both in political debate and in the media, on the need for a national response to the covid- emergency (a need well understood by our participants, as seen in the section "results"). again, an attitude that stands in sharp contrast with any shift toward tribalism. thus, assuming that the need for public trust prompted the high levels of institutional trust manifested by participants, we propose to interpret their other responses within the broad framework of motivated reasoning (kunda, ) and cognitive dissonance theory (festinger, ) : as the chosen path to pursue the paramount goal of personal and public safety, trusting public authorities became in turn a necessary instrumental goal, thus coloring all other attitudes expressed by the respondents; more precisely, it prompted them to actively look for reasons to justify their (unavoidable) trust in public authorities, in order to minimize cognitive dissonance. indeed, the need for trust experienced by italian citizens during the covid- emergency was at odds with their widespread attitude of distrust toward the very same public authorities they now needed to rely upon in the face of the outbreak: this, we argue, produced a massive and sudden shift in their perception of those public authorities, to better accommodate the new reality they had to deal with. in this interpretation, the trust boom observed in the survey was not produced by any collective epiphany on the actual qualities of the public institutions involved, but rather by a cognitive realignment of individual attributions to the current needs citizens were experiencing. all of a sudden, italian citizens found themselves pressured to rely on some key public authorities in ways and to a degree never experienced before, at least since the worst days of world war ii. regardless of how well these authorities behaved in the first stages of the covid- crisis, italians opted to re-frame their attributional states in a way that made this novel institutional trust justified, thus flipping the usual causal connection involved in acts of trust: it is not a case of detecting the appropriate qualities in public authorities and therefore deciding to trust them, but rather an instance of having first the need to trust those authorities and then justify such trust by assuming that these authorities would manifest the qualities required to warrant that trust. this is also justified and supported by the implicit pact with which public authorities communicated the need for this responsible and trusted attitude toward them as decisive for the achievement of the common purpose. it is worth noting that our reliance on motivated reasoning to explain some of these survey data is very different from the most common use of this notion in recent studies on public opinion: although originally conceived in much broader terms (kunda, ) , motivated reasoning in recent decades has become more and more associated with political ideology, with several studies investigating how partisan affiliations affect and filter our beliefs on matters of public interest (e.g., redlawsk, ; slothuus and de vreese, ; kahan, ; bolsen et al., ) . in fact, the same approach has been applied, with mixed results, to the public reaction to the covid- pandemic, e.g., looking at how political partisanship affected people's ability to discriminate between reliable information and fake news (pennycook et al., ) , timeliness in the adoption of restriction measures (rosenfeld, ) , health behaviors (kushner gadarian et al., ) , and compliance with social distancing guidelines (rothgerber et al., ) and stay-athome regulations (goldstein and wiedemann, ) . while the relevance of politically grounded motivated reasoning provides an interesting perspective on public opinion dynamics, other predictors have been found to be more relevant in explaining some of the target phenomena (e.g., fake news vulnerability, see pennycook and rand, ) ; more to the point, this is not the type of motivated reasoning we are discussing here. on the contrary, our data show no effect of political partisanship on trust attributions toward italian public authorities in charge of coordinating the covid- response, including those that did have a clear political connotation, e.g., the national government. instead, we appeal to the notion of motivated reasoning in relation to a manifestly non-partisan goal, i.e., public safety, and the related need to trust public authorities to be able to ensure such goal: this is the kind of motivated reasoning we argue influenced responses in our sample, independently from the political affiliation of either the survey participants or the relevant public authorities. alongside the preservation of consistency in citizens' beliefs toward public authorities, there is also another, more emotional path through which a need for trust may generate broader shifts in public perception. as noted by many trust theorists (luhmann, ; gambetta, ; batson, ; hardin, ) and also described in the socio-cognitive model adopted here (falcone and castelfranchi, a; castelfranchi and falcone, ) , a fundamental function of trust is to allow both individuals and groups to face uncertainty, to moderate it and deal with it. trusting someone or something immediately reduces the perception of risk; in this sense, trust offers the advantage of a subjective sense of safety, before and without being able to reach that safety objectively. it allows us to face the risk and take it, partially by giving us control over part of that risk, since trusting implies actively choosing to expose ourselves to a risk, i.e., the risk of having our trust betrayed (mayer et al., ) . this is why koller ( ) individuated risk as a key determinant of trust, in the sense that a risky situation may bias people toward trustworthiness when assessing potential allies in facing such risk: "to the degree that the individual fears the occurrence of an event of negative valence (...) he exaggerates the subjective probability of an event of positive valence, which implies that he expects the interaction partner to behave promotively" (koller, , p. ) . this is very much in line with the higher levels of trust we observed in the most vulnerable age groups and in the italian regions most affected by the covid- outbreak (see section "gender effects"). in the context of a health emergency such as the covid- pandemic, this subjective dimension of trust becomes particularly apparent: consider how physicians and nurses in italy turned overnight from marginalized workers in a distrusted field to the most revered national heroes. the individual and collective gain of this sudden change of perception is obvious: faced with the danger of contracting a deadly virus, the belief that your life will be in the hands of trusted professionals is incredibly valuable, not only for the unlucky few that will actually have to rely on those professionals, but for everybody, since it greatly helps in calming down their fear and anxiety. in this perspective, the trust boom recorded in our survey should be considered not only as a merely intellectualistic attitude but also as a response with deep emotional undertones: this is the type of trust that is not only cognitively justified, but also felt, insofar as it provides us with the calmness needed to remain productive under the extreme stress of a pandemic. it is worth noting that emphasizing the motivated nature of institutional trust during a pandemic is not the same as treating this newfound trust in italian public authorities as a fiction, just a desperate figment of the imagination of a population looking for solace from a terrible crisis. nothing could be farther from the truth: precisely because this institutional trust was experienced as a matter of necessity by the italian people, it is also genuinely (and dramatically) authentic. italian citizens, during those terrible days in early march , truly believed that public authorities would prove themselves worthy of their trust-possibly for the first time after many decades of increasing institutional distrust. yet, it is a very fragile belief, because it is massively based on assumptions: should the public authorities subsequently fail to prove themselves equal to the task at hand, this huge "trust credit" would come due, producing an even bigger backlash in terms of the gap between citizens and institutions. this would indicate the clear failure of an "alliance" in which citizens have invested their trust in public authorities. on the other hand, an actual demonstration of trustworthiness by the public authorities during the covid- emergency may engender a more durable and long overdue step change in institutional trust in italy. as the nobel prize joseph stiglitz put it in a recent interview to the italian newspaper la repubblica ( april ), we should "not waste this crisis, " since it opens up genuinely new opportunities for rethinking the fabric of our societies. what is more, respondents in our sample were fairly optimistic on the future of trust relationships with their institutions, with scientists, and among themselves, while expressing reservations on the adequacy of the current economic model (see section "descriptive statistics"). however, optimism is, by its very nature, a delicate thing, so the danger of experiencing a "trust crack" right after the initial trust boom is as real as ever. indeed, other ongoing research on the relationship between institutional trust and public response to the covid- emergency may invite a bleaker outlook on how things will unfold: in their comparison of data from european countries, oksanen et al. ( ) highlighted a negative correlation between institutional trust prior to the crisis and the delay in introducing restrictions to curtail contagion-the less trust was manifested in public authorities before the covid- outbreak, the more time passed after the first confirmed virus-related death and the introduction of containment measures. while we do not dispute the role of institutional trust as a protective factor against virus outbreaks (already well documented with ebola, see blair et al., ; vinck et al., ) , we are skeptical of the particular correlation observed by oksanen et al. ( ) , since it does not take into account the fact that different european countries were affected by the covid- outbreak at different times: in particular, italy, france, and spain [all "late intervention countries, " according to oksanen et al. ( ) ] were among the first countries to record severe outbreaks, and much of the measures later adopted by other countries were largely based on the evidence coming in from these first, unwilling testbeds for the public response to the virus. this is confirmed by the same data used by oksanen et al. ( ) : in terms of absolute dates, italy was among the first countries to endorse all the five types of interventions considered in their study, much earlier than many others that are instead regarded as "early adopters." moreover, the alleged correlation considers only the adoption of some form of interventions, without discriminating between countries that adopted all of them (like italy) or just a few, sometimes even only one (as in the case of sweden). this is probably why subsequent data do not seem to support the proposed correlation: for instance, sweden, one of the countries with one of the highest levels of institutional trust before covid- , as of may , has a very high ratio to the number of deaths per million inhabitants (among the top six nations in the world); similarly, belgium, where containment measures were adopted much more promptly than in italy according to oksanen et al. ( ) , in early may had the world's highest number of covid- confirmed deaths per million inhabitants. for all these reasons, we are not persuaded that prior institutional trust was the main factor determining timely adoption of containment measures by public authorities: while early intervention remains critical in facing virus outbreak, in the case of covid- , we believe that this was determined mostly by other factors, e.g., where the outbreak manifested sooner in europe. looking at the main predictors of trust highlighted by our regression analyses, respondents exhibited a matter-of-fact, evidence-based attributional strategy toward public authorities: consistently with the socio-cognitive model of castelfranchi and falcone ( ) , competence, intentionality, trustworthiness as information sources, and the perceived adequacy of the proposed interventions were the most relevant factors in justifying trust in public authorities. the relevance given to the role of public authorities as information sources is also consistent with the significant weight that information has in shaping participants' institutional trust, based both on their own self-report and on regression analysis (see sections "descriptive statistics" and "regression analyses"): this highlights the importance of feedback and control for trust. even when trust on public authorities is perceived as a necessity by citizens, they try to retain a measure of control over it, by monitoring the quality of institutional information channels. equally suggestive are some of the factors that failed to impact institutional trust in our sample: most notably, the amount of personal sacrifice imposed upon participants by the restrictions introduced by the government. significantly, this dimension did not affect citizens' trust in public authorities, contrary to what would be reasonable to expect under different circumstances: this, in turn, provides further support to our interpretation of the observed trust boom as a matter of necessity-insofar as public safety is the paramount goal, the severity of the necessary costs are immaterial in modulating institutional trust. this provides a nice illustration of the complex and context-dependent nature of feedback mechanisms on trust attributions: whether or not a certain observable feature of the situation (in this case, personal costs) will affect trust depends on its role within a broader attributional process, which cannot be oversimplified as a single feedback loop (for discussion, see falcone and castelfranchi, ) . finally, it is worth stressing that the main predictors of trust remained stable both geographically and temporally: nonetheless, controlling for region of residence allowed us to notice a more focused mindset for trust attribution in the most affected regions, whereas comparing responses before and after the new restrictions introduced in italy on march highlighted a leveling effect of these measures, which made us realize the national character of the covid- crisis to everybody, including citizens living in areas with only minor outbreaks. this last point underscores a common pattern to many of our main results: a shift from the particular to the general in how institutional trust is granted and justified by citizens, apparently caused by the unique circumstances of the covid- pandemic. as we discussed in section "results, " the responsibility of dealing with this emergency was clearly assigned to the national government, whereas regional and local authorities were perceived as marginal; moreover, high confidence was granted to public institutions, largely ignoring their political affiliation, unlike what happened in other countries, e.g., the united states (goldstein and wiedemann, ; kushner gadarian et al., ) , and without concern for any further agenda they might serve (in fact, trust in public authorities was paralleled by distrust in the various political parties, including those currently in power); consistently with this mindset, collectivistic reasons for institutional trust trumped individualistic concerns, and the perception of a common effort toward shared goals overshadowed any personal sacrifice that may be required to individuals and groups (this also relates to the fact that personal health itself obliges to look and reflect primarily on collective health, on which the former strictly depends); finally, confidence in each other's compliance with general rules was high, and the future outlook on trust was positive for public institutions, science, and civic society, not so much for the overall model of development. in short, participants responded to this survey not as individuals calculating trust based on likelihood of personal gains or losses (the standard economic view of trust), but rather as members of a collective subject, jointly engaged in facing a problematic situation. this tendency to make common cause against a shared concern is one of the most valuable assets any society can leverage to fight a public crisis, so in this sense, our data paint a positive picture of how italian citizens responded to the covid- emergency, as far as trust in public authorities is concerned. however, as repeatedly stressed above, this asset is also incredibly delicate, especially in a country with a complex and thorny history of institutional distrust, like italy. hence, a crucial research priority for future research, both in the short run and in the long term, is to keep monitoring how trust dynamics between citizens and public authorities will be affected by the next stages of the covid- pandemic: in fact, while our data suggest a generally positive reaction in the early phases of the emergency, they provide no guarantee of the fact that such trend will continue in the same direction. on the contrary, as mentioned, things could either turn for the best, as our respondents chose to believe, or turn for the worst, should public authorities fail to live up to their citizens' high expectations. all datasets generated for this study are included in the article/supplementary material. this study complied with all the ethical guidelines and standards for online surveys with human participants, in accordance with the local legislation and institutional requirements. the participants provided their written informed consent to participate in this study and were free to quit the survey at any time. rf led the design of the survey. ec, as, and sf performed data analysis. rf and fp wrote most of the manuscript. all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. evaluating covid- public health messaging in italy: self-reported compliance and growing mental health concerns the altruism question: towards a social social-psychological answer homophily and polarization in the age of misinformation public health and public trust: survey evidence from the ebola virus disease 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outbreak, social response, and early economic effects: a global var analysis of cross-country interdependencies date: - - journal: j popul econ doi: . /s - - - sha: doc_id: cord_uid: q dqsl n this paper studies the social and economic responses to the covid- pandemic in a large sample of countries. i stress, in particular, the importance of countries’ interconnections to understand the spread of the virus. i estimate a global var model and exploit a dataset on existing social connections across country borders. i show that social networks help explain not only the spread of the disease but also cross-country spillovers in perceptions about coronavirus risk and in social distancing behavior. in the early phases of the pandemic, perceptions of coronavirus risk in most countries are affected by pandemic shocks originating in italy. later, the usa, spain, and the uk play sizable roles. social distancing responses to domestic and global health shocks are heterogeneous; however, they almost always exhibit delays and sluggish adjustments. unemployment responses vary widely across countries. unemployment is particularly responsive to health shocks in the usa and spain, while unemployment fluctufations are attenuated almost everywhere else. after being identified in december in wuhan, china, the novel coronavirus (sars-cov- ) initially spread in the hubei region and later across mainland china. although the rest of the world soon learned about the first publicly known cases, several countries did not perceive an immediate risk for their populations. starting in january , the epidemic spread outside china, first in thailand, south korea, japan, and in the usa, and in many cases it was connected to recent travelers to the country. in europe, italy reported its first official community-based case on february , and, very quickly, clusters of cases developed in the lombardy region. it was later discovered that the virus had been circulating in lombardy since at least early january (cereda et al. ) and, possibly, since december. by mid-march, the vast majority of countries in the world had multiple cases, with the centers of the outbreak moving first to europe and later to the usa. most countries responded by requiring their populations to adhere to some form of social distancing to reduce the rate of infection and lessen the strain on healthcare providers. responses, however, have been widely heterogeneous. italy reacted with a few-days delay after the outbreak and then implemented restrictive stay-athome policies. a minority of countries initially experimented with laxer restrictions, either based on a misguided attempt to have their populations achieve herd immunity on their own (the uk, which soon moved away from the policy), or because of an unwritten "social contract" with citizens rather than enforcement from policymakers (sweden). others acted quickly and decisively to attempt to eradicate the disease before it became widespread (new zealand). the spread of coronavirus has highlighted the importance of interdependencies across different regions. depending on business links and other existing relationships, the virus rapidly moved across borders. perceptions about the crisis and social behavior responded generally with lags, but they were also likely affected by observed experiences abroad. countries had the opportunity of learning from others about social adjustments that were more or less effective in containing the disease. the main objective of this work is to study these global interrelationships in the early response to covid- shocks. in particular, this paper exploits information about social networks across countries to study interdependencies in the number of disease cases, in the perceptions of their citizens about coronavirus risk, and in their social responses. i also provide some preliminary evidence on the early economic effects of the pandemic by looking at a potential leading indicator of unemployment. i include in my sample countries and use a variety of data sources. to capture the extent of pairwise country social connections, i use data obtained from facebook, which measure the total number of friendships across pairs of countries as a fraction of the total number of combined users in the two countries. this social connectedness indicator allows me to have a measure that can account for different types of relationships: regular friendships, business links, family ties, relations based on older and more recent patterns of immigration, and tourism flows. social networks can help explain the transmission of covid- cases across borders, and they are likely to represent a superior measure compared with the use of geographic distance alone. other contemporaneous papers make a similar observation (e.g., kuchler et al. ) . at the same time, social networks not only can potentially explain patterns of disease contagion, but they can also help account for spillovers in ideas and behavior. controlling for the country-specific dynamics of covid- cases, people's risk perceptions may respond differently and also be affected by the experience and perceptions of individuals in their networks of social connections, including those residing abroad. the same is true for responses in terms of social distancing: individuals who had large connections to countries where the virus outbreak and the social distancing responses were already happening may have learned from their early experiences, taken the epidemic more seriously, and responded similarly. to measure the actual social distancing response in each country, i exploit a novel dataset made available by google through its country-specific social mobility reports. finally, i use internet data from google trends to measure coronavirus risk perceptions and to have a real-time, daily indicator of unemployment. i estimate a global var model to study the transmission of pandemic health shocks both domestically and globally. in my global framework, for each country, covid- cases can affect risk perceptions about the virus, which can trigger a social distancing response. as a result of social distancing or general uncertainty, unemployment may increase. the model allows me to treat all these variables as endogenous. this is necessary since social distancing, for example, is likely implemented in response to rising numbers of covid- cases, but it also itself has an impact on the future number of cases. moreover, domestic variables in the model are also allowed to respond to foreign aggregates. the foreign variables enter each domestic model with weights that depend on the matrix of social connections. the relevant foreign aggregate for each country is different, since the patterns of connections are unique to the country. in the gvar literature, the domestic models can be estimated separately as conditional vars. all endogenous variables can then be stacked together to form a large-scale global var; it is then possible to track the responses of all variables to each shock in each country. through the use of a connectivity matrix (my social connection matrix), the global var model offers a relatively simple and parsimonious way to deal with potentially complex interactions across different variables and countries. main results my estimates highlight the importance of interdependencies and social networks in the transmission of coronavirus cases, in the increase of risk perceptions, and in social distancing behavior. domestic variables, for the vast majority of countries, are significantly affected by foreign aggregates, constructed with weights based on the strength of social connections across countries. for example, as documented in brynildsrud and eldholm ( ) , the first cases in nordic countries (in their case, norway, but likely similar in neighboring countries) were due to travelers returning from vacations in lombardy. to the extent that some of these tourism patterns increase the probability of facebook links as well, which i believe reasonable, my measure will allow me to track likely routes for the spread of the disease. the gvar model has been proposed by pesaran et al. ( ) and is surveyed in chudik and pesaran ( ) . given the role played by italy and the usa as centers of the outbreak in different phases of the epidemic, i study how variables in the rest of the world respond to coronavirus shocks originating in these countries. i document strong and significant responses of risk perceptions and social distancing to the italy covid shock almost everywhere in the world. countries also respond to the subsequent us shock, although with a smaller magnitude. spillovers from spain and the uk also play a sizable role. the countries' responses to foreign and their own domestic coronavirus shocks are heterogeneous. i can, however, reveal some common patterns. the countries that respond with social distancing do so with a delayed and sluggish adjustment. they seem to learn from the experience of other countries, but they display an adaptive behavior: they do not adjust their habits instantly; instead, they gradually reduce their social mobility, which reaches a negative peak almost a week after the shock. in the opposite direction of causality, changes in social distancing lead to a decline in the growth rate of covid- cases. the implications of the pandemic for unemployment also vary significantly by country. labor markets in the usa and spain are the most negatively affected, with large expected increases in unemployment rates. but large spikes in unemployment are not inevitable since most other countries seem to experience much more contained fluctuations. the results suggest that different institutional features can partly insulate the corresponding populations from the worse effects of large exogenous shocks. due to the historical importance of the covid- pandemic, research related to the disease and its effects has been growing swiftly. many papers use the leading model in epidemiology, the sir (or, alternatively, the extended seir) model based on kermack and mckendrick ( ) , to simulate the evolution of the disease (e.g., ferguson n and et al ( ) ). in economics, a number of recent papers have adopted a similar framework and developed the theory further by adding relevant trade-offs between health and economic costs (e.g., eichenbaum et al. ( ) , alvarez et al. ( ) , jones et al. ( ) ). this paper, instead, takes a different route by providing empirical evidence related to the social response to the outbreak, and using an alternative framework. in contrast to studies using the sir model, i do not aim to predict the evolution of the number of infected individuals in a population; my focus lies more on explaining the social responses to the original health shocks around the world. other recent works investigate the determinants of different approaches to social distancing. gupta and et al. ( ) find that social distancing responses do not necessarily correspond to policies mandated by state and local governments. painter and qiu ( ) and adolph et al. ( ) find that political beliefs affect compliance with social distancing indications in the usa. andersen ( ) finds evidence of substantial voluntary social distancing, and he also shows that it is affected by partisanship and media exposure. in light of these results, my approach does not use data on mandates, but it exploits, instead, the actual decline in mobility, as measured using location tracking technologies. qiu et al. ( ) focus on the early months of the pandemic. they provide empirical evidence on the transmission of coronavirus cases across cities in china between january and february. they estimate how the number of new daily cases in a city is affected by the number of cases that occurred in nearby cities and in wuhan, over the previous weeks. they show that social distancing measures reduced the spread of the virus, whereas population flows out of wuhan increased the risk of transmission. my paper stresses the importance of modeling cross-country interrelationships to understand the evolution of the next phase of the pandemic. a recent work by zimmermann et al. ( ) shares a similar goal. they find that countries that are more globalized are affected by the pandemic earlier and to a larger extent. therefore, they discuss how early measures that temporarily reduce inter-country mobility would be beneficial. outside of the recent covid- literature, my paper also provides a contribution to the literature on gvar models (see chudik and pesaran , for a survey) . most papers in the literature consider macroeconomic applications and study the global spillovers of policy and other shocks (e.g., pesaran et al. ; chudik and fratzscher ; dees et al. ) . others have studied interdependencies in housing markets (holly et al. ) , firm-level returns (smith and yamagata ) , and a variety of other applications (di mauro and pesaran ; pesaran et al. ). the effect of foreign variables is usually assumed to depend on trade balances across countries. my framework, instead, introduces a different connectivity matrix, based on social networks, which can be promising for a different set of applications. therefore, my paper is also connected to recent papers that propose the use of facebook connections to measure social networks across locations (bailey et al. ) . finally, i measure risk perceptions and fears of unemployment using google trends data. this approach has become more and more popular and is now exploited in different fields to measure people's attention (da et al. ) , in forecasting and nowcasting economic variables (see the various examples discussed in choi and varian ) , and to track the spread of diseases (e.g., ginsberg et al. ; brownstein et al. ) in the absence of easily observable private information. askitas and zimmermann ( ) discuss how internet data can be useful for empirical research in a variety of social science applications and, in particular, for research about human resource issues (askitas and zimmermann ( ) and simionescu and zimmermann ( ) provide evidence directly related to the unemployment rate). the paper exploits a variety of newly available datasets to study the interrelationship between health shocks originating from the covid- pandemic, people's real-time perceptions about coronavirus risk, the extent of their social distancing response, and unemployment. i investigate the connections among these variables both within countries, and across borders, by studying contagion and spillovers internationally. the data are collected on a sample of countries. those include current oecd member countries, candidate countries that applied for membership, and the countries that the oecd defines as key partners (brazil, india, indonesia, south africa). the countries account for % of global gdp (besides china, the main omission is russia, which accounts for about %) and % of global population; they also account for % of coronavirus cases in the sample period. data on novel covid- cases each day for each country are made available by johns hopkins university's center for systems science and engineering (csse). the estimations use either the growth rate or, as a robustness check, the number of daily cases. the epidemiology literature stresses the importance of social distancing to contain the spread of the virus, by reducing the basic reproduction number r (the expected number of secondary infections produced by a single infection in a population where everybody is susceptible) and flattening the curve of infected individuals. the response has been different across countries, either in terms of policies, enforcement, or voluntary reductions in mobility. therefore, it is important to have accurate data on actual social distancing by different populations to track the implied health and economic effects. to this scope, i use daily time series indicators on social mobility made available by google. the indicators are obtained using aggregated, anonymized data from gps tracking of mobile devices, for users who opted in to "google location history." the data measure the change in the number of visits and length of stay at different places compared with a baseline. for each day of the week, mobility numbers are compared with an historical baseline value, given by the median value for the corresponding day of the week, calculated during the -week period between january and february , . the data are reported for five place categories: grocery and pharmacies, parks and beaches, transit stations, retail and recreation, and residential. in addition to the official number of covid- cases, which may be an imperfect measure of the pervasiveness of the virus in the population, i also measure the population's risk perception about coronavirus. the risk perception is measured using daily data on web searches from google trends. i use the search results for the whole "topic" category; therefore, the indicator also includes all related search terms, such as "coronavirus symptoms," "coronavirus treatment," "coronavirus vs. flu," and so forth. finally, i similarly use an indicator of unemployment to measure the initial economic effects of the outbreak. given that actual unemployment data are typically available only at monthly frequency and that their release is lagged by more than a the full list of countries is as follows: australia, austria, belgium, brazil, canada, chile, colombia, costa rica, czech republic, denmark, estonia, finland, france, germany, greece, hungary, india, indonesia, ireland, israel, italy, japan, south korea, latvia, lithuania, luxembourg, mexico, netherlands, new zealand, norway, poland, portugal, slovak republic, slovenia, south africa, spain, sweden, switzerland, turkey, uk, the usa. the only country that has been removed from the oecd list is iceland, since google mobility data were not available. for non-oecd key partners, i exclude china, since for my sample the numbers of cases had already declined (google mobility data would also be unavailable for the country). google llc "google covid- community mobility reports." https://www.google.com/covid / mobility/ month, i also exploit google trends data about unemployment as a variable that can be used to have early and real-time indications of the official variable. as before, i use google searches about the unemployment topic (again, including all searches related to unemployment, such as "unemployment benefits," "unemployment insurance," "how to apply for unemployment," "losing my job," and so forth). askitas and zimmermann ( ) and choi and varian ( ) , among others, show that unemployment searches can help predict initial unemployment claims and the unemployment rate. more recently, askitas and zimmermann ( ) and simionescu and zimmermann ( ) document how internet data can be useful for nowcasting and forecasting the unemployment rate in a diverse set of countries. my unemployment variable can, therefore, be interpreted as a real-time signal for unemployment, or, alternatively, as a measure of people's perceptions, attention, or fears, about unemployment over the time period that i study. finally, i measure international social connections using facebook's social connectedness data. the index uses active facebook users and their friendship networks to measure the intensity of connectedness between each pair of locations. the measure of social connectedness between two locations i and j is given by: where fb connections i,j denotes the number of friendship connections between region i and j , and fb users i , fb users j denote the number of facebook users in i and j . the social connectedness index, therefore, measures the relative probability of a facebook connection between any individual in location i and any individual in location j . the data used in this paper refer to the measure calculated for march . bailey et al. ( ) proposed the measure to study the effects of social networks across us counties. other current papers are uncovering the link between social networks and the diffusion of covid- (e.g., kuchler et al. ) . the measure can be preferred to alternatives based simply on inverse geographic distance, since it can provide a more accurate account of business relations, tourism patterns, and family or friendship ties, across different areas. i argue here that the strength of social connections can also affect information about the outbreak and social distancing responses. as bailey et al. ( ) show, facebook friendship links between the usa and other countries, for example, are strongly correlated both with bilateral migration patterns and trade flows. they regress social connectedness on geographic distance, the number of residents with ancestry in the foreign country (as an indicator of past migration), and on the number of residents born in the foreign country (indicating current migration), and show that all three are strongly significant. friendship connections also lead to statistically significant increases in both exports and imports between the usa and the foreign country. for italy, the strongest social connections are with switzerland and slovenia, followed by austria, germany, spain, belgium, and the uk. distance is clearly a determinant of social networks, but not the only determinant. social connections are stronger between italy and australia, italy and the usa, and italy and canada, than between italy and turkey, although the latter is geographically much closer. for the usa, as expected, the most socially connected countries are mexico and canada, followed, at lower levels, by ireland and israel. the usa have strong connections with australia and new zealand, which would be downplayed based on a pure measure of distance. figure shows, instead, the social distancing response across a sample of major countries in the sample (for easiness of exposition, i show the experiences of out of countries in the figure). mobility declined by % or more in italy, france, spain, and new zealand. while in some countries, the adjustment was abrupt (e.g., new zealand, france, spain), it was slower and more gradual in others, such as the uk (where the response starts a few days later) and the usa; their overall declines in mobility were also more modest. sweden is an outlier in europe, as it maintained only small fluctuations of mobility around the historical mean. japan and korea observed their first cases earlier; therefore, their social distancing responses during this period appear more limited. in many european countries and in the usa, mobility returns to its historical average by the beginning of june. to model global interdependencies in the spread of the disease and countries' responses, i follow the gvar approach proposed in pesaran et al. ( ) and surveyed in chudik and pesaran ( ) . assume that there are n units, representing countries in this case, and for each unit, the dynamics is captured by k i domestic variables. for each country i, the k i × vector x i,t of endogenous variables includes four domestic series: the growth rate of covid- cases, the risk perception about covid- , the change in social mobility, and the perception about unemployment. the vector of domestic variables is modeled as: for i = , , ..., n, and where il , i , and il denote matrices of coefficients of size k i × k i and k i × k * i , where k * i denotes the number of "foreign" variables included in the vector x * i,t , and ε i,t is a k i × vector of error terms. in the empirical analysis, i select the optimal number of lags p i and q i for each country using schwartz's bayesian information criterion (bic). for each country i, therefore, domestic variables are a function of their p i lagged values, possibly of the contemporaneous values and q i lagged values of foreign, or global, variables. the foreign variables x * i,t are k * i × cross-section averages of foreign variables and they are country-specific: the matrixw i has size k × k * i and contains countryspecific weights, with diagonal elements w ii = . my approach uses the extent of social connections across country borders from the facebook social connectedness index dataset to measure the weights. gvar models assume that the variables x * i,t are weakly exogenous. this corresponds to the popular assumption in open-economy macro models that the domestic country is treated as "small" in relation to the world economy, i.e, it does not affect global variables. this assumption can be easily tested for all the variables. for cases in which a domestic variable has an unduly large effect on global variables, weak exogeneity will not be invoked there and the foreign variable, instead, will not be included in that var. the estimation works in two steps. first, varx* (that is, vars with weakly exogenous foreign variables) models can be estimated for each country separately. since i study the social responses to the covid- outbreak, i include in the domestic var also the risk perception variable, in addition to the number of cases. i believe that changes in the number of confirmed coronavirus cases may lead to different risk perceptions in the different countries, which, in turn, can affect people's willingness to adhere to stay-at-home orders or to voluntarily engage in social distancing. as a measure of economic consequences, i choose an indicator of unemployment. other options that are available at daily frequencies include stock returns, interest rates, and electricity data. stock returns and interest rates are inferior indicators of economic activity in this period as they were largely influenced by government and central banks' emergency interventions. electricity data would be appropriate, but they have been made available only for a small selection of european countries (mcwilliams and zachmann ). in the analysis, the number k * i is also equal to , as the vector x * i,t contains the country-specific global counterparts for the same variables in x i,t , i.e., the growth rate of covid- cases, coronavirus risk perceptions, social mobility, and unemployment. second, the estimated country models are stacked to form a large gvar system, which can be solved simultaneously. domestic and foreign variables are stacked in the the model in eq. can be rewritten as: where gives: substituting into eq. and stacking all the unit-specific models yield: where with g invertible, as it is in this case, the gvar is given by: with f l = g − g l . the gvar solution can be used to trace the impact of shocks on the variables of interest, both domestically and globally. to find the impulse response to shocks, i adopt the generalized impulse response function (girf) approach, proposed by koop et al. ( ) , pesaran and shin ( ) , and also used in pesaran et al. ( ) . the vector of girfs is given by: where j indexes the different shocks, h denotes the horizon for the impulse response function, i t = x t , x t− , ... denotes the available information set at time t, and where √ σ jj indicates that the magnitude of the shock is set at one standard deviation of the corresponding ε j,t . the gvar specification can be seen in relation to a number of econometric alternatives: spatial vars, panel vars, and dynamic factor models. spatial vars are very strongly connected. they also assume a connectivity matrix, which is usually based on geographic distance. the main difference between the two approaches lies with the structure of correlations: as discussed at length in elhorst et al. ( ) , spatial vars may be preferred when correlations across units are extremely sparse, for example, when a unit is only affected by few bordering units ("weak," or local, crosssectional dependence). the gvar is meant to capture stronger interrelationships, with dense connectivity matrices, where each country unit is affected, in different ways, by several other countries, or by an aggregate measure ("strong" crosssectional dependence). spatial vars can also be seen as a particularly restricted case of a gvar model. the approach can similarly be seen as a particular form of panel var. the main advantage here is that, through the weight matrix w i , this approach exploits knowledge about social networks and uses that knowledge to inform the magnitude of cross-country interdependencies. panel vars often impose the same coefficients for each unit, shutting down static and dynamic heterogeneity, as well as neglecting cross-country interdependencies. an exception is provided by canova and ciccarelli ( ) : they introduce a factor structure in the coefficients to solve the curse of dimensionality. their approach is particularly useful when there is no a priori knowledge that can be exploited about the spillovers. in this case, the extent of social networks can be, instead, exploited to provide some information about the relative strength of interdependencies. finally, the gvar has relations with dynamic factor models. as chudik and pesaran ( ) show, the gvar specification approximates a common factor across units, and it extracts common factors using structural knowledge. the model is particularly suited to account for potentially complex patterns of interdependencies across countries. at the same time, the gvar specification does so while maintaining simplicity and parsimony. the dimensionality issue is resolved by decomposing a large-scale var into a number of smaller scale vars for each unit, which can be estimated separately, conditional on the dynamics of weakly exogenous foreign variables. the interdependencies are not left entirely unrestricted, since it would be unfeasible to estimate all the parameters, but they are given a structure based on knowledge of the data. in the benchmark analysis, i estimate the gvar model using daily data from february to april , . the dates are chosen based on availability of google social mobility data at the time the paper was written. the exogeneity assumption is relaxed where it appears unlikely: for covid cases, i do not include foreign variables in the model for the usa, italy, and spain, since they may be endogenous. those countries, at different times, have accounted for a large share of global cases. i allow the covid variable for all other countries to be affected by foreign series. i also allow risk perceptions in each country, as well as social distancing outcomes, to be affected even contemporaneously by corresponding variables in different countries. finally, i assume that domestic unemployment perceptions are affected by foreign unemployment perceptions, but not within the same day. this assumption is not important for the results (which are robust), but it is motivated by the idea that the unemployment data are driven more by country-specific, than across-the-border, factors. i test the weak exogeneity assumptions for all foreign variables, and they are never rejected in the data. recently, some studies have emphasized the importance of superspreaders in the transmission of the virus (e.g., adam et al. , who study clusters in hong kong). beldomenico ( ) discusses how sars-cov- appears to start by spreading gradually in a region, until transmission is triggered by a possible cascade of superspreader events, and cases explode. as a result, the pattern of transmission can become highly heterogeneous. here, i focus on numbers of cases aggregated at the country level. my framework can account for heterogeneous responses across countries. however, even if the weak-exogeneity tests suggest that domestic countries do not affect global variables in a statistical sense, it is conceivable that, with superspreaders, covid infections can transmit very quickly, and do so even between country pairs with a limited degree of social connections. my identification assumption, however, requires that the impact of a superspreader from country i on the total number of global cases remains small enough. first, to study the magnitude of global interdependencies, table shows the contemporaneous effects of foreign variables on domestic variables, for each country. the table reports the estimated coefficients, alongside the associated standard errors. domestic variables are significantly affected by the country-specific foreign aggregates, computed using the matrix of social connections as country-by-country weights. the results indicate that the international spread of covid- cases can be, in part, explained by existing social networks across country borders. moreover, the contagion not only relates to the number of cases and the spread of the disease, but it also affects the spread of perceptions and social behavior. both the measure of risk perceptions about coronavirus and the social distancing responses are significantly influenced by developments in the rest of the world. only few countries do not show a statistically significant response to global conditions. risk perceptions do not rise in response to increasing international distress only in brazil, south africa, and turkey. it is likely that their populations initially underestimated the likelihood of the pandemic reaching them, as they were farther from the epicenters. most countries also gradually learn from each others' social distancing responses. among the few exceptions, japan and korea are not significantly affected by foreign experiences: they implemented social distancing earlier than other countries, but they already relaxed many of the restrictions before the period that i consider. these results highlight the importance of considering global interrelationships and social connections in understanding the transmission of the virus and societal responses. my results add to those in zimmermann et al. ( ) , who investigate the role of globalization during the pandemic. countries with a higher index of globalization had faster transmission speed and higher infection rates, although they responded better to the challenges by achieving lower fatality rates. international travel and migration play key roles in the transmission. their paper, therefore, discusses the benefits of inter-country distancing, based on the imposition of temporary travel restrictions. my empirical results point to similar policy implications: since the table reports the estimated gvar coefficients with the associated standard error shown below in parentheses. significance at the % level is denoted by ***, at the % level by **, and at the % level by * coronavirus cases spread internationally as a result of existing social networks, early border closures and travel restrictions can be effective. i study the global responses to shocks from italy and the usa since these countries played outsized roles in different phases of the pandemic. figures and show the impulse response functions for all countries in the sample for the risk perception and social distancing variables to a one-standard-deviation covid shock originating in italy. risk perceptions increase, with some sluggish impulse responses across countries of coronavirus risk perception to a covid- growth rate shock originating in italy adjustment, almost everywhere in the world in response to the initial shock from italy. the responses typically reach their peak about - days after the original shock. the response is more delayed in brazil, india, and south africa. as seen in the previous section, these countries are less influenced by global variables in this period. populations in neighboring european countries, as well as in the usa, australia, and canada, instead significantly update their perceptions. the overall effect is much smaller in sweden, finland, turkey, israel, and lithuania. again, japan and south korea do not seem to significantly respond, as they experienced their outbreaks earlier than the rest of countries. similarly, most countries respond with reductions in social mobility. the social distancing response, however, is already delayed and sluggish in italy, with a negative impulse responses across countries of social mobility to a covid- growth rate shock originating in italy peak in mobility occurring days after the shock. other european countries, such as france, switzerland, and the uk, do not seem to adjust at all for the initial - days, after which they gradually reduce their social mobility as well. the patterns are similar everywhere: after the situations worsen in one country, the others do not immediately learn from its experience and change their behavior. instead, they appear to behave more adaptively, by only gradually altering their habits in response to the evolving situation. one issue to consider is whether the joint declines in social mobility are driven by policies that happened at the same time. my measure of actual mobility captures both the effects of mandates and those of voluntary distancing. i use data on the government response index made available for different countries through the oxford covid- government response tracker (oxcgrt)'s website and discussed in thomas hale et al. ( ) . i regress the google mobility indicator on a constant and on the government response index for each country. figure shows the estimated coefficients for the sensitivity of mobility to the government response, and the resulting r for each country's regression. the results clarify that measures based on actual mobility carry additional information that goes beyond what can be captured by looking only at the implemented policies. for many countries, mobility responds negatively to policy restrictions, with r coefficients falling in the . - . range. the explanatory power is particularly strong in mexico and new zealand. but simply using policy responses would miss the extent of social responses in many other countries, where the explanatory power is closer to (as in korea, netherlands, and scandinavian and baltic countries). the focal point of the pandemic later moved to the usa, at least starting from mid-march. figure displays the effects on coronavirus risk perceptions in the rest of the world to a us coronavirus risk shock. i consider the risk perception shock for the usa, rather than the one based on the number of cases, since testing was initially fig. relation between voluntary social distancing and government lockdown policies. the results are based on the regression social mobility t = β + β govt. response t + ε t for each country. the top panel shows the estimated coefficient β , the second the regression r the spillovers in risk perceptions are again statistically significant, but much smaller in magnitude than those observed in response to the corresponding italian shock. the same is true for responses of social mobility to a us coronavirus risk shock, shown in fig. . for many countries, i observe a slight increase in social distancing, including for the usa themselves. in terms of policy implications, the results highlight the importance of rapid interchanges of information: the rest of the world can learn from policies and behaviors that seemed to work in the countries that were reached early by the virus. the results show that perceptions about the pandemic spread to different countries. the resulting the responses to the pandemic have been heterogeneous across countries. figure overlaps, for a selection of countries, the impulse responses of social distancing and unemployment to the country's own coronavirus risk shock. i single out the responses for italy, spain, the uk, the usa, sweden, and japan, since they characterize somewhat different approaches to the crisis. the populations of italy and spain sharply decreased their social mobility after the domestic coronavirus shock. the responses reach their maximum effects after - days, and they last for weeks. their behavior suggests that even in the countries that were most affected by the virus, their social distancing responses, while substantial, have been unnecessarily delayed. japan displays a smaller, and more sluggish, response. the usa and the uk are also characterized by negative and statistically significant adjustments in mobility, but their responses are many order of magnitudes smaller than the ones observed in italy and spain. finally, it is well documented that sweden adopted a more permissive approach, by letting its citizens adjust their ita spa uk usa swe jap fig. impulse response functions of coronavirus risk perceptions and unemployment to the country's own coronavirus risk shock behavior without the same strict enforcement that was observed in other countries. the response for sweden, accordingly, does not show any significant decrease in mobility to the country-specific risk shock. turning to the early estimates about potential economic effects, i show the responses of the real-time unemployment indicator to each country-specific coronavirus shock. the figure shows that unemployment does not necessarily need to skyrocket in response to health shocks. unemployment insurance claims have reached record levels in the weeks after the outbreak in the usa. the impulse responses are consistent with the behavior of unemployment claims, revealing an extremely large response of the google unemployment indicator. unemployment is also set to considerably increase in spain. the country has a large share of workers on temporary contracts, who are more likely to become unemployed due to the uncertainty generated by the pandemic. other countries in the sample, however, as well as the vast majority of countries not shown in the figure, appear more successful in insulating their labor forces from the crisis. even if the recessionary effects on output are likely to be large almost everywhere, for most countries, early indicators of unemployment suggest that local labor markets are not going to experience the same turbulence as those in the usa. so far, the analysis has focused on the direction of causality that goes from covid cases to social and economic responses. here, i provide evidence on the opposite direction: the effects of social distancing on new covid cases. figure shows the impulse responses of the growth rate of covid- cases in different countries to a social distancing shock, measured as a one-standard-deviation decline in social mobility. social distancing leads to declines in the growth rate of coronavirus cases in the days after the shock. the only country in the figure that does not show a negative response is the uk, for which social distancing has, in fact, been much slower to start. the results reaffirm the importance of social distancing, whether through mandatory policy or voluntary behavior, in reducing the spread of the virus. while in epidemiology, the benefits of social distancing are usually modeled as changes in the parameters of a sir model, here i show that the effects can be uncovered also in a simpler linear framework. moreover, the results regarding unemployment, presented in the previous section, suggest that social distancing does not necessarily have to translate into high unemployment rates. a prompt social distancing response, coupled with labor institutions that attenuate the impact of business cycles, can successfully limit the health shocks from the pandemic, without causing extensive economic damage. the empirical analysis, so far, has been based on data up to mid-april. i now update the dataset to include the most recent months. after april, the social distancing efforts were successful in most of europe: the number of daily cases in italy, spain, germany, france, and most neighboring countries, declined; as a result, the countries started to relax most restrictions on mobility. the global centers of the virus moved instead to the americas, with us cases still remaining high, and with brazil's situation rapidly deteriorating. the situation also worsened considerably in india. to incorporate data for this second phase, i re-estimate the gvar model for the more recent sample between april and june (the last day of availability of google mobility data at the time of writing). the results are reported in tables and . table shows the values of the peak responses for the impulse response functions of coronavirus risk perceptions in each country in the sample to corresponding coronavirus risk shocks from seven countries: italy, the usa, spain, uk, brazil, chile, and india. these countries are selected as they had large number of cases at different times, during the sample. table reports similar information (in this case, the size of the largest negative responses across horizons) for the social distancing responses, instead. to compare the role played by the different countries, i show the results for both the first phase, starting in mid-february and ending in mid-april, and for the second phase, from mid-april to mid-june. most countries were significantly affected by italy's shocks during the first phase. risk perceptions particularly increased in spain, the uk, and the usa. higher risk perceptions led to a much larger decline in social mobility in spain (− . ), though, than in the other two countries (− . and − . , respectively). in the second phase, italy's role diminished, and many countries reacted instead to shocks from the usa, spain, and the uk. although cases exploded in brazil, chile, and india, between april and june, the spillovers from these countries to the rest of the world have remained more limited. the largest effects may be detected in neighboring countries: for example, the largest increase in risk perceptions in response to shocks in brazil and chile is observed in colombia. the effects on social mobility are somewhat larger, but far from the values obtained in response to shocks from spain and the uk, for example. the results suggest that, in most countries, public perceptions and behavior respond to global, not only to domestic, variables. the impact of individual countries, instead, varies over different phases of the pandemic and depending on the extent of social connections. overall, this paper's results highlight the importance of interconnections to understand not only the spread of the virus, but also adaptation and gradual learning in importing ideas and behavior from other countries. risk perceptions and the willingness to engage in social distancing by the populations of most countries significantly respond to the corresponding variables in socially connected countries. i stress the role of existing social networks across borders in the transmission of health shocks, perceptions about the risk of the disease, and ideas regarding the merit of social distancing. the results reveal heterogeneous responses across countries to their own domestic coronavirus shocks. a common feature in all responses is that individuals responded with a lag and only gradually reduced their social mobility. this observation is consistent with epidemiological models that include adaptive human behavior, such as the model presented in fenichel and et al ( ) . that research stresses the role of public policies based on informing and motivating people to reduce person-to-person contacts. this may be particularly important for countries in which citizens have weaker social connections to the rest of the world, and in which, policymakers may delay in implementing mitigation policies. institutional differences among the countries' labor markets are likely responsible for substantially different increases in unemployment. the lower degree of employee protections in the usa and the large share of temporary workers in the spanish economy are likely to account for the far worse outcomes in these countries. everywhere else, fluctuations in unemployment have remained more subdued. there are some possible limitations related to the data series used in the analysis. unemployment indicators based on internet data may be more or less accurate depending on the country: as discussed in simionescu and zimmermann ( ) , their predictive power for actual unemployment may depend on the internet penetration in the country, and on demographic variables, such as the age composition of internet users. internet use may also vary across the income distribution, particularly in less economically developed countries. perceptions about coronavirus risk may not be captured equally well in all countries in the sample. the matrix of social connections based on facebook friendships may be subject to similar problems: facebook users may have different average income and age than the population as a whole, and such friendships may capture to a larger extent personal, rather than business, links. my sample of countries necessarily excludes others (for example, china), which may be important in terms of social connections. their omission may potentially lead to an omitted variable bias in the var regressions. this section assesses the sensitivity of our estimates to alternative data and econometric choices. the benchmark estimation used data on covid- cases transformed into daily growth rates. i can examine the sensitivity of the results to using the number of new daily cases instead. table reports the estimated interdependencies corresponding to those previously shown in table . to save space, the results are shown for a subset of six countries. the estimates remain similar, with the exception of a smaller spillover of global risk into the domestic italian risk perception variable. also, in the benchmark estimation, the conditional country-specific models corresponded to vars with the addition of weighted foreign aggregates. another option sensitivity check i) repeats the estimation using the level of new daily covid- cases rather than their growth rate; case ii) estimates conditional vector-error-correction models rather than a var for each country; case iii) computes changes in social mobility excluding the series related to residential mobility often used in the gvar literature is to allow for long-run relationships and estimate vector error correction (vecm) models instead. the results shown in table , as well as all the main findings, remain in line with those discussed so far. finally, the google mobility indicator was computed by taking the average of mobility changes across all available categories. it can be argued that the relevant social distancing measure that matters for health outcomes should exclude residential mobility. therefore, i repeat the analysis by constructing social mobility, but now excluding the residential component. again, the results remain substantially unchanged. i estimated a global model of countries to examine the interconnections in coronavirus cases and in social and economic responses during the first months of the covid- pandemic. the results suggest that social connections across borders are helpful to understand not only the spread of the disease, but also the spread in perceptions and social behavior across countries. initial shocks from italy affected risk perceptions about coronavirus in most countries in the world. many of them responded by significantly reducing their mobility. populations in most countries, however, displayed a degree of behavioral adaption: they did not change their habits instantly, but only gradually over time. shocks from the usa, spain, and the uk also had significant effects later on. a subset of countries did not respond much through social distancing to global or domestic shocks. as a result, they do not show the same reduction in the growth rate of covid- cases in response to social distancing that is observed in other countries. the original health shocks, either directly, or through increased uncertainty and social distancing, have economic effects. while i do not have data at high frequency on realizations of the unemployment rate, i exploit daily data on an indicator that has been shown to predict actual unemployment quite accurately: unemployment from google searches. the response of unemployment across countries is very heterogeneous. in the usa, unemployment skyrockets. this is consistent with the response of initial unemployment claims in the country. the same happens in spain, with a large increase of unemployment in response to health shocks. in other countries, the responses are more muted, as public programs intervened to provide subsidies to employers and employees to protect existing employment relationships. clustering and superspreading potential of severe acute respiratory syndrome coronavirus (sars-cov- ) infections in hong kong, preprint pandemic politics: timing state-level social distancing responses to covid- . medrxiv, . . early evidence on social distancing in response to covid- in the united states google econometrics and unemployment forecasting the internet as a data source for advancement in social sciences simple planning problem for covid- lockdown, nber working papers social connectedness: measurement, determinants, and effects do superspreaders generate new superspreaders? a hypothesis to explain the propagation pattern of covid- digital disease detection-harnessing the web for public health surveillance high covid- incidence among norwegian travellers returned from lombardy: implications for travel restrictions estimating multicountry var models the early phase of the covid- outbreak in lombardy, italy predicting initial claims for unemployment insurance using google trends predicting the present with google trends identifying the global transmission of the - nancial crisis in a gvar model infinite dimensional vars and factor models theory and practice of gvar modelling search of attention exploring the international linkages of the euro area: a global var analysis the gvar handbook: structure and applications of a macro model of the global economy for policy analysis spillovers in space and time: where spatial econometrics and global var models meet impact of non-pharmaceutical interventions (npis) to reduce covid mortality and healthcare demand. mar fenichel ep et al ( ) adaptive human behavior in epidemiological models detecting influenza epidemics using search engine query data tracking public and private responses to the covid- epidemic: evidence from state and local government actions annalena pott ( ) variation in us states responses to covid- the spatial and temporal diffusion of house prices in the uk optimal mitigation policies in a pandemic: social distancing and working from home nber working papers contribution to the mathematical theory of epidemics impulse response analysis in nonlinear multivariate models the geographic spread of covid- correlates with structure of social networks as measured by facebook bruegel electricity tracker of covid- lockdown effects, bruegel datasets political beliefs affect compliance with covid social distancing orders forecasting economic and financial variables with global vars modelling regional interdependencies using a global error-correcting macroeconometric model generalised impulse response analysis in linear multivariate models impacts of social and economic factors on the transmission of coronavirus disease (covid- ) in china big data and unemployment analysis, glo discussion paper series , global labor organization (glo) firm-level return-volatility analysis using dynamic panels inter-country distancing, globalisation and the coronavirus pandemic acknowledgments i would like to thank the editor, klaus f. zimmermann, and three anonymous referees for comments and suggestions that substantially improved the paper. i outline here the steps for the estimation of the gvar model (see also smith and galesi ): . first, the connectivity matrix w (of size × in this case) is constructed using facebook's social connectedness index data. for each country i, i fix w i,i = (the domestic country is not used for the construction of the foreign variable) and i calculate the weights w i,j , i = j , as the social connectedness between countries i and j as a fraction of the sum of connectedness between country i and each country in the sample, sci i,j / n j = sci i,j . therefore, the resulting connectivity matrix has columns that sum to . . country-specific foreign variables are then constructed as x * i,t = n j = w i,j x j,t , using the weights w i,j , for each reference country i. . i estimate conditional varx* (that is, a var with a foreign, weakly exogenous, variable) models, as specified in expression eq. . the models can be estimated separately for each country by ols. i choose lag length also separately for each of them based on schwartz's bayesian information criterion (bic). in most cases, the data select short lag lengths (p and q equal to or ) as optimal. i did not find consistent patterns of seasonality in the data. therefore, we do not perform any seasonal adjustment before the estimation. the benchmark estimation considers varx* models. the robustness section experiments with vecmx* specifications, which allow for cointegrating relationships both within the variables in x i,t and between variables x i,t and x * i,t . in that case, for each domestic vecmx*, the cointegration rank is selected based on johansen's trace statistics. . after being estimated independently, the domestic vars are stacked together as shown in eq. . the global var is "solved" for all the k = n i= k i endogenous variables, as shown in ( )-( ). . i check the moduli for the system eigenvalues and confirm that they are all within the unit circle. . i compute generalized impulse response functions following koop et al. ( ) , as shown in expression ( ) as. the response to a one standard-deviation shock is given, for each, where e j is a selection vector, composed of zeros, except for an element equal to to select the shock of choice. the matrix r h is the matrix of coefficients in the gvar's moving average representation: x t = ε t + r ε t− + r ε t− + .... i use bootstrapping to compute the impulse response error bands. key: cord- -fio cjj authors: nan title: peripheral nerve society meeting july – , sitges, barcelona, spain date: - - journal: j peripher nerv syst doi: . /jns. sha: doc_id: cord_uid: fio cjj nan the peripheral nerve society was founded in from two groups of academic investigators, peripheral nerve study group and peripheral neuropathy association of america, interested in the basic biology and function of the peripheral nervous system and its application to the clinic. their invite only biennial meetings involved - attendees in cloistered settings organized by shoestring and local initiative. from this, we have grown remarkably. we now have an annual meeting of over people including meetings within the meeting for the special interest groups in inflammatory, diabetic and hereditary neuropathy. with this substantial growth and the success of jpns, the journal of the peripheral nervous system the society continues to flourish. has proven to be a year full of exciting changes for the society. pns has transitioned from a biannual, to an annual meeting. next year, the meeting will be taking place at the renaissance baltimore harborplace hotel from - july in baltimore, maryland. the development of a new website has been completed, please visit www.pnsociety.com to see the new face of the society. finally, pns has adopted new executive staff. with their guidance and the leadership of an active and diverse board of prominent professionals in the field the peripheral nerve society continues to grow and anticipates more exciting changes in the year to come. the peripheral nerve society provides annual meetings, teaching courses, guidelines, and other resources to aid in the education of members. becoming a member of pns means collaborating with prominent global professionals in the field to develop and provide the best treatments for people with peripheral nerve diseases and setting standards of care within the field. please participate in our future by joining the pns, volunteering for a project aligned with your interests and sending your ideas for the future to the executive office, or board member. peles e . department of molecular cell biology, rehovot, israel. two schwann cell-dependent mechanisms control the presence of na + channels at the nodes of ranvier: i. clustering of the nodal complex by glia-derived proteins and ii. restriction of nodal proteins within the nodal gap by the paranodal junctions. these mechanisms depend on specific cell adhesion molecules that mediate the contact between myelinating glia and their underlying axons at the forming nodes and the paranodal junction. during myelination, na + channels initially clustered at heminodes that border each myelin segment. this process requires gliomedin, nrcam and neurofascin (nf ), three cell adhesion molecules (cams) that mediate the interaction between schwann cell microvilli and the axon. na + channels clustering activity of gliomedin is tightly regulated by two distinct and functionally opposing proteolytic events. while the clustering activity of gliomedin is enhanced by its shedding from the surface of schwann cells by a furin protease, its activity is negatively regulated by bone morphogenetic protein /tolloid-like (bmp /tld), and tolloid-like (tll ) metalloproteinase. cleavage by these enzymes restricts the activity of gliomedin to the nodal area and prevents the formation of ectopic clusters along axons that are devoid of myelin segments, as well as below the myelin internodes. hence, proteolytic processing of gliomedin facilitates, yet limits, the clustering of na + channels to specific sites along the axon in a timely manner. furthermore, axon-glial contact mediated by gliomedin and nf at the nodes, not only plays a role in na + channel clustering during development, but also contributes to the long-term maintenance of na + channels at nodes of ranvier. in addition to clustering by gliomedin, the distribution of na + channels is restricted between two growing myelin segments by the flanking paranodal junction. at this site, axon-glia contact is mediated by a distinct set of cell adhesion molecules (i.e., caspr, nf and contactin) that also delineate the underlying axonal and glial cytoskeleton. this paranodal junction-dependent restriction of na + channels to the nodes is mediated by the spectrin-based paranodal axonal cytoskeleton. illa i . neuromuscular unit, neurology department, hospital santa creu i sant pau, universitat autònoma de barcelona, barcelona, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune disorder of the peripheral nerves with clinical and immunological heterogeneity. currently, the diagnosis of cidp is based on clinical and electrophysiological criteria and does not take into consideration the presence of immune biomarkers. several autoantibodies against proteins of the node of ranvier in patients with cidp have now been described. these antibodies define specific cidp subtypes sometimes referred to as nodopathies and can have diagnostic and prognostic implications. anti-contactin (cntn ) antibodies. we have described the presence of antibodies to cntn in a small subset of patients with cidp. these patients shared a phenotype and have poor response to ivig. the anti-cntn antibodies are predominantly igg . pathological studies from skin and sural nerve biopsies of patients show morphological changes in the paranodes. experimental data supporting the pathogenicity of anti-cntn igg antibodies include: a) demonstration in vitro that the antibodies disrupt the binding of the cntn -caspr complex to neurofascin- (nf ); b) intraneural injections of antibodies progressively and specifically disrupt the paranodal axo-glial junction; and c) chronic infusion of antibodies induced clinical and electrophysiological worsening in animals with experimental autoimmune neuritis (ean). anti-nf antibodies. antibodies to neurofascins were first reported in patients with guillain-barré (gbs) and cidp and subsequently, antibodies specific to the nf isoform were found in a small group (< %) of patients with cidp. studies by us and confirmed by others have demonstrated that patients with cidp and anti-nf antibodies have a distinct phenotype that often includes a low-frequency tremor and poor responses to ivig. the autoantibodies are predominantly of the igg subtype. the passive transfer of monoclonal anti-neurofascin antibodies (which recognize all neurofascin isoforms) to mice with ean strongly exacerbated the severity of the pathology, but no studies have yet demonstrated that patient-derived anti-nf igg antibodies are pathogenic. a pathogenic role of the antibodies is however supported by sural nerve biopsies from patients with cidp and anti-nf antibodies that showed paranodal demyelination in the absence of inflammation, the loss of septate-like junctions and, the interposition of cellular processes between the paranodal loops and the axolemma. these alterations are reminiscent of those found in nfasc-null mice suggesting that anti-nf antibodies may specifically disrupt the nf -cntn -caspr complex at the paranodes. antibodies to other nodal proteins. recently neurofascin- and neurofascin- were reported as the main targets of autoantibodies in five patients with igg reactivity against the nodes of ranvier; the antibodies were predominantly igg . these patients presented with clinical features distinct from those in patients with anti-nf igg antibodies. four of these patients had subacute onset of sensory ataxia without tremor. the presence of anti-caspr antibodies has been reported in two patients with inflammatory neuropathies, one classified as cidp, the other as gbs. both patients had intense neuropathic pain. the skin biopsy from both patients showed paranodal disruption. some patients whose sera show nodal or paranodal reactivity in teased nerve fiber preparations have antibodies against other nodal proteins, such as gliomedin or neuronal cell adhesion molecule (nrcam) . the skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. indeed some mechanoreceptors can detect even nanometer-scale movements. movement is transformed into electrical signals via the gating of mechanically-activated ion channels at sensory endings in the skin. the sensitivity of piezo mechanically-gated ion channels are controlled by stomatin-like protein- (stoml ), which is required for normal mechanoreceptor function. under pathophysiological conditions following nerve injury or diabetic neuropathy the slightest touch can produce pain. it is at present unclear whether peripheral changes in sensory mechanotransduction may underlie hypersensitivity associated with neuropathic pain. here we have examined the role of the stoml modulation of piezo channels in mechanoreceptors and nociceptors to under pathophysiological conditions. we recently developed small molecules that act as inhibitors of stoml function. peripheral application of stoml inhibitors can alleviate hypersensitivity in models of neuropathic pain. our data strongly suggest that tactile evoked pain in models of peripheral neuropathy may be at least partly driven by sensitization of sensory mechanotransduction driven by stoml . coleman m . john van geest centre for brain repair, cambridge, uk. axons are lost early in many neurodegenerative disorders of peripheral and central nervous system. the degeneration of transected axons (wallerian degeneration) can be slowed tenfold by overexpression of a variety of nad-synthesizing enzymes, such as isoforms of nmnat or the related mutant fusion protein, wld s . wallerian degeneration is also delayed by deletion of tlr adapter protein sarm , a protein recently reported to promote nad degradation. it is important to understand fully the mechanism of wallerian degeneration because related mechanisms contribute to axon loss in a number of disease models, including models of peripheral neuropathies, parkinson's disease, multiple sclerosis and glaucoma. new data also suggest a role in hereditary spastic paraplegia. while depletion of nad is an attractive hypothesis for the mechanism of wallerian degeneration, especially as nad can be increased by dietary methods, it cannot explain a number of key observations. fk , an inhibitor of nampt, blocks the nad salvage pathway and strongly depletes nad, including within axons. however, instead of killing axons as the nad hypothesis would predict, it does precisely the opposite: it phenocopies the protective effect of wld s . moreover, ectopic expression of the bacterial enzyme nmn deamidase, a protein absent in mammals, protects injured axons both in transgenic mice and in primary neuronal cultures, but it has no effect on nad levels either under basal conditions or in degenerating nerves. these observations fit better with a proposed toxic role for the nad synthesis intermediate nmn, a model that can also explain the protective effect of wld s . a full understanding of the pathway should identify a number of points where intervention could be a treatment for multiple axonopathies. as with any medical discipline, expansoin of knowledge about the fundamental science behing a disorder of the human nervous system comes part and parcel with a change in our understanding of the epidemiology of any given disorder or groups of disorders. recent advances in our fundamental understanding of inflammatory neuropathies of the peripheral nervous system have been accompanied by drastic changes in our understanding of the neuroepidemiology of these disorders -the specific populationss affected by peripheral neuropathies, as well as the varying importance / contributions of select peripheral neuropathies to the overall burden of peripheral nervous system (pns) disease, and how this shift in epidemiological understanding influences the clinical approach to diagnosis and management of patients with pns disease. the past few decades have witnessed a paradigm shift in many aspects of pns disease diagnosis and treatment; from the association of human immunodeficiency virus (hiv)-associated neuropathies; to the increassing recognition of hereditary / familial peripheral neuropathies; to the increased recognition of specific neuropathies such as multifocal motor neuropathy with conduction block. in addition, timely events such as the recent, and increasingly irrefutalbe evidence for a link between zika virus and a guillain-barré syndrome, and the rather unexpected resurgence of peripheral neuropathies due to previously 'exotic' etiologies such as lepromatous neuropathy require prompt clinical attention. this plenary session aims to describe the evolving neuroepidemiology of peripheral nervous system disorders, and how these changes may influnece the clinical approach to the diagnosis, prognostication, and treatment of otherwise 'unusual' periphal nerve diseases. oxaliplatin chemotherapy for colorectal cancer is seriously limited by neurotoxic side effects which are not fully understood. oxaliplatin-induced peripheral neurotoxicity (oipn) comprises an acute syndrome and a chronic sensory neuropathy. the acute symptoms, notably cold hyperalgesia, have been attributed to transient ion channel dysfunction, and the worse they are the more severe the chronic neuropathy that ensues. we designed a combined in vitro and in vivo project, using neurophysiology to better understand the pathogenesis of oipn. in the in vitro study, differentiated f cells (rat drg neurons x mouse neuroblastoma n tg- cell line) were incubated for and hours in . m oxaliplatin, and their electrophysiological properties studied by patch-clamp. the treated f cells showed relatively depolarized resting membrane potentials, significantly decreased firing frequencies, and increased sodium current densities. moreover, a decrease in erg (ether-à-go-go-related gene) potassium current was also evident. in the in vivo study, we applied nerve excitability testing (net) to a wistar rat model of oipn. to investigate the acute syndrome, we compared behavioural and neurophysiological data of animal cohorts (controls and oipn rats, n= each) before and after oxaliplatin administration ( mg/kg, iv). twenty-four hours after the injection we observed differences between the groups in behaviour (cold plate test, p= . ) and in superxcitability of motor axons (p= . ). to investigate the chronic neuropathy, we compare a control group (n= ) with a treated group (n= , oxaliplatin mg/kg twice weekly x weeks, iv). both groups are studied with behavioural, neurophysiological (sensory and motor nerve conduction studies, net), and pathological (caudal and sciatic nerve, skin biopsy, drgs) methods. data are collected at baseline, end of treatment and weeks after treatment; to obtain a full net profile of all significant changes. in this highly translational approach to oipn, the in vivo net changes in the acute and chronic rat models can be matched on the one hand to findings from in vitro experiments, and on the other to clinical data, since net is also easily applied in humans. of these results. in denmark there is a unique situation to conduct epidemiological studies facilitated by the danish civil registration and the danish national hospital registry (dnhr). this enables us to identify all gbs patients in denmark in a given period. from the same period as the igos cohort was included we have identified all gbs patients admitted to or seen in outpatient's clinics of hospitals in denmark (september st to december st ). records from the population based danish cohort will be reviewed for demographic and clinical data and compared to the patients included in igos from denmark, as well as with the igos europe/america cohort. during this period patients from denmark have been included in igos. the danish group is comparable to the europe/america group not counting the danish patients (n= ) of the igos cohort in regard to sex and age at entry, gbs disability score at nadir, and percentage of patients needing mechanical ventilation. in the danish igos group % are males, the median age is ( - ) years, the mean(sd) gbs disability score at nadir . ( . ), and % of the danish group needed mechanical ventilation. in the europe/america group % are males, the median age is ( - ), mean(sd) gbs disability score at nadir . ( . ) and % of the group needed mechanical ventilation. at the meeting we will compare and present data from the danish population based cohort as well as epidemiological data. chronic inflammatory demyelinating polyneuropathy (cidp) is a common autoimmune disease of the peripheral nervous system (pns) that causes sensorimotor impairment. mice with a dominant autoimmune regulator gene (aire) g w mutation on the non-obese diabetic (nod) background (nod.aire gw/+ mice) develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. in sapp, demyelination is caused primarily by th t cells; however, the contributions of nerve-resident cells such as schwann cells are poorly understood. we identified a population of non-hematopoietic, integrin alpha + (itga +) cells in the pns that increases in frequency and number during sapp. these itga + cells coexpress numerous schwann cell markers including sox , p , s b, myelin protein zero, and peripheral myelin protein , suggesting that itga + cells are schwann cell-like. additionally, during sapp, these itga + cells upregulate the extracellular matrix protein periostin (postn), which has recently been shown to promote macrophage recruitment and activation in inflammatory disease and cancer. our data indicate that macrophages are pathogenic during sapp. therefore, we hypothesized that itg a+ cells promote macrophage recruitment during sapp via postn production. to test this hypothesis, we performed in vitro chemotaxis assays. conditioned media from nod.aire gw/+ nerve promoted significantly more macrophage chemotaxis than conditioned media from postn −/− nerve. furthermore, postn recombinant protein was sufficient to induce macrophage chemotaxis in vitro. our findings show that itg a+ schwann cell-like cells mediate macrophage chemotaxis by upregulating postn during sapp and suggests postn as a novel target for the treatment of cidp. "wear-off" frequency will be analyzed by assessing the proportion of subjects with any given degree of gs and rods intracycle fluctuation and the proportion of cycles in which gs and r-ods fluctuation occurs. to determine the extent of "wear-off" the degree of difference between maximum and minimum gs, r-ods, tugs, onls, and vas scores will be analyzed. currently subjects from different sites have been enrolled ( sites eligible for enrollment). this interim study report will provide preliminary representative data, demonstrating ivig "wear-off" effects on gs and other outcome measures. by better understanding the frequency and extent of ivig treatment-related fluctuations we expect that these results will help facilitate development of cidp treatment optimization strategies. we also expect that this information will be important in forming hypotheses to be tested in future studies (for example, comparing different dosage intervals, optimal ivig taper guidelines, or assessing the long-term outcome of short-term cycle to cycle clinical fluctuations). neuropathic pain is a frequent feature of peripheral neuropathy causing a significant impact on patients' quality of life and health care costs. resolving the genetic architecture of painful neuropathy will lead to better disease management strategies, risk stratification, and counselling. therefore, we aim to develop a reliable technique to rapidly and accurately re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. whole exome sequencing of thousands of samples remains expensive for clinical use. several targeted enrichment approaches are currently available to selectively enrich for genomic regions of interest. in this study, we compared the sensitivity, specificity, targeting efficiency, reproducibility of performance and cost effectiveness of truseq ® custom amplicon-next generation sequencing (tsca-ngs) and molecular inversion probes-next generation sequencing (mips-ngs) methods. for both methods, we constructed a targeted enrichment kit to capture the coding and exon-flanking intron sequences of nine sodium channel genes (scn a, scn a-scn a, and scn b- b) expressed in nociceptive neurons. probes were designed for the two methods using their respective informatics pipelines. in total, patients with diabetic and idiopathic neuropathy were tested by both methods. among the patients, patients were tested previously by sanger sequencing for scn a-scn a. approximately kb was captured and sequenced. % of the targeted regions showed an average coverage of ≥ x in tsca-ngs, and % in mips-ngs. we managed to identify potential pathogenic mutations and polymorphism variants by mips-ngs and tsca-ngs. moreover, we observed a perfect agreement ( %) between sanger sequencing data and those obtained using mips-ngs and tsca-ngs. both ngs approaches showed user-friendly software to design probes and exhibited a similar on-target efficiency. although the overall coverage per region varied across different dna samples, it was sufficient to detect any variant in these regions. mips-ngs has more versatile assay design, demonstrated a high degree of flexibility with probes re-placement and > x cheaper than tsca-ngs. mips-ngs is a reliable, flexible, and inexpensive method to detect genetic variations in thousands of patients. in our centers, this technology is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients. alonso-jiménez a , , belvis-nieto r , diaz-manera j , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; centro para la investigación biomédica en red para enfermedades raras, ciberer, madrid, spain; neurology department, hospital universitario dexeus, barcelona, spain. most acute demyelinating polyneuropathies have an immune-mediated pathogenesis and are included within the guillain-barré syndrome spectrum. occasionally, other mechanisms such as metabolic, infectious or toxic may lead to gbs-like presentations. thermatrim ® and pura alegria ® are different brands of the same illegal slimming product that is sold through online vendors and in which the exact composition is unknown. here we present a patient with an acute demyelinating polyneuropathy secondary to the intake of the slimming product "pura alegría". a year-old woman with no remarkable medical history reported days history of distal numbness in her feet that progressed in one week to her knees and her left hand. she had had an upper respiratory tract infection ten days prior to these symptoms. the neurological examination showed absent distal vibratory and arthrokinetic sensations and arreflexia in lower limbs, decreased vibratory sensation in her hands and a ataxic gate. the lumbar puncture showed . g/l of proteins with no cells. the emg fulfilled diagnostic criteria for acquired demyelination. intravenous immunoglobulin therapy was started but the symptoms kept worsening and corticosteroids were started. the patient mentioned then the slimming product. a brain mri showed diffuse leukoencephalopathy that was asymptomatic. steroids and the pura alegria slimming products were withdrawn and the patient recovered completely after one year of follow-up. "pura alegría", "thermatrim" and "thermatrim plus" are slimming products that were forbidden in spain after several cases of acute leukoencephalopathy and acute polyneuropathy. exact composition is unknown although the spanish drug agency detected the pesticide malonoben, a tyrosin kinase inhibitor, among the components. since nine cases of pura alegria/thermatrim neurological toxicity, including leukoencephalopathy and polyneuropathy have been described. all cases had good outcomes after treatment withdrawal, although recovery is slow and may be incomplete. our case highlights the need to carefully consider drug toxicity, including dietary supplements, in the differential diagnosis of gbs specially when evolution does not follow typical patterns. alvarez s , klein d , martini r , moldovan m , , krarup c , . center for neuroscience, university of copenhagen, denmark; department of neurology, developmental neurobiology, university hospital würzburg, germany; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. charcot-marie-tooth neuropathy type a (cmt a) resulting from peripheral myelin protein kda (pmp ) overexpression is the most common hereditary motor and sensory neuropathy in humans. the transgenic pmp (pmp tg) mouse line c carrying copies of the human pmp gene, has a slowly progressing neuropathy phenotypically like cmt a with thin and abnormally thick myelin profiles and supernumerary schwann cells. in addition, pmp tg nerves showed activated macrophages leading to axon-myelin compartment disruption and maldistribution of k+ channels (kohl b et al, am j pathol. ; : ) . the aim of the present study was to investigate the motor axon excitability in pmp tg versus wt littermates. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle and "threshold-tracking" the plantar compound muscle action potential (cmap). at age months, when the post-developmental maturation was nearly complete in the wt, the pmp tg cmap showed an increase in latency by %. the cmap amplitude was decreased by %, although the mean motor unit size (mscan method) appeared unchanged indicating a lack of collateral sprouting. furthermore, pmp tg showed abnormalities in both passive cable properties and voltage dependent parameters. at age month, the cmap latency of pmp tg was increased by % as compared to wt. in contrast to this marked conduction slowing along the tibial nerve from to months of age, the progression of excitability changes localized at ankle appeared modest. nevertheless, when pooling data from to months, the increase in pmp tg latency was correlated (spearman p< . ) with an increase in accommodation half-time during depolarizing electrotonus (+ % of threshold) from to ms and a reduction of the late subexcitable period of the recovery cycle from to % of threshold, both changes consistent with a redistribution of k+ currents consistent with the maldistribution of k+ channels. our data suggest that in the pmp tg cmt a model, a functional, thus potentially reversible abnormality in k+ channel distribution, accumulates along the nerve and aggravates the conduction impairment due to impaired myelin formation and maintenance. alvarez s , krarup c , , moldovan m , . center for neuroscience, university of copenhagen, denmark; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. mice heterozygously deficient of myelin protein p gene (p +/−) show a mild progressive dysmyelinating neuropathy, with conduction slowing and impaired excitability, phenotypically similar with charcot-marie-tooth disease type b (cmt b). we found that in p +/− the accumulating myelin abnormalities were paralleled by progressive changes in voltage-dependent motor axon function resulting in neurotoxic membrane depolarization (rosberg mr, et. al. neurobiol dis. : ) . the aim of this study was to investigate the relationship between demyelination and motor axon function in p +/−. demyelination of the right sciatic nerve by topic lysophosphatidylcholine (lpc) application was carried out in p +/− and wild-type (wt) mice, in year (mature) and years (aged) groups. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle (distal to lpc demyelination) and "threshold-tracking" the plantar cmap responses. live imaging studies by cellvizio (mauna kea technologies, paris, france) confocal laser endomicroscopy were carried out in transgenic mice expressing the fluorescent reporter yfp in peripheral nerve axons under the thy promoter. in mature wt the sciatic morphological and electrophysiological demyelinating features following lpc could be readily observed at hours but disappeared by weeks. no morphological changes could be observed at the tibial level. consistently, no conduction or excitability changes could be observed at the right tibial neve level as compared to the left tibial nerve in wt. in contrast, in p +/− the motor axon function was impaired at the tibial nerve level at weeks after sciatic lpc demyelination. in mature p +/−, although the cmap amplitude appeared preserved, the distal motor latency was prolonged whereas the excitability measures showed reduced deviations during threshold electrotonus and increased refractoriness at the expense of superexcitability of the recovery cycle, both consistent with membrane depolarization. furthermore, in aged p +/− the delayed tibial conduction was associated with a drop in cmap amplitude and a prolongation of the strength-duration time constant. taken together these data suggest that focal demyelination aggravates membrane dysfunction along the entire motor axon in p +/− providing a novel experimental model to explore the link between demyelination and axonal membrane dysfunction in cmt b. amass l , li h , gundapaneni b , schwartz j , keohane d . pfizer inc., new york, ny, usa; inventiv health inc., burlington, ma, usa. a number of factors can influence disease progression in transthyretin familial amyloid polyneuropathy (ttr-fap), a rare, fatal, hereditary amyloidosis. this analysis evaluated the specific role of baseline neurologic severity on neurologic disease progression in ttr-fap. a predictive model was created based on longitudinal data from val met patients who participated in the tafamidis (a selective ttr stabilizer) clinical development program. data from the intent-to-treat population of the double-blind, placebo-controlled registration study (tafamidis group, n= ; placebo group n= ) and its two consecutive open-label extension studies in which all patients received tafamidis were used. the second extension study is ongoing, but a formal, prospectively-planned interim analysis was conducted with the cut-off date of december , . this analysis focused on the first months of treatment for the overall study cohort analyzed. the neuropathy impairment score-lower limbs (nis-ll) was used to assess neurologic functioning at baseline and at subsequent study visits. a linear mixed-effects model for repeated-measures (mmrm) analysis, with baseline nis-ll, treatment, and their interactions with time as fixed effects, was used, and the slope and intercept for each patient were included as random effects. patients were primarily caucasian with early-stage neurologic disease (baseline nis-ll mean [standard deviation]: tafamidis, . [ . ]; placebo, . [ . ] ). across both groups, disease progression increased with increasing levels of baseline severity (nis-ll) (p< . ). however, the predicted magnitude of change from baseline to month for tafamidis was consistently less than that for placebo across a range of observed baseline nis-ll values, suggesting a disease-modifying effect of tafamidis. similar findings were observed for the nis-ll muscle weakness subscale. this mmrm analysis in patients with val met ttr-fap demonstrates that disease progression strongly depends on baseline neurologic impairment and highlights the disease-modifying effect of tafamidis across a range of baseline levels of neurologic severity. clinicaltrials.gov identifiers: nct , nct , nct . amino h , misawa s , sekiguchi y , shibuya k , watanabe k , suichi t , kuwabara s . department of neurology, chiba university, chiba, japan. guillain-barré syndrome (gbs) is a potential life threatening neurological disorder and respiratory insufficiency is one of the critical complications. eramus gbs respiratory insufficiency score (egris) is a method for predicting the chance of respiratory insufficiency in gbs. however, clinical characteristics and courses can vary for subtypes of gbs, whose occurrences differ for each region: acute inflammatory demyelinating polyneuropathy (aidp) is very common in european countries, whereas acute motor axonal neuropathy (aman) is frequently seen in asian countries. the aim of this study is to investigate the usefulness of egris in japan, where aman is more common than in the netherlands. clinical and electrophysiological profiles of consecutive gbs cases, who visited our hospital within days after symptoms onset between and , were reviewed. of the gbs patients, % were classified as aidp and % as aman according to the electrodiagnosis criteria by ho and colleagues. higher egris scores correspond to higher risk of respiratory insufficiency in total of the gbs patients, as well as in aidp patients. however, in patients with aman, egris scores did not always match the chances of respiratory insufficiency: up to % of the patients with low risk of egris showed respiratory failure, whereas only % of the patients with high risk of egris needs intubation/mechanical ventilation. in aman, associations with mechanical ventilation were seen for rapid progression (shorter duration between onset and hospital admission), more decreased vital capacity, and more frequent autonomic involvement. egris is useful also for japanese gbs patients. however, for aman patients, it should be used with discretion. another score to predict respiratory insufficiency might be required in asian countries. anandan c , litchy wj , laughlin rs , leep hunderfund an , naddaf e . mayo clinic, rochester, usa. in patients with suspected ulnar neuropathy, nerve conduction studies (ncs) are commonly requested to help with diagnosis and localization. however, routine ncs are often normal or not localizing. ulnar ncs recording from the first dorsal interosseous muscle (ncs-fdi) is thought to increase the diagnostic yield of electrodiagnostic testing, although not commonly considered. we developed a quality improvement strategy to routinely perform ulnar ncs recording from the abductor digiti minimi muscle (ncs-adm) as well as ulnar ncs-fdi in all patients referred for suspected ulnar neuropathy. we utilized the dmaic (define, measure, analyze, improve, control) model of process improvement to define our problem and create a map of the current process for ulnar neuropathy diagnosis in our electromyography laboratory. we determined baseline performance via review of distal sensorimotor polyneuropathy (dpn) is the most common complication of diabetes and risk factors beyond hyperglycemia have proven important particularly in type diabetes (t dm). only few prospective studies from early-stage t dm exist. we aimed to study the development of dpn during the first years after a screening-based diagnosis of t dm. from the addition-denmark study participants were eligible for this study. dpn was assessed by the michigan neuropathy screening instrument questionnaire (mnsi) at four time-points during follow-up. dpn was defined by a mnsi score ≥ . participants ( %) were positive in mnsi at baseline and thus excluded from this study. by kaplan-meier plot we evaluated the cumulative incidence of dpn and in cox proportional hazard models we calculated hazard ratios (hr) for the intervention groups in the addition trial and for various covariates proposed to influence the development of dpn. models were adjusted in steps for intervention group, age, sex, baseline mnsi, lipid-lowering and anti-hypertensive treatment. this study cohort consists of participants ( % men) with a median age of . years (p ;p : . ; . ) and median baseline hba c of . (p ;p : . ; . ). a cumulative incidence of % was seen during years of diabetes. there was no statistically significant difference in hr between the intervention groups or by sex but a significantly higher hr of . ( %ci: . ; . ) was seen for age (per year). the highest hr was found for a history of cardiovascular disease (myocardial infarction or stroke) up to ten years prior to the diabetes diagnosis with a hr of . ( . ; . ). weight, waist circumference, body-mass index and methylglyoxal (log transformed) showed modest but statistically significant associations with incident dpn with standardized hrs of . ( %ci: . ; . ), . ( %ci: . ; . ), . ( %ci: . ; . ) and . ( %ci: . ; . ) respectively. this study demonstrates a fairly low cumulative incidence of dpn in people with screen-detected t dm and provides evidence that macrovascular disease, obesity and oxidative stress are important risk factors for dpn even at the earliest stages of t dm. andermann syndrome, also known as agenesis of the corpus callosum and peripheral neuropathy (accpn), is an autosomal recessive disorder with a broad spectrum of mild to severe neuromuscular and psychiatric consequences. the gene variants causing disease were first identified in french-canadian families. in the present study, we intended to phenotype and genotype a series of non-french-canadian familial cases presenting with charcot marie tooth disorder associated with agenesis/dysgenesis of the corpus callosum. for this purpose, seven families, of consanguineous marriage, were studied. patients were clinically and para-clinically investigated using mri and electrophysiology (mncvs). for some, a sural nerve biopsy was taken. microsatellite markers around the accpn locus were used in two large families; followed by sanger sequencing of all the exons and intron-exons boundaries of the gene, in one patient from each of the families. the age at onset of the disease was at birth in the patients from the largest consanguineous family ( affected individuals). the biopsy from one patient showed a severe demyelinating neuropathy with many hypomyelinated fibres and mostly secondary axonal changes. these finding were compatible with electrophsiological data where the mncvs are of demyelinating range. dysgenesis of the corpus callosum in one patient and agenesis in another sib were revealed by mri. we identified one homozygous truncating mutation in this family. interestingly, no causative variant was found in a patient from another family and showing homozygous haplotype. two different heterozygous variants were identified at one hit in two patients from two non-consanguineous families. genetic investigations will be continued to identify the possible second hit. in the remaining families, no variant was found. the negative family cases will be subjected to ngs. at this stage, it is tempting to speculate on the genetic heterogeneity of accpn in our series. baba m , suzuki c , ogawa c , tomiyama m . department of neurology; diabetes center, aomori prefectural central hospital, aomori, japan. in we introduced a staging system of severity of diabetic polyneuropathy (dpn) by nerve conduction study (ncs) of the lower limb: sensory ncs of the sural nerve and motor ncs of the tibial nerve. the system consists of five stages; ncs- (normal): no abnormalities, ncs- (mildly abnormal): presence of delay of mcv, scv, minimal f-wave latency, or positive a-wave, ncs- (moderately abnormal): decrease in sural snap less than uv, ncs- (severely abnormal): decrease in plantar muscle-cmap to - mv, ncs- (ultimately abnormal): plantar muscle-cmap lost or less than mv with trace of sural-snap. to examine validity of the system, we conducted -year prospective observation on development of diabetic foot (df) by the ncs staging system. in addition, occurrence of ischemic heart disease (ihd) and stroke (is), and death of neuro-vascular events were also counted. n - , we carried out ncs in diabetics, and categorized them by the ncs staging system: % was ncs- , % was ncs- , another % was ncs- , % was ncs , and % was ncs- . we then followed them and prospectively counted the occurrence of df, ihd and is in patients (mean age ys). the occurrence of df during the following years was; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- : %. occurrence of any of df, ihd and/or is was as follows; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- %. there was no death from nsc- , − , and − groups (n= ), while two from ncs- group (n= ) were found dead in a bed or on a driver's seat, and other two from ncs- group (n= ) died of sudden cardiac arrest or infection after foot amputation. in summary, the present ncs grading system seems to work satisfactory not only for diagnosis of severity of the current dpn, but also for prognostic prediction of the dpn-related foot and vascular events. bacon c , feely sme , shy me . university of iowa hospital, iowa city, ia, usa. for patients with charcot-marie-tooth disease, also known as hereditary motor and sensory neuropathy, the rasch modified cmtnsv is a validated measurement of symptoms and impairment. the score is comprised of nine parameters of a clinical examination, including nerve conduction studies (ncs) . each parameter has an individual score ranging from zero to four, with the composite score having a maximum of prior to rasch modification. for patients who do not complete a ncs, the cmt exam score (cmtesv ) is used, with a maximum score of before rasch modification. one parameter of the cmtesv is the vibration sense. using a rydel tuning fork, a care-giver measures vibration sense in a patient's feet, ankles, and knees and a score is determined by the severity of reduced vibration sense. in cmt a patients, % received a score of out of , noted by a reduced vibration sense at the knee. in order to capture a more sensitive vibration measurement, we tested ways of taking the total raw score of the rydel tuning fork at each point of vibration sense, the toe, ankle, and knee bilaterally. scores range from zero to , with reflecting full vibration sense. in this modified measurement, vibration sense scores varied in wider distribution. for cmt a patients captured in this studied, a modified vibration sense score results in a potentially more sensitive total cmtesv score. bae dw , an jy . st. vincent's hospital, the catholic university of korea, suwon, korea. diabetic lumbosacral radiculoplexus neuropathies (dlrpn) are usually subacute painful, monophasic, asymmetrical lower limb neuropathies with incomplete recovery due to ischaemic injury and microvasculitis. the diagnosis relies mostly on clinical suspicion and characteristic electromyographic findings. however, in acute phase, neuroimaging has more important diagnostic significance than electrophysiological studies. here we describe mri and ultrasound findings in a -year-old woman with dlrpn who was diagnosed with diabetes three years ago, but has not received any other treatment. she had a -day history of acute-onset severe pain with weakness of muscles innervated by left femoral and obturator nerve and decreased sensation in left l -l dermatome. nerve conduction studies showed reduced amplitude in left femoral nerve and electromyography showed only increased insertional activity in left iliopsoas muscle and no volitional activity in muscles innervated by left femoral and obturator nerve. ultrasound revealed increased cross-sectional area (csa) of left femoral and lateral femoral cutaneous nerve. mri showed enhancement in left l , nerve roots and proximal femoral nerve and increased signal intensity in left iliacus and iliopsoas. we diagnosed her with dlrpn and started corticosteroid. nerve ultrasound has not been previously reported in a patient with cabin air in commercial airliners originates from aircraft engines or auxiliary power units. this bleed air may occasionally be contaminated with hydraulic fluids and engine oil that contains a number of potentially hazardous chemicals including tricresyl phosphate (tcp). over the last years reports are emerging about aircrew members that experience symptoms such as tingling or burning of extremities in addition to headache, and vertigo. this "aerotoxic syndrome" is controversially discussed in the literature and has been attributed to exposure to organophosphate contaminated cabin air. since tcp has been associated with peripheral neuropathy we aimed to determine the frequency of peripheral neuropathy among frequent flyers. civilian air crew members and frequent flying passengers (m:f = : , median age years, median exposition time in aircrafts of . hours) were examined at the university hospital of cologne or at the frankfurt airport (iata code fra) by a detailed questionnaire of past medical conditions, a standardized neurological examination and nerve conduction studies of sural, tibial, and ulnar nerves. we identified subjects with clinical and electrophysiological evidence for large fiber peripheral neuropathy. incidence of peripheral neuropathy was not correlated to exposition time in aircrafts. in addition subjects showed signs of ulnar neuropathy, subjects reported abnormal vibration sensation, subjects suffered from gait imbalance and individuals reported tingling of extremities. our study shows a . % prevalence of large fiber peripheral neuropathy among frequent flyers. comparison of these data with prevalence rate in an age-matched control group will reveal a possible association of chronic exposure to cabin air and risk for peripheral neuropathy. the high incidence of the symptom tingling in our cohort warrants further studies to determine the risk for small fiber neuropathy in this condition. intravenous immunoglobulin (ivig) are human igg derived from plasma pools of healthy donors. although there are studies in literature evaluating their effectiveness in different pathological animal models, there are no data about their possible role on bortezomib (btz)-induced peripheral neuropathies. female wistar rats were treated following a preventive schedule (btz and ivig co-treatment for and weeks) and a therapeutic schedule ( weeks of btz treatment followed by a -week ivig-btz co-treatment). caudal nerve conduction velocity (ncv), plantar and dynamic tests were performed at different time points. animals were sacrificed after ws (acute phase) or ws (chronic phase) and tissue samples (dorsal root ganglias -drg-, sciatic nerve, caudal nerve, skin) were collected for morphological, morphometrical and immunohistological analysis.in the preventive schedule, ivig was not able to rescue caudal ncv reduction caused by btz neither after nor after weeks of co-treatment. same results were observed in the therapeutic schedule. on the other hand, the evaluation of mechanical allodynia and cold hyperalgesia showed that ivig injection protected from btz effect in both treatment schedules. morphometric analysis evidenced that, even if not statistically significant only the preventive schedule has a tendency to protect the caudal nerve from btz damage. this result is consistent with the morphological evaluation of the nerve. also, intra-epidermal nerve fibers density was preserved in the preventive schedule but not in the therapeutic one. finally, sciatic nerve and drg macrophage infiltration levels tended to be reduced in the therapeutic schedule and were brought back to ctrl (rats not treated or injected with ivig alone) levels in the preventive one. in conclusion, we were able to demonstrate for the first time that ivig treatment especially used as preventive treatment option may reduce btz-induced neuropathic painful pointing out the possible role of inflammation in the pathogenesis of this invalidating pathology. this work was supported by kedrion spa. we studied the prevalence, the molecular cause and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the north of england. detailed neurological and electrophysiological assessments and next generation panel testing or whole exome sequencing were performed in patients with clinical symptoms of distal hereditary motor neuropathy (dhmn, patients), axonal motor neuropathy (motor cmt , patients) or complex neurological disease predominantly affecting the motor nerves (hmn plus, patients) . the prevalence of dhmn is . affected individuals per . inhabitants ( % confidence interval: . - . ) in the north of england. causative mutations were identified in out of index patients ( . %). the diagnostic rate in the dhmn subgroup was . %, which is higher than previously reported ( %). we detected a defect of neuromuscular transmission in cases and identified potentially causative mutations in patients with demyelinating multifocal motor neuropathy. many of the genes were shared between dhmn and motor cmt , indicating identical disease mechanisms therefore we suggest changing the classification and include dhmn also as a subcategory of cmt. abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies. barohn rj , gajewski b , pasnoor m , brown l , herbelin l , kimminau k , jawdat o , parks c , shlemon p , dimachkie mm and the pain-controls study team . the university of kansas medical center, kansas city, ks, usa. cryptogenic sensory polyneuropathy (cspn) is a common slowly progressive neuropathy that affects adults and presents with significant neuropathic pain for which multiple medications have been tried including antiepileptics, antidepressants, topicals and narcotics. a web based survey among neuromuscular experts suggested pregabalin as being more effective than other medications, however there are presently no comparative studies to assess the most effective medication. the objective of this study was to determine which of the pharmaceutical therapies (pregabalin, duloxetine, nortriptyline or mexiletine) is most effective for neuropathic pain and best tolerated in cspn. to achieve this objective we performed a prospective randomized open labelled comparative effectiveness adaptive design study of cspn patients through the patient centered outcomes research institute (pcori). cspn patients who fulfilled the inclusion and exclusion criteria were enrolled into this study. patients underwent a baseline neurological evaluation and randomly assigned to one of the neuropathic medications for months. the primary outcome is the change in likert-like pain scale. the secondary outcomes included nih pain interference scale, nih fatigue interference scale, nih sleep disturbance scale, sf- and adverse events. the outcome measures are performed at baseline, month , and . statistical analysis using bayesian adaptive design developed by berry consultant software will be performed to determine winner and losers (winner = greater than point improvement in pain). total number of patients to be enrolled is . recruitment has been challenging and a number of recruitment techniques have been used. to date, there have been patients screened, patients randomized from us sites. anticipated completion of enrollment by june and end of final patient assessment by september . interim analysis performed after first patients completed their months as part of bayesian adaptive design analysis and occurs every weeks. the distribution of randomization of patients to the medications at last adaptive design randomization was . % to medication , % to medication , . % to medication and . % to the th medication. this study may give physicians and patients evidence for future management of cspn patients. transthyretin amyloidosis (attr), which encompasses a group of disorders with significant clinical variability, is caused by transthyretin (ttr) derived amyloid deposition. the clinical aspects of autonomic nervous system involvement in attr are only partially known. the ongoing, multinational, longitudinal, observational transthyretin amyloidosis outcomes survey (thaos) provides the opportunity to expand our understanding of dysautonomia in attr. data from all symptomatic subjects enrolled in the thaos registry with a diagnosis of attr (cut-off date: january , ) were assessed for the presence and temporal course of autonomic symptoms, genotype and phenotype associations, and clinical burden according to the frequency and severity of symptoms. of symptomatic subjects enrolled in thaos, ( . %) had autonomic symptoms at enrollment including: gastrointestinal ( subjects, . %), urinary ( , . %), erectile dysfunction ( , . %), orthostatic hypotension ( , . %), xerophthalmia ( , . %) and dyshydrosis ( , . %). subjects with autonomic manifestations, compared with those without, were younger (mean age [standard deviation, sd] of . [ . ] vs . [ . ] years), with a longer duration of attr symptoms ( . [ . ] vs . [ . ] years). autonomic dysfunction was less common with wild-type attr ( of subjects, . %) than in mutation groups: val met ( / , . %); non-val met/non-cardiac ( / , . %); and "cardiac mutations" (val ile, leu met, thr ala, or ile leu mutations; / , . %). similarly, time (mean [sd] , years) from first attr symptoms to onset of autonomic symptoms, was longest for wild-type attr ( . [ . ] ) followed by "cardiac mutations" ( . [ . ]), non-val met/non-cardiac ( . [ . ]), and val met ( . [ . ] ). autonomic symptoms were present at disease onset in over a third of subjects ( , . %) . autonomic dysfunction was less frequent in subjects with cardiac phenotype ( of subjects, . %), than with mixed ( / , . %) or neurologic ( / , . %) phenotypes. the burden of autonomic symptoms (mean [sd] ) varied by genotype, val met ( . [ . ] , non-val met/non-cardiac ( . [ . ], "cardiac mutations" ( . [ . ]), wild-type attr ( . [ . ]), and by phenotype, mixed ( . [ . ]), neurologic ( . [ . ]), cardiac ( . [ . ]). dysautonomia is common, and a significant burden, in subjects with hereditary forms of attr. its prevalence is higher in val met than in other genotypes, and in the neurologic or mixed phenotypes. the objective of this study is to assess the usefulness of motor unit number index (munix) technique in charcot-marie-tooth type a (cmt a) disease and to test correlation between munix and clinical impairment. munix technique was performed in abductor pollicis brevis (apb), abductor digiti minimi (adm) and tibialis anterior (ta) muscles in the non-dominant side. a munix sum score was calculated by adding munix of these muscles. muscle strength was measured using the mrc (medical research council) scale. disability was evaluated with several functional scales including cmt neuropathy score version (cmtnsv ) and overall neuropathy limitation scale (onls). cmt a patients with known pmp gene duplication were enrolled. the munix of the adm, apb and ta muscles were correlated with the mrc of the corresponding muscle (p< . ). munix sum score was correlated with clinical scales: cmtnsv (r=− . , p< . ), onls (r=− . , p< . ). in conclusion, munix correlates with muscle strength and clinical measurements of disability in cmt a patients. the munix technique evaluates motor axonal loss and correlates with disability. the munix sum score may be a useful outcome measure of disease progression in cmt a. the objective of this study was to assess the usefulness of mri in charcot-marie-tooth type a (cmt a) disease and to test correlation between muscle fat fraction and clinical impairment. mri was performed in the non-dominant lower limb of cmt a patients and healthy controls. fat fraction of tibialis anterior muscle, cross section area and volume of sciatic nerve were determined. muscle strength of dorsiflexion was measured using a dynamometer. disability was evaluated with cmt neuropathy score version (cmtnsv ). cmt a patients with known pmp gene duplication were enrolled. fat fraction of tibialis anterior muscle was significantly increased in patients compared to healthy controls. it was correlated with muscle strength (r=− . , p< . ) and cmtnsv score (r=− . , p< . ). cross section area and volume of sciatic nerve were significantly increased in patients compared to healthy controls. in conclusion mri fat fraction correlates with muscle strength and clinical measurement of disability in cmt a patients. it may thus be a useful outcome measure of disease progression in cmt a. basta i , peric s , cobeljic m , bjelica b , bozovic i , kacar a , nikolic a , rakocevic stojanovic v , stevic z , lavrnic d . neurology clinic, clinical center of serbia, school of medicine, university of belgrade, belgrade, serbia. there is a complete lack of data about epidemiological and clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) in serbia and surrounding countries. furthermore, there is a striking scarcity of information about quality of life (qol) in cidp patients and all qol studies have been conducted in countries with high standards of health care. in august we have designed the iness (inflammatory neuropathy study of serbia) in order to comprise as many patients with cidp from serbia, republic of srpska (bosnia and herzegovina) and montenegro covering population of more than nine million people. our first aim is to analyze overall impact of cidp on physical, mental and social areas of life measured with generic, symptom specific and disease specific questionnaires -sf- , inqol and cap-pri, respectively. furthermore, we aim to analyze influence of the disease on patients' working status and presence of depressive mood measured by beck's inventory. following features of patients are included: sociodemographic data, clinical aspects of the disease, level of disability, severity of sensory symptoms, presence of comorbidities, electrophysiological characteristics, as well as fatigue, autonomic symptoms and neuropathic pain. we intend to define the most significant predictors of decreased qol in order to focus on patients with the highest risk and to improve care of cidp. we also want to see if cidp patients in complete remission as per clinical findings still have reduced quality of life. we have recruited patient so far and we expect to include around subjects overall. we will present the first data of the study at the pns meeting . beaudonnet g , prud'hon s , cauquil c , labeyrie c , not a , bouilleret v , adams d . neurophysiology chu bicêtre, le kremlin bicêtre, france; neurology chu bicêtre, le kremlin bicêtre, france. familial amyloid neuropathy (fap) is a life-threatening disease of autosomal dominant inheritance due to transthyretin (ttr) gene mutation, a liver-produced protein. current treatments slow down its natural course and are indicated from the very first objective symptoms. we aimed to evaluate two neurophysiological markers: sympathetic skin response (ssr) and heart rate variability (hrv) in the early detection of sympathetic damages due to fap. ssr and hrv were assessed in ttr gene mutated patients with neither clinic nor electroneuromyographic abnormalities and controls matched on gender and age. cases were recruited consecutively from current care in the french reference center for rare diseases of bicetre university hospital. ssr was recorded on the two palms and on the sole of the left foot with to stimulations between and ma. hrv was registered during three conditions of seconds each: normal breathing, deep breathing ( cycles of seconds of inspiration and seconds of expiration) and valsalva manoeuver during seconds. valsalva ratio, defined by the ratio between the longest and shortest rr intervals, was significantly higher in the control groups after bonferroni correction (means of . and . , respectively, p< . ). there was no significant difference between the two groups for any ssr parameter, although means of amplitudes were systematically higher in controls than among cases. our results confirm that autonomic nervous fibers are damaged early in both clinical and electroneuromyographic asymptomatic patients mutated on the ttr gene. valsalva ratio seemed to be the most discriminative marker. long-term follow-up with test repetition and confrontation with cardiologic assessment will help to precise how these tests could be used in current care. they might help to identify high risk patients to propose them an appropriate early treatment and could be used to follow treatment efficacy. familial amyloidotic polyneuropathy (fap) was originally characterized by andrade as an axonal neuropathy which subsequently was found to be associated with a number of mutations in the plasma protein transthyretin (previously named prealbumin). it is now recognized that cardiomyopathy may be a significant factor in a majority of patients with the hereditary form of transthyretin amyloidosis (fap) and many of the transthyretin (ttr) mutations are associated with cardiomyopathy with no or minimal signs of peripheral neuropathy. attr-wt also called senile cardiac amyloidosis and senile systemic amyloidosis is recognized as late-onset, usually in the th or th decade of life, and the fact that the majority of patients are males. transthyretin neuropathy proven by nerve biopsy has been rarely reported in this population. here we report our experience with patients having attr-wt characterized by cardiomyopathy but also with varying degrees of peripheral neuropathy. clinically, the neuropathy appears as typical axonal or mixed axonal/demyelinating neuropathy as is seen in fap. pathologically, two types of ttr deposition have been found, ( ) intraneural ttr amyloid deposits as seen in fap are present in some patients and ( ) other patients have extensive vascular deposition of amyloid in both perineural arteries and veins without deposits within nerve trunks. in conclusion, peripheral neuropathy may definitely be a part of the attr-wt clinical presentation and with the increase in numbers of attr-wt cardiomyopathy patients being identified, it is important to ascertain whether any evidence of peripheral neuropathy is due to the amyloidosis and not to compounding syndromes such as diabetes mellitus type ii. besora s , santos c , izquierdo c , martinez-villacampa m , simó m , bruna j , , velasco r , . neuro-oncology unit, hospital universitari de bellvitge-institut català d́oncologia, ĺhospitalet, barcelona, spain; medical oncology department, institut català d́oncologia, ĺhospitalet, barcelona, spain; department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. oxaliplatin (oxa) is the first-line chemotherapy agent in the treatment of colorectal cancer (crc). oxa-induced peripheral neuropathy is the most frequent long-term side-effect. retreatment with oxa is frequently considered in patients as salvage treatment. patients receiving oxa-based chemotherapy regimen at least twice at our institution between and were reviewed. the aim of this study was to investigate whether retreatment with oxa increases the risk of developing or worsening previous oxa-induced peripheral neuropathy. the severity of neuropathy was measured by national cancer institute-common toxicity criteria (nci), total neuropathy score (tns) © and nerve conduction studies. one hundred twenty-five crc patients were included. median age was years. after first-line oxa-based chemotherapy, . % of patients developed neuropathy according nci, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. severity of neuropathy was grade ( . %), grade ( %), and grade ( . %). median time to retreatment with oxa was months. frequencies of neuropathy before retreatment were as follows: . % grade , . % grade , and . % grade . after retreatment, severities of neuropathy were . % grade and . % grade . no patient developed grade . . % of patients did not develop neuropathy. peripheral neuropathy was the reason for stopping prematurely treatment after first-line and retreatment in . % and . % of patients, respectively. worsening of previous nci score was observed in . % of patients. the great majority of patients ( . %) remained within the same nci score than before retreatment after median [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients that did not develop neuropathy after first treatment (n= ), only and patients developed grade and , respectively, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients who initially developed grade and neuropathy, no differences in tnsc © scores just before and after finishing retreatment with oxa were identified ( [ - ] vs [ - ], p= . ). retreatment with oxa in crc patients is a feasible option even in patients who developed moderate or severe neuropathy previously. lack of worsening of previous neuropathy is observed in the great majority of patients. neurological monitoring of patients candidates to retreatment with oxa should be considered. bessaguet f , sturtz f , magy l , , desmouliere a , bourthoumieu s , demiot c . ea -myelin maintenance & peripheral neuropathy, faculties of medicine and pharmacy, university of limoges, limoges, france; department of neurology, reference center for rare peripheral neuropathies, university hospital of limoges, limoges, france. prolonged pressure and the resulting local ischemia are widely accepted as the primary etiology of skin pressure ulcers (pus) but precise mechanisms of their formation remain unclear. in this study, we wanted to study the potential role of sensory small nerve fibers in regulation of inflammation during pus formation. to achieve this goal, we developed a mouse model of a purely sensory neuropathy and this was induced by resiniferatoxin (rtx). in this model, seven days after a single injection of rtx ( g/kg; ip), mice present a thermal and mechanical hypoalgesia associated with large substance p (sp) and calcitonin gene-related peptide (cgrp) depletion without neurodegeneration. this model mimic quite well what is observed in early stages of sensory nerve fiber defect. studies have shown that sp and cgrp are involved in cutaneous inflammation regulation. in fact, these neuropeptides are released by sensory fibers and are pro-inflammatory mainly through recruitment of immune cells and vasodilation. thus, we studied gene expression of pro and anti-inflammatory cytokines by a rna array approach during pus formation in control and rtx mice. seven days after a single injection of rtx, epidermis, dermis and subcutaneous tissue layer were pinched with magnetic plates during hours. pressure induced a stage pus. gene expression was evaluated in each compressed area h after pressure. results showed mainly a down-regulation of gene expression in pus of rtx mice compared to control mice. a decrease of cgrp/sp in skin sensory small fiber increased pus formation associated with an increase of interleukins (il)- , il- , il- and il- expression and a decrease of il- expression in rtx mice. supplementary experiments with rt-qpcr for each cytokine will be necessary to confirm these preliminary results. these observations suggest a cgrp/sp role in regulation of cytokines expression during pus formation. the new inflammatory profile exhibited in this study might help in the design of new treatments improving the quality of life of neuropathic patients prone to developing bedsores. bhattacharyya bj , jayaraj nd , belmadani a , ren d , rathwell ca , hackelberg s , miller rj , menichella dm . department of neurology, northwestern university, chicago, il, usa; department of pharmacology, northwestern university, chicago, il, usa. painful diabetic neuropathy (pdn) is one of the most common and intractable symptoms of diabetes, affecting % of diabetic patients. the hallmarks of pdn are neuropathic pain and small fiber degeneration, manifested by the loss of dorsal root ganglion (drg) nociceptor axons. neuropathic pain is associated with nociceptor hyper-excitability in the absence of physiologically appropriate stimuli. in states of neuropathic pain, drg nociceptors become increasingly responsive to a variety of excitatory influences, including inflammatory cytokines. in particular, we have shown that stromal cell derived factor- (sdf- ) and its receptor cxcr are necessary for the generation of neuropathic pain in mouse models of pdn. however, the molecular mechanisms leading to the hyper-excitability of drg nociceptors in pdn are unknown, as are the mechanisms leading to small fiber degeneration. this fundamental gap in our knowledge represents a critical barrier to progress in developing novel therapeutic approaches for pdn. the objective of this study is to identify the molecular cascade linking cxcr /sdf- chemokine signaling to drg nociceptor hyper-excitability, neuropathic pain, and small fiber degeneration in pdn. drg nociceptors can be identified by a series of molecular markers, including expression of the sodium channel na v . . indeed, > % of na v . -expressing drg neurons are nociceptors. feeding mice a high fat diet (hfd) for several weeks induces glucose intolerance, obesity, and mechanical allodynia, a particular pain hypersensitivity associated with pdn. using the hfd model combined with dreadd receptor technology, we have shown that reducing excitability of na v . -expressing neurons prevents and reverse neuropathic pain, neuronal calcium overload, mitochondrial dysfunction, and small fiber degeneration. furthermore, we have shown that cxcr receptors are necessary for neuropathic pain and small fiber degeneration in pdn. taken together these data demonstrate that na v . nociceptor hyperexcitability in pdn is driven through the activation of cxcr receptors. inhibition of hyperexcitability can prevent and reverse the development of pdn. furthermore, these observations will advance our understanding as to how changes in excitability, calcium influx, and mitochondrial dysfunction in nociceptors contribute to neuropathic pain and small fiber degeneration in pdn, which is a critical barrier to progression for effective and disease modifying treatment for pdn. bianco m , terenghi f , gallia f , nozza a , scarale a , fayoumi mz , giannotta c , morenghi e , nobile-orazio e . poems (polyneuropathy, organomegaly, endocrinopathy, m protein and skin changes) syndrome is an unusual multisystem disease with neurological disability due to a severe disabling polyneuropathy, with high mortality by multiorgan failure. peripheral blood stem cell transplantation (pbsct) is considered the treatment of choice for poems while lenalidomide is the most promising therapy for patients not eligible for pbsct. the aim of the present study was to compare the long-term effects on clinical, biological and neurophysiologic parameters in patients with poems treated with lenalidomide or pbsct. the clinical, biological and neurophysiologic data were reviewed in poems patients treated with pbsct (n: ) or lenalidomide (n: ). the mrc sumscore on muscles, onls scale, vegf serum levels and nerve conduction studies were assessed before (t ) and after (t ) and years (t ) of treatment and the differences were compared using anova. combining the two groups of patients, there was a significant improvement after treatment in the mean mrc sumscore (t = ± ; t = ± ; t = ± ; p = . ), in the mean onls score (t = . + . ; t = . + . ; t = . + ; p = . ), in the ulnar mean distal motor latency (t = . ± . msec; t = . ± . msec; t = . ± . msec; p = . ), distal compound muscle action potentials amplitude (t = . ± . mv; t = . ± . mv; t = . ± . mv; p = . ), motor conduction velocity (t = . ± . m/sec; t = ± m/sec; t = . ± . m/sec; p= . ) and serum vegf levels (t vs t : p = . ; t vs t : p = . ). the difference was also significant when we separately analyzed patients treated with lenalidomide and pbsct and there was no difference between the two groups in any of the analyzed parameters. treatment with pbsct and lenalidomide significantly and similarly improved clinical, biological and neurophysiologic parameters in patients with poems syndrome up to two years. since pbsct may not be suitable for all patients, lenalidomide may represent an effective and a valuable alternative in these patients or in those relapsing after pbsct inducing a prolonged clinical, biological and neurophysiologic improvement. bis d , tao f , abreu l , sleiman p , hakonarson h , zuchner s and inherited neuropathy consortium. dr. j.t. macdonald department for human genetics, hussman institute for human genomics, university of miami, miami, fl, usa; center for applied genomics, the children's hospital of philadelphia, philadelphia, pa, usa. inherited peripheral neuropathies are clinically and genetically heterogeneous diseases that can cause distal muscular atrophy and sensory loss. alleles in over one hundred different genes have been shown to cause peripheral neuropathies; yet, greater than % of axonal neuropathy patients do not receive a genetic diagnosis. large scale exome studies are now beginning to be sufficiently powered to perform mutational burden analysis. this approach compares damaging allele frequencies of cmt cases with a control group to identify additional causes for neuropathies. this approach will also identify genes that require an oligogenic inheritance to cause a phenotype. in a deviation from the classic linkage-based and heuristic variant filtering approaches to gene identification, we are performing burden analyses in a large cohort of cmt families. in known neuropathy genes, we saw that neuropathy cases carried on average . rare, non-synonymous variants, while unrelated non-neuropathy controls harbored . variants (p= . e- , mann-whiney u-test). enrichment of rare, non-synonymous variants in cmt disease genes within inherited peripheral neuropathy cases suggests the presence of multiple weaker alleles in individual patients. we also performed an unbiased exome-wide gene-based burden analysis and ranked genes after multiple testing correction. several new candidate genes were identified that need further follow up conformational studies. a number of known cmt and related genes were observed in the list of top candidates. we are currently analyzing additional aspects of this sample and are actively seeking more cmt exomes to enlarge our study. in summary, statistical methods traditionally reserved for more 'common' phenotypes' increasingly are becoming available for rare disease genetics. c bl/ mice were treated with bortezomib alone or in combination with monastrol. neuropathic changes were assessed by nerve conduction studies and histological analysis. analysis of axonal morphology was performed with light and electron microscopy. anti-neoplastic properties of monastrol alone and in combination with bortezomib were assessed in different blood cancer cell lines. prolonged treatment with bortezomib induced a sensory neuropathy in mice. significant changes in axonal morphology correlated with reduced function of peripheral nerves. the administration of monastrol substantially ameliorated morphological features of axonal alterations and sensory neuropathy. cytotoxicity studies in blood cancer cell lines showed no interference of monastrol with the cytostatic effects of bortezomib. our data indicate that monastrol may alleviate bortezomib induced neuropathy. the favorable cytotoxic profile of monastrol makes it an interesting candidate as neuroprotective agent to prevent bortezomib-induced neuropathy. boczonadi v , meyer k , gonczarowska-jorge h , , bartsakoulia m , roos a , , bansagi b , zahedi rp , talim b , bruni f , kaspar b , , lochmüller h , boycott km , müller js , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; rinch, columbus, oh, usa; leibniz-institute für analytische wissenschaften-isas-e.v., dortmund, germany; capes foundation, brazil; department of pediatrics, hacettepe university children's hospital, ankara, turkey; dbbb,university of bari aldo moro, bari, italy; department of neuroscience, the ohio state university, columbus, oh, usa; department of genetics, cheo, university of ottawa, ottawa, canada. while autosomal dominant mutations in gars, encoding the glycyl-trna synthetase, have been identified in patients with charcot-marie-tooth peripheral neuropathy (cmt d) and distal spinal muscular atrophy type v (dsma-v), autosomal recessive mutations cause mitochondrial disease affecting skeletal muscle and heart. gars is a bi-functional enzyme and it is responsible for normal protein translation both in mitochondria and the cytoplasm. in this study we have focused on the mitochondrial function of the gars by investigating a mouse model (gars c r) , human fibroblasts and induced neuronal progenitor cell lines (inpcs). mild mitochondrial abnormalities were detected in skeletal muscle of the gars c r mice while no other tissues were affected. control and patient fibroblasts harboring gars mutation were directly converted into inpcs. we identified tissue specific impairment of mitochondrial function in neuronal cells carrying not only recessive but also dominant gars mutations, suggesting neuron-specific effects of mitochondrial alterations.comparative proteomic analysis of inpcs showed significant changes in mitochondrial proteins. furthermore, the reduction of the vesicle-associated membrane protein-associated protein b (vapb) and its downstream pathways in gars-deficient inpcs suggests that altered mitochondria-associated endoplasmic reticulum (er) membranes (mam) may also contribute to the motor neuropathy. boczonadi v , king ms , bansagi b , roos a , , eyassu f , borchers c , lane m , ramesh v , lochmüller h , pyle a , griffin h , smith ac , chinnery pf , , alan j robinson aj , edmund rs kunji ers , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; mitochondrial biology unit, medical research council, cambridge; leibniz institute of analytic sciences (isas), dortmund, germany; uvic-genome bc proteomics centre, vancouver, canada; paediatric neurology, royal victoria infirmary, newcastle upon tyne foundation hospitals nhs trust, newcastle upon tyne, uk. members of the mitochondrial carrier family (slc ) transport nucleotides, keto acids, amino acids, fatty acids, co-factors and inorganic ions across the mitochondrial inner membrane. several inherited diseases with very variable clinical presentations are associated with dysfunctional mitochondrial carriers. we report a patient with childhood-onset spinal muscular atrophy and mitochondrial myopathy caused by a homozygous mutation in slc a , encoding the mitochondrial oxodicarboxylate carrier (odc). the mutation renders the carrier dysfunctional and, consequently, -oxoadipate cannot be imported into the mitochondrial matrix. computer modelling of the metabolic defect caused by the mutation predicted that the impaired transport leads to accumulation of -oxoadipate, pipecolic acid and the known neurotoxin quinolinic acid, which were precisely confirmed by targeted metabolomics in serum and urine. exposure of -oxoadipate and quinolinic acid reduced the level of mitochondrial complexes in sh-sy y cells in-vitro suggesting a possible pathomechanism. here we demonstrate that -oxoadipate and quinolinic acid are toxic for spinal motor neurons and their increased levels may contribute to neuropathy. and families have been reported. interestingly, most mutations target the same amino acid residue (k e, k m, k n, k t) in the highly conserved -crystallin domain of the hspb protein. the spectrum of diseases caused by mutations in the hspb gene was recently expanded to distal myopathy. hspb is ubiquitously expressed, but is highly expressed in motor neurons and muscles. the hspb is a chaperone that participates in clearing misfolded poly-q containing proteins such as mutant huntingtin and ataxin- involved in respectively huntington's disease and spino-cerebellar ataxia. hspb directly interacts with the co-chaperone bag and their role in chaperone-assisted selective autophagy is well described. to delineate the molecular deficits and functional consequences of hspb mutations we generated a knock-in (ki) mouse model for the k n missense mutation mimicking the neuropathy phenotype. we observed that homozygous mutant mice (hspb k n/k n ) develop a progressive axonopathy, with decreased compound motor action potential amplitudes, and loss of large and medium myelinated axons. this results in locomotor deficits with an impaired performance at the rotarod and grip strength tests. at the ultrastructural level, the hspb -ki model displays severe signs of axon degeneration and a clear myofibrillar myopathy, as observed in some patients with hspb mutations. interestingly, hspb positive aggregates were found in the sciatic nerve and gastrocnemius muscle of our mutant mice. additionally, our model allowed us to generate hspb knock-out (ko) mice using the same targeting vector. strikingly, the homozygous hspb -ko animals do not show any sign of axonopathy and display a much milder myopathy than the hspb -ki animals. these data suggest that part of the pathomechanisms is due to toxic gain-of-function of the mutant protein. boutahar n , reynaud e , nasser y , camdessanché jp , antoine jc . university hospital, saint-etienne, france. dysimmune sensory neuronopathies (snn) depend on neuron cell death induced by an inflammatory reaction in dorsal root ganglia. we have recently identified the intracellular tyrosine kinase (trk) domain of the fibroblast growth factor receptor- (fgfr ) as the target of antibodies in a subset of patients with non paraneoplastic snn. fgfr is one of the four fgfrs and has been involved in sensory neurons maintenance during development and cell death induction after axotomy. fgfrs ligand fixation results in the activation of several intracellular pathways through adaptator protein interactions with the trk domain. in particular ras activation may lead to cell proliferation or apoptosis through erk / or p map kinase signaling. the p mapk pathway is also involved in neuronal cell death induced by nmda and ampa receptor activation. as fgfr is a cell surface protein, human antibodies may interfere with the receptor functioning as a growing number of evidence has showed with other cell surface antibodies in neurological diseases. to test this hypothesis we developed an in vitro model using fvbn mice cortical neurons culture exposed to a rabbit polyclonal antibody reacting with the trk domain of fgfr . comparatively to normal rabbit iggs, the fgfr antibody induced neuron cell death in a dose dependent manner. neuron cells were exposed to fgfr antibody concentration leading to - % cell death in absence or presence of the p mapk inhibitor sb and the expression of fgfr , glur subunit of ampa receptors and nr subunit of nmda receptor was measured by quantitative rt-qpcr. the fgfr antibody induced an upregulation of fgfr while the glur and nr subunits were modulated. these changes were prevented in presence of sb . these preliminary results indicate that anti-fgfr iggs may interfere with the functioning of the intracellular domain of the protein and the expression of nmda and ampa receptors through the p map kinase pathway. this model may be used to test the effect of human anti-fgfr iggs in vitro. braathen gj , tveten k , holla Øl , busk Øl , hilmarsen ht , svendsen m , høyer h . section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. next-generation sequencing (ngs) has during the last years entered the clinical diagnostics. ngs has proven to be very efficient in the diagnostics of disorders where multiple genes can be involved. our ngs-based targeted gene panel consists of hereditary neuropathy genes, i.e. mostly charcot-marie-tooth genes. this study is a retrospective study of clinic samples received between may and february . we describe the identified novel likely pathogenic sequence variants, according to international guidelines. in this period we identified novel, not previously described, likely pathogenic sequence variants in the following genes: aars, fgd , gan, hint , litaf, lrsam , mme, mpz, nefl, pmp , sbf , sh tc and yars. there is now a large range of genes causing hereditary peripheral neuropathies and many likely pathogenic sequence variants. likely pathogenic sequence variants are not only identified in old well established neuropathy genes but also in the newer genes like mme. modelling brown-vialetto-van laere syndrome in c. elegans mutations in slc a and slc a , which encode the riboflavin transporters rfvt and rfvt . patients with rfvt deficiency exhibit proximal and distal limb weakness, sensory ataxia, diaphragmatic paralysis, optic atrophy, sensorineural deafness and bulbar palsy. riboflavin is critical for the biosynthesis of flavin mononucleotide and flavin adenine dinucleotide, essential cofactors for carbohydrate, amino-acid and lipid metabolism. mutations in slc a reduce or abolish rfvt expression resulting in impaired riboflavin uptake into sensory and motor neurons. high-dose riboflavin treatment can improve or stabilise a patient's condition, however the optimum dose and long term effects of riboflavin treatment, and disease pathomechanisms remains poorly understood. to further understand the pathophysiological consequences of slc a mutations, we propose developing an animal model for bvvl. caenorhabditis elegans (c. elegans) are small round transparent nematodes extensively used for studying the genetics and molecular biology of neurodegenerative diseases. there are two c. elegans riboflavin transporter genes, rft- and rft- . based on protein sequence homologies and expression profiles for both genes, rft- is the ortholog of rfvt . the expression of rft- is regulated by riboflavin availability and knock-down of the rft- gene by sirna perturbs c. elegans development. our aim is to develop a knock-in c. elegans model of bvvl. human rfvt and c. elegans rft- protein sequences were aligned with clustal omega to identify conserved amino acid residues associated with bvvl mutations. the amino acid involving the l p rfvt mutation is conserved in rft- (residue l ). to create our model, we will introduce the l p rft- mutation into the rft- locus in c. elegans genomic dna using crispr/cas- technology. this bvvl c. elegans model will allow us to explore the pathogenic consequences of rfvt deficiency underlying motor nerve degeneration and to evaluate drug therapy regimes by determining the optimal riboflavin dose and treatment initiation, and trialing other compounds that may improve benefits seen with riboflavin supplementation. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune-mediated inflammatory disease of the peripheral nervous system. cidp can present chronic progressive or relapsing-remitting courses and can predominantly affect motor but also sensory nerve fibers causing weakness of proximal and distal muscles. it represents the most common chronic autoimmune neuropathy and is pathologically characterized by focal inflammatory-mediated demyelination followed by axonal degeneration. recently, we have developed a new animal model for cidp, the chronic-ean, induced in lewis rats by active immunization with s-palmitoylated p ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) peptide. this model fulfills electrophysiological criteria of demyelination with axonal degeneration, a result confirmed by immunohistopathology. the late phase of the chronic disease is characterized by an accumulation of il- + cells and macrophages in sciatic nerves and as well as high serum il- levels. it is a reliable and reproducible animal model for cidp that can now be used for translational drug studies for chronic human autoimmune-mediated inflammatory diseases of the peripheral nervous system, particularly cidp, for which, there is a real need for new immunotherapies. the aim of this study was to test the therapeutic efficacy of ivig and a recombinant fc fragment (fcrec) in this new cidp animal model. treatments with ivig and fcrec proved effective in preventing further progression of cidp in rats. the therapeutic treatments not only decreased the maximal clinical scores of the cidp rats compared to albumin treatment but also abolished the disease chronicity. interestingly, a better efficacy of fcrec treatment compared to ivig was demonstrated at histological level, with the myelinated fibers well preserved and the greatly reduced accumulation of macrophages and il- + cells in sciatic nerves. ivig and fcrec therapeutic activities in this model can also be followed by measurement of antibodies in the serum and could therefore be used as biological markers. the current study provides for the first time direct evidence that ivig is effective in the treatment of cidp rats and suggests that a novel fcrec compound is more effective than ivig. it will contribute to the development of more effective and safer drugs for the treatment of autoimmune peripheral neuropathies like cidp. each time, one measurement with supramaximal stimulation according to the conventional ncs and another measurement with % enhanced stimulation intensity were conducted. we analyzed the results with special consideration of minimal and maximal fiber velocities and of the velocity with maximal fiber proportion. as a result, we detected statistically significant differences between cidp patients and controls in all these parameters. compared with controls, cidp patients had slower minimal and maximal fiber velocities and also the spectrum of motor conduction velocities was definitively shifted to slower velocities. slower minimal conduction velocities could be detected in some cidp patients by using the enhanced stimulation intensity. interestingly, in some cidp patients the conventional ulnar ncs were normal, but the collision technique showed fibers with a conduction velocity of less than m/s, indicating demyelination. to our knowledge, this is the first study using hopf's collision technique systematically in cidp patients. significant differences between the variation of ulnar motor nerve conduction velocity of cidp patients and controls could be detected. this method also showed signs of demyelination in some cidp patients with normal ulnar ncv. by enhancing stimulation intensity above the threshold of supramaximal stimulation in conventional ncs, the collision technique may be even more valuable. the clinical course of guillain-barré syndrome (gbs) is highly variable and some patients may develop treatment-related fluctuations (trfs) as an indication of ongoing disease activity and temporary treatment effect. other patients present as gbs, but subsequently develop repetitive relapses as an indication of acute-onset chronic inflammatory demyelinating polyneuropathy (a-cidp). this distinction is important because treatment may differ. we determined the frequency and clinical presentation of patients with gbs-trf and a-cidp in the first patients included in the international gbs outcome study (igos) with a follow-up of at least months. thirty-eight patients ( %) were excluded because of alternative diagnoses. of the remaining patients, ( %) had at least one trf, ( %) had a-cidp. preliminary analysis showed no significant differences between the groups (total-gbs, gbs-trf and a-cidp) for sex, age, sensory symptoms, cerebrospinal fluid results and mechanical ventilation. a-cidp patients had a median age of years (iqr - ), % was male, and all patients were treated in the acute phase of the disease (intravenous immunoglobulins (ivig) ( ), plasma-exchange (pe) ( ) or methylprednisolone (mp) ( )). gbs-trf patients has a median age of years (iqr - ), % was male and all patients received treatment (ivig ( ) and pe ( ). gbs-trf patients showed more antecedent events ( % versus %, p= . ), a higher gbs disability score (≥ ) at nadir ( % versus %, p= . ) and less frequently developed ataxia ( % versus %, p= . ) than patients with a-cidp. onset to nadir was longer in a-cidp than in gbs-trf ( days (iqr - ) versus days (iqr - ), p= . ) and the total gbs group ( days (iqr - ), p= . ). the time until the first clinical deterioration tends to be longer in the a-cidp patients (median days (iqr - ) versus median days in the gbs-trf group, not significant). the diagnosis a-cidp was made after a median of days . in conclusion, this first analysis identified distinctive characteristics of gbs-trf and a-cidp in support of a different pathogenesis that may help with early identification of these disorders in clinical practice. additional results will be presented at the conference. chronic inflammatory demyelinating polyneuropathy (cidp) is a disorder with a highly diverse clinical presentation, electrophysiological phenotype, response to treatment and outcome. this heterogeneity may indicate the presence of distinct subtypes of cidp, which may have a different pathogenesis and require more personalized treatment. the international cidp outcome study (icos) is a prospective, observational, international multi-center study that aims to describe this variation in clinical and electrophysiological subtypes and to define the clinical and biological determinants of these subtypes, disease activity, treatment response and outcome. in addition icos aims to provide an infrastructure for conducting new (therapeutic) studies in cidp, similar to the international gbs outcome study (igos). all patients fulfilling the efns/pns ( ) diagnostic criteria for cidp can be included in icos, independent of age, duration and severity of disease or treatment. we collect information on neurological deficits, diagnostic characteristics, various validated clinical outcome measures, previous and current treatment and we collect biomaterials (dna, cerebrospinal fluid, nerve biopsies and repeated serum samples). icos was started as a pilot study in dutch university centers. by february , patients were included in icos, patients recently diagnosed with cidp and previously diagnosed patients. included were ( %) males and ( %) females with a median age of years (iqr - ). the current cohort consists of classic (sensory-motor) cidp, madsam and pure motor variants. of the patients diagnosed in the past, patients ( %) were treated (intravenous immunoglobulins (ivig) ( ), prednisolone ( ), subcutaneous immunoglobulins (scig) ( ), dexamethasone ( ), plasma-exchange ( ) and ivig with methylprednisolone (mp) ( )). the recently diagnosed patients all received treatment (ivig ( ), dexamethasone ( ), plasma-exchange ( ) and patients were treated in a pilot study with ivig with methylprednisolone (mp)). the protocol has been evaluated and adjusted and will be shared with other researchers via the inflammatory neuropathy consortium and igos consortium. our aim is to include at least cidp patients worldwide with a minimum follow-up period of years. dominant mutations in glycyl trna synthetase (gars) cause inherited axonal neuropathy (charcot-marie-tooth type d). mutations in the mouse gars gene cause a similar phenotype, and represent valid disease models. we routinely use two mouse strains, one with a severe neuropathy, and one with a milder, later onset neuropathy. using these mouse models, we are exploring the mechanisms through which gars mutations cause peripheral axon degeneration. efforts include ribosome tagging to isolate ribosome-associated mrnas specifically from motor neurons, and non-canonical amino acid tagging to visualize and isolate newly synthesized proteins. taking advantage of the anatomy of motor neurons, we are able to analyze cell bodies separately from peripheral axons in both of these approaches. in addition, we are using these mouse models for preclinical studies testing gene therapy approaches to treat cmt d. consistent with our previous genetic studies in mice, knockdown of the mutant transcript, while preserving sufficient levels of the wild type, is a very successful approach when administered before the onset of neuropathy. we are now testing this approach in mice after the onset of symptoms, and in mice carrying a mutant gars allele associated with human disease. these studies are potentially translational, and also address mechanistic questions such as the timing and cell autonomy of the pathophysiology. burnor e , yang l , hao z , patterson k , quinn c , scherer ss , lancaster e . the university of pennsylvania, philadelphia, pa, usa; the second xiangya hospital, central south university, hunan, china. autoantibodies to two isoforms of neurofascin (nf and nf ) have been reported in patients with guillain-barre syndrome (gbs) or chronic inflammatory demyelinating neuropathy (cidp). it is not clear which of these responses reliably distinguish autoimmune neuropathies from other severe neuropathies, which responses are transient versus persistent, or which responses may target multiple isoforms of neurofascin. in addition, approximately % of neuropathy patients have no known cause, and it is unknown whether a subset of these patients may have autoantibodies to neurofascins. we have studied cohorts of patients with autoimmune neuropathy (n= ), genetic neuropathy (n= ), and idiopathic neuropathy (n= ) for igg and igm responses to neurofascins. neurofascin antibodies were found in ( %) of patients with autoimmune neuropathy, and ( . %) idiopathic neuropathy patients, but only % ( of ) in patients with genetic neuropathy. follow-up serum samples were available for positive cases. persistent responses were associated with chronic neuropathy while transient responses were seen in gbs or with remission of cidp. most patients had responses specific to either nf of nf . however, a particularly severe, treatment-resistant form of cidp, approaching a locked-in state, was seen in a patient with a unique response to all three isoforms of neurofascin (nf , nf , nf ). treatment of this patient with rituximab resulted in clinical improvement and resolution of the neurofascin antibody response. in conclusion, autoantibodies to neurofascins distinguish autoimmune neuropathies from severe genetic neuropathies, but the clinical phenotype may depend on the persistence and isoform specificity of the immune response. antibodies to the common domains shared by nf and nf may portend a severe but treatable neuropathy. a subset of idiopathic neuropathy patients may have an autoimmune mechanism. burns j , , sman ad , cornett kmd , wojciechowski e , , walker t , menezes mp , , mandarakas mr , rose kj , , bray p , , sampaio h , farrar m , , refshauge km , raymond j and the fast study group. university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia; university of new south wales, sydney, australia. exercise has undisputed benefits for human health and potentially as a treatment for neuromuscular disease. but there is also a risk of harm due to overwork weakness. we report the results of a -month randomised, double-blind, sham-controlled trial evaluating progressive resistance exercise of foot dorsiflexor muscles in pediatric cmt. sixty patients ( cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx ) aged - years were randomly assigned to undergo -weeks ( sessions) of moderate-intensity progressive resistance exercise or sham exercise. the primary endpoint was change in isometric dorsiflexion strength between groups assessed by hand-held dynamometry (expressed as a z-score based on age-and sex-matched normative reference values, positive values indicate an improvement in strength). the primary safety endpoint was change in muscle and fat volume of the muscles responsible for dorsiflexion by mri between groups. secondary outcomes were function (balance, long jump, -min walk test), walking ability ( d gait analysis), self-reported ankle instability, parent-reported quality of life (physical summary, psychosocial summary and global impression of change scores), and adverse events. fifty-five ( %) children completed the trial. adherence was comparable between exercise ( %) and sham ( %) groups. while patients experienced muscle soreness during training in the exercise group, adverse events did not differ between groups. the mean z-score for dorsiflexion strength increased in the exercise group by % at -months (from − . ± . to − . ± . ) and decreased in the sham group by %, mirroring the natural history of cmt (from − . ± . to − . ± . ). between-group ancova-adjusted difference at -months was . ( %ci, . to . ; p= . ). the mean scaled scores for mri muscle volume and fat volume were comparable between groups at months (p> . ). the global impression of change scores favoured the exercise group at -months (p= . ) and -months (p= . ). there was no other measurable effect of exercise. pre-specified subgroup analyses according to age ( - years vs. - years) showed a larger treatment effect with exercise in adolescents. targeted progressive resistance exercise was effective at halting progression of dorsiflexion weakness without detrimental effect on muscle morphology or other signs of overwork weakness in children with cmt. byung-nam y , yoon-ho h , suk-won a , seok-jin c , jung-joon s . inha university hospital, incheon, south korea; seoul metropolitan government boramae medical center, seoul national university, seoul, south korea; joong ang university hospital, seoul, south korea; seoul national university hospital, seoul, south korea. a years old female patient visited the hospital with weakness in both upper limbs from months under the suspicion of motor neuron disease with the finding of extensive denervation changes on electromyography. whole spine mri showed ventral nerve rootlet enhancement in the c and t bundles. the emg had confirmed active denervation changes in the muscles innervated by bilateral c ∼c roots. the cerebrospinal fluid culture test showed a protein (csf) elevation to mg / dl. she got a steroid pulse therapy. she had the symptoms of dry mouth and dry eyes during the recent years. a salivary gland scan test was performed for the possibility of sjögren's syndrome, and as a result, absorption of the contrast agent in both parotid and submandibular glands was decreased. the shammer test showed mm on the right side and mm on the left side. she was diagnosed of sjögren's syndrome. after weeks, overall strength improvement was observed, and bilateral shoulder abduction was improved to mrc grade or higher. the follow-up spine mri also showed that the initially seen ventral nerve root enhancement was disappeared. the case had visited the hospital with major symptoms of weakness and atrophy of the muscles, showing similar pattern to motor neuron disease, and was diagnosed as inflammatory polyradiculopathy and confirmed as primary ss during differential diagnosis. this case suggests that primary ss may induce inflammatory polyradiculopathy, which shows motor symptoms as major symptoms rather than sensory symptoms, and that a fast and accurate diagnosis is needed in terms that it can be treated with steroids and appropriate immune suppressive agents. traumatic injuries to peripheral nerves are frequent. however, effective pharmacological treatments are lacking. curcumin, a polyphenol found in rhizomes of curcuma longa, has been shown to develop antioxidant, anti-inflammatory, and neuroprotective properties. however, due to its poor hydrosolubility and its extensive metabolism, the use of large curcumin doses is required for therapeutic purpose. the aim of the present study was to investigate the effects of a local infusion of a low curcumin dose on nerve regeneration and functional recovery after sciatic nerve injury in rats. the experiments were conducted in g sd male rats submitted to unilateral sciatic nerve crush at d . curcumin was solubilized in polyethylene glycol and continuously administered using osmotic pumps ( . mg/day until d ) with a catheter delivering the drug near the lesion site. functional analyses using von frey, beam walking, static sciatic index (ssi) and grip strength tests were carried out at d (reference test) and every week after injury (d , d , d , d and d ). in addition, an evoked electromyogram was performed at d and d . after euthanasia (d ), nerve and muscle samples were collected and analyzed by light and electron microscopy. functionally, a significant improvement of the mechanical sensitivity (+ %) was observed at d in the curcumin-treated group (n= ) vs. vehicle group (n= ). in curcumin-treated group, skillful walking and finger spacing of the ipsilateral paw (ssi) were fully restored respectively at d and d contrary to vehicle group. furthermore, curcumin treatment improved the grip strength recovery (+ % at d ). the electrophysiological results indicated a full recovery of motor nerve conduction velocities (mncv) after days of curcumin treatment, while mncv remained altered in vehicle group ( % of the mncv at d ). morphometric analysis of nerve sections using g-ratio showed an improvement in the thickness of the myelin sheath in curcumin treated animals (+ % vs. vehicle group). histological investigation of gastrocnemius muscle indicated decreased neurogenic lesions in curcumin group. proteomic analysis is currently under investigation to understand the mechanisms involved in curcumin effects. our data could lead to the development of new therapeutic strategies in peripheral nerve regeneration using low doses of curcumin. previous studies suggest that the metabolic syndrome (mets) is associated with distal symmetrical polyneuropathy (dsp), and that diabetes, pre-diabetes, and obesity are the main metabolic drivers. the aim of this study is to investigate the association of mets components with retinal and cognitive function in a bariatric surgery cohort prior to surgery. patients were recruited from the adult bariatric surgery clinic at the university of michigan and lean controls from a research website (no mets components based on ncep/atpiii definition). participants underwent extensive metabolic phenotyping including a glucose tolerance test and fasting lipid profile. dsp was defined using the toronto consensus definition of probable clinical neuropathy. retinal function was measured with frequency doubling technology perimetry (average mean deviation), and cognitive function with the nih toolbox (composite score). univariate linear regression models were used to evaluate the association between mets components and retinal and cognitive function. to date, we have recruited bariatric surgery participants and lean controls. in the bariatric population, the mean (sd) age was ( . ) with % female compared with a mean age of . ( . ) with % female in the lean group. in the bariatric group, . % had diabetes, . % pre-diabetes, and . % normoglycemia. the dsp prevalence was % in lean controls, . % in normoglycemic, . % in pre-diabetic, and . % in diabetic bariatric participants (p< . for trend). retinal function was . ( . ), − . ( . ), − . ( . ), and − . ( . ) (p= . for trend), and cognitive function was . ( . ), . ( . ), . ( . ), . ( . ) (p= . for trend) in these same groups for lean controls, normoglycemics, pre-diabetics and diabetics, respectfully. pre-diabetes (− . , %ci: − . ,- . ) was the only mets component associated with retinal function, and waist circumference was the only one associated with cognitive function (− . , %ci − . ,- . ). dsp and retinal function, but not cognitive function decline with worsening glycemic status. similar to previous data for dsp, pre-diabetes and obesity are associated with retinal and cognitive function respectively. interestingly, while clinical dsp is common in this population, clinical retinopathy and dementia are not, indicating that dsp may be the first metabolic complication in the morbidly obese. and anti-cntn antibody-positive cases were confirmed by cba and were of igg subclass in half of them. by cba we identified additional / ( %) anti-caspr seropositive patients, whose isotype is currently being tested. sera of anti-nfasc and anti-cntn igg seropositive patients and patients with anti-caspr antibodies stained paranodes by indirect immunofluorescence on mouse teased nerve fibers. of note, seronegative patients for known antibodies showed reactivity against node and/or paranodes. compared to other seronegative cipd patients, seropositive patients had more frequently subacute onset of the neuropathy and a younger age at onset, particularly for nfasc or caspr antibodies. weakness was more severe and was often associated with proprioceptive loss, sensory ataxia and tremor. neuropathic pain was not a feature of caspr -seropositive patients. frequent findings were increased distal motor latencies and temporal dispersions on nerve conduction study and a higher protein level in csf. finally seropositive patients tended to have a higher disability and showed worst response to ivig. rituximab was effective in one patient with anti-nfasc antibodies and two patients with anti-cntn antibodies showed good and persistent recovery after cyclophosphamide. prevalence of antibodies was % in italian cidp patients and their presence was associated with distinctive clinical features. their determination, followed by characterization of igg subclass in positive cases, has clear clinical impact, by helping to guide therapeutic choices. the reactivity against nodal and paranodal components in sera from patients without known antibodies suggests that other targets could play a role in the autoimmune response in cidp and they still need to be identified. mutations in amynoacyl trna synthetases (arss), enzymes that catalyse the covalent attachment of amino acids to their cognate trna, are responsible for autosomal dominant cmt , intermediate cmt (cmt-i) and dhmn. we report the case of a male of italian ancestry who first presented with bilateral ankle clonus at three months, followed by toe walking and ankle instability. the ankle clonus subsided during adolescence. in the third decade he developed progressive walking difficulties followed by distal sensory loss. neurological examination at the age of revealed a steppage gait, distal lower limb weakness, decreased pinprick to the ankles, and reduced vibration sensation at the knees. reflexes were brisk in the upper limbs, reduced at the knees and absent at the ankles. muscle tone was increased in the lower limbs and plantar responses were extensor. nerve conduction studies revealed an axonal neuropathy. brain and spinal cord mri were normal. sanger sequencing of pmp , gjb , mpz , gdap and mfn were negative. sureselect focused exome sequencing (agilent technologies, santa clara ca, usa) demonstrated a c. g>a, p.arg his mutation in aars. the p.arg his mutation in aars has previously been reported in families with intermediate or axonal motor-sensory neuropathy, and in one case was associated with sensory-neuronal deafness. cns involvement has not previously been described with this mutation. mutations in aars have been associated with a range of phenotypes including cmti, cmt and dhmn with variable age on onset ranging from to years (mean years). of note, the aars p.gly arg mutation has been reported in a family with cmt and pyramidal signs. this study provides further evidence that pyramidal tract involvement can be an early feature of cmt n due to mutations in aars, further expanding the spectrum of arss-associated phenotypes. small fibres in the skin are vulnerable to damage in metabolic or toxic conditions such as diabetes mellitus or chemotherapy resulting in small fibre neuropathy and associated neuropathic pain (np). whether injury to the most distal portion of sensory small fibres due to a primary dermatological disorder can cause np is still unclear. recessive dystrophic epidermolysis bullosa, (rdeb) is a rare condition in which mutations of proteins of the dermo-epidermal junction lead to cycles of blistering followed by regeneration of the skin. damage is exclusive to the skin and mucous membranes, with no known direct compromise of the nervous system. it is increasingly recognised that most rdeb patients experience daily pain, the aetiology of which is unclear but may include inflammation (in the wounds), musculoskeletal (due to atrophy and retraction scars limiting movement) or np. in this study we investigated the incidence of np and examined the presence of nerve dysfunction in rdeb patients. around three quarters of patients presented with pain of neuropathic characteristics which had a length dependent distribution. quantitative sensory testing of the foot revealed striking impairments in thermal detection thresholds combined with an increased mechanical pain sensitivity and wind up ratio (temporal summation of noxious mechanical stimuli). nerve conduction studies showed normal large fibre sensory and motor nerve conduction however skin biopsy showed a significant decrease in intraepidermal nerve fibre density. autonomic nervous system testing revealed no abnormalities in heart rate and blood pressure variability however the sympathetic skin response of the foot was impaired and sweat gland innervation was reduced. we conclude that chronic cutaneous injury can lead to injury and dysfunction of the most distal part of small sensory fibres in a length dependant distribution resulting in disabling np. these neuropathic pain (np) following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons which begins to normalise over time. we investigated the composition and distribution of shaker type potassium channels (kv channels) within the nodal complex of myelinated axons following injury. at the neuroma that forms after damage, expression of kv . and . (normally localised to the juxtaparanode) was markedly decreased. in contrast kv . and . , which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in the rat and in humans. within the dorsal root (a site remote from injury) we also noted a redistribution of kv channels towards the paranode. blockade of kv channels with dtx after injury reinstated hyperexcitability of a-fibre axons and enhanced mechanosensitivity. changes in the molecular composition and distribution of axonal kv channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. members of the francophone anti-mag cohort group are listed in "appendix". appendix polyneuropathy with anti-mag igm antibodies is classically progressive, predominantly sensory, and distal with ataxia and sometimes postural-intention tremor. we assessed clinical, biological, electrophysiological, and histopathological features in patients with igm gammopathy and anti-mag antibody titres higher than btu. we focused on characteristics of patients according to the anti-mag antibody titres at diagnosis. we retrospectively and prospectively analysed standardized report forms of patients from fourteen french-speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset and last follow-up was . years ( . - . ). about . % of patients presented with a "variant" clinical phenotype independently of anti-mag titres (< or ≥ btu). this included acute or chronic sensorimotor polyradiculoneuropathies, paucisymptomatic sensory polyneuropathy and multifocal neuropathy. at the most severe disease stage, . % of patients were significantly disabled. anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in respectively , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. sixteen percent of patients did not meet the electrodiagnostic criteria of definite demyelinating neuropathy, independently of anti-mag titres (< or ≥ btu). nerve biopsy, performed in nineteen patients, provided support to the diagnosis of anti-mag neuropathy in some particular issues (low titres of anti-mag, unusual clinical or electrophysiological phenotype). we assessed the degree of probability (probable, possible or uncertain) that patient neuropathies are directly related to anti-mag antibodies, according to anti-mag titre, electrophysiological data and nerve biopsy characteristics if available. it appears uncertain in patients with low anti-mag titres ( . % of the whole population). the clinical phenotype didn't appear to be different according to anti-mag antibody titre. many of the patients with low anti-mag titres presented "genuine" anti-mag neuropathy as demonstrated by edx studies, clinical presentation and sometimes nerve biopsies. for a small proportion of these patients, a direct relation between neuropathy and anti-mag antibodies is uncertain due to atypical clinical presentation, axonal neuropathy pattern or nerve biopsies, and positivity of antigangliosides antibodies. we assessed therapeutic management, response to immunotherapies and adverse events in a cohort of patients presenting with igm gammopathy and anti-mag antibody titres higher than btu. we retrospectively and prospectively analysed standardized report forms of patients from french speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset to diagnosis and last follow-up were respectively . ( - ) and . years ( . - . ). anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. seventy eight percent (n = ) of patients received immunotherapies. transient response to ivig or plasma exchanges at six month was observed in less than and % of patients respectively. chemoimmunotherapies and rituximab were more frequently administered in the group of patients with malignant hemopathies (n = ) compared to mgus (n = ) (mean lines of therapy = . , range - , sd . versus . , range - , sd . , p = . ). more than % of patients (n = ) received rituximab monotherapy. clinical worsening, mostly transient and reversible, was observed in / patients after rituximab. clinical response to rituximab at months and/or during - months follow-up period was observed in . % of patients, and correlated with anti-mag titre ≥ btu ( / responder versus / non-responder patients, p = . ). at - months follow-up, responder patients presented shorter symptom duration compared to non-responders, though not significant after logistic regression ( . years, range . - . , sd . in responder patients versus . years, range . - . , sd . , in non responder patients, p = . ). in some cases, electrodiagnostic studies were recorded during rituximab treatment follow-up and showed in responder patients a clear improvement of motor conduction velocities. these data may support another clinical trial to study benefice of rituximab in anti-mag neuropathies early in the disease. it raises issues about the value of incorporating electrodiagnostic parameters as end-points. schwann cells (scs) are essential for axon integrity and myelination in the uninjured pns. after pns injury, scs function as "first responders" , undergoing phenotypic re-programming and orchestrating many processes that lead to functional nerve repair. receptors in scs that contribute to sc repair programs remain incompletely understood. we identified a member of the ionotropic glutamate receptor (igr) family, n-methyl-d-aspartate receptor (nmda-r), in scs that is upregulated after nerve injury and acts as a co-receptor with ldl-receptor related protein (lrp ). lrp is a well-known regulator of the sc response to pns injury. herein, we used pcr to profile igr expression in cultured rat scs. obligate receptor subunits required for assembly of nmda-rs, ampa-rs, and kainate receptors were identified. treatment of rat scs with - microm glutamate or . - . microm nmda robustly activated akt and erk / . the response was transient and bimodal; glutamate concentrations greater than microm failed to activate cell-signaling. phosphoprotein profiling demonstrated other cell-signaling and transcription factors regulated by glutamate in rat and human scs, including p s -kinase, glycogen synthase kinase- , ribosomal s kinase, c-jun, and creb. activation of cell-signaling by glutamate in scs was blocked by eliminating calcium from the medium, by the selective nmda-r antagonist, mk , and by genetic silencing expression of the obligate nmda-r nr subunit. phosphoinositide -kinase/pi k functioned as an essential upstream activator of both akt and erk / in glutamate-treated scs. by activating pi k and erk / , glutamate promoted sc migration. glutamate ( microm) or nmda ( microm) injected into crush-injured sciatic nerves robustly activated p-erk / in both myelinating and non-myelinating scs in vivo. these results identify igrs as potentially important cell-signaling receptors in scs that may promote axon-glial interactions. understanding the function of sc nmda-r is important given current efforts to develop nmda-r-targeting drugs for patients with pain, depression, and alzheimer's disease. while frequently overlooked in a therapeutic context, scs are extremely important in the pathogenesis of chronic neuropathic pain. if these drugs modulate the activity of sc nmda-r and sc physiology, the response to pns injury may be altered and the possibility that neuropathic pain develops increased. candayan a , atkinson d , durmus tekce h , parman y , jordanova a , battaloglu e . department of molecular biology and genetics, bogazici university, istanbul, turkey; center for molecular neurology, antwerp university, antwerp, belgium; istanbul medical school, istanbul university, istanbul, turkey. charcot-marie-tooth (cmt) disease is a group of inherited peripheral neuropathies affecting one in individuals worldwide. the disease presents both clinical and genetic heterogeneity. so far, mutations in genes and loci are associated with cmt with autosomal dominant, autosomal recessive, x-linked and mitochondrial inheritance. despite the advances in genetic testing, approximately % of all cmt patients worldwide remain without a molecular diagnosis. we have investigated unrelated cmt patients of turkish descent, in all of which pmp duplication has been excluded previously. we used multiplex amplification of specific targets for resequencing (mastr) assay to sequence exonic regions of common cmt genes. recurrent mutations were identified in cases in mfn , gjb , mpz and hint genes. we have also identified novel variants in cases in mfn , pmp , gars, aars, ighmbp and gdap genes, all of which are very rare or not present in the variation databases and are predicted to be pathogenic by in silico tools. familial segregation analyses are ongoing for novel variations. mfn and gjb genes were the most commonly mutated causative genes in this cohort. cases without molecular diagnosis after the mastr testing are candidates for further analyses such as whole exome sequencing or whole genome sequencing. outcomes of the current study and our previous experience with turkish cmt patients suggest a high genetic heterogeneity. our insight is that different genetic strategies or larger panels are essential to determine the causes underlying cmt especially in regions where rare recessive types of the disease can be observed due to high frequency of consanguineous marriages. capoccitti g , giannini f , ginanneschi f , casali s , insana l , rossi a . university of siena, siena, italy. several efforts have been made to elaborate new electrophysiological criteria for early diagnosis of guillain-barré syndrome (gbs) and to differentiate demyelinating (aidp) from axonal (aman/amsan) forms. the aims were to verify the diagnostic power of total cmap (tcmap) duration, firstly applied in gbs field. this parameter was compared with commonly used neurophysiological measures, including negative phase cmap duration (ncmap), and was added to modified rajabally criteria. we reviewed the clinical and electrophysiological data of patients with gbs (level or of brighton clinical criteria). each patient underwent at least two neurophysiological studies, the first within weeks, the second between and weeks from symptom onset. at least four motor and three sensory nerve conduction studies were recorded for each test. regarding early diagnosis, the binary logistic regression model with multiple variables, including ncmap duration, showed that the features of predictive model presenting greater significance (p < . ) were tcmap duration, sural sparing and a-waves. among these, tcmap duration showed greater significance (p = . ). the tcmap was diffusely prolonged in aidp compared to aman/amsan, already in first examination and confirmed in the second one. roc analysis for tcmap duration in aidp vs. aman/amsan showed: cut-off . ms, auc . , ppv . %. we propose the tcmap duration as a new useful electrophysiological measure for early diagnosis of "generic" gbs and for early differentiation between aidp and aman/amsan. moreover, the prolongation of tcmap, the presence of a-waves and sural sparing represent a strongly diagnostic predictive triad of aidp. aifm (apoptosis-inducing factor, mitochondrionassociated- ) has captured great attention from biomedical researchers due its critical role in the regulation of cell apoptosis. this flavoprotein is typically located in the mitochondrial intermembrane space where it is associated with respiratory chain complex-i. upon a cell-death insult, aifm is cleaved into a kd protein that is released into the cytosol. the kd peptide may enter the nucleus to trigger chromosome condensation and fragmentation, initiating a caspase-independent pathway of apoptosis. however, this nuclear translocation may be blocked by cytosolic heat-shock protein- (hsp ) that binds with the fad domain (aa - ) of aifm . mutations in aifm gene have resulted in several clinical phenotypes, including a family with cmtx (glu val). clinical deficits in these patients usually involve multiple organs. in this study however, we identified a family with a novel missense mutation (phe leu) in aifm that developed a late-onset sensory motor axonal polyneuropathy by nerve conduction criteria. the proband and affected siblings exhibited distal muscle weakness and atrophy with normal cognitive and cranial nerve functions. there was no obvious phenotype from other organs. interestingly, this phe leu mutation affects a highly conserved amino acid at the center of the fad domain. we hypothesize that this mutation impairs the binding between aifm and hsp , leading to an enhancement of cell-death signaling. this family therefore provides a unique opportunity to explore how altered apoptotic signaling affects peripheral nerve system. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr chronic inflammatory demyelinating polyneuropathy (cidp) can be the key symptom leading to diagnosis of associated lymphoma. patients with diagnosis of cidp according to efns/pns criteria associated with b cell lymphoproliferative disorders (bld), in one center, between november and november were included. demographical, clinical, nerve conduction, immunological, histological data and response to treatment were recorded retrospectively. eight patients ( men), median age yo were included. onset of polyradiculoneuropathy was either chronic (n= ) or acute (n= ). neurological condition led to diagnosis of bld in all but one case, because of onset (n= ) or worsening of neuropathy (n= ). clinical presentation was that of cidp in patients or pseudo-canomad in and plexopathy in one. lymphoma type was: lymphoplasmacytic (n= ), diffuse large b cell (n= ), small lymphocytic (n= ), marginal zone (n= ), unclassified small b cell (n= ). only patients presented with lymphadenopathies. bld was diagnosed in all patients on myelogram or bone marrow biopsy, performed because of cytopenia (n= ), atypical (n= ) or severe (n= ) neuropathy. monoclonal gammapathy was identified in / patients (igm n= , igg n= ). neuromuscular biopsy was performed in patients and disclosed endoneural infiltration in . anti-neuronal or antigangliosides antibodies were positive in patients. none of the patients presented a systemic autoimmune disease, hemolytic anemia associated with bld (n= ). immunomodulating treatment was administered in all patients (ivig n= , plasma exchange n= , steroids n= ) and immunosuppressants (n= ). immunochemotherapy for lymphoma was initiated because of lymphoma type or severity in cases, in cases because of the associated neuropathy. median follow-up was of months after treatment initiation. four out of patients treated within months of neurological onset improved as well as one out of patients whose preexisting neurological condition had worsened. two patients presented neurological relapse during progression of lymphoma. two patients died. unusual presentation of cidp -i.e., rapid progression or treatment failure -should lead to further testing for associated lymphoma. because general symptoms and lymphadenopathy often lack, diagnosis requires analysis of bone marrow with lymphocyte phenotyping. early treatment with immunochemotherapy was associated with better prognosis in our series. cardiac scintigraphy is a useful tool for the diagnosis, prognosis and pre-symptomatic early detection of familial amyloidosis-associated neuropathies three main types of familiar amyloidosis: transthyretin (ttr), apolipoprotein a and gelsolin. cardiac involvement is a leading cause of morbidity and mortality; one new described mutation strongly related with isolated cardiac amyloidosis is the ttr val ile. the discovery of tests that allow early diagnosis of cardiac involvement in amyloidosis and to infer about the etiology of the disease is of major importance. in a cohort of patients with different types of familiar ttr amyloidosis, we aimed to assess the role of mtc- , -diphosphono- , -propanodicarboxylic acid ( mtc-dpd) in detecting myocardial amyloid infiltration. we enrolled four patients diagnosed with late familiar amyloidosis, which mutations were documented at deoxyribonucleic acid analysis: three patients with ttr val met mutation and one patient with ttr val ile mutation. three patients were asymptomatic for cardiac involvement and one patient (val ile mutation) had a previous diagnosis of heart failure. myocardial uptake of mtc-dpd scintigraphy was semiquantitatively and visually assessed at five minutes and three hours.the uptake of mtc-dpd highly demonstrated amyloid in cardiac area in two out the three cases of ttr val met and in ttr val ile. ttr val ile case presented the highest uptake due to the exclusive deposition of amyloid in cardiac area resulting in severe heart failure. in hereditary transthyretin-related amyloidosis, including the mutations ttr val met and val ile, mtc-dpd cardiac scintigraphy can identify infiltration even in asymptomatic individuals, allowing an early diagnosis of cardiac compromise in this group of diseases. we can consider that this non-invasive test would be a tool with potential importance in the diagnosis, prognosis and pre-symptomatic early detection of cardiac amyloidosis, giving emphasis on its applicability in familial forms of amyloidosis. cervellini i , galino j , zhu n , birchmeier c , bennett dl . ndcn university of oxford, oxford, uk; max-delbrück-center for molecular medicine, berlin, germany. erk/mapk pathway has a critical role in pns development since its involvement in many physiological processes. sustained erk / mapk activation in schwann cells enhances myelin growth during development and overcomes signals ending myelination leading to a continuous myelin production. however, strong activation of erk has also been shown to cause schwann cells dedifferentiation and demyelination in vivo. our aim was to investigate whether a mild activation of this signalling pathway in adult schwann cells (scs), by expression of gain of function mek dd allele, could have a beneficial role in remyelination and regeneration after injury. erk/mapk activation in adult scs in plpcreer t ;mek dd mutant mice, did not affect myelination during development. following sciatic nerve injury, wallerian degeneration was enhanced in mutants pushing towards a dedifferentiation stage of scs as previously described. however, mapk activation was detrimental during regeneration with a delay in functional recovery and a negative impact in both myelinated and non-myelinated fibres compared to controls. one month after injury the total number of axons in mutant sciatic nerves was half of the controls. although no differences in g-ratio have been found in the two groups, mutants presented a higher number of myelinated axons showing myelin disruption with start of myelin decompaction, lack of cajal bands, abundant sc processes surrounding axons and a shorter sc elongation, as seen by decreased internodal distance. in addition, we found a negative effect of mapk activation also in small diameter axons with the presence of abnormal remak bundle structures, reduced number of c-fibres/remak bundle and a significant decrease in intra epidermal nerve fibres density in the skin. we concluded that mild mapk activation has a different role in development and remyelination where negatively affects axon survival, myelin stability, remak bundle formation and small fibres regeneration. cervellini i , galino j , zhu n , bao lu , bennett dl . ndcn, university of oxford, oxford, uk; harvard medical school, boston, ma, usa. neprilysin (nep) is an endopeptidase which has been of interest due to its potential role in neurodegeneration and pain as a consequence of its ability to degrade amyloid and substance-p respectively. nep expression is not limited to cns and it has been reported to be expressed in schwann cells, nodes and schmidt-lanterman incisures. our interest in this gene was related to recent findings that have associated homozygous and heterozygous nep mutations with charcot-marie-tooth type- . in old mice lacking nep subtle morphological changes have been reported. our aim was to determine whether nep expression was modulated by nerve injury and to investigate its role in axon regeneration and re-myelination. we find that nep gene expression was decreased after nerve crush and furthermore was dependent on the growth factor (and pro-myelin signal) neuregulin- . in control mice nep expression was transiently reduced and returned to baseline at day after injury, in neuregulin- knock-out (ko) mice, in which re-myelination was impaired, the expression was still decreased at day . in assessing behavioural measures of locomotor and sensory function one month after sciatic nerve crush, nep ko mice showed a functional regeneration comparable to wt, as seen by sciatic functional index measurement, beam and toe spreading tests. the only significant difference we observed between wt and ko was in the sensorial test, showing ko mice recovering faster in the pinch test by days after crush. the results for all the tests at baseline did not differ between the two groups. detailed histological analysis of nerve repair was undertaken using electron microscopy. there was no difference between wt and ko in total axon number, g-ratio, axon diameters and other myelin features one month post crush. in summary, although nep expression is regulated by nerve injury in a neuregulin- dependent fashion this endopeptidase is dispensable for axon regeneration and re-myelination after nerve injury in the rodent. diabetes neuropathy is a common complication of diabetes, and neuropathic pain has a detrimental impact on quality of life. this study investigated sensory nerve excitability properties to elucidate the axonal changes of diabetic neuropathy. a total of diabetes patients ( type ii, and type i) were enrolled in this study. clinical assessment, nerve conduction studies, and nerve excitability testing data were analyzed to determine axonal dysfunction in diabetic neuropathy. among those patients, seventeen subjects had complained of spontaneous painful sensation over feet or hands (painful cohort), and seventy-eight patients had no sensory symptoms or decreased the sensation over foot (non-painful cohort). sensory nerve excitability of the painful cohort showed reduced late subexcitability (p= . ), increased superexcitability (p= . ) in compared to the non-painful cohort. there is no difference in disease duration, blood glucose levels (hba c) between these two cohorts. these findings suggested the possible pathogenesis of painful sensory axons might be hyperpolarized or slow potassium channels dysfunction. these insights our further understanding of painful diabetic neuropathy, and may provide a basis for neuroprotective or therapeutic approaches for painful polyneuropathy. the main purpose of this study was to assess the clinical feasibility of diffusion tensor imaging (dti) for the diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp). between march and december , we prospectively enrolled patients with definite cidp according to the efns criteria and two control groups: healthy volunteers matched on age and sex and patients with cmt- a. using a -t magnetic resonance imaging scanner, we obtained dti scans of brachial plexus of these groups and prepared fractional anisotropy (fa) maps, and compared these values between groups. adc values and cervical nerve roots diameters on stir sequences were evaluated too. two neuroradiologists, blinded to clinical informations, reviewed mri studies independently. in all patients with cidp, we also performed clinical evaluation and electroneuromyography. significantly decreased fa values (p< . ) and increased adc values were observed in cidp patients compared with healthy subjects. there is no significant difference between cidp and cmt group. inter-observer concordance was excellent for fa values ( c= . ; p< . ) and moderate for adc values ( c= . ; p< . ) and cervical nerve root diameters ( c= . ; p< . ). there is a significant correlation between fa and disease duration (r = − . , p < . ), inclusion mrc score (r = . , p < . ) and between fa mesured on c c and incat score at inclusion (r = − . , p < . ). no significant correlation is observed between fa and electrophysiological indices. compared with healthy subjects, cervical nerve root diameters were significantly increased (p< . ) in patients with cmt and cidp. contrary to fa values, moderate level of concordance was found between inter-observers measurements of diameters (cclin = . ). our preliminary data prove the clinical feasibility and reproductibility of dti for the evaluation of plexus and cervical nerve roots in patients with cidp. cheng yj , teng a , goh ejh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. the sural-sparing pattern of the sensory nerve action potentials (snap) of guillain-barré syndrome (gbs) has been attributed to greater immunological injury of the blood-nerve barrier at its most vulnerable regions. we asked if entrapment sites, such as median nerve at the wrist, are more predisposed than the distal nerve endings to such injury. we compared the median snap with radial snap measured antidromically at digit in gbs patients whose nerve conduction study showed the sural-sparing pattern. the terminal nerves at digit are of similar length, but those of median nerve are prone to compression, often subclinically, at the carpal tunnel while those of radial nerve are not. we defined the sural-sparing pattern as a greater decrease in median and or ulnar snap than that of the sural, compared to age and height-matched normal controls. a total of gbs and miller fisher patients from our institution's database were studied. patients had the sural-sparing pattern, of whom had pre-existing carpal tunnel syndrome. of the remaining patients with sural-sparing, had abnormal median snap at digit , while had both abnormal median and radial snaps at digit . none had isolated abnormality of the radial digit snap. among the cases that had abnormal median and radial snaps at digit , the mean percentage decrease when compared to age and height matched norms was greater in median nerve compared to radial nerve ( % and % respectively). of the patients without sural-sparing pattern, had normal snaps; patient had inexcitable sensory nerves while the other had a length-dependent decrease in snap. in the latter patient, unlike those with sural-sparing, there was no differential decrease of median snap over radial snap at digit . our findings suggest that the disruption of blood nerve barrier at entrapment sites rather than the distal nerve endings may underlie the pathophysiology of the sural-sparing pattern seen in gbs. chiba a , uchibori a , gyohda a . kyorin university, tokyo, japan. serum igg anti-gq b antibody is the most specific biomarker for fisher syndrome and its related disorders (fs-rd), but approximately - % of the patients are seronegative for it in conventional assays (gq b-seronegative). some molecules need ca + cation to interact with their ligands, and antibodies with such a property (ca + -dependent antibodies) are reported. we have found that such a ca + -dependency is also present in igg anti-gq b antibody, and majority of gq b-seronegative patients with fs-rd have this type of antibodies. in patients with final clinical diagnoses as fs-rd (fisher syndrome, guillain-barré syndrome with ophthalmoplegia, bickerstaff brainstem encephalitis, and acute ophthalmoplegia), were seropositive for igg antibodies against gq b-related antigens (isolated gq b in , and gq b-conatinig complexes in two) in conventional elisa using phosphate-buffered saline. in the remaining patients, eight ( %) turned positive for igg antibody against gq b-related antigens (isolated gq b in seven and gq b-conatinig complexes in one) in elisa using ca + -added tris-buffered saline. the reaction strengths increased depending on ca + concentration, and reached to nearly maximum level in the physiological concentration. all the patients with the ca + -dependent antibodies were also positive for igg antibody against gt a-related antigens, suggesting that the terminal disialo residue common to both the gangliosides would be important as an epitope also for the ca + -dependent antibodies. in the patients with ca + -non-dependent antibodies, only two showed increased titers of igg anti-gq b antibody by adding ca + , and showed significantly decreased titers. this difference in the effect of ca + -addition between ca + -dependent and ca + -non-dependent antibodies suggests that ca + would not be just an enhancer of the antigen-antibody reaction. there are four single bonds between the two pyranose rings in the terminal disialo, and those rotatable bonds make it possible for the disialo structure to take various conformations. a molecular model shows that the distance between two minus-charged carboxy groups in the disialo could vary from nearly zero to approximately , pm and that the disialo would take specific conformations, if divalent ca + cation, which size is approximately pm in diameter, interacts with these two minus-charged groups. the ca + -dependent antibodies might recognize such particular conformations of gq b. charcot-marie-tooth disease (cmt) is a genetically and clinically heterogeneous disorder with variable inheritance modes. it is characterized by loss of muscle tissue and touch sensation, predominantly in the feet and legs but also in the hands and arms in the advanced stages of disease. as several molecules have been reported to have therapeutic effects on cmt, depending on the underlying genetic causes, exact genetic diagnostics have become important for executing personalized therapy. aminoacyl-trna synthetase (arss) genes encode enzymes responsible for charging trna with corresponding amino acids. arss are ubiquitously expressed, essential enzymes responsible for performing the first step of protein synthesis. specifically, arss attach amino acids to their cognate trna molecules in the cytoplasm and mitochondria. recent studies have demonstrated that mutations in genes encoding arss can result in neurodegeneration, raising many questions about the role of these enzymes in neuronal function. mutations in six cytoplasmic ars genes have been reported as the cause of cmt. this study was performed the whole exome sequencing to identify genetic defects in korean cmt patients from unrelated families. variants were sorted with cmt gene list that includes almost genes were related cmt neuropathy, and additionally sorted wes data as ars genes. capillary sequencing for family members and more than controls revealed five novel mutations, c. g>a (p.d n), c. c>t (p.s f), and c. c>a (p.p h) in gars; c. c>a (p.p t) in mars; _ ga>at (p.d i) in yars gene in each family. the mutation sites were well conserved between different species and each mutation were located in the well-conserved catalytic domain or between two catalytic domains or anticodon-binding domain. in silico analysis predicted all mutations may affect protein function. clinical features were similar to those reported in other countries, but differed in terms of age at onset and degree of disability. we believe that those novel ars mutations are the underlying causes of the each family. a -year-old man presented with a -year-history of weakness in biltearal upper limbs. he was complaining of intermittent fasciculation of upper and lower limbs with gradually worsening of paresthesia for years. dysphagia and dysarthria were also presented years ago. there was no patient affected muscle weakness and bulbar symptoms in his family members. in neurological examination, the patient had weakness in biltereal upper and lower limbs (mrc grade ) and prominent distal sensory loss were combined in length dependant pattern. deep tendon reflexes were absent on bilataral biceps and knee joints. in nerve conduction study, there was consistent with demyelinating sensorimotor polyneuropathy. molecular diagnostic analyses those spinobulbar muscular atrophy (sbma) and mutation related to peripheral myelin protein (pmp ) gene were performed and confirmed expansion of expansion of a polymorphic cag in androgen-receptor (ar) gene and deletion of pmp gene. smba, also known as kennedy disease, is an adult-onset, x-linked recessive trinucleotide, polyglutamine (poly-g) disorder caused by expansion of a polymorphic cag tandem-repeat in exon of ar gene on chromosome xq - . charcot-marie-tooth disease (cmt) is the most common hereditary neuropathies and cmt cases with motor conduction velocities(mcvs) of upper limb below m/s are defined as demyelinating (cmt ) and those with mcvs above m/s are defined as axonal (cmt ). most families with cmt linked to duplication of pmp gene on the short arm of chromosome ( p . ), called cmt a. the reciprocal deletion of pmp gene is a responsible genetic defect in % of hereditary neuropathy with liability to pressure palsy (hnpp). these "classical" phenotypes of cmt a and hnpp have been considered which are determined by different mutation mechanism of the same gene. however, an overlap of cmt a and hnpp due to pmp gene deletion was reported that suggestion the phenotype of hereditary neuropathies may differ variably. herein, we report a patient who simultaneously presented clinical and electrophysiologic features of smba and cmt a with genetical confirmation of cag expasion and deletion of pmp gene. charcot-marie-tooth type a disease (cmt a) is an inherited peripheral neuropathy stemming from overexpression of pmp protein in schwann cells due to the duplication of the pmp gene. this leads to abnormal schwann cell differentiation and dysmyelination, eventually leading to axonal loss and muscle wasting. no approved treatment is currently available for cmt a. we conducted a systems biology level analysis of the signaling network putatively underlying the processes driving cmt a pathology. based upon this, we identified and tested three repurposed drugs -baclofen, naltrexone and sorbitol -alone and in combination to determine their ability to rescue aberrant myelination in cultures derived from cmt a transgenic rats overexpressing pmp gene. to this end, we studied a validated in vitro co-culture model of sensory neurons and schwann cells adapted to -well culture plates. this model allows measurement of the appearance of myelin proteins as an index of the physiological process of in vivo myelination. total myelin length was quantified with an automatic image analyzer following pmp immunostaining. we first determined the full dose-response curves of single drugs, emphasizing their promyelinating activities. we then tested binary combinations of very low and inactive doses of each drug and compared these to the activity of the combination of the three, namely pxt . whereas combination of any two drugs was not significantly active at the doses tested, combination of all three produced a synergistic improvement in myelination. these findings clearly demonstrate the necessity of using pxt over its single components and highlight the value of pleiotropic combinational repurposing of drugs at low doses as a novel approach for rapid drug development in cmt a and other disorders. autonomic dysfunction is frequently observed in guillain-barré syndrome (gbs) and affects approximately % of the patients. it has been shown that the sweating function can be impaired in gbs. the aim of the present investigation is to summarize the current knowledge on sweating disturbances in gbs patients. we have used appropriate terms to systematically search for references published until and indexed in the following databases: medline, embase, lilacs and cochrane. the inclusion criteria were a diagnosis of gbs and a description of the methods used to test the sudomotor function. the search was limited to the english language. relevant information about study design, methods of assessing the deficit of sweating, patient's characteristics and main results were collected. we selected original references for the final analyses. the majority of the studies were cross-sectional in nature and there were two longitudinal studies. the severity of sweating impairment varied according to the applied method, ranging from normal to almost sympathetic nervous system failure. in seven research papers, the sympathetic skin response was used to evaluate the sudomotor function in patients, and approximately % demonstrated abnormal results. however, researchers used different stimulation protocols and parameters to interpret their results. regarding whole-body sweating test, four research papers applied the thermoregulatory sweating test in patients and they showed areas of anhidrosis on the lower limbs in all of them. eight patients presented sweating impairment on the upper limbs and abdominal wall. results on the sudomotor axon reflex test suggested a length-dependent pattern of sweating loss according to one case report. in another study, eight gbs patients were tested only on the distal leg and foot dorsum and the authors proposed an association between post-ganglionic sudomotor function and antiganglioside antibodies. the present literature review showed that the studies of sweating disturbances in gbs patients included only small cohorts. future studies with larger patients sample sizes are necessary to investigate the patterns of sweating loss in gbs and their changes along the follow up. funding: grants # / - and # / - , são paulo research foundation (fapesp). sensory neuronopathy (sn) represents a rare subgroup of peripheral neuropathies characterized by degeneration of primary sensory neurons at the dorsal root ganglia on the spinal cord. depending on the neuronal population affected, its clinical presentation may manifest as gait ataxia, proprioceptive sensory loss and positive and negative sensory symptoms. although a few reports have mentioned areas of anhidrosis in sn, we were not been able to find previous case series studies on the sweating function in sensory neuronopathies. the aim of the present investigation was to study the whole-body distribution of sweating on both anterior and posterior surfaces in patients diagnosed with sn. quantitative sensory testing for cold and warm sensation threshold (method of levels) was performed on the dorsum of the hands and feet in a randomized order. we tested the thermoregulatory sweating using a sweat chamber ( to ∘ c air temperature and % relative humidity). the oral and skin temperature was monitored and the test time did not exceed min. in order to study the sudomotor axon reflex we employed the q-sweat device on standardized body sites. the test was performed on both sides, simultaneously. we included seven patients (three male; mean age . years) with a mean disease duration of . months (range - ) and a confirmed diagnose of sn. patients presented an asymmetrical loss of cold and warm threshold on hands or feet compared with healthy control (p< , ). regarding the tst results, we found a striking variation of sweating disturbances, ranging from small areas of anhidrosis on the trunk to complete failure of the sympathetic nervous system. two patients underwent the axon reflex test and there was an asymmetrical and mostly distal pattern of sweating loss in one of them and a distal-symmetrical on the second one. our findings indicate a great variability of sweating losses in sn, not overlapped to the sensory loss areas. currently, we are testing more patients in order to confirm our results. funding: grants # / - , # / - and # / - , são paulo research foundation (fapesp). diet, exercise, and inflammation are established modulators of peripheral nervous system function, including pain. prior work examining exercise consistently demonstrates a benefit on heightened pain from a number of acute and chronic pain models. in the present work, we investigated several parameters of peripheral nerve function relevant to pain in rats bred for high (high capacity runners, hcr) or low running capacity (low capacity runners, lcr). the longtime selective breeding of these rat substrains has created divergent intrinsic aerobic capacities and predisposition of metabolic conditions between lcr and hcr rats. examination of the role of sex in the development of chronic pain has established key differences in males and females. to understand gender specific differences, this study focused on female rats to understand the role of metabolic status and peripheral nerve function in females. our analysis identified numerous parameters of peripheral nerve function relevant to pain and neuropathy that are different among lcr and hcr female rats. lcr female rats display reduced hind paw mechanical sensitivity, increased hind paw intraepidermal nerve fiber density and trka-positive epidermal axons, increased numbers of langerhans and mast cells in the hind paw dermis, and increased overall fat mass relative to body weight compared to female hcr rats. examination of sensory and motor nerve conduction velocities, thermal sensitivity, and mrna expression of selected genes relevant to peripheral sensation found no differences between hcr and lcr females. together these results suggest that a genetic component of aerobic capacity and metabolic status can influence sensory sensitivity and specific aspects of inflammation and immune responses in peripheral tissues, which may lead modify the animal's responses to tissue damage and painful stimuli. the lcr and hcr rat model will provide a useful model in the future to assess the involvement of metabolic status in the development of pain. ivig is often considered treatment of first choice in chronic inflammatory demyelinating polyradiculoneuropathy (cidp) because of its rapid onset of action and its relatively safe long-term adverse event profile. clinical trials published so far focused on a loading dose of . g/kg ivig and/or a standard maintenance dosage of . g/kg ivig once every weeks, but have not investigated different dosing options. this study is a prospective, double-blind, randomized, parallel group, multi-center phase iii efficacy study and will be conducted in centres in canada, eu, russia, ukraine and australia. adult patients with definite or probable cidp according to the efns/pns criteria will be enrolled and randomized : : to receive either . g/kg or . g/kg or . g/kg ivig (panzyga ® ) for seven maintenance infusions at -week intervals during the dose-evaluation phase. the starting loading dose will be . g/kg ivig (panzyga ® ) for all patients. primary objective: efficacy measured as percentage of responders (decrease in adjusted incat score by at least point) in the . g/kg ivig (panzyga ® ) arm (given every weeks) at week as this should corroborate the existing and published evidence on efficacy of ivig in cidp. secondary outcome: percentage of responders at week in the . g/kg and . g/kg ivig (panzyga ® ) arms relative to baseline and compared to the . g/kg arm. the procid study aims to confirm published clinical results obtained with the . g/kg standard dose and will in addition evaluate one higher and one lower maintenance dose, with the aim to offer cidp patients a more adequately dosed and effective treatment policy. * este medicamento no se encuentra comercializado en españa. understanding the rate of disease progression in patients with charcot-marie-tooth disease (cmt), both within and between subtypes is important for clinical prognosis and is crucial for clinical trial design. due to the progressive nature of cmt, intervening at the earliest stages of the disease is a priority. measuring progression of a disease with both motor and sensory deficits requires a multi-item composite scale. the cmt pediatric scale (cmtpeds) is a well-tolerated psychometrically robust -item scale measuring fine and gross motor function, strength, sensation and balance for children and adolescents aged - years with cmt. the aim of this study was to determine the rate of disease progression of children and adolescents within and between genetic subtypes of cmt. ( female) participants aged - years enrolled in the inherited neuropathies consortium were included in this study. demographic, anthropometric and diagnostic information were collected at baseline and -year follow-up. disease progression was measured with the cmtpeds. on average cmtpeds scores progressed at a rate of . points over -years ( % change from baseline, p< . ). there was no difference in rate of disease progression between males and females. of the most common genetic subtypes, participants with cmt a/pmp duplication progressed by . points ( % change from baseline, p< . ), nine participants with cmt b/mpz mutation progressed by . points ( % change), six participants with cmt a/mfn mutation progressed by . points ( % change) and seven participants with cmt c/sh tc mutation progressed by . points ( % change). participants with cmt a progressed faster than those with cmt a (p= . ). children with cmt a progressed consistently during childhood and adolescence while children with cmt b and cmt a progressed faster during childhood than adolescence. overall, children with cmt progress at a significant rate over -years according to the cmtpeds. understanding the rate at which affected children deteriorate is essential for adequately powering clinical trials of disease-modifying interventions. queen square, london, uk; department of neurology, university of iowa carver college of medicine, iowa city, ia, usa; department of neurogenetics, the national hospital for neurology and neurosurgery, ucl institute of neurology, london, uk; department of molecular neuroscience, ucl institute of neurology, london, uk and national hospital for neurology and neurosurgery, queen square, london, uk. in recent years, targeted ngs panels have changed the diagnostic work-up in patients with inherited neuropathies. however, there is limited data on the impact of targeted ngs panels on the diagnosis of cmt patients in everyday practice. the aim of this study was to investigate the impact of targeted ngs panels on the diagnosis of cmt across two tertiary referral centres in the united kingdom (london) and united states (iowa). in london, patients with a diagnosis of cmt (previous pmp duplication and common cmt genes excluded in appropriate cases) underwent targeted ngs panel sequencing covering genes associated with cmt and additional genes associated with hsp or als. a variable number of genes, ranging from to , were analysed depending on the clinical phenotype of the patients. a definite molecular diagnosis was achieved in cases ( %) including pathogenic and likely pathogenic mutations in sh tc ( cases), gjb ( cases, including cases with mutations in the promoter and 'utr regions), gdap ( cases), fgd ( cases), aars ( cases), ighmbp ( cases), mpz ( cases), nefl ( cases). vus were further identified in patients. the diagnostic rate was higher in demyelinating cmt cases ( / , %), compared to cases with axonal cmt ( / %), dhmn ( / , %) and hsn ( / , %). in iowa, patients were investigated by ngs panels covering to genes associated with cmt. a molecular diagnosis was reached in / ( %), and in particular / ( %) demyelinating and / ( %) axonal cmt cases. the most frequent genes identified were gjb ( cases), mfn ( cases), sh tc ( cases) and ighmbp ( cases). vus were identified in patients, including cases with novel variants in aars, warranting additional testing such as segregation of the variant in the family or functional validation studies. in clinical practise, targeted ngs panels represent an effective approach for the diagnosis of cmt. the lower diagnostic rate in london is likely to be due to prior sanger sequencing and exclusion of mutations in common cmt genes in this patient population. more than genes are known to cause cmt and an even larger number are known to cause peripheral neuropathy as part of a more complex neurological disorder. despite the use of custom panels, a significant proportion of patients with inherited neuropathy have no molecular diagnosis. the aim of this study was to investigate the diagnostic yield of a disease-associated gene exome (sureselect focused exome, agilent technologies, santa clara ca, usa) in the diagnosis of cmt and in cases with complex neurological syndromes associated with neuropathy. thirty-one patients with molecularly undiagnosed inherited neuropathy were analysed with sureselect focused exome sequencing. six patients had a more complex phenotype including learning difficulties, cerebral white matter changes, ataxia and pyramidal tract involvement. a genetic diagnosis was achieved in / ( %) of cases by detecting a mutation in cmt-associated genes mpz ( cases), aars, nefl, bscl , bicd and trpv . of note, six cases had mutations in genes which are not covered by currently available diagnostic targeted ngs panels, including kif a, polg, mme ( cases), dnajb , and a novel candidate gene. the average coverage was higher compared to the usual coverage of whole-exome sequencing; % of the targets were covered at x or more, and % of the targets were covered at x or more. this study provides evidence that the sureselect focused exome is a useful tool for the diagnosis of cmt and complex neurological disorders and provides further insight into the phenotypic spectrum of genes associated with inherited neuropathy. changes in the cis-regulatory sequences of a gene's untranslated regions (utr) are increasingly recognised as a significant cause of inherited disease in humans. for example, variants in the non-coding region of gjb account for % of all mutations in our cohort of cmtx patients. one of the biggest challenges in analysing the large number of non-coding variants in a gene is identifying those that are disease-causing and those which are polymorphisms. the aim of this study was to implement a reliable method for the in-vitro functional validation of non-coding variants in the promoter and 'utr regions of gjb . in our cohort of cmtx patients we have previously identified seven mutations (c.- _ inst, c.- g>a, c.- t>g, c.- t>c, c. t>c, c.- g>c, c.- c>t) in the promoter region and one novel mutation in the 'utr (c. c>t), which were considered likely to be pathogenic based on the clinical phenotype, segregation in affected family members and absence in control databases. we have now generated a luciferase-based reporter system and optimised it in a hela cell line. mutations in the promoter region were generated by site-directed mutagenesis using a commercially available gjb promoter clone (genecopoeia). our preliminary results show a reduction of luciferase activity for the c.- t>c and c.- _ inst mutations compared to the wild-type promoter. this difference was increased when transcription factors sox or egr were co-transfected with the t>c and c.- _ inst mutations respectively. validation of other variants is currently ongoing. if successful, our study will provide a useful tool for the validation of mutations in non-coding regions of gjb . moreover, it will constitute a proof-of-principle approach to the functional validation of non-coding variants in other cmt genes known to cause disease by a loss of function. recessive and dominant mutations in leucin-rich repeat and sterile alpha-motif-containing (lrsam ) have been associated with cmt p. lrsam is a ubiquitin e ligase containing a ring domain in its c-terminal, which is crucial for correct protein folding and ubiquitination activity. to date, the majority of dominant mutations reported have resulted in a frame shift disrupting a major portion of the ring-domain, although point mutations in this domain have also been described. the aim of this study was to report the prevalence, clinical features and genetic findings of patients with cmt p in our centre. we performed targeted next-generation sequencing in genetically undiagnosed cmt patients and identified cases with heterozygous mutations in lrsam ( . %) from unrelated families. the mutations identified included frameshift insertions and deletions, a non-frameshift deletion and non-sense and missense point mutations. all of the mutations were novel and were located in or flanking the ring domain. the average age of disease onset was in the rd decade but an earlier onset was reported in two cases. four had a positive family history in keeping with autosomal dominant inheritance. symptoms at presentation were heterogeneous and encompassed distal numbness, unsteadiness, distal weakness of upper or lower limbs and foot deformities. positive sensory symptoms, including tingling and shooting pains, and cramps were also frequently reported. neurological examination showed mild to moderate distal atrophy and weakness, with early ankle plantar flexion involvement in three patients. loss of vibration and reduced joint position sense were often prominent in the lower limbs and appeared to be disproportionate to the degree of weakness and impairment of pinprick sensation. ankle jerks were absent but knee and upper limb reflexes could be normal or brisk. after an average disease duration of years, all but one patient was able to walk independently. nerve conduction studies showed a sensory and motor axonal neuropathy with normal conduction velocities. our study highlights that mutations in lrsam are a relevant cause of cmt and are associated with prominent large fibre sensory loss. in recent years, the implementation of ngs panels for the molecular diagnosis of cmt has increased the number of patients with a genetic diagnosis. nevertheless the interpretation of a particular variant as disease causing can be challenging especially when multiple variants are identified in a single patient. we report two illustrative cases of such challenges. the first index case was born of non-consanguineous healthy parents. he presented with falls in early childhood. over the years he developed foot deformities and progressive length dependent weakness. he had multiple orthopaedic operations to his feet. the past history was also notable for kyphoscoliosis, sensorineural deafness from the age of and bilateral cataracts. nerve conduction studies at the age of revealed a demyelinating neuropathy consistent with the clinical phenotype of cmt . his younger brother had a similar, although more severe phenotype. a litaf (c. c>t,p.pro ser) mutation was identified by sanger sequencing and was present in both affected brothers but also in the unaffected sister. ngs for cmt -associated genes was therefore performed and identified two compound heterozygous pathogenic mutations detected in sh tc (c. c>t, p.arg *; delg, p.arg serfs* ), which segregated with the disease in the family. the second case describes a brother and sister with early onset demyelinating cmt associated with scoliosis and cranial nerve involvement. the male proband underwent ngs and a single previously reported pathogenic intronic splice-site mutation in sh tc (c. - a>c) was found. relative read-depth analysis of ngs was performed to look for possible copy number variants in sh tc , thus identifying a deletion of exon , which was confirmed by long pcr. cruz-velásquez gj , miramar-gallart md , alarcia-alejos r , roche-bueno jc , rodríguez-valle a , capablo-liesa jl . neurology service, university hospital miguel servet, zaragoza, spain; genetics unit, clinical biochemistry service, university hospital miguel servet, zaragoza, spain. charcot-marie-tooth disease (cmtd) defines a clinical and genetically heterogeneous group of inherited peripheral neuropathies characterized by chronic motor and sensory impairment. the type cmtd- also known as cowchock syndrome, is the product of the mutation in the apoptosis inducing factor mitochondria associated gene (aifm ). it is a slowly progressive, recessive, x-linked disease characterized by axonal neuropathy, deafness, and cognitive impairment. our purpose is to describe new cases, brothers, children of non-consanguineous parents, with a characteristic phenotype and a new mutation in the aifm gene. both siblings present from childhood, progressive weakness in lower limbs with diffuse amyotrophies. needing tenotomy before the year due to equinovarus foot. likewise they develops sensory deafness and one of them requires unilateral support at and the other wheelchair at , this one need a pacemaker for an atrioventricular block at . with brain functions and normal language, sensorial deafness, proximal and distal weakness in the four limbs with intense amyotrophies, predominantly distal. tactile and painful sensitivity decreased in glove and sock pattern. an extensive metabolic and biochemical study was normal. the electroneurography demonstrates an axonal neuropathy without response in most of the nerves explored. the electromyography shown a myogenic pattern with distal predominance. brain mri was normal in both cases. through a genetic study by exoma targeting genes associated with cmt and inherited related neuropathies was identified the homicigosis mutation in the aifm gene (p.glu lys), located in the chromosomal region xq . (cmtx ). cowchock syndrome is a rare entity, with few cases described in the literature. the in silico analysis indicates in of the predictors used (provean, sift, polyphen , lrt, mutationtaster, mutationassessor and condel) , that it is a deleterious variant. we audited all patients with peripheral neuropathy caused by a paraprotein, who received treatment and attended king's college hospital peripheral nerve service between - . patients were identified retrospectively from our database of patients attending the peripheral nerve outpatient clinic. clinical information was obtained retrospectively from hospital electronic patient records. we excluded patients with poems syndrome or in whom the neuropathy was not felt to be caused by the paraprotein. we identified patients with a diagnosis of paraproteinaemic neuropathy. we excluded four who did not fulfill the diagnostic criteria and eleven who had received no treatment or were under diagnostic study. we included patients in the final audit. ( %) had igm paraprotein. the haematological diagnosis was monoclonal gammopathy of undetermined significance (mgus) in %, waldenstroḿs macroglobulinaemia %, and lymphoma or plasmacytoma %. after treatment, overall ( %) patients improved neurologically, ( %) stabilised, and ( %) worsened. in the patients who received more than one type of treatment, we analysed outcomes according to the most powerful treatment received. patients received rituximab alone of which ( %) improved, ( %) stabilised and ( %) worsened. nine patients received rituximab combined with cyclophosphamide or bendamustine, of which ( %) improved, ( %) stabilized and ( %) worsened. eight patients received intravenous immunoglobulin, of which ( %) improved, ( %) stabilized and ( %) worsened. four patients received other chemotherapy, of which improved, stabilised, worsened. two patients received corticosteroids and both worsened. there was improvement in / ( %) with mag antibodies and / ( %) without. there was improvement in / ( %) with mgus and / ( %) with haematological malignancy. there was improvement in / ( %) with kappa light chains and / ( %) with lambda. factors associated with better outcome (by univariate analysis) were negative mag antibodies, kappa light chain, and haematological malignancy. there was no significant difference between treatments in the proportions who improved. cumberbatch m , cox a . addenbrooke's hospital, cambridge, uk. treatment of patients with autoimmune neuropathies such as chronic inflammatory demyelinating polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) has centred on the use of intravenous immunoglobulin (ivig). however, ivig therapy is associated with systemic side-effects, treatment wear-off effects and regular hospital attendance. subcutaneous immunoglobulin (scig) is an efficacious alternative that can be flexibly dosed, self-administered at home and avoids the 'peaks' and 'troughs' observed with ivig. these factors may combine to improve patient satisfaction and alleviate hospital capacity issues. here, we report on clinical and patient experience of switching from hospital-based ivig to home-based manual push scig for the treatment of cidp and mmn. this was a clinical case series of patients ( cidp, mmn; mean age . years) who were clinically stable on ivig and wished to switch to manual push scig. starting scig dose was equivalent to the final ivig dose for each patient (mean . g/kg week). clinical efficacy (medical research council sum score, -m walk, modified inflammatory neuropathy cause and treatment score, overall neuropathy limitations scale, romberg test) and patient-reported outcomes ( -item short form health survey , life quality index [lqi] ) were assessed at baseline and at regular intervals until the final visit ( - months after switching). at baseline, patients cited 'convenience' as their primary reason for switching to scig. eight patients completed the full assessment period and successfully undertook administrations at home (via hospital-at-home service in cases). dose adjustments, based on clinical need, were required in patients. treatment efficacy and patient quality of life, measured by sf- , were maintained after switching to scig; overall patient satisfaction, measured by lqi, increased from % to %. in the lqi, 'convenience', 'travel time/cost' and 'interference-work' were significantly improved (p< . ) after switching to scig therapy. adverse events included mild erythema and localised swelling, as expected for a ml subcutaneous injection. these findings suggest that manual push scig therapy is a viable alternative to ivig for patients with cidp and mmn, as it maintains disease stability, is more convenient for patients and may help ease hospital capacity concerns. cumberbatch m , soares regua d , cox a . addenbrooke's hospital, cambridge, uk. intravenous immunoglobulin (ivig) is used to treat a number of chronic autoimmune neurological diseases. in most centres, infusions are given at slow rates as there is a perception that this reduces the risk of adverse events (aes). this results in longer in-patient admissions, or frequent day case attendances, impacting on both the patients' quality of life and hospital capacity. however, there is little evidence to suggest that slow infusion rates are required. we used the manufacturer recommendations to optimise infusion rates and reduce the time patients spend in hospital. we report a retrospective audit which describes the impact of different ivig infusion rates on patients' clinical condition. the audit comprised three -month assessment periods: january-june (cohort ; infusion rates of . ± . ml/kg/hr, n = ); january-june (cohort ; . ± . ml/kg/hr, n = ) and july-december (cohort ; . ± . ml/kg/hr, n = ). clinical data were reviewed to determine: patient demographics, duration of infusion; time spent in hospital; and incidence of aes. the three cohorts were well matched in terms of patient demographics ( patients were treated in all treatment periods). cohorts and had significantly shorter treatment episodes than cohort ( . and . vs. . hours, p< . ), spent less time on the unit over the month period ( . and . vs. . hours, p< . ) and had fewer admissions/patient ( . and . vs. . , p< . ). the overall incidence of confirmed aes (mainly headaches) was similar across the cohorts (cohort : %; cohort : %; cohort : %). these findings indicate that increases in ivig infusion rate are well tolerated and significantly reduce treatment time, which benefits patients and offers potential cost savings and reduced pressures on hospital capacity for healthcare providers. cunningham me , yao d , meehan gr , barrie ja , willison hj . university of glasgow, glasgow, uk. one mechanism of injury in the acute motor axonal neuropathy (aman) form of guillain-barré syndrome (gbs) is the attack of peripheral nerve axons by anti-ganglioside antibodies (agabs). rodent models have demonstrated that that binding of these antibodies activates the complement cascade, resulting in the insertion of the terminal component, membrane attack complex (mac) into the axonal membrane. complement activation also results in the release of anaphylatoxins, which are known to recruit phagocytic immune cells to the site of injury. our current in vivo mouse model of agab and complement-mediated injury are acute and severe, resulting in respiratory distress over several hours of such magnitude to warrant termination of experimental procedures. to observe and potentially target immune cell infiltration following agab and complement-mediated injury, a subacute model extending over days is required. here, we demonstrate the development of such a model. to compare differences in immune cell infiltration subacutely under control and injury conditions, mice with endogenous expression of egfp in monocytes and macrophages underwent a modified agab and complement-mediated injury, resulting in a less severe phenotype than previously published models. six days following injury, immune cells in the diaphragm were compared by immunofluorescence and flow cytometry. flow cytometry found overall presence of neutrophils was significantly increased in the diaphragm. macrophages were also increased in injured mice, although did not achieve statistical significance at this timepoint. these results were reflected in immunofluorescent staining of the diaphragm where egfp+ macrophages were quantified surrounding the neuromuscular junction (the primary injury target in this model). the development of an extended mouse model of agab and complement-mediated injury is important, since acute models do not take into consideration either the late-term effects of complement-mediated activation at the nerve membrane, or the recovery phase. future studies will look at the effect of inhibiting complement activation on the presence of immune cells in distal motor nerves. family- is a large australian family with an autosomal dominant form of dhmn (dhmn : omim % ) -a group of length-dependent neurodegenerative disorders affecting the lower motor neurons leading to chronic disability. we recently reported a novel . mb chromosomal insertion within the dhmn locus which we hypothesise is likely to cause disease by dysregulating the expression of one or more nearby genes. studying gene dysregulation in peripheral nerve disease is challenging as the relevant tissues (spinal cord and peripheral nerve) are not easily accessible in patients. therefore, alternative strategies are needed to elucidate the disease mechanisms and pathways involved in peripheral nerve degeneration. to address this problem, we have devised a two-tiered strategy to assess dysregulation of candidate genes using patient lymphoblast and fibroblast cell lines. these cell lines can be easily established, are minimally invasive to obtain, and will harbour the natural mutation and genetic background of patients. our strategy firstly uses lymphoblast gene expression profiles as an initial screening tool to prioritise candidate genes for assessing altered expression. differentially expressed genes will then be modelled in c.elegans where behavioural and nerve morphology can be assessed. using rt-pcr, we have screened dhmn candidate genes in patient and control lymphoblast cell lines. eighteen candidate genes were expressed in lymphoblasts. twelve of the eighteen genes were prioritized for further analysis based on expression in both lymphoblast and neural tissues. quantitative analysis using qrt-pcr taqman assays revealed that ube c, was differentially expressed between patients and controls. it is important that patterns of differential expression can be recapitulated in neural cell-specific models. as part of our second strategy, we have generated patient and control induced pluripotent stem cell derived motor neurons (ipsc-mns) from reprogrammed fibroblasts. using this model, we will perform rna-seq and qpcr experiments to examine disease-relevant alterations in gene expression in neural tissue. we predict that utilization of these two strategies will shed light on the pathogenic mechanisms underlying the dhmn insertion and provide useful insights of pathways leading to peripheral nerve degeneration. the outcome of guillain-barré syndrome remains unchanged since plasma exchange and intravenous immunoglobulin were introduced over years ago. pathogenesis studies on gbs have identified the terminal component of complement cascade, the membrane attack complex, as a key disease mediator and thus a therapeutic target. the inhibition of complement in guillain-barré syndrome (ica-gbs) trial looked at the first use of c pathway inhibition with eculizumab in humans with gbs in a randomised, double-blind, placebo-controlled trial. its primary outcome was to look at safety and tolerability of administration concomitantly with ivig and in the context of severe (gbs disability score or greater) disease. participants were recruited for a month period, with regular follow up. subjects were screened, with ( %) being randomised. the two main causes for failure to proceed were participant concerns around eculizumab side effect profile, specifically the meningitis risk, and also intercurrent infection precluding treatment. five received eculizumab for four weeks, alongside standard intravenous immunoglobulin treatment, with receiving placebo. the safety outcomes, monitored via adverse events capture at each trial visit, showed eculizumab to be well tolerated and safe when administered in conjunction with ivig. the most common adverse events were mild derangement in transaminases or infection. there were no infusion reactions. primary and secondary efficacy outcomes were captured via gbs disability scores, mrc sum scores, rasch overall disability scores and overall neuropathy limitation scores. for the primary efficacy outcome at weeks after recruitment, of placebo and of eculizumab-treated subjects had improved by or more grades on the gbs disability score. all patients had improvements in other measured parameters. this trial highlights the challenges in recruiting acutely unwell patients, due to time constraints and intercurrent infection. although the small sample size precludes a statistically meaningful analysis, these pilot data indicate further studies on complement inhibition in gbs are warranted. charcot-marie-tooth disease (cmt) affects about one in . people. currently more than genes have been identified, with the most different phenotypes. the majority of cases in western countries are autosomal dominant and classified as demyelinating and axonal according to electroneuromyography (enmg). the clinical condition is characterized by weakness and predominant sensory changes in the feet and hands. sometimes there are different phenotypes. recently, variants in heterozygotes in the hars gene (histidil-trna synthetase) have been described associated with cmt called type w.to report a case of cmt-sensitive phenotype with a probable new mutation in hars gene (p.leu arg).a male patient, adopted son, caucasian, drug addict, for three years suffered pain in lower limbs, of great intensity, refractory to drug treatment. the examination showed retrognathism, abolition of patellar and achilleas reflexes, painful and thermal anaesthesia and apalesthesia in the feet.the enmg showed reduced sensory action potentials in sural and superficial fibular nerves. laboratory investigations for painful polyneuropathy of thick and fine fibres was normal. sural nerve biopsy revealed axonal predominance neuropathy. exome sequencing revealed a mutation in the hars gene with a pathogenic variant in heterozygosity, with replacement of the amino acid leucine at position by arginine. our patient, although we did not know the antecedents, presented painful polyneuropathy, whose genetic research, although not unequivocal, indicated a variant called cmt w. few cases of this variant were described, with several mutations. our case revealed mutation hitherto unknown (p.leu arg). we conclude by the importance of a thorough genetic evaluation, in cases of sensory polyneuropathy of unknown cause. small fiber neuropathy (sfn) is a condition that affects the small a -and c-fibers, leading to severe neuropathic pain and autonomic dysfunction. several sodium channel gene mutations have been found in patients with sfn, with scn a-gene mutations being the most frequent. because current available sodium channel blockers are not selective for na v . , these treatments often result in numerous side effects. lacosamide is an anticonvulsant that targets specific sodium channels with a slow-inactivation state, while sparing those with normal activity. several mutations of the scn a-gene with an impaired slow-inactivation of na v . have been found in patients with sfn. therefore, a positive effect of lacosamide on pain reduction in these patients is expected. the primary objective of this study was to determine the effect of lacosamide versus placebo on pain in subjects with scn a-associated sfn. secondary objectives were to determine the effect of lacosamide on autonomic symptoms, sleep interference, and quality of life, and to examine the safety and tolerability. the lacosamide-efficacy-'n'-safety in sfn (lenss) study was a randomized, placebo-controlled, double-blind, crossover-design study. subjects were randomized to start with lacosamide and end with placebo or vice versa. during both of the two phases of the study, the subjects were treated for a period of eight weeks of mg bid, preceded by a titration period, and ended by a tapering period. patients filled in a pain diary twice daily and scored a set of validated questionnaires on autonomic symptoms, sleep interference, and quality of life at multiple study visits. in total patients with scn a-associated sfn were included between november and february . the subjects had a median age of years, ranging from to years. sixty percent of the patients were female. the final results of the study, including the primary and secondary outcomes, will be presented. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces must be in intimate contact with nerve fibres to have a proper function, but it has been shown that this is compromised due to the foreign body reaction (fbr). this fbr is the first response of the nonspecific immune system against an implanted device and is characterized by a first inflammatory phase followed by a second antiinflammatory and fibrotic phase. this process results in the formation of a tissue capsule around the interface causing function loss due to the physical separation between the active sites of the electrode and nerve axons. taking this into account, here we have tested several anti-inflammatory drugs such as dexamethasone, ibuprophen and maraviroc to reduce macrophage activation as well as clodronate liposomes to reduce monocyte/macrophage infiltration. moreover, sildenafil have been administered as an antifibrotic drug to reduce collagen deposition in a fbr model with longitudinal parylene c-based intraneural devices implanted in rat sciatic nerve. briefly, animals were systemically treated with dexamethasone, ibuprophen, sildenafil, maraviroc or clodronate liposomes for two weeks, and nerve damage, inflammatory reaction and matrix thickness around the implant were assessed. treatment with dexamethasone, ibuprophen or clodronate liposomes significantly reduced the inflammatory response in the nerve in comparison to saline group while sildenafil or maraviroc had no effect on iba positive cells infiltration in the nerve. however, only dexamethasone was able to significantly reduce the matrix deposition around the implant after two weeks of treatment. these results support the idea that inflammation triggers the foreign body response in peripheral nerves and a potent anti-inflammatory treatment with dexamethasone could have a beneficial effect on lengthening intraneural interfaces lifespan. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces functionality decreases over time, among other factors, due to the foreign body response (fbr), which encapsulates the implanted devices and physically separates the active sites from the nervous tissue. here we have studied the fbr to parylene c or polyimide thin devices implanted in rat sciatic nerves, assessing thickness of the tissue capsule, signs of inflammation and nerve damage. we have characterized the responsible cells of this response and several molecular mediators over months of implant to find differences between the fbr to both materials. after weeks of implant, the inflammatory response due to the surgery was already decreased, whereas in the implanted nerves it reached its highest levels to then decrease at chronic time points. besides, the amount of foreign body giant cells (fbgc), as a result of macrophage fusion, found in the tissue capsule around the implant also increases progressively to reach a maximum after weeks. on the other hand, molecular analysis of the environment revealed a peak of inflammatory cytokines during the first day of implant to return to standard levels thereafter. however, an increase on ccls molecules was found at later time-points for both materials. with regard to the capsule thickness, all the devices were surrounded by a tissue deposition which appeared soon after the implantation. however, in the case of polyimide devices, the tissue capsule showed a peak weeks after the implant and signs of remodeling thereafter, while the parylene c devices showed a second increase from to weeks in comparison to polyimide devices. immunohistochemical and electron microscopy analysis revealed two different cell types implicated in the fbr in nerve to both materials: macrophages, in close contact with the interface, and fibroblasts which appear after weeks surrounding the tissue capsule. although further analyses are needed to elucidate the differences in the fbr to parylene c and polyimide polymers, these results can help to determine therapeutic targets in order to reduce this response and to improve the intraneural interfaces lifespan. delmont e , antoine jc , paul s , boucraut j , attarian s . referral centre for als and neuromuscular diseases, marseille, france; referral centre for neuromuscular diseases, saint etienne, france; immunology laboratory, saint etienne, france; immunology laboratory, marseille, france. peripheral neuropathies with antibodies against myelin associated glycoprotein (mag) are chronic sensory neuropathies characterized by the presence of an igm monoclonal gammopathy and high levels of anti-mag antibodies. these antibodies recognize a specific epitope called human natural killer (hnk ) shared by nk lymphocytes and several components of the peripheral nerve (mag, p , pmp , sgpg, phosphocan). recently an elisa test has been developed to detect antibodies against hnk epitope. our objectives were to determine the sensitivity and the specificity of anti-hnk antibodies in the diagnosis of anti-mag neuropathy and to know if these antibodies were correlated with the severity of the disease. anti-hnk antibodies were assessed in anti-mag neuropathies and in negative controls: chronic inflammatory demyelinating polyradiculoneuropathies (cidp), miller fisher syndromes, sensory neuronopathies, length-dependant axonal sensory polyneuropathies, healthy controls. in anti-mag neuropathies, were recorded age, disease duration, incat sensory sum score (iss), overall neuropathy limitation scale (onls), rasch-built overall disability scale (rods), mrc sum score, anti-mag antibodies titer, peak dosage of the igm monoclonal gammopathy. anti-hnk antibodies were measured with gangliocombi™ mag elisa test and anti-mag antibodies with anti-mag autoantibodies elisa test both from buhlmann company. anti-hnk antibodies were positive in / anti-mag neuropathies, and in / controls (sensitivity %, specificity %). in anti-mag neuropathies, anti-hnk titer was correlated with sensory deficiency evaluated with the iss score (r= . , p= . ) and with disability evaluated with the rods (r= − . , p= . ) and onls scales (r= . , p= . ). anti-hnk titers were not related to age, disease duration, mrc sum score, anti-mag antibodies titer, peak dosage of the paraproteinemia. anti-mag antibodies titers were associated with none of the characteristics of the patients with anti-mag neuropathy. anti-hnk antibodies have good sensitivity and specificity in the diagnosis of anti-mag neuropathy. compared to anti-mag antibodies, their value is that their titers are related to the disease severity. these results need to be confirmed in a larger prospective cohort. chronic inflammatory demyelinating polyradiculoneuropathy (cidp)is a heterogeneous and treatable immune-mediated disorder that critically lacks biomarkers to support diagnosis. recent evidences indicate that paranodal proteins (contactin- , contactin-associated protein- , and neurofascin- ) are the targets of autoantibodies in a subset of patients with cidp showing distinct clinical presentations. particularly, these biomarkers appear to have clinical relevance and help to orientate therapeutic choice. here, we examined five patients presenting an igg reactivity against the nodes of ranvier and the axon initial segment. using a proteomic approach, cell-based assays and elisa, we identified neurofascin- (nfasc ) and neurofascin- (nfasc ) as the main targets of autoantibodies at the nodes of ranvier. four patients displayed predominantly antibodies of the igg subclass, whereas one patient presented igg antibodies that activated the complement pathway in vitro. these antibodies recognized different epitopes than the previously described anti-neurofascin- igg suggesting different pathogenic functions. accordingly, patients with anti-nfasc / igg showed a distinctive clinical presentation. most patients had a severe phenotype associated with conduction block or decreased distal motor amplitude. tremors or neuropathic pain were not observed. four patients presented with a subacute-onset and sensory ataxia. of interest, the neuropathy occurred concomitantly with nephrotic syndromes in two patients and with an igg -related retroperitoneal fibrosis in one patient. this suggested that autoantibodies could be responsible for the occurrence of both disorders. intravenous immunoglobulin and corticosteroids were effective in three patients, and one patient improved following cyclophosphamide and rituximab treatment. clinical remission was found to correlate with the depletion of anti-nfasc / antibodies and the loss of igg reactivity toward the nodes of ranvier. in addition, recovery of conduction block and of distal motor amplitude were observed following remission and suggested a nodo-paranodopathy. our data demonstrate that nodal antigens are the target of autoantibodies in a subgroup of patients with cidp. this emphasizes that the pathogenic mechanisms involved in chronic immune-mediated demyelinating neuropathies are broad and may include dysfunctions of the nodes of ranvier. mutations in the neurofilament heavy (nefh) gene have been recently identified as a rare cause of autosomal dominant, axonal charcot-marie-tooth disease (cmt ). the clinical spectrum of this condition remains to be delineated. we report two french families with an axonal, predominantly motor, dominantly inherited form of cmt caused by two previously unreported mutations in the nefh gene. twelve patients belonging to two different families were included in the study. they displayed an axonal motor and sensory neuropathy, with no mutations in known axonal cmt genes. a remarkable feature in all patients was the early involvement of proximal muscles of the lower limbs, occurring approximately to years after the onset of motor deficit. proximal weakness affected predominantly the iliopsoas muscle, whereas quadriceps and hamstring muscles were relatively preserved. muscle weakness and muscle wasting progressed rapidly, with most of the patients requiring walking assistance after years of disease evolution. three patients in family had brisk reflexes. nerve-conduction velocity studies displayed evidence of a motor and sensory axonal neuropathy predominantly affecting the lower limbs. original deletions of nucleotides near the end of the coding sequence of nefh were identified: in family , c. _ del (p.lys argfs* ), and in family c. _ del (p.lys glyfs* ) causing a frameshift. interestingly, this frameshift leads to the loss of the terminating codon and to the translation of additional amino acids encoding a cryptic amyloidogenic element, suggesting that this type of mutations could induce protein aggregation. consistently, we showed that overexpression of the mutated forms of nefh in a human neuroblastoma cells induced the formation of protein aggregates. we also observed that it triggered caspase activation and apoptosis. using electroporation of chick embryo spinal cord, we confirmed in vivo that mutated nefh formed aggregates and triggered apoptosis of spinal cord neurons. altogether, this suggests that these mutations in nefh cause protein aggregation and neurotoxicity in neurons expressing nefh. progressive loss of such neurons would explain the early motor involvement and the pyramidal signs observed in some patients. our results provide a physiological explanation to the presence of cmt and als clinical features in affected patients. del valle j , , delgado-martínez i , righi m , santos d , , cutrone a , bossi s , d'amico s , micera s , , navarro x , . neuroprosthetic devices that are aimed to restore sensorimotor limb function of amputee patients require highly selective electrodes designed to establish a tight relationship with the nerve, allowing the bidirectional transduction of signals between nerve fibres and the interface and enabling close-loop control from the user. differently from extra-or intraneural interfaces, regenerative nerve electrodes are designed to enable electrical interface with regrowing axonal bundles of injured nerves, aiming to achieve high selectivity for recording and stimulation. however, most of the developed designs pose an obstacle to the regrowth mechanisms due to low transparency and cause an impairment of the nerve regeneration. in this work, we present a novel double-aisle planar regenerative electrode, a new type of highly transparent, non-obstructive regenerative electrode, which allows the selective stimulation and recording of separated nerve fascicles. the design consists of a thin and flexible double-sided electrode longitudinally inserted across a conduit thus creating two separated aisles in which regenerating fascicles can independently regrow after nerve transection. electrodes implanted in acutely transected nerves of rats showed the capability of selectively stimulating and recording different fascicles inserted in the aisles. moreover, chronic implantation of the electrode in a nerve gap of mm after sciatic nerve section allowed for fascicle regeneration and reinnervation of distal muscles as confirmed by the high number of myelinated axons inside each aisle, good biocompatibility, and adequate nerve conduction. in addition, three and six months after implantation, independent stimulation and recording of each separately regenerated fascicle were possible. our results demonstrate the potential contribution of the doubled-aisle regenerative electrode to selectively interface different fascicles of an injured nerve with no deleterious effects on nerve regeneration. therefore, this multi-aisle regenerative electrode may be suitable for neuroprosthetic applications, such as prostheses for the restoration of hand function after amputation or severe nerve injuries. demichelis c , garnero m , franciotta d , cortese a , callegari i , mancardi gl , schenone a , leonardi a , benedetti l . department of neuroscience, rehabilitation, ophthalmology, genetics, maternal and child health, university of genoa and irccs aou san martino-ist, genoa, italy; laboratory of neuroimmunology, irccs, "c. mondino" national neurological institute, university of pavia, pavia, italy; u.o. neurology, asl imperiese, imperia, italy. querol et al. showed that neurofascin (nf ) antibodies identify a chronic inflammatory demyelinating polyradiculoneuropathy (cidp) phenotype characterized by severe polyradiculoneuropathy, poor response to intravenous immunoglobulins (ivig), and disabling tremor. neurological improvement after therapy with rituximab has been previously reported in three patients with cidp with igg anti-nf antibodies. herein we describe the acute-onset of a case of cidp positive for nf igg antibodies resistant to conventional therapies and responsive to rituximab. the patient is a year-old woman who presented acute onset ataxia and gait disturbances; her symptoms progressed over two weeks and distal weakness, numbness and paresthesias appeared too. the nerve conduction study was suggestive for a motor-sensory polyradiculoneuropathy mainly demyelinating. the cerebrospinal fluid analysis showed elevated protein level and normal cellular count. the patient was initially diagnosed with guillain-barré syndrome (gbs) and treated with plasma exchange without improvement. an ivig cycle was started with a partial relief but at the time of admission to the rehabilitation center the patient still had a marked weakness in all four limbs. after six months she presented a further clinical deterioration and she was restricted to wheelchair. there was no response to additional treatment with ivig, while pulse corticosteroid treatment determined a significant clinical improvement. during the next months, despite the maintenance of steroid therapy, the patient presented a progressive deterioration and she was again restricted to wheelchair. postural and intention tremor appeared at upper limbs and became progressively more disabling. anti-nf ab dosage resulted positive. rituximab was administered at a dosage of mg/m /weekly for weeks. after three months the tremor improved, allowing her to eat independently and the patient was able to walk with bilateral support. antibodies anti-nf were negative. after six months she walked without support and she was able to stitch crochet. as previously reported, in this case a cidp positive for igg nf developed severe polyradiculoneuropathy with predominant distal weakness, ataxia, disabling tremor and resistance to conventional therapies. interestingly the onset was gbs-like. the correct identification of these cidp subtypes has diagnostic, prognostic and therapeutic implications. rituximab con be useful in these patients. demir Ö , yazıcı t . department of neurosurgery, university of gaziosmanpaşa school of medicine, tokat, turkey; department of neurosurgery, kent hospital, giresun, turkey. it is still challenging problem to maintain motor and sensory functions of peripheral nerve after nerve transection. after the nerve injury, calcium concentration in the damaged area increases. then the calcium ions act like cytotoxic agents in the damaged area. nifedipine is calcium channel blocker. we aimed to investigate the effects of nifedipine on nerve regeneration by modulating calcium in the damaged area. twenty-four swiss albino male rats were divided into two groups. left sciatic nerve transection surgery was performed to the all rats in both groups. then the all transected nerves were sutured primarily with epineural interfascicular method. in the experimental group, the anastomosis sites were wrapped with a piece of gel foam soaked into diluted nifedipine solution. in the control group, the anastomosis sites were wrapped with a piece of gel foam soaked into saline solution. we evaluated the effect of nifedipine by using functional, electro-physiological and histopathological studies after the surgeries. in the postoperative second week, walking test was performed and sciatic function index was calculated. in the postoperative third week electroneuronography (enog) was performed. there are significant differences between two groups. nifedipine improved nerve recovery functionally (p< . ) and electro-physiologically (p< . ). in the postoperative fourth week, we performed histopathological examination. in the experimental group with nifedipine there were more organized axons that reached the aim. we conclude from these results that nifedipine is an effective nerve protective agent when used locally at the anastomosis site after the transection of the nerve. the lack of effective, disease-modifying therapies for cmt highlights the need for novel preclinical models suitable for drug discovery. studies in rodent models of cmt tend to be time-consuming, and findings so far have translated poorly into clinical trials. primary and induced pluripotent stem cell (ipsc)-derived neuronal cultures are an established model of neurological diseases. however, due to the random distribution of neuronal bodies and neurites that happen when plating these cells, this system is not ideal to investigate axonal, length-dependent processes like peripheral neuropathies and particularly cmt. to optimize this well-established model system, we developed a robust human platform to study axonal morphology and physiology based on motor neuron neurospheres. we differentiated motor neurons from human induced pluripotent stem cells, purified them by magnetic sorting and cultured them in suspension until they formed neurospheres. floating neurospheres can be maintained in agitation for months as a reliable source of motor neurons. after neurospheres are platted, axons rapidly grow out of them in a radial fashion, resembling dorsal root ganglia cultures. this configuration allows for a better visualization of axons in imaging studies and for continued axonal growth over at least a -day period. axons grew at an average rate of micrometers/day and reached up to cm in length. neurospheres can be fixed and stained allowing for morphological analysis and investigation of protein distribution in axons. this system is also ideal for time-lapse imaging to study axonal transport of organelles and neurofilament kinetics. lastly, our motor neuron neurosphere system lends itself well for high content screening platform. neurospheres can be plated in -well plates where multiple compounds can be tested and the axons easily imaged by a high content screening microscope. in summary, we developed a new platform to investigate motor axons in vitro, which are particularly useful to study length-dependent processes such as inherited peripheral neuropathies and may facilitate the identification of new therapeutic compounds using high content screening systems. mutations in the neurofilament light chain (nfl) gene cause autosomal dominant axonal charcot-marie-tooth neuropathy (cmt e). nfl is a major component of the neuronal cytoskeleton, and is believed to function in conjunction with nfm and nfh to provide structural support for the axon and regulate axon diameter. despite the significant advances in understanding its biological basis, there is still no effective, disease-modifying therapy for cmt e, in part due to the paucity of preclinical models suitable for drug discovery. the development of novel preclinical platforms that can faithfully mimic mechanisms of axonal degeneration in vitro would be an essential and valuable resource to better understand the biology of cmt e and identify potential targets for therapy development. to address this, we generated control and cmt e patient-derived motor neurons and cultured them in suspension until they formed neurospheres. immunostaining of cmt e neurospheres with nfl and tubb antibodies revealed numerous areas of nfl accumulation in n s cmt e axons, resembling the accumulations of mutant nfl protein seen in the processes of catecholaminergic neuronal cell line cad overexpressing several nfl mutants. further analysis demonstrated that areas of nfl accumulation were also immunopositive for nfh, pnfh and nfm and that at least nfl and nfm co-localized in the same areas of deposits. taken together, these results demonstrate that abnormal axonal neurofilament distribution is a feature of cmt e ipsc-derived motor neurons and involve all three neurofilament subunits. we also developed an image analysis routine to allow for automatized quantification of neurofilament distribution. preliminary quantification of nfl signal intensity revealed that axons from patients have a weaker nfl signal compared to control axons, but present several signal peaks above the range observed in controls, which related to the areas of nfl accumulation. these results suggest that nfl accumulates in certain regions of cmt e axons but is reduced in the areas with no accumulation. these findings can be readily adapted into a high content screening platform and will be used to identify compounds able to reverse this axonal phenotype. in summary, we identified a strong axonal phenotype in human cmt e motor neurons with potential as a screening platform for drug discovery. nodal and paranodal proteins have been identified as antigens in peripheral inflammatory neuropathies, however the frequency and clinical relevance of antibody responses against these targets remain poorly investigated in gbs. patients with acute onset inflammatory neuropathies were identified by exploration of the local databases of the departments of neurology and the institute of neurology of the medical universities in innsbruck and vienna. patient data, electrophysiological classification and presence of anti-gangliosid antibodies were retrospectively retrieved by review of patient records. only patients with typical clinical presentation and electrophysiological results consistent with one of the subtypes of gbs were included in the study. among forty-nine patients, thirty-five were classified as aidp, six aman, three amsan, three mfs, and two pharyngo-cervico-brachial gbs. of the included patients had anti-ganglioside-antibodies. ten patients with the initial suspicion of aidp had a disease duration of more than months and were reclassified as cidp. all patient and twenty sera of control patients with non inflammatory polyneuropathy were screened by an optimized tissue based assay using rat brains for immune responses against surface antigens, and by cell-based assays with transfected hek cells for antibodies against contactin (cntn ), contactin (cntn ), contactin-associated-protein (caspr ) and neurofascin- (nf ). in the tissue based assay some of the patients showed a light neuropil staining. none of gbs patient's sera had antibody reaction to cntn , cntn , caspr or nf in cell-based assays. among the cidp patients, two patients demonstrated reactivity against cntn with similar clinical presentation as previously described. none of the control patients had any antibody reaction to the performed tests. our results suggest that antibody responses to cntn , cntn , caspr or nf are absent in austrian gbs patients, although more patients will be screened to substantiate these preliminary results. furthermore, it remains to be established whether antibodies against cntn may predict a chronic course in acute onset inflammatory neuropathies. a few variants of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described with a frequency of - %. their relation and possible evolution into typical-cidp remain unclear, as is their treatment response possibly because of differences in diagnostic criteria. we used the data from a web-based database on italian patients with cidp to determine the frequency and characteristic of these variants, the possible evolution into typical-cidp, and their treatment response. all the patients were assessed at study entry and the disease course before inclusion was analyzed. by february- , we included patients ( men, women), aged - years (median years) with a mean disease duration of . years (range . - years) and complete data available from . based on the clinical data and our revised diagnostic criteria, patients ( %) were classified to have atypical cidp at onset and for the following two years including with dads ( %), with motor cidp ( %), with sensory cidp and cisp ( . %), with lewis-sumner syndrome ( . %), and with recurrent cranial neuropathy. at study entry, patients ( %) had progressed into typical cidp after - years (median years) while ( %, % of total) still had atypical cidp after . - years (median years) with a similar proportion of progression ( - %) within each group. the diagnosis of atypical cidp at entry fulfilled efns/pns criteria in ( %). csf studies were diagnostic in / ( %) patients, nerve biopsy in / ( %), and nerve imaging in / ( %) tested patients. similarly to typical cidp, % of treated patients with atypical cidp improved after treatment with a proportion of response varying from % to % in the different forms. most patients with sensory or motor cidp had however an unsatisfactory response to steroids. this study shows that he proportion of patients with atypical cidp varies during the course of the disease with almost % of the patients evolving into typical cidp within years from onset. in addition, response to treatment is frequent in atypical cidp even if not all the forms respond to the same therapies. only few studies investigated the frequency of antecedent events and comorbidities in patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and little is known on the role of possible predisposing factors, dietary, and lifestyle habits, on the onset and progression of the disease. we used the data from a web-based database on italian patients with cidp to determine the frequency of antecedent events and comorbidities and the possible role of predisposing factors including lifestyle and dietary habits and exposure to toxic agents, using a structured questionnaire. partners of patients served as controls. impairment was evaluated using the mrc sumscore and disability with incat and r-ods scales. logistic regression was used to calculate odds ratio (or) with % confidence interval (ci) for the risk of cidp. sex and disease-duration were included as covariates. by february- , patients were enrolled, with complete data on patients for antecedent events and comorbidities and patients and controls for lifestyle habits. ninety-two patients ( %) reported an antecedent event, mostly infection or vaccination ( %). one or more comorbidity were present in % of the patients including hypertension ( . %), thyroid disorders ( %) and diabetes ( . %) and in % influenced the choice of initial therapy. exposure to toxic environmental agents (odds ratio [or] = . ; % ci, . - . ), cigarette smoke (or = . ; % ci, . - . ), and dietary supplements (or = . ; % ci, . - . ) were associated with a higher risk of cidp while rice consumption was associated with a reduced risk (or = . ; % ci, . - . ). concerning disease severity, more severely affected patients more frequently consumed raw-meat (or = . ; % ci, . - . ) and white meat (or = . ; % ci, . - . ), while rice (or = . ; % ci, . - . ) and soft drink consumption (or = . ; % ci, . - . ) and physical activity were associated with lower disability (or = . ; % ci, . - . ). this study confirms that comorbidities are frequent in patients with cidp and often influence the choice of initial therapy. in addition preliminary data show that toxic exposure and some lifestyle and dietary habits may influence the onset and progression of cidp. doppler k , schuster y , weishaupt a , sommer c . department of neurology, university hospital würzburg, würzburg, germany. autoantibodies against the paranodal protein contactin- have recently been described in patients with cidp. in most patients, autoantibodies of the igg subclass are predominant and are supposed to be pathogenic. the role of igg anti-contactin- is so far unclear. in the present study, igg of three different patients, one with igg anti-contactin- , one with a low titer of igg anti-contactin- and one with a high titer of igg anti-contactin- , and of controls were injected into the sciatic nerves of lewis rats. nerve conduction studies of the injected nerve and motor and sensory testing were performed before and after injection. conduction blocks and motor deficits were detectable in the two patients with high titers of igg and igg , not in the patient with low titers. the percentage of conduction blocks was . % in rats injected with igg of the igg patient and % in those injected with igg . motor deficits were detectable in both patients with conduction blocks but were most apparent in the patient injected with igg of the igg patient. no differences in sensory testing were observed. conduction blocks and motor deficits improved after five days and were normal after seven to eight days. our data give the first evidence of pathogenicity of igg anti-contactin- autoantibodies, not only igg . igg of the igg patient induced a more severe clinical and electrophysiological phenotype compared to the igg patient. remarkably, this reflected the clinical phenotype of the patients, as the igg patients showed an acute-onset of sensorimotor symptoms at the time of blood withdrawal whereas the igg patient presented with a more chronic course of disease. doppler k , frank f , koschker a-c , reiners k , sommer c . department of neurology, university hospital würzburg, würzburg, germany; endocrinology and diabetes unit, department of medicine i, university hospital würzburg, würzburg, germany. axoglial dysjunction and paranodal demyelination have been discussed as potential mechanisms of nerve fiber damage in diabetic neuropathy. studies on human tissue are limited, as nerve biopsies are invasive and only rarely performed in patients with confirmed diabetic neuropathy. skin biopsy has recently been suggested as a good tool to analyze paranodal and nodal changes of myelinated fibers. in the present study, we analyzed the paranodal and nodal region in myelinated fibers of skin biopsies of patients with diabetic neuropathy, patients with diabetes mellitus without neuropathy, and normal controls. immunofluorescence of skin sections with antibodies against caspr, neurofascin, sodium channels and myelin basic protein was performed to assess paranodal/nodal architecture, segmental demyelination and myelinated nerve fibers. staining with antibodies against protein gene product . was used to quantify unmyelinated nerve fibers. we found an increase of elongated ranvier nodes and a dispersion of neurofascin at the distal leg in patients with diabetes mellitus with and without neuropathy and at the finger in patients with diabetic neuropathy. an increased dispersion of caspr was only found in biopsies of the finger in patients with diabetic neuropathy. our data show that skin biopsy is an appropriate tool to analyze nodes of ranvier in patients with diabetes mellitus. structural nodal changes are detectable in diabetic neuropathy, and even in diabetic patients without neuropathy. dourado me , fernandes u , vital al , ramos e , urbano jc , sena a , queiroz jw , jeronimo smb . federal university of rio grande do norte, natal, brazil. the erasmus gbs outcome score (egos) is a validated prognostic model that uses acute phase and easy-to-obtain clinical characteristics to determine outcome at months in patients with gbs. this study aims to assess the validity of egos in rio grande do norte, brazil, and to compare with another european study. data collected prospectively from a cohort of patients with gbs of rio grande do norte, brazil, between june and august , was assessed. ninety patients were excluded for missing data or diagnoses of miller fisher syndrome and atypical forms of gbs. to calculate the egos, the gbs disability score was assessed in the second week of disease and at months. to compare this study with the european one in independent group proportions, we used the student's t-test, being considered statistically significant p< , . the patients included were divided in four groups based on egos. thus, patients had egos between and ; had egos between . and . ; had egos and had egos between . and . in the first, second, third and fourth group, ( %), ( . %), ( . %) and ( . %) of the patients were unable to walk independently after six months of the disease, respectively. overall, of the patients analyzed, ( . %) had poor outcomes in this study. in the european paper, based on the same group division, of ( . %), of ( %), of ( %) and of ( %) were unable to walk independently. comparing both studies, the patients of this study were younger, more seriously ill in the first weeks and with more sensitive deficits. there were no difference relative to sex, cranial nerves deficits and presence of anti-gangliosides antibodies. using the student's t-test for ability to walk after months according to egos stratification, we achieve in the first group p= . ; in the second p= . ; in the third p= . ; and in the fourth p< . .the egos did not have a good capacity to predict the ability to walk after months of gbs in rio grande do norte, brazil. historically, guillain-barré syndrome (gbs) epidemics are rarely seen. between and , we treated and followed cases of gbs in the state of rio grande do norte with a yearly incidence of . / , . no seasonality was observed. the mean age of the patients was years (range, - ), with % of the cases younger than years. demyelinating variant was the most frequent subtype of gbs. in march , the first report of autochthonous transmission of zika virus (zikv) was determined in natal, brazil. later that month, we documented an increase in incidence of gbs in natal, brazil. the incidence in was of . / . . of the cases of gbs diagnosed in , were diagnosed from march through may, which coincided with the outbreak of zikv in natal, brazil. eighteen patients ( % of the cases) had a history of rash and fever prior to onset of gbs symptoms, with the median age of years (range, - ) . the electroneuromyography studies of these patients indicated that ( . %) had acute inflammatory demyelinating polyneuropathy, ( . %) had acute motor axonal neuropathy, and ( . %) was inconclusive. the mean time from onset of zikv infection symptoms to onset of the gbs were days (range, - ). the mean time of nadir was days (ranged, - ) . cranial neuropathies were present in patients ( . %). nine patients were bedridden ( %) and ( . %) required mechanical ventilation. the mean protein content of the central spinal fluid was . g/l, with the white blood cell count below /mm in all patients. they were all treated with intravenous igev. they all improved quickly. anti-gm was negative in all patients. rt-pcr was negative for dengue, chikungunya and zika. serum mac-elisa igm for zika and dengue was made in patients and it had % of positivity. prnt for zika and dengue had % positivity. in summary, we report a geographically and temporally defined cluster of gbs associated with an outbreak of acute rash in the state of rio grande do norte, brazil. as the prevalence of diabetes mellitus continues to increase worldwide, diabetic complications represent a growing burden to patients and society. distal symmetrical polyneuropathy (dsp) is a common complication that affects up to % of diabetic patients. dsp reduces patient quality of life due to chronic pain, ulcerations, and may lead to lower extremity amputations. despite its high prevalence, the mechanisms underlying diabetic dsp are poorly understood and several mechanisms are believed to play a role. we hypothesize that diabetic dsp arises from microvascular complications characteristic to diabetes. specifically, capillary dysfunction -disturbances in capillary flow patterns -is a likely candidate to explain development of dsp, as it can limit oxygen and nutrient delivery to nervous tissue, causing nerve dysfunction and damage, and thus development of dsp. we will study this hypothesis utilizing the state-of-the-art blood flow imaging techniques to visualize and quantify endoneureal blood flow and then link these findings with measures of dsp (e.g. nerve conduction velocity; intra-epidermal nerve fibre density) in animal models. we will include several animal models of diabetic dsp caused by either type or type diabetes. two photon microscopy and optical coherence tomography allow visualisation and quantification of capillary transit times and blood flow within peripheral nerves at high resolution. we hypothesise that changes in blood flow patterns and subsequent impairment of nutrient and oxygen delivery to nervous tissue precede the onset of diabetic dsp. if our experiments support this prediction, we will attempt to develop interventions that improve capillary blood flow to prevent or delay the development of dsp. duman o , saracoglu m , haspolat s , bozkurt o . department of child neurology, akdeniz university hospital, antalya, turkey. axonal polyneuropathies are very heterogeneous group of diseases which are very rarely seen during infantile age. some of them may be accompanied by developmental retardation, severe muscle weakness and progressive course. we aimed to present two cases of axonal sensorymotor neuropathy with infantile onset and atypical course. our -year-old boy patient was admitted to our clinic for progressive gait loss since one month. he was the first offspring of consanguineous parents with normal prenatal and natal history. electromyography revealed axonal sensorymotor polyneuropathy. metabolic and cerebrospinal fluid (csf) examinations for etiology were all normal. brain and spinal magnetic resonance imaging (mri) were normal. he had partial benefit from oral steroid treatment. in the course of disease along with four neuropathy attacks he had significant benefit from serial intravenous immunoglobulin treatments in two years clinical course. a month-old girl who is the first offspring of nonconsanguineous parents was admitted to the clinic for acute tetraparesis. axial sensorymotor polyneuropathy was detected in the electromyography. metabolic and cerebrospinal fluid (csf) examinations were normal. she had three more acute polyneuropathy attacks during ivig cessation period. both patients revealed with serial immunoglobulin treatments but unresponsive to riboflavine treatment. we aimed to discuss our rarely seen and the pathogenesis is not completely understood cases' course. serial ivig treatment may be helpfull for such patients' treatment. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominantly inherited disorder caused by mutations of the transthyretin (ttr) gene. the mutant amyloidogenic transthyretin protein causes the systemic accumulation of amyloid fibrils that result in organ dysfunction. ttr-associated fap is a progressive and fatal disease, if left untreated, and should be considered in the differential diagnosis of any person presenting with a progressive polyneuropathy, particularly with accompanying autonomic involvement. the clinical, electrophysiological, histopathological, and genetic characteristics of patients from turkey ( female, male) from eleven families with polyneuropathy and mutations in ttr were evaluated. two patients had no family history of ttr-fap and were considered as sporadic cases, and the remainders were familial cases displaying an autosomal dominant inheritance pattern. sequence analysis of the ttr gene revealed five mutations (p.val met, p.glu gln, p.gly glu, p.glu gly and p.gly glu). most common mutation was p.val met (in unrelated families). mean age at disease onset was . ± . years (range - years). the most commonly reported initial complaint was paresthesia in the feet (asymmetric in three patients). four patients ( male) with the p.glu gln mutation presented with carpal tunnel syndrome. two patients with the p.gly glu mutation showed episodes of dysarthria and hemiparesis, consistent with this genotype. seven patients died during the follow-up period as a result of systemic involvement. this study suggests that our cohort of ttr-fap patients from turkey exhibits clinical and genetic heterogeneity. ebenezer gj , liu y , judge dp , cunningham k , truelove s , carter nd , sebastian b , byrnes k , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; division of cardiology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. effect of amyloid deposition on cutaneous nerves was assessed in subjects with pathogenic ttr variants and control subjects. three groups of subjects each including ttr-fap patients, age/gender-matched healthy subjects and disease controls as well as ttr mutation carriers without neuropathy (ttr-nopn) and with al-amyloid underwent neurological examination and mm skin biopsies. micron sections were stained with anti-pgp . , anti-ttr and congo red. amyloid burden with imagej, intraepidermal (ienfd) sweat gland (sgnfd) and pilomotor densities (pmnfd) measured and correlations between amyloid burden, fiber subtype, neuropathy impairment score-ll (nis-ll) and nis sensory subscore were evaluated. ienfd, sgnfd, and pmnfd were all significantly reduced in ttr-fap patients vs. healthy controls while mutation carriers had intermediate reductions. lower nerve fiber densities were associated with nis-ll (p< . ). congo red staining revealed brilliant red amyloid deposits with apple-green birefringence within dermal collagen, sweat glands, and arrector pili muscles. amyloid infiltration was observed in the endoneurium and perineurium of small fiber sensory and autonomic nerves that innervate sweat glands and arrector pili muscles. cutaneous amyloid deposition was detected in % of ttr-fap and not in healthy or disease controls subjects. both al and / ttr-nopn subjects were congo red positive. amyloid burden was inversely correlated with ienf (p< . , r=− . ) sgnf (p< . , r=− . ), pmnf (p= . , r=− . ) distal leg densities, and correlated with nis-ll (p= . , r= . ) and nis sensory subscore (p= . , r= . ). wild-type ttr staining was less prominent in pathogenic ttr carriers. the diagnostic sensitivity and specificity to detect amyloid in skin were % and % in ttr-fap. the repeat measurement of the amyloid burden from the same section with imagej was r = . , p< . and different sections from the same biopsy was r = . and p< . . we conclude that endoneurial amyloid contributes to sensory and autonomic nerve injury. amyloid burden correlated strongly with sensory/autonomic axon densities and nis-ll. skin punches offer a convenient alternative to establishing a tissue diagnosis. amyloid burden is an attractive biomarker marker for ttr-fap and treatment effect. the study was supported through a grant from pfizer. ebenezer gj , truelove s , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. we investigated differences of unmyelinated sensory nerve fibers in the distal limb among healthy-weight subjects (bmi < kg/m ) and overweight/obese subjects (bmi ≥ kg/m ), aged - years. subjects underwent neurological examination and mm skin punches from distal leg (dl), thigh (dt), and proximal thigh (pt) sites, from which micron sections were stained with anti-pgp . antibody; intraepidermal nerve fiber density (ienfd; fibers/mm) and epidermal thickness were assessed. a second dl biopsy was processed for electron microscopic examination and both thick and thin sections were examined for ultrastructural changes. multivariable linear regression models were used to assess the effect of age, gender, height and weight. after controlling for height, age, and obesity, females were found to have lower distal leg ienfd (− . ; p= . ). increasing age and height were significantly associated with decreasing dl ienfd, with decreases of − . fibers/mm per years (p<. ) and − . fibers/mm per cm (p< . ), respectively. even after controlling for height, being overweight/obese was associated with reduced dl ienfd, with . fibers/mm lower dl enfd than healthy-weight individuals (p=. ). these findings remained consistent across distal thigh and proximal thigh enfd, though not all associations remained significant. the epidermis was thicker in obese subjects across the lower limb, most pronounced at the distal leg (μm, mean± sd, healthy-dl: ± . , dt: . ± . , pt: . ± . , obese-dl: . ± . , dt: ± . , pt: . ± . ). under em very few intact dermal nerve bundles were identified at the proximal thigh sites. the atrophic and degenerating axons were seen with perineurial infiltration by dense collagen in obsess/overweight subjects but not age/gender matched controls. obesity further accelerates attenuation of epidermal nerve fibers across the lower limb even after controlling for other associated factors. echaniz-laguna a , geuens t , petiot p , péréon y , adriaenssens e , haidar m , capponi s , maisonobe t , fournier e , dubourg o , degos b , salachas f , lenglet t , eymard b , delmont e , pouget j , juntas morales r , goizet c , latour p , timmerman v , stojkovic t . strasbourg university hospital, strasbourg, france; peripheral neuropathy group, vib department of molecular genetics and institute born bunge, university of antwerp, antwerpen, belgium; lyon university hospital, lyon, france; nantes university hospital, nantes, france; hôpital de la pitié-salpétrière, paris, france; nice university hospital, nice, france; marseille university hospital, marseille, france; montpellier university hospital, montpellier, france; bordeaux university hospital, bordeaux, france. in this study, we describe the phenotypic spectrum of distal hereditary motor neuropathy caused by mutations in the small heat shock proteins hspb and hspb and investigate the functional consequences of newly discovered variants. among unrelated patients with distal motor neuropathy, we identified mutations in hspb ( index patients/ ; . %) and hspb ( index patients/ ; . %) genes. patients have slowly progressive distal ( %) and proximal ( %) weakness in lower limbs, mild lower limbs sensory involvement ( %), foot deformities ( %), progressive distal upper limb weakness ( %), mildly raised serum creatine kinase levels ( %) and central nervous system involvement ( %). we found a broad range of disease onset with some patients presenting with foot drop at the age of years, and others presenting symptoms only after years. disease progression was slow in all patients, and even with a disease duration of more than years patients were still able to walk. none of our patients were wheelchair dependent. muscle pathology, nerve pathology and electrophysiology showed in all cases a slowly progressive, mostly symmetrical and predominantly distal motor axonal neuropathy. mild sensory involvement was observed upon nerve conduction studies, mostly the lower limbs, in % of cases. we identified hspb and hspb mutations, including respectively and not previously reported. transmission was either dominant ( %), recessive ( %) or de novo ( %). three missense mutations in hspb (pro ser, gly asp, gln arg) cause hyperphosphorylation of neurofilaments, while the c-terminal mutant ser leu triggers protein aggregation. two frameshift mutations (leu fs, ala fs) create a premature stop codon leading to proteasomal degradation. two mutations in hspb (lys met/asn) exhibited increased binding to bag . we demonstrate that hspb and hspb mutations are a major cause of inherited motor axonal neuropathy. mutations lead to diverse functional outcomes further demonstrating the pleotropic character of small heat shock proteins. eftimov f , querol l , rajabally ya and on behalf of all participants of the st enmc workshop. academic medical center, amsterdam, the netherlands; hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; aston university, birmingham, uk. although chronic inflammatory demyelinating polyneuropathy (cidp) is a treatable neuropathy further research is urgently needed to define the diagnostic clinical and electrophysiological boundaries of cidp and its subtypes, and to define the role of biomarkers in supporting the diagnosis, monitoring disease activity and predicting response to treatment and outcome. in recent years, several national registries and biobanks have been developed to enable systematic data collection in cidp. an international registry with large number of patients is needed to allow answering many important questions and develop validated prognostic models to predict outcome in individual patients with cidp. at the inflammatory neuropathy consortium (inc) meeting in , the inc members agreed that a european neuromuscular center (enmc) workshop would be the ideal setting to reach a consensus on the infrastructure of database and biobanks. the st enmc workshop will take place on may - , with participants representing different countries. primary objective of the workshop is to reach a consensus on inclusion and exclusion criteria, core sets and recommended sets of clinical data, diagnostic data and follow-up points and a manual of operations for collection of biomaterials. a secondary objective is to construct an infrastructure to allow sharing data between different databases and biomaterials. conclusions of the consensus meeting and outline of further perspectives will be presented at the peripheral nerve society meeting in . eichinger kj , burns j , cornett k , bacon c , shepherd m , mountain j , sowden j , shy r , shy me , herrmann dn . clinical outcome assessments that measure functional ability are important endpoints for clinical trials. dr. burns has led the development/validation of a functional outcome assessment (cmtpeds) for individuals with charcot marie tooth disease (cmt) ages - years in the inc rdcrn. the cmtpeds is reliable and sensitive to change. however a validated functional outcome measure (fom) for adults with cmt is needed. our data in - year-olds indicated that the cmtpeds could be modified for adult use. however, some items of the cmtpeds (e.g. balance beam and jumping) have floor effects in adults with cmt. we have developed an adult cmt-fom modeled on the cmtpeds, and refined based on literature review, patient interviews, a large-scale cmt patient survey and expert opinion. the cmt-fom is a performance-based scale comprising items that are combined to form a composite score to quantify functional ability of adults with cmt. the cmt-fom shares items with the cmtpeds. four items were added to measure functional abilities relevant to adults (sit to stand, meter walk/run, stair climb, and timed up and go test). the cmt-fom scoring mirrors the cmtpeds. to generate a score ranging from - , raw item scores are converted to z scores, based on age-and sex-matched normative reference values form the norms project and categorized to a - likert along a continuum of impairment levels. we have conducted a pilot study of the cmt-fom in adults with cmt a ( male, female, age . ± . yrs) of differing severity (cmt exam score (cmtes) range - ). the cmt-fom is feasible, individuals were able to complete all items, and takes minutes to perform. the mean cmt-fom score was . ± . (range - ). concurrent validity of the cmt-fom is supported by an association with the cmtes (r = . ). the overall score did not demonstrate floor or ceiling effects. in summary the adult cmt-fom is well-tolerated and captures upper and lower limb strength, dexterity, balance, speed, ambulation and endurance. the cmt-fom requires validation in a large longitudinal cohort, prior to application in clinical trials. estilow t , glanzman am , burns j , cornett kmd , menezes mp , shy r , moroni i , foscan m , pagliano e , pareyson d , laurà m , bhandari t , muntoni f , reilly mm , finkel rs , sowden j , eichinger k , herrmann dn , shy me , yum sw , ramchandren s and on behalf of the inherited neuropathies consortium. cmt is associated with progressive impairment of the hands. reducing this impairment by treating children who are in the early stages of the disease is crucial. studies assessing measures of cmt hand function in children and their associations with patient-reported outcomes are lacking. we analyzed the upper extremity items from the cmt pediatric scale (cmtpeds) and pediatric cmt quality of life scale (pcmt-qol) in children ages - years enrolled in the inherited neuropathies consortium, to explore the relationships between measures of hand function (impairment, activity, and activities of daily living), and patient-reported outcomes. weak grasp ( %), hand pain ( %), and tremor ( %) were prevalent impairments. performance on activity level tasks, the nine hole peg test ( hpt) and functional dexterity test (fdt), were impaired in % and % of the cases, respectively. patients reported difficulty "sometimes" to "always" in opening a jar/lid ( %), zipping/buttoning ( %), writing ( %), carrying a plate without spilling food ( %) and putting on shoes ( %). patients reporting tremor showed significant differences on the hpt (p=. ). grip strength was shown to have a moderately significant correlation with performance on the fdt (r=. ; p<. ). stepwise multiple linear regression showed that grip strength (beta=−. ; p<. ), hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to open a jar (adjusted r =. ; p<. . grip strength (beta=−. ; p<. ), and fdt (beta . ; p<. ) were predictive of ability to carry plate without spillage (adjusted r =. ; p<. ). grip strength (beta=−. ; p<. ), hpt (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to put on shoes (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to zip/button (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and hpt (beta . ; p<. ) were predictive of ability to use a pen/pencil (adjusted r =. ; p<. ). children with cmt present with frequent limitations in adl performance impacting qol. the upper limb measures of the cmtpeds are associated with hand performance and interventions to improve grip strength and reduce pain should be investigated further with respect to their impact on improving function, and ultimately qol. evans me , morrow jm , wastling s , sinclair cdj , fischmann a , shah s , emira ak , hanna mg , yousry ta , thornton js , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; neuroradiological academic unit, ucl institute of neurology, london, uk; university of basel hospital, basel, switzerland. responsive outcome measures are needed in charcot-marie-tooth disease (cmt) to allow adequately powered clinical trials to test novel therapeutics. we have shown high responsiveness of mri quantified intramuscular fat accumulation in calf muscles of cmt a patients over months. the aim of the present study was to assess the responsiveness and longitudinal validity of quantitative mri over a year follow-up period. we undertook two further sets of quantitative mri, myometric and clinical assessments in the original mrc centre cmt a quantitative mri cohort. mri sequences included fat quantification using the point dixon fat-water separation method, t quantification and magnetisation transfer imaging. of the patients with genetically confirmed cmt a were assessed at baseline ( male, mean age . ± . years), underwent repeat assessments a median on months (data already published), underwent repeat assessments at a median of months, and underwent a final assessment at a median of months. the primary outcome measure currently being analysed is mean calf muscle fat fraction at a single axial slice a fixed distance distal to the knee joint. results of this analysis and correlation with clinical measures will be presented at the peripheral nerve society meeting. fainmesser y , dori a , , drory ve , . department of neurology and neuromuscular service, tel-aviv medical center, tel-aviv, israel; department of neurology, sheba medical center, ramat gan, israel; sackler faculty of medicine, tel aviv university, israel. the use of anabolic drugs by those who wish to increase lean body mass is widespread and not well supervised medically. a years old man was referred for evaluation of a slowly progressive sensory and motor disturbance in the distribution of the left ulnar nerve. his symptoms began months after repeated self-injections of anabolic steroids and vitamin e into the biceps and triceps brachii muscles bilaterally. his examination showed increased muscle mass of the injected muscles with a hard-rubbery consistency, atrophy and weakness of left interossei, mild weakness of the bilateral biceps brachii and sensory loss in an ulnar nerve distribution. nerve conduction studies showed a left ulnar neuropathy with reduced motor and sensory response amplitudes and denervation in the left first dorsal interosseus, as well as mild myopathic changes with significantly reduced insertional activity in both biceps muscles. mri of the soft tissues of the arms showed massive fibrosis and infiltration of fat in the arm muscles compressing the ulnar and median nerves on the left. neurolysis of the left ulnar nerve was performed, and the ulnar nerve was found enclosed in a fibrotic mass throughout the entire length of the upper arm. the brachial artery and the median nerve were similarly enclosed in fibrotic tissue and were released. a biopsy of the affected muscles showed muscle necrosis and fibrosis. the patient was treated with physiotherapy and losartan with only mild improvement in the consistency of the muscles, but without clinical improvement in ulnar nerve function, and worsening of nerve conduction. this case illustrates severe peripheral nerve damage due to entrapment in massive muscle fibrosis following improper intramuscular injection of anabolic steroids for cosmetic purposes. fabrizi gm , testi s , høyer h , braathen gj , squintani g , bertolasi l , ferrarini m , taioli f , cabrini i , pancheri e , cavallaro t , tonin p . department of neuroscience, biomedicine and movement, university of verona and department of neuroscience, aoui verona, italy; section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. inherited diseases of nerve and muscle may overlap phenotypically or coexists as facets of the same disorder. two families whose probands were initially diagnosed with a lower motor-neuron (lmn) syndrome and a hereditary distal motor neuropathy (dhmn) turned out to represent a vcp (valosin-containing protein)-related syndrome and a gne (udp-n-acetylglucosamine -epimerase/n-acetylmannosamine kinase)-related "distal myopathy" . both conditions shared the presence of rimmed vacuoles (rv) in muscle biopsies. in family , the year-old male proband had a lmn syndrome manifesting at age years (wasting/weakness, cramps, fasciculations of thigh muscles, later involving the distal upper limbs). his year-old brother had lower limb weakness and diffuse pain in bones/joints since age . the father was diagnosed with a "muscular dystrophy" thirteen years before dying a years; by age years he had developed a behavioural frontotemporal dementia (ftd). electrodiagnosis (edx) disclosed myopathic changes with ongoing denervation together with a mild sensory-motor axonal polyneuropathy in the proband, and a chronic sensory-motor axonal polyneuropathy in the brother. mri showed fatty replacement of weakened muscles in both siblings and diffuse changes of bones consistent with paget disease (pd) in the elder sibling. in family , a year-old african woman was affected by weakness and wasting starting at the distal lower-limb muscles at age years and soon progressed proximally; parents were not consanguineous and two sisters out of six siblings, deceased in their thirties for post-partum complications, had a similar disease. edx mainly disclosed a neurogenic process with ongoing denervation. muscle biopsies from the three family- patients and from the family- proband showed a myopathy with rv. next-generation sequencing demonstrated a heterozygous c. c>t change of vcp leading to a known pathogenic p.arg cys substitution in all family- patients, and two compound heterozygous c. g>a and c. g>a changes of gne in the family- proband leading to known p.ala thr and p.ala thr substitutions. vcp is known to cause autosomal dominant amyotrophic lateral sclerosis- , charcot-marie-tooth disease type y or inclusion body myopathy (ibm) with pd and ftd (ibmpfd); gne causes the autosomal recessive nonaka myopathy (alias ibm ). both cases emphasize the clinical and neurophysiological heterogeneity of those disorders. fargeot g , vandendries c , labeyrie c , viala k , theaudin m , adams d . neurologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neuroradiologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neurologie, groupe hospitalier universitaire pitié salpêtrière, paris, france. the diagnosis of cidp is often challenging, especially when electrophysiological signs of demyelination are lacking. plexus mri has documented nerve abnormalities in small series of typical cidp but its contribution in patients with no electrophysiological signs of demyelination remains to be proved. we report the results of plexus mri in a serie of patients suspect of having cidp without efns pns definite electrophysiological criteria. we did a retrospective study of patients consulting in kremlin bicetre and pitié salpétrière hospital. lumbar or brachial plexus mri (or both) were performed and we assessed nerve trophicity, t -stir signal intensity and gadolinium enhancement as well as the topography of abnormalities. a consensus diagnosis was made by a group of experts (based on clinical data and other supportive criteria) and allowed us to classified patients in "cidp" or "other diagnosis". the practical contribution of plexus mri to the diagnostic algorithm has been studied. diagnosis of cidp was made in patients. mri was abnormal in % of cidp patients and showed nerve roots hypersignal/hypertrophy/enhancement in respectively . / . / . % of patients. the pattern of abnormalities was often asymetrical ( . %), diffuse ( . %) or multifocal ( . %). after unblinding, the mri confirmed the diagnosis of experts in . % of patients and changed the diagnosis in % of patients. plexus mri has shown to be useful in our serie to confirm the diagnosis of experts or to modify it in patients ( %) . the "classical" pattern described in definite cidp (diffuse nerve root hypertrophy and hypersignal) was documented in % of our cidp patients whereas less typical pattern (focal or multifocal abnormalities, hypersignal without hypertrophy) was found in %. plexus mri seems usefull when facing patients suspected of having cidp when electrophysiological criteria are not met: both symetrical diffuse or asymetrical multifocal patterns can be found and should be always correlated to the clinical examination and other supportive criteria. the specificity of such abnormalities remains to be studied. feely sme , rebelo a , abreu l , tao f , bacon c , zuchner s , shy me . university of iowa, iowa city, ia, usa; dr. john t. macdonald department of human genetics and hussman institute for human genetics, miller school of medicine, university of miami, miami, fl, usa. mutations in bcle-associated athanogene (bag ) have been shown to cause a distal myofibrillar myopathy and cardiomyopathy that can severely affect children or only affect adults depending upon the particular mutation. children with severe cardiomyopathy and myopathy have also developed axonal peripheral neuropathy, consistent with the known localization of bag in neurons as well as in muscle. we have identified two large autosomal dominant families with adult onset charcot-marie-tooth disease (cmt ) with the identical novel missense mutation pro ser, a codon previously shown to cause severe or mild myopathy depending on the amino acid substitution. these families expand the phenotypes caused by mutations in bag to include cmt and provide an additional example of adult onset cmt that may previously have been diagnosed as chronic idiopathic axonal neuropathy (ciap). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. myelin protein zero (mpz), expressed only by myelinating schwann cells, has sequence alterations that have previously been reported to cause demyelinating, intermediate, and axonal forms of charcot marie tooth (cmt) disease. we describe a rare duplication in mpz which is causing an early onset, demyelinating form of cmt in our patient. patient was a product of a normal pregnancy and delivery. early milestones were delayed. she began walking at months of age. she was not able to keep up with her peers and was the slowest runner. she started wearing smos at years of age and afos by years of age. she was diagnosed with scoliosis at years of age and started wearing a brace at years. her examination at years of age showed that her fdi, apb, and adm were all / bilaterally. weakness in her lower extremities included / foot eversion and / great toe dorsi flexion. she had tight heel cords bilaterally. pinprick sensation was reduced throughout in her upper and lower extremities and absent at her toes bilaterally. vibration sensation with a rydel tuning fork was absent at her toes and ankles and reduced at her knees and fingers. nerve conduction studies were performed and revealed no responses in all sensory nerves tested with the exception of the radial nerve which had normal latency and mildly slowed conduction velocity ( m/s). prolonged latencies, demyelinating range slowing (between - m/s), and low cmap amplitudes in almost all segments of the median and ulnar motor nerves were also observed. these findings were consistent with a hereditary sensorimotor demyelinating polyneuropathy. she was diffusely areflexic and her total cmt pediatric score (cmtpeds) was / which is in the severe range. her cmt neuropathy score (cmtns) was / in the moderate range. the duplication of mpz has previously been identified as a rare cause of cmt b. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. mutations in egr cause a severe, demyelinating form of cmt, cmt d. we describe a novel mutation in egr which led to extreme variability in severity in a family. proband was a year old girl who was the product of a normal pregnancy and delivery. early milestones were on time. problems with walking started at years of age. at . she was unable to use stairs, run, or jump. at she was wearing bilateral afos. by she was using a wheelchair and started breathing assist at night. she lost arm lift but could still hold a pen. by she could not ambulate independently and breathing assist was required day/night. she lost the ability to write and developed a head drop. by years she could not sit up. on exam her head and neck muscles were / . upper limbs, deltoids, biceps, wrist ext/flex, finger ext, fdi, and adm were / bilaterally. triceps were / ; finger flexors and apb were / bilaterally. lower limbs were / and she had contractures on the right. sensory examination was normal. she was diffusely areflexic. ncvs were absent. both the cmtns and cmt pediatric score were severe at / and / . exome sequencing revealed a r l variant in egr which was likely pathogenic. the probands' mother also had this mutation. she had no reported symptoms at the age of with strength at / throughout with the exception of left foot eversion which was +/ and great toe dorsi flexion which was -/ bilaterally. sensory exam showed a decrease of vibration and pinprick sensation at left toe. she had a normal gait, tandem gait and could toe walk, but not heel walk. she was diffusely areflexic. ncvs showed absent sensory responses. motor ncvs showed her latencies were prolonged and velocities reduced ( - m/s). cmtns was mild ( / ). additional family members who had the r l mutation were evaluated including maternal grandmother who was moderately impaired ( / ) and maternal aunt who was severe ( / ). no other mutations that could cause another known neuropathy or myopathy were identified and mitochondrial sequencing was normal. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). fisgun a , luan x , hoke a . johns hopkins university, baltimore, md, usa. molecular mechanisms that underlie slow distal axonal degeneration seen in chemotherapy induced peripheral neuropathy (cipn) are unclear. however, several identified molecular targets suggest shared mechanisms with wallerian degeneration. since spontaneous mutation in wlds mice and genetic deletion of sarm gene lead to slow wallerian degeneration, we asked if wlds or sarm knockout (ko) mice are resistant to distal axonal degeneration induced by several chemotherapy agents. we chose chemotherapeutic drugs from different classes of agents, paclitaxel (taxane), cisplatinum (platin-based drugs) and bertozomib (proteasome inhibitor), to model cipn in mice. primary outcome measure was evaluation of epidermal nerve fibers in the hind paw plantar footpads. secondary outcome measures included thermal sensation and nerve conduction studies. sarm ko mice were almost % protected against development of sensory neuropathy but the protection in wlds mice was partial. this study confirms the pivotal role sarm plays in mediating axonal degeneration and identifies inhibition of sarm activity as a potential therapeutic target for prevention of cipn. florio f , scapin c , ferri c , feltri ml , wrabetz l , d'antonio m . myelin biology unit, san raffaele scientific institute, milan, italy; hjkri-university of buffalo, ny, usa. myelin protein zero is the most abundant structural protein in myelin of the pns. in humans, more than mutations in p are associated with hereditary neuropathies. deletion of serine causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in mice (wrabetz et al., ) . p s del protein is misfolded and is retained in the er where it gives rise to a dose-dependent unfolded protein response (upr) (pennuto et al., ) . the upr results in the activation of transcriptional and translation control programs that reduce protein synthesis and increase the folding and degradative capacity of the cell. usually, when this first response is not sufficient the cells may activate apoptosis resulting in cell death. however, in p s del schwann cells there is no cell death suggesting that these cells may respond differently to chronic stress. transcriptomic analysis performed on p s del nerves showed increased expression of transcription factors normally present only in the early phases of differentiation such as c-jun, sox and id (d' antonio et al. ) . in order to understand the role of the expression of these factors in the peripheral nerve myelination we used ex vivo and in vivo approaches and we showed that sox and id act as negative regulators of myelination. these results suggest that the expression of sox and id may contribute to the hypomyelination observed in p s del mice. as such, we reasoned that their ablation could ameliorate the phenotype. surprisingly, the ablation of these factors in the p s del mouse severely worsens the neuropathy bursting schwann cell differentiation and increasing the expression of both p wild type and mutant allele with concomitant exacerbation of the upr. this suggests that the overexpression of early differentiation factors in schwann cell under chronic er-stress is an adaptive mechanism that limits differentiation and reduces the expression of toxic proteins. intravenous immunoglobulin (ivig) is the treatment of choice for the guillain-barré syndrome (gbs). the working mechanism of ivig in gbs is undefined, but most likely all potential effects are dose dependent. the pharmacokinetics (pk) and pharmacodynamics (pd) of ivig in gbs are highly variable between patients and a rapid consumption or clearance of ivig is associated with poor recovery. in the current study we developed a model to predict the pk of a standard dosage of ivig ( . g/kg for consecutive days) in individual patients with gbs. non-linear mixed-effects modelling (nonmem) was used to construct a model based on a cohort of gbs patients, with a total of sequential serum igg levels. the final model accurately describes the day to day increment in igg levels during the -day course and the initial rapid fall and graduate decline to steady-state levels thereafter. we explored several potential covariates that improved the predictive capabilities and decreased the between-subject variation in the model. the model including these covariates were evaluated successfully (bootstrap analysis) and through numerous simulation studies each based on (simulated) gbs patients. in conclusion, a first accurate and robust nonmem model for the pk/pd of standard ivig treatment in gbs was developed. the model can be used to predict the pk in individual patients applying a few simple baseline characteristics. in addition, the effect of different treatment regimens of ivig in gbs on a population pk/pd level can be simulated. this modeling technique is a new tool to optimize the pk in individual patients and the study design for new trials with ivig in gbs. forese mg , pellegatta m , rivellini c , podini p , quattrini a , previtali sc , taveggia c . axonal neuregulin (nrg ) type iii is an essential instructive signal for peripheral nervous system (pns) myelination, as its expression determines whether axons are myelinated as well as the thickness of the myelin sheath. we recently demonstrated that gamma-secretase cleavage of nrg type iii generates an axonal intracellular fragment, which translocates in the nucleus to upregulate the expression of the prostaglandin d synthase (l-pgds) gene in neurons. l-pgds catalyzes the conversion of prostaglandin h into prostaglandin d (pgd ). we also showed that specific inhibition of l-pgds activity impairs in vitro myelination. accordingly, myelin in l-pgds null mice is noticeably thinner, thus indicating that l-pgds is a new modulator of developmental pns myelination. previous studies have shown that prostaglandins are involved in the process of wallerian degeneration (wd) and axonal regeneration after injury. thus, to determine whether l-pgds and pgd could be important in pns regeneration and remyelination, we performed sciatic nerve crush injury in months old l-pgds null and wild type control mice and we analysed nerves by morphologic, biochemical, histological and molecular approaches at different time points (t) after crush. we focused on three phases: degeneration (t -t ), axon regeneration (t -t ) and remyelination (t ). our results indicate that in l-pgds null mice the amount of myelin proteins synthesized after crush as well as the number of remyelinated fibers do not change, suggesting that l-pgds might be dispensable for remyelination. however, we observed an increased number of macrophages in null nerves during regeneration (t ), possibly as a consequence of an increase in the blood-nerve barrier (bnb) permeability, indicating potential alteration in the regeneration process in l-pgds null mice. these results suggest that l-pgds could have a different role in developmental pns myelination and after injury. whether other prostaglandins and synthases might compensate for l-pgds activity is currently under investigation. fornasari be , , raimondo s , , ronchi g , , crosio a , budau ca , el soury m , muratori l , , tos p , battiston b , geuna s , , gambarotta g . department of clinical and biological sciences, university of turin, italy; neuroscience institute cavalieri ottolenghi, turin, italy; microsurgery unit, health and science city, cto, turin, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. to repair nerve gaps following severe peripheral nerve injuries, chitosan tubes were proved to give good results, comparable with those obtained with nerve autografts, the gold standard technique. to further improve peripheral nerve regeneration using chitosan tubes, a conduit enrichment strategy was developed using longitudinal skeletal muscle fibres, which have been previously shown to be good fillers in the "muscle in vein" experimental paradigm, where they played a trophic and a structural role. to this aim, rat median nerve gaps were repaired using two different conduits: mm chitosan tubes filled with a longitudinal piece of pectoralis major muscle ("muscle in tube") and hollow chitosan tubes. samples were harvested at early time points ( , , , days) for biomolecular and morphological analysis, and later ( months) for stereological analysis. autologous nerve grafts were used as gold standard positive control in the early time points. biomolecular analysis carried out on in vitro degenerating muscle and on "muscle in tube" at early time points show that the muscle produces high levels of soluble isoforms of neuregulin , a key factor for schwann cell survival and activity, usually released by schwann cells after nerve injury. functional assay and stereological analysis carried out on the distal part of regenerated nerve months after nerve repair, show no significant differences in the regeneration outcome between hollow chitosan tube and "muscle in tube" groups. therefore, we conclude that for short gaps (≤ mm), both hollow chitosan tube and "muscle in tube" are good techniques to repair nerve defects and we suggest that the "muscle in tube", which spontaneously releases neureg-ulin , might be a promising strategy to promote regeneration when the gap is longer or the repair is delayed in time. foucquier j , bertrand v , jouve e , truillet r , mandel j , laffaire j , blin o , magy l , lehert p , , hajj r , guedj m , cohen d , attarian s . pharnext, issy-les-moulineaux, france; aix marseille université, aphm, marseille, france; hôpital dupuytren, limoges, france; university of melbourne, melbourne, vic , australia; faculty of economics, louvain, belgium. charcot-marie-tooth disease type a (cmt a) is a rare disease belonging to a group of inherited, progressive, chronic motor and sensory peripheral neuropathies. the charcot-marie-tooth neuropathy score (cmtns) (shy et al., ) and the overall neuropathy limitations scale (onls) (graham & hughes, ) are considered as the main clinical scales for evaluating progression of disability associated with cmt. as cmt a is a slowly progressive neurodegenerative disease, the choice of endpoints and their ability to monitor small changes over time remain a major concern for clinical drug development. with this in mind, we studied a cohort of french cmt a patients with a follow-up ranging from months to . years resulting from the merge of two multicentre clinical trials (micallef et al., ; attarian et al., ) and a non-interventional study (unpublished) . the sensitivity to change of both cmtns and onls were assessed using a mixed effect model estimating annual progression with time in years, cmtns or onls baseline value as covariates, study centre as a fixed factor and patients as a random effect to account for the repeated measures. disease progression was estimated to be + . points per year on the cmtns (p = . ) and + . points per year on the onls (p = . x − ), both corresponding to a deterioration of impairment and disability. while both endpoints have similar and favourable properties, our set of observations led us to conclude that the onls could be more promising to monitor disease progression in cmt a. frasquet m , , lupo v , mas f , , vílchez r , chumillas mj , , espinós c , sevilla t , , . hospital universitari i politècnic la fe, valencia, spain; instituto de investigación sanitaria la fe, valencia, spain; centro de investigación príncipe felipe, valencia, spain; eresa, valencia, spain; centro de investigación biomédica en enfermedades raras (ciberer), valencia, spain; departamento medicina, universitat de valencia, valencia, spain. mutations in the bicd gene are a cause of dominant spinal muscular atrophy, lower extremity predominant (smaled). we report six patients belonging to three spanish families who carry three different novel mutations in the bicd gene. we describe clinical, electrophysiological and magnetic resonance imaging (mri) data. we provide results of muscle biopsy of one patient and skin biopsy for the study of epidermal nerve fiber density (enfd) of other two patients. genetic diagnosed was reached using a gene panel for genetic testing of cmt and dhmn. three novel mutations in the bicd gene that segregated with the disease were detected: p.val gly; p.tyr his and p.s l. the most frequent clinical phenotype consisted of mild weakness in proximal muscles of lower limbs combined with foot deformities. one patient had prominent sensory symptoms and abnormalities on sensory examination. other two patients had minor sensory abnormalities on examination. in one patient sensory and motor nerve action potentials were reduced, in the rest of patients electrophysiological studies showed normal motor and sensory nerve responses, with chronic denervation predominantly in muscles of lower limbs. mri studies at the level of tight and calf were performed in all patients. the most affected muscles were rectus femoris, vastus lateralis and medial gastrocnemius. mri studies at the level of feet were obtained from five patients and showed that there was not fatty infiltration in intrinsic foot muscles. mri at the level of pelvis muscles performed in four patients showed marked fatty infiltration of gluteus medius muscle in two of them. muscle biopsy performed in one patient showed myopathic features. skin biopsy was performed in two patients of the same family. in the older patient, who had minor sensory abnormalities on examination, there was a marked reduction of enfd that followed a length-dependent pattern. in conclusion, we report three new pathogenic mutations in the bicd gene. in our study we include mri findings at the level of pelvis and feet, which allow us to better define the pattern of muscle involvement related with this gene. our results also raise the subject of a possible sensory involvement in the disease. hereditary neuropathy with liability to pressure palsies (hnpp) is an autosomal dominant disorder that usually results from deletions in the pmp- gene. the neuropathy is unique in that it manifests with recurrent mono-neuropathies at common sites of compression. while spontaneous recovery from episodes of nerve injury usually occurs, it is often incomplete, and over time patients may develop a length dependent polyneuropathy. given the relapsing/remitting nature of hnpp symptoms, standard clinical scores, such as the cmt neuropathy score, are not effective at capturing the severity or progression of the disease. a specific tool is therefore needed for measuring clinical severity of hnpp in preparation for emerging clinical trials. in the current study, we evaluate a new pilot measure, the hnpp severity score (hnpps). the score is composed of patient reported questions addressing current and prior sensory and motor symptoms, and the impact of symptoms on quality of life, followed by items based on a motor examination. total scores vary from - , with higher scores indicating increased disease severity. in this study, the hnpps was administered to patients with genetically confirmed hnpp at the ucl institute of neurology. subjects included males and females with a mean age of years (+/− , range - ). the mean hnpps was . points (+/− . , range - ) and the data did not demonstrate major skew. the cronbach alpha for the hnpps was . , and items based on the physical examination showed the least variability. a modest correlation was observed between the hnpps and the cmt examination scores (pearson correlation . , ci . - . ). we conclude that the hnpps may be a useful measure of clinical severity in hnpp, and should be refined in larger patient cohorts. fridman v , sillau s and on behalf of the inherited neuropathies consortium (inc) . university of colorado hospital, aurora, co, usa; university of iowa hospitals and clinics, iowa city, ia, usa. the most common of the hereditary neuropathies (hn) is cmt a, an autosomal dominant demyelinating neuropathy that results from duplications in the pmp- gene. recent advances in defining the pathomechanisms of the disease have led to an increasing number of potential therapies; however, the absence of reliable natural history data and the paucity of sensitive clinical outcome measures have been barriers to effective clinical trials. the charcot marie tooth neuropathy score (cmtns) was developed to quantify impairment and measure progression in hn. it was observed that while the score discriminates well between mildly and severely impaired patients, it tends to cluster together patients in the middle range of severity. to improve the score's sensitivity, rasch analysis-based weighted category responses were developed. we report a longitudinal study of weighted cmtns and cmt examination scores (cmtes) over a three-year time frame in patients with cmt a. baseline, one year, two year and three year wcmtnsv /wcmtesv scores were available for / , / , / and / patients respectively. mean wcmtns (sd)/wcmtes (sd) scores were as follows: . ( . )/ . ( . ) at baseline, . ( . )/ . ( . ) at one year, . ( . )/ . ( . ) at two years and . ( . )/ . ( . ) at three years. a mixed regression model showed significant change in wcmtns and wcmtes at years (mean change from baseline at years was . points (p=. ) for wcmtns and . points (p=. ) for wcmtes. significant change as compared to baseline was also seen at years (mean change from baseline . points (p= . ) for wcmtns and . points (p= . ) for wcmtes. we conclude that weighted cmtnsv scores show change over the first three years of the inherited neuropathies consortium natural history study and are a helpful measure of progression in cmt a. fridman v , novak p , david w , macklin ea , mckenna-yasek d , walsh k, oaklander al , brown r , hornemann t , eichler f . massachusetts general hospital, boston, ma, usa; university of massachusetts medical school, worcester, usa; university hospital zurich, zurich, switzerland. hsan is an autosomal dominant, severe sensory motor polyneuropathy caused by mutations to serine palmitoyl-coa transferase. mutations shift the substrate preference from serine to alanine leading to formation of neurotoxic -deoxysphingolipids ( -deoxysl). treatment with high-dose l-serine has been shown to reduce -deoxysl accumulation and improve neuropathy in transgenic hsan mice. we report a two-year, delayed-start, placebo-controlled clinical trial evaluating the safety and efficacy of oral l-serine in hsan . eighteen hsan subjects were equally randomized to l-serine ( mg/kg/d) or placebo for year. at -weeks, the placebo group crossed-over and all participants took open-label l-serine for one additional year. sixteen subjects completed their -week visit, and no serious adverse events related to l-serine were reported. participants randomized to l-serine experienced a decline in charcot marie tooth neuropathy scores (cmtns) over year relative to placebo (− . units, % ci − . to − . , p = . ). both groups improved in the second year of the study, with a diminished difference in cmtns at weeks (− . units, % ci − . to . , p= . ). skin biopsies from the distal leg site were largely devoid of intra-epidermal nerve fibers (ienf), but at year, a greater increase in ienf density was seen in participants on l-serine versus those on placebo (median change of vs. fibers/μm , p= . ). -deoxysl levels declined among participants on l-serine versus those on placebo after one year of treatment ( % decrease in deoxysphinganine vs. % increase on placebo, p < . ), and placebo participants experienced similar declines in -deoxysl levels after crossing over to l-serine. we conclude that l-serine is a safe and potentially efficacious treatment for hsan . fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the relationship between guillain-barré syndrome (gbs) and malignancy is uncertain. under the diagnostic criteria of paraneoplastic neurological syndrome (pns) by euronetwork, , neuropathy with no definite onconeural antibodies identified due to gbs has been classified as "non-classical" paraneoplastic disorder. we retrospectively analyzed data of consecutive patients admitted with gbs from birmingham, u.k. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to calculate the relative cancer risk using different definitions and determined characteristics of malignancy-associated gbs. patients were classified according to definitions: ) all cases of malignancies excluding preceding diagnoses > year before gbs and with no evidence of malignant disease activity at the time of gbs diagnosis, and ) malignancies diagnosed > years post-gbs onset as per pns criteria. total number of gbs patients with malignancy was ( . %). a total of patients ( . %) fulfilled requirements for inclusion as malignancy-associated gbs as per existing criteria for pns. associated malignancy consisted of angioimmunoblastic t-cell lymphoma ( ), poorly differentiated squamous cell carcinoma of nasal septum ( ), gastric adenocarcinoma ( ), hepatocellular carcinoma due to hepatitis b ( ), rectal carcinoma with liver metastasis ( ) and myelodysplastic syndrome ( ). malignancy was globally commoner in our gbs cohort compared to the general population (odds ratio: . ; ci: . - . ; p = . ). however, this was unconfirmed if paraneoplastic criteria were applied. gbs patients with cancer were significantly more likely to be older (p = . ), hyponatremic (p = . ) and demonstrate more electrophysiological axonal loss (p < . ). cerebrospinal fluid (csf) protein levels were lower in the malignancy group (p = . ) and neurological improvement was less likely (p = . ). in-patient mortality was significantly higher in patients with malignancy (p < . ). none of the patients in the malignancy group had positive anti-ganglioside antibodies or anti-neuronal antibodies (anti-hu, yo, ri, crmp ). we conclude global cancer risk is higher in gbs than in the general population, although definition-dependent. application of the strict published criteria for paraneoplastic syndrome reduced the number of cases and suggested absence of a link. malignancy requires consideration in elderly, hyponatremic subjects with normal csf protein, severe electrophysiological axonal loss who fail to improve post-treatment. fu liong h , aude-marie g , anne-catherine an , emilien d , julien c , shahram a , yusuf r . anti-myelin associated glycoprotein antibody (anti-mag) neuropathy is a slowly progressive neuropathy resulting in disability through distal sensory more than motor deficits and tremor. there are currently no evidence-based treatments for anti-mag neuropathy and the effect of the disease on quality of life (qol) in this patient population is unknown. our objective was to assess determinants of qol in patients with anti-mag neuropathy. the sf- questionnaire was assessed in patients, from marseille, angers (france) and birmingham (united kingdom). routine clinical evaluations included mrc sum score, incat sensory score, inflammatory rasch built overall disability score (i-rods), ataxia score, jamar grip dynamometry, timed meter-walk, neuropathic pain symptom inventory (npsi) score, and fatigue severity score (fss). there were males and females. mean age was . years (sd: . years). mean disease duration was . years (s.d.: . years). there were no significant differences between the french and u.k. cohorts in terms of gender distribution, age, disease duration, anti-mag antibody titre. all physical assessments, including mrc sum score (p= . ), jamar grip dynamometry (p= . ), onls (p= . ), irods (p= . ), incat sensory score (p= . ), -meter timed walk (p= . ), ataxia score (p= . ), tremor score (p= . ), were comparable. prevalence and/or severity of pain (p= . ), fatigue (p= . ), restless legs syndrome (p= ) and cramps (p= . ) were also similar. physical component summary (pcs) and mental component summary (mcs) of the sf questionnaire were significantly lower than in reported normal subjects of both countries (p< . ). all sf- qol domains correlated with i-rods, except mcs for which significance was however approached (p= . ). pcs correlated with mrc sum score, ataxia score, timed m-walk, tremor, jamar grip dynamometry, npsi pain score, fss and level of social support. mcs correlated exclusively with fss and level of social support. in multivariate regression, pcs was associated independently with i-rods (p< . ) and npsi pain score (p= . ), whereas mcs was associated independently with fss (p= . ). qol is accurately predicted in anti-mag neuropathy by the i-rods and fss, lending support to their use in clinical and research settings. effective measures to improve qol should include tremor and neuropathic pain treatment, fatigue management and improved social support. fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the electrophysiological diagnosis of guillain-barré syndrome (gbs) is dependent on a number of abnormalities affecting different parameters in peripheral nerves on nerve conduction studies (ncs). diagnostic sensitivity varies with different electrodiagnostic criteria, practices as well as extensiveness of nerve study. however, the most efficient method of performing electrophysiology for guillain-barré syndrome (gbs) is unknown. we retrospectively analyzed electrophysiological data of consecutive gbs patients from birmingham, uk ( - ), studied ≤ weeks post-onset. we first identify abnormal nerves from various regions which produced gbs electrodiagnosis using the recently described criteria by rajabally et al. we subsequently used pre-established hypothetical nerve conduction study protocols to determine the potential optimal method of achieving electrodiagnosis. we found the sensitivity of electrophysiology for each gbs subtype was dependent on the upper and lower limb nerves tested. in acute inflammatory demyelinating polyneuropathy (aidp), abnormalities were predominant in the arms, whereas leg abnormalities predominated in axonal gbs. in aidp, the most common abnormal parameters were distal motor latency ( . %) and conduction block ( . %), and the most frequently affected nerve was the median ( . %). prolonged f-waves were present in % and f-waves were absent in %. mcv was the least frequently abnormal ( . %) with demyelinating range slowing, significantly lower than cb or dml prolongation (p< . in both cases). in axonal gbs, reduced motor amplitudes ( . %) and conduction block ( . %) were the most common parameters, and the most frequently abnormal nerve was the tibial ( . %). f-waves were absent in . %. . % of all motor nerves were unexcitable, significantly more common in lower limb nerves compared to upper limb nerves (p= . ). on comparison of different hypothetical ncs protocols ( unilateral protocols with nerves, with nerves as well as the protocols with -nerves and with exclusive bilateral upper limb and lower limb testing), unilateral -nerve (median, ulnar, common peroneal and tibial) testing produced the highest diagnostic sensitivity for both aidp ( . %) and axonal ( . %) gbs. electrodiagnostic sensitivity in gbs is thus dependent on nerves tested and parameters considered. each subtype preferentially involves specific nerves and parameters. these findings may help per-procedure interpretation, improve electrodiagnostic sensitivity, and reduce patient discomfort. funakoshi k , nagashima t , kokubun n , hirata k , yuki n . department of neurology, dokkyo medical university, tochigi, japan; department of neurology, mishima hospital, nagaoka, japan. recently, igg anti-ganglioside complex (gscs) antibodies have been reported in patients with guillain-barré and miller fisher syndrome. some researchers (nobile-orazio et al., ) reported igm anti-gscs antibodies in multifocal motor neuropathy (mmn) and other chronic immune-mediated neuropathies. in ten of eleven mmn patients with anti-gm antibody, there was a decreased reactivity to gm /gd a compared to single antigen gm . similarly, in one of two chronic inflammatory degenerative polyradiculoneuropathy (cidp) patient with anti-gm antibody, there was a decreased reactivity to gm /gm , gm /gd a, gm /gd b, gm /gt b compared to gm . these relationships were defined negative interaction. in one cidp with anti-gd b antibody, anti-gm /gt b and -gm /gt b reactivity increased although gm and gm were negative (positive interaction). in japan, on the other hand, this correlation remains unclear. sera were investigated from one mmn patient ( y/o male) with anti-gm , and anti-gd a antibody, one cidp patient ( y/o female) with anti-gm , and one subacute sensory motor polyradiculoneuropathy ( y/o male) with anti-gd b, and anti-gq b. igm antibodies to gscs gm /gd a were tested with a mixture of gm and gd a (each pmol/well) as antigen. anti-gm /gd a antibodies were judged to have positive interactions when the optical density was . greater than the sum of the antibodies against individual gm and gd a, and negative interactions when the optical density was % or less compared to the antibodies against individual gm or gd a. antibodies to at least one combination of two of the six gangliosides (gm , gm , gd a, gd b, gt b, and gq b) were similarly tested and judged for positive or negative interactions. in one mmn, anti-gm /gd a had negative interactions. in one cidp, anti-gm /gd a, and anti-gm /gq b had negative interactions. in one subacute sensory motor polyradiculoneuropathy, various anti-gscs antibodies including gd b or gq b had negative interactions. in the cidp patient mentioned above, anti-gm /gt b or anti-gm /gt b had no positive interactions. the relationship between this attenuated reactivity presumably driven by adjacent gangliosides and the mechanism of chronic immune-mediated neuropathies needs to be clarified. galino j , cervellini i , zhu n , stöberl n , fricker fr , lee g , hütte m , lalli g , bennett dl . the nuffield department of clinical neurosciences, john radcliffe hospital, university of oxford, oxford, uk; wolfson card, king's college london, guy's campus, london, uk. schwann cells (scs) are the myelinating cells in peripheral nerve system and are important for normal peripheral nerve development and repair. rala and ralb are small gtpases that have been implicated in neural tube closure, neurite branching and have been described as upstream effectors of proteins involved in cell migration and membrane dynamics. due to their potential role in sc function here we investigated, by genetic ablation in transgenic animals of one or both gtpases, their importance in sc function in adulthood and in nerve repair. we showed that ral gtpases are dispensable for sc function in the naïve state. however ral signalling (provided by rala or ralb) is required for effective remyelination of axons following nerve injury. moreover, absent ral signalling produced defects in axon reinnervation of distal organs and a delay in motor function recovery after nerve injury. we also studied the ral dependent cellular mechanisms that may be responsible for impaired sc remyelination and noted abnormal sc lamellipodia formation that prevent normal axial and radial axon remyelination. this work demonstrates for the first time a novel mechanism for ralgtpases that controls sc lamellipodia formation and their importance in normal sc function during peripheral nerve repair. garcía-lareu b , , , ariza l , cobianchi s , , chillón m , , , , navarro x , , , bosch a , , . tumor necrosis factor (tnf) alpha has been implicated in the pathogenesis of diabetic peripheral neuropathy (dpn), among other inflammatory demyelinating diseases and neuropathic pain. tnf alpha is a pro-inflammatory cytokine that can act at several stages in the demyelination process. it is produced by schwann cells (scs) in the peripheral nervous system (pns) after nerve injury and released into the local environment to attract and activate macrophages at the site of injury, contributing to wallerian degeneration. in vivo studies demonstrated a local inflammation in the sciatic nerve of rats after injection of tnf alpha, followed by demyelination and axonal degeneration. furthermore, the application of tnf alpha resulted in acute mechanical hiperalgesia, a main characteristic of neuropathic pain and therefore tnf alpha is postulated as a biomarker for painful changes after nerve injury. with the aim to characterize tnf alpha effects in chronic neuropathic pain and in diabetic neuropathy, a transgenic mouse model overexpressing tnf alpha cdna under the peripheral myelin protein p promoter was generated. here we characterized the overexpression of tnf alpha in myelinated scs at different stages of myelination (postnatal days , and ) showing that high levels of tnf alpha in sciatic nerve leads to the downregulation of the major pns myelin proteins (p , mbp, pmp , mag) compared to wild type mice, correlating with the loss of structured myelin and an increase in p ntr, a marker for immature and non-myelinated scs in the sciatic nerve. iba staining showed high levels of macrophage infiltration in both sciatic nerve and spinal cord tissues, compared with wild type animals. stress conditions were induced by sciatic nerve crush surgery after which recovery and subsequent remyelination were delayed in the transgenic mice, as evaluated by the sciatic functional index (sfi) and electrophysiological tests. on the other hand, mechanical and thermal nociception seemed to be unaltered, with or without lesion. this model could be helpful in the characterization of the role tnf alpha in pain development, injury and dpn as well as in developing efficient therapeutic strategies to modulate such pathological conditions. garcía-sobrino t , , blanco-arias patricia , , vidal-lijó mp , quintáns bea , , sobrido mj , , pardo j , . department of neurology, hospital clínico, santiago de compostela, spain; neurogenetics research group, instituto de investigaciones sanitarias (idis), santiago de compostela, spain; genomic medicine group (u ), centre for biomedical network research on rare diseases (ciberer), spain; department of neurophysiology, hospital clínico, santiago de compostela, spain. charcot-marie-tooth (cmt) disease is a genetically heterogeneous group of hereditary motor and sensory neuropathies. more than genes were involved in the disease pathogenesis. the objective of this study was to assess the genetic distribution of cmt disease in galicia (northwestern spain). patients were diagnosed as cmt if they had a consistent neurological and/or neurophysiological examination or if they had sensory motor neuropathy with a positive family history. a total of cmt adult patients ( % females) were evaluated with a median age of [ - ] years. the molecular diagnosis was achieved in patients ( %) with a higher success in cmt ( %) than cmt ( %). globally, pmp duplication was the most frequent finding ( %), followed by mutations in mpz ( %), mfn ( %), gjb ( %) and gdap ( %). in cmt , with expception of the pmp duplication, pathogenic variants in egr , nefl and mpz gene were most common. pathogenic variants in mfn and gdap accounted for % of cmt patients. several patients referred to our institution as cmt were diagnosed as hereditary motor neuropathy (hmn) and pathogenic variants in the bscl gene were the most frequent. pathogenic variants not previously related to cmt were identified in mpz, mfn , gjb , egr , nefl and sh tc genes. sporadic or autosomal recessive (ar) cmt accounted for the % of all diagnoses. the genetic epidemiology of cmt in galicia follows a similar pattern to other populations, although some remarkable features are axonal gdap and demyalinating egr pathogenic variants as well a seemingly elevated proportion of ar cases. mutations in mpz gene are associated to a wide range of phenotypes. the r c, in particular, is associated to a severe and early onset disease. some cmt patients respond to immunosuppressive or immunomodulatory treatment, some of them harbouring mpz mutations. we present the case of a -year-old brazilian female with a severe cmt b (r c) that presented an acute episode of worsening after a febrile exanthematic disease. she complained of tingling, stopped walking without support, and could not raise her arms. her emg showed an asymmetrical reduction of motor cv, reduced amplitudes, severe temporal dispersion and possibly conduction block. csf protein was mg%, with normal cell count. diagnosed as gbs, she received ivig. curiously, she markedly improved, regaining the functional status of youth, recovering functions that she had long lost. on emg, motor amplitudes improved significantly, but cv remained the same. reviewing carefully her past history, we identified some motor fluctuations along her adult life. at age , while pregnant, she developed positive sensory symptoms, became unable to get up from the chair and could not walk without assistance. after delivery, she improved, but did not return to baseline. one year later, she presented a new transitory functional worsening after an influenza illness. her parents were healthy but two out her children have a severe cmt. her first daughter never walked and died at the age of six. six months after treatment with ivig, she faced a new relapse. we pulsed with corticosteroids, with great response. she remains stable to date, four months after treatment. the episodic fluctuations and the evident response to treatment clearly suggest an associated immunomediated process. the fast improvement of the amplitude with maintenance of cv suggests that another mechanism in addition to demyelination and axonal degeneration is involved. we propose the existence of an associated channelopathy. funded by cnpq, faepa, pronas (ministry of healthy) gbs is an acute, immune-mediated neuropathy that comprises three major subtypes: aidp, aman, and amsan. on clinical grounds, this distinction is based on nerve conduction studies (ncs): aidp is a demyelinating neuropathy, aman is an axonal motor neuropathy, and amsan, an axonal sensory and motor neuropathy. recently, it has been demonstrated that axonal gbs, in addition to axonal degeneration, may present conduction block (cb) that can progress to axonal regeneration or revert, characterising a reversible conduction failure (rcf), a finding usually associated to good prognosis. patients presenting axonal cb are frequently diagnosed as having aidp, a mistake that can be avoided with two sets of ncs, one as early as possible and the second after three weeks of disease. following these recommendations, we retrospectively classified a brazilian group with gbs followed in our institution that had been submitted to two ncs sets. from september to january , patients fulfilled clinical criteria for gbs at clinical hospital of ribeirão preto and had more than one ncs accomplished. at least four motor nerves (median, ulnar, peroneal, and posterior tibial) and five sensory nerves (medial, ulnar, radial, sural, and peroneal superficial) were evaluated in each examination, according to routine procedures. first ncs revealed patients with aidp ( %), cases of axonal gbs ( aman, amsan, %), and patients with equivocal results ( %). at follow-up study, patients ( %) had their classification changed. the main shifts were from aidp to axonal group and from equivocal results to aidp. aidp increased to % (n = ), axonal gbs increased to % (n = , aman, amsan), and there were no more equivocal patients. although the majority of studies have shown that aidp is more frequent in western countries while axonal gbs predominates in eastern countries, we found a different pattern of distribution in our population, with predominance of the axonal subtypes. the considerable increase of axonal gbs at follow-up studies reinforces that serial emg are mandatory for accurate diagnosis of gbs subtypes. prospective studies are now being carried out in order to confirm these results. in amyloid light chain (al) amyloidosis, misfolded light chain (lc) accumulates and causes progressive peripheral neuropathy (pn) and progressive failure of critical organs such as the heart and kidneys. progressive ascending sensorimotor neuropathy is often a related clinical finding. the deposition of toxic lc amyloid in peripheral nerves is associated with paresthesias, pain, muscle weaknesss, orthostatic hypotension, and diarrhea or constipation in approximately % of patients with al amyloidosis. there are no approved treatments for al amyloidosis. current therapeutic approaches for al amyloidosis are intended to limit lc production but do not directly target misfolded lc deposited as amyloid in organs. we report phase / trial results of neod , an investigational monoclonal antibody that targets misfolded lc and may neutralize circulating lc aggregates and clear insoluble deposits. trial inclusion criteria were one or more plasma cell-directed treatment completed before enrollment, partial or greater hematologic response to any previous therapy, and persistent organ dysfunction. neod was administered intravenously every days during a dose-escalation phase ( patients; . , , , , , , or mg/kg in a + study design) and an expansion phase ( patients; mg/kg). we assessed safety, tolerability, pharmacokinetics, immunogenicity, organ responses based on consensus criteria, and pn responses using the neuropathy impairment score-lower limb (nis-ll). neod treatment was not associated with dose-limiting toxicities or serious adverse events, drug-related discontinuations, or antidrug antibody development in any patient (n = ). of patients with measurable pn at baseline (n = ), % achieved pn response based on the nis-ll score after months of treatment, which resulted in a median % nis-ll score reduction (mean baseline nis-ll, . ). in a best response analysis, % of cardiac-evaluable patients (n = ) and % of renal-evaluable patients (n = ) met respective criteria for organ response. improvements in neuropathy have not been previously shown in al amyloidosis. these results demonstrated that monthly neod infusions were safe, well tolerated, and associated with responses across three different organ systems. gervais cbl , ross ma , goodman bp , khoury ja , muzyka i , smith be . mayo clinic in arizona, scottsdale, az, usa. this work describes clinical, examination and electrophysiologic findings in a cohort of patients with length dependent sensorimotor peripheral neuropathy (ldsmpn) with ventral abdominal sensory loss. ldsmpn affects the longest nerve fibers, namely those innervating structures in the feet and hands. what is less well appreciated is that length dependent involvement of sensory nerve fibers in ldsmpn from the thoracic segments gives rise to ventral abdominal sensory loss. consecutive patients seen for ldsmpn (n= ) were evaluated for the presence or absence of ventral abdominal sensory loss. demographic variables, symptoms and quantitative neurologic findings (neuropathy impairment score [nis]) were examined using descriptive statistics. final diagnoses were noted. ventral abdominal sensory loss to pinprick (which was asymptomatic in all patients tested) was documented in / ldsmpn patients ( . %), mean age was . years (range - ), m:f gender ratio was . ( : ), mean nis was . (range - ), ncs/emg abnormalities were found in / patients (the remaining showing objective evidence of small fiber sensory involvement). ldsmpn patients without ventral abdominal sensory loss (n= ) had a mean age of . (range - ), m:f of . ( : ), and mean nis of . (range - ). no patient ( / ) had dorsal torso sensory loss between the shoulder and buttock levels on either side. diagnoses of the ldsmpn patients with vs. without ventral abdominal sensory loss included charcot marie tooth or other hereditary neuropathy (n= vs. ), abnormal carbohydrate metabolism (n= vs. ), idiopathic (n= vs. ), hypothyroidism (n= vs. ), inflammatory (n= vs. ) and other (n= vs. ). ventral abdominal sensory loss appears to be common in patients diagnosed with ldsmpn of a variety of causes including inherited neuropathies; in addition to those innervating distal limb territories, distal sensory fibers from the thoracic region represent another category of length dependent involvement in ldsmpn; ) the clinical examination of ldsmpn should include the ventral abdomen. gibbons c , garcia j , casasola m , freeman r . beth israel deaconess medical center, harvard medical school, boston, usa. objective: to determine the relationship between measures of autonomic function, electrochemical sweat conductance (esc) and intra-epidermal (ienfd) and sudomotor nerve fiber density (sgnfd). background: structural and functional measures of small fiber neuropathy have been studied in patients with diabetes, but little information comparing these techniques exists. design/methods: we studied patients with diabetes (ages ± yrs, gender f) and healthy control subjects (age ± yrs, gender f). subjects underwent examination scores (nis-ll), quantitative sensory testing, autonomic testing (heart rate variability, valsalva maneuver, tilt test), esc, and millimeter punch skin biopsies at the distal leg and distal thigh for ienfd & sgnfd. results: there were strong correlations between exam scores (nis-ll) and biopsy ienfd (r=− . , p< . distal leg; r=− . , p< . distal thigh), and sgnfd (r=− . , p< . distal leg; r=− . , p< . distal thigh). moderate correlations were noted between exam scores and qst (r values . - . , p< . ), ienfd and qst (r= . - . ). modest correlations were noted between esc and parasympathetic function (r= . - . , p< . ). modest correlations were noted between esc and ienfd (r= . , p< . , but only at the distal leg) and esc and nis-ll (− . , p< . ). no correlations between exam scores and autonomic function were noted. no correlations were detected between esc and sgnfd (r=− . - . ) or esc and sympathetic adrenergic function (r= . ). conclusions: differences between tests are expected based on our understanding of the pathophysiology and natural history of diabetic neuropathy. exam scores, qst results and biopsy results do correlate, and are consistent with prior studies. in contrast, there was little relationship between tests of autonomic function and exam scores, similar to prior studies and our understanding of autonomic function and differences in p. esc had little correlation with either exam scores or biopsy ienfd or sgnfd. there was a modest correlation between esc and parasympathetic function. the exact relationship between esc and diabetic peripheral neuropathy is not clear, and further research studies are needed to determine the role this technique has in clinical practice. objective: to determine the optimal tissue thickness of skin biopsy sections for studies of cutaneous nerve fibers. background: although analysis of intra-epidermal nerve fiber density (ienfd) is routinely reported using μm thick tissue sections, many recent studies of peripheral alpha-synuclein deposition use or μm frozen sections, or μm paraffin embedded tissue sections. design/methods: we compared the results of biopsies from patients with parkinson's disease (pd), using tissue sections each of , and μm thickness. tissues were stained with pgp . and stained for phosphorylated alpha-synuclein (p-syn). the total number of dermal structures (hair follicles, sweat glands, pilomotor muscles) were quantified, nerve densities analyzed, and the frequency of p-syn positive results. we also studied μm paraffin embedded tissue samples from patients with pd. results: in the biopsies of patients with pd there were no differences in the number of sweat glands, hair follicles or pilomotor muscles in , or μm sections. there were significantly fewer blood vessels noted in and μm sections compared to μm sections (p< . ). ienfd and sgnfd declined with tissue thickness (p. . , all) and there was increased variability in results in thinner tissue sections. there was a highly significant reduction in p-syn positive sections in thinner tissue sections (p< . , all tissues compared to μm sections). paraffin embedded tissue sections had significantly lower nerve densities and positive p-syn results (p< . ) compared to all frozen tissue sections of , and μm thickness. thinner tissue sections carried a greater risk of false positive result or indeterminate results due to difficulty interpreting overlap with pgp . . conclusions: tissue sample thickness plays a critical role in interpretation of skin biopsy results. thinner tissue sections, or paraffin embedded tissue sections, do not provide equivalent data and significantly underestimate nerve densities and positive alpha-synuclein results with increase false positive results despite similar numbers of dermal tissue structures. ace- is an investigational protein therapeutic that acts as a localized ligand trap for myostatin and other negative regulators of muscle growth. local injection of ace- into the gastrocnemius muscle of wild-type, mdx, and sod mice produced dose-dependent increases in muscle mass and force without systemic effects. in a phase single-center, double-blind, placebo-controlled dose escalation study in post-menopausal women, unilateral injections of ace- into the rectus femoris (rf) or tibialis anterior (ta) muscle were generally safe and well tolerated. mean percent changes from baseline in muscle volume of the injected muscle were + . % in the rf and + . % in the ta at the highest dose administered with minimal changes observed in the contralateral side and placebo-treated subjects. frequent related aes (≥ %) included injection site pain, pain in extremity, injection site discomfort, and muscle twitching, with similar incidence in ace- and placebo-treated groups. all aes were grade - and reversible. together, these preclinical and clinical results support further studies of ace- in myogenic and/or neurogenic diseases with focal loss of muscle strength and function, including cmt. a phase study is ongoing in facioscapulohumeral muscular dystrophy (fshd). study a - is an ongoing multicenter, two-part, phase study to evaluate the safety, tolerability, pharmacodynamics, efficacy, and pharmacokinetics of ace- in patients with cmt and cmtx. part is open-label and will enroll up to dose-escalating cohorts ( patients per cohort); part is randomized, double-blind, and placebo-controlled, and will enroll an additional patients. ace- will be administered bilaterally to the ta muscle once every three weeks for up to five doses. a safety review team will meet periodically throughout the study to review safety data and make dosing recommendations, including the recommended dose level for part . eligible patients must have genetically confirmed cmt or cmtx with mild-moderate weakness in ankle dorsiflexion. safety and tolerability will serve as the primary outcome for part , muscle volume evaluated by mri for part . additional outcome measures of interest include strength by quantitative muscle testing, function by motor tests, and quality of life by the cmt-health index questionnaire. our study was aimed to evaluate the functional and morphological consequences of cellular and humoral responses in chronic inflammatory demyelinating neuropathy (cidp), using extensive standardized high-resolution sonography (hrus) and nerve conduction study (ncs) protocols in incident treatment-naive patients. we enrolled consecutive, newly diagnosed, treatment naive patients with cidp. in addition to all relevant clinical examinations, all patients underwent a standardized ncs and extensive hrus protocol, of median, ulnar, tibial, fibular and sural nerves. we assessed standard nerve and fascicle size, and echogenicity. we found focal sonographic enlargements in multiple nerves and nerve segments with and without ncs abnormalities. the degree of nerve hypertrophy was not associated with presence of ncs features of demyelination, i.e. / ( %) of median nerve segments showed enlargement without strong decrease in motor conduction velocity and / ( %) hypertrophic median nerve segments revealed no conduction block. a lower distal cmap of median nerve was related with lower mrc sums-scores (p < . ). we found no correlation between age, disease duration or mrc sum-score and nerve size. cellular and humoral responses in cidp may lead to nerve enlargement along the length of nerves, that can be detected by hrus, whereas ncs allows identification of its' specific focal disruption in nerve function. the most common complication of diabetes is peripheral neuropathy, which has prevalence as high as % and is characterized by damage to neurons, schwann cells and blood vessels within the nerve. the concept of schwannopathy as an integral factor in the pathogenesis of diabetic neuropathy is re-emerging, and it is now known that schwann cells cultured in hyperglycemic environments underproduce neurotrophins and exhibit loss of axonal associations, further indicating a non-optimal glial cell activation and function. furthermore, the increased expression of p ntr in myelin sheaths around fibers that are susceptible to axonal degeneration in diabetic neuropathy suggest an important role for this molecule in disease progression. with this project, it is our main goal to evaluate how disruption of p ntr signaling in the schwann cells affects the pathophysiology of diabetic neuropathy. by using a fluorescent live/death cell viability assay, our preliminary data indicate that wild-type schwann cell cultures present increased cell death rate h after stimulation with high levels of glucose. the p ntr has a highly recognized role in the activation of death signals and when absent, we observed that schwann cells are significantly more resistant to apoptosis in hyperglycemic conditions. the role of p ntr receptor signaling in neuron-schwann cell communication and myelination under in vitro diabetic conditions was investigated with primary schwann cell-sensory neuron co-cultures. after eight days of ascorbic acid stimulation, both under euglycemic and hyperglycemic conditions, myelination was assessed by confocal microscopy using specific markers for neurons and myelin. results highlight a compromised ability of wild-type schwann cells to myelinate axons when exposed to a hyperglycemic environment, which was even intensified in co-cultures with schwann cells lacking the p ntr . to complement the in vitro studies, we are modeling type diabetes in a p ntr schwann cell conditional ko mouse model and plan to investigate nerve mrna expression profile to disclose genetic regulation depending on this receptor signaling and its modulatory role in endoneurial hypoxia and neuroinflammation. the results from this project will provide an integrated vision of how impaired schwann cell activity guides neuropathy progression. gondim faa , barreira aa , cruz mw , cunha fmb , de freitas m , frança mc jr , marques w jr , nascimento ojm , oliveira asb , pereira rc , pupe c , rotta ft , schestatsky p . panelists on behalf of the scientific department of peripheral neuropathy, brazilian academy of neurology, brazil. neuropathy is one of the most common neurological manifestations of several diseases and sfn has been progressively receiving more attention in the medical literature. the aim of this study is to generate a set of recommendations to define and diagnose sfn in brazil. a group of neurologists, members of the scientific department of peripheral neuropathy from the brazilian academy of neurology reviewed a preliminary draft prepared by the first author that was distributed by email. the panelists got together on . . at the city of fortaleza, brazil, to discuss and finish the text for the first submission of the manuscript. sfn can be defined as a subtype of neuropathy characterized by selective involvement of unmyelinated or thinly myelinated sensory (sometimes also autonomic) fibers. it is usually characterized by sensory (pain/dysesthesias/pruritus) or combined sensory and autonomic complaints, associated with an almost entirely normal neurological examination (except for sensory changes). electromyography is normal. a growing list of medical conditions has been linked to sfn, although there is no evidence-based literature to support the use of any specific set of screening tests to diagnose the etiology of sfn (the panelists will suggest a basic screening panel). sfn may also serve as a fallacious but useful terminology to uncover discrepancies in the normal values from different neurophysiology laboratories. in brazil, skin biopsy is not usually performed and initial forms of leprosy may have predominant small fiber involvement. there are several tests to demonstrate involvement of small sensory and autonomic fibers. skin wrinkling test, sympathetic skin responses & heart rate variability (conducted on emg machines) and thermoregulatory sweat test may be low-cost screening alternatives. after the final meeting on . . , we finished the first draft for submission to arquivos de neuropsiquiatria (together with a translation to portuguese as supplementary material), the official journal of the brazilian academy of neurology to serve as a source for the definition and diagnosis of sfn in brazil. the final draft will be submitted after presentation at the pns meeting in barcelona on . . the lower cranial nerves (lcn) include the paired glossopharyngeal, the vagal, the accessory and the hypoglossal nerves. these are involved in the execution of swallowing, speech, phonation, tasting, as well as sensory and autonomic functions. lcn can be affected in central and peripheral nervous system diseases. this study investigates the use of ultrasound (us) to detect lesions of the peripheral course of lcn. in addition mri can be used in unilateral lcn local lesions to demonstrate indirect signs of nerve lesions such as muscle atrophy. the patients were examined in supine position with the neck in maximum extension with either a regular or a portable us system (logiq e and logiq e ge healthcare, milwaukee, wisconsin) and high frequency transducers ( to mhz). according to the detected pathology longitudinal or transversal images were recorded. a series of exemplary observations are demonstrated. in one case a paraganglioma in the glossopharyngeal nerve at the carotid sinus was found. in another patient an us of the thyroid gland revealed a nerve tumor, which was later identified to be a schwannoma. in another case multiple neurofibroma were identified in the vagal nerve during a routine neurofibromatosis screening. us has become also important in investigations of lesions of the accessory nerve, following damage by surgery. nerve continuity and scar formation can be distinguished. lesions of the hypoglossal nerve can be caused by tumor infiltration. in one patient an infiltration of the nerve by a squamous cell carcinoma, and in another case by a low grade sarcoma were detected. in addition mri demonstrated an atrophy of the tongue. identification and assessment of the lcn from the point of exit of the skull to their endpoint can be pursued by us techniques, which have been described for the individual nerves. us is easily applicable and -in addition to the "static" image of the mri -allows assessing nerve positions during different movements, as well as detecting muscle movements as fasciculations. mri can also be used to detect not only more proximal lesions, but also changes of the skeletal muscle by denervation. grümme l , hattenhauer st , wolffram k , kleinschnitz c , mausberg ak , stettner m . university hospital essen, essen, germany; heinrich-heine-university, düsseldorf, germany. in chronic inflammatory demyelinating polyneuropathy (cidp), t cells are suspected to play a crucial role in myelin destruction. cd and cd t cells contribute to the inflammatory process, while the exact mechanism of myelin damage is still under debate. to elucidate the molecular interaction of t cells and myelin sheets, we compared neuritogenic and control cells in a live imaging in vitro model. the myelinated fibres of rat dorsal root ganglia (drg) served as model for the peripheral nervous system. drgs of embryonic (e ) lewis rats were cultured; myelination was initiated after one week in vitro and continued for two additional weeks. lewis rats immunized with p and neuritogenic t cells of the lymph nodes were obtained ten days after immunization. control t cells were prepared from healthy rats. for vital tracking, neuritogenic t cells were stained with orange cell tracker, while the control cells were labelled with cfse. myelin detection in vital cultures was assessed by incorporation of c fatty acids conjugated with a fluorophore into the myelin layer. experiments were performed in a conditioned microscope chamber, the two t cell populations were added simultaneously to the drg culture and migration was tracked using live cell imaging. we observed differing migration patterns for neuritogenic and control t cells. the velocity as well as the directionality was altered. after initial contact, the non-neuriotgenic t cells in close proximity to myelin subsequently decreased over time, while the numbers of neuritogenic t cells close to myelin remained stable. long term incubation with neuritogenic t cells affected the myelin integrity in regard to the intermodal length as well as the myelin ratio. further experiments will elucidate the specific effects of neuritogenic t cells to decipher the role of t cells during inflammation in the pns, which could be useful in developing targeted therapies. gundapaneni b , sultan mb , keohane dj , schwartz j . inventiv health inc., burlington, ma, usa; pfizer inc., new york, ny, usa. transthyretin familial amyloid polyneuropathy (ttr-fap) is a rare, life-threatening disorder caused by protein destabilizing ttr gene mutations, broadly classified as val met (the most common mutation worldwide) and non-val met genotypes. tafamidis, a highly-specific ttr-stabilizer, is the only medicine approved to delay neurologic progression in ttr-fap. the objective of this post-hoc analysis was to compare the effects of tafamidis on neuropathy progression in patients with val met and non-val met genotypes. val met patients were participants in a randomized, double-blind, placebo-controlled clinical trial of tafamidis, while non-val met patients were participants in an open-label tafamidis study. patients were grouped into three cohorts: val met tafamidis (n= ); val met placebo (n= ); and non-val met tafamidis (n= ). baseline disease severity and change in disease severity from baseline to month was assessed using the neuropathy impairment score-lower limbs (nis-ll). the effect of tafamidis in the val met and non-val met cohorts versus the val met placebo cohort was determined using a mixed-effects model for repeated measures (mmrm). at baseline, patients in the non-val met cohort were older, had longer symptom duration, and more advanced neurologic impairment than the val met cohorts. at month , the baseline-adjusted mean ± standard error change in nis-ll was comparable between the val met tafamidis and non-val met tafamidis cohorts ( . ± . and . ± . , respectively). these changes were smaller than that observed in the val met placebo cohort ( . ± . ; p= . vs val met and p= . vs non-val met) indicating less disease progression. based on predicted values from the mmrm analysis, the size of the change in nis-ll across the full range of baseline nis-ll scores was remarkably similar in the val met tafamidis and non-val met tafamidis cohorts and was consistently smaller than that observed in the val met placebo cohort. moreover, in all three cohorts, as baseline nis-ll increased, the predicted level of disease progression also increased. in conclusion, while controlling for baseline disease severity, tafamidis delayed disease progression to a comparable extent in val met and non-val met patients. the similar trajectories of disease progression across val met and non-val met patients suggest that these two genotype groups may be more similar than previously considered. clin-icaltrials.gov identifiers: nct ; nct . gutmann l , shy m . university of iowa, iowa city, ia, usa. post tetanic potentiation (ptp) is a physiological phenonmenon seen with disorders of the neuromuscular junction (nmj). it is caused by the influx of ca++ into the terminal axon during the tetanus resulting in an increased number of acetylcholine (ach) vescicles released by each axonal action potential. in myasthenic syndromes it results in improved nmj function by increasing the probability of achieving a large enough end plate potential to generate a muscle action potential. ptp has different appearances depending on whether the nmj defect is pre-synaptic or post-synaptic. in pre-synaptic defects (the most common being lambert-eaton syndrome) there is an increased amplitude of the muscle action potential after a tetanus that persists up to minutes. prolonged ptp has now been reported in disorders, one on a genetically determined neuropathy/myasthenic basis and the other on an acquired toxic origin. prolonged ptp up to minutes was reported in families with a motor neuropathy (having leg weakness and foot deformites) and a congenital myasthenic syndrome caused by a heterozygous mutation of the synaptotamin ii (syt ) gene (c t>g p.pro [p.asp ala] and c g>a [p.pro leu]). electrophysiological testing showed features of a presynaptic defect with prolonged ptp persisting up to minutes. the same phenomenon has been noted in the acquired pre-synaptic defect caused by botululinum toxin. the ptp continued up to minutes. syt is the synaptic vescicle calcium sensor in the terminal axon, allowing for fusion of ach containing vescicles with the presynaptic membrane and the synchronous release of ach. the fusion requires a complex assembly process involving snare proteins. both the syt gene mutation and botulinum toxin affect normal syt function, the mutation by altering amino acids in the calcium-binding domain and the toxin by binding to syt as well as gangliosides gd a and gt b on the neural membrane. the mechanism for the ptp prolongation remains unknown. prolonged ptp appears to be a unique physiological abnormality resulting from altered syt . this phenomenon has been decribed to occur in syt mutations causing congenital motor neuropathy/myasthenic syndrome and botulism. the abnormality may represent a physiological marker for a presynaptic nmj defect involving altered syt . hachisuka a , , senger j , curran m , chan km , . division of physical medicine and rehabilitation, university of alberta, edmonton, canada; department of rehabilitation and medicine, university of occupational and environmental health, kitakyushu, japan; division of plastic surgery, university of alberta, edmonton, canada. background: motor unit number estimation (mune) techniques are valuable tools in neuromuscular disease. among them, the multiple point stimulation (mps) is one of the most common used. contamination by distant single motor unit potentials (smups) generated by neighboring muscles is a potential confounding factor. this is particularly problematic in ulnar neuropathy, one of the most common neuropathies in humans. reason being that the ulnar nerve innervates the majority of hand muscles. the goals of this study are to test the hypotheses that ) distant smups all have an initial positive deflection and ) elimination of smups generated by distal muscles will significantly lower the mune results in the hypothenar muscles. methods: to address the first hypothesis, we tested subjects by stimulating their median nerve while recording smups simultaneously over the hypothenar and thenar muscles. for the second hypothesis, we carried out mps mune of the hypothenar muscles using multi-channel recordings placed over ulnar innervated intrinsic muscles across the hand. when a smup with an initial positive deflection was detected at the hypothenar electrodes, its original was systemically tracked through all the recording channels. results: in the first series of experiments, in accordance with the dipole theory, all smups recorded at the hypothenar recording electrodes had an initial positive polarity. in the second series of experiments, of the studies carried out in subjects, distant smups generated by muscles other than those in the hypothenar eminence represented ± . % (mean±sd) of the overall sample. mune calculated using only smups generated by the hypothenar muscles was ± , compared to ± if all smups were included (p < . ). the extent of increase in mune was highly correlated with the proportion of distant smups found in each study (r = . , p < . ). conclusion: in contrary to some studies suggesting that smups from distant muscles could have an initial negative deflection, we found all smups from distant muscles had a positive deflection. exclusion of those smups from the sample had a significant impact on the mune results. hagiwara w , konno s , kihara inoue m , fujioka t . toho university, tokyo, japan. matrix metalloproteinase (mmp) plays crucial roles in developing immune-mediated neuritis as guillain-barré syndrome (gbs) and its animal model experimental autoimmune neuritis (ean). to investigate the intraneural expression of mmps during ean and the effect of a phosphodiesterase- inhibitor cilostazol (clz) on it, ean rats were treated with either mg/kg/day of clz or vehicle from one day post immunization (dpi). to induce ean female lewis rats were immunized with synthetic peptide from bovine p protein. cauda equina (ce) were removed in several time points, total rna was extracted and reverse-transcribed to obtain cdna that was subjected to real-time pcr analysis for expression of mmp- , mmp- and tissue inhibitor of matrix metalloproteinase- (timp- ) messages. mmp- and mmp- messages peaked at dpi, that is presymptomatic phase of ean. all rats developed motor paralysis at dpi, mmp- and mmp- messages subsided at this moment. however, timp- message reciprocally increased at dpi, persisted through dpi. treatment of clz suppressed motor paralysis of ean significantly. mmp- message peaked at dpi and mmp- message peaked at dpi. on the other hand, both messages at dpi were suppressed compared to untreated ean rats. timp- message in clz treated rats peaked at dpi coincided with motor paralysis peak. both mmp- and mmp- messages might result in subsequent upregulation of timp- that finally downregulates mmps activity and inflammatory process. clz treatment suppressed and delayed expression of mmps and facilitate timp- expression, resulting suppression of ean. the precise mechanism of expression of mmps and timp- remain unclear, however, that mmp- and mmp- messages peaked at dpi suggests involvement of pro-inflammatory cytokines such as tumor necrosis factor-alpha or interleukin- . clz might suppress these cytokines resulted in mmps down regulation. mmp- less affected by clz and thus stimulated timp- expression at dpi. clz might rational treatment for immune-mediated neuropathy via mmps modulation although further investigation especially in-vivo study is needed. haidar m , de winter v , asselbergh b , bouhy d , timmerman v . peripheral neuropathy research group, vib, university of antwerp, antwerp, belgium. the small heat shock protein hspb (hsp ) gene is ubiquitously expressed and encodes for a chaperone protein with essential cellular functions. our lab was the first to identify missense mutations in hspb responsible for axonal charcot-marie-tooth neuropathy (cmt f). since then we became interested in understanding the physiological functions of hspb and its association with cmt neuropathies. we demonstrated the involvement of hspb in microtubule stability. because of the link between autophagosome formation and its intracellular transport, and microtubules stability, we believed that the macro-autophagy process could be regulated by hspb . macro-autophagy is a cellular housekeeping process during which autophagosomes target, envelop and degrade aberrant protein aggregates and damaged organelles. there is strong evidence for an essential role for autophagy in the maintenance of neuronal homeostasis; hence its impairment can lead to a neuropathic condition. our data indicate that macro-autophagy is disrupted by hspb cmt-causing mutations. combining novel microscopy and interactomics techniques we unravelled the way different cmt-causing mutations in hspb impair the autophagic pathway. our data present the impairment of autophagy as a possible pathomechanism for cmt-causing hspb mutations. hajjar h , gautier b , berthelot j , gonzalez e , gess b , young p , tricaud n . institute of neurosciences of montpelllier, inserm, university of montpellier, montpellier, france; universitätsklinikum münster, klinik für schlafmedizin und neuromuskuläre erkrankungen, münster, germany. cmt a, the most common of charcot-marie-tooth diseases, results from the duplication of peripheral myelin protein (pmp ) gene. this gene encodes for a small protein of kda, pmp , mainly produced by schwann cells and the excess of pmp leads to demyelination. there is no cure for this disease but one approach for a treatment is gene therapy. a transgenic rat model exists for cmt a, which possesses copies of the mouse pmp gene. our goal is to provide a proof of principle for gene therapy in peripheral nerves using this rat model of cmt a. our strategy is to reduce the overexpression of mouse pmp protein in rats schwann cells using short hairpin rnas (shrnas). shrnas are small non-coding rnas that specifically bind to targeted mrnas resulting in their degradation. we tested for the efficiency of several shrnas targeting mouse pmp in vitro to find two shrnas that reduce pmp levels. the shrnas have been cloned in an adeno-associated serotype (aav ) viral vector together with green fluorescent protein in order to detect infected cells. aav was selected for its high transduction rate of myelinating schwann cells, for its good diffusion and low immunogenicity. we plan bilateral injections in the sciatic nerve of control and diseased rats. the efficiency of this gene therapy will be checked by assessing muscle strength (grip test), way of walking (catwalk), mobility (rotarod) and nerve conduction velocity of treated cmt a rats versus non-treated. the process of myelination and myelin maintenance in schwann cells will be analyzed by biochemistry and electron microscopy. biochemical tests include western blot for pmp protein expression in sciatic nerve, immunohistochemistry for pmp protein expression in myelinating schwann cells and pcr for mrna pmp expression. if the therapy is successful in rats, it could possibly be later on used in clinical trials. ultrasound is a widely-used tool in diagnosing carpal tunnel syndrome (cts). several different methods for sonographical evaluation of median nerve damage exist, such as calculating the ratio of cross sectional areas (csa) of median nerves in various sites. this enables detection of actual nerve swelling proximal to the carpal tunnel as an expression of median nerve damage inside the carpal tunnel (as seen in cts). in comparison to other diagnostic methods no data exist about the prevalence of cts like changes in an unselected population. a series of non-selected fresh cadavers were examined. arms of fresh, non-embalmed whole body cadavers were examined. the medical record did not allow to obtain conclusive information on the peripheral nerves. using a regular ultrasound system with a mhz transducer, median nerves were identified and tracked along their course in the forearms. csa measurements of the median nerves were performed at two sites in each arm: .) halfway between the elbow joint and wrist, .) directly proximal to the carpal tunnel. csa ratio was calculated with the following formula: csa ratio = csa wrist (cm ) csa forearm (cm ) csa ratio < . was found in ( . %) arms, csa ratio ≥ . in ( . %) arms and csa ratio ≥ in ( %) arms. csa ratio ≥ . was detected in . % of women and % of men. the overall mean (± sd) age was . ± . years. men ( . ± . ) were significantly younger than women ( . ± . ; p = . ). a weak but significant correlation between age and csa ratio was found in women (spearman -− . ; p= . ), but not in men (p= . ). the mean bmi for csa ratio ≥ . was . ± sd . . based on a csa ratio ≥ . as a criterion for cts, the present ultrasound results are consistent with the average cts prevalence reported in previous studies, which were obtained with electrophysiological methods. this study on a large unselected series of cadavers confirms the comparability of both methods. instrumental activities of daily living (badl and iadl), falls and gait patterns in the large, prospective, population based rotterdam study. in total, participants of this study (mean age years, % women) underwent a polyneuropathy screening involving a symptom questionnaire, neurological examination and nerve conduction studies. screening yielded four groups: no, possible, probable and definite polyneuropathy. participants were interviewed about badl (stanford health assessment questionnaire), iadl (instrumental activities of daily living scale) and frequency of falling in the previous year. in a random subset of participants, gait was assessed with an electronic walkway (gaitrite). associations of polyneuropathy with badl and iadl were analyzed continuously with linear regression, and dichotomously with logistic regression. history of falling was evaluated with logistic regression and gait changes were evaluated with linear regression. we found that participants with definite polyneuropathy had more difficulty in performing badl and iadl than participants without polyneuropathy. polyneuropathy related to worse scores of all badl (especially walking) and three iadl components (housekeeping, traveling, and shopping). participants with definite polyneuropathy were two times more likely to fall, and these falls more often resulted in injury. participants with polyneuropathy had worse gait parameters on the walkway, including lower walking speed and cadence, and more errors in tandem walking. in summary, chronic polyneuropathy is strongly associated with significant impairment in daily life. recognition of polyneuropathy and related disability is very important in order to inform, support and possibly treat patients, and to prevent future falls and dependence in daily functioning. patients with polyneuropathy often suffer from tingling sensations, numbness, weakness and pain. these symptoms are used in several screening questionnaires, most of which were developed for high-risk patient groups, such as individuals with diabetes mellitus. in most tools equal weights are applied to all symptoms, while some might be more informative than others. we evaluated the diagnostic value and frequency of occurrence of individual symptoms of chronic polyneuropathy and constructed and validated a simple screening questionnaire that can reliably help to diagnose polyneuropathy in low-risk patient groups. in a multi-step procedure, we initially compiled a twelve-item questionnaire concerning symptoms of polyneuropathy. the questionnaire was completed by polyneuropathy patients and controls (headache, transient ischemic attack, multiple sclerosis). we calculated the sensitivity, specificity and likelihood ratios of each individual symptom. next, stepwise multivariable logistic regression was used to create a compact model, able to discriminate cases from controls using only the most informative symptoms. a simple scoring system was subsequently developed based on the regression coefficients of this reduced model. external validation was subsequently conducted in a population of cases with chronic idiopathic axonal polyneuropathy and controls without polyneuropathy. performance was assessed with discrimination (area under the curve, auc), and calibration. numbness and tingling feet were most frequently reported by polyneuropathy patients and had the highest sensitivity. feeling as if walking on cotton wool and allodynia of the feet had the highest specificity. multivariable logistic regression yielded a model that contained these four symptoms, complemented with balance problems and tingling hands. based on this regression analysis, the erasmus polyneuropathy symptom score (e-pss) was created, a score ranging from to . this polyneuropathy symptom score had a good performance (auc . ) in de derivation set and proved to be valid in the external population (auc . ). in this study, we created a simple, validated polyneuropathy symptom score (e-pss) that takes both the individual value of only six different symptoms and its frequency into account. this tool can be helpful as screening instrument in clinical practice and for future studies on polyneuropathy. multiple sclerosis (ms) is traditionally viewed as a central nervous system disease. to date, there is no unequivocal evidence implicating involvement of the peripheral nervous system (pns). this study aims to prove whether the pns is additionally affected and if so, to detect, localize and quantify these peripheral nerve lesions in patients with multiple sclerosis (ms) by applying high-resolution mr-neurography (mrn) with large anatomical coverage in combination with standard electrophysiological and neurological tests. we prospectively enrolled patients with confirmed ms (> years), two patients with clinically isolated syndrome (cis), and age-/sex-matched healthy volunteers. any other potential causes for a concomitant polyneuropathy were excluded. all ms patients underwent detailed neurological and electrophysiological testing. tesla mrn with large anatomical coverage from lumbar plexus and spinal nerves down to ankle level was performed in all participants by using fat-saturated, t -weighted turbo-spin-echo (tse) sequences (tr/te / ms) and a dual echo tse sequence for t -relaxometry (tr ms; te /te / ms). a d t -weighted, fat-saturated space sequence (tr ms; effective te ms) was used for imaging of the lumbar plexus. manual segmentation of spinal/sciatic/tibial/peroneal nerves was performed on a total of , axial slices. besides evaluation of nerve t w-signal, detailed quantification of nerve lesions by analyzing morphometric (nerve caliber) and microstructural markers (proton-spin-density and t -relaxation-time) was conducted. mean lesion load at thigh level was higher in ms ( . ± . ) vs. controls ( . ± . ;p< . ). nerve proton-spin-density was also higher in ms (tibial/peroneal: . ± . / . ± . ) vs. controls (tibial/peroneal: . ± . / . ± . ;p< . ). in contrast, t -relaxation time was significantly higher in controls (tibial/peroneal: . ± . / . ± . ) vs. ms (tibial/peroneal: . ± . / . ± . ;p< . ). proximal tibial and fibular nerve caliber was also significantly higher in ms (tibial: p< . ; fibular: p= . ). for the first time, pns lesions in ms patients could be visualized and quantified in vivo by high-resolution mrn. lesions are indicated by an increase of proton-spin-density and a decrease of t -relaxation-time. nerve caliber as a morphometric criterion also significantly increased. this proof-of-concept study may offer new insights into the pathomechanism of ms and might have future implications on therapeutic approaches. immunoglobulin g (igg) fc-gammars confer diverse effector functions by linking the cellular and humoral arms of the immune system that has been involved in the pathogenesis of guillain-barré syndrome (gbs). in the post-polio era, the polymorphisms of fc-gammar and their relevant knowledge have become one of the main targets for new therapeutic strategies for the treatment of gbs patients. differences in severity and frequency of gbs subtypes found between south-asian and western populations can be attributed to their genetic susceptibility. therefore, we aimed to determine fc-gammar polymorphic alleles (fc-gammariia: h /r ; fc-gammariiia: v /f ; fc-gammariiib: na /na ) and their possible link with gbs on the currently available large gbs cohort in bangladesh. fc-gamma r polymorphisms of gbs patients and healthy controls were genotyped using sequence-specific pcr. for validation, we carried out the sequencing of some samples for fc-gammariia and fc-gammariiia alleles. no significant differences were found regarding the distribution of fc-gammar genotypes and allele frequencies in gbs patients and controls. fc-gammar-h/h- genotype was significantly predominant in patients with severe disease compared to patients with mild disease (p= . , or, . ; % ci, . - . ). no other significant associations were found in gbs patients for candidate alleles and disease severity. fc-gammariiia-f/f- was found to be significantly predominant in anti-gm antibody positive gbs patients compared to anti-gm antibody negative patients (p= . , or, . , % ci, . - . ). fc-gammariiia-v alleles were significantly higher in patients with poor prognosis when compared to patients with good outcome (p= . , or, . , % ci, . - . ). no significant association of fc-gammariiib genotypes and alleles were found with gbs patients, disease severity and disease outcome. extensive subgroup analysis revealed no significant association in genotype and allele frequencies between aman and aidp subtype. in conclusion, igg fc-gammar polymorphisms do not constitute significant risk markers for susceptibility to gbs, however homozygous fc-gammariia-h might be involved in the severe form of gbs. in addition, fc-gammariiia-f/f- might play an important role in the molecular mimicry against nerve gangliosides in gbs. further studies that enroll a large number of patients (e,g. igos) are required to confirm the present findings from different geographical areas. hayes jm , o'brien pd , backus c , feldman el . department of neurology, university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common complication observed in patients with impaired glucose tolerance and type diabetes. male mice fed a high fat diet (hfd) develop metabolic impairments and pn serving as an appropriate animal model to study pn development and progression. it is well documented that female mice fed a hfd display a degree of protection against hfd-induced metabolic changes with mice retaining relatively normal insulin sensitivity. this protection is attributed to differences in fat accumulation and to the anti-diabetic effects of estrogen. based on these sex-dimorphisms we hypothesized that hfd-fed female mice would also exhibit resistance to developing pn. in the present study male and female c bl /j mice were fed either a standard diet ( % kcal fat; sd) or a high fat diet ( % kcal fat; hfd) from wk. at wk, wk and wk, neuropathy phenotyping was performed on all groups complemented with longitudinal metabolic assessments including insulin tolerance testing (itt). neuropathy phenotyping consisted of hindpaw latency to heat stimulus, motor and sensory nerve conduction velocities (ncvs), and terminal intraepidermal nerve fiber (ienf) counts. assessment of insulin resistance through itt demonstrated that during early hfd feeding, female hfd-fed mice exhibited relatively normal insulin responsiveness, while male hfd mice exhibited insulin resistance. despite this finding, wk female hfd mice displayed a similar pattern of pn to that of their male counterparts, with similar fold-changes in hindpaw latency and sensory and motor ncvs. therefore, although female hfd-fed mice exhibit resistance to hfd-induced metabolic changes, they display a pn comparable to male hfd-fed mice suggesting that systemic insulin resistance does not mediate pn. further studies are underway investigating the role of insulin signaling in the peripheral nerves of female hfd-fed mice. anti-mag (myelin-associated glycoprotein) neuropathy is a disabling autoimmune peripheral neuropathy caused by monoclonal immunoglobulin m (igm) autoantibodies that recognize the carbohydrate epitope hnk- (human natural killer- ). this glycoepitope is highly expressed on adhesion molecules, such as mag, present in myelinated nerve fibers. since the pathogenicity and demyelinating properties of anti-mag autoantibodies are well established, current treatments aim at a reduction of autoantibody levels. however, the therapies applied so far are primarily immunosuppressive and lack selectivity and efficacy. we therefore hypothesized that a significant improvement of the disease condition could be achieved by selectively neutralizing the pathogenic anti-mag antibodies with carbohydrate-based ligands mimicking the natural hnk- glycoepitope. in an inhibition assay, a mimetic (mimhnk- ) of the natural hnk- epitope inhibited mag-binding by pathogenic igm antibodies from patient sera, however only with micromolar affinity. therefore, considering the multivalent nature of the mag-igm interaction, polylysine polymers of different sizes were substituted with the mimetic. with the most promising polylysine glycopolymer pl (mimhnk- ) the inhibitory effect on patient sera was improved by a factor of up to , per epitope, consequently leading to a low nanomolar inhibitory potency. since clinical studies indicate a correlation between the reduction of anti-mag igm levels and clinical improvement, an immunological surrogate mouse model for anti-mag neuropathy, producing high levels of anti-mag igm, was developed. the observed efficient removal of these antibodies with the glycopolymer pl (mimhnk- ) represents a first step towards an antigen-specific therapy for anti-mag neuropathy. hinder lm , mendelson f , backus c , feldman el . university of michigan, ann arbor, mi, usa. in the united states % of children and young adults are obese and at risk of developing prediabetes. prediabetic patients largely develop the same macro-and microvascular complications as patients with type diabetes, including peripheral neuropathy (pn). moreover, recent clinical data suggest normoglycemic obese patients develop pn. central obesity, characterized by excess fat storage in visceral white adipose tissue, leads to systemic metabolic dysfunction largely due to an imbalance between pro-inflammatory/anti-inflammatory adipokine production. subcutaneous adipose tissue is considered 'benign', but adopts a visceral-like phenotype in response to metabolic stress, with reduced thermogenicity, reduced brown adipose identity, and increased pro-inflammatory gene expression. the popliteal adipose tissue (pat) depot, corresponding to subcutaneous adipose, is adjacent to the peripheral nerve affected in pn, contains the lymph node for lymphatic drainage of the hind limb, and expands following local, sterile hind paw inflammation. the aim of the current study was to characterize pat changes in the high fat diet (hfd) mouse model of obesity and pn, and consider its contribution to peripheral nerve dysfunction. we previously reported c bl /j mice fed % hfd from - wk develop obesity, 'prediabetes' and pn; and switching mice back to a standard diet from - wk improves metabolic and pn phenotypes. at and wk pat was bilaterally dissected and the lymph node removed. the left pat was processed for histomorphometry, and the right pat for rt-qpcr. at wk hfd was associated with a significant shift in adipocyte size-frequency distribution, with a greater number of larger adipocytes. switching the hfd mice back to standard chow from - wk restored the size-frequency distribution towards age-matched controls. rt-qpcr was performed to assess changes in thermogenicity (ucp ), brown adipose identify (cidea) and sterile inflammation (saa ). at and wk hfd pat had reduced thermogenicity and brown adipose identify, and increased sterile inflammation. this switch towards a visceral-like phenotype was reversed in the hfd mice switched back to standard chow. in summary, hfd-induced changes in pat histomorphometry and adipose identity closely associate with pn phenotype. these preliminary data suggest a potential role for pat-nerve signaling in pn. hinder lm , backus c , hayes jm , feldman el . university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common and debilitating complication of obesity and diabetes that triggers pain and loss of sensation. substantial nerve damage occurs in many patients prior to noticeable symptoms and no treatments are currently available; therefore, there is a critical need to identify treatment strategies that impact the underlying disease pathogenesis. our in vivo fluxomics data in the bks-db/db mouse model of type diabetes (t dm) and pn suggest that 'metabolic reprogramming' occurs in the t dm nerve to downregulate mitochondrial oxidative phosphorylation of substrates derived from glycolysis and fatty acid beta-oxidation. therefore, we hypothesize that distinct systemic metabolic alterations occur in obesity and diabetes which induce tissue-specific metabolic reprogramming within the peripheral nerve, altering fuel utilization and ultimately leading to tissue dysfunction. we contend that identifying conserved bioenergetic profiles across mouse models of pn will provide insight into key pn mechanisms. the current study utilized two mouse models of pn: the % high fat diet (hfd) mouse model of obesity and prediabetes at wk of age ( wk hfd), and the leptin receptor-deficient bks-db/db model of t dm at wk of age. mitochondrial function was determined in primary dorsal root ganglia (drg) neurons and sural nerve tissue from both models using the seahorse xf analyzer. resting mitochondrial oxidative metabolism was upregulated in drg neurons from mice with pn, with increased resting atp production and maintained mitochondrial coupling. in contrast, resting atp generation was decreased in sural nerve from mice with pn, with decreased coupling efficiency. relative spare respiratory capacity was attenuated in both drg neurons and sural nerve from mice with pn, indicating that mitochondria were less able to increase respiration in response to an energetic challenge. moreover, mitochondrial copy number was unchanged in drg neurons, but decreased in sural nerve tissue of mice with pn compared with respective controls. these data suggest a change in absolute number and function of sural nerve mitochondria, and a conserved cross-model proximal-distal bioenergetic profile in pn. we are currently exploring the relationship between these changes and pn pathogenesis. hoang ttn , umapathi t . hospital, ho chi minh city, vietnam; national neuroscience institute, singapore. ho chi minh city (hcmc) is the biggest metropolitan city in southern vietnam. its population is more than ten million. adult patients with neurological disorders are seen at six city public hospitals, including , hospital. there has been no systematic study of guillain barré syndrome (gbs) at hcmc or in vietnam in general. we are in the process of starting a prospective gbs database that we hope to expand to the other public hospitals at hcmc. here we describe our experience from . we saw gbs patients at , hospital. most of the cases were admitted in the rainy season, from late april to november, when mosquito-borne flavivirus infections are more common. patients were seen in the first week of illness and reported antecedent fever. two patients had diarrhea. diagnoses were made largely on clinical features, cerebrospinal fluid analysis and nerve conduction study. clinical findings include limb weakness, numbness and vii cranial nerve palsy. extraocular eye movements were affected in one patient. none had respiratory involvement severe enough to require artificial ventilation or intensive care. there were no pure miller-fisher syndrome cases; we suspect this might be related to the mild deficits that did not prompt hospitalization. nerve conduction studies showed typical features such as loss of f waves and abnormal blink reflex. the electrophysiology of patients' was dominated by demyelinating changes, one case was largely axonal and the remaining patient had normal electrodiagnostic study. repeat nerve conduction studies were not feasible because of limited resources. neurologists use corticosteroids as the main treatment. intravenous immunoglobulin and plasma exchange are costly and not reimbursed by medical insurance. we are currently preparing to systematically study gbs in southern vietnam, specifically with regards to possible role of antecedent flavivirus infections. we are also exploring the possibility of using low volume plasma exchange as a feasible cost-effective therapeutic modality. about % of patients with guillain-barré syndrome (gbs) treated with intravenous immunoglobulin (ivig) or plasma exchange deteriorate after initial improvement or stabilization -a phenomenon that is termed treatment-related fluctuation (trf). it is important to distinguish acute onset cidp (a-cidp) from gbs-trf during early course of the disease, because their therapeutic strategies and prognoses are different. herein, we describe a patient with gbs-trf, but with an extended progression phase that exceeds weeks. a -year-old woman was admitted due to acute onset progressive leg weakness and diplopia that had developed weeks prior (onset, d ). on neurological examination, right facial palsy was also observed. lower extremity weakness was moderate in proximal and distal muscles (mrc grade iv) with absent knee and ankle jerks. sensory examination revealed no abnormality in all modalities. she denied any recent diarrhea or upper respiratory infection, and vaccination. albumino-cytologic dissociation was noted in csf analysis; white blood cell count of / l and protein level of . mg/dl. nerve conduction study revealed demyelinating sensorimotor polyneuropathy with prolonged distal latency and conduction blocks. anti-ganglioside antibodies (gm igm/g, gd b igm/g, and gq b igm/g) were all negative. following ivig treatment, she was discharged with considerable improvement (d ). days later, she was re-admitted due to deterioration of leg weakness and hand clumsiness (d ). after another ivig treatment, she was discharged with clinical improvement (d ). days later, however, she was admitted again (d ) due to another considerable deterioration with four extremity weakness being worst at this time (mrc grade ii-iv in upper extremity, grade ii in lower extremity). a-cidp was considered given the progression phase exceeding weeks, but, we decided to give another treatment with ivig instead of a switch to corticosteroids because of uncertainty regarding distinction between a-cidp and gbs-trf. she was significantly improved following ivig treatment, and finally discharged (d ). thereafter, there has been no further deterioration during long-term follow-up of year. conclusively, this is a rare case of gbs with extended progression phase and trf. we propose that this could be referred to as subacute inflammatory demyelinating polyradiculoneuropathy (sidp) with trf. hsieh s , chao c . national taiwan university hospital, taipei, taiwan. transthyretin (ttr)-related familial amyloid polyneuropathy (fap) constitutes a major etiology of adult-onset hereditary neuropathies worldwide, in particular, a mutant ttr of ala ser (ttr-a s) in taiwan, the most common cause of acquired genetic neuropathy with adult onset (> years of age) of taiwanese patients. fap is a pan-modality neuropathy involving motor, sensory, and autonomic components of the peripheral nervous system with early involvement of small fibers as a major symptom. the early symptoms of fap are sometimes minimal and difficult to ascertain, mainly related to the fact that conventional electrophysiological examinations were not sensitive enough to detect small fiber neuropathy. skin biopsy with quantification of intraepidermal nerve fibers (ienf) has become one of the standard approaches to diagnose small fiber sensory neuropathy based on pathological documentation of nociceptive nerve degeneration. to explore the issue of early biomarkers in fap, we performed skin biopsy and compared ienf density with parameters of nerve conduction studies (ncs) and quantitative sensory testing (qst) on subjects ( men, aged . ± . years) with genetic confirmation of ttr-a s: patients and carriers. the ienf densities were significantly reduced compared to the age-and gender-matched controls in carriers ( . ± . vs. . ± . fibers/mm, p = . ) and patients ( . ± . vs. . ± . fibers/mm, p = . ). the latter was consistent with our previous report (neurology, : - , ) . the abnormal rate of ienf density was significantly higher than that of ncs and qst, respectively. in conclusion, there was significant skin nerve degeneration in carriers with ttr-a s. compared with qst and ncs, ienf density assessment had the highest abnormal rate and highest sensitivity to detect neuropathic changes in the early stage of fap. charcot-marie-tooth type p (cmt p) has been associated with frame-shift mutations in the ring domain of lrsam (an e ligase). this study describes families with a novel missense mutation of lrsam gene and explores pathogenic mechanisms of cmt p. this american family with dominantly inherited axonal polyneuropathy reveals a phenotype similar to those in previously reported non-us families. the affected members in our family co-segregated with a novel missense mutation cys arg that alters a highly conserved cysteine in the ring domain. this mutation leads to axonal degeneration in the in vitro neuronal cell-line. moreover, using protein mass spectrometry, we identified a group of rna binding proteins (including fus, a protein critically involved in motor neuron degeneration) that interacted with lrsam . the interactions were disrupted by the cys arg mutation, which resulted in reduction of intranuclear rna-binding proteins. a knockin mouse of cys arg has been created for further explorations of cmt p mechanisms and therapeutic development. together, our findings suggest that the mutant lrsam may aberrantly affect the formation of transcription machinery. given a similar mechanism has been reported in motor neuron degeneration of amyotrophic lateral sclerosis, abnormalities of rna/rna-binding protein complex may play a role in the neuronal degeneration of cmt p. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr ). this double-blind, multicenter, parallel-group trial randomized ( : ) adult participants (n= ) with chronic inflammatory demyelinating polyradiculoneuropathy being treated with intravenous immunoglobulin (ivig) or corticosteroids to . mg fingolimod (n= ) or placebo (n= ) once-daily. previous treatment was discontinued (ivig) or tapered (corticosteroids). in the total trial population, there was no significant difference between the groups in the primary outcome, time-to-first confirmed worsening (≥ -point increase on the adjusted inflammatory neuropathy cause and treatment [incat] scale vs. baseline), or time-to-first any worsening (confirmed on incat assessment or unconfirmed). no significant difference between the two treatment groups was shown on secondary outcomes; change from baseline in grip strength and rasch-built overall disability scale (r-ods) at six months or trial end. analyses of pre-specified subgroups were performed for primary and secondary outcomes. this trial evaluated the efficacy and safety of fingolimod in chronic inflammatory demyelinating polyradiculoneuropathy (cidp). corticosteroids, intravenous immunoglobulin (ivig) and plasma exchange are recognized treatment options but no other immunomodulators demonstrated efficacy in a controlled trial. fingolimod has been shown to be efficacious and is approved for the treatment of relapsing multiple sclerosis. results from experimental autoimmune neuritis in rats suggested that it might show an effect in cidp. in this double-blind, multicenter, parallel-group trial, cidp participants receiving ivig or corticosteroids were randomized to once-daily fingolimod . mg or placebo ( : ). participants were stratified by inflammatory neuropathy cause and treatment disability (incat) scores and prior treatment. previous ivig treatment was discontinued after one final course before randomization. previous corticosteroid treatment was tapered over weeks. the primary outcome was time-to-first confirmed worsening (≥ -point increase on the adjusted incat score versus baseline). secondary outcomes included change in grip strength and rasch-built overall disability scale (r-ods) score from baseline to month and at trial end. the trial was stopped for futility by an independent data monitoring committee after a pre-planned interim analysis based on pre-specified criteria. in all, participants received fingolimod (ivig: , corticosteroids: ; age: . ± . years [mean±standard deviation]; male: . %); received placebo (ivig: , corticosteroids: ; age: . ± . years; male: . %). the percentage ( % confidence interval) of participants free from confirmed worsening at the trial end was not significantly different between fingolimod ( . % [ . %- . %]) and placebo ( . % ( . %- . %); p= . ). in the first days, approximately % participants experienced worsening. at trial end, approximately % participants had no worsening. there was no significant difference after six months or at the trial end (whichever occurred earlier) in the secondary endpoints. adverse events were reported in / and / participants in the fingolimod and placebo group, respectively. there were no deaths. nine participants in the fingolimod group and in the placebo group had serious adverse events. adverse events leading to trial drug discontinuation occurred in ( %) participants on fingolimod and none on placebo. no new safety signals emerged in this trial. acknowledgment: the authors consulted for or were employed by the study sponsor novartis pharma ag, basel, switzerland. myelin sheath enwraps non-nociceptive mechanoselective abeta-afferents transmitting touch/vibration sense. a prominent reduction in the mechanical stimulus required to evoke a withdrawal response in rodents, a phenomena interpreted as mechanical allodynia, arises due to peripheral nerve/myelin damage. evidence has emerged that nerve injury-induced mechanical allodynia depends on the adaptive immune/t cell activity in female but not male rodents. having previously demonstrated both the release of the cryptic - peptide regions of myelin basic protein (mbp - ) following sciatic nerve chronic constriction injury (cci) and the direct, robust and t cell-dependent ability of the pure mbp - peptides to induce mechanical allodynia after injection into the intact sciatic nerve, we hypothesized that mbp - contributes to sexual dimorphism in mechanical allodynia. the pure mbp - wild-type (wt), its histidine (his) mutant or scramble peptides were administered into an intact sciatic nerve fascicle in male and female rats or mice, followed by von frey testing. intra-sciatic mbp - -wt peptide induced robust and lasting allodynia in females. in contrast, males responded with a brief and mild decline in mechanical sensitivity for one day post-injection of both wildtype and control peptides. the algesic ability of mbp - -wt was diminished in the his mutant. we here present the molecular changes in the sciatic nerve, drg and the spinal cord after the intra-sciatic mbp - injection in male and female animals. in addition, using the biotin-labeled mbp - peptide and the hrp-labeled goat anti-rat igg/igm antibodies, we developed an elisa to quantitatively assess seropositivity for the specific anti-mbp - peptide igm/igg autoantibodies in female and male rats post-cci. human serum from female patients with multiple sclerosis was used for control. our work corroborates the findings of sexual dimorphism of mechanical hyperpathia and suggests its potentially autoimmune nature in females. iijima m , nishi r , ikeda s , kawagashira y , koike h , sobue g , katsuno m . nagoya university, nagoya, japan. non-obesity diabetic (nod) b - knockout (ko) mice are characterized by chronic and progressive neuritis and expected as models of immune-mediated neuropathies, especially cidp. hindlimb-predominant weakness due to inflammatory demyelination followed by axonal degeneration begins from around twenty week-age in all female mice until thirty week-age. to clarify the efficacy of immunoglobulins as immune-regulating therapeutics and the similarity of pathogenesis of human cidp, we injected intraperitoneally human-derived immunoglobulins (ipig, mg/mg bw/week) and saline as a control to totally forty female mice. clinical and pathological estimations in sciatic nerves were performed in time series. as a result, the ipig-treated group was protected from weight loss which could be related to axon loss followed by muscle atrophy as well as inflammatory demyelination between twenty-five week-age and thirty week-age compared to the control. in addition, the pathological findings in sciatic nerves showed that ipig apparently suppressed inflammatory infiltrates. about the subsets of inflammatory infiltrates, while macrophages (cd +) and lymphocytes (cd +) highly existed and suggested to play a main role in the neuritis until thirty week-age, only macrophages naturally disappeared after thirty week-age without any therapeutic induction. immunoglobulins effectively suppressed only macrophages although that did not suppress cd + lymphocytes. in conclusion, nod b - ko mice respond to immunoglobulins in a similar manner to human cidp and this efficacy is due to the suppression of macrophage-dominant pathogenesis. therefore, macrophage-derived pathogenesis is for the main target of immunoglobulin therapy and we should focus on the lymphocyte-derived pathogenesis which might plays an important role in non-responders to immunoglobulins. ikeda s , nishi r , kawagashira y , iijima m , koike h , katsuno m , sobue g , . department of neurology, nagoya university graduate school of medicine, nagoya, japan; research division of dementia and neurodegenerative disease, nagoya university graduate school of medicine, nagoya, japan. chronic inflammatory demyelinating polyneuropathy (cidp) is an acquired immune-mediated polyradiculoneuropathy that is characterized by heterogeneous clinical manifestations. typical cidp is defined as neuropathy manifesting in a progressive manner, stepwise manner, or with recurrent symmetrical proximal and distal weakness and sensory impairment in all four limbs. although they occur at a lower proportion than the so-called typical cidp, atypical forms, such as multifocal acquired demyelinating sensory and motor (madsam), distal acquired demyelinating symmetric (dads), pure sensory, pure motor, and focal, are considered to cidp subtypes. thus far, pathological features characterizing each clinical subtype have not been fully elucidated. we analyzed clinical and pathological correlations in consecutive cidp patients who underwent sural nerve biopsy and fulfilled the definite or probable efns/pns criteria. there were male and female patients. the age at biopsy was . ± . (mean ± sd) years, and the duration from the onset of neuropathy to biopsy was ± months. fifty-five percent (n = ) of the patients were classified as having typical cidp. regarding atypical cidp, madsam (n = , %), dads (n = , %), and pure sensory (n = , %) subtypes were the major subtypes, while pure motor (n= , %) and focal (n= , %) subtypes were rare. no significant difference was found among these subtypes in terms of sex, age at biopsy, and disease duration. sural nerve biopsy specimens revealed that the densities of large myelinated fibers significantly decreased in the madsam subtype than in the other subtypes (p = . ). in addition, the variation in nerve fibers among fascicles was more conspicuous in the madsam subtype than in typical cidp (p= . ). patients with the dads subtype tended to show the formation of onion-bulbs. in conclusion, pathological findings of sural nerve biopsy specimens were different among the cidp subtypes. further studies are needed to clarify mechanisms leading to different pathological features. small volume plasma exchange (svpe) can be an affordable and potentially effective alternative form of plasma exchange. svpe is the repeated removal of small volumes of supernatant plasma over several days via sedimentation of patient whole blood. the aim of this study is to assess the clinical feasibility and safety of svpe in patients with gbs in low-income countries. twenty adult patients with gbs diagnosed as per the criteria for gbs of the national institute of neurological and communicative disorders and stroke (ninds) were enrolled for svpe at a centre in bangladesh. serious adverse events (sae) were defined as the number of patients developing severe sepsis associated with the central venous catheter (cvc) or deep venous thrombosis in the limb where the cvc is placed for svpe. the svpe procedure was considered safe if less than of svpe-treated gbs patients have a sae, and feasible if eight litres of plasma could be removed in at least of svpe-treated gbs patients. among the cases who received svpe, ( %) patients were male and the age range between to yrs. all the patients were quadriplegic and bedbound at enrolment for svpe with a median mrc score of (iqr, - ). cranial nerve involvement, autonomic dysfunction and requirement for assisted ventilation were observed in ( %), ( %) and ( %) patients respectively. electro physiologically ( %) patients were motor axonal and ( %) patients were sensory-motor demyelinating type. during the svpe none of our patients experienced sae and one patient experienced central line associated blood stream infection. common adverse effects were transient intravenous fluid responsive hypotension during the svpe sessions in ( %), cv catheter insertion site hemorrhage in ( %) and hypersensitivity reaction to fresh frozen plasma in ( %) patients. there was no hypo-albuminemia, anemia or electrolyte imbalance observed in most patients ( %) treated with svpe. improvement in one or more grade of the gbs disability score at four weeks after the onset of svpe was observed in ( %) patients. in conclusion svpe can be a safe, feasible and cost effective alternative to standard pe in the developing countries. guillain-barré syndrome (gbs) is a descriptive disease entity defined by a set of clinical, electrophysiological and laboratory criteria. various clinical phenotypes exist that may be triggered by different antecedent infectious events. although the disease appears to affect primarily the elderly in developed countries, but, scenario is different in developing countries. bangladesh has made an impressive progress towards the eradication of poliomyelitis, and no new cases have been reported since . gbs, an acute polyradiculoneuropathy, is the most frequent cause of acute flaccid paralyis. the crude incidence rate of gbs in < years of age reported here appears to be . × to × higher than that reported in the literature. we conducted a hospital based observational study including patients fulfilling the national institute of neurological disorders and stroke (ninds) criteria for gbs patients between and in dhaka medical college hospital, dhaka, bangladesh. detailed clinical, electrophysiological, serologic and microbiological data were obtained. gbs affected predominantly in young adults males (m/f= : ) living in rural areas. antecedent events were recorded in > % of patients; frequent events being gastroenteritis (> %) and upper respiratory tract infection ( %). more than % of the patients were bed-bound (gbs disability score ) at entry and about % patients required mechanical ventilator. about % patients did not receive specific treatment either intravenous immunoglobulin (ivig) or plasmapheresis due to high expensive treatment cost. % patients had died during hospitalization. % of patients had an axonal variant of gbs and evidence for a recent c. jejuni infection ( %). c. jejuni infection was significantly associated with serum antibodies to the gangliosides gm and gd a, axonal neuropathy, and greater disability. in conclusion, the majority of the patients do not receive standard treatment with ivig in view of its high price. therefore, we developed low-cost treatment strategies and conducted a safety and feasibility trials for small volume plasma exchange (svpe) on gbs patients in bangladesh. in future, it is essential to conduct a phase ii clinical trial to assess the efficacy of svpe for low-in-come countries. prognosis of guillain-barré syndrome (gbs) has not improved in last two decades. current therapies (intravenous immunoglobulin, ivig and plasma exchange, pe) had been proved to be effective on two third of patients in developed world. unpredictable and poorly understood clinical course of gbs hamper treatment development. in bangladesh, most patients affected by gbs cannot afford specific treatments with ivig or pe instead most of them receive only supportive care. therefore, we aimed to compare the outcome of ivig treated patients with supportive care patients in improvement of gbs disability score and mrc sum score by using world's largest gbs cohort in bangladesh. we conducted a prospective observational study enrolling gbs patients between and from dhaka medical college hospital and national institute of neuroscience and hospital, dhaka, bangladesh. only gbs patients ( %) received standard ivig treatment. in current analysis, ivig treated patients and age, sex and severity matched controls (supportive care only) were considered. outcome of both groups were compared using fisher's exact or chi square test and survival analysis were performed by kaplan meier method using log rank test. among patients (cases and controls), male/female ( / ), median age years, % patients were bed-bound, one-fourth patients required mechanical ventilation and % were axonal. we did not found any significant differences of treatment outcome in both cases and control groups in gbs disability score (week : p= . , months: p= . ) and mrc sum score (week : p= . , months: p= . ). survival analysis revealed, the differences of time required for independent locomotion, improvement of one gbs disability score and improvement of mrc score were not statistically significant between treatments (ivig) and supportive care patients. in conclusion, our analysis showed that standard dose of ivig use has no considerable advantage to improve specific outcome measures among gbs patients in bangladesh. as the phenotype of gbs in bangladesh is different from developed world; therefore, an efficacy trial for ivig is needed for developing countries like bangladesh or new targeted therapeutic strategies can append beneficial effects for gbs patients. isose s , , watanabe k , omori s , sekiguchi y , beppu m , shibuya k , amino h , suichi t , misawa s , kuwabara s . graduate school of medicine, chiba university, chiba, japan; national hospital organization chiba east national hospital, chiba, japan. diabetic neuropathy is a frequent cause of neuropathic pain, suggesting the small-fiber involvement. additionally, persistent peripheral pain-related inputs could cause neuronal hyperexcitability and complex interactions of the nociceptive pathways, i.e., central sensitization. to investigate the pathophysiology of neuropathic pain in diabetic neuropathy, we studied pain-related evoked potentials (preps) after selective intraepidermal electrical stimulation (ies) to a-deltaand c-fibers in diabetes patients with neuropathic pain (n= ) and without neuropathic pain (n= ). we also conducted a longitudinal study to assess changes in preps and pain profiles in patients with neuropathic pain months after the start of treatment with duloxetine. this study is registered with the umin clinical trials registry, umin . ies was applied in the hand and foot, and preps were recorded from the cz electrode referenced to the linked earlobes. we evaluated prep latencies, amplitudes, and amplitude ratios of preps after c/a-delta -fiber stimulation. in the conventional nerve conduction studies, patients with neuropathic pain significantly showed conduction slowing and decreased snap amplitudes in the median and sural nerves compared with those in patients without neuropathic pain. in pain-related sep studies, there were no significant differences in prep amplitudes and latencies after a-delta -or cfiber stimulation between the patients with neuropathic pain and without it. prep amplitude ratios after c/a-delta -fiber stimulation tended to increase in patients with neuropathic pain compared to patients without pain. after the treatment with duloxetine, c/a-delta -prep amplitude ratios were significantly decreased after both hand and foot stimulation, and as for numerical rating scale (nrs) scores as the intensity of pain. patients with less pain relief showed the tendency of higher c/a-delta prep amplitude ratios before treatment compared to patients with better pain relief. the correlation between reduction of c/a-delta prep amplitude ratios and nrs reduction did not reach statistical significance. this pain-related sep study demonstrated that abnormal cortical response in patients with neuropathic pain could improve after the treatment with duloxetine, this might reflect the cortical hyperexcitability as a central sensitization. this study is funded by shionogi & co., ltd. the sponsors played no role in the design and management of the study, collection and analysis of data, interpretation of the results, or the writing of the writing of the report. peripheral nerve injury is commonly associated with traumatic injury which is often amenable to surgery. despite improved methods in surgical repair, functional recovery remains a challenging clinical problem that often leads to significant morbidity in patients. alternative therapies that could augment surgical repair may be beneficial in functional outcomes. neuroinflammation is a complex pathway with different cellular components and cytokines that are activated following peripheral nerve injury. macrophages are responsible for the breakdown of debris following injury as well as promotion of regenerative signals. macrophage polarization is the process by which macrophages take on phenotypically distinct functions based on the local environment and signaling cues. exercise has been shown to drive macrophage polarization from a pro-inflammatory m phenotype towards an anti-inflammatory m phenotype in numerous tissues, but remains uninvestigated in the peripheral nervous system. the purpose of our study was to identify how exercise affects macrophage polarization, motor and sensory function, and neuroregeneration following sciatic nerve crush. c bl/ mice underwent sciatic nerve crush injury and were then given access to running wheels (exercised) or not given access to running wheels (sedentary) for weeks. exercised mice ran an average of . km per night. injured exercised mice were protected from the development of thermal hyperalgesia when compared to injured sedentary mice. exercised mice had fewer paw slips on beam walk testing compared to sedentary mice. no differences were measured in mechanical sensitivity or motor coordination and balance assessed by rotarod. while motor nerve conduction velocities were significantly reduced for injured mice compared to uninjured controls, motor nerve conduction velocities from injured exercised animals were significantly higher than injured sedentary animals suggesting improved nerve recovery with exercise. injured sciatic nerves from exercised mice demonstrated increased m macrophages compared to sciatic nerves from injured sedentary mice. the behavioral changes and altered macrophage polarization correlated with increased epidermal nerve fiber density, improved myelination, and increased in vitro neurite outgrowth from injured exercised animals. therefore, exercise alters macrophage polarization towards an anti-inflammatory phenotype which improves repair and recovery of the injured peripheral nerve. jack mm , shah k , everist b , reyna j , hylton p . university of kansas medical center, kansas city, ks, usa. diffusion tensor imaging (dti) has long been used to evaluate the location and integrity of white matter tracts in the brain. dti uses quantitative data and directionality of water diffusion to determine axonal connectivity of the nervous system. the technology has only recently been utilized in limited settings in the peripheral nervous system due to challenging technical factors and lack of widespread availability. magnetic resonance (mr) neurography or peripheral neurography is a technique which uses diffusion to differentiate between intraneural and perineural tissues. it allows for fascicle patterns to be visualized particularly in the setting of peripheral nerve sheath tumors. peripheral nerve sheath tumors of various pathologies cause surrounding nerves to be involved or displaced in a range of directions. this technique helps determine the anatomic location of these nerve fibers in relation to the mass, which is particularly helpful at distinguishing neuromas from schwannomas. this data is invaluable to the surgeon to ensure a safe and low morbidity operation. while this technology has benefit particularly with surgical planning, it has been underutilized due to the challenges of requiring complex software to produce fiber tracks and the inability to translate these images into the operating room. here, we utilized brainlab software that is commonly available and utilized in surgical suites to produce images of the radial nerve fiber tracts with an associated peripheral nerve sheath tumor prior to surgical resection. while the software is commonly used in the central nervous system, it has not been reported to have been used in the peripheral nervous system. this software offers a high usability and produces anatomically correct and reliable fiber tracts. this technique overcomes the reliance on highly specialized software and extensive training required for use that most other tractography software has. utilizing peripheral neurography in this case allowed for complete surgical resection without postoperative deficits. this data offers clinicians an option to investigate peripheral nerve fibers in various pathologic states, to plan appropriate operative trajectories to peripheral nerve pathology, and to improve surgical outcomes for patients with peripheral nerve sheath tumors. jacobsen b , parry g , allen j , walk d , muley s , ortega e . barrow neurological institute, phoenix, az, usa; university of minnesota, minneapolis, mn, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is an immune mediated neuropathy that is responsive to immunomodulatory agents such as glucocorticoids, intravenous immunoglobulin (ivig) and plasma exchange (pe). the specific immunopathogenic mechanisms of cidp remain unclear but there is increasing interest in nodal proteins as a site of the immune attack. even though the majority of patients respond to one of the aforementioned immunomodulatory agents there are some who are unresponsive or incompletely responsive to these first line agents and other more aggressive treatments may be necessary. cyclophosphamide and stem cell transplantation may be effective but are associated with considerable morbidity. anecdotal reports suggest that rituximab may be beneficial for some patients that fail first-line therapy, especially if they have antibodies to nodal proteins. we present four patients with intractable cidp who responded to rituximab. one of the three patients had diabetes. disease duration prior to starting rituxan was short ( months) in two patients and longer and months in the other two. all patients had failed treatment with glucocorticoids and ivig, and in two, plasma exchange and ivig-pe were also ineffective. two of the four patients were quadriparetic and non-ambulatory. two gm doses two weeks apart of intravenous rituximab were instituted in all patients. all patient tolerated the treatments well without adverse effects. all patients responded within four weeks and continued to improve at six months. other immunomodulatory agents were successfully tapered but not totally discontinued. it remains unclear whether antibodies to nodal proteins were present in these patients. in conclusion, although rituximab efficacy remains uncertain on the basis of randomized controlled clinical trials, it may be beneficial in selected patients otherwise intractable to first-line treatments. further studies are necessary to better understand which patients may benefit most from rituximab and where in the treatment algorithm rituximab should be applied. neuropathic pain is a chronic condition seen in patients suffering a direct injury to the peripheral or central nervous system or an indirect injury due to, e.g., diabetes or multiple sclerosis. current treatment options fall short of preventing or completely relieving patients of their pain. for years, research has focused on understanding the role of neurons in neuropathic pain pathogenesis while overlooking the role of supportive cells in general and satellite glial cells (sgcs) in the dorsal root ganglion in particular. these cells not only buffer the neuronal microenvironment they are also involved in controlling the electrical activity flowing through the neurons and in neuropathic pain pathogenesis. the aim of this project is to understand the role of sgcs in neuropathic pain development and thereby aid the identification of new drug targets. to purify the sgcs from adult mice we optimized a fluorescently activated cell sorting (facs) protocol. the success of our purification method was confirmed using qrt-pcr and visual inspection of the sorted cells. finally, we are running rna sequencing on sgcs after peripheral nerve injury to compare their transcriptome to that of uninjured cells at different time points. the results from our study are likely to deepen our understanding of how sgcs contribute to the development and maintenance of neuropathic pain. guillain-barrésyndrome (gbs) is an immunemediated disorder in the peripheral nervous system (pns) triggered by molecular mimicry against nerve gangliosides. one of the cell surface receptors (fas)-ligand (fasl) interaction transmits apoptotic signal to eliminate the auto-reactive b and t-cells, which generates cross-reactive antibody against nerve cells. host genetic polymorphism of fas and fasl may alter their expression and induce aberrant apoptotic response to develop gbs. therefore, we determined the single nucleotide polymorphisms (snps) of both fas receptor (− g/a and - a/g) and fasl ligand (− c/t) in gbs patients (n= ) as well as healthy controls (n= ) using the lightcycler technique. serum level of soluble form of fas and fasl was measured using commercially available sandwich elisa kit. comparison of genotype, allele and haplotype frequencies was done with the gbs subgroups based on the clinical and serological data. ag heterozygote (p= . , or= . , % ci= . - . ) and polymorphic g-allele (p= . , or= . , % ci= . - . ) of fas receptor - a/g promoter snps were significantly associated with anti-ganglioside (gm ) antibody positive gbs patients. in addition, − g-allele (p= . , or= . , % ci= . - . ) and - g/- g haplotype (p= . , or= . , % ci= . - . ) were predominantly associated with the axonal variant of gbs patients. serum soluble form of sfas (median levels pg/ml vs. pg/ml, p= . ) and sfasl (median levels pg/ml vs. pg/ml, p= . ) were found to be elevated in anti-gm antibody positive gbs patients compared to anti-gm negative patients. no significant association was found in genotypic distribution between gbs patients and healthy controls. in conclusion, fas/fasl promoter snps are not a susceptible factor for gbs but could be one of the influencing factors to develop cross-reactive anti-ganglioside antibodies in gbs patients in bangladesh. furthermore, functional studies with a larger sample size (using cohort like international gbs outcome studies-igos) are required to explain the immune pathogenic role of these snps for gbs patients. jang sy , yoon ba , shin yk , yun sh , jo yr , park ji , shin kj , kim jk , park ht . dong-a university, busan, south korea; inje university, busan, south korea. myelination is essential for the proper function of the nervous system. schwann cells, which form the peripheral myelin sheath, have the unique ability to dedifferentiate and to destroy the myelin sheath under various demyelination conditions. during schwann cell dedifferentiation-associated demyelination in wallerian degeneration after axonal injury, schwann cells exhibit myelin and junctional instability, down-regulation of myelin gene expression and autophagic myelin decomposition. however, in inflammatory demyelinating neuropathy, it is still unclear how schwann cells react and contribute to segmental demyelination before myelin scavengers, macrophages, are activated for myelin clearance. here, we show that schwann cell dedifferentiation-associated demyelination is a mechanism involved in the initial demyelination observed in a mouse model of inflammatory demyelinating neuropathy using ultrastructural, biochemical and microarray analyses. myelin uncompaction and myelin membrane instability generated by dedifferentiated schwann cells lead to autophagolysosome-dependent cytoplasmic amputation between the axon-containing myelin sheath and the schwann cell body, resulting in the formation of the "myelin corpse", thereby allowing macrophages to phagocytose the myelin corpse in the end stage of segmental demyelination. we found myelin corpse formation in inflammatory demyelination to be a process similar to the myelin rejection during wallerian degeneration, which appeared to be dependent on schwann cell autophagolysosome activation since schwann cell-specific atg knockout mice exhibited delayed myelin rejection following nerve injury. finally, lysosome inhibition in schwann cells not only prevented segmental demyelination but also delayed the progression of clinical stages by suppressing the myelin corpse formation in inflammatory demyelinating neuropathy. thus, our findings indicate that demyelination by schwann cells and macrophages might be part of a process that includes sequential divisions of labor with respect to myelin rejection and digestion, respectively. in conjunction with previous studies showing schwann cell dedifferentiation and autophagy in toxic and hereditary neuropathies, the concept of "schwann cell dedifferentiation-associated demyelination" provides insight into the development of possible therapeutic strategies to prevent schwann cell demyelination in peripheral demyelinating neuropathies. methods: electrophysiological data, the charcot marie tooth exam score (cmtes), and bmi from patients with known cmt a were obtained and analyzed. results: when controlled for age, bmi does not affect studies of ulnar motor nerve conduction in ctm a patients, but rather specific components of the cmt exam scores (cmtes, loss of pinprick sensation and motor strength in the lower extremities). discussion: bmi and clinical components of the cmtes are correlated, but it is uncertain which is the primary effect -i.e., whether the reductions in pinprick sensation and motor strength in the lower extremities lead to a higher bmi, or higher bmi results in these signs. introduction: charcot-marie-tooth disease type c (cmt c) is a rare, dominantly inherited neuropathy caused by mutations in the lipopolysaccharide-induced tumor necrosis factor (litaf) or small integral membrane protein of the lysosome/late endosome (simple) gene. methods: we present a case series comprised of patients in whom cmt c is caused by a gly ser substitution in the encoded protein. we focus on clinical presentation, electrodiagnostic analyses, and our findings in the context of previously described cases. results: the gly ser mutation causing cmt c is a mild form of cmt, as patients walked on time, had less weakness than those with charcot-marie-tooth disease type a (cmt a), had a charcot marie tooth neuropathy score (cmtns) indicative of mild disease, and had faster ulnar and median motor nerve conduction velocities compared to those with cmt a. discussion: the g s mutation in litaf seems to be clinically indistinguishable from a mild presentation of cmt a. jha mk , russell k , lee y , rothstein jd , morrison bm . johns hopkins university, baltimore, md, usa. peripheral nerves are highly dependent on metabolic energy to maintain both basic cellular functions such as axon transport, na+/k+ ion gradients, and myelination, as well as to support regeneration following injury. though glucose certainly provides some metabolic support, our recent studies have shown that monocarboxylates, such as lactate, pyruvate, and ketone bodies, also contribute to recovery from peripheral nerve injury. monocarboxylate transporters, particularly mct , are the predominate transporters for monocarboxylates in the peripheral nerve. in a recent publication, we found that mct heterozygous null mice, which express % less mct in all cells, have slowed nerve regeneration and reduced myelination following sciatic nerve crush. this study was limited by the global reduction of mct , which is widely expressed in schwann cells (sc), dorsal root ganglia (drg) neurons, endothelial cells, macrophages, and perineurial cells within the regenerating peripheral nerve. to better understand the mechanism by which mct contributes to normal nerve function and nerve regeneration, we produced and validated conditional mct null (mct loxp) mice that allow selective deletion of mct from scs, drg neurons, endothelial cells, or macrophages through mating to cell-specific cre lines. we are currently quantifying peripheral nerve development, aging, and regeneration in each of these mouse lines. following sc-, but not drg-, specific mct knockdown, sensory peripheral nerves develop demyelination by months of age, manifest by reduced myelin thickness, increased g-ratio, and reduced conduction velocity. studies are ongoing in cultured scs to determine the mechanism for demyelination. neither sc nor drg knockdown of mct impairs nerve regeneration following sciatic nerve crush. these results suggest that sc-specific mct is critical for maintaining myelin in sensory, but not motor, peripheral nerves as they age. they also suggest that mct expression in peripheral nerve cell types, other than sc and drg, is important for nerve regeneration. ongoing studies are determining the contribution of mct in other peripheral nerve cell types, particularly endothelial cells and macrophages, to normal development and regeneration following injury. our results will clarify the role of lactate and its transporter, mct , in peripheral nerve function, potentially suggesting novel targets for demyelinating neuropathies or nerve injuries. small fiber neuropathy (sfn) is a condition in which the smallest nerve fibers are affected, characterized by neuropathic pain and autonomic dysfunction. according to international criteria, sfn diagnosis is based on clinical symptoms in combination with abnormal temperature threshold testing (ttt) and/or reduced intraepidermal nerve fiber density (ienfd) in skin biopsy. skin biopsy is moderately sensitive, invasive and the process is time consuming and expensive. ttt is a widely available diagnostic tool, but lacks specificity. previous studies introduced stimulated skin wrinkling (ssw) as an objective, non-invasive diagnostic tool to detect sympathetic nerve dysfunction in sfn by means of a categorical assessment. however, our unpublished data has shown that inter-observer reliability of categorically assessed ssw is quite low. in this current study we will use a new digital method for ssw quantification: the digit wrinkle scan© (dws©). the primary study objective is to define normative values for dws© expressed as total wrinkle length per fingertip surface (mm/mm ). subsequently we investigate the applicability of dws© in patients with definite sfn, based on abnormal iefnd and/or ttt, determining the dws© sensitivity and specificity, as well as its validity. for this cross-sectional study, we will include healthy participants and patients diagnosed with sfn. eligibility is based on meeting the inclusion and exclusion criteria and providing written informed consent. skin wrinkling is induced by emla (eutectic mixture of local anesthetics) cream© application and captured by taking pictures. the primary outcome measure is total length of wrinkles per mm as shown on the photographs, which will be calculated by a new software program. patients are stratified according to age and gender. based on the results of healthy participants, normative values will be defined. inter-and intra-observer reliability will be determined. in the sfn group, additional correlation analysis will be conducted to determine the correlation between dws© and different outcome measures (sfn-symptom inventory questionnaire, visual analogue pain scale, neuropathic pain scale, sfn-rasch-built overall disability scale, ienfd and ttt). we expect to provide digitally quantified ssw (dssw©) normative values that can be used in clinical practice in the diagnostic workup for sfn. cmt , characterized by axonal degeneration, is an inherited motor and sensory neuropathy accounting for about % of total cmt patients. the cmt subtype shows on its own, a vast genetic heterogeneity with more than mutations in known genes rendering the identification of relevant drug targets and therapies very challenging. so far, only hdac inhibitors have shown promising results in mouse models for hspb mediated axonal cmt (cmt f), albeit such a single gene approach may have a limited relevance at clinical levels owing to the limited number of patients per genotype. in this study, we investigated common causative molecular players of cmt associated axonal degeneration. for this, an itraq based proteome analysis was performed on five patient's derived lymphoblasts bearing different cmt causal mutations alongwith respective age and gender matched unaffected controls. software-assisted interpretations of the obtained data led us to identify two proteins which were significantly downregulated in cmt patients compared to controls. these two proteins were then validated using western blotting and qpcr on patient derived lymphoblasts and fibroblasts. our results prompted us to unveil whether these two proteins can be used as potential biomarkers for identifying cmt patients. therefore, through a europe-wide collaboration, we constructed a cohort of cmt patients and healthy controls. these two proteins exhibited significant downregulation in this cohort suggesting a potential new role of these proteins as cmt biomarkers. remarkably, we were also able to validate the significant decrease in ineurons (neurons differentiated from patient derived ipscs) strengthening the importance of our finding and also suggesting the relevance of these proteins in the pathogensis of axonal cmt. this will be the first study involving multiple cmt causal genes at once, thereby holding the potential to offer new drug targets and potential biomarkers with wider application both clinically and pharmaceutically. mesenchymal stem cells (mscs) represents a valuable source of stem cell therapy, can differentiate into various cell types. we investigated of the neuromuscular potential of human tonsil-derived mscs (t-mscs) for neuromuscular regeneration in trembler-j mice that is considered to be a model for charcot-marie-tooth disease type a (cmt a diseases), which is involving hereditary motor and sensory peripheral neuropathies. the t-mscs differentiated toward skeletal myocytes, as evidenced by increased expression of skeletal muscle-related markers (including troponin i type , and myogenin) and the formation of myotubes in vitro. in situ transplantation of t-msc-derived myocytes (t-myocytes) into gastrocnemius in trembler-j mice, a mouse model of cmt a, enhanced motor function, as identified with recovery by a compound muscle action potential (cmap) amplitude. and the regenerated shape of the sciatic nerve and skeletal muscle by immunochemistry, without the formation of teratomas. furthermore, the expression levels of nerve growth factor (ngf) and glial cell line-derived neurotrophic factor (gdnf) were significantly increased in t-myocyte compared with t-mscs in vitro. these results indicate that the transplantation of t-myocyte can be a therapeutic option of cell therapy for the neuromuscular regeneration in hereditary peripheral neuropathy, comprising cmt a disease. kagiava a , karaiskos c , richter j , tryfonos c , lapathitis g , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory, the cyprus institute of neurology and genetics, nicosia, cyprus; department of molecular virology, the cyprus institute of neurology and genetics, nicosia, cyprus; neurology clinics, cyprus school of molecular medicine, the cyprus institute of neurology and genetics, nicosia, cyprus. x-linked charcot-marie-tooth disease (cmt x) is a common form of inherited demyelinating peripheral neuropathy resulting from mutations affecting the gap junction protein connexin (cx ). using a cx knockout (ko) mouse model of the disease, we have shown that targeted expression of virally delivered cx results in morphological and functional improvement. since patients with cmt x express mutant forms of cx in schwann cells, that could potentially interact with virally delivered wild type (wt) cx through dominant-negative effects, we also treated mutant mice expressing the t i, r w and n d mutations associated with cmt x on a cx ko background. all three mutants were localized in the perinuclear compartment of myelinating schwann cells consistent with retention in the er (t i) or golgi (r w, n d) with loss of physiological expression in non-compact myelin areas. following intrathecal delivery of the human gjb gene we could detect the virally delivered wt cx correctly localized in the non-compact myelin areas only in t i/cx ko mutant mice, but not in the other two mutants, suggesting dominant effects of the r w and n d mutant but not of the t i mutant. gjb treated t i/cx ko mice showed improved motor performance, along with lower ratios of abnormally myelinated fibers and reduced numbers of inflammatory cells in all tissues examined compared to mock-treated animals. in contrast, gjb treated r w and n d mutant mice showed only slight but not statistically significant improvement. this study provides additional proof of principle for a clinically translatable gene therapy to treat cmt x even in the presence of endogenously expressed cx mutants, since at least one er-retained cx mutant did not interfere with the expression of virally delivered cx allowing a therapeutic benefit similar to cx ko mice. however, golgi-retained mutants may interfere with virally delivered wt cx and other approaches besides gene addition may be needed for effective treatment. funding: muscular dystrophy association (grant mda to kak). kaida k , kadoya m , koike h , iijima m , takazaki h , ogata h , moriguchi k , shimizu j , nagata e , takizawa s , chiba a , yamasaki r , kira j-i , sobue g , ikewaki k . national defense medical college, tokorozawa, japan; nagoya university graduate school of medicine, nagoya, japan; kyushu university, fukuoka, japan; university of tokyo, tokyo, japan; tokai university school of medicine, isehara, japan; kyorin university, tokyo, japan. antibodies to a glial protein, neurofascin (nf) have recently been identified in approximately % of patients with chronic inflammatory demyelinating polyneuropathy (cidp), which are igg -predominant. igg anti-nf -associated cidp may be a distinct subtype from typical cidp in terms of clinical features and response to immunotherapy. however, a diagnostic criterion of anti-nf -associated cidp has not established yet. to develop optimal criteria and design the best treatment plan for the anti-nf -associated cidp, procedures for determining anti-nf antibodies should be simplified, prevalent, and reproducible, as well as being accurate. cell-based assay (cba) has hitherto been utilized for determining antibodies to nf in sera from patients, results of which have been confirmed by immunohistochemistry (ihc) using teased nerve fibers from rodents. these methods are the most reliable techniques, while not necessarily easy-to-use and easy to maintain in most laboratories. in the present study, we aimed to validate the diagnostic utility of a conventional enzyme-linked immunosorbent assay (elisa) for determination of anti-nf antibodies and the igg subclass. sera from patients with efns/pns criteria-met cidp were examined with elisa using human recombinant nf . to verify elisa results, ihc on rat sciatic nerves, western blot (wb) and cba using nf -transfected and naive hek cells were conducted. the human nf -based elisa clearly distinguished between anti-nf antibody-positive and -negative sera. fifteen cidp patients ( %) were igg anti-nf antibody-positive, which were confirmed by wb, ihc and cba studies. none of disease controls or healthy subjects had positive results. twenty-five sera randomly selected from anti-nf -negative cidp sera were also negative on cba. the anti-nf activities on elisa were significantly positively-correlated with those on cba (p < . ). analyses of clinical and laboratory findings showed that anti-nf -associated cidp was characterized by younger onset, distal dominant phenotype, tremor, sensory ataxia, higher protein levels in cerebrospinal fluid, and poor response to ivig, which were consistent with those in previous studies. this elisa combined with determination of the igg subclass is a simple and reliable method for initial screening for anti-nf antibodies. the genetic abnormality responsible for x-linked charcot-marie-toothy neuropathy subtype cmtx was recently identified by whole genome sequencing to be a kb insertion into chromosome xq . . the clinical profile of cmtx in childhood is not well described. we reviewed the clinical characteristics, neurophysiological profile and cmt pediatric scale (cmtpeds) assessments of children with genetically confirmed cmtx . cmtx was characterized by early onset, and early and progressive hand weakness. most affected children were symptomatic within the first two years of life. the most common presentation was with equinovarus foot deformity in the first year of life. cmtpeds analysis in these children revealed that cmtx progressed more rapidly ( . ± . points/ year, n= ) than cmt a and cmtx . grip strength in the second decade of life in most affected males was two standard deviations below age-and sex-matched normative reference values. the most severely affected individual was wheelchair bound at years of age and two individuals had no movement in the small muscles of the hand in the second decade of life. there was only a single symptomatic female identified and she had mild signs. nerve conduction studies showed a demyelinating sensorimotor neuropathy with motor conduction velocity in eight children while one child had a length-dependent sensorimotor axonal neuropathy. understanding the unique phenotype of cmtx is essential for directing genetic testing, as the cmtx insertion will not be detected on the snp microarrays, multi-gene panels or whole-exome sequencing currently used for the diagnosis of cmt. the early onset of disease coupled with rapid progression means that many children with cmtx will have severe disability within the first two decades of life and hence early diagnosis is needed for early commencement of rehabilitation. kapoor m , catania s , sarri-gonzales s , lunn mp , manji h , reilly mm , carr as . centre for neuromuscular diseases, national hospital for neurology and neurosurgery, london, uk; department of neurophysiology, national hospital for neurology and neurosurgery, london, uk. the conventional dosing of ivig in cidp and mmn is based on treatment trials that used bolus and maintenance dosing of ivig between - . g/kg. there are rare published articles reporting the efficacy of higher maintenance ivig doses. we present three cases of inflammatory neuropathies, who are currently stabilized on ivig doses of mg- mg/kg of ivig per month, refractory to standard dose ivig and other immunosuppressants. the first case is a year-old-lady with cidp who presented with episodes of ascending sensory disturbance, weakness, and diplopia. she had activity related fluctuations and pre-dose deterioration on g/kg/month ivig. she then had an acute deterioration with mrc sum score dropping from to even with additional plasma exchange. her bilateral foot drop (mrc grade - ) and fluctuations persisted with an increase of ivig to . g/kg/month. she is now clinically stable (ankle dorsiflexion mrc grade - , mrc sum score ) on mycophenolate and g ivig weekly ( . g/kg/month). case is a -year-old male fitness instructor with mmn and sjogren's syndrome. he presented with recurrent proximal and distal weakness that responded to g/kg of ivig and deteriorated with iv methylprednisolone. he had peri-dose fluctuations, intermitted proximal weakness, and persistent foot drop (ankle dorsiflexion mrc grade - ) at . mg/kg/month, worsening to mrc grade - on . g/kg/month and fluctuating between mrc grade - on . g/kg/month. an increase of ivig to g weekly ( . g/kg/month), has resulted in mmn rods scores of / , improved distal power and return to full capacity at work. case is a -year-old man with predominantly upper limb cidp. he received g/kg ivig without any benefit, had no response to doses of plasma exchange, doses of cyclophosphamide or dose of rituximab between and . since , he has received g/kg/month ivig with improvement of mrc sum score from to . these cases highlight that some patients require a much higher than conventionally prescribed dose of ivig, and that these doses are tolerated over years without serious adverse events. idiopathic rapid eye movement sleep behaviour disorder (irbd) has been identified as a precursor of alpha-synucleinopathies, such as parkinson's disease, dementia with lewy bodies, multiple system atrophy. several studies linked changes in cutaneous innervation with central nervous system pathology in neurodegenerative disorders. recently small fiber neuropathy and alpha-synuclein deposition in the skin found to be a potential biomarker in parkinson's disease. we evaluated the epidermal innervation of irbd patients and age and sex-matched controls from skin punch biopsies from the distal leg using pgp . immunohistochemistry. furthermore, a battery of clinical examinations were performed on patients and controls alike, including structured interviews, clinical motor and non-motor questionnaires and rating scales (e.g. unified parkinson's disease rating scale [updrs], non-motor symptoms questionnaire [nms-quest] and beck depression inventory, epworth sleepiness scale, evaluation of cognitive and olfactory functioning as well as blood samples. irbd patients, compared to controls, showed a significant reduction in intraepidermal nerve fiber density (p = . ), whereas the axon swelling ratio, did not differ between groups. patients with irbd reported non-motor symptoms more frequently than controls (updrs i, nms-quest). olfaction and daytime sleepiness differed between both groups, whereas there were no differences regarding cognition. these in vivo findings demonstrate small fiber neuropathy in irbd patients that are associated with non-motor symptoms indicating that peripheral abnormalities may occur early in irbd. they warrant larger scale longitudinal studies in order to investigate their prognostic value. katz j , lewis r , spatafora d . california pacific med center, san francisco, usa; cedars-sinai medical center, los angeles, usa; neuropathy action foundation (naf), santa ana, usa. multifocal motor neuropathy (mmn) is a rare condition that affects . in every , individuals worldwide and is associated with motor dysfunction and moderate to severe disability. the neuropathy action foundation conducted a global survey to determine the impact of mmn on patient quality of life (qol) and gaps in patient/provider educational needs. the first global mmn qol survey was an item internet questionnaire available between january and july , . the survey focused on three primary areas: timely and accurate diagnosis, the efficacy of treatment, and the impact of the disease on patients qol. the survey was completed by patients from countries. the majority of respondents said they were diagnosed between the ages of and years ( . %), more than % reported that it took more than one year to be diagnosed and more than % reported that it took - years or longer to be accurately diagnosed. with respect to treatment options : . % reported receiving intravenous immune globulin and . % reported receiving subcutaneous immune globulin therapy. other therapies being used to treat mmn were gabapentin ( . %), and pregabalin ( %). almost half ( . %) said that mmn often impacts their overall schedule. half of the participants reported that mmn often or always interferes with their employment; % had difficulty typing on a computer or using a telephone, . % had trouble concentrating, and . % said they had to work really hard to pay attention or else they would make a mistake. this is the first assessment of mmn from a patient's perspective. the survey highlighted critical issues relating to the diagnosis, management, and impact on the qol of individuals with mmn. the data also identified gaps and insights in provider education relating to proper diagnoses and management of the condition from a patient's perspective. katz j , levine t , dimachke m , barohn r . forbes norris center, san francisco, ca, usa; phoenix neurological institute, phoenix, az, usa; kansas university medical center, kansas city, ks, usa. in the united states, cidp cases are submitted to insurance companies to determine whether ivig therapy is appropriate. this is done using specified diagnostic criteria, which reduce diagnosis to a boolean analysis, where a disease can only be present or absent. this leaves no room for uncertainty, even when it truly exists. boolean criteria are useful for clinical trials, but fall short where real decisions are made under uncertainty and based on perceived cost/benefit analysis. this project attempts to elucidate root causes of uncertainty and to find solutions to this dilemma. we asked cidp experts to select a single diagnosis in cases where ivig was approved using the submitted case records. while there was agreement on many cases, in the five most "uncertain" cases no more than reviewers agreed on a single condition, who chose up to four separate entities. among these, at least three reviewers diagnosed an immune neuropathy in all five. the root cause of the disagreement, to a large degree was unclear documentation (aunts) which consisted of pasted, missing, and disorganized data. reviewers missed useful information, admitting it was too difficult to fully parse records. to resolve uncertainty, reviewers admitted to discounting certain reported datum to help fit the entity they suspected, such as reported therapeutic responses or certain electrodiagnostic/exam findings. other disagreement, however, reflected the complexity of neuropathy diagnosis, such as knowing if improvement was due to natural history or treatment, unawareness of rare presentations, or analyzing a true uncle (complex case). reviewers used bayesian (select most likely diagnoses from a list) and fuzzy logic (compare best fits to base cases). when the "best" diagnosis did not fit the base case, they had to re-interpret the data. improving review procedures requires eliminating aunts by collecting all key information and simplifying how records are presented. it also needs more advanced data methods to analyze common and rare borderline presentations (uncles like mama v pma, cidp v cspn, etc..), developing diagnostic algorithms that address real uncertainty, educating prescribers and patients on process, and creating systems that measure outcomes longitudinally after induction or tapering of therapy. keisuke y , miyuki m , motoi k , susumu k . department of neurology, faculty of medicine, kinki university, osaka, japan. anti-ganglioside antibodies are closely associated with clinical phenotype and specific symptoms in acute immune-mediated neuropathies. igg anti-gq b antibodies are specifically associated with miller fisher syndrome (mfs), bickerstaff brainstem encephalitis (bbe) and guillain-barré syndrome (gbs) with opthalmoplegia (gbs-op). in addition, ganglioside complexes (gscs) containing gq b also can be targets in such diseases, and might be associated with the clinical features. however, factors regulating clinical phenotype in those gq b-associated antibodies-positive diseases have not yet been known. for investigating the differences of antibody reactivities among those diseases, we examined, using combinatorial glycoarray, igg antibodies to ten individual glycolipids [gm , gm , gm , gd a, gd b, gq b, galactocerebroside (gal-c), lactosylceramide (laccer), ga , sulfatide] and glycolipid complexes consisting of two of the glycolipids listed above in sera from patients with gbs-op who were positive for anti-gq b antibody by elisa (gbs-op-gq b), patients with mfs with the clinical triad (opthalmoplegia, ataxia, and areflexia), and patients with bbe. by combinatorial glycoarray, overall sensitivity of antibodies to gq b and gscs containing gq b was . % ( / ) in gbs-op-gq b, . % ( / ) in mfs, and . % ( / ) in bbe, respectively. there were no significant differences in antibody reactivities between mfs and bbe. it is notable that antibodies to gscs containing gd b were more frequently found in gbs-op-gq b patients than in mfs or bbe patients (e.g., gd b/sulfatide: p= . and p< . , respectively). presence of the antibody reactivities to gscs containing gd b may possibly be related with clinical features of gbs-op-gq b, including frequent need of artificial ventilation. kennedy r , , , carroll k , , paterson k , ryan mm , , , mcginley jl , . royal children's hospital, parkville, australia; university of melbourne, parkville, australia; murdoch childrens research institute, parkville, australia. problems with walking and footwear fit are often reported by children and adolescents with charcot-marie-tooth disease (cmt). a cross-sectional, case controlled study of gait was conducted in children with cmt and typically developing (td) children. gait was assessed barefoot and in two types of the participants' own typical footwear; optimal (e.g. athletic shoes) and suboptimal (e.g. slip-on footwear). the aims were to determine differences in spatio-temporal (s-t) gait variables between children with cmt and td children; and to investigate the effect of footwear choices. twenty-nine independently ambulant children aged - years with confirmed genetic or clinical diagnoses of cmt, and age and gender matched td children participated (mean age . years; males). exclusion criteria included developmental disorders, other neuromuscular conditions or musculo-skeletal diseases that could affect gait, and lower limb injury or surgery in the preceding months. assessment included s-t gait patterns, footwear characteristics, metre run, and cmtpeds. gait was assessed at self-selected speed with an electronic walkway (gaitrite™), with trials for each condition. the primary gait variable assessed was speed; other variables included step length, step time, cadence, base of support width and step-to-step gait variability. across all footwear conditions children with cmt walked more slowly ( regeneration of cutaneous unmyelinated axons is known to be slowed in dm-patients and after -months, the density of intraepidermal nerve fibers (ienf) does not return to baseline levels after chemical or mechanical axotomy. however, the long-term outcome of regeneration in dm or control subjects is not known. additionally, it is not clear if the regeneration of sensory distal axons is length-dependent. here we measured the rate of axonal regeneration -months after chemical denervation using a capsaicin model in dm patients (n= / dm /dm ) without neuropathy, and controls. dm skin punches were performed at distal thigh at baseline, -hours post-capsaicin, and at , , and days. blood glucose and hgba c were measured at baseline, , and days. healthy controls had skin punches at both distal leg and proximal thigh at baseline, after capsaicin chemical axotomy, and days , , and . regeneration rate was significantly higher at the thigh in healthy controls ( . fibers/mm/day ( % ci: . - . fibers/mm/day) compared to dm (p= . ), but no difference between dm ( . fibers/mm/day % ci: . - . fibers/mm/day) or dm ( . fibers/mm/day % ci: . - . fibers/mm/day) (p= . ). comparing regeneration rate at different time intervals, showed that regeneration was significantly slowed between day and dm patients, while it continued with the same rate in controls. blood glucose or hga c had no effect on regeneration rate. ienfd returned to baseline in controls by -months ( % of baseline) while it is did not in dm subjects, %/ % (dm /dm ) of ienfd baseline, (p= . dm vs. controls). there was no difference in regeneration rate ienfd %-baseline by -months at distal leg and proximal thigh in controls (p= . ). these results suggest that the rate and outcome of regeneration is independent of the length of the axon. additionally diabetic patient have incomplete nerve regeneration after months regardless of diabetes type or the level of glycemic control. regeneration of axons slowed down over time in patients with dm and reached a plateau after days. kiessling p , lledo-garcia r , watanabe s , langdon g , tran d , bari m , christodoulou l , price g , smith b , byrnes w , brock m , jolles s . ucb pharma, monheim, germany; ucb pharma, slough, uk; ucb pharma, braine-l' alleud, belgium; ptx solutions ltd, london, uk; ucb pharma, raleigh, durham, usa; university hospital of wales, cardiff, uk. ucb is a humanised high-affinity monoclonal igg antibody developed to bind human neonatal fc receptor (fcrn), selectively inhibiting igg salvage and recycling. conditions such as myasthenia gravis (mg) are characterised by pathogenic igg autoantibodies; inhibition of fcrn may provide a suitable therapeutic approach. this phase i, double-blind, dose-escalating, first-in-human study (nct ) evaluated the safety and pharmacology of ucb . forty-nine healthy adults (mean age years, range - ) were randomised and received a single dose of intravenous (iv) or subcutaneous (sc) placebo (n= and n= , respectively), or a single dose of iv or sc ucb ( , or mg/kg; n= per dose, per administration). subjects were followed up until day . one placebo iv subject did not complete the study. twenty-seven of subjects ( %) receiving ucb , and / ( %) receiving placebo, reported ≥ treatment-emergent adverse event (teae) of mild/moderate intensity. severe teaes occurred in four subjects, all in the ucb mg/kg iv group (headache [n= ], back pain [n= ]). no serious aes occurred. incidence of infections was similar with ucb and placebo. the most frequently reported infection was nasopharyngitis. treatment-related teaes were reported by % of subjects receiving ucb and % receiving placebo: the most common in the ucb -treated groups were headache ( / ; %) and vomiting ( / ; %); these occurred more frequently with the iv than sc route. non-linear increases in ucb plasma concentration-time profile with increasing dose were observed with ucb . serum igg was reduced in a dose-depended manner with ucb iv and sc: decreases from baseline to day with ucb iv were . %, . % and . % for , and mg/kg doses, respectively, and . %, . % and . %, with ucb sc doses, respectively. these data indicated that the fcrn inhibitor ucb effectively reduced serum igg, with sc administration generally better tolerated than iv. further to these observations, the efficacy, safety and tolerability of ucb sc for chronic-intermittent treatment of moderate-to-severe mg are being evaluated in an ongoing phase ii, multi-centre, randomised, double-blind, placebo-controlled study (eudract - - middle east respiratory syndrome (mers) has a high mortality rate and pandemic potential. however, very little information has become available on this syndrome since it first erupted in . this study aimed to evaluate the frequency of neurological complications and their clinical presentations in mers. we reviewed the medical records of all patients who were diagnosed with laboratory-confirmed mers coronavirus (cov) infections and subsequently admitted to a single reference center for mers treatment during the outbreak in korea. in total, patients ( . %) reported neurological symptoms during or after mers-cov infection. the potential diagnoses in these cases included bickerstaff's encephalitis overlapping with guillain-barré syndrome, critical illness polyneuropathy or other toxic or infectious neuropathies. neurological complications did not appear concomitantly with respiratory symptoms, but were instead delayed by - weeks. neuromuscular complications were not rare in mers-cov-infected patients, and they may have previously been underdiagnosed. understanding neurological manifestations is important in an infectious disease like mers, because evaluation is frequently limited during treatment, but it can interfere with prognosis and sometimes require modification of treatment. kim hj , hyun jk , kim tu . dankook university, cheonan, south korea. the diagnosis of carpal tunnel syndrome (cts) is based on clinical symptoms, physical examinations and supported by nerve conduction study (ncs). ultrasonographic examinations can be performed to assess peripheral nerves with less discomfort and the surrounding anatomic structures. while the usefulness of ultrasonography (usg) in the cts has been reported, no study to date has compared the diagnostic utility of various usg findings for cts. we investigated the correlation of various usg findings to the clinical symptoms/signs and ncs findings in patients with cts. twenty-eight hands ( patients) with cts based on electrodiagnostic criteria and clinical symptoms such as tingling sensation or pain in the first to third fingers, burning sensation, paresthesia and weakness of hand grip power. all subjects were examined with usg. cross-sectional area (csa) and flattening ratio (fr) of the median nerve was calculated at level of radio-ulnar joint, pisiform and hamate. swelling ratio of the median nerve and palmar displacement of the flexor retinaculum was also calculated. clinical assessment was conducted using the boston carpal tunnel questionnaire (bctq) scale and historical-objective (hi-ob) scale. the analysis of correlation between usg findings and clinical symptom scales/ncs findings was performed using correlation analysis. the csa of the median nerve at level of radio-ulnar joint was significant correlated with bctq scale, hi-ob scale, distal motor latency, and conduction velocity (cv). the csa of the median nerve at level of pisiform was significantly correlated with hi-ob scale, distal motor latency, and cv. the fr of the median nerve at level of radio-ulnar joint was significantly correlated with bctq scale, hi-ob scale, distal motor latency, and cv. the swelling ratio of the median nerve was also significantly correlated with distal motor latency and cv. in patients with cts, csa of the median nerve at level of radio-ulnar joint was most closely related to ncs findings and clinical symptoms. so, csa of the median nerve at radio-ulnar joint might be a complementary tool for the diagnosis of cts. kitaoji t , tsuji y , ashida s , yamada t , ishii r , tanaka a , mizuno t . department of neurology, graduate school of medical science, kyoto prefectural university of medicine, kyoto, japan. the case was a -year-old woman. first, she noticed paresthesia in the right plantar eight months before admission and in the left plantar four months before admission. three months before admission, she developed muscle weakness in her feet. the muscle weakness and paresthesia extended to the lower legs in a few months. twenty days before admission, she experience difficulty in walking. on admission, the muscle weakness was observed in the legs, especially in the right tibial anterior muscle (ta). there was severe sensory disturbance and loss of deep tendon reflex in the legs. she had trouble walking due to the weakness and sensory aphasia. in nerve conduction study (ncs), conduction block was observed between the ankle and popliteal in both tibial nerves. the blood level of angiotensin converting enzyme (ace) was elevated. cerebrospinal fluid analysis was normal. there was no enhancement in the lumbar nerve roots shown on mri. gallium- scintigraphy showed hot spots on bilateral hilar lymph nodes and mediastinal nodes and biopsy of mediastinal nodes showed non-caseating epithelioid granuloma. therefore, we diagnosed her with sarcoid peripheral neuropathy by sarcoidosis. by using the nerve ultrasound, partial spindle-shaped nerve enlargement was observed at the part of conduction block in the left tibial nerve. we started the treatment with methyl prednisolone ( mg, days) and oral prednisolone therapy ( mg/kg/day). after treatment, the paresthesia and muscle weakness in the legs had gradually improved. the partial enlargement in the left tibial nerve also improved on the -hospital day. in ncs, the conduction block improved, however, the compound muscle nerve potential of tibial nerve decreased because of axonal damage. this partial spindle-shaped nerve enlargement by using ultrasound has never been reported in sarcoid peripheral neuropathy before. the nerve ultrasound may be useful for evaluation of therapeutic effect of sarcoid peripheral neuropathy. peripheral nerve injuries are still debating problems in the world because of poor recovery. there is absolutely a need for new therapeutic agents to improve outcome by altering nerve regeneration. there are some studies in the literature about some therapeutic agents that used in the cases of peripheral nerve injuries. despite these studies, an agent with clinical use has not been presented yet. in this experimental study, we aimed to analyze the effects of curcumin (cur) in the cases of peripheral nerve injuries. forty rats were randomly and equally divided into four groups. the first group was control group. rats in this group were not operated. right sciatic nerve injuries were performed to the other groups. the second group was operation group with no therapeutic agent. the third group was operation and local cur applied group. the fourth group was operation and systemic cur applied group. electrophysiological evaluations were performed with electroneurography (enog) before and after the surgeries. systemic use of cur although caused improvement in the enog values but could not make a positive contribution to the nerve regeneration statistically. additionally local use of cur made negative effect to the nerve regeneration statistically. according to our statistical results we could not recommend cur as a nerve protective agent. klein cj , wu y , jentoft me , mer g , spinner rj , dyck pjb , dyck pj , mauermann ml . mayo clinic, rochester, usa. intraneural perineurioma is a hypertrophic peripheral nerve tumor having immunoreactivity to epithelial membrane-antigen, negative for s- . the origin of perineurial cells is debated to be similar to meningeal cells. ip does not metastasize, but motor deficits accumulate over time from tumor growth in nerve and plexus. after schwannomas and neurofibromas, perineuriomas are the most common nerve tumor of young adults. a chromosome q deletion has been reported in one patient. we identified ip cases from our previously published clinical cohort with available flash frozen ip tissue for dna isolation. wes with cnv analysis and cgh microarray analysis (agilent x k superprintg ) were performed on extracted dna; had available germline dna (lymphocytes and buccal tissue). we compared the exome data against online and in-house control data (∼ , ) examining variants less than . frequencies, predicted damaging or nonsynonymous. wes identified three novel, heterozygous, damaging mutations in tumor necrosis factor receptor-associated factor (traf ) in of ( %) cases; p.l p (n= ), p.h r (n= ) and p.s r (n= ). mutations were within the wd domain, p.l p, p.h r within exon and p.s r within exon , and mapped to a limited region of traf with protein structure modeling. two of cases ( . %) showed macroduplications/deletions on multiple chromosomes, including chromosome , confirmed with cgh microarray analysis and cnv results from exome data analysis. four of ( %) had no discovered mutation. age of onset or severity did not correlate with type of mutations. this study provides strong evidence that traf is a specific tumor driver of ip. mutations in traf are also linked to benign intracranial meningiomas suggesting a shared pathogenesis and close origins of perineurial and meningeal cells. study supported by: mayo foundation and the mayo center of individualized medicine. klein i , , bobylev i , , lehmann hc , . university hospital cologne, cologne, germany; center for molecular medicine cologne (cmmc), cologne, germany. peripheral neuropathy is a common side effect of paclitaxel. clinical evidence suggests that the delivery mechanism of paclitaxel formulations influence time course and severity of paclitaxel induced peripheral neuropathy. in a preclinical model we studied access, distribution and toxicity of two paclitaxel formulations (nanoparticle albumin-bound paclitaxel (nab) and solvent-based paclitaxel) in the peripheral nervous system (pns). c bl/ mice were treated with mg/kg or mg/kg of nab-paclitaxel or solvent based paclitaxel. kinetics of paclitaxel in neurons was assessed by a newly established immunostaining technique. neurotoxicity was evaluated by functional assays and nerve morphology. paclitaxel accumulated mostly in dorsal root ganglia, whereas distal nerve segments showed only low uptake of paclitaxel. treatment of mice with the two paclitaxel formulations resulted in paclitaxel uptake mostly in nf + larger fiber neurons. in ib +, and cgrp+ small fiber neurons, paclitaxel was less frequently detected. nab-paclitaxel was incorporated more rapidly compared to solvent-based paclitaxel but neurons also showed a faster clearance of nab-paclitaxel compared to solvent based paclitaxel. functional assays and nerve conduction studies indicated that nab-paclitaxel was less neurotoxic compared to solvent-based paclitaxel. this is the first study that characterizes in detail the access of nab-paclitaxel and solvent based paclitaxel into the pns. our findings have important implications to understand the pathomechanisms of paclitaxel induced neurotoxicity and to develop neuroprotective strategies by preventing access of paclitaxel to the pns. the aim of our study was to investigate the etiology of neuropathy in patients with rheumatoid arthritis (ra). subjects were neuropathy patients with ra admitted to our department. laboratory investigations, nerve conduction studies (ncs) and sural nerve biopsy were performed. mean patient age was . years (range, - years), and mean disease duration was . years (range, - years). clinical diagnosis for neuropathy was rheumatoid vasculitis (rv) in patients, rv with acute motor axonal neuropathy (rv-aman) in and chronic inflammatory demyelinating polyneuropathy with ra (ra-cidp) in . rheumatoid factor ( / ) and rheumatoid arthritis particle agglutination ( / ) was high and c tended to be lower in the rv group (rv and rv-aman). anti-ganglioside antibodies were examined in patients, with positive results in . an rv-aman case was diagnosed with motor-dominant clinical presentation and the presence of anti-galnac-gd a immunoglobulin (ig)g antibody. no other cases with rv were examined for anti-ganglioside antibodies. positive results for anti-gm and gm igg antibody were seen in one ra-cidp patient. we evaluated sural/median (s/m) ratio) for sensory nerve action potential (snap). s/m ratio was low in rv cases ( / ) and high ( / ) in immune-mediated cases, suggesting a so-called normal sural abnormal median pattern in immune-mediated neuropathies. the rv-aman case showed a moderate value in s/m ratio. nerve biopsy revealed thinly myelinated nerve fibers in ra-cidp cases compatible with demyelination, while the rv group showed the typical pathology for necrotizing vasculitis. rv cases were treated with prednisolone (psl), intravenous methylprednisolone, intravenous cyclophosphamide and increased psl dose. ra-cidp and rv-aman were treated with intravenous ig. in conclusion, neuropathy in ra can be divided into vasculitic and immune-mediated groups. ncs, and the s/m ratio of snap with some laboratory parameters in particular, may be of use in differential diagnosis and deciding treatment strategies. koh s , wong shj , loh kw , chng ysk , pawa c , ei ma , lee bjh , subramaniam t , umapathi t . national neuroscience institute, singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university singapore, singapore; tan tock seng hospital, singapore; khoo teck puat hospital, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful autonomic neuropathy that develops with abrupt improvement in glycaemic control. typically, type or dm patients on insulin or oral hypoglycaemic agents (ohga), present with painful neuropathy and autonomic dysfunction within weeks of rapid improvement in glucose control. current emphasis to achieve good glycaemic control rapidly may inadvertently increase incidence of tind, hence the impetus to understand risk of over-zealous glycaemic control. we therefore set out to study the occurrence of tind in a dm cohort of a tertiary hospital. we screened all patients who had two hba c measurements between and . during this period, approximately patients were seen per year. we found patient-encounters that showed hba c decrease of ≥ % over months or ≥ % over months. we then used a structured checklist of tind symptoms to shortlist cases. these case-encounters were scrutinised and classified as; 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or uncertain temporal relationship to decrease in hba c; unlikely tind: alternative explanation exists for symptoms. only one case was deemed 'probable tind'-a middle-aged man with newly diagnosed type dm who presented to emergency department with palpitations and worsening 'frozen feet' sensation that disturbed sleep. his hba c decreased by . % in weeks. his symptoms improved within a month with neuropathic pain medications and resolved months later. he also developed maculopathy and proliferative retinopathy. ten months later, he developed significant proteinuria. four other cases were classified as 'possible tind' while the remaining were unlikely tind. our study is limited by retrospective design and reliance on hospital records. nevertheless, our findings suggest that tind is uncommon in a general cohort of dm patients. on the other hand, the number of patients with painful neuropathy and acute dysautonomia symptoms contemporaneous with rapid decline in hba c raises the intriguing possibility that forme fruste of tind exists and one should interrogate the rate of hba c decline in dm patients with these symptoms. koike h , kadoya m , kaida k , ikeda s , kawagashira y , iijima m , kato d , ogata h , yamasaki r , matsukawa n , kira ji , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; national defense medical college, tokorozawa, japan; nagoya city university graduate school of medical sciences, nagoya, japan; kyushu university, fukuoka, japan. we investigated the morphological features of chronic inflammatory demyelinating polyneuropathy (cidp) with autoantibodies directed against paranodal junctional molecules, particularly focusing on the fine structures of the paranodes. sural nerve biopsy specimens obtained from cidp patients with anti-neurofascin antibodies and patient with anti-contactin antibodies were assessed. these antibodies were examined using sera obtained from patients with cidp who fulfilled the criteria of the european federation of neurological societies/peripheral nerve society. thirteen cidp patients without these antibodies were also examined to compare pathological findings. characteristic light and electron microscopy findings in transverse sections from patients with anti-neurofascin and anti-contactin antibodies indicated a slight reduction in myelinated fiber density, with scattered myelin ovoids, and the absence of macrophage-mediated demyelination or onion bulbs. teased-fiber preparations revealed that segmental demyelination tended to be found in patients with relatively high frequencies of axonal degeneration and was tandemly found at consecutive nodes of ranvier in a single fiber. assessment of longitudinal sections by electron microscopy revealed that detachment of terminal myelin loops from the axolemma was frequently found at the paranode in both anti-neurofascin and anti-contactin antibody-positive cidp patients compared with antibody-negative cidp patients. patients with anti-neurofascin antibodies showed a positive correlation between the frequencies of axo-glial detachment at the paranode and axonal degeneration, as assessed by teased-fiber preparations (p < . ). in conclusion, paranodal dissection without classical macrophage-mediated demyelination is the characteristic feature of patients with cidp with autoantibodies to paranodal axo-glial junctional molecules. koike h , ikeda s , takahashi m , kawagashira y , iijima m , misumi y , ando y , ikeda si , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; kumamoto university, kumamoto, japan; shinshu university hospital, matsumoto, japan. peripheral neuropathy is the cardinal feature of familial amyloid polyneuropathy (fap), but its mechanism has not been fully elucidated. we used electron microscopy to examine schwann cells and endoneurial microvessels. sural nerve biopsy specimens from fap patients with transthyretin val met mutation were assessed. patients were consisted of early onset cases from endemic foci and late onset cases from non-endemic areas. loss of nerve fibers with or without neighboring amyloid deposition was a common feature. the amount of amyloid deposition was greater relative to the extent of nerve fiber loss in early onset cases than in late onset cases. the atrophy of schwann cells, particularly nonmyelinating cells, that were apposed to amyloid fibrils was more conspicuous in early onset cases than in late onset cases. the numbers of endothelial cell nuclei, endothelial cell profiles, and occluded microvessels were significantly increased in the fap patients compared with patients with nutritional/alcoholic neuropathies (p < . , . , and . , respectively). findings suggestive of the disruption of blood-nerve barriers, such as the loss of tight junctions and the fenestration of endothelial cells, were also more frequently found in the fap patients (p < . ), irrespective of the presence or absence of amyloid deposition. in conclusion, these findings suggest that direct insult of amyloid fibrils causes schwann cell damage resulting in the predominant loss of small-fiber axons characteristic of early onset cases. in addition, vasculopathy may also participate in the pathogenesis of neuropathy, particularly in late onset cases. kolb n , smith ag , singleton jr , beck s , howard d , dittus k , karafiath s , mooney k . university of vermont, burlington, vt, usa; university of utah health, slc, ut, usa. chemotherapy induced peripheral neuropathy (cipn) is a major cause of morbidity due to numbness, pain, and gait instability. this prospective study compares the current standard care for cipn symptom management to a new care delivery model which utilizes an automated symptom tracking program paired with a nurse practitioner led intervention triggered by moderate to severe symptoms. all participants beginning taxane or platin based chemotherapy called a telephone based automated symptom tracking program daily (symptom care at home -sch) to report chemotherapy related numbness and tingling. sch tracked the presence and severity of neuropathic symptoms and their interference with activities of daily living (adls) on a - scale. participants were randomized to two groups. the usual care (uc) group was advised to call their oncology provider for recommendations on symptom management. in the nurse practitioner (np) group, when symptom severity was ≥ participants received automated self care strategies and a call from a nurse practitioner to provide treatment recommendations based on consensus guidelines. patients participated in the study. mean duration of follow up was . ± . days with . ± . calls. the np group had fewer days with any neuropathic symptom ( . % ± . vs. . % ± . , p= . ), with moderate to severe neuropathic symptoms ( . % ± . vs. . % ± . , p< . ) or days of distress from neuropathic symptoms ( . % ± . vs. . % ± . , p= . ). on days with moderate to severe symptoms participants also reported burning ( . ± . %), weakness ( . ± . %), balance problems, ( . ± . %), and tripping ( . ± . %). there was no significant difference between groups in the interference in adls (np . ± . vs. uc . ± . , p= . ). overall the automated telephone system effectively identified neuropathy symptoms and their severity. compared to usual care in which patients must independently reach out to their care team for symptom management, sch is effective in decreasing symptom prevalence, severity and distress. kouton l , kremer l , tard c , morales r , kuntzer t , attarian s , boucraut j , delmont e . referral centre for als and neuromuscular diseases, marseille, france; neurology department, strasbourg, france; neurology department, lille, france; neurology department, montpellier, france; neurology department, lausanne, switzerland; immunology laboratory, marseille, france. antibodies against proteins of the node of ranvier have been recently described in severe chronic inflammatory demyelinating polyradiculoneuropathies (cidp). they target paranodal proteins, namely contactin (cntn ) and neurofascin (nf ). cell-based assay and elisa are available in research, but no gold standard technic is admitted for the detection in routine of these antibodies. our objective was to evaluate if flow cytometry analysis is an efficient technic to detect antibodies against ctn and nf in a large cohort of cidp patients. flow cytometry analysis were performed on a bd facs-diva. human embryonic kidney (hek) cells were transfected either with nf or cntn . sera were diluted / . antibodies anti-cntn or nf were revealed using fitc conjugated anti human igg antibodies. delta mfi (mean fluorescence intensity) was calculated as mfi of transfected cells less mfi of non-transfected cells. measures were normalized using positive controls and negative controls from healthy blood donors. sera of cidp patients from different french neuromuscular referral centres were analysed with flow cytometry. respective delta mfi were (standard deviation ) and (standard deviation ) for antibodies against nf and cntn in cidp antibodies negative patients. antibodies against nf were found in patients (respective mfi , , ) and against cntn in two other patients (respective mfi and ). isotype of these antibodies was igg in patients and igg and igg in the remaining patient. all the patient had severe cidp. four patients had poor response to intravenous immunoglobulins (ivig) and have been treated with immunosuppressive drugs. as usually reported, the patient with anti-nf antibodies had postural tremor. flow cytometry seems effective to detect antibodies against nf and cntn . compared to other assays, benefits of flow cytometry are: to analyse a large number of sera in the same time and to give objective numerical results expressed in mfi that can be compared to the results of other samples. further studies are needed to confirm that flow cytometry can be the best test to assess antibodies against cntn and nf in routine. the mechanism by which intravenous immunoglobulins (ivig) improves peripheral nerve function in multifocal motor neuropathy (mmn) is unknown. the rapid clinical improvement following ivig could be related to blocking complement deposition on gm epitopes, change in ion-channel properties of affected motor axons, or both. the present study investigated median nerve motor excitability parameters at ∘ c just before ivig administration as well as at the peak of clinical improvement in patients with mmn. the investigated nerves were characterized either by conduction block (n= ), demyelinative slowing without block (n= ), or motor axon loss (n= ). the results of motor excitability testing in mmn showed no difference between pre-and post ivig recordings. clinical assessment of apb muscle showed increase in mrc score in patients and decrease in patient after ivig administration. in patients mrc score of the apb remained the same. those findings indicate that clinical changes following ivig administration are not related to excitability parameters of affected motor axons in mmn. where impulse conduction is blocked or markedly slowed in motor axons but is normal in sensory axons. sensory symptoms or signs are usually absent but have occasionally been reported in skin areas innervated by nerves with prominent motor axon loss. although the mechanism of selective motor involvement in mmn is unresolved, it may be related to differences in antigenic properties between motor and sensory axons or differences in biophysical properties. the objective of the present study was to compare ion-channel activity in both motor and sensory axons of nerves affected by mmn. affected nerves had to have motor conduction block, demyelinative slowing on motor ncs, or motor axon loss, whereas sensory ncs had to be normal. we performed excitability tests of motor and sensory axons in affected median nerves of mmn patients and healthy controls at ∘ c. conditioning and test stimuli were delivered at the median nerve at the wrist; cmaps were recorded from the thenar muscle and snaps from the rd digit. results of motor excitability testing in mmn showed fanning-out of threshold electrotonus, decreased i/v slope, and increased superexcitability, all compatible with persistent hyperpolarization of resting membrane potential in motor axons. sensory excitability testing in mmn showed decreased subexcitability but was otherwise normal. this may indicate minimal involvement of sensory axons in mmn. krarup c , , moldovan m , , alvarez s , ciano c , pisciotta c , pareyson d . university of copenhagen, copenhagen, denmark; rigshospitalet, copenhagen, denmark; fondazione irccs istituto neurologico c. besta, (incb), milan, italy. mutations in the gene coding for myelin protein zero (mpz, p ) are associated with different forms of charcot-marie-tooth (cmt) disease. we describe a family harboring a frameshift mutation (c. dela / p.asp thrfster ) in the p gene, predicted to result in a nonfunctional p truncated very early in the extracellular domain. this offered the rare opportunity to assess the consequences p deficiency in absence of the potential gain-of-function effects of the mutations itself. conventional conduction studies and multiple measures of nerve excitability by "threshold tracking" were carried out in heterozygote parents (aged and ) and their two homozygote sons (aged and ). in the homozygous patients, all distal limb cmaps and snaps were absent. for neurophysiological assessment, the spinal accessory nerve was stimulated at the neck and cmap was recorded over the upper trapezius muscle. eight normal subjects, mean age , were used as control. the two sibs showed a severe phenotype with early onset, severe scoliosis, complete loss of distal movements and relevant proximal weakness, cmt examination score (cmtes) - / ; both heterozygous parents had very mild adult-onset neuropathy with cmtes < / . control subjects had a trapezius cmap with a latency of . ms and an amplitude of . mv. heterozygotes had a mild cmt type b phenotype, with a cmap latency of . ms and an amplitude of . mv whereas the homozygotes had a severe neuropathy with a cmap latency of . ms and an amplitude of . mv. consistently, the homozygotes had a more severe impairment in excitability with a rheobase of . ma as compared to . ma in the heterozygote and . ma in controls. deviations in excitability measures were similar to our previous reports in p +/− and p −/− mice. mathematical modeling, indicated both altered passive cable properties due to dysmyelination and depolarizing features with increased na+ currents. our data suggest that p deficiency is associated with impaired axonal na+ channel function, arguing for the translational value of na+ channel blocker treatments as found in p null mouse models. krishnarajah s , divino v , mallick r , dekoven m . csl behring, king of prussia, pa, usa; quintilesims, fairfax, va, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is a rare neurological disorder of the peripheral nervous system. the objectives of this retrospective real-world study were to compare demographic and clinical characteristics among cidp cases and matched controls and to assess cidp treatment utilization. adults newly diagnosed with cidp between / / and / / were identified in the quintilesims pharmetrics plus health plan claims database (first diagnosis date termed the index date). eligibility requirements were: confirmation of cidp (second cidp diagnosis or initiation of cidp therapy) within year of initial diagnosis, continuous health plan enrollment in the months prior to diagnosis (the pre-index) and the years following diagnosis (the follow-up), and no cidp diagnosis or use of cidp therapy in the pre-index. a total of , cidp cases met the study eligibility criteria. cases were direct-matched to controls based on age, gender, region, health plan, and payer type at index, and pre-index charlson comorbidity index score. the final sample consisted of cases matched to controls (both: mean [sd] age . [ . ]; . % male; . % commercially-insured). alternative pre-index diagnoses among cases included inherited neuropathies ( . %) and chronic acquired polyneuropathies ( . %). in the pre-index, neuropathic pain ( . % vs. . %), back pain ( . % vs. . %), and use of opioids ( . % vs. . %) and anti-convulsants ( . % vs. . %) were significantly higher among cases compared to controls (p< . for all). median total pre-index healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . ). over the follow-up, median total healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . [mean $ , and $ , ] ). cidp-related therapy costs accounted for . % of total healthcare costs for cases. the majority of cases ( . %) initiated cidp therapy over the follow-up, in a mean of . ( . ) days from initial diagnosis. half ( . %) of cases initiated treatment with corticosteroids only, while . % initiated ivig only. over the follow-up, . % of cases used any corticosteroid, while . % used any ivig. our findings suggest a substantial clinical and economic burden of cidp compared to matched controls. corticosteroids and ivig were most commonly used to treat cidp. kronlage m , baeumer p, pitarokoili k , schwarz d , schwehr v , godel t , heiland s , gold r , bendszus m , yoon ms . department of neuroradiology, heidelberg university hospital, germany; department of neurology, st. josef hospital, ruhr university of bochum, germany. objective: to evaluate large coverage magnetic resonance neurography (mrn) in chronic inflammatory demyelinating polyneuropathy (cidp). methods: in this prospective study patients with cidp and healthy controls were examined by a standardized mrn protocol at tesla. lumbosacral plexus was imaged by a t -weighted d-sequence ( mm isotropic voxel size); peripheral nerves of the upper and lower extremity by axial t -weighted turbo-spin-echo sequences ( . x . mm in-plane resolution). lesions were characterized by nerve cross sectional area (csa) and t -weighted signal (nt ). additionally, t -relaxometry of the sciatic nerve was performed using a multi-spin-echo sequence. all patients received a complementary electrophysiological exam. results: patients with cidp exhibited increased nerve csa and nt compared to controls (p < . ) in a proximally predominating pattern. roc analysis revealed best diagnostic accuracy for csa of the lumbosacral plexus (auc = . ) and nt of the sciatic nerve (auc = . ). csa correlated with multiple electrophysiological parameters of demyelinating neuropathy (f-wave latency, nerve conduction velocity) of sciatic and median nerve, while nt only correlated with f-wave latency of sciatic and not median nerve. t -relaxometry indicated that mr-signal increase in cidp was due to increase in proton-spin-density (p < . ), and not increase in t -relaxation time. conclusion: both nt and csa might aid in diagnosis of cidp, but csa correlates more robustly with electrophysiological parameters. since best diagnostic accuracy was shown for proximal nerve locations, mrn may be a useful complementary tool in select cidp cases. kühnemund j , wetzel c , bégay v , moshourab r , lewin gr . mdc & bih, berlin, germany; mdc, berlin, germany; charité, berlin, germany. damage of peripheral sensory nerves due to diabetes, herpes zoster infection, chemotherapy or trauma can cause chronic neuropathic pain. common symptoms include increased pain sensation (hyperalgesia), touch-induced pain (allodynia), paresthesia and spontaneous pain. we currently have poor understanding about the underlying molecular mechanisms at the peripheral level and treatment of patients suffering from neuropathic pain is inadequate. recent meta-analysis studies show that common first-line medications only yield nnts (numbers needed to treat) between . to . . it is still unclear to what extent allodynia can be attributed to changes in the physiological properties of intact sensory afferents. in this study, we aimed to elucidate whether changes occur in intact sensory afferents that innervate the plantar skin of the hind-paw following induction of neuropathic symptoms. this question was of particular interest considering that blocking mechanotransduction in the skin can alleviate mechanical hypersensitivity in neuropathic pain models (wetzel et al nature neuroscience ( ): - ). we used the chronic constriction injury (cci) model in mice which is behaviourally characterized by robust mechanical hypersensitivity. we made electrophysiological recordings from primary afferent neurons which had intact axons passing through the constriction to innervate the plantar skin using an ex-vivo skin-nerve preparation. we were able to record from myelinated afferent fibers and unmyelinated c-fibers with receptive fields in the control uninjured plantar skin as well as plantar skin of cci mice. we will present evidence that changes in the mechanosensitivity of sensory fibers innervating the glabrous skin may contribute to the symptoms of neuropathic pain. neuropathology, national hospital for neurology and neurosurgery, london, uk; irccs foundation "carlo besta" neurological institute, milan, italy; neurogenetics unit, national hospital for neurology and neurosurgery, london, uk; nuffield department of clinical neurosciences, oxford, uk; department of clinical neurophysiology, norfolk and norwich university hospital, uk. hsn secondary to sptlc / is a rare slowly progressive neuropathy resulting in marked sensory loss, especially nociception and significant motor deficit. despite most of the patients having the same c w mutation in sptlc , there is marked heterogeneity in the phenotype. l-serine oral supplementation has been suggested as potential therapeutic candidate however the lack of outcome measures is a major limiting factor in the initiation of a clinical trial. we undertook a natural history study to identify outcome measures that are responsive enough to be used in a clinical trial. the assessments used were cmt neuropathy score (cmtns version and cmtns version rasch modified), mri of calves and thighs, computerised myometry, quantitative sensory testing (qst), comprehensive neurophysiological assessment, proximal thigh skin biopsy for intra-epidermal nerve fibre density (ienfd), plasma dsl levels and patient based questionnaires (neuropathic pain symptom inventory and sf- v ). standardised response mean, srm (mean change/standard deviation of change) was used to compare responsiveness between tests. patients were recruited: with sptlc (c w) and with sptlc mutations. when analysed as a whole cohort, proximal calf mri fat fractions showed the most significant change over months. ienfd, plasma dsl levels, npsi and sf- v showed minimal change or the change was not in the clinically expected direction. for subsets of the remaining assessments which showed the highest responsiveness, the cohort was sub-divided into mild-moderate (cmtns ≤ ) and severe (cmtns> ) subgroups. in the mild-moderate subgroup, the greatest improvement in responsiveness was seen in computerised myometry (ankle plantarflexion: srm=− . and ankle eversion: srm=− . ). in the severe subgroup, qst (vibration detection thresholds on hands: srm=− . and face: srm=− . and pressure pain threshold on the face: srm= . ) and proximal calf mri fat fractions (srm range= . - . ) showed the greatest improvement. focusing on subgroups classified according to disease severity improved the responsiveness of some tests into the highly responsive range with mri still being the best outcome measure. this will reduce the number of participants required to power a clinical trial and might be a possible solution for designing a clinical trial for a rare, slowly progressive disease with a heterogeneous phenotype. kugathasan u , clark aj , suriyanarayanan s , laurá m , wilson e , , kalmar b , greensmith l , , hornemann t , reilly mm * , bennett dlh * . hsn secondary to sptlc / mutation is a slowly progressive sensory motor neuropathy leading to profound sensory loss with variable but often severe motor deficit. the genes sptlc and encode for the essential enzyme serine palmitoyltransferase (spt) which catalyses the rate limiting step in the sphingolipid de-novo biosynthesis. mutations in these two genes alter the substrate specificity of spt leading to the synthesis and accumulation of atypical metabolites called -deoxysphingolipids ( -deoxysl). plasma levels of -deoxysl are raised in hsn patients. deoxysphingolipids have been shown to be toxic in avian and by our group, in mammalian primary drg and motor neuron cultures. firstly, this study looked at the effects of -deoxysl on the survival and neurite integrity of human ipsc derived sensory neurons following exposure to different concentrations of deoxysphingolipids. later, we determined if there was autonomous -deoxysl production in hsn patient ipsc derived sensory neurons. sensory neurons were differentiated from human ipscs using a combination of small molecular inhibitors. deoxysphingolipids were found to be neurotoxic in this model after hours of treatment. in control lines, there is a significant reduction in neuronal survival following treatment with both -deoxysphinganine ( -deoxysa) and -deoxymethylsphinganine ( -doxmethsa). a clear dose-dependent increase in the expression of the axonal injury marker, atf , is seen with both -deoxysphngoid bases. in both instances, -deoxysa is more neurotoxic than -doxmethsa, which is similar to the findings in avian and mammalian primary neuronal cultures. autonomous -deoxysl production is seen in ipsc derived neurons obtained from three different hsn patients with the levels being significantly greater than that seen in multiple control lines. this is the first study to demonstrate that human ipsc derived sensory neurons can be used as an in-vitro model for hsn , providing a great opportunity both to probe the pathomechanisms mediating deoxysphingolipid toxicity and to test potential therapeutic agents. recent studies have demonstrated an association between autoantibodies directed against antineurofascin- (anti-nfasc ) and a subpopulation of cidp patients characterized by sensory ataxia, tremor and poor response to ivig treatment. here we report on the clinical features of three patients who developed acute changes in their phenotype during the course of their neuropathy, a potential clue to recognize a neuropathy with predominantly humoral dysimmunity. case reports: our index patient had developed sensory changes over weeks, followed by irregular locomotion, and muscle weakness with general areflexia. a first run of ivig improved the patient in a week, but he relapsed within days. a second ivig course with prednisone again normalized deficits within days. ivig runs and rituximab were still necessary to treat a nd , and then a rd relapse before obtaining complete improvement. the course of the neuropathy was months. two other patients were encountered with more chronic courses but in whom periods of worsening or improvement suggested a relapsing-remitting neuropathy, either spontaneously or following immunomodulating treatments. in all three, extensive work-ups were negative, anti-nfasc igg were positive, and detailed repeat nerve conduction studies demonstrated fluctuating conduction blocks. discussion: our report underscores that in chronic cidp patients a relapsing-remitting course could be encountered as a key feature of anti-nfasc neuropathy. this not yet described characteristic course could be of value when deciding using rituximab instead of immunomodulating treatments. kusunoki s , morikawa m , kuwahara m , ueno r , samukawa m , hamada y . faculty of medicine, kindai university, osaka-sayama, japan. anti-glycolipid antibodies are often detected in sera from patients with autoimmune neuropathies, such as guillain-barré syndrome (gbs), chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and multifocal motor neuropathy (mmn). not only individual glycolipid antigens but also mixtures of two different glycolipids (glycolipid complexes) are sometimes recognized by serum antibodies. to investigate antibody activities against large number of glycolipid complexes in serum samples from patients with gbs, mmn, and cidp, we examined igm and igg antibodies against glycolipids [gm , gm , gd a, gd b, gq b, galnac-gd a, lm , galactocerebroside (gal-c), asialo-gm (ga ), and sulfatide] and glycolipid complexes consisting of two different glycolipids listed above, by using combinatorial glycoarray. serum was obtained from patients with gbs, patients with cidp and patients with mmn, all in the acute or relapsing phase. serum was also obtained from healthy controls and patients with other neurological diseases. we investigated the relationships between the clinical features and presence of those antibodies. high titers of igg antibodies were detected almost exclusively in gbs patients. in contrast, igm antibodies were frequently present in mmn and gbs. among the anti-glycolipid complex antibodies in gbs, anti-gm /sulfatide, anti-ga /sulfatide, anti-gm /gd a, and anti-gq b/sulfatide igg antibodies were common ( , , , and patients, respectively). igg antibodies against antigens containing gm were significantly correlated with pure motor gbs (p < . ) and those against antigens containing gq b were significantly correlated with gbs with ophthalmoplegia (p < . ). in seven of the patients with anti-gq b/sulfatide complex antibodies, the antibodies were specific to the gq b/sulfatide complex rather than the individual gq b and suldfatide antigens. moreover, four patients did not have antibodies other than those to the anti-gq b/sulfatide complex. in patients with mmn, igm antibodies to antigens containing gm or galnac-gd a were present in % and . %, respectively. glycoarray is efficient for detecting antibodies against numerous glycolipid complexes in immune-mediated neuropathies. we need further investigations on other immune-mediated diseases using larger number of antigens. kuwabara s , misawa s , sekiguchi y , susumu kusunoki and the jet-gbs study group . department of neurology, chiba university, chiba, japan; department of neurology, kindai university, osaka, japan; japanese eculizumab trial for guillain-barré syndrome, chiba, japan. guillain-barré syndrome is a monophasic immune-mediated neuropathy, but a substantial number of patients with severe disease have poor recovery, even if treated with immunoglobulin. recent studies suggest that complement activation plays a pivotal role in gbs-associated axonal degeneration, and eculizumab is a monoclonal antibody that specifically binds to complement component and inhibits complement activation. jet-gbs is an investigator-led, phase , randomized, placebo-controlled trial conducted in hospitals, this trial aims to investigate the safety and efficacy of eculizumab for treatment of severe gbs. patients were randomly assigned ( : ) to treatment with immunoglobulin plus either eculizumab ( mg/day; n= ) or placebo (n= ) once weekly for weeks. the primary outcome measures are safety and efficacy (the proportion of subjects who regain their ability to walk independently at week ). the secondary outcome measures included the proportion of subjects who were able to walk independently at week , and other measures such as mrc sum scores, nerve conduction parameters. enrollment for the trial began in august , and follow-up of the last patient was completed in october . analyses will be made in april , and the results will be presented at this meeting. this trial is registered with clinicaltrials.gov identifier: nct , and funded by the japanese agency for medical research and development, and alexion pharmaceuticals inc. neurofascin , a paranodal protein in peripheral nerve, is a target antigen for autoantibodies in a subset of chronic inflammatory demyelinating polyneuropathy (cidp). anti-neurofascin antibody-positive cidp is characterized by onset at younger age, tremor, and refractoriness to ivig. we have treated four patients with anti-neurofascin antibody-positive cidp at kindai university hospital. anti-neurofascin antibody was detected by both elisa and cell-based assay in those patients. igg subclass was predominantly igg in all four cases. the median age of the patients at admission was . years [range: - years]. among the four patients, three had tremor, and two had severe cerebellar ataxia. cerebrospinal fluid protein levels were remarkably increased [median: . mg/dl, range: - mg/dl]. although ivig treatment was administered in all four patients, the responses were poor or partial. in contrast, plasma exchange (pe) was performed in all four patients and the clinical symptoms dramatically improved in two of them. corticosteroids were also effective in those two patients. sural nerve biopsy was performed in all four patients. although sensory nerve action potentials of the sural nerves from those patients were not evoked, the transverse semithin sections of sural nerves from three patients revealed only slight or mild loss of myelinated fibers. partial paranodal demyelination was observed in teased-nerve fibers from three patients. in addition, abnormal paranodal lesions such as loss of the transvers bands were observed by electron microscope in all four patients. those electron microscopic findings were not observed in control patients with anti-neurofascin antibody-negative cidp. anti-neurofascin igg antibody-positive cidp shows distinctive clinical and pathological features. labeyrie c , besson f , vandendries c , cauquil c , beaudonnet g , not a , durand e , adams d . neurologie adulte, chu bicêtre, le kremlin bicêtre, france; médecine nucléaire, chu bicêtre, le kremlin bicêtre, france; clinique bizet, paris, france; unité de neurophysiologie clinique, chu bicêtre, le kremlin bicêtre, france. study of the proximal portion of the peripheral nervous system (pns) is difficult because less accessible to electrophysiological exploration and biopsy. the indications of mri are increasing in proximal neuropathies to analyse morphology of the roots, plexus and proximal nerves: integrity of the nerve bundle, inflammation or infiltration of these structures. pet-computed tomography (pet-ct) can detect infiltration of the proximal segments of the pns, with hypermetabolism of roots, plexuses and large nerve trunks. pet-ct is also useful in detection of solid neoplasm which can infiltrates pns and primary nerve sheath tumors. however, spatial resolution of pet -ct scan is limited to explore the roots and plexuses for moderate hypermetabolisms. we believe that a mild hypermetabolism could be highlighted in inflammatory neuropathies or in mild tumor infiltrations. we performed a fusion of whole body pet (performed because of suspected neoplasia) and plexus mri (pet-mri) images in ten patients with a peripheral neuropathy for which we suspected proximal involvement. the pet-ct and mri were first read separately, then with merge of the images by a nuclear physician and a neuroradiologist. five out of ten patients presented with hypermetabolism of pns (hmpn+) on pet-mri: root (n= ), and/or dorsal root ganglia (n= ). median sul of lesions was . . among the hmpn+ patients: hypermetabolism was already apparent on pet-ct in cases, the merge invalidated abnormalities seen on pet-ct in patients and mri detected additional lesions, not visible on pet-ct, in one patient. all hmpn+ patients had hypersignal and or hypertrophy of pns on mri either diffuse (n= ), or multifocal (n= ). among hmpn-(no hypermetabolism) out of ( %) had abnormal mri ( multifocal and diffuse). gadolinium enhancement was found in all patients receiving gadolinium in the hmpn+ (n= ) and only in / patients in the hmpngroup. final diagnoses of hmpn+ patients were neurolymphoma in one ( %), and idiopathic cidp in the others ( %) with histological proof on nervous root biopsy in one of them. final diagnosis in hmpn-patients was cidp in out of and sequelae of neuropathy in relation to lymphoma without relapse for the last one. cidp was more disabling (onls≥ ) in hmpn+ than in hmpn-group % vs. %. pet-mri could be helpful to detect a proximal inflammation of the pns, and to plan further testing. further studies are needed to evaluate the prognostic value of hmpn+. lancaster e , li j , liem r , scherer ss . perelman school of medicine, university of pennsylvania, philadelphia, pa, usa; columbia university school of medicine, new york, ny, usa. heterozygous nefl n s/+ mutant mice are the first animal model of a charcot-marie-tooth disease e (cmt e). people with this mutation have a severe, early onset axonal neuropathy. axons in the mutant mice have reduced number of neurofilaments and decreased diameters. they also show early onset of tremor and abnormal hindlimb clasping behavior. we measured the compound action potentials (caps) from tails every weeks from the same cohort of nefl n s/+ mutant mice and their wt littermates from to weeks. even at the age of weeks, the amplitude of mutant caps was only ∼ % of wt caps ( ± . microv, n= vs ± . microv, n= ), and the caps stayed substantially smaller than those in wt mice for weeks ( ± . microv, n= vs ± . microv, n= ). the conduction velocity and the duration of caps in mutants were slower and wider compared to those of wt. separate cohorts of mice (n= mutants and n= wt) were sacrificed at different time points for analysis by light microscopy. in caudal nerve of mutant mice, the number of axons was significantly reduced compared to that of wt at weeks and, by weeks, it was reduced more than %. this model system may be useful for preclinical studies of treatments for cmt e since the animals show progressive neuropathy over weeks, which can be objectively measured electrophysiologically and anatomically. despite more than cmt genes identified today, at least % of cmt patients do not carry a mutation in any of these genes. progress in gene identification in recent years suggests that there are still many more cmt disease genes to be discovered; however, it has become rare to gather support from multiple large families that allow for conclusive linkage analysis. we have studied multiple extended dominant cmt families with linkage support for a gene at chromosome p . . originally a czech family yielded a two point lod score of . at this locus and a family from southern italy showed a lod score of . . whole exome sequencing of multiple family members identified missense mutations in the gene atpase na+/k+ transporting subunit alpha (atp a ). atp a has not been associated with human diseases thus far. in expression studies on teased fiber preparations we already confirmed predominant expression in the nodes of ranvier of peripheral nerves. through collaborative efforts in the inherited neuropathy consortium and beyond we identified five additional multigenerational families via exome or sanger sequencing resulting in a total of seven unique segregating missense changes: leu arg, ile thr, ala thr, asp phe, pro ala, pro thr and asp ala. five of these mutations fall into a remarkably narrow motif associated with the sodium binding structure of atp a , flanking the flexible hinge motif. functional studies in different model systems (mammalian cell lines, xenopus oocytes, patient fibroblast lines) are underway to determine whether a loss or a dominant gain-of-function represents the disease mechanism. taken together, we show strong support for a major new dominant cmt gene, atp a . this finding represents a new pathway and an attractive new target for therapy development in axonal cmt. laurá m , ramdharry g , , singh d , kozyra d , skorupinska m , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; school of rehabilitation sciences, st george's university of london/kingston university, uk; royal national orthopaedic hospital, stanmore, uk. charcot-marie-tooth (cmt) disease is the most common inherited peripheral neuropathy. foot deformities are frequent complications and orthopaedic surgery is often required. however there are no evidence based guidelines on the type or timing of the surgery. only few studies have described the long-term results of surgical procedures and evidence regarding optimal surgical management of these patients is lacking. we prospectively studied surgical management of cmt patients attending our centre. we collected data and assessed cmt patients before and after surgery. data included: history of ankle instability, pain, skin condition, details of physiotherapy and orthotic management, assessment of lower limb strength, charcot-marie-tooth examination score (cmtes), foot posture index, ankle dorsiflexion range of movement and specific questionnaires (foot index and manchester-oxford foot questionnaire, modified fatigue severity scale and modified falls efficacy scale), details of surgical procedures. patients were assessed yearly after surgery. so far patients ( males and females, age range - ) have been evaluated prior to surgery. all patients but one had genetically confirmed cmt ( cmt a, cmtx, cmt a). patients have been assessed after year, patients after years, patients after years and patient after years from surgery. a wide range of surgical procedures were performed by one dedicated orthopaedic surgeon. preliminary results showed reduction of number of falls in / ( %) patients and improvement of callosities in / ( %) patients at year follow up. there was also significant improvement of alignment of the operated foot (p= . ) and pain (p= . ). there were no significant changes in measures of strength and ankle range of motion. further analysis on a larger number of patients will be important to determine the long-term outcome of surgery. data acquired from this study will help develop orthopaedic intervention guidelines and identify areas for further research. lavigne-moreira c , oliveira mf , marques vd , onofre ptbn , dos santos acj , nascimento ojm , barreira a , marques w jr . division of neuromuscular diseases and neurogenetics, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; department of neurology, fluminense federal university, rio de janeiro, brazil. fap associated to ttr mutations is defined as a length-dependent axonal sensory and motor polyneuropathy that at early stages affects mainly the small nerve fibers, associated to autonomic and thermo-algesic sensations. the expected emg pattern was that of an axonal sensory and motor polyneuropathy, but in fact several unexpected patterns may be found. in this study we present the results found in a brazilian population with ttr mutation. patients were divided in three groups: ttr-met of early onset, ttr-met of late onset and ttr non-met . in the first group (ttr-met of early onset), ( . %) examinations were normal, ( . %) were axonal, were demyelinating fulfilling pidc criteria, one suggested a predominately motor polyneuropathy and the final one presented a lower motor neuron disease pattern. in the second group (ttr-met of late onset), ( . %) had an axonal pattern, ( . %) had an intermediate cv, ( . %) had a demyelinating pattern, had a lumbossacral pattern and the final one had no definite pattern. among the non-ttr met , two patients had the ttr -asp tyr, one presented an axonal pattern, while the second presented initially an axonal pattern, that changed to a demyelinating pattern in the second examination. patients with ile val mutation presented an axonal neuropathy associated to cts. most patients with demyelinating or intermediate pattern were treated with corticosteroids or ivig, with no satisfactory results. this small series of patients shows clearly that fap-ttr is associated to several emg patterns in addition to the expected sensory and motor axonal polyneuropathy. this variability is present in the same family and in the same patient in different occasions. clinicians should be alert to these possibilities to do not delay diagnosis and treatment. identifying the mechanisms involved in this variability could improve our knowledge of this intriguing disease. lavin tm . greater manchester neurosciences centre, salford royal hospital, manchester, uk. subcutaneous immunoglobulin (scig) has evidence from small trials for its use following intravenous igg (ivig) loading in chronic inflammatory demyelinating polyneuropathy (cidp). potential advantages of scig include stable igg levels and reduced complication rates. scig de novo can be considered as igg levels gradually rise with subcutaneous delivery and maybe beneficial for patients for whom complications are a concern. we present cases of cidp who were initiated on subcutaneous immunoglobulin g ( g/kg/month, % scig, hizentra, csl behring) following corticosteroids; without intravenous igg loading. patient is year old with a history of ihd with a progressive lower limb sensory changes and sensory ataxia over years. neurophysiology confirmed sensory demyelinating changes. he did not respond to corticosteroids. concerns were raised about the potential risk orf adverse cardiac events with iv immunoglobulin. scig therapy was introduced at g weekly. initial therapy has been well tolerated and objective measures (rods-cidp score, jamar grip strength, hole peg) have remained stable through initiation, and at months. patient is yr old male with a past history of branch retinal vein thrombosis. he developed a sensorimotor polyneuropathy over years with neurophysiology fitting efns criteria for cidp. given concerns regarding previous thrombosis, a trial of scig was given at g/week, with resolution of sensory ataxia and improvement in objective markers (jamar grip strength, rods-cidp score, hole peg) at months. unfortunately an adverse event occurred of an urticarial skin reaction. patient is a yr old male who presented with motor predominant cidp. there was no response to corticosteroids but improvement following plasma exchange. due to a past history of transient ischaemic attack, scig was started. there has been a positive response after months with improvement in walking distances and stable objective markers (rods-cidp score, grip and pinch strength). our cohort remained neurologically stable during initiation of scig, with adverse reaction. our experience would support further trials in this area regarding the efficacy of scig compared to ivig in both the short and long term. diabetic peripheral neuropathy is the most common and debilitating complication of diabetes and it is associated to neuropathic pain and non-traumatic amputations. despite the economic burden and human costs, nowadays there is not a specific treatment to cure diabetic peripheral neuropathy. hyperglycaemia and dyslipidemia-mediated oxidative and endoplasmic reticulum stress has been linked to diabetic peripheral neuropathy, among other altered pathways. in order to study in more detail the molecular mechanisms that lead to the development of diabetic peripheral neuropathy we set up in vitro models of the disease using nsc- (motoneurons) and med . (sensory neurons) cell lines or primary cultures of dorsal root ganglia and motoneurons exposed to saturated fatty acids. it has been reported that palmitic acid is able to induce endoplasmic reticulum stress and oxidative stress in multiple cell types including schwann cells and myenteric neurons, resulting in a good model of dyslipidemia-mediated stress. here we found that palmitate induces upregulation of the molecular chaperone bip/grp and the ccaat-enhancer-binding protein homologous protein (chop) mrnas and a dose-dependent downregulation of the apoptosis marker bcl- in primary cultures. moreover, palmitic acid induces loss of neuron dendrites and cell death at high doses and upregulation of heme oxygenase (ho- ) and chop at lower doses in the nsc- cell line. we are currently characterizing multiple molecules implicated in oxidative stress, endoplasmic reticulum stress and inflammation pathways in these cell types to find new therapeutic targets to treat type diabetic sensorimotor polyneuropathy, that will be subsequently validated in the db/db mouse model by pharmacological or gene therapy strategies. lee bjh , ohnmar o , wong j , koh sj , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful peripheral neuropathy and autonomic dysfunction that occurs within weeks of tight glycaemic control. therapy with either insulin or oral hypoglycaemic agents (ohga) may result in tind. the quantum and rate of decline in hba c predicts development and severity of tind. both type i and ii dm patients are prone to this iatrogenic complication. besides the expected morbidity associated with painful somatic and autonomic neuropathy, tind patients also develop life-threatening eye complications such as maculopathy. with greater awareness, we picked up at least typical cases of tind in recent few months. however, there was a fourth possible tind case, whom we feel deserves special attention. the circumstances surrounding this case are common and may go under-recognised in acute hospitals. a year-old man with -year history of poorly controlled type ii dm was admitted with a partial left middle cerebral artery stroke. one month before admission, his hba c was . %. his hospitalization was prolonged because of his considerable disability that required rehabilitation. he stayed weeks. his blood sugar was difficult to control with hyperglycaemic episodes requiring ohgas and insulin. the highest capillary blood glucose recorded was mmol/l. he also had episodes of hypoglycaemia. three weeks into hospitalization, he developed severe orthostatic hypotension. he came to our attention two months later when he was sent for autonomic screening tests for severe postural hypotension, in spite of therapy with fludrocortisone and midodrine. his hba c was . %. he had marked orthostatic hypotension suggestive of sympathetic dysfunction. we were not able to discern if he had a painful neuropathy because of his aphasia. there was no recent ophthalmology review. we suggested a diagnosis of possible tind. he was discharged with lower doses of ohga. however, day after discharge, he attended the emergency department for a syncopal episode. this case raises intriguing questions on the safety of glucose control paradigms commonly employed in patients with acute stroke and myocardial infarction as well as the possible role of major fluctuations in blood glucose in the development of tind. lee hs , kim sm . presbyterian medical center, jeonju, korea; yonsei university college of medicine, seoul, korea. the occurrence of peripheral neuropathy by a tumor within or compressing a nerve in neurofibromatosis (nf) type and is relatively well known. however, nf presenting with demyelinating polyneuropathy unrelated to tumor masses is rarely reported. we report a rare presentation of an nf patient with demyelinating polyneuropathy of subacute onset. a -year-old woman was referred to our hospital with paresthesia on both limbs months before. she was healthy and had no family history. the symptoms worsened over the next months, tremor and weakness in both limbs occurred. although she treated as a chronic inflammatory demyelinating polyneuropathy (cidp) with intravenous steroid and immunosuppressant in another hospital, symptoms were getting worse. bifacial numbness and left eyeball pain occurred weeks ago and she visited our hospital. on neurologic examination, bilateral facial sensory hypoesthesia and symmetrical both limbs weakness (mrc grade iv+) were observed. decreased touch and pinprick sensation on both limbs were observed like stocking-glove distribution. the reflexes were sluggish. on nerve conduction study, sensorimotor demyelinating polyneuropathy with conduction blocks and temporal dispersions was observed. bilateral r , r responses were prolongation on facial blink test. mri of cervical spine and brain revealed contrast enhancing tumor-like enlargement of multiple nerve roots and cranial nerves. nerve biopsy was performed on right supraorbital nerve, neurofibromatous changes were observed. the symptom deterioration was stopped without treatment. gene test dose not revealed nf type and . on review of the literature, polyneuropathy in nf patient can result from tumor masses within the proximal nerve roots, or along the peripheral nerve, or in the extramedullary lesion affecting neighboring nerve roots. thus, axonal type polyneuropathy and focal amyotrophy have been reported in nf. demyelinating polyneuropathy has not been reported before in our knowledge. although the etiology of demyelinating polyneuropathy in nf requires further clarification, some authors claim that unknown local toxic or metabolic influences of the endoneurial pathological cells on adjacent nerve fibers. in this patient, atypical symptoms were observed in cidp such as cranial nerve involvement and stocking-glove sensory distribution. in this case, a nerve biopsy can be helpful the accurate diagnosis. lee jy , yoo jh , kang dk , bae js . department of neurology hallym university, seoul, korea. acute disseminated encephalomyelitis (adem) is an uncommon post-infectious inflammatory demyelinating disorder of central nerve system, while guillain-barre syndrome (gbs) is a prototype of acute post-infectious peripheral neuropathy. previous reports regarding the coexistence of these relatively rare diseases suggest that certain immunogenicity within central and peripheral nerves may share a common autoimmune process during the disease course. a previous healthy years old man was admitted because of fever, headache, nausea and myalgia in department of infectious disease. two weeks before admission, he suffered from watery diarrhea for days and spontaneously recovered. at initial presentation, he had high fever( ∘ c), headache and myalgia and intermittent horizontal diplopia. a few days after admission, he began to complain of drowsy mentality, bilateral extremities weakness, especially lower limbs, dysarthria, bilateral facial paralysis, urinary retention, dyspnea. on neurologic examination, he had mildly drowsy mentality, symmetric muscle weakness scoring of on bilateral hip, knee flexion and finger extension, but he had no sensory symptoms. he showed gazed evoked nystagmus with no extraocular muscle palsies. deep tendon reflexes were not present. pulmonary function test revealed a severe restrictive pattern. csf studies disclosed a dissociative increase of protein contents ( mg/dl) without pleocytosis. anti ganlioside antibody assay identified an anti-gt a igg positivity in his serum. nerve conduction study (ncs) showed prolonged motor terminal latencies and slow motor conduction velocity on multiple nerves. in contrast, sensory ncs revealed no abnormal findings. imaging studies unexpectedly revealed apparently symmetrical lesions across bilateral brainstem and basal ganglia suggesting a diagnosis of adem. after both ivig and high dose steroid treatment, he remarkably recovered from disturbed mental state and motor weakness. about month after the symptom onset, he could walk with assistant aid and discharge to other hospital for rehabilitation. our case suggest that certain component of autoimmunity simultaneously result in both cns and pns inflammation. specific immunological mechanism is remained to be elucidated. although we could not conclude whether cellular component or humoral component is dominant for our case, the presence of anti gt a antibody suggest a role of humoral mechanisms. the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. leitao amf , , araújo df , marques h , pamplona l , , souza mh , , nbraga ll , , gondim faa , . the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. the classic guillain barré syndrome (gbs) is characterized by motor weakness, hyporreflexia, but limited sensory deficits. sensory variants involving either small or large fibers or both are unusual and represent a diagnostic challenge. we described patients presenting with the sensory variant of gbs and retrospectively analyzed the clinical and electrophysiological findings of patients fulfilling the criteria for sensory gbs according to oh et al. criteria. six patients were identified (mean age years: range - years). four had a previous infection. they all consulted due to distal painful paresthesias and allodynia. on examination the patients presented normal strength and normal cranial nerves through the course of the disease with reduced knee and ankle reflexes in patients. distal hyperesthesia to pinprick was identified in and one of them additionally had hyperhidrosis and constipation. two additional patients presented hypoesthesia to pinprick and temperature. one patient had distal proprioceptive sensory loss with sensory ataxia. csf albumin cytological dissociation was present in patients. nerve conduction studies (ncs) identified a sensory motor demyelinating neuropathy in patients. among the with normal ncs, had abnormal cold and warm threshold in their qst evaluation. all patients received symptomatic treatment for the neuropathic pain and only two ivig therapies. longstanding pain, fatigue or both were persistent findings in patients after a mean follow up of months. in conclusion the sensory variant of gbs is both an infrequent presentation and a diagnostic challenge. longstanding pain and fatigue are common persisting findings. the epidemic of zika virus (zikv) throughout the americas and asia, and the subsequent rise in reported cases of guillain-barré syndrome (gbs) caused worldwide concern. as of january , countries have reported evidence of mosquito-borne zikv transmission and in of these countries, a sudden increase of gbs has been reported. moreover, case studies and a case-control study further indicate that zikv may trigger gbs. however, accurate diagnosis of both zikv and gbs in many of these studies is disputed, and a comprehensive description of the clinical phenotype of gbs related to zikv is lacking. the international gbs outcome study (igos) is a prospective observational study on the factors determining the onset, clinical course and outcome of gbs. at present, a research consortium of centers from countries has included patients in igos. our aim is to investigate zikv-related gbs in igos as is already occurring in colombia. in igos-zika, data on clinical features and ancillary investigations will be collected in zikv endemic areas according to the igos protocol with some modifications. first, igos-zika has a case-controlled study design to define the association between gbs and zikv and other arboviruses. second, urine samples will be collected and additional questions on preceding events will be asked, focusing on arbovirus infections. third, a more limited follow-up is required. our aim is to recruit additional centers via the inflammatory neuropathy consortium (inc) and centers in all arbovirus endemic regions that are willing to participate. the focus of igos-zika will be on the accuracy of the diagnosis of both gbs and zikv and on defining the associated clinical phenotype, course and outcome. igos-zika provides the opportunity to combine data and biobanks from various geographical regions using a standardized protocol and to compare these data with data and biosamples already collected in igos. studying these cases will help to optimize diagnostics and care for gbs patients in arbovirus endemic countries and provides a unique opportunity to further understand the pathogenesis of gbs. moreover, this study design and network can be used to adequately respond to other future viral epidemics related to gbs. lerat j , cintas p , dzugan h , , magdelaine c , , sturtz f , , lia as , . service de biochimie et génétique moléculaire -chu de limoges, limoges, france; service de neurologie et d'explorations fonctionnelles -chu de toulouse, toulouse, france; ea -université de limoges, limoges, france. pharc syndrome is an autosomal recessive neurodegenerative pathology leading to demyelinating polyneuropathy, hearing loss, cerebellar ataxia, retinis pigmentosa and early-onset cataract. these various symptoms can occur at different ages, so that pharc syndrome can be a differential diagnosis of charcot-marie-tooth disease (cmt) associated with deafness. only abhd mutations have been reported in patients. we described the th mutation and compared our results to the literature data. we analysed by next generation sequencing (ngs) strategy using a targeted cmt and associated neuropathies -gene panel the dna of a -year old male who has suffered from demyelinating sensory and motor polyneuropathy and ataxia since the age of . bilateral sensorineural deafness was diagnosed at the age of five. bilateral congenital cataracts were operated on at the age of . a new large complex homozygous mutation, with one deletion of seven base pairs and one insertion of base pairs, was detected. by analyzing our patient data and those of the literature, we evaluated that, in pharc syndrome, sensorineural deafness always occurs as the first feature in late teens. the ophthalmological symptoms are cataracts that occur at a mean age of yo and then retinis pigmentosa at a mean age of . demyelinating sensory-motor polyneuropathy is the most variable characteristics, which occurs in the thirties. we report the first large complex homozygous mutation in pharc syndrome, which is certainly under-diagnosed. therefore, it seems interesting to include abhd in the panels of the five symptoms, especially deafness ones. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a chronic disabling disease that often improves with immune therapy. to date, the most reliable diagnostic criteria for cidp are the efns/pns revised criteria, with a reported sensitivity of % and specificity of %. we implemented a web-based database to collect data from patients with cidp followed by italian centers with expertise on cidp to determine the frequency and characteristic of cidp and it variants, the diagnostic criteria used for their diagnosis, the possible evolution into typical cidp, the association with specific anti-nerve antibodies, and their response to therapy. all the patients were evaluated at the time of inclusion and will be followed for two years to monitor their outcome and response to therapy. by february we included patients with cidp and variants ( men, women), aged - years (median ) with a mean disease duration of . years (range . - years). based on clinical symptoms, cidp was defined as typical in % and atypical in %. the diagnosis of typical cidp fulfilled efns/pns criteria in % of the patients while nerve conduction studies were not diagnostic in % (grouped as clinical cidp) or not available in %. we analyzed the frequency of supportive criteria for the diagnosis of cidp in patients with clinical cidp and found that increased csf proteins, demyelination or cell infiltration on nerve biopsy and imaging abnormalities consisting with cidp on us or nmr were present in %, % and % of the patients, respectively. a relapsing course was present in % of patients with clinical cidp, increasing the reliability of the diagnosis for cidp. in addition an improvement after one or more therapies was reported by % of the patients, with a positive response to ivig in %, steroids in % and plasma exchange in %, similarly to what observed in patients fulfilling efns/pns criteria. in % of the patients with clinical cidp two or more supplementary criteria for cidp were present. this study on a large population of patients is providing useful information that may help to revise the current diagnostic criteria for cidp. li j , cannell m , suragani r , pearsall r , kumar r . acceleron pharma inc, cambridge, usa. charcot-marie-tooth (cmt) is the most common hereditary peripheral neuropathy and is characterized by demyelination and/or axonal damage of peripheral nerves and muscle weakness. foot drop, steppage gait, and foot deformities are a typically seen in cmt patients. consequently, falls are commonly reported in these patients. improvement of dorsiflexor muscle function to prevent falls may improve quality of life and activities of daily living in patients with cmt. ace- , a locally-acting ligand trap that binds growth and differentiation factors (gdfs) and activins, has previously been shown to increase muscle mass and force in both duchene muscle dystrophy (dmd) and amyotrophic lateral sclerosis (als) mouse models. in the current study, we evaluated the therapeutic effects of ace- to improve muscle strength in the trembler (tr-j) mouse model of cmt a. these mice harbor a mutation in the peripheral myelin protein (pmp ) known to cause cmt a. seven-month old (b .d -pmp tr-j /j) mice were administered ace- ( g, twice weekly) intramuscularly to one of the unilaterally tibialis anterior (ta) muscle for weeks. the contractility of the ta muscle was evaluated during isometric contraction. all data were compared to the uninjected contralateral control hind-limb. after weeks of ace- treatment, ta muscle mass was increased by % (p< . ) and its physiological cross-sectional area was increased by % (p< . ). the increase in muscle mass correlated with an increase in strength, with maximum tetanic force and twitch force improved by % (p< . ) and % (p< . ), respectively. in addition, temporal properties during isometric contraction, such as maximum rate of contraction and relaxation, were accelerated by % and % respectively (p< . ) in the ace- -treated ta muscle compared to its contralateral hind-limb. pathological and biochemical assessment of ace- -treated mice showed enlarged myocyte area (+ %, p< . ) and reduced atrogin- mrna expression (− %, p< . ). together, these results demonstrated that ace- attenuates the degree of muscle atrophy and also improves muscle function in a mouse model of cmt a. the current study provides proof of concept for the use of ace- as a therapy for cmt to improve dorsiflexor muscle function and alleviate foot drop. we have generated a rat model of charcot-marie-tooth disease a (cmt a) harboring the p.arg trp mfn mutation, whose human counterpart results in a severe, early-onset axonal neuropathy. the mutation was made using zinc finger nuclease-mediated genome editing in fertilized rat eggs. a large cohort of mutant and wt littermates were characterized behaviorally and found to develop multiple motor deficits that worsened over time. nerve conductions of the tail (caudal nerve) was performed on a separate cohort of mutant (n= ) and wt littermates (n= ) every weeks from to weeks. mutant rats showed a progressively decline in the amplitude of the compound action potential after weeks, whereas the amplitude progressively increased in their wt littermates. separate cohorts of rats were sacrificed at , , and weeks and analyzed by light microscopy. in mutant rats, there was a reduced density of myelinated axons and active axonal degeneration in distal but not proximal nerves, and in the fasciculus gracilis of the cervical spinal cord at and weeks. these findings were not present in the -week-old cohort of mutant rats, or in wt rats at or weeks. a genetically authentic animal model of cmt a that develops a progressive, length-dependent axonal neuropathy will be a valuable tool for examining the pathogenesis and treatment of cmt a. lindborg ja , niemi, jp , defrancesco a , zigmond re . case western reserve university, cleveland, usa. traditionally the role of immune cells in nerve degeneration and regeneration has focused on the infiltration of inflammatory monocytes into the distal nerve after nerve injury and the phagocytosis by the resulting macrophages of myelin and axonal debris, thereby clearing a path for regenerating axons. therefore, it was surprising when we discovered that in ccr knockout (ko) animals, in which the entry of these inflammatory monocytes does not occur, that wallerian degeneration precedes normally. we now report that the reason for this is that neutrophils and schwann cells compensate for the decrease in macrophage accumulation. furthermore, nearly complete depletion of circulating neutrophils by systemic injection of an antibody to ly g leads to an inhibition of myelin clearance both in ccr ko and in wild type animals. on the other hand, we have demonstrated a second site of macrophage accumulation in wild type animals, namely around axotomized sensory neurons in dorsal root ganglia (drgs). blockade of that accumulation, for example as occurs in ccr ko animals, leads to a dramatic impairment of nerve regeneration. to examine the relationship between macrophages and regeneration further the monocyte chemokine ccl was overexpressed in drg neurons in intact animals by viral infection using an aav containing the ccl coding sequence. the resulting overexpression of ccl led to the accumulation of macrophages in drgs even though no injury had taken place and subsequently to an increase in the intrinsic growth capacity of the sensory neurons. examination of changes in gene expression in the drgs in these animals revealed increased expression of the cytokine leukemia inhibitory factor and an increase in its downstream signaling pathway that involves the phosphorylation and nuclear translocation of stat . strikingly, pharmacological blockade of stat activation inhibited the increase in the neurons' growth capacity produced by the virus. these results reveal unexpected interactions between immune cells and neurons facilitating nerve degeneration and regeneration and could lead to therapies to improve regeneration after injury or in disease. lin j , , qiao k , , huang j , , zhao cb , , lu jh , . institute of neurology, fudan university, shanghai, china; department of neurology, huashan hospital, fudan university, shanghai, china. we used terminal latency index (tli) as a tool in differentiation between poems syndrome and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). comparison of median and ulnar nerve conduction studies including motor conduction velocity (mcv), distal motor latency (dml) and terminal latency index (tli) were studied in poems patients, matched cidp patients and normal controls. in this cohort, the average age at evaluation was . ± . years old in poems group and that of cidp patients was . ± . years old. except the ulnar terminal latency index in cidp group, poems and cidp patients demonstrated prolonged distal latencies, low conduction velocities and increased terminal latency indexes compared with the normal group. reduced conduction velocities and higher terminal latency indexes in poems group than in cidp group was found. increased tli was demonstrated in . %(median nerve) and . %(ulnar nerve) poems and that in cidp patients was . %(median nerve) and . %(ulnar nerve). decreased tli was found in . %(median) and . %(ulnar) cidp patients and none in poems. temporal dispersion (td) and conduction block (cb) were more often seen in cidp patients with increased tli than that in poems. compared with cidp and poems showed greater slowing of the intermediate nerve segments and relatively more uniform demyelination. about % cidp demonstrated more distal conduction slowing and more td and cb especially in those with increased tli. terminal latency index combined with td and cb may be helpful in differentiating poems from cidp. lin y , sung j , chang t , jowy t . department of neurology, taipei municipal wanfang hospital, taipei, taiwan. the purpose of our study is to exam whether electrophysiology changes could be detected in prediabetes patients and to discover the possible mechanism of nerve injury in prediabetes stage. we analysis and compare the nerve excitability test data between prediabetic patients and age-matched normal control subjects. prediabetes is defined by american diabetes association (ada) as one of the three following: hba c . % to . %, fasting glucose mg/dl to mg/dl, and hour oral glucose tolerance test to mg/dl. patients with radiculopathy, myelopathy, entrapment neuropathy such as carpel tunnel syndrome, and polyneuropathy were excluded. the strength-duration time constant (sdtc) and superexcitability showed significant difference (p< . ) between two groups. we also find increased threshold electrotonus in depolarization (ted) and reduced relative refractory period (rrp) and refractoriness in . msec. these early changes in prediabetic patient are similar in nerve excitability feature of diabetic patients. however, the above changes are not found in motor axonal excitability test. our data supports that nerve excitability test may be a useful, non-invasive, and less time dependent tool to detect peripheral nerve injury in prediabetic stage. the sensory axons are more vulnerable than motor axons. superexcitability is the most sensitive parameter in prediabetes. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominant disorder caused by mutations of ttr gene and is associated with variable penetrance. ttr-fap is rare, except for endemic areas. this is a retrospective study of ttr-fap patients diagnosed at our center between - . we identified four families with different ttr mutations. in one family with v a mutation, nine family members over four generations diagnosed with ttr-fap was followed since . in the other families the index cases with different mutations were identified between - . affected family members with v a mutation developed severe progressive polyneuropathy with cachexia, with onset of the disease between ages and . three patients presented with marked visual symptoms (one patient underwent vitrectomy). nine patients died to years after disease onset. two patients (sisters) underwent liver transplantation -one died after years of disease at age , second is years old and wheelchair-bound as her symptoms continue to progress. the ttr mutations diagnosed in the index cases of three other families are: d v, f l and v m. they all presented with similar clinical picture of late-onset ttr-fap with predominant progressive axonal sensory, motor and autonomic polyneuropathy. all three index cases were men, the onset of symptoms was between - years with numbness and paresthesia in the feet followed by weakness and autonomic dysfunction. all had excessive weight loss resulting in cachexia and were diagnosed with cardiomyopathy. no patient suffered from visual symptoms. all three patients progressed to stage ii of ttr-fap -walking with assistance. time to diagnosis was . - years. due to advanced stages of their disease these patients were not suitable for therapy with tafamidis or the liver transplantation. the ttr d v (p.d v) was confirmed as a de novo mutation, which is uncommon in ttr-fap. in the remaining families carriers of ttr mutations were identified and are followed up. pedigree analysis of the family with f l mutation revealed affected members who with high probability died from ttr-fap. our study suggests that patients with ttr-fap in poland exhibits clinical and genetic heterogeneity. liu x , fan d . department of neurology, peking university third hospital, beijing, china. objective: to identify the gene mutation of chinese charcot-marie-tooth pedigrees and investigate the correlation among the clinical manifestation, electrophysiology and mechanism of different genotype. methods: we included pedigrees with cmt enrolled in our hospital from january, to december . we recorded clinical features, cmtns and electrophysiological data at diagnosis. the patients underwent mutation analysis of pmp , cx , mpz, mfn , hspb , hspb using mlpa, dhplc and sanger gene sequencing. results: we found pmp duplication pedigrees ( . %), cx pedigrees ( . %), mfn pedigrees ( . %), mpz pedigrees ( . %) conclusions: in chinese han population, the proportion of pmp duplication is relatively low, the majority of clinical manifestation is classical cmt. axonal cmt can show isolated lower extremity injury, with central nervous system involvement. hmn may be an underestimated clinical types, the identification should be done with caution in differential diagnosis. liu y , liu b , sebastian b , wozniak km , wu y , slusher b , polydefkis m . johns hopkins school of medicine, baltimore, usa. chemotherapy-induced peripheral neuropathy (cipn) is a common dose-limiting toxicity in the treatment of many cancers. most cipn studies preferentially focus on sensory fiber loss and dysfunction. here, we compared the structural and functional recovery of autonomic fibers in sweat glands (sweat gland nerve fiber density, sgnfd) and sensory fibers (intra-epidermal fiber density, ienfd) in mouse footpads after exposure to a maximum tolerated dose (mtd) of several common chemotherapy agents. additionally, we assessed footpad sweat production as a functional correlate to sgnfd reductions. female balb-c mice ( -animals/group) were treated with a mtd of four anti-tubulin drugs: paclitaxel (pca, mg/kg), ixabepilone (ixa, mg/kg), eribuline (erib, . mg/kg), vinoelbine (vino, mg/kg), or corresponding placebo given intravenously, mwf for two weeks. recovery was assessed at -hours, , , , , and weeks following the last dose. footpads were processed to visualize epidermal nerve fibers using pgp . and autonomic nerve fibers with tyrosine hydroxylate and pgp . . ixabepilone-treated mice experienced significant reductions in sgnfd at hrs, while ienfd nadir occurred at a later time point, -weeks. the recovery to baseline levels occurred more quickly for ienfd ( -weeks) than sgnfd ( -weeks). in contrast, vinorelbine and eribuline treated mice experienced a maximum deficit in sgnfd and ienfd at hrs and sgnfd recovery was slower ( -weeks) compared to ienfd ( -weeks). pca-treated animals showed more severe ienfd and sgnfd deficits compared to the other agents with both ienfd and sgnfd not recovering completely until -months. reductions in th-sgnfd were comparable or more pronounced to decreases in pgp . -sgnfd for all agents and timepoints. pca-treated animals demonstrated reductions in footpad sweat droplet number thereby providing a functional correlate. together, these data indicate that in mouse models of cipn, autonomic nerve fibers are affected more severely than sensory nerve fibers, and also recover more slowly than intraepidermal nerve fibers. autonomic dysfunction may be an important and under-appreciated consequence of chemotherapy exposure. the pi -kinase vps (pik c ) synthesizes phosphatidylinositol -phosphate (pi p), a lipid critical for both endosomal membrane traffic and macroautophagy. human genetics have implicated pi p dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating charcot-marie-tooth (cmt) peripheral neuropathy. here, we investigated the role of vps , and pi p, in mouse schwann cells by selectively deleting vps in this cell type. vps -schwann cell knockout (vps scko ) mice show severe hypomyelination in peripheral nerves. vps −/− schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. upon reaching the promyelinating stage, vps −/− schwann cells are significantly impaired in the elaboration of myelin. nerves from vps scko mice contain elevated levels of the lc and p proteins, indicating impaired autophagy. however, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in vps scko mice is caused by impaired autophagy. endosomal membrane traffic is also disturbed in vps −/− schwann cells. we investigated the activation of the erbb / receptor tyrosine kinases in vps scko nerves, as these proteins, which play essential roles in schwann cell myelination, are known to traffic though endosomes. in vps scko nerves, erbb was hyperphosphorylated on a tyrosine known to be phosphorylated in response to nrg exposure. the overall level of erbb was also decreased during myelination. our findings suggest that the loss of vps alters the trafficking of erbb / through endosomes. abnormal erbb / signaling may contribute to the hypomyelination observed in vps scko mice. lombardi r , devaux j , cortese a , dacci p , benedetti l , demichelis c , lauria g . irccs foundation "carlo besta" neurological institute, milan, italy; aix-marseille université, marseille, france; irccs c. mondino national neurological institute, pavia, italy; university of genoa and irccs aou san martino-ist, genoa, italy. the recent identification of igg anti-neurofascin (nfascin) antibodies in a group of patients has widened the spectrum of presentation for chronic inflammatory demyelinating polyradiculoneuropathy (cidp). these patients can be distinguished by disabling tremor, poor response to intravenous immunoglobulin and distal and sensory disturbances. cell-adhesion molecule nfasc and cntn are expressed on the paranodal junction (pnj) of nodes of ranvier, and play key roles on sodium channel clustering and glia-axon interactions. quantification of unmyelinated intraepidermal nerve fibers (ienf) is a useful parameter employed in small nerve fiber pathology diagnosis. in addition the immunohistochemistry evaluation of dermal nerve fibers allows to examine morphological changes of myelin sheath and ranvier nodes structure. we performed immunofluorescent colocalization studies using antibodies to visualize axons (protein-gene-product . , neurofilament, tubulin), sheath of myelin (myelin-basic-protein) and specifically nodal/paranodal/juxtaparanodal structures (pannfascin, nfascin , nfascin , caspr, cntn , potassium and sodium channels) in skin tissues from seronegative and seropositive cidp patients. we analyzed axon and myelin sheath damage, abnormal nodal-paranodal-juxtaparanodal architecture and morphometric parameter as internodal length of ranvier nodes. our results on skin biopsies from three igg nfasc -positive cidp patients revealed complete loss of nfasc staining at the paranodes, asymmetrical paranodes and widening of the nodes of dermal myelinated nerve fibers. one igg cntn -positive cidp patient showed abnormal nodal/paranodal immunostaining with different features as compared with igg nfasc -positive patients suggesting specific changes. however, such alterations were not found in four seronegative cidp patients. our data support the hypothesis that examining specific axonal and myelin markers could provide diagnostic and prognostic clues on nodo-paranodopathies. the goal of this study is attempt a possible correlation between the presence of serum autoantibodies and structural changes in nodal/paranodal regions of dermal nerve fibers in cidp patients. knowledge of autoantibodies expression in the peripheral myelinated nerves of cidp patients could serve for stratifying patients and potentially guiding personalized treatments. lunati a , lerat j , dzugan h , , rego m , magdelaine c , , bieth e , calvas p , cintas p , gilbert-dussardier b , goizet c , journel h , magy l , toutain a , urtizberea j , sturtz f , , lia as , . charcot-marie-tooth disease is one of the most frequent inherited peripheral neuropathies ( / ). so far, mutations in more than genes have been identified causing either the demyelinating form (type ) or the axonal form (type ). duplication of pmp gene is the most frequent cause of autosomal dominant demyelinating form. autosomal recessive demyelinating form is often due to sh tc gene mutations. patients suffer then from early severe neuropathy starting in the first decade. scoliosis and deafness are often observed. we analysed patients suffering from peripheral neuropathy, by multiplex-ligation-dependant-probe-amplification (mlpa), followed by targeted next-generation-sequencing (ngs) using a -gene custom panel designed for the diagnosis of charcot-marie-tooth and associated neuropathies. mutations of interest were verified by sanger sequencing. diagnosis was positive for patients. as expected, the most frequent mutation was the pmp duplication detected in patients. deletion of pmp was observed in patients and pathogenic point mutations were detected in patients. sh tc gene appeared to be the most frequently mutated with nine patients diagnosed. associated with known mutations, four new mutations have been identified: two nonsense mutations and two missense mutations. all these patients presented deafness and/or scoliosis. sh tc appears to be an important gene involved in charcot-marie-tooth disease, often associated with deafness and /or scoliosis. it is important to pay attention to these associated symptoms in charcot-marie-tooth patients in order to guide their diagnosis and to improve their medical care. lupo v , , frasquet m , , sánchez-monteagudo a , , barreiro m , alberti ma , casasnovas c , quintáns b , , , camacho a , domínguez c , sedano mj , pelayo al , pardo j , sobrino t , sobrido mj , , , sevilla t , , espinós c , . mme (membrane metalloendopeptidase) mutations, inherited in an autosomal recessive fashion, have been recently identified in japanese probands (higuchi et al. ) . thus, mme has been included in the list of cmt genes as a new autosomal recessive axonal form, cmt t (mim ), and moreover, it is considered a strong candidate for the genetic diagnosis of unsolved late-onset cmt cases. in fact, few months later auer-grumbach et al. ( ) reported european probands with autosomal dominant late-onset cmt and mutations in mme. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy using an updated version of our custom gene panel, which includes mme. in this study, we report probands with cmt , intermediate cmt or dhmn/cmt and homozygous or compound heterozygous mutations in mme, and proband with cmt and a heterozygous mutation in mme. we have identified different type of mutations: novel splice donor variant, frameshift, nonsense and missense mutations. the two nonsense changes, p.trp * and p.arg *, and the splice donor variant c. + g>a, were detected in homozygous or compound heterozygous state in patients, while the frameshift mutation, p.pro leufs* , was detected in homozygous or heterozygous in the remaining patients. strikingly, the two nonsense and the frameshift mutations had been previously reported as causative for autosomal-dominant cmt t (auer-grumbach et al. ) . out of three missense mutations, one is novel (p.his tyr), and two are reported in control databases (p.asn lys and p.arg trp). this study shows that the autosomal recessive cmt t is common in spanish population, and moreover, it suggests that screening of mme using gene panel testing could help to improve diagnosis of unclarified inherited peripheral neuropathies cases. funds: isciii (pi / , pi / ); fundació per amor a l' art. inherited peripheral neuropathies (ipns) encompass a group of disorders highly heterogeneous, clinically and genetically. charcot-marie-tooth (cmt) disease is closely related to distal hereditary motor neuropathy (dhmn) or distal spinal muscular atrophy (dsma), and some patients show additional signs associated with amyotrophic lateral sclerosis (als). targeted gene panel and exome sequencing are considered to be powerful and cost-effective tools for diagnosis of these disorders. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy: families were investigated by exome sequencing and, cases were tested using different updated versions of a gene panel . each version comprises , or ipn genes, respectively and it shows a high coverage performance: percentage of analyzable target base with > coverage was , %. both exome and gene panel capture libraries were based on sureselect capture technologies (agilent technologies), and sequencing was performed in miseq or hiseq illumina equipment. we have identified novel genes and novel mutations in known genes, broadening the phenotypical spectrum associated with ipns. exome sequencing has allowed us to identify causative gene in % of familiar or sporadic cases: morc , aars, bscl , kif a, gars, egr , fig , dnajb , drp , ighmbp , dao, sod , fig . gene panel testing has been mostly performed in sporadic cases, and it has allowed us to identify either disease-causing or candidate mutations in % of cases: kif a and bicd were the most common genes mutated. update gene panels neuro and neuro have revealed novel mutations in genes recently associated to cmt and cmt disease: mme and pmp , respectively. in sum, both strategies have helped us to achieve a more accurate clinical and genetic reclassification of these disorders, an impossible challenge using conventional sequencing methods. our study expands the clinical phenotype previously associated to known ipn causing-gene, and emphasizes that gene panels should be considered as a first diagnosis method for unclarified ipn patients. funds: isciii (pi / , pi / , pi / ); fundació per amor a l' art. magy l , mathis s , goizet s , tazir m , vallat j-m . department and laboratory of neurology, national reference center for rare peripheral neuropathies, chu limoges, france; department of neurology, chu bordeaux, france; department of medical genetics, chu bordeaux, france; department of neurology, chu algiers, algeria. charcot-marie-tooth (cmt) disease is a hereditary neuropathy with a relatively homogeneous phenotype but is genetically heterogeneous. moreover, nerve conduction studies distinguish different forms, adding another level of complexity. the current classification of cmt being difficult to understand for physicians, scientists and patients, we presented and published a proposal for updating this classification, based on inheritance, nerve conduction findings and gene/mutation involved. inputs from colleagues prompted us to conduct a survey in order to try to reach some consensus about our proposals. we conducted an internet survey between october and december . the link to complete the survey was sent several times by email with an introduction to more than people. participants were contacted through the emailing list from the last cmt meeting in venice (september ) and additional physicians and scientists who are involved in cmt care and research were contacted as well. one hundred seven people from various countries (mainly france, italy and the usa) answered the survey. most ( %) of the participants were between and years of age, % being physicians and % being scientists. the vast majority ( %) considered the proposal constituted an improvement over the historical classification whereas % wanted to keep the old one. about the order of information, % of participants thought the mode of inheritance should come first, whereas % felt the phenotype should be placed at the beginning. ninety-one percent of people thought cmt should be kept as a generic name for hereditary sensory and motor neuropathy. for pure sensory neuropathy, % favoured hsn over hsan although % thought the opposite and % of participants felt dhmn should be kept for distal motor neuropathy. about nerve conduction findings, % of participants thought the intermediate phenotype has to be kept and % favoured our proposal to replace " " by "de" (for demyelinating) and " " by "ax" (for axonal). finally, % of responders thought that genetic information should be included in the classification of cmt. overall, our proposal of a new classification received a very good appreciation from physicians and scientists implicated in the care of patients with hereditary neuropathy. mallik r , hubsch a , gaida a , barnes d . csl behring, kop, usa; csl behring, bern, switzerland; csl behring, ottawa, canada. the risk of hemolytic events (hes) with intravenous immunoglobulin (ivig) therapy appears to be linked to the isoagglutinin (anti-a and anti-b) level of the specific ivig product. using published anti-a and anti-b titers for seven ivig products and corresponding he rates reported to the eudravigilance database, we developed a mathematical model to predict the risk of he to patients receiving ivig products of given anti-a and anti-b levels. modeling was performed separately for the risk to patients with blood groups a, b, ab and o and the overall population risk was estimated assuming a blood group distribution of % a, % b, % ab and % o. applying the prediction model, we calculated the he risk for an ivig product produced via a chromatographic process (privigen ® , csl behring) a) without any isoagglutinin reduction measures ( ) ( ) ( ) ( ) ( ) ( ) ( ) , b) with an anti-a donor screening program eliminating approximately % of donors with high anti-a titers ( ) ( ) ( ) , c) incorporating an anti-a/anti-b specific immunoaffinity chromatography (iac, igisolo tm ) step in the manufacturing process (since ) and d) with both measures (b and c) combined; as well as for an ivig product produced with a cohn-like cold ethanol fractionation process (carimune ® nf/sandoglobulin ® , csl behring). isoagglutinin titers in ivig products, measured by european pharmacopoeia direct assay, were provided by dr c bellac, swissmedic, bern, switzerland. the predicted risk was highest with the chromatographically purified ivig without isoagglutinin reduction ( . cases expected per kg ivig used). anti-a donor screening reduced the predicted risk to . cases/ kg. a greater risk reduction was predicted with the iac isoagglutinin reduction step ( . cases/ kg). the combination of both methods produced little benefit ( . cases/ kg) versus iac alone. the predicted hemolytic risk with ivig produced by cohn-like ethanol fractionation was low ( . cases/ kg). an observational cohort study to confirm these hemolytic risk reductions is in progress. at present, the observed hemolytic risk for anti-a donor screening appears consistent with the prediction calculated by the model; results for iac isoagglutinin reduction are expected in . the cmt infant scale (cmtinfs) is an outcome measure of functional ability for young infants and children aged < years. cmtinfs aligns with the cmt pediatric scale and cmt neuropathy score to measure disease severity across the lifespan. to measure gross motor and fine motor function, cmtinfs comprises of two subscales: gross motor (e.g. head control, crawling, walking, jumping and hopping) and fine motor function items (e.g. grasping, reaching, tearing paper and buttoning). overall and subscale-specific function is expressed as a z-score based on normative reference values (positive z-scores indicate poorer function). a total of controls aged - months (mean age , sd m) have been assessed across australia (n= ), thailand (n= ) and usa (n= ). total cmtinfs z-scores did not differ significantly between sites (australia vs thailand) (p= . ) or gender (p= . ). data collection is ongoing and infants aged < years are eligible for inclusion. to date, infants ( % male) aged - months (mean age , sd m) with a range of cmt subtypes ( cmt a, cmt d, cmt c, cmt x and unidentified gene) have been assessed with cmtinfs. mean total z-score for infants with cmt ( . , sd . , range: − . - . ) was significantly higher than controls ( . , sd . , range: − . - . , t=− . , p= . ). differences between affected infants and controls were larger in infants older than months. infants with cmt a (cmtinfs z-score . , sd . ) and cmt c (z-score . ) were less affected than cmtx (z-score . ) and cmt d (z-score . ). the gross motor function subscale differed significantly between cmt cases and controls ( . , sd . vs . , sd . ; p=. ) and a significant difference was also observed for the fine motor function subscale ( . , sd . vs . , sd . ; p= . ). reliability, factor and rasch analysis of the cmt-infs is underway to assess validity. initial results support the sensitivity of cmtinfs in distinguishing between infants with and without cmt. preliminary analyses also suggest the scale is sensitive to genetic subtype. with increased power, cmtinfs promises to become a useful outcome measure of disease severity and function in infants with cmt. manso c , querol l , mekaouche m , illa i , devaux j . aix-marseille université, marseille, france; universitat autónoma de barcelona, barcelona, spain. contactin- , contactin-associated-protein- (caspr ), and neurofascin- (nfasc ) are essential for the formation of paranodal axoglial junctions. igg autoantibodies to contactin- , caspr , and nfasc are associated with subsets of patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp) presenting with common clinical features. anti-contactin- igg autoantibodies have been shown to be pathogenic and to affect the paranodal axoglial junctions in vivo and in vitro. by contrast, the pathogenic effect of anti-nfasc igg have not been demonstrated. here, we purified anti-nfasc igg from cidp patients' plasma and investigated their effects after passive transfer. to determine whether these antibodies can pass the paranodal barrier, we performed intraneural injections of anti-nfasc igg autoantibody. by contrast to anti-contactin- igg , anti-nfasc did not penetrate the paranodal regions after intraneural injections, but bound to the surface of the schwann cell. to perform chronic exposure, lewis rats were implanted with intrathecal catheter and anti-nfasc igg were administrated in a daily manner during three weeks. igg to nfasc , but not control igg , induced progressive clinical deteriorations characterized by gait ataxia and hindlimb paraparesis. these deteriorations were associated with nerve activity loss in motor spinal nerves and with a selective loss of the paranodal specialization characterized by the disappearance of the caspr /contactin- /nfasc complex at paranodes. the passive transfer of anti-nfasc igg thus seem to induce similar pathogenic effects as the anti-contactin- igg . however, the pathogenic mechanisms leading to paranode disappearance appear different. our findings indicate that igg directed against nfasc are pathogenic and further show that these antibodies are reliable biomarkers of a specific subset of cidp patients. better fit the needs of the consortium and improve patient experience. these enhancements include creating mobile friendly webpages, updating enrollment form content, access to a customized dashboard, and the ability of registrants to explore their data in comparison to other registrants. we will review these enhancements in depth, and demonstrate their impact on the growth and development of the rdcrn inc contact registry. martinez c , hubsch a , watson dj , shebl a , wallenhorst c , simon tl . institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl bering ag, bern, switzerland; csl behring llc, king of prussia, usa; csl bering gmbh, marburg, germany. hemolytic anemia (ha) is a complication of intravenous immunoglobulin (ivig) treatment, particularly in patients receiving high dose ivig for immune modulation, such as guillain-barré syndrome or chronic inflammatory demyelinating polyneuropathy. the primary mechanism for the increased risk is believed to be passive acquisition of anti-blood group a and b antibodies (isoagglutinins) from the ivig product. to reduce the quantity of isoagglutinins, an anti-a donor screening was implemented for the ivig privigen ® from - and donors with high titers were excluded from contribution to pooled plasma. anti-a donor screening was replaced since with an immunoaffinity chromatography step, which decreases isoagglutinins to a greater extent, but no data are available to test its clinical effectiveness. to test the effectiveness of the donor screening, two cohorts of patients treated with privigen ® before and after start of donor screening were identified from a hospital-based administrative database of us hospitals with in-and outpatient discharge diagnoses, procedures, drug utilization and laboratory tests between / and / (period ) and between / and / (period ). privigen ® dose per kg body weight was estimated from the daily quantity administered and age-and sex-specific us population body weight estimates. ha within days of privigen ® use was assessed from manual records review and the incidence rate of ha in the two periods compared. incidence rate ratios (irr) of ha were adjusted for sex, age, treatment setting, indication and dose per kg body weight using period as reference. the incidence rate of ha was . / , person-days ( % confidence interval: . - . ) in period ( has in , person-days) and . ( . - . ) in period ( has in , person-days). the adjusted irr was . ( . - . ). significantly less ha risk was found with high dose (≥ . g/kg body weight) privigen ® , irr . ( . - . , p= . ). we conclude that anti-a donor screening and exclusion of donors with high anti-a titers from plasma pools is associated with a decreased risk of ha with ivig. matsumoto a . department of neurology, keijinkai jozanki hospital, sapporo, japan. subacute myelo-optico neuropathy (smon) is the intoxication of clinoquinol with main clinical symptoms of paresthesia and spasticity of legs. these symptoms have been considered to be elicited by the disturbance of spinal cord and peripheral nerve as the intoxication of clinoquinol. however, as to the patients with smon who have been still living after the onset of disease, the examination of nerve conduction velocities are in normal ranges and the clinical symptoms of peripheral neuropathy are not observed now. in order to investigate whether the peripheral neuropathy were observed in the early stage of smon, we investigated the longtidunal changes of electrophysiological results in patients who could examine the nerve conduction studies from early stage of smon until the present time. as to the disturbance of pyramidal tract functions (myelopathy) in smon patients, the central motor conduction times were calculated by transmagnetic stimulation of motor cortex, cervical roots and lumbar roots. the peripheral nerve conduction velocities of sensory nerve were examined with the sural nerves. as the results, in patients with smon who could examine the electrophysiological examination from the early stages of smon until to years later, the central motor conduction times of leg muscles from motor cortex to lumbar roots were prolonged in the smon patients compared to the normal cases. these results suggest the presence of disturbances of conduction velocities of spinal cord. conduction velocities of sensory nerve velicities (sncvs) showed the delayed sncvs of sural nerves( - m/sec) at the first examination from the onset of - years. however from to years later, sncvs of these cases were covered to - m/sec. from these electrophysilogical examinations, it was suggested that the presenting main symptoms of smon were the disturbance of myelopathy, and the disturbance of peripheral nerve function had been recovered after onset of smon being elapsed a long time, natural killer (nk) cells are part of our innate immune system with regulatory and effector functions. they comprise the first line of defence in the recognition and destruction of virus-infected and pathologically altered cells. different studies suggest that the treatment with intravenous immunoglobulins (ivig) has an immunomodulatory effect on nk cells. ivig is a first-line treatment for various autoimmune diseases in particular in chronic inflammatory demyelinating polyneuropathy (cidp). the lack of a predictive marker for ivig responsiveness in cidp avoids the early preservation of non-responding patients. to better understand the effect of ivig in patients with cidp, we tested whether ivig treatment altered the nk cell status. additionally, we analysed if the alteration in the populations may serve as a surrogate marker in predicting the outcome of ivig treatment. using semi-quantitative pcr and flow cytometry in the peripheral blood of patients with cidp, we analysed the effects of ivig on the nk cell population before treatment initiation and h after first dose and correlated the changes with the reponsiveness to ivig. ivig administrations induced a reduction in the expression of several typical nk cell genes. interestingly, this ivig-induced reduction of nk cells was reversible four weeks after the ivig treatment. flow cytometry data revealed that ivig reduced the cytotoxic cd dim nk cell population, while regulatory cd bright nk cells remained almost unaffected or were even increased. interestingly, we found that the observed effects on nk cells almost exclusively occurred in cidp patients who responded to ivig therapy. correlation between the changes in the nk cell population and treatment efficiency suggests a crucial role for nk cells in the immunomodulatory mechanism of ivig. further studies are warranted to investigate whether the differences in the nk cell status of patients with cidp represent a reliable surrogate marker in predicting the outcome of ivig therapy. mccray b , sullivan j , woolums b , aisenberg w , lloyd t , sumner c . johns hopkins university, baltimore, usa. mutations in transient receptor potential vanilloid (trpv ), a calcium-permeable non-selective ion channel, cause charcot-marie-tooth disease type c (cmt c). trpv is unique in that it represents the only membrane-expressed ion channel in cmt and thus a potential therapeutic target. previous work has suggested that trpv mutations lead to gain of channel function and toxicity in cultured cells. neuropathy-causing mutations of trpv largely cluster in the cytosolic ankyrin repeat domain (ard) that is known to mediate protein-protein interactions, suggesting that pathogenesis may be related to disruption of such interactions. in order to identify trpv -interacting proteins, we performed two unbiased proteomics screens and identified multiple cytoskeletal-modifying proteins including syndapin- , a neuronal protein known to promote axonal outgrowth by influencing the actin cytoskeleton. in cultured cells, we have shown that trpv and syndapin- co-localize to highly dynamic actin-rich cellular processes and together stimulate robust neurite extension, but this facilitation of neuritogenesis is impaired by disease-causing mutations in trpv . we have also shown that over-expression of syndapin reduces trpv -mediated calcium influx in cultured cells and rescues toxicity of mutant trpv . in addition, syndapin over-expression suppresses mutant trpv phenotypes in a drosophila model of trpv -related neuropathy. further, we have demonstrated that treatment of drosophila with a specific trpv channel antagonist ameliorates trpv mutant toxicity. together, our data highlight the importance of trpv interaction with cytoskeletal proteins such as syndapin- in the pathogenesis of cmt c. specifically, our results suggest that mutations in trpv disrupt the normal role of trpv in regulation of cytoskeletal dynamics and that interactions with the cytoskeleton reciprocally modulate trpv channel function and influence toxicity of mutant trpv . mcgonigal r , yao d , barrie ja , crawford c , willison hj . university of glasgow, glasgow, uk. guillain-barré syndrome (gbs) is in part mediated by anti-gm ganglioside antibodies induced by preceding infections. anti-gm antibodies target plasma membrane gm that is extensively distributed in both glial and axonal membranes, particularly at the node of ranvier. antibodies deposited at this site in models of gbs are associated with complement deposition, conduction block, structural disruption of ion channels and macrophage infiltration. the wide distribution of the gm ganglioside target leads to unwanted complexity in ascribing pathological outcomes to injury of cell-specific membranes, in particular unravelling the consequence of paranodal schwann cell membrane injury on axonal function, and vice versa. to overcome this impasse, we have generated transgenic mice through glycosyltransferase manipulation that express gm exclusively in neurons or glia, thus allowing us to very specifically target and injure axonal or glial membranes with a single anti-gm ganglioside antibody. through this route we can create mouse models of both the axonal and demyelinating forms of gbs, induced by a single anti-gm antibody, thus creating otherwise highly comparable conditions. here, we show anti-gm antibody binding is restricted to the nodal axolemma in galnact −/− -tg(neuronal) mice and conversely to paranodal loops in galnact −/− -tg(glial) mice. when anti-gm antibody and a source of complement is added to a nerve-muscle ex vivo injury paradigm, there is a loss of axonal integrity (i.e. loss of neurofilament immunolabeling) when the neuronal membrane is targeted in galnact −/− -tg(neuronal). conversely, axonal integrity is maintained when the paranodal membranes are decorated by antibody and complement products ex vivo in galnact −/− -tg(glial) mice. in a passive immunisation model in vivo, galnact −/− -tg(neuronal) mice acutely develop weakness, respiratory dysfunction, associated complement deposition, and degenerative pathology in distal axons. in contrast, galnact −/− -tg(glial) mice have significantly fewer abnormalities under the same acute conditions. these data indicate the high vulnerability of axonal membranes to acute injury and underline the importance of developing specific axonal protection strategies. in summary, targeting the nodal axolemmal or glial membranes allows us to study associated nodal pathology, and determine the downstream consequences on function and axon fate, currently a major area in gbs clinical research. memon a , madani s , schultz l , grover k , arcila-londono x , sripathi n , ahmad bk . neuromuscular division, department of neurology, henry ford hospital, detroit, michigan, usa. objective: to differentiate sensory electrophysiology, tli and treatment response in patients with paraproteinemic cidp. background: low tli has been reported as a useful electrophysiological marker for mag-cidp. to our knowledge comparison of sensory electrophysiology and tli of paraproteinemic cidp subgroups have not been previously reported. methods: retrospective review(january -december ) of patients with cidp fulfilling electrophysiological criteria(aan ad hoc subcommittee and albers and colleagues).cidp patients with diabetes(n= ) were excluded. patients were divided into idiopathic (n= ) and paraproteinemic cidp(n= ). paraproteinemic cidp sub-groups: mag( ), non-mag( ) and igg( ) were compared to idiopathic cidp( ). these groups were compared for demographics, history of cancer, csf protein, sensory conductions, tli measurements and response to treatment using chi-square tests for binary and categorical variables and t-tests for continuous measures. results: there was a higher proportion of females in idiopathic-cidp compared to non-mag-cidp ( % vs %). idiopathic group having a higher proportion of patients on monotherapy( % vs %) and combination therapy( % vs %) compared to non-mag. higher mean csf protein compared to mag-cidp(p= . ) was seen in the idiopathic. the difference between idiopathic and igg-cidp was significant for overall rx response(p= . ) and rx response in patients with follow-up(p= . ). for both variables, patients in the idiopathic group had a higher proportion of patients on combination therapy and lower proportion of no treatment offered compared to patients in the igg-cidp. % of non-mag-cidp patients had a history of cancer vs % of mag-cidp. none of the other differences were significant. there were no group differences in sensory electrophysiology and tli. conclusions: sensory electrophysiology and tli may have no value in differentiating paraproteinemic cidp. csf protein is higher in idiopathic cidp compared to mag-cidp. idiopathic-cidp has a higher proportion of females compared to non-mag-cidp and a higher proportion of patients on combination therapy compared to igg-cidp. cancer screening should be considered in patients with non-mag-cidp. memon a , madani s , ahmad bk , schultz l , grover , arcila-londono x , sripathi n . department of neurology, henry ford hospital, detroit, michigan, usa. introduction: sensory electrophysiology and terminal latency index (tli) differences have been described in various cidp sub-groups. objective: evaluate electrophysiology, tli and treatment response in idiopathic and diabetic cidp. methods: retrospective review of patients with cidp who underwent electrodiagnostic evaluation (january -december . patients fulfilled electrophysiological criteria described by ad hoc subcommittee of american academy of neurology (aan) and albers et al. we excluded patients( ) with acute inflammatory demyelinating neuropathy, hereditary sensorimotor neuropathy, vasculitis and polyneuropathy with paraproteinemia. patients were divided into idiopathic( ) and diabetic( ) groups. these groups were compared for age, sex, history of cancer, csf protein, response to treatment, sensory response abnormalities and tli measurements using chi-square tests for binary and categorical variables and t-tests for continuous measures. all testing was at the alpha= . level. results: group differences for age, sex, history of cancer, csf protein and treatment response were not significant. comparing tli values in measurable responses, the difference between the two groups for tibial tli was significant (p= . ), with idiopathic group having a lower mean as compared to the diabetic. tli values differences for median, ulnar and peroneal nerves were not significant. the difference in abnormal rates of sensory responses was significant for the sural nerve with the idiopathic group having a lower rate compared to the diabetic group ( % vs %, p< . ). no differences were noted for the ulnar, median and radial nerves. tibial tli and sural sensory responses have some value in differentiating the two groups. larger prospective studies are needed to confirm our findings. ivig is an important treatment option for cidp. although recommended by treatment guidelines, little is known about the consequences of temporary ivig withdrawal to assess ongoing immunoglobulin need. path is a randomized, double-blind trial of the subcutaneous immunoglobulin (scig) igpro (hizentra ® , csl behring) in cidp. before scig randomization, subjects underwent periods of ivig withdrawal ( weeks or until pre-determined indications of clinical deterioration) and ivig restabilization (igpro ; privigen ® , csl behring). subjects not showing deterioration during withdrawal period were withdrawn from study. to proceed to scig randomization, subjects had to achieve "cidp stability" (no relevant change in incat score at last two restabilization visits and at least the same total score as at screening). subjects entered the ivig withdrawal period. ( %) of these qualified for igpro restabilization; subjects ( %) were not ivig dependent, and ( %) were withdrawn for other reasons. one subject withdrew consent before igpro dosing. at the end of the igpro restabilization period, cidp stability was achieved in % of subjects ( did not reach stability, were withdrawn for other reasons). post-study follow-up information was available for / subjects who did not reach stability: ( %) had improved to baseline clinical status and had not, meaning at least % of the subjects improved to their pre-study status. during the restabilization period, / subjects ( %) improved in at least one of the predefined outcome measures. on average, subjects improved by . points in incat total score, . points in i-rods centile score, kpa in mean grip strength (dominant hand), and . points in mrc sum score. improvement occurred with a median of days after the first igpro dose in one or more efficacy outcome measures and in % of cases after the third igpro maintenance infusion. headache and nasopharyngitis were the most frequently reported adverse events (aes) during restabilization. aes deemed causally related were mostly mild or moderate. no unexpected aes or laboratory or vital sign findings associated with igpro occurred during the study. in summary, igpro reversed neuromuscular disability and improved activity/participation after previous clinical deterioration during an ivig withdrawal period. pmp duplication is the most frequent cause of charcot-marie-tooth disease (cmt). since it discovery, more than genes have been identified to be potentially responsible for cmt disease. however only single nucleotide variations (snvs) or small indels have been described. this could be due to the new sequencing strategy (ngs), especially ngs by amplicon sequencing, for whose few convenient tools are available and easily usable to detect cnvs responsible for inherited disease. to overcome this problem, we designed "cov'cop", a user-friendly tool able to detect cnvs among amplicons sequencing data. using the run's coverage file provided by the sequencer, "cov'cop" simultaneously analyzes all the patients of the run using a two-stages algorithm containing correction and normalization levels and provides an easily understandable output, showing with various colors, potentially deleted and duplicated amplicons. we validated our method on several datasets, including those of our targeted ngs panel screening genes known to be involved in cmt and close pathologies. cov'cop detected easily pmp duplication and deletion in our patients, confirmed by mlpa. in addition, cov'cop permitted the detection of new cnvs different from the pmp duplication, in cmt patients. we confirmed these cnvs by quantitative pcr and cgh array. we present here one of these cnvs: the duplication of aars gene detected in cmt patients and we discuss the pathogenicity of this new cnv. additional cnvs responsible for cmt disease are probably still to be discovered and we believe that cov'cop will help molecular geneticists to rapidly identify them. poems (polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscraisia and vascular endotherial (vegf) overproduction. although number of therapeutic interventions for plasma cell dysorders have been applied to poems syndrome, there have been no randomized clinical trials. this phase / double blind, randomized, placebo comtrolled trial was performed to investigate the safety and efficacy of thalidomide for patients with poems syndrome who are not eligible for stem-cell transplantation. the primary endpoint was the reduction rate of serum vegf concentrations at weeks in intention to treat analysis. additional outcomes of long-term extension study included progression-free survival. twenty-five poems patients were randomly assigned to either thalidomide plus dexamethasone or placebo plus dexamethasone from nov , , to july , . one patient in the placebo group was excluded from analyses because of a protocol violation. the adjusted mean serum vegf reduction rate at weeks was . (sd, . ) in the thalidomide group compared with − . ( . ) in the placebo group (p= . ). the kaplan-meier rate of progression-free survival at months was . in the thalidomide group, as compared with . in the placebo group (hr, . ; % ci, . to . ). in the randomized study period, mild sinus bradycardia was more frequent in the thalidomide group than in the placebo group ( % vs %; p= . ). thalidomide suppresses serum vegf concentrations and lengthened pregression-free survival in poems patients who are ineligible for stem cell transplantation. although thalidomide treatment has a risk of bradycardia, the benefits would exceed the risk. this study is registered with the umin clinical trials registry, umin . montes-chinea ni , coutts m , vidal c , courel s , rebelo a , abreu l , zuchner s , saporta, ma , . department of neurology, university of miami, miami, usa; department of human genetics, university of miami, miami, usa. autosomal dominant mutations in synaptotagmin- (syt ), a synaptic vesicle protein that functions as a calcium sensor for neurotransmission, have been previously linked to presynaptic neuromuscular junction (nmj) dysfunction and motor neuropathy in two families. both pathogenic mutations (asp ala and pro leu) were located in the c b domain of syt , which is essential for neurotransmitter release at the nmjs. we report a family with a new missense mutation in the c b domain of syt and a similar phenotype characterized by a slowly progressive, predominantly motor neuropathy and evidence of presynaptic nmj dysfunction on nerve conduction studies. the index case is a year-old woman with gradually progressive weakness of her extremities. she had normal developmental milestones, but was found to have bilateral high arched feet and hammertoes and occasional falls around the age of . she gradually developed progressive leg weakness, worsening bilateral hand cramping, weak handgrip, and only mild paresthesias on distal extremities. family history is remarkable for similar symptoms reported by her maternal grandfather, two maternal uncles, her mother and a younger sister. her neurological exam revealed inability to walk on heels or toes, significant distal lower extremity weakness and absent ankle deep tendon reflexes. cranial nerve examination and coordination were normal and there were only non-specific sensory changes in the lower extremities. emg/ncs revealed normal sensory responses throughout; however, motor nerve evaluation demonstrated globally reduced amplitudes with a > % increment after brief isometric contraction. further electrophysiological evaluation with slow ( hz) repetitive nerve stimulation of the right ulnar motor nerve revealed a % decremental response in amplitude and a > % increase in amplitude immediately after a one-minute period of sustained muscle contraction, which rapidly extinguished after one minute. voltage-gated calcium channel (vgcc) antibodies and a chest ct were normal. targeted sanger sequencing revealed an ile lys mutation in syt , which is located in the c b domain and is predicted to impair protein function. syt -related neuropathy is a rare disease, but should be suspected in patients presenting with a combination of pre-synaptic nmj dysfunction (resembling lambert-eaton myasthenic syndrome) and a predominantly motor neuropathy, especially in the context of a positive family history. charcot-marie-tooth (cmt) disease affects roughly in , individuals and is described as an inherited peripheral neuropathy primarily affecting distal muscles. limited studies detail patient-reported impact of muscle weakness on functional activities. this anonymous survey was developed with input from clinical experts and patient interviews and aimed to better understand the prevalence and impact of various cmt clinical manifestations on patients' lives. the survey was administered online to the hereditary neuropathy foundation's (hnf) patient contact database and is ongoing through june . here we present preliminary data on patient characteristics and disease impact for cmt patients collected february - , . respondents were mostly female ( %) and mostly from the us ( %). median age (range) at symptom onset was years ( - years), at diagnosis was years ( - years), and at present was years ( - years). the sample was representative of all cmt types (cmt , , , ,and x). the most common physical and clinical manifestations of cmt were problems with balance ( %), ankle weakness/foot drop ( %), loss of feeling or abnormal sensation in the lower leg/foot ( %), and hand muscle weakness ( %). maintaining balance, walking long distances, and climbing up and down stairs were key challenges associated with ankle weakness/foot drop. foot drop was considered by % to be the primary factor contributing to falls, which averaged . falls to ground per month. of those with foot drop, % had bilateral weakness. a majority of respondents ( %) used some form of assistive device for mobility, including ankle-foot orthotics/below-the-knee leg braces ( %), canes/walking sticks ( %), and custom foot orthotics/inserts ( %). the most common drug therapy included pain and anti-inflammatory medications ( %). foot surgery was the most common surgical procedure received ( %) and toe surgery was the most common surgery considered ( %). key symptoms that affected quality-of-life "very much" included problems with balance ( %), ankle weakness (foot drop) ( %), and fatigue ( %). these data suggest a high prevalence of lower leg muscle weakness; therefore, therapies aimed at improving ankle weakness and the resulting foot drop and imbalance may be beneficial to patients' daily functioning and quality of life. morano m , , gambarotta g , ronchi g , , cillino m , fornasari be , , fregnan f , , tos p , cordova a , moschella f , geuna s , , raimondo s , . department of clinical and biological sciences, university of turin, orbassano (to), italy; neuroscience institute of the "cavalieri ottolenghi" foundation (nico), university of turin, orbassano (to), italy; plastic and reconstructive surgery. department of surgical, oncological and oral sciences, university of palermo, palermo, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. nerve fiber regeneration and complete functional recovery after peripheral nerve injury do not always occur and can be influenced by many factors including patient age, gender, lesion site, injury severity, size of the gap between damaged nerve stumps and time interval that elapses before performing surgical repair. the poor outcome occurring after a long delay can be due to loss of the neuron ability to regenerate, loss of the schwann cell ability to support regeneration and, of course, progressive muscle atrophy. the aim of this study was to investigate the nerve regeneration after delayed repair and to study the degenerative processes of the denervated distal nerve stump and denervated muscle. in particular, the analyses were focused on the role of nrg/erbb system, that is expressed both in nerve and in muscle tissue, during degenerating and regenerating processes. functional recovery analysis performed after nerve repair showed that only the group repaired immediately and not the groups repaired with a delay of or months, recovered partially. nevertheless, morphological analyses demonstrated that, despite the delay, the nerve fibers are still able to regenerate, even if they are fewer and smaller than the immediate repaired group. moreover, the analysis of the nrg /erbb system showed a significant decrease of soluble nrg in both degenerating and delayed-repaired nerves. the poor outcome after delayed nerve regeneration might be explained by schwann cell impairment and the consequent ineffective support for nerve regeneration. as regards denervated muscle analysis, results showed that erbb receptors expression is related to the innervated state of the muscle, with an upregulation of erbb clearly associated with denervation state. interestingly, nrg isoforms are differently regulated depending on the type of nerve injury. future experiments will be needed to address the in vivo efficacy of different isoforms of nrg both in injured nerve and denervated muscle. we planned a multicenter, prospective, randomized, single blind, controlled study to evaluate the efficacy and safety of an innovative rehabilitation protocol based on the use of treadmill training in a cmt a population. the protocol required that subjects were blindly randomized into two treatment groups, spe (three months of respiratory, proprioceptive and stretching exercises) or trespe (the same treatment plus aerobic training at the treadmill). subjects were evaluated at baseline (t ), after three month of treatment (t ) and further three months of follow up free of therapy (t ). the full assessment included: -mwt (primary outcome measure), -mwt, walk- , short physical performance battery (sppb); lower limbs dynamometric strength evaluation; berg balance scale (bbs); cmt neuropathy score; medical outcomes study short form (sf ). a total of subjects (mean age of . ± . years) were recruited. at t we found a significant improvement in both groups in the -mwt (p< . ), -mwt (p< . ), bbs (p< . ) and sppb (p< . ), while at t only the -mwt was still significantly improved (p< . ) in the spe group. no significant differences between groups were observed for any of the outcome measures. performances on walk did not significantly change during follow-up (p= . ). concerning the sf , we did not observe consistent changes during follow-up or consistent differences comparing the two treatments. in conclusion, this multicenter, prospective, randomized, single blind, controlled study shows that the combination of respiratory, proprioceptive and stretching exercises has a positive impact on the performance of cmt patients, especially regarding walking tests. the aerobic exercise at the treadmill, is well tolerated but apparently does not add any further improvement to the conventional treatment. we speculate that the relatively low clinical severity of the patients, due to the selection criteria, may have prevented a positive effect of treadmill exercise. müller m , dohrn m , romanzetti s , reetz k , gess b . university rwth aachen, department of neurology, aachen, germany. muscle mri is increasingly used in neuromuscular patients to detect changes in muscle volume, muscle fat infiltration and edema. muscle mri are mostly analyzed by qualitative means as quantitative analysis is time-consuming and not well established. here, we developed a novel method for semi-automated segmentation of muscle mri data sets. based on axial t -weighted dixon mri stacks, muscle volumes were quantified by an adapted water-shed algorithm. muscle volumes of thighs and calves were determined separately and the ratio of thigh/calf was calculated. myopathy, neuropathy patients and healthy controls were included in the study. muscle volumes determined by semi-automated segmentation were very similar to manually segmented data sets, differences being < %. this was the case for patients as well as healthy controls. the time-saving effect of automated segmentation was very strong ( vs min. per patient). muscle volumes of the thigh and also of the calf of myopathy patients showed a highly significant difference (p< . ) compared to healthy subjects. in neuropathy patients there was a just significant difference (p< . ) of muscle volumes compared to healthy patients that did not sustain in bonferroni's multiple comparison test. the ratio of thigh/calf muscle volume was significantly different comparing patients with myopathy and neuropathy (p< . ). subgroup analyses of different groups of myopathy patients showed highly significant differences (p< . ) in myositis, limb-girdle-muscular dystrophy and metabolic myopathy, compared to healthy patients, but no significant differences in-between these groups. taken together, the data shows that automated segmentation of muscle mri allows for exact and fast quantification of muscle volumes in neuromuscular patients. higher patient numbers are necessary to test differences between specific disease groups. further studies should also address the possible use as a marker of disease progress for clinical studies or therapy monitoring. murakami t , nishimura h , nagai t , hemmi s , kutoku y , sunada y . department of neurology, kawasaki medical school, kurashiki, japan; department of pathology, kawasaki medical school, kurashiki, japan. familial amyloidotic polyneuropathy (fap) is an autosomal dominant hereditary systemic amyloidosis caused by mutation of transthyretin (ttr) gene, and usually shows sensory dominant polyneuropathy and autonomic neuropathy at the initial stage. the pathogenesis of neuropathy is not well understood, and explained by several mechanisms, including such as mechanical compression, vessel occlusion, ttr toxicity and schwann cell dysfunction. we describe a sporadic patient with late-onset fap due to ttr e k. she noticed dysesthesia first in the foot at age . the symptoms were slowly progressive, and abnormal sensations were extended up to the both upper arms and the both knees at age . distal muscle weakness and atrophy was also observed in the extremities. she noticed difficulties in walking and frequent diarrhea. echocardiogram revealed diffuse left ventricular hypertrophy, suggesting cardiac amyloidosis. amyloid deposits were not detected in the endoneurim or perineurium of the sural nerve years after the onset of the disease, but a marked loss of myelinaed and unmyelinated nerve fibers was observed in it. ttr-derived amyloid deposits were confirmed in the peroneous brevis muscle, salivary gland and heart tissue. dna analysis revealed the heterozygote mutation, p.e k (e k)/c. g>a, of ttr gene, and she was diagnosis as fap. these findings suggest that the proximal parts of peripheral nervous system might be strongly involved by ttr aggregates or amyloid fibrils. blood-nerve barrier in the distal part of peripheral nerves could be preserved until later in the patient. several biopsy sites other than nerve may be helpful and necessary for diagnosis of ttr amyloidosis in mild or late-onset fap as our case. amyloidgenic element (cae) encoded by the ' utr. this study also identified a de novo c. _ dup frameshift mutation predicting p.lys glnfs* in nefh from a cmt family with atypical clinical symptom of proximal dominant weakness. this mutation is located near the previously reported frameshift mutations, suggesting a mutational hot spot. these relatively frequent deletion/duplication events with this resign might be caused by the putative hairpin structure. patient's lower limb mri revealed a marked hyperintense signal changes in the hip muscles than those in the thigh or lower leg muscles. this study also observed an anticipation pattern of earlier onset ( yrs old for mother to yrs old for daughter) and more severe symptoms in later generation. therefore, this study suggests that the stop loss and translational elongations by the ' utr of the nefh mutations may be relatively a frequent genetic cause of axonal peripheral neuropathy with the specific characteristics of proximal dominant weakness and an anticipation pattern. neil j , haghi ashtiani b , choumet v , musset l , léger jm . department of immunology, pitié-salpêtrière hospital (ap-hp), paris, france; department of neurology, national referral center for rare neuromuscular diseases, pitié-salpêtrière hospital (ap-hp), paris, france; institut pasteur, emerging diseases epidemiology unit, paris, france. the myelin-associated glycoprotein (mag) is a transmembrane glycoprotein localized in periaxonal shwann cells and oligodendroglial membranes of myelin sheaths. mag contains a carbohydrate epitope (hnk- ) that is a target antigen in autoimmune peripheral neuropathy associated with monoclonal igm gammopathy. in these neuropathies, numerous studies report the absence of correlation between the titers of anti-mag antibodies and the disease course. anti-mag titers and igm level at diagnosis are not always associated with disease severity and there is not good correlation between pre-and post-treatment anti-mag titers in patients who respond clinically to immunomodulators. mag belongs to siglec- a family and the linkage of sialic acid to the underlying sugars is an important determinant of siglec binding. mag shows high affinity for alpha- , -linked sialic acid ( , -sa).moreover, human monoclonal igm possesses heavy chain glycosylation sites at asn , , , and with sialylated olidgosaccharides and high-mannose type oligosaccharides. igm may bind to mag via these glycan epitopes as an alternative and additional route of antigen binding other than through the fab v regions. this mag-glycans igm interaction may be clinically neutral but could lead to an overvaluation of the biological results. in this study, we analyzed sera from patients with igm reactivity against mag: of them had an anti-mag neuropathy with various degrees of severity, and the last one had igm monoclonal gammopathy, strong serum anti-mag reactivity but no neurological disease. igm were extracted and purified from these sera by affinity chromatography. for each batch, an aliquot was digested by jack bean alpha-mannosidase and anti-mag reactivity was performed by elisa and indirect immunofluorescence (iif), before and after demannosylation. these extracts, tested in an iso quantitative way with regard to the original serum, showed a decrease of activity (elisa) and intensity (iif) after demannosylation. furthermore, elisa anti-mag was carried out in sera from patients with igm monoclonal gammopathy without neurological impairment: of them ( . %) showed a significant biological response. taking into account the fact that anti-mag antibodies are pathogenic (in animals models), these results support the hypothesis of neutral intermolecular interactions between igm and mag. ng cjb , ng jph , tay lb, t , umapathi . yong loo lin school of medicine, national university of singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. patients with postural orthostatic tachycardia syndrome (pots) have marked tachycardia with no associated decrease in pbp. we asked if the reflex tachycardia, which corresponds to postural dizziness in these patients, could be a result of a decrease in casp. two male patients, and years of age, with clinical features typical of pots went through a complete battery of autonomic screening tests. the sympathetic and parasympathetic responses were normal other than a heart rate increase of and beats per minute, respectively, on standing. there was no significant decrease or increase in pbp on standing for and minutes. casp was measured non-invasively by a device bpro r that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure waveform from which a numerical casp value is derived. the casp measurements for both patients did not decrease on standing for and minutes. our preliminary observation suggests that the basis of tachycardia in pots patients may not be a decrease in central blood pressure. we are proceeding to systematically study more pots patients to corroborate the above observation. we are also trying to compare the difference between pbp and casp in pots and age, gender-matched normal controls. the major limitation of the study is the model-based mathematical derivation, rather than direct measurement, of casp. we are proceeding to systematically stud y more pots patients to corroborate the above findings. ng jph , ng cjb , t umapathi . lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university of singapore, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. orthostatic hypotension (oh) is a prominent component of autonomic dysfunction (ad), the consequent hypoperfusion of vital organs responsible for considerable morbidity and mortality. these structures are exposed to casp rather than pbp. we sought to understand the relationship of casp to peripheral blood pressure (pbp) in patients with autonomic dysfunction exposed to orthostatic stress. we reviewed autonomic function tests of patients tested at our laboratory over a -year period. the patients were divided into cohorts: ( ) no ad and no oh ( ) no ad, with oh ( ) mixed ad ( ) parasympathetic dysfunction ( ) sympathetic dysfunction. casp was measured non-invasively by a device, bpro r , that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure-wave form from which a numerical casp value is derived. the difference and ratio of casp to pbp was recorded at rest and after minutes of standing. out of patients had complete data and a definitive final diagnosis. cohorts - had , , , , patients respectively. mean casp-pbp difference in cohorts - were − . , − . , − . , − . , − . respectively at minutes, and − . , − . , − . , − . , − . respectively at minutes. mean casp/pbp ratio in cohorts - were . , . , . , . , . , . respectively at minutes, and . , . , . , . , . respectively at minutes. there was no significant difference in the response of casp and pbp to orthostatic stress across abnormal cohorts to and in comparison with the "normal" cohort (p= . to . ). there was also no relationship to symptoms, namely postural dizziness. in conclusion, autonomic dysfunction does not seem to affect the casp-pbp relationship as measured by non-invasive means. the absence of true normal controls, the exclusion of significant number of patients because of incomplete data and the model-based mathematical derivation rather than direct measurement of casp are limitations that we aim to address in follow-on studies. niu jw , guan hz , cui ly , guan yz , liu ms . the department of neurology, peking union medical college hospital, chinese academy of medical sciences, beijing, china. we aimed to explore the correlation between afterdischarges in motor nerve conduction studies and clinical motor hyperexcitability in patients with voltage-gated potassium channels (vgkc) antibodies. six patients with positive serum antibodies to contactin-associated protein-like (caspr ) or/and leucine-rich glioma-inactivated protein (lgi ) were recruited, including with autoimmune encephalitis, and with cramp-fasciculation syndrome. electromyography (emg), nerve conduction studies (ncs) and f waves were performed, and afterdischarges were assessed. one patient was followed up. five patients had clinical evidence of peripheral motor nerve hyperexcitability (myokymia or cramp), and four of them had abnormal spontaneous firing in concentric needle electromyography. prolonged afterdischarges following normal m waves were present in all six patients, including the two patients who had no emg evidence of peripheral nerve hyperexcitability (pnh). in the patient who was followed up, afterdischarges disappeared after treatment with intravenous immunoglobulin (ivig). afterdischarges in motor nerve conduction study might be more sensitive than needle electromyography for detecting peripheral motor nerve hyperexcitability in patients with vgkc antibodies, and could disappear gradually in accordance with clinical improvement and reduction of antibodies. in our research, multiple sites measurement of cross sectional areas (csa) by ultrasound was performed to differentiate charcot-marie-tooth type a (cmt a) and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). twenty-eight patients with cidp, patients with cmt a, and healthy controls (hc) were recruited prospectively. consecutive ultrasonography scanning was performed from wrist to axilla on median and ulnar nerves. csas were measured at predetermined sites of each nerve. cmt a had significantly larger csas at all sites of median and ulnar nerves (all p< . ). in cmt a, csas increased gradually and homogeneously from distal to proximal along the nerve, except potential entrapment sites. cidp displayed three different morphological patterns, including mild enlargement in patients, prominent segmental enlargement in , and slight enlargement in one, among which different treatment responses were observed. all patients with mild nerve enlargement treated with intravenous immunoglobulin (ivig) were responsive ( / ), while less than half of those with prominent segmental enlargement ( / ) were responsive (p< . ). the patterns of csa enlargement were different in cmt a and cidp patients. consecutive scan along the nerve and multiple sites measurement by ultrasound could supply more detailed morphological feature of the nerve and help to differentiate cidp from cmt a. guillain-barre syndrome (gbs) mainly consists of acute inflammatory demyelinating polyradiculoneuropathy (aidp) and acute motor axonal neuropathy (aman). in aman, conduction block (cb) could be reversible or followed by axonal degeneration. we aimed to identify the correlation between existence of cb and the functional outcome for patients with gbs. gbs patients were prospectively recruited for serial electrophysiological tests and disability evaluation. all patients received treatment of intravenous immunoglobulin (ivig), and their disabilities were evaluated on the hughes functional grading scale before and month after treatment. patients were classified into aidp, aman, equivocal or normal according to electrodiagnostic criteria described by rajabally et al. aman patients who had follow-up nerve conduction studies were further classified into three groups. group was typical aman without conduction block, group had reversible conduction block, group had conduction block and subsequential axon degeneration. electrophysiological study results showed aidp, aman, equivocal and normal. probable or definite conduction block was observed in aidp patients and aman patients. aman with cb had higher reduction of hughes grade at one month ( . ± . vs . ± . ,p= . ), and lower percentage of patients with slow recovery (unable to walk independently at six months) ( % vs %, p= . ) compared with aman without cb. there were no significant differences between aidp with cb and without cb, in the reduction of hughes grade at one month. among the aman patients who were followed up, were typical aman without cb (type ), had reversible cb (type ), had cb and subsequential axon degeneration (type ). hughes grades at nadir were similar, while patients with reversible cb (type ) had the largest hughes grade reduction at one month (type - . vs type - . vs type - . ). none of the patients with axon degeneration (type ) showed rapid recovery, while % of those with reversible cb (type ) had rapid recovery (improvement by two or more hughes grades within four weeks after onset). electrodiagnosis of aman with conduction block, especially reversible conduction block, might be a marker of good recovery. we report a follow-up study of nerve ultrasound in a patient with primary neurolymphomatosis. a -year-old female presented with -months history of asymmetric limb pain, paresthesia, and weakness. electrodiagnostic studies and spinal cord mri showed an axonal neuropathy involving cervical and lumbosacral root, brachial plexus and left median nerve. detection of malignant b lymphocytes by cytology and flow cytometry of cerebral spinal fluid confirmed the diagnosis of b-cell non-hodgkin lymphoma. nerve ultrasound showed dramatic enlargement of upper, middle and lower trunks of left brachial plexus (cross sectional area-csas mm , mm , mm respectively), middle trunk of right brachial plexus (csa mm ), and proximal part of left median nerve (csa - mm ). five months later, after five chemotherapy of rituximab and high-dose methotrexate, and intrathecal injection of cytosine arabinoside and dexamethasone, the patient had clinical improvement. nerve ultrasound also showed alleviation of nerve enlargement. the csas of upper, middle and lower trunks of left brachial plexus were mm , mm , mm respectively; the csa of middle trunk of right brachial plexus was mm ; the csa of proximal part of left median nerve was - mm . peripheral nerve ultrasound could help locate the distribution of nerve involvement, and reveals disease progression. nolano m , provitera v , stancanelli a , caporaso g , saltalamacchia am , borreca i , lullo f , califano f , lanzillo b , iodice r , manganelli f , barone p , santoro l . irccs "salvatore maugeri" foundation, institute of telese terme (bn), italy; "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; center for neurodegenerative diseases (cemand), department of medicine and surgery, neuroscience section, university of salerno, italy. a peripheral nerve involvement has been demonstrated in pd with the evidence of a small fiber pathology as possible intrinsic feature of the disease and a higher occurrence of large fiber neuropathy in patients longtime treated with l-dopa. however the role that disease itself and ldopa have on small and large fiber pathology in pd is still debated. we studied morphology and function of cutaneous innervation, in idiopathic pd patients ( male, aged . ± . ), including naïve and l-dopa treated subjects without electrophysiological signs of neuropathy, with the aim to assess and characterize small and large fiber involvement and the effect of l-dopa on it. all patients underwent a screening to rule out potentially neurotoxic conditions such as glucose intolerance, dysendocrinopathies, vitamin e, b and folic acid deficiency, hepatic or renal failure, hiv or connective tissue disorders. skin biopsies were obtained from thigh, leg and fingertip from the more affected side and bilaterally from thigh and leg in patients. samples were processed with indirect immunofluorescence technique using primary antibodies to mark different sensory and autonomic fiber populations. density of intrapapillary myelinated endings (ime), meissner's corpuscles (mc) and epidermal nerve fibers (enfs) was obtained as well as a semi-quantitative assessment of sudomotor, pilomotor and vasomotor innervation. further evaluation included sympathetic skin response, quantitative sensory testing and dynamic sweat test. morphological and functional findings were compared with data extracted from our age and sex stratified normative dataset. ienf, ime, mc densities were lower (p< . ) compared to controls in both naïve and l-dopa treated patients without differences between them except for mc density that was lower in l-dopa treated subjects ( . ± . vs . ± . /mm ). a loss of autonomic nerves was also found in both groups compared to controls. significant abnormalities (p< . ) of thermal sensory thresholds, tactile thresholds, mechanical pain perception and reduced sweating output were present and similar in both groups. our work confirms in pd an intrinsic peripheral nerve pathology involving both small and large fibers. small fiber pathology isn't affected by l-dopa treatment while sensory large fibers involvement, already present in naïve patients worsens with ldopa treatment. noto y , garg n , li t , timmins hc , park sb , shibuya k , kiernan mc . brain and mind centre, the university of sydney, sydney, australia. the diagnosis of amyotrophic lateral sclerosis (als), a progressive, fatal neurodegenerative disorder defined by combined upper and lower motor neuron involvement, remains clinically based. the purpose of this study was to determine the ultrasound appearance of peripheral nerves in als patients, and to investigate whether parameters such as distal/proximal ratios of nerve cross-sectional areas (csas) may effectively differentiate disease mimics from als. nerve ultrasound of the median, ulnar, and tibial nerves was performed in als patients compared to mimic patients ( patients with peripheral nerve hyperexcitability syndromes (pnhs) and patients with multifocal motor neuropathy (mmn)). comparison of nerve and the distal/proximal ratios was undertaken by ultrasound and compared across clinical and neurophysiological parameters.compared to normal controls, csa of the median nerve at the upper arm was decreased in als (p < . ). in comparison to als mimic disorders, csa at the proximal site of the median, ulnar and tibial nerve and the forearm/upper arm ratio of the median and ulnar nerves had diagnostic values. in addition to csa of the median, ulnar, and tibial nerves, the median and ulnar nerve forearm/upper ratios may provide a useful marker in for the diagnosis of als. approximately in , individuals are diagnosed with charcot marietooth (cmt) disease, making it the most common hereditary peripheral neuropathy. there is no documented cure for cmt, however, many of those affected, report difficulty with mobility, imbalance, and weakness of the feet and hands. in the general population, patients reporting difficulty walking, falls and/or fear of falling, and poor strength are often referred to physical (pt) and occupational therapists (ot) to skillfully address the impairments and help restore function and quality of life (qol). for patients diagnosed with cmt this is unfortunately not the norm, often leaving patients without any skilled guidance on managing their functional impairments. patients ( males) with a mean age of . years ( - ), went through a progressive and skilled pt intervention over the course of months. the program included: therapeutic activities and exercise, neuromuscular reeducation, and manual techniques to address the documented deficits. patients were progressed through the program based on the borg scale. each patient was assessed prior to and post the commencement of the program with the berg balance scale, minute walk test, timed up and go, sit to stand in seconds, foot self selected and fast gait speed, the lower extremity functional scale, activities balance confidence form, upper extremity functional index, oswestry, and the sf- qol measure. the patients were seen by the same skilled pt - a week for weeks. all but patients improved in all measures taken, indicating an improvement in function and overall quality of life. participants reported a total of falls in the months prior to the initiation of the study and only fall was reported during the month pt intervention. this study makes a strong case for the utilization of skilled pt to address deficits in patients with cmt. additionally, the utilization of objective, valid and reliable outcome measures in this population may help healthcare practitioners establish baseline function and response to change. a large randomized control trial is recommended to study the effects of a specific pt intervention on outcome measures in patients with cmt. ogata h , fujita a , yamasaki r , matsushita t , kira ji . department of neurology, neurological institute, graduate school of medical sciences, kyushu university, fukuoka, japan. clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) patients with autoantibodies against neurofascin (nf) , one of the paranodal proteins, have been elucidated while the relation between anti-nf antibody levels and long-term clinical course in these patients still remains elusive. we retrospectively collected clinical, electrophysiological and immunological data of three anti-nf antibody-positive cidp patients. they were all males and their ages at onset were , , and years old. their clinical severity was evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale. anti-nf antibody levels were measured by flow cytometry using hek cell lines stably expressing human nf . after immunotherapies of various combinations, including intravenous immunoglobulin, plasmapheresis, corticosteroids and other immunosuppressants, were introduced, their clinical parameters were gradually improved. decreased or absent dtrs were normalized and grip strength was increased by more than kg. hughes functional scale scores were decreased by at least one point compared with those at nadir. ncs findings of all three patients also showed obvious amelioration. for example, their f wave latencies in the right ulnar nerve were improved from to ms, from to ms, and from to ms, respectively. anti-nf antibody levels after treatment were decreased in two patients whose pre-and posttreatment sera were available. when dose of oral prednisolone was being tapered, they experienced re-exacerbation of clinical parameters, especially dtrs and grip strength. their ncs findings and serum anti-nf antibody levels were also deteriorated. exacerbation of these laboratory data in one patient preceded his clinical fluctuation, which suggests that ncs and serum anti-nf antibody levels could be used as early disease activity markers. in this case series, not only clinical but also laboratory data support a notion that anti-nf antibody-positive cidp patients were reactive to combined immunotherapies including corticosteroids. even though various treatments were administered to them, efficacy of oral corticosteroids seemed to be dose-dependent. optimal disease activity markers and immunotherapies for long-term maintenance of remission in anti-nf antibody-positive cidp should be identified. evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale, after starting immunotherapies, ohnmar o , kamyw , ng lfp , t umapathi . university of medicine , yangon, myanmar; singapore immunology network, a*star, singapore; national neuroscience institute, singapore. singapore's zika virus (zikv) outbreak started in late august . over a period of months, we studied patients enrolled into our institution's prospective guillain-barré syndrome (gbs) database for relationship to zikv infection. we also studied gbs controls that were seen before the established outbreak and non-gbs controls. the index cases tested negative for zikv pcr in blood and urine. we proceeded to test zikv igg, igm, dengue virus (denv) igg and igm, and neutralization assays against zikv and denv. one patient with anti-gq b igg positive miller fisher syndrome had detectable zikv igm and zikv igg. the serum showed low titre denv igm and denv igg. follow-up serum at about months showed increase in zikv igg. we believe this patient has zikv-gbs. one patient with acute motor sensory axonal neuropathy and another with acute inflammatory demyelinating polyneuropathy had high zikv igm but low denv igm and igg. another patient with mfs showed high levels of zikv and denv igm but low igg. the latter two patients had gbs before the zikv outbreak in singapore. we suspect these patients could have zikv-gbs, but are awaiting convalescent sera for confirmation. two patients seen during the outbreak had detectable levels of zikv igg but serial testing showed a decline after a period of - months. the initial and follow-up sera showed raised denv igm and igg levels in one and raised igg levels in the other. in addition, both had stronger neutralizing capacity against denv than zikv suggesting that the initially detectable zikv igg levels was due to cross reactivity with previous denv infection. four patients, gbs control and non-gbs controls also showed serological response consistent with previous exposure to denv. one normal control showed nil exposure to both viruses. in summary, using various overlapping serological methods we diagnosed definite and suspect zikv gbs cases. our findings highlight ) insensitivity of blood and urine pcr to diagnose zikv-gbs ) the problems of interpreting zikv serology from cross-reaction with denv ) serial serology increases diagnostic accuracy. okar sv , ergunay k , bekircan-kurt ce , , erdem-ozdamar s , , tan e , . department of neurology, hacettepe university, ankara, turkey; virology unit, department of medical microbiology, hacettepe university, ankara, turkey; neuromuscular disease research laboratory hacettepe university, ankara turkey. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated polyradiculoneuropathy. it is believed that acute inflammatory demyelinating poliradiculoneuropathy is caused by t-cell mediated autoimmune response targeting peripheral nerve myelin. molecular mimicry plays a role in the pathogenesis of some gbs cases. this mechanism has been well demonstrated in the acute motor axonal neuropathy (aman) variant, in which autoantibodies to camphylobacter jejuni share epitopes with peripheral nerve gangliosides. this molecular mimicry mechanism can be attributed to some cases with atypical triggers such as zika virus or west nile virus infections. moreover there are accumulating clinical data for vector borne viral infections triggering gbs. we evaluated vector-borne viral infections in our gbs and aman patients. eight patients with gbs, two patients with aman and as a control group, seven patients with normal pressure hydrocephalus were included. gbs and aman was diagnosed with clinical, electroneuromyograpic and cerebrospinal fluid (csf) findings. cerebrospinal fluid serum and urine samples were examined for vector borne viral infections via generic flavivirus and phlebovirus pcr. we also documented our patients prognostic scores such as modified erasmus gbs outcome score (megos) and modified erasmus gbs respiratory insufficiency score (megris). the mean age of the patients was , ( - years) , six of them were female. all csf, serum and urine samples of our patients and control patients were negative for flavivirus and phlebovirus families. the preliminary results of our study in this our small cohort did not show any correlation between the vector-borne viral infections and gbs. further studies with broad number of patients are needed for more suggestive results. neuromyotonia (nmt) consists of spontaneous motor unit activity that reflects increased peripheral nerve excitability, leading to involuntary, persistent muscle activity, visible as muscle twitching at rest, with generalized, easily provoked cramps. since the first electrophysiological (emg) description of nmt by denny-brown and foley ( ) , there has been discussion about the origin of the abnormal electrical activity recorded in needle emg studies. we studied two patients. patient 's nmt is aggravated by cold, and he has an associated non-progressive mild polyneuropathy with demyelinating features. he is now -year-old and has been followed in our centre for years. he is negative for anti-vgkc antibodies. firstly, he was treated with carbamazepine and phenylhydantoin with poor response, but he has shown major improvement on intravenous immunoglobulin (ivig) during the last years. patient is a year-old man with nmt, followed for year, with high titters of anti-vgkc antibodies ( pmol/l; normal< ). he improved on ivig during the last months. screening for neoplasia was unremarkable in both patients (negative anti-neuronal antibodies, in particular anti-hu, anti-yo and anti-ri antibodies; normal computerized tomography scan of the chest and abdomen). in addition to routine studies, we tested synchronicity to spontaneous discharges in different motor units in simultaneous recordings made with two needle electrodes in the first dorsal interosseus muscle. time-locked fasciculations in these double recordings would represent abnormal ectopic activity initiated in a nerve trunk with ephaptic stimulation of a nearby axon. in patient , this research protocol was applied once, years after regular ivig treatment. patient was investigated before and year after ivig. both patients improved after ivig, mirrored by a striking decrease in the amount of spontaneous activity on emg. moreover, our technique did not detect synchronous spontaneous activity (time-locked fasciculations) on the second assessment, although this was predominant before treatment in patient . in nmt, abnormal discharges originate both in distal axonal branches and in more proximal segments. it appears that ivig is more effective in blocking antibody activity in proximal axonal segments, perhaps related to factors such as blood-nerve barrier, temperature or differing ion channel distributions. celiac disease (cd) is a chronic, multisystem and immune-mediated disorder characterized by small-bowel sensitivity to dietary gluten in genetically predisposed individuals. neurological manifestations may occur in about % of patients, including peripheral nerve involvement. recent growing evidence strongly suggests that peripheral neuropathy in cd may be autoimmune and associated with anti-ganglioside antibodies. a -year-old woman presented with slowly progressive weakness of her right hand and fingers extensors. medical and family history were unremarkable. on examination, muscle strength (medical research council-mrc) was scored for right wrist and finger extensors, for right abductor pollicis longus and for right ankle dorsiflexion. right triceps brachii and brachioradialis reflexes were weak, but normal elsewhere. the remainder of the neurological examination was normal. neuroaxis magnetic resonance was unremarkable, specifically with no gadolinium-enhancing lesions. motor and sensory nerve conduction studies were normal. no conduction block or abnormal temporal dispersion was found. needle electromyography showed severe neurogenic changes with abnormal spontaneous activity in right radial-innervated muscles, and chronic neurogenic changes in homolateral tibialis anterior, peroneus longus and extensor digitorum brevis. ganglioside antibody testing was positive to igg anti-gm antibody, but negative to anti-gm and other anti-ganglioside antibodies. additional blood tests were unremarkable, in particular cryoglobulin testing was negative. intravenous immunoglobulin improved weakness, as right extensors of the wrist and fingers scored (mrc). a monthly treatment was initiated, which after months was changed to every weeks to preserve function. a diagnosis of mama was established. later, cd was diagnosed in her daughter due to chronic diarrhoea. our patient underwent anti-tissue transglutaminase antibodies determination and small-bowel biopsy after years disease' duration, and a diagnosis of cd was made. gluten-free diet was started, but her neurological picture did not change after six months. in our case the presence of igg anti-gm antibody may support a causal link between mama and biopsy-confirmed cd, and the lack of response to gluten-free diet may be explained by chronic axonal injury induced by memory t-cells. this case broadens our knowledge about neurological manifestations in cd, raising a probable association with purely axonal multifocal motor neuropathies and anti-ganglioside antibodies. Õunpuu s , , pogemiller k , acsadi g , pierz k , , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa. orthopaedic surgical intervention of the foot and ankle is performed in persons with charcot-marie-tooth (cmt) for improving foot pain, ankle instability, orthosis fitting/comfort issues and shoe wear. the impact of these surgeries on ankle function during gait is not known. therefore, the goal of this study was to measure gait changes in ankle function following surgical intervention by means of computerized motion analysis. fourteen patients with cmt ( ± years pre and ± years post-operative) with bilateral orthopaedic surgery were included. all patients had a plantar fascia release plus some combination of other soft-tissue (posterior tibialis and tendo-achilles lengthenings; extensor hallucis longus, peroneus longus and anterior tibialis transfers) and bony procedures (metatarsal and cuboid osteotomies). all patients completed two gait analyses (pre and on average . years post-surgery) during barefoot walking using a standardized d motion analysis protocol. the changes in ankle kinematics and kinetics and temporal-spatial parameters were analyzed in reference to a control group of patients with cmt without intervening surgeries with . years between gait analyses as well as normal reference data. the surgical group showed a significant increase in height between the pre and post-operative analyses and no changes in walking velocity (pre: ± , post: ± , normal: ± cm/sec). similarly, the ankle kinematics and kinetics showed no changes as a result of surgery. however, there was a trend for increased peak ankle dorsiflexion (pre: ± , post: ± , normal: ± degrees). as in the surgical group, the control group showed an increase in height but no simultaneous increase in walking velocity (test : ± , test : ± cm/sec). the control group also showed no changes in ankle kinematics and kinetics suggesting that the impact of the disease is, for the most part, not noticeable over . years. the results show that surgical intervention that primarily addresses foot alignment does not negatively impact ankle kinematics and kinetics during gait. the comparison with the control group supports this finding. however, there are some other findings such as the increase in peak dorsiflexion during stance in some patients that will need to be examined further in larger cohorts. Õunpuu s , , pogemiller k , pierz k , , , acsadi g , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa. ankle-foot-orthoses (afos) are commonly prescribed for patients with charcot-marie-tooth (cmt) to improve gait and reduce ankle instability and falling. there are no studies that examine bracing outcomes using objective evaluations. the purpose of this study was to examine the impact of various afo designs on gait parameters in children and adolescents with cmt. we predicted that the afos would improve excessive and delayed peak dorsiflexion in stance and equinus in swing. fifteen patients (mean age ± years) were analyzed barefoot and with their prescribed afos by means of a standardized motion analysis protocol. a full clinical examination was also completed including strength and passive range of motion measures. the afos included solid, hinged and posterior leaf spring (tapered, less supportive) designs. sagittal plane ankle kinematics and kinetics and temporal-spatial parameters were analyzed in comparison to normal controls. walking velocity improved from ± to ± cm/sec demonstrating the functional benefits of afos. ankle plantar flexion angle at initial contact improved from − ± to − ± degrees demonstrating improvement in drop foot in swing and at initial contact reducing the risk of tripping. however, the degree of peak ankle dorsiflexion in stance remained the same and excessive at ± degrees barefoot and ± degrees with afos suggesting that in many patients the afo design was not sufficiently supportive. peak ankle plantar flexor moment showed improvement from . ± . to . ± . nm/kg highlighting the improved base of support provided by the afos that compensate for ankle plantar flexor weakness. however, peak power generation was reduced from . ± . to . ± . w/kg indicating that some of the available ankle strength was impeded with the afos. the results of this study suggest that orthoses can provide improved gait outcomes which will improve overall function. however, there are individual differences in patient impairment (strength, range of motion and bony deformity) and associated gait presentation that need to be accounted for in afo design. afos do not always function as intended due to the complex interaction between patient impairment, orthosis stiffness and orthosis design. motion analysis can assist in identifying the specific afo needs for an individual with cmt. painous c , lópez-pérez ma , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, barcelona, spain; hospital san pedro, logroño, la rioja, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune neuropathy with a heterogeneous clinical spectrum. specific autoantibodies define different clinical phenotypes. cidp with nf antibodies constitutes a specific cidp subset in which a high incidence of postural and intention tremor has been described and that responds poorly to immunoglobulins. we report a patient with an anti-nf positive cidp that presented head, voice and limb tremor that improved with immunotherapy. a -year-old man with unremarkable medical history, presented at the age of with progressive distal weakness and paraesthesia. numbness, gait ataxia and action tremor involving voice, head and limbs appeared sequentially. the emg showed features of acquired demyelination fulfilling cidp diagnostic criteria. a first course of intravenous immunoglobulins was ineffective. the weakness, ataxia and tremor worsened significantly, needing two walking aids first and becoming wheelchair bound later on. he received six plasmapheresis cycles that were also ineffective and oral corticosteroids ( mg/kg) were started with mild improvement. after corticosteroid tapering the patient developed a severe relapse and was referred to our centre. five plasma exchange cycles followed by rituximab ( mg/m , doses) were added to the corticosteroids. three months later the weakness, ataxia and tremor, including the head and voice tremor, started to improve significantly. six months later the patient presented a significant improvement and was able to walk unaided. the anti-nf antibody titres fell from / pre-rituximab to be undetectable. limb tremor is known to occur in patients with inflammatory neuropathies and, specifically in anti-nf + cidp patients but, to our knowledge, this is the first report of a cidp patient presenting with head and voice tremor ever reported. the improvement of tremor with immunotherapy strongly suggest that anti-nf autoantibodies are involved in its pathogenesis, expanding the phenotype of anti-nf specific clinical features. palaima p , peeters k , l. pelayo-negro a , garcía a , gallardo e , garcía-barredo r , de vriendt e , infante j , berciano j , jordanova a . vib and university of antwerp, center for molecular neurology, molecular neurogenomics group, antwerp, belgium; university hospital "marqués de valdecilla", santander, spain. charcot-marie-tooth disease type g is an autosomal dominant and slowly progressing inherited neuropathy which was first described over years ago. it has been attributed to a single spanish family consisting of individuals with affected members spanning four generation. initially, the genetic defect was linked to a . mb region in q -q . . however, extensive sequence and structural variant analyses using whole genome sequences (wgs) of three affected individuals did not reveal any known or novel genetic causes within the region. over the last decade, serial clinical re-evaluation of the entire pedigree was performed leading to changes to the affection status of seven individuals redefining the disease-linked region to chr q . -q . (z max = . at theta = ). additional family members were then submitted for whole exome sequencing. this has led to the identification of a novel missense variant in the e ubiquitin-protein ligase lrsam (p.cys tyr) that was previously not covered by wgs. the variants co-segregated with disease and were absent from controls. other mutations that are known to disrupt the ring domain of lrsam have been previously reported to cause both autosomal dominant and recessive cmt type p (cmt p). unlike prior reports, we demonstrated that the mutation does not influence overall protein levels of lrsam , nor of its ubiquitylation target tsg in patient derived lymphoblasts. transcriptomics analysis identified significant upregulation of another e ubiquitin-protein ligase (nedd l) and of a key regulator of axonal degeneration (tnfrsf ). notably, magnetic resonance imaging of lower-limb musculature systematically showed fatty atrophy in both clinical and subclinical mutation carriers emphasizing its use for the identification of mildly affected members. our findings demonstrate that the isolated genetic entity cmt g is caused by a missense mutation in lrsam and should be reclassified as cmt p. moreover, we reveal novel molecular players associated with lrsam dysfunction, and highlight pathways and therapeutic targets shared with amyotrophic lateral sclerosis and alzheimer disease. damage to peripheral nerves is a prerequisite for neuropathic pain. however, it remains unclear why some neuropathy patients have pain, but others with identical nerve conduction, qst and ienfd do not. we propose to examine the distribution of trpv and its co-localization with cgrp and sp in epidermal nerve fibers in patients with and without neuropathic pain. we aimed to define the expression pattern of trpv in normal healthy subjects first, and study the co-localization of trpv with cgrp and sp in normal controls and patients with painful neuropathy. skin biopsies from neuropathy patients and normal subjects were utilized. anti-trpv antibody generated in our university with support from ninds-funded dept. of neuroscience monoclonal core (ns ) was used. combined immunohistochemistry were performed to identify co-expression of trpv with cgrp, sp and pgp . . the distribution of trpv in controls revealed a proximal to distal gradient similar to that observed for ienf labeled by pgp . . trpv staining was more intense in nerve terminals in the epidermis. combined immunostaining revealed that % of pgp . labeled fibers in the epidermis were trpv +, while % of cgrp+ fibers trpv +. patients with pain had a higher density of trpv + fibers compared to that of patients with numbness. a greater proportion of cgrp+ fibers ( %) in the painful patients were trpv +. expression of trpv in controls exhibits a distal to proximal gradient. trpv expression and co-localization with cgrp were altered in neuropathic pain patients, suggesting that this receptor plays an important role in pathological states. activation of the nop-receptor (nop-r) by its endogenous ligand nociceptin/orphanin fq or non-peptide agonists modulates nociception and analgesia in neuropathic pain models. the wide distribution of nop-r and its endogenous ligand represents an attractive treatment target. since pain is the most prominent feature in pc, we defined expression of the nop-r in plantar skin biopsies and assessed whether alterations exist in pc-affected vs pc-unaffected and anatomically matched control skin. skin biopsies from k a pc and control subjects were immunohistochemically stained for nop-r. combined immunostaining for nop-r with pgp . , neurofilament h (nfh) and cgrp was used to define nop receptor expression in the epidermis and upper dermis. robust nop-r was detected in epidermal keratinocytes and a subset of pgp . + fibers in both epidermis and dermis. staining was inhibited through pre-incubation with a nop-r blocking peptide and western blot analysis using homogenized human skin tissue demonstrated a band at ∼ kd consistent with nop-r molecular weight. nop-r expression occurred in dermal nfh+ a beta-fibers in all groups though no cgrp+ fibers co-expressed nop-r. pc-affected skin had slightly lower nop-r expression than in pc-unaffected skin and a similar pattern in anatomically matched locations from healthy control subjects was observed. nop-r is expressed in human plantar skin epidermal keratinocytes as well as a subset of epidermal and dermal nerve fibers. these fibers are pgp . +, cgrp-and many co-express nfh. nop receptor is a viable pharmaceutical analgesic target in pc patients irrespective of its slight down-regulation as compared to pc-unaffected skin. this work was supported by grünenthal gmbh. pareyson d , stojkovic t , leonard-louis s , reilly mm , laurà m , parman y , battaloglu e , tazir m , bellatache m , bonello-palot n , sacconi s , guimarães-costa r , attarian s , latour p , megarbane a , schenone a , ursino g , sabatelli m , luigetti m , santoro l , manganelli f , quattrone a , valentino p , murakami t , scherer ss , dankwa l , shy me , bacon cj , herrmann dn , pisciotta c , previtali s , bolino a . milan, italy; paris, france; london, united kingdom; istanbul, turkey; algiers, algeria; marseille, france; nice, france; lyon, france; genoa, italy; rome, italy; naples, italy; catanzaro, italy; kurashiki, japan; philadelphia, usa; iowa city, usa; rochester, usa. cmt b and b are characterized by recessive inheritance, early onset, severe course, slowed nerve conduction, myelin outfoldings, association with loss-of-function mutations in myotubularin-related protein- and − (mtmr , mtmr /sbf ), respectively, involved in the metabolism of ptdins p and ptsins( , )p phosphoinositides, key regulators of membrane trafficking. in a multicentre retrospective study to better characterise cmt b, we collected a minimal dataset of information including cmtes/cmtns on cmt b patients, cmt b ( unrelated families) and cmt b ( families). cmt b patients were younger and with earlier onset than cmt b : last visit was performed at a mean age of years (sd . ; range - ) for cmt b and years ( . ; - ) for cmt b ; disease onset occurred at a mean age of . years ( . ; - ) in cmt b as compared to . years ( . ; - ) in cmt b ; delay in motor milestones occurred in / cmt b and / cmt b subjects. eleven cmt b patients became chair-bound, whereas all cmt b subjects but one are still ambulant, although with afos for patients and requiring unilateral support in two cases. both disease types are characterised by vocal cord involvement ( / cmt b and / cmt b ); respiratory involvement was seen almost exclusively in cmt b patients (n= , four requiring non-invasive ventilation and one tracheostomy, as compared to one cmt b patient on niv at age ); two cmt b subjects and an affected cmt b relative not included in the present study died of respiratory complications. glaucoma (n= ) and buphthalmos (n= ) occurred only in cmt b . cmtes/ cmtns scores were higher in cmt b patients in spite of their younger age, indicating more severe disease: cmt b = cmtes mean . (n= ; sd . ; range - / ), cmtns mean . (n= ; sd . ; range - / ,; cmt b = cmtes mean (n= ; sd . ; range - / ), cmtns mean (n= ; sd . ; range - / ). our data confirm that cmt b is more severe than cmt b . interestingly, mtmr interacts with mtmr . mtmr is a catalytically active phosphatase, whereas mtmr is a catalytically inactive protein, known to increase mtmr enzymatic activity. thus, in cmt b nerves a residual enzymatic activity of mtmr may result in a less severe clinical phenotype as compared to cmt b . the charcot-marie-tooth disease (cmt) national registry is fully operative (https:www.registronmd.it) with collection of clinical/genetic information (minimal dataset) and reporting of clinical scales; cmt patients have registered thus far and have chosen one of the reference centers; information collected during the ad hoc visit have been entered in the registry for of them. registered patients have the chance to participate to a study that requires filling online self-reported questionnaires related to five important issues: disease course and complications during pregnancy; use, efficacy and tolerability of orthotics and assistive devices; outcome of surgery for skeletal deformities; safety of anesthesia; occurrence of sleep disorders (including evaluation of fatigue, anxiety and depression). by february , patients and relatives/friends (as healthy controls) have filled the questionnaires. we are performing a first explorative analysis of results, but data collection on all questionnaires will be prolonged until novemebr , , to obtain a larger sample. pregnancy: / cmt women had at least one pregnancy; complications ranging from mild to severe occurred in / pregnancies vs / in controls. cmt worsened in pregnancies ( patients) with no recovery in instances. prenatal diagnosis was performed in / pregnancies. satisfaction related to surgical procedures for foot deformities, assessed with vas (score - ), was . ± . (n = ). repeat surgery was required in / instances. sleep: the epworth sleepiness scale questionnaire revealed abnormalities of sleep in / cmt patients ( %) and in / controls ( %). the vast majority of cmt subjects ( / ; mean . ± . ), but also of controls ( / ; . ± . ) were not good sleepers according to the pittsburgh sleep quality index (psqi) (range - , good sleep). fatigue: scores of modified-fatigue impact scale (range - , no fatigue) were higher for cmt (mean ± . ) than controls (mean . ± . ). hospital scale for anxiety and depression: / cmt subjects had mild-to-severe anxiety and / mild-to-severe depression as compared to / and / controls, respectively. data analysis on orthotics and anesthesia is ongoing. in conclusion, the first data analyses confirm that there are problems related to all the five domains explored, that will need to be specifically addressed in patients' care. supported by telethon-uildm grant gup . charcot-marie-tooth disease (cmt) is the most frequent motor and sensory peripheral neuropathy and is known to have the uniform demyelination. the traditional definition of conduction block is the reduction of negative peak of compound muscle action potential (cmap) of proximal segment relative to adjacent distal segment more than %. in this study, we tried to find the frequency of conduction block in cmt a patients and to investigate the differences of the clinical manifestation. we enrolled unrelated cmt a patients ( males and females) with pmp duplication and undertook nerve conduction studies from to . stimulation sites were wrist, elbow and axilla for median nerve study. eleven patients ( . % of all enrolled patients) had ncs features suggestive of conduction block. compared to cmt a patients without conduction block, functional disability scale was significantly higher in cmt a patients with conduction block (p < . ). however, onset age and disease duration were not different between cmt a patients with and without conduction block. in addition, conduction block was more frequently observed in distal segments than proximal segments. we suggest that the frequency of conduction block in cmt a patients is not low, and there is some heterogeneity of demyelination in cmt a patients. also, in cmt a patients, the conduction block might have relationship with clinical disability. park jg , park ms . yeungnam university college of medicine, daegu, korea. the subacute combined degeneration (scd) is diagnosed by a characteristic clinical manifestation, spinal mri, and decreased serum vitamin b levels. we report a case of scd with elevated homocysteine and methlymalonic acid level in the situation of spurious elevation of vitamin b concentration. an years old man presented with progressive gait disturbances and sensory disturbances. about a year ago, he felt both hands and feet paresthesia and gait disturbances. four months before the visit, nerve conduction tests were performed elsewhere due to symptoms. polyneuropathy was found. the laboratory test showed megaloblastic anemia. however, serum vitamin b concentration was increased to pg / ml (normal: - pg / ml). his symptoms progressed gradually. at presented, neurological examination showed spasticity of the lower limb without weakness, and vibration and position sensation were decreased in both lower limbs, and showed ataxic gait. spinal cord mri showed a lesion with a long t high signal intensity from c to t in the posterior column of the spinal cord. further laboratory test were added, then while folate was normal level but homocysteine at . umol/l (normal: - umol/l), and methylmalonic acid at . um / l (normal: - . um / l) were increased. finally, scd caused by vitamin b deficiency was diagnosed. in addition, gastric endoscopy was performed to find the cause of vitamin b deficiency and chronic atrophic gastritis was found. after cobalamine treatment for months, hemoglobin level was improved and his symptoms were all improving with a little gait disturbances. false-elevation of vitamin b level could lead to delayed diagnosis and cause irreversible changes in the nervous systems. one of the most commonly used methods to measure vitamin b concentrations is competitive-binding assay, which may result in normal levels even with vitamin b deficiency due to the effect of anti-intrinsic factor antibodies. when vitamin b deficiency is suspected, even if the vitamin b concentration is normal, additional tests of homocysteine and methylmalonic acid should be considered. parman y , durmus h , deymeer f , oflazer-serdaroglu p , battaloglu e . istanbul university, istanbul faculty of medicine, department of neurology, istanbul, turkey; bogazici university, istanbul, turkey. hereditary motor and sensory neuropathies, also called cmt neuropathies, are the most common group among the hereditary neuropathies. cmt subtypes are grouped by axonal, demyelinating or intermediate phenotype, or by autosomal-dominant (ad), autosomal-recessive (ar) or x-chromosomal inheritance. cmt- is considered axonal and characterized by distal muscle wasting, mild sensory loss and normal or near-normal nerve conduction velocities. mutations in more than genes are known to be able to cause autosomal dominant (ad) or recessive (ar) cmt- to date. the genetic heterogeneity in charcot-marie-tooth (cmt) is a challenge for genetic diagnostics. clinical clues and frequencies of mutations in cmt genes from large cohorts may help to develop strategies for efficient genetic testing. here, we present the clinical, electrophysiological and genetic features of patients from turkey, diagnosed as genetically confirmed cmt- in the department of neurology, istanbul faculty of medicine our clinic between and . genetic testing was performed for gdap by dna sequencing and samples from patients were exome sequenced (wes). twenty-one male and ten female patients from unrelated families were investigated. segregation was ar in eight, ad in six families and two were isolated cases. intraand interfamilial variability in the age of onset with a range of - (mean . ± . years) and disease progression rate were striking. slowly progressive weakness and atrophy of distal muscles in the feet and/or hands were the most common presenting symptoms. mfn was found in four unrelated ad kinship and in unrelated ar kinships and was most common gene mutated among whole cohort. gdap was the most commonly mutated gene among ar families ( / ). wes revealed further mutations in aars, nefl, kif b and hspb , however, the causative mutation could not be identified in known cmt genes in about % of patients. our data indicates the marked intra-and inter-familiar phenotype variability in cmt- as previously described in literature. many more genes causing arcmt- remain to be discovered. pasnoor m , veerapaneni k , murphy r , statland jm , kimple d , hamasaki a , glenn md , herbelin l , barohn rj , jawdat o , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. electrodiagnostic (edx) studies involving electromyography/nerve conduction study (emg/ncs) are useful for diagnosis of neuromuscular disorders. although these are safe procedures, emg and ncs often evoke anticipated pain and anxiety. there are no published research studies to support the notion that providing detailed information to patients prior to these procedures would reduce procedural pain and ameliorate anxiety levels afterwards. the objective of this study was to perform a prospective survey to assess anticipated pain and anxiety in patients presenting for edx studies, design a detailed patient education form, and assess the change in perceived pain and anxiety pre and post procedure in the standard of care (soc) education versus the printed detailed education instrument group. after completing a brief pain/anxiety questionnaire, patients were randomly assigned to either soc verbal education group or to read the detailed education form. another brief questionnaire was completed post procedure. seventy-eight patients were enrolled at the time of abstract submission, in intervention and in soc groups. mean age of patients enrolled was ± years. pain was anticipated by % patients as a visual analog scale (vas) score mean of . ± . with no significant difference between both groups (p= . ). post-procedural pain was reported in % of patients with mean pain score of . ± . and no significant difference between the two groups (p= . ). anxiety pre-procedure was reported by % patients with a mean likert-like score of . ± . score in all cases (p= . between both groups). post-procedural anxiety frequency was reduced to % with mean level of . ± . (p= . between both groups). most patients reported pain with both emg and ncs ( %). we found that edx testing evokes moderate pain in most and in some cases moderate anxiety level. the detailed education intervention did not attenuate the frequency or severity of pain. post-procedural anxiety was expectedly reduced in severity and frequency in both groups with a suggestion that anxiety severity is lesser in the detailed education group but numbers were small. further studies with more detailed questionnaire and perhaps web-based education in larger population may be useful to reduce pain/anxiety in patients presenting for edx studies. pasnoor m , roach c , barohn rj , statland j , jawdat o , dick a , glenn m , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. diagnostic criteria for chronic inflammatory demyelinating polyneuropathy (cidp) have been designed for use in clinical trials. there is limited data on the comparative diagnostic utility of these criteria in the clinic setting and it is unknown if some of these criteria are better for predicting treatment response. the objective of this study is to compare the sensitivity of various diagnostic criteria and review treatment response. after obtaining local irb approval, we performed a retrospective chart review of cidp patients seen at the university of kansas medical center between and . we abstracted the clinical, electrodiagnostic, and treatment information. we determined the frequency of patients fulfilling each of the following criteria: efns , aan, saperstein and incat. we defined treatment response based on treating physician's impression of change, patient-reported functional improvement or one point grade change in the mrc grade. fifty-three cidp patients charts were reviewed, were excluded due to missing data. mean age was . , and ( %) were female. elevated csf protein was seen in / ( %). twenty-eight ( %) fulfilled efns definite criteria, ( . %) efns probable, ( . %) aan, ( . %) incat, and ( . %) the saperstein criteria. treatment response in patients who fulfilled efns definite/probable criteria included / to ivig, / to iv or oral corticosteroids, / to mycophenolate mofetil; among patients who fulfilled aan criteria, these were respectively / , / and / ; among those meeting incat criteria / , / and / ; and for saperstein criteria it was / , / and / . half to two-thirds of patients responded to plex based on different criteria. while all cidp criteria can similarly predict ivig treatment response, the efns criteria are most sensitive for the clinical diagnosis of cidp. pellegatta m , canevazzi p , forese mg , podini p , quattrini a , taveggia c . division of neuroscience and inspe, axo-glia interaction unit, san raffaele scientific institute, milan, italy; division of neuroscience and inspe, experimental neuropathology unit, san raffaele scientific institute, milan, italy. axonal nrg type iii is an essential instructive signal for pns myelination, since its expression determines whether axons are myelinated and also the thickness of the myelin sheath. previous studies have shown that secretases' cleavage controls nrg type iii activity at post-transcriptional level. specifically, the beta-secretase bace- cleavage of nrg type iii promotes myelination, whereas the alpha-secretase tace inhibits pns myelination by inactivating nrg type iii. after a damage, the axon located distally to the site of injury degenerates and loses the myelin sheath, following a process termed as wallerian degeneration. unlike the cns, axons in the pns can regenerate and regrow. several studies have shown that regeneration is favored by the trans-differentiation of schwann cells, which start to create a permissive environment for axonal regrowth. in addition, it has been shown that nrg regulates the early stages of the dedifferentiation process and is essential for creating a permissive environment to regeneration. similarly, the beta secretase bace- promotes pns regeneration and remyelination. in the present study, we analyzed the role of the alpha-secretase tace during wallerian degeneration. our data indicate that tace is upregulated in sciatic nerves after injury, distally to the site of damage. since tace is expressed in schwann cells and axons, we analyzed the role of both glial and neuronal tace during degeneration and regeneration processes. thus, we performed crush injury in sciatic nerves of two different transgenic lines carrying a conditional deletion of tace either in schwann cells (p -cre//tace flox/flox ) or in neurons (chat-cre//tace flox/flox ). the analyses of the different phases of the wallerian degeneration in these animals suggest a role of glial tace during the early stage of the process. in fact, we observed an increased number of myelinated axons only in p -cre//tace flox/flox mice, while we did not detect substantial differences in mutant chatcre//tace flox/flox mice. these results suggest that even during regeneration tace has an inhibitory role, as deletion of tace in schwann cell likely induces the activation of a neuro-protective program that we are currently investigating. péréon y , nizon m , küry s , besnard t , quinquis d , boisseau p , magot a , mussini jp , mayrargue e , barbarot s , bézieau s , isidor b . reference centre for neuromuscular disorders; dept. of genetics; dept. of paediatric surgery; dept. of dermatology, university hospital, nantes, france. the hereditary sensory and autonomic neuropathies (hsan) constitute a clinically and genetically heterogeneous group. hsan are associated with sensory dysfunction, altered pain and temperature perception with varying degrees of autonomic dysfunction, and abnormal small fibers neurodevelopment. more than ten genes have been described so far in the hsan group. original classification encompassed four entities, but additional subgroups continue to be described. hereditary sensory neuropathies (hsan) type ii are characterized by autosomal recessive inheritance, onset at birth and self-mutilating behavior. until now, one homozygous frameshift variant, c.[ _ del], p.(lys phefs* ), was reported in arl ip (adprribosylation-like factor -interacting protein ) in a consanguineous family. patients presented with spastic paraplegia, diffuse sensory and motor polyneuropathy and acromutilation. the disorder was classified as autosomal recessive spastic paraplegia spg . here, we described a new patient with congenital insensitivity to pain, sensory neuropathy, self-mutilation, and spastic paraplegia. whole exome sequencing showed a homozygous frameshift variant c. [ _ del], p.(lys phefs* ) in arl ip . the protein harbours reticulon-like short hairpin transmembrane domains and has a role in endoplasmic reticulum shaping. the variant causes an additional c-terminus hydrophobic domain which could alter its function. arl ip interacts with atlastin- responsible for spg a and hsan type d. this report highlights the role of arl ip in pathophysiology of insensitivity to pain and spastic paraplegia. péréon y , nguyen a-l , leclair-visonneau l , fayet g , nguyen j-m , magot a . laboratoire d'explorations fonctionnelles, hôtel-dieu, nantes, france; département de statistiques médicales, hôtel-dieu, nantes, france. carpal tunnel syndrome (cts) is responsible for sensory and/or motor symptoms that may be increased by wrist flexion or extension (phalen sign). previous studies have shown that short duration (e.g. min or less) flexion or extension was not responsible for significant changes of median nerve conduction parameters. the objective is the study was to determine how prolonged extension (reverse phalen's test) affects median and ulnar nerve conduction parameters in normal subjects. after providing informed consent, normal subjects ( females, age rank - years) underwent motor and sensory testing of both median and ulnar nerves from the dominant side. edx testing was performed using standard techniques with wrist in neutral position, then during passive wrist extension ( ∘ ) for minutes, with sequential recording of both motor action potential (cmap) and sensory action potential (snap) latencies and amplitudes every minutes after extension onset. ulnar nerve conduction parameters remained unchanged in all the subjects. regarding median nerve, groups of subjects could be individualized: subjects with no changes; subjects with only significant decrease of snap amplitude (− %); subjects with both snap amplitude and velocity decrease; subjects with both cmap and snap significant changes. when present, changes did not appear before min, with snap decrease being the earliest observed abnormality. prolonged wrist extension was responsible for median nerve edx parameter modifications in / normal subjects. sensory fibers were firstly affected, corresponding to the chronology clinically encountered in cts. based upon these results, the same protocol will be used in patients presenting with mild clinical cts, in order to try and identify an additional edx technique being useful in the diagnosis of cts. one could also wonder whether the subjects with the most important changes would be more prone to present cts in the future. perez-siles g , , drew a , , ellis m , kidambi m , takata r i , speck-martins c e , hagerman k a , siskind c e , day j w , ginzberg m , nicholson g , , , kennerson m l , , . northcott neuroscience laboratory, anzac research institute, sydney, australia; sydney medical school, university of sydney, sydney, australia; molecular medicine laboratory, concord repatriation general hospital, sydney, australia; sarah network rehabilitation hospitals, brasilia, df, brazil; department of neurology, stanford health care, stanford, ca, usa; pediatric neuromuscular unit, wolfson medical center, holon, israel. mutations in the atp a gene cause of x-linked hereditary distal motor neuropathy (dhmnx). to date, missense mutations (t i and p s) in this copper transporting atpase are the only confirmed mutations causing dhmnx in two independent families. next generation sequencing (ngs), including whole exome and whole genome sequencing, is increasing the rapid detection of variants in genes known to cause disease, however the absence and size of additional families, make it challenging to determine the pathogenic or benign status of variants identified. we have recently identified new variants in atp a in patients with progressive peripheral neuropathy, suggesting further genetic heterogeneity of dhmnx. two of these new variants, pe v and pm v, are located at highly conserved amino acids within domains of atp a (a-domain and p-domain, respectively) that are critical for the catalytic cycle of the copper transporter. we have also identified a third variant (c. - a>g) located bases upstream exon that is predicted to abolish a conserved branch-site, a consensus intronic sequence necessary for the processing of immature rna during splicing. our recent investigations using both patient fibroblasts and an atp a conditional knock in mouse model for dhmnx expressing atp a t i , the orthologue of the human atp a t i mutation, showed reduced atp a protein levels and defective retrograde trafficking of atp a, as pathological hallmarks of dhmnx atp a causative mutations. to elucidate if the newly identified pe v, pm v and c. - a>g atp a variants are pathogenic, we have systematically assessed patient fibroblasts harbouring these mutations for altered parameters previously found in the pathogenic t i atp a mutation. we predict that functional characterisation of atp a using patient fibroblasts harbouring newly identified variants will provide the evidence to ascertain whether these variants are disease causing. establishing a systematic functional readout for atp a variants will improve accuracy of genetic counselling and patient management of dhmnx in those cases where genetic evidence is limited. this study represents a complementary and necessary approach to the use of ngs, for validating the pathogenic status of variants identified and for expanding the genetic heterogeneity of dhmnx. month of birth (mob) have been defined as a risk factor for more than diseases. for instance, susceptibility for multiple sclerosis (ms) seems to be associated with being born in may, while lower risk of ms is observed in those born in november. there are no studies that assessed potential association between mob and a risk for guillain-barré syndrome (gbs). we sought to determine if the risk of gbs is associated with the mob. study included gbs patients diagnosed in serbia, montenegro and republic of srpska in the period from january , until december , . mob of patients and of general population were compared. patients with gbs had tendency to be born in october ( % increase compared to general population, p= . ) and were less likely to be born in june ( % decrease, p= . ). when consider specific seasons, gbs patients were more likely to be born in winter months ( % increase, p< . ). no associations were found between month/season of birth and disease severity. results of this pilot study showed that gbs patients are more likely to be born in cold months, and less likely to be born in june. this might be explained by higher exposure to different pathogens during pregnancy in cold months. in accordance with this, it is well known that early exposure to infective agents reduces risk of allergic and autoimmune diseases. further studies are needed to test our findings in different cohorts and in regions with different climate. these results may shed new light on the disease pathogenesis. majority of patients with guillain-barré syndrome (gbs) tend to have a successful recovery, but in number of them significant long-term consequences may negatively affect their quality of life. aim of this research was to analyze the outcome of the disease one year after the acute episode of gbs. among patients diagnosed with gbs in seven tertiary centers in serbia, montenegro and republic of srpska during , subjects were retested after one year ( % males, mean age ± years). functional disability of patients was estimated based on the gbs disability scale (gds). severe form of the disease (gds score - ) was registered in % of patients at admission, % at nadir, and % on discharge. after one year follow-up period, % of patients had no symptoms of gbs, % had mild symptoms, % was able to walk but not to run, % needed unilateral support during walking, while % was wheelchair bound or bedridden. lethal outcome was registered in three patients during acute phase of gbs and in four more during one-year follow-up. paresthesias were present in % of gbs patients, musculoskeletal pain in %, and fatigue in % one year after acute phase of the disease. factors associated with the worse functional outcome (gds grade above ) after one year were: age above , preceding respiratory infection, and worse gds on discharge. one year after the onset of the disease, significant number of our gbs patients had neurological impairments including sensory symptoms, pain, fatigue and muscle weakness which may significantly affect patients' everyday functioning. phetteplace je , saade d , bacon c , shy me . university of iowa hospitals and clinics, iowa city, ia, usa. charcot-marie-tooth (cmt) disease, also known as hereditary motor and sensory neuropathy, is a heritable peripheral neuropathy caused by genetic mutations in many different genes. cmt type a is the most common form of cmt. individuals affected with cmt a commonly have distal muscle atrophy, foot deformities, abnormal gait, and reduced nerve conduction velocities caused by demyelination of the peripheral nerves. cmt a is known to be caused by a duplication of the pmp gene at p . . the proposed disease mechanism of cmt a is believed to be increased gene dosage of the pmp gene, although the exact function of pmp is not known. there have been previous reports in the literature of individuals who carry a homozygous duplication of pmp , therefore having four copies of the gene. however, many of these reports have focused on adult patients who symptoms may not vary significantly from individuals who harbor only a duplication of pmp . a month old male presented to the university of iowa hospital due to a family history of cmt a and concern for delay in independent ambulation. he was known to have both a maternal and paternal family history of cmt a, although the families were believed to be unrelated. on exam he was unable to walk independently, but could take a few steps when his hands were held. per report he began crawling and scooting at months of age. he was not found to have any structural abnormalities or atrophy in his upper or lower extremities. electrodiagnostic testing revealed a demyelinating neuropathy. only one nerve was tested, as the patient was unable to tolerate additional shocks. the peroneal nerve was found to have a nerve conduction velocity of m/s. based on the clinical presentation, electrodiagnostic examination and family history deletion and duplication testing for pmp was performed. this testing revealed a homozygous duplication of pmp , which has been previously reported to cause cmt a. this case supports the gene dosage disease mechanism, as our patient appears to be more severely affected than a typical infant or toddler affected with cmt a. piscosquito g , saveri p , provitera v , stancanelli a , ciano c , magri s , taroni mpz mutations are associated with the demyelinating early-onset charcot-marie-tooth (cmt) type b and with the axonal later onset cmt i/cmt j. paresthesias/dysesthesias and pain have been already reported in some cmt types and also in cmt i/j. we report patients ( from families, sporadic cases,) carrying the mpz c. dela mutation (p.asp thrfster ). this is predicted to be a loss-of-function mutation, leading to p haploinsufficiency. most of the subjects have genetic ancestry from puglia region (southern italy). two patients carried the mutation in homozygosity, showing a severe phenotype: onset at birth, severe scoliosis, proximal weakness, distal limb plegia, no autonomous walking, cmt examination score (cmtes) - / . all the other heterozygous patients had mild adult-onset (mean age , range - ) neuropathy, walked without aids or ankle-foot orthoses, had cmtes < / . five showed mild foot deformities, / had foot and / hand weakness (mrc never less than / ), / sensory loss in lower limbs (ll). paresthesias/dysesthesias (mostly tingling and burning in type) localized in hands and ll were reported by / subjects, neuropathic pain by / . in the homozygous patients, all cmaps and saps were absent. in all other subjects, motor conduction velocities (cv) were > m/s (range - m/s) and sensory cv > m/s (range - m/s). cmaps as well as saps were normal or moderately reduced in amplitude, indicating only slight axonal loss. we confirm that heterozygosity is associated with very mild cmt, whereas homozygosity causes a very severe neuropathy. our data suggest that paresthesias and neuropathic pain are very common and should be considered as part of the phenotype. although the clinical picture suggests a small fiber neuropathy, no such evidence is provided in the current literature. in the attempt to further investigate this field, we found in a -year-old male patient of our series that all thresholds were increased at quantitative sensory testing, and several abnormalities of small nerve fibers were present in skin biopsy. apparently, p aneuplody causes neuropathy and small fiber involvement through a mechanism different from other mpz missense mutations. poitras t , chandrasekhar a , singh v , martinez j , zochodne dw . neuroscience mental health institute, and division of neurology, department of medicine, university of alberta, edmonton, alberta, canada. a microarray mrna analysis of dorsal root ganglia examined in long-term diabetic mice yielded an unexpected upregulation of a unique set of proteins known as major urinary proteins (mups & ). originally, this family of barrel shaped proteins was described as including mediators of pheromone secretion in the urine of rodents, apparently unconnected to other functions. we identified its unanticipated expression in sensory neurons of adult mice and rats and explored its potential impact on the properties of these neurons. analysis of rat and mouse sensory neurons showed widespread and pan-neuronal cytosolic mup expression, not only in the cell body, but also in distal sensory projections located in the dermis of the footpad. to assess the role that mup might offer in the growth behaviour of adult neurons, we examined sirna-induced knockdown of mup on pre-injured dissociated drg primary cultures. mup knockdown in both species showed a significant increase in neurite outgrowth following mup sirna treatment when compared to a scrambled sequence control. mice treated with streptozotocin (stz) model features of human type diabetic polyneuropathy, including decreases in multi-fiber motor and sensory measurements, and changes in thermal sensation in the extremities. to explore the potential role of mup upregulation specifically in diabetes, we examined the impact of non-viral intranasal injection of mup sirna on indices of neuropathy in chronic type diabetic mice. following treatment, mice treated with mup sirna showed improvement in both motor and sensory nerve conduction velocities compared to scrambled controls. taken together, this data suggests that mup plays a growth-suppressive role in both diabetic and non-diabetic neurons and may also contribute to the electrophysiological and behavioral abnormalities associated with diabetic polyneuropathy. the repurposing of mups in sensory neurons identifies an interesting role for a supposed pheromone protein. [supported by cihr, cda and adi] enhancement of their outgrowth properties (singh et al, ) . here we asked whether similar activation, using low dose capsaicin, might similarly ramp up the regenerative activity of sensory neurons. using rat adult primary sensory neurons, we identified a dose dependent relationship between capsaicin exposure and their outgrowth with enhancement at lower doses and expected neurotoxicity at higher doses. doses that enhanced outgrowth were nonetheless associated with rises in intraneuronal calcium. we next tested whether a similar impact of trpv activation in vivo might exist. we used a high sciatic crush injury mouse model to assess the impact of capsaicin at low ( um) or high ( mm) doses applied directly at the site of axon outgrowth after injury. by days following the single application of capsaicin, low doses of capsaicin showed more rapid recovery of thermal, but not mechanical sensation, whereas high dose capsaicin showed no significant difference compared to vehicle treated mice. in parallel with the neurotoxicity observed in vitro, the high dose capsaicin resulted in a decrease in sensory nerve conduction velocity, while the low dose showed no significant difference when compared to the vehicle treated animals. there was no impact on motor axon recovery. taken together, subtoxic trpv activation with intraneuronal calcium mobilization, as applied to regenerating sensory axons, enhances their outgrowth after injury, and improves behavioural recovery similar to that following extracellular electrical stimulation. [supported by cihr] hereditary transthyretin (hattr) amyloidosis is a rapidly progressive, life-threatening disease caused by ttr gene variants, resulting in ttr misfolding and amyloid deposition in multiple organs, including peripheral nerves. hattr amyloid polyneuropathy has prominent small fiber involvement characterized by neuropathic pain and dysautonomia. measurement of intraepidermal and sweat gland nerve fiber density (ienfd, sgnfd) in skin biopsies has been useful in identifying small fiber abnormalities in hattr. we report the effect of patisiran, an investigational rnai therapeutic for the treatment of hattr, over a -month period on ienfd, sgnfd and dermal amyloid content from the phase open-label extension (ole) study. skin biopsies (distal leg, distal thigh) were obtained every months for up to years within the phase ole study (patisiran . mg/kg iv, q w, nct ) and analyzed in a blinded manner by a central laboratory. all analyses are exploratory; nominal p-values are reported without adjustment for multiple testing. twenty-seven patients enrolled; preliminary data indicated patisiran was generally well tolerated; resulted in sustained mean serum ttr lowering of ∼ % for > months and a mean . -point decrease in mnis+ (n= ). twenty-four patients underwent serial skin punch biopsies. at baseline, mean ienfd was . and . fibers/mm, while mean sgnfd was . and . m/mm for distal thigh and distal leg, respectively. amyloid was detected in ∼ % of skin biopsies, mean baseline dermal amyloid content . % and . % for distal thigh and distal leg, respectively, which inversely correlated with ienfd and sgnfd. overall, ienfd was stable throughout the -year treatment. sgnfd increased over time relative to baseline, reaching statistical significance at , , and months for distal thigh (p< . , all time points) and at months for distal leg (p= . ). dermal amyloid content decreased over time and reached statistical significance at , and months for distal thigh (p< . , all time points) and at , , , and months for distal leg (p< . , all time points). in summary, improvements in sgnfd and dermal amyloid content observed over a -year period suggest that these parameters have the potential to serve as biomarkers of response to patisiran treatment. polydefkis m , neuhaus s , doherty l , ebenezer gj . department of neurology, johns hopkins university, baltimore, md, usa. misdiagnosis and delayed identification of an etiology for sensory neuropathies hampers adequate management and delays initiation of therapy. we present several cases that illustrate variable presentations of amyloid neuropathy (an) and an complicated by amyloid arthropathy. a -year-old former college athlete developed painless difficulty walking. he was unable to keep up with family members during routine outings and subsequently was unable to navigate uneven ground during a hike, something he had previously excelled at. balance decreased, making bicycling difficult. neurological evaluation -months after symptom onset led to a diagnosis of peripheral neuropathy. a reversible neuropathy panel was normal though a c was elevated ( . %). over several months, his strength/sensation deteriorated despite diet improvement, losing lbs and a c improvement ( . %). upon referral, emg revealed a sensorimotor neuropathy with markedly reduced/absent sural, peroneal and tibial motor amplitudes. there was mild orthostasis. nis was . skin biopsies revealed severe ienfd, sgnfd reductions and positive congo red staining, confirmed with birefringence. a sural nerve biopsy confirmed amyloid deposits. genetic testing revealed the ttr-fap variant leu his. there was no history or suspicion for a family history of amyloid. a -year-old male with a history of controlled diabetes x years developed abrupt onset of rls months after a lymphoma diagnosis. treatment with rituximab led to radiographic resolution of the lymphoma though the patient developed progressive peripheral neuropathy confirmed by ncv/emg. new-onset early satiety, mild orthostasis and ed emerged. skin biopsies demonstrated a lichenoid pattern of amyloid deposition consistent with aa-amyloid. a -year old woman with leu his ttr-fap since developed progressive leg weakness, reduced ability to walk and low back pain. emg demonstrated progression of her sensorimotor neuropathy and superimposed multilevel radiculopathy. spine mri demonstrated severe lumbar stenosis requiring surgical decompression. postoperatively, she regained strength, walking more easily. congo red staining of vertebral bone demonstrated widespread positive staining with birefringence consistent with amyloid arthropathy. these cases illustrate to wide sprectrum of amyloid neuropathy presentation, an example of amyloid arthropathy-induced spinal stenosis and the utility of skin biopsy to diagnose and manage amyloid neuropathy. the differentiation between hereditary neuropathy and chronic inflammatory demyelinating polyneuropathy (cidp) in children is especially significant because of completely different treatment possibilities and prognosis in these conditions. the aim of the study was to compare electrophysiological abnormalities in a group of children and young adults with demyelinating neuropathy of chronic or subacute onset. retrospective analysis of clinical and nerve conduction study (ncs) data included children and young adults with charcot-marie-tooth neuropathy x type (cmtx ), children with charcot-marie-tooth neuropathy type a (cmt a) and children with cidp. in our study / cmtx had both axonal and demyelinating changes in ncs study. the aan and efns electrophysiological cidp criteria were fulfilled in / cmtx , / cmt a and / cidp patients. additionally patients with cmtx are classified with efns criteria as "probable/possible cidp". a distal compound muscle action potential (dcmap) was > ms in all cmt a and / cidp patients but none with cmtx . abnormal median/normal sural snap (amns) parameter was observed in // cidp and / ctmx patients but not any cmt a patient while abnormal sural/normal median snap (asnm) parameter was found in / patients with cidp. a difference between conduction velocities (cv) of two corresponding nerves > m/s was seen in / cmtx and / cidp patients but no one with ctm a. one patient with cmtx had especially conspicuous difference between nerve conduction in lower limbs, with axonal neuropathy and demyelinating features, and upper limbs with no changes. one -years-old patient with genetically confirmed cmtx had no abnormalities in ncs. electrophysiological data of cmtx , cmt a and cidp indicate diffuse demyelination, however in cmtx axonal changes coexist, seen in / patients, and asymmetrical abnormalities between corresponding upper and lower limb were observed in / patients. our study has showed that duration of dcmap is useful not only in diagnosing an inflammatory neuropathy but also in differentiating cmtx from cidp. however presence of the not homogenous abnormalities in ncs in patients with cmtx may mimic inflammatory neuropathy. prada v , mori l , francini l , accogli s , ursino g , gemelli c , schizzi s , grandis m , bellone e , mandich p , schenone a . department of neurosciences, rehabilitation, ophthalmology, genetics and maternal and child health, university of genoa, genoa, italy. the overwork weakness (ow) problem in cmt disease has been debated for long time. it has been reported that the non-dominant hand (ndh) of patients with cmt disease is stronger than the dominant hand (dh) as a result of ow and some authors verified this hypothesis using mrc on different muscles (van pomeren, ). more recently, piscosquito et al. ( ) found that the ow phenomenon does not exist using a dynamometer and the hole peg test, a dexterity test. we recruited cmt patients and healthy controls. we evaluated the strength of the -pinch and of the hand-grip with a dynamometer, the opposition ability with the thumb opposition test (tot) and applied an instrumental testing of hand function using the sensor engineered glove test (segt), which previously demonstrated its sensitiveness to measure severity of hands dysfunction in cmt patients. tot was significantly higher in the ndh compared to dh (ndh= , ± , ; dh= , ± , , p< . ) in cmt patients, instead there was no difference in dh and ndh in healthy controls. in the evaluation of -pinch and of the hand-grip, ndh performed slightly but not significantly better than the dh ( -pinch: dh= , ± , n; ndh= , ± , n; ± , n; ndh= , ± , n, p:ns) . in normal controls we confirmed the % rule (noguchi & demura, ). finally, segt results were similar between the ndh and dh in cmt patients but in normal controls there was a better performance in the dh. in conclusion, this study supports the existence of the overwork weakness in cmt, evident in every measurements. dexterity and overall ability of the hands impaired in the dh, probably because of compensating movements, compared with the healthy controls in the weaker hand. finally, our results support the importance of avoiding supramaximal exercises and educating the cmt patients to prevent incorrect movements, which may overload the hand muscles. prasad k , ohnmar o , teng a , cheng yj , umapathi t . national neuroscience institute, singapore; yangon general hospital, yangon, myanmar; yong loo lin school of medicine, national university of singapore, singapore. antecedent infections that have been linked to guillain-barré syndrome (gbs) include campylobacter jejuni, haemophilus influenza, epstein-barr virus, cytomegalovirus, mycoplasma pneumoniae and influenza a virus. in south-east asia, the burden of mosquito-borne flavivirus infections is considerable. even in a small country like singapore, with a population of only . million and in spite of excellent sanitation and public health measures, dengue virus (denv) infections can exceed , a year. both symptomatic and asymptomatic denv infections are associated with gbs. zika virus (zikv) infections were detected in singapore in late august . doctors working in south-east asia anecdotally report that gbs cases increase after rainy season when mosquitos breed. we are currently planning studies to estimate the hitherto unknown burden of gbs associated with denv, zikv and other mosquito-borne viruses in this region. as a preliminary step, we reviewed the gbs cases seen at our institution in for evidence of denv and zikv. three out of the tested cases were positive for denv serology. one of these was also positive for denv pcr. all these patients had symptomatic dengue. two had acute inflammatory demyelinating polyneuropathy (aidp) and acute motor axonal neuropathy (aman) subtype of gbs. none the patients tested were positive for zikv pcr in serum and urine. one patient, with miller fisher syndrome (mfs), had serological evidence of recent zikv. he did not have clinical zikv symptoms. in mfs cases, the initial detectable zikv igg was later proven to be due to cross reactivity with previous denv infection. in cases ( amsan, aidp, mfs) the initial zikv igm was raised. we await analysis of convalescent sera to decide if they indeed had zikv or the results were related to previous and co-infection with denv. our preliminary findings indicate that flavivirus infections may account for at least some of the gbs cases in singapore. the lack of symptoms in some cases and the interactions between zikv and denv antibodies make accurate diagnoses challenging. prior r , , benoy v , , vanden berghe p , van den bosch l , . ku leuven-university of leuven, department of neurosciences, experimental neurology and leuven research institute for neuroscience and disease (lind), leuven, belgium; vib-center for brain & disease research, laboratory of neurobiology, leuven, belgium; laboratory for enteric neuroscience, translational research center for gastrointestinal disorders, k.u. leuven, leuven, belgium. charcot-marie-tooth disease (cmt) is the most common inherited neurodegenerative disorder of the peripheral nervous system. it is divided into two main subtypes, a demyelinating subtype (cmt type ) and an axonal form (cmt type ). cmt is a length-dependent disease, in that it effects the longest neurons in the body, thus the muscles in the peripheral regions are affected first and foremost. moreover, the majority of cmt patients share a classical phenotype with shared pathological hallmarks, such as distal muscular atrophy, reduction in nerve conductions, etc. but also molecular pathological hallmarks, like the breakdown in the transport of organelles and vesicles in neurons in a process called axonal transport. currently, there is no cure or effective treatment available to cmt patients. histone deacetylase (hdac ) has been shown to be a key regulator in axonal transport. moreover, inhibiting hdac has been demonstrated to stabilize microtubules, which act as molecular tracks for motor proteins and facilitates axonal transport of cargos. our labs current focus is on cmt type a, the main cause of cmt, which is caused by a duplication of a segment of chromosome p . containing the gene encoding peripheral myelin protein (pmp ). pmp is mainly expressed by schwann cells, the cells that myelinate neurons in the peripheral nervous system. in the work presented, the commercial mouse schwann cell-line, sw cells, was transfected to overexpress pmp using a lentivirus vector system to mimic that of cmt a patient schwann cells. the overexpression of pmp was confirmed using immunofluorescence and western blot techniques. these transfected sw schwann cells were then co-cultured with primary mouse dorsal root ganglion neurons (drgs) isolated from adult mice. these co-cultures where then analysed after weeks in vitro for axonal transport. the investigated groups were: a co-culture of drgs + sw cells, a co-culture of drgs + sw cells transfected with pmp , and a co-culture of drgs + a sw cell-line transfected with green fluorescent protein using a lentivirus vector system, and a monoculture of drgs. furthermore, this work demonstrates that the overexpression of pmp in schwann cells can impair axonal transport in co-cultured drgs in comparison to the other co-culture groups. moreover, these axonal transport defects were able to be rescued by the treatment of a hdac inhibitor, tubstatin a. to conclude, selective hdac inhibitors have been shown to be a beneficial treatment for a number of cmt subtypes in preclinical studies in our lab and offer as a viable treatment for a currently, untreatable debilitating disease. provitera v , caporaso g , stancanelli a , piscosquito g , di caprio g , saltalamacchia am , santoro l , nolano m . "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; department of neurosciences, reproductive and odontostomatological sciences, university 'federico ii' of naples, naples, italy. the observation of rate and patterns of cutaneous nerves regrowth after mechanical or pharmacological distal axonotmesis, has proven to be an excellent tool to study human nerve regeneration in normal and pathological conditions. most of the observations, however, are referred to small fibers, possibly due to the availability of excellent models of reversible distal cutaneous small fiber axonopathy. no such model is available to study the regeneration of myelinated fibers in human. we studied regrowth of cutaneous large fibers in fingertip of patients with carpal tunnel syndrome after surgical decompression. we recruited patients (m/f / ; age . ± . years) with carpal tunnel syndrome candidate to surgery. patients underwent clinical and electrophysiological evaluation, quantitative evaluation of discriminative threshold at rd fingertip. in the same site, patients also underwent mm punch skin biopsy. skin sections were stained by immunohistochemical techniques and cutaneous innervation was analyzed by confocal microscopy. meissner corpuscles and their myelinated afferences were assessed following previously published procedures. twelve months after surgery, patients repeated functional evaluation and underwent a second skin biopsy two mm apart from the first one. mean density of mcs/mm was . ± . at time and . ± . at follow-up, mean density of myelinated endings was . ± . at time and . ± . at follow-up. however, not in all patients regeneration occurred. based on the variation of mc density between time and follow-up we were able to identify patients in which active regeneration had occurred. in this subgroup mean density of mcs/mm was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . in the same group, mean density of myelinated endings was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . we describe morphological patterns associated to nerve regrowth in the biopsies of patients in which nerve regeneration occurs. we propose an in vivo model to study regeneration of large myelinated fiber endings in human skin. in addition to the count of nerve endings, the identification of patterns associated to nerve regeneration can increase the diagnostic yield of skin biopsy. the most common type of charcot-marie-tooth disease is caused by a duplication of pmp , leading to dysmyelination, axonal loss and progressive muscle weakness (cmt a). currently, no approved treatment for cmt a patients is available. among others, preclinical therapeutic approaches aim to correct the pmp overexpression in order to ameliorate axonal loss and muscle weakness. we previously reported that the novel polytherapeutic drug pxt , a low-dose combination of baclofen, naltrexone and sorbitol, improved dysmyelination and axonal loss after long-term application in adult pmp transgenic rats, a known animal model of cmt a. interestingly, after short-term application in young cmt a rats we observed a long-lasting prevention of muscle weakness as well but without obvious effects on dysmyelination and axonal loss. improved distal motor latencies in the electrophysiological recordings and a shift in the axonal diameter distribution raised the hypothesis that therapy may improve the neuromuscular endplate pathology as another therapeutic target for cmt a. here, we report a preclinical trial in adult cmt a rats treated from postnatal week for consecutive weeks as the most effective regimen in order to facilitate a deeper understanding of the mode-of-action of pxt . long-term therapy effects were confirmed by an ameliorated behavioral phenotype, improved distal motor latencies and also nerve conduction velocity in the electrophysiological recordings. histological analysis revealed an increased number of neuromuscular endplates, which were lowered to wildtype level after pxt treatment. further investigations include detailed analysis of peripheral nerve myelin thickness, internodal and nodal length, terminal axonal sprouting at the neuromuscular endplate and muscle fiber subtyping in treated cmt a rats. charcot-marie-tooth disease (cmt) is a rare hereditary neuropathy for which novel treatments are being developed and urgently needed. only few clinical trials and natural history studies have been performed. evaluation of intervention efficacy is hampered by slow progression and lack of sensitive outcome measures. the interindividual disease variability is high and prognostic disease measures are not established for cmt patients. previously, we have identified skin-derived disease and progression biomarkers in a large european and us-based cmt a cohort. within the german charcot-marie-tooth disease network (cmt-net, www.cmt-net.de) we aim to establish easier accessible biomarkers in blood of cmt a patients. blood analysis is less invasive and can be repeatedly performed during clinical trials and routine patient care. we have started clinically assessing approximately young, adolescent and adult cmt a patients at clinical centres (münster, münchen, göttingen) including a large set of clinical outcome measures (cmtnsv , onls, mwt, mwt, walk , hpt, sf- , fss, psqi, ess, bdi-ii). we will sample blood and skin tissue once a year at , and months for an observational period of years. tissue samples will be analyzed on the transcriptional, translational and epigenetic level. we envision that the diagnostic and progression biomarkers may be used to measure therapeutic effects within clinical trials and later in clinical routine monitoring. this is the first trial testing "circulating" biomarkers from blood in a prospective observational trial in cmt a patients. igm peripheral neuropathy is a slowly progressive and heterogeneous disease with symptoms ranging from mild foot numbness and minor imbalance to severe neuropathic pain and sensory and motor dysfunction. international consensus regarding assessment and treatment of patients with igm peripheral neuropathy is lacking. this might be caused by the repeated use of suboptimal outcome measures, the small trial sizes with low numbers of treated patients, the indolent disease course needing a longer follow-up period to capture relevant changes, or the possibility that administered treatments were not aggressive enough. the imagine study is an international, multi-centre, prospective, observational cohort study, which will result in a unique collection of a large number of prospectively collected and highly standardized clinical data, and a biobank from a large population of well-defined patients with igm peripheral neuropathy. the main objective is to describe in detail the variation in clinical subtypes, clinical disease course, past and current practice of treatment, antibody titres, and clinical picture at the various levels of assessing outcome. patients being at least years, and fulfilling the international criteria for igm peripheral neuropathy are eligible. exclusion is primarily based on concomitant diseases influencing peripheral nerve function. several study parameters measuring weakness, sensation, activity and participation, ataxia, pain, and quality of life are of interest. in february an enmc kick of meeting is organized, bringing together an igm peripheral neuropathy study group. the meeting will focus on the development of a core set of outcome measures to be used in future studies in igm peripheral neuropathy. the imagine study started in the netherlands in september , and to date patients were included. the imagine study starts in the united kingdom, italy, and france in the beginning of . centres recruiting at least patients with igm peripheral neuropathy are eligible to participate in the study. during the pns global recommendations from the enmc meeting, as well as the first results of the imagine study, will be presented. puget s , paolantonacci p , burlot l lfb biomédicaments, les ulis, france; lfb biotechnologies, les ulis, france. in dysimmune neuropathies patients (mmn, cidp, gbs), ivig have become the gold standard due to their efficacy and tolerability. acute renal impairment, a rare but serious adverse event, can be induced by all ivig. its incidence is not determined precisely for each ivig. it mostly occurs in patients at risk, with pre-existing conditions (prior renal insufficiency, diabetes, mellitus, arterial hypertension, elderly > years old, dehydratation, hypervolemia, sepsis, paraproteinemia or concomitant use of other nephrotoxic drugs) and treated by immunomodulatory doses. as the number of these patients increases over the years, the choice of ivig in association with the precautionary measures will be decisive to reduce or avoid the occurrence of this adverse event. from s to s, the nephrotoxicity of ivig has been explained by tubular toxicity related to osmotic nephrosis. sucrose, a stabilizer of some ivig, has been implicated as one of the causes of this. even if several new ivig without sucrose are used, renal impairment cases have been described and studies have highlighted that sucrose is not the only ivig stabilizer associated with tubular damage. however, in a recent study, renal impairment cases ( / ) have occurred in cidp elderly patients treated by free-sucrose ivig. the occurrence of renal failure seemed to be low (only / ) for patients treated with sucrose-ivig in association with precautionary measures (hydratation, adaptation of dosage according body mass index, rate infusion). otherwise, since s, cases of renal impairment secondary to hemolysis have been increased with some ivig but would be rare for full ethanolic fractionated products as they have been identified to generate very low occurrence of hemolysis. the occurrence of renal impairment related to ivig seems to be multifactorial, need to find out all causes (mechanisms of different stabilizers, hemolysis, and concentration of ivig). registries would be helpful for physicians to define the incidence, relative risk of ivig-related renal impairment and the right ivig in association with precautionary measures in these patients at risk. the published ultrasound (us) studies on chronic inflammatory demyelinating polyradiculoneuropathy (cidp) report diffusely increased cross-sectional area (csa) of nerves. these data have not been correlated with histological patterns. to date, no further information about us ultrastructural nerve modification has been provided because of limited frequency range ( - mhz) of the us probe. the aim of this preliminary study is to evaluate the correlation between histological findings from sural nerve biopsies and us patterns found with uhfus ( mhz) from sural nerve at the ankle. four patients with cidp underwent uhfus nerve evaluation of clinically affected sural nerve before undergoing a sural nerve biopsy. us findings included: cross-sectional area of the nerve, connective tissue depth and changes in echogenicity of fascicles. those data were then compared to the histological findings obtained in transversal and longitudinal sections. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened and in two of the four patients hyperechogenicity of fascicles was observed. also, thanks to the ultra-high-resolution of the probe, a direct correlation of the histological and us images was possible, so as a direct measurement of internal microstructure of the nerve. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of sural nerve can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue, as confirmed by the histological study. in particular, we have observed a hyperechogenicity of fascicles in most severe patients; in those cases, histology confirmed an increase of endoneurial depth. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed and will be presented during the meeting. puma a , cambieri c , panicucci e , desnuelle c , raffaelli c , sacconi s . high-resolution ultrasound (hfus, mhz) of peripheral nerves is a valuable non-invasive, painless complement to neurophysiology, especially in the workup of cidp. nevertheless, the current spatial resolution of echographic images doesn't allow a detailed study of the nerves. ultra-high frequency ultrasound (uhfus, mhz) is a new tool with a - times higher spatial resolution than traditional hfus. the aim of this preliminary study is to evaluate sensory and motor nerves structural characteristics found with uhfus in patients with definite cidp. seven patients with cidp underwent uhfus nerve evaluation of median, ulnar, peroneal and sural nerves, bilaterally. us findings included: cross-sectional area of the nerve, connective tissue depth, nerve vascularization and changes in echogenicity of fascicles. patients also underwent electroneurography (eng) and plexus mri. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened. two of the seven patients presented an epineural hypervascularization, observed at the doppler evaluation; none of them was treated by iv immunoglobulines. echographic changes were present even in the absence of mri abnormalities (root hypertrophy). eng characteristics correlated with the us patterns. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of nerves can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue. in particular, uhfus allows a more detailed study of nerves, demonstrating that the structural abnormalities hit connective tissue, while -conversely to previous studies -fascicles anatomy seems to be spared. we did not show nerve vascularization except in non-treated patients. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed. purcell l , , wojciechowski e , , gibbons p , , jamil k , , menezes, m , , burns j , . sydney children's hospitals network (randwick and westmead), new south wales, australia; university of sydney, new south wales, australia; universiti kebangsaan, kuala lumpur, malaysia. - % of children with cmt are reported to have hip dysplasia. we aimed to investigate the relationship between radiological hip dysplasia indicators and walking pattern in children with cmt. thirty children ( female; . ± . yrs; ± . cm; ± . kg cmt a, cmtx , cmtx , cmt f, cmt a) underwent d gait analysis ( dga), and had an anterior-posterior pelvis radiograph within months of assessment. radiographs were reviewed by two orthopaedic surgeons, and the reliability of measures of dominant limb hip health via radiograph was assessed. of the measures, measures had an intraclass correlation coefficient > . between raters, and the more experienced surgeon's measures were used for further analysis. the measures of acetabular index (ai), centre edge angle (cea), neck shaft angle (nsa), medial joint space, head width, lateral uncoverage, migration percentage and triradiate were used to investigate correlations with kinematic and kinetic dga variables of the pelvis and dominant limb hip, knee and ankle in planes, temporal spatial parameters and gait profile score. dga data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model. gait data were compared to typically developing children ( female; . ± . yrs, ± . cm, ± . kg). five of affected children had a migration percentage > ∘ , representing % of our sample. maximum hip abductor moment in terminal stance was significantly lower than normative reference values, and was moderately correlated with of the radiographic measures (ai r=− . , p= . ; nsa r=− . , p= . and medial joint space r=− . , p= . ). walking speed (normalised to leg length) was correlated with medial joint space (r= . , p= . ) head width (r=− . , p= . ) and triradiate (r=− . , p= . the ganglionopathies are a unique group of disorders with a varied etiology which includes autoimmune disorders, paraneoplastic syndromes and toxin induced causes. it has been observed that despite extensive investigations the cause of a sensory neuronopathy is often idiopathic in approximately - % cases. electrodiagnostic criteria to diagnose a possible ganglionopathy requires at least one sensory action potential absent or three sensory action potentials < % of the lower limit of normal in the upper limbs plus less than two nerves with abnormal motor nerve conduction study in the lower limbs. ulnar sensory-motor amplitude ratio values (usmar) lower than . is useful in differentiating ganglionopathy from axonal length-dependent neuropathy. we performed a retrospective analysis of patients who were either admitted or evaluated in the outpatient department in department of neurology, nizam's institute of medical sciences, with a possible ganglionopathy as per the diagnostic criteria proposed by camdessanchéet al. consecutive patients who sought treatment during a year period with a clinical pattern of sensory neuronopathy were analysed retrospectively. we reviewed the electrodiagnostic studies of patients and calculated the ulnar sensory-motor amplitude ratio values in comparison with patients with a clinical and electrophysiological diagnosis of diabetic axonal sensorimotor neuropathy. the clinical profile of a ganglionopathy from the indian subcontinent comprised of a prominent sensory ataxia as the initial presenting clinical manifestation with a predilection towards involvement of large-fiber sensory modalities. electrophysiologically a pattern of absent snaps was characteristically encountered with a significant value of usmar less than . , enabling differentiation from axonal length-dependent neuropathy. rahman im , jahan it , nahar s , khalid mm , jahan i , hayat s , , , islam z . guillain-barré syndrome (gbs) is a post-infectious autoimmune polyradiculoneuropathy where immune response is triggered by molecular mimicry between glycolipid antigens expressed by an infective agent such as campylobacter jejuni (c. jejuni) and human peripheral nerve gangliosides. cd molecules are mhc-like structures specialized in capturing and presenting a variety of glycolipids to antigen-specific t lymphocytes. the objective of this study was to investigate the possible role of coding region polymorphisms of cd genes in the pathogenesis of gbs in bangladeshi population. single nucleotide polymorphisms (snps) of exon of cd a (* /* ) and cd e (* /* ) in well defined gbs patients and healthy controls were studied to delineate their effect in developing gbs. we genotyped exon of both cd a (cys/tryp) and cd e (gln/arg) genes through pcr-rflp. to validate these findings, direct sequencing of pcr product was performed for at least samples for each position. we found no differences in genotypes and allele frequencies of both genes in gbs patients compared to controls. however, compared to control individuals with cd a* /cd e* haplotype were . times more likely to develop gbs, whereas individuals with cd a* /cd e* haplotype had a reduced relative risk by . times. a positive association of cd a* /cd e* haplotype was observed only in axonal form of gbs ( . % vs. . %, p = < . ). haplotype cd a* /cd e* was prevalent among anti-gm antibody positive gbs patient compared to anti-gm antibody negative patients ( . % vs. . %) though it was not statistically significant. snps in cd a and cd e were not associated with antecedent c. jejuni infection, disease severity and disease outcome at months of follow up. in conclusion, cd polymorphisms are not a susceptibility or disease causing factor in gbs. conversely, increasing knowledge of this field may offer new dimension for the research to elucidate better answer for disease pathogenesis and also contribute to conduct high power meta-analysis. extensive genetic testing was performed on a unique patient, who received national media attention due to her severe cmt phenotype. the index patient was never able to walk independently, had difficulty breathing and swallowing and demonstrated aspiration by a barium swallow study. nerve conduction velocity testing revealed velocities of m/s in her upper extremity. she required continuous positive airway pressure (cpap) for an upper respiratory infection and developed bilateral vocal cord paralysis. we used the combined brief assessment of motor function (bamf) scale to evaluate her disability. her fine motor scale was a / , her upper extremity gross motor scale was a / and her lower extremity gross motor scale was / . unfortunately, she died at age in her sleep, presumably from respiratory arrest. the proband's father was asymptomatic, however, his neurological exam showed pes cavus bilaterally and mild atrophy of the first dorsal interossei muscles in his hands. his nerve conduction velocities were slowed to m/s in the upper extremities and his cmt neuropathy score (v ) was / . we performed whole exome and subsequently whole genome sequencing in the trio. comprehensive structural variant analysis for copy number variations, large deletions, and recombinations was completed by a combination of software tools. known cmt genes were excluded as the underlying cause and the only viable candidate gene remaining was sh tc . we showed expression of sh tc in peripheral nerve and schwann cells. sh tc is a paralogue to sh tc , which causes the recessively inherited dysmyelinating form cmt c. sanger sequencing confirmed the variants in our family, p.v m (c. g>a, chr : ) and p. a p (c. g>c, chr : ) , and showed the segregation of the heterozygous variations. we hypothesize that the heterozygous paternal allele had a minimal effect and let to a very mild or subclinical form of peripheral neuropathy. of interest, the recessive sh tc gene has also been shown to cause mild aberrant forms of peripheral nerve degeneration in carriers of heterozygous disease alleles. in summary, this paper will present genetic and molecular evidence from an extensive n= study proposing a novel cmt gene. charcot-marie-tooth (cmt) is an important cause of morbidity worldwide. it is a heterogeneous disease manifesting as progressive weakness, wasting and loss of feeling in a length-dependent pattern. there are an increasing number of cmt-related genes in the literature. more recently, recessive mutations in the hint gene have been reported as causative of predominant motor axonal neuropathy associated with neuromyotonic discharges on emg. one of the characteristics of autosomal recessive cmt is it varying frequency in different populations and ethnic groups. we sought to evaluate the frequency of mutations in the hint gene in a brazilian cohort with axonal hereditary neuropathy. all patients included in this study born within south east area of brazil. the group consists of consecutive patients with axonal neuropathy screened for recessive or sporadic axonal neuropathy in the neurogenetics laboratory of clinical hospital of ribeirão preto. direct sequencing of the coding region of hint gene was done. among patients screened, were suspected of having recessive axonal neuropathy without neuromyothonic discharges, and two have axonal neuropathy associated with neuromyothonic discharges on emg. we did not find any disease causing mutations among our patients. some previously reported studies reported a high frequency of mutations in the hint gene among recessive axonal neuropathies in some european countries. our results demonstrated that frequencies of mutation underlying genetic hereditary neuropathies are different between different ethnic groups and have implications for the organization of services management of cmt, for genetic counseling, and for gene flow in different world populations. funded by cnpq, fapesp, faepa, pronas (ministry of health). rodriguez-menendez v , ballarini e , malacrida a , , ceresa c , semperboni s , , meregalli c , cavaletti g , nicolini g . school of medicine and surgery, experimental neurology unit, university of milano-bicocca, monza, italy; phd program in neuroscience, university of milano-bicocca, monza, italy. bortezomib (btz) is a proteasome inhibitor widely used for multiple myeloma treatment. btz induced peripheral neuropathy (bipn) is the most frequent adverse effect. bipn in humans, is a dose dependent painful sensory neuropathy characterized by nerve axonopathy and a tendency to recover in the follow-up period after btz withdrawal. bipn affects principally dorsal root ganglia (drg) and different rodent models have shown alterations in sensory neurons, small unmyelinated axons, large myelinated axons, axonal mitochondria and schwann cells. in this work, we evaluated the effects of btz in vitro on drg neurons isolated from adult mice. our interest focused on dystonin, a protein able to interact with all the three components of cytoskeleton (microtubules, microfilaments and intermediate filaments) and able to bind map b (a mt-associated protein) through which can influence golgi apparatus morphology. there are different neuronal isoforms of dystonin and in particular dystonin-a is able to modulate tubulin acetylation and stability through interaction with map b. it is noteworthy to underline that map b expression in the mature nervous system is restricted to sensory neurons. western blot analysis demonstrated that a treatment with nm btz induces a statistically significant decrease in tubulin acetylation. this result does not go along with our previous study where we observed a tubulin polymerization increase after btz treatment. therefore, we have decided to focus our interest on soma organelle organization that is well known to be dependent from cytoskeleton structure. immunofluorescence images showed an altered distribution of acetylated tubulin in soma cytoplasm after hour treatment. moreover, confocal analysis of cis golgi (gm ) demonstrated that in btz treated neurons the normal golgi structure is lost showing a spot-like non perinuclear labeling distribution. additionally, dystonin distribution seemed comparable to that of cis golgi apparatus suggesting that btz could induce a change in dystonin localization which in turn affects golgi organization, probably through map b.these data suggest that the cytoskeleton alterations, induced by btz, could probably cause wrong maturation and trafficking alteration of golgi vesicles consequently impairing the correct sensorial function. romão tt , aleixo bfl , wedemann d , herculano fgn , prado h , cal h , pupe c , bittar c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. acute motor and sensory axonal neuropathy (amsan) is a clinical variant of guillain-barré syndrome (gbs) which has rarely been reported in association with human immunodeficiency virus (hiv) and neurosyphilis. we describe a case of a -year-old woman who started with muscle weakness in the left leg followed by weakness in the right leg and in upper limbs in one month. motor symptoms were followed by urinary and fecal incontinence. no preceding symptoms, as diarrhea or fever were referred. neurological examination showed global areflexia with impairment of vibratory and tactile modalities, as well as loss of proprioception. pinprick sensation was moderately compromised. no impairment of higher mental functions was observed. other causes of neuropathies were ruled out. cerebrospinal fluid (csf) examination showed cell/mm , increased protein level, normal glucose level and negative vdrl test. blood analysis showed hiv and positive serology tests, vdrl : and positive t. pallidum hemagglutination assay. igm cytomegalovirus antibody was negative, cd was in normal range and hiv load was undetectable. cranial and spinal cord magnetic resonance imaging revealed extensive involvement of the posterior spinal cord tracts which supports the diagnosis of neurosyphilis. nerve conduction studies showed a significant reduction in compound motor action potentials amplitudes, particularly at lower limbs, and absent sural nerve responses. electromyography revealed positive waves and fibrillation at lower limbs. treatment with methylprednisolone, human intravenous immunoglobulin and ceftriaxone has been settled, resulting in improvement of upper limbs paresis and sphincters dysfunction. antiretroviral therapy was initiated. this amsan case is associated with central nervous system involvement, encompassing an encephalomyeloradiculopathyrelated to hiv/t. pallidum co-infection. c.jejuni is the main etiological agent associated with amsan, but there are some reports of hiv/cmv co-infected patients who presented this gbs variant. facing a patient with amsan, mainly when cns involvement is present, it is of crucial importance to undergo investigation of an ongoing infectious process, such as hiv and syphilis, considering the impact of early treatment on prognosis. roodbol j , , yiu e , doets a , , de wit mcy , monges ms , van nes s , catsman-berrevoets ce , van den berg b , , jacobs bc , and on behalf of the igos-kids consortium. only a few prospective cohort studies so far have investigated the clinical presentation and course of guillain-barré syndrome (gbs) in children using age-adjusted outcome scores. in we started a study based on the igos protocol but adjusted for children with gbs (igos-kids). (pediatric)neurologists from argentina, australia and the netherlands joined forces to determine the clinical and biological determinants of disease progression and recovery in gbs in children from different geographical areas. for each age category specific pain scales are used including the comfort score for children < years old, pain faces revised age - years old and numerical rating scale in children ≥ years old and outcome measures. a new strength score was developed: gbs kids score. this score will be used and validated in addition to the mrc-sum score. the onls, r-ods and fss are also be used in igos-kids. age-dependent quality of life questionnaires were added to the igos-kids protocol, namely the pedsql and the pedsql multidimensional fatigue scale. these scales are available for children ≥ years old. an activity score validated for neuromuscular disorders for children ≥ years old was also added. currently there are children included in igos-kids, eight children from australia and five from the netherlands. additional clinicians and researchers interested in gbs in children are most welcome to participate in igos-kids. prognostic models have been developed to predict the highly variable clinical course of guillain-barré syndrome (gbs) in adults. the clinical course of gbs is equally variable in children, but the current prognostic models have not been validated in children. in this study, we aimed to identify in children with gbs the characteristics at hospital admission that predict the clinical severity and outcome. the study was conducted in two patient cohorts from europe: one (largely) german cohort of children and one dutch cohort of children. clinical information was obtained regarding preceding infection, first symptoms, neurological deficits at admission and nadir, results of additional investigations (cerebrospinal fluid and nerve conduction studies). clinical severity, course and outcome was defined by the gbs disability score, especially the ability to walk unaided, at a follow-up of month, months, months and months after onset of symptoms. univariate and multivariate regression analyses were performed initially on the two separate cohorts. combined the cohorts consisted of children (age median years, range - years) including boys ( %). the median duration between onset of symptoms and hospital admission was days (range ). pain at admission was remarkably frequent and present in ( %) children. in the combined cohorts, children ( . %) developed respiratory failure and one child died. multivariate regression analysis showed that in both cohorts strong predictors of respiratory failure available at hospital admission were cranial nerve involvement, a higher gbs disability score and a shorter period in days between onset of symptoms and admission. this information was used to develop and validate a prognostic model for children with gbs that will be presented at the conference. in conclusion, based on the analysis of two independent cohorts of patients the predictors of respiratory failure and clinical recovery were identified and validated. similar factors were identified for adult patients, although the prognosis in children in general is better than in adults. this information will be used to develop a simple prognostic model for current clinical practice to predict the chance of respiratory failure and outcome in children with gbs. the negative trials of vitamin c in cmt a have highlighted the lack of sensitive outcome measures in charcot-marie-tooth disease (cmt). neurofilaments are abundant neuronal cytoskeletal proteins and their concentration in blood is likely to reflect axonal breakdown. we therefore examined plasma neurofilament light chain (nfl) concentration as a potential biomarker in cmt. blood samples were collected from patients with cmt and age matched healthy controls over a -year period. disease severity was measured using the weighted cmt examination and neuropathy scores. plasma nfl concentration was determined using an in house developed simoa assay based on the nfl antibodies from the nf-l light elisa kit (umandiagnostics). plasma nfl concentrations were significantly higher in cmt patients (median: . pg/ml; iqr: . - . ) than in controls (median: . pg/ml; iqr . - . , p< . ) and correlated with disease severity as estimated by the weighted cmt examination (n= , r= . , p< . ) and neuropathy scores (n= , r= . , p= . ) . concentrations were also significantly higher when subdividing cmt patients by genetic subtype; cmt a (n= , p< . ), sptlc (n= , p< . ) and gjb (n= , p= . ) or into demyelinating, cmt (n= , p< . ) or axonal, cmt (n= , p< . ) forms compared to healthy controls. there was no significant difference in the plasma nfl concentration after year in patients with cmt (n= , mean difference − . pg/ml, % confidence interval − . - . ) or healthy volunteers (n= , mean difference − . pg/ml, % confidence interval . - . ) which is unsurprising as cmt is a slowly progressive disease in which the rates of axonal degeneration are likely to be constant and elevated. in summary, we have shown that plasma nfl levels are significantly raised in patients with cmt and that they correlate with disease severity. this is of relevance not only for the field of cmt but for peripheral neuropathies in general, and suggests that plasma nfl holds promise as a biomarker of peripheral neuropathy in both routine practice and clinical trials. neurolymphomatosis (infiltration of the peripheral nervous system by lymphoma cells) is a rare and usually devastating condition belonging to the spectrum of lymphoma-associated neuropathies. cerebrospinal fluid examination with cytologic examination, flow cytometry and clonality testing by pcr may show malignant cells especially when nerve root involvement is prominent. however, nerve biopsy remains a useful tool to confirm the presence of malignant cells invading the nerve. neuropathological features are important and pcr-based immunoglobulin or t-cell receptor clonality testing on nerve fragments may add notable value for the diagnosis of neurolymphomatosis, although this has not been systematically investigated. we retrospectively studied clinicopathological data and clonality results of nerve biopsy samples in patients with nl from centres, performed between and . among patients with nl, we found % of b-cell lymphoma and one t-cell lymphoma. nl was the first manifestation in , % of patients. the main clinical pattern was symmetrical progressive sensorimotor polyneuropathy in % of patients and pain was a prominent feature in %. clonality testing showed a monoclonal rearrangement in ( %) cases, oligoclonal rearrangement and no amplification in ( %). the main histological pattern was perivascular infiltration of predominant b-cells in the epineurium, without signs of vasculitis. the extent of axon loss was highly variable between patients. various chemotherapeutic regimens were used and the median overall survival was months. only one case showed a monoclonal pattern among control nerve samples from patients with other types of neuropathy including vasculitis and cidp. overall, we found that clonality testing on nerve biopsy specimens may provide decisive information on the presence of neurolymphomatosis, with a sensitivity of . %, a specificity of . %, a positive predictive value of . % and a negative predictive value of . %. we confirm the utility of nerve biopsy for the diagnosis of nl and show the great yield of pcr-based clonality testing to assess the malignant feature of peripheral nervous system lymphoid infiltrates. despite an accurate diagnosis, neurolymphomatosis still remains a devastating disease with an overall poor prognosis. in the last years, cumulative data have shown that patients with amyotrophic lateral sclerosis (als) and mouse models of the disease present loss of small epidermal and dermal nerve fibers and sensory dysfunctions, in addition to the classical motor symptoms. our objective is to characterize this small fiber neuropathy and to clarify if axonal loss involves all sort of fibers equally, or if there is some specificity. for this purpose, we performed an immunohistochemical characterization of total intraepidermal nerve endings (protein gene product; pgp . ), peptidergic (calcitonin gene-related peptide; cgrp) and nonpeptidergical nerve epidermal endings (isolectin b ; ib ) of the sod g a mouse at different stages: presymptomatic stage ( weeks), disease onset ( weeks) and in symptomatic stage ( weeks). the sympathetic sweat gland innervation was immunolabeled for vasoactive intestinal peptide (vip) from very early stage ( weeks) to disease onset ( weeks). the results showed a marked reduction of the intraepidermal nerve fibers already in the presymptomatic stage compared to the wildtype littermates (p< . ). this axonal loss affected more markedly the nonpeptidergic axons from the disease onset stage ( % axonal loss, p< . ), whereas no significant differences were found in the cgrp positive fibers ( . % axonal loss). a reduction of the sympathetic innervation of the sweat glands was also found from the disease onset stage ( % axonal loss, p< . ). in summary, we have found that nonpeptidergic and sympathetic innervation of the skin are predominantly affected in the sod g a mouse model. these findings suggest that this specificity could be used as an accessible biomarker for the disease. hereditary and inflammatory neuropathies are peripheral nerve disorders of different pathophysiology whose identification is crucial for therapeutic approach. diagnosis of cmt is easy when there is a family history, disease course is slowly progressive, neurophysiological findings are homogeneously abnormal. cases of cmt patients with inflammatory-like phenotypes leading to a misdiagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described. the presence of "red flag signs", such as slowly progressive decline, mild sensory symptoms, minimal electrophysiological progression and no response to therapy should prompt re-evaluation of the diagnosis of cidp. the introduction of nerve neuroimaging has contributed to the diagnostic work-up. we describe cmt patients ( men, women, mean age . ± . yrs, mean neuropathy duration . ± . yrs) with genetically confirmed cmt, who were initially diagnosed with cidp. neurophysiology showed demyelinating features in patients, the remaining patients had axonal features. csf analysis showed albumin-cytological dissociation in patients, oligoclonal igg bands were present in patient. nerve ultrasound in patients with demyelinating neuropathy showed diffuse increased cross-sectional area. mr-neurography in one patient confirmed diffuse nerve hypertrophy and increased signal intensity, supporting the hypothesis of a overlap syndrome. all patients were treated with immunomodulatory therapies. among patients with demyelinating features, patients underwent ivig for months, without benefit; the remaining were treated with steroids, showing temporary improvement. the patients with axonal neuropathy complaining of a progressive history, underwent ex adjuvantibus plasma exchange and ivig, without benefit. given the lack of benefit from therapies, screening for hereditary neuropathies was performed. among patients with demyelinating neuropathy, had cmt a, cmt b, cmt d. among the patients with axonal neuropathy, one was diagnosed with cmt k. in patients an overlap syndrome was present. several clinical and laboratory features (csf protein elevation), might contribute to the misdiagnosis of cidp in cmt patients. refractoriness to immune-modulatory treatment should rise the suspicion of a hereditary neuropathy. an overlap cmt/cidp syndrome may be considered, in front of acute/subacute deterioration and/or proximal muscles involvement. two patient with an overlap syndrome showed benefit after either steroids or ivig. ruiz m , tripodi sm , campagnolo m , zara g , salvalaggio a , ruggero s , toffanin e , anglani m , briani c . neurology, department of neuroscience, university of padua, padua, italy; neuroradiology, padua hospital, padua, italy. facial diplegia is a rare regional subtype of guillain-barrè syndrome characterized by rapidly progressive bilateral facial palsy in absence of other cranial neuropathies, ataxia or limb weakness. the diagnosis is based on history, clinical examination, laboratory data. the outcome of this variant appears to be better than that of classical gbs. although about % of gbs patients clinically exhibit facial nerve involvement, there are only few reports that demonstrate mri gadolinium facial nerves enhancement. a -year-old previously healthy man sought neurological advice for the acute onset of right perioral paresthesias and hyposthenia at the extensor hallucis longus bilaterally. in the previous month, during a trip to china and japan, he had suffered from gastroenteritis. at neurological examination he presented with bilateral extensor hallucis longus hyposthenia. ankle jerk reflexes were absent. one week later, he developed bilateral facial palsy. neurophysiology disclosed absence of activity in the facial nerves innervated muscles bilaterally and signs of mild partial denervation at right l metamer. csf analysis showed increased protein levels ( mg%) and . leucocytes/ l. serological tests for infectious agents were negative and serum levels of angiotensin converting enzyme were normal. antibodies to monosialoganglioside gm of igg isotype were positive ( / ). t brain mri showed, after gadolinium administration, marked bilateral enhancement of the facial nerves in their extra-and intra-canalicular segments. t lumbosacral mri scan ruled out the presence of disc diseases as well as signal modifications within conus medullaris and cauda equine nerve roots. the patient was treated with iv immunoglobulins ( . g/kg/die for days) with benefit on facial weakness. at follow-up examination months later, the patient presented a further improvement of the facial diplegia and neurophysiology disclosed a partial recovery of activity in the facial nerves, with persistent axonopatic damage. bilateral extensor hallucis longus hyposthenia persisted. in conclusion, we report on a regional subtype of guillain-barrè syndrome with the curious association of facial diplegia and bilateral extensor hallucis longus hyposthenia, and igg anti-ganglioside antibodies, that are not commonly described in facial diplegia. moreover, we provide t brain mri evidence of facial nerve involvement. rumora ae , tabbey ma , lograsso g , dolkowski j , haidar j , lentz si , feldman el . department of neurology, university of michigan, ann arbor, usa; department of internal medicine, division on metabolism, endocrinology and diabetes, university of michigan, ann arbor, usa. diabetic neuropathy (dn) is the most common complication of diabetes affecting up to % of diabetic patients. the pathogenesis of dn in type diabetes is directly related to the metabolic syndrome associated with the western diet composed of elevated levels of long chain saturated fatty acids (sfas) and low levels of medium chain sfas. long chain sfas are associated with insulin resistance and dyslipidemia while medium chain sfas have been associated with decreased lipid accumulation and improved mitochondrial function. since dn is primarily a disorder of the sensory dorsal root ganglion (drg) neurons, we sought to evaluate the impact of sfa hydrocarbon chain length on mitochondrial trafficking mechanisms that are critical for distributing atp throughout drg axon to provide energy for synaptic transmission. we hypothesize that sfas with longer hydrocarbon chains will impair mitochondrial trafficking whereas medium length sfas will not impact mitochondrial movement along the drg axon. in this study, we examined the impact of sfa hydrocarbon chain length on mitochondrial trafficking, directionality and velocity in primary mouse drg neurons. drg neurons were treated with increasing concentrations of long chain sfas, stearate and palmitate, or medium chain sfa, laurate, ranging from . to micromolar for twenty-four hours. drg neurons treated with long-chain sfas, palmitate and stearate, showed a significant decrease in the percentage of motile mitochondria whereas medium chain sfa, laurate, does not alter mitochondrial motility. we next assessed whether motile mitochondria in drg neurons treated with palmitate or stearate exhibited altered directionality or velocity of mitochondrial trafficking. palmitate and stearate treatments resulted in a trending decrease in the number of mitochondria trafficking in both anterograde and retrograde directions. furthermore, . to micromolar palmitate and stearate induced a significant decrease in mitochondrial velocity. laurate treatment, on the other hand, retained directionality and velocity of mitochondrial trafficking. these results suggest that hydrocarbon chain length of saturated fatty acids plays an important role in regulating mitochondrial trafficking mechanisms in dorsal root ganglion neurons. impaired mitochondrial trafficking in drg neurons exposed to elevated levels of long-chain sfas may play a critical role in the progression of dn. dyslipidemia is a critical factor that contributes to the development of diabetic neuropathy (dn). the progressive nerve damage associated with dn correlates with the dyslipidemic state characterized by elevated circulating levels of harmful saturated fatty acids (fas) and low levels of beneficial unsaturated fas. in dn, primary sensory dorsal root ganglion (drg) neurons exhibit energy dyshomeostasis and mitochondrial dysfunction, however, little is known about the differential impact of saturated and unsaturated fas on mitochondrial mechanisms in drg neurons. mitochondrial trafficking is an essential mechanism for transporting mitochondria throughout drg axons to provide cellular atp for neuronal function and neurotransmission. since mitochondrial trafficking is regulated by metabolic flux, we sought to determine whether saturated fa, palmitate, and unsaturated fa, oleate, have differential effects on mitochondrial trafficking in drg neurons. we evaluated mitochondrial trafficking patterns and the mitochondrial membrane potential (mmp) in primary drg neurons treated with physiologically relevant concentrations of palmitate, oleate, and combinations of both fas. primary drg neurons treated with . to micromolar palmitate induced a significant and dose-dependent reduction in the percentage of motile mitochondria. these palmitate treatments also induced a dose-dependent reduction in mitochondrial velocity but did not impact the directionality of mitochondrial movement. alternatively, . to micromolar oleate treatments did not impair the percent of motile mitochondria, the direction of mitochondrial movement, or mitochondrial velocity. since palmitate and oleate have differential effects on mitochondrial motility, we next assessed whether oleate could counter the inhibitory effect of palmitate on mitochondrial trafficking. surprisingly, oleate/palmitate mixtures at ratios of : or : prevented palmitate-induced impairment of mitochondrial trafficking. we assessed the impact of palmitate and oleate on mmp by staining palmitate and oleate-treated drg neurons with tetramethylrhodamine methyl ester. drg neurons treated with micromolar palmitate exhibited an increase in the percentage of depolarized mitochondria while mitochondria in oleate-treated drg neurons retained mmp. interestingly, drg neurons treated with : oleate/palmitate mixtures also maintained polarized mitochondria. these results suggest that saturated and unsaturated fas have a distinct impact on mitochondrial trafficking mechanisms in drg neurons and that equimolar ratios of oleate/palmitate can prevent impairment of mitochondrial trafficking. of g alpha- in m r overexpressed sensory neurons significantly reversed the m r-induced reduction in relative levels of total neurite outgrowth (mean±sd: . ± . (control) vs . ± . (g alpha- knockdown), p< . , n= neurons). further, treatment of m r overexpressing neurons with mt or pirenzepine sequestered g alpha- / proteins at the m r and significantly reversed the impaired cytoskeleton-mediated reduction in total neurite outgrowth (mean±sd: . ± . (control), ± (+ nm mt ), ± . (+ micromolar pirenzepine); n= , and neurons, respectively; for treated groups p value= . vs control by one-way anova). mt and pirenzepine also significantly restored mitochondrial trafficking and abundance of mitochondria in the distal neurites. our findings suggest a novel mechanism in which modulation of m r influences tubulin polymerization, mitochondrial distribution in nerve terminals and controls axonal outgrowth and regeneration. funded by cihr and nih. muscarinic receptors are a group of five g-protein coupled receptors (gpcrs) that are targeted by drugs for the treatment of several human pathophysiological conditions. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) antagonists of the muscarinic acetylcholine type receptor (m r) induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt et al., ) . one of the major cellular effectors activated by gpcrs is extracellular signal-regulated kinase (erk). the erk signaling cascade regulates a variety of cellular processes including growth and proliferation. both g protein and beta-arrestin mediated signaling pathways can lead to erk activation by phosphorylation through different kinases. activated erk in turn can phosphorylate about cellular substrates, thereby mediating diverse functions. in this study, we have analyzed beta-arrestin recruitment, as part of the receptor internalization process induced by agonist/antagonist binding. in addition, we studied phosphorylation of erk by mt and pirenzepine using isoelectric focusing with phospho-erk specific antibodies and a variety of cell-based assays including beta-arrestin and g protein (gnas/gnasl/gnaq/gna /gna /gna ) knockout cell lines. our study revealed that beta-arrestin is recruited to the m r upon mt and pirenzepine treatment. treatment with nm mt and micromolar pirenzepine significantly increased the dual phosphorylation of tyr and tyr residues of erk / in primary rat sensory neurons (p< . ) in comparison to muscarinic agonist carbachol ( micromolar) and general antagonist atropine ( micromolar). we have identified multiple distinct phosphorylation events on the m r by isoelectric focusing that are specific to mt and pirenzepine induction. further, we have shown that mt and pirenzepine-mediated erk phosphorylation is dependent on both g protein and beta-arrestin recruitment to m r. finally, we reveal that increased erk phosphorylation by mt and pirenzepine significantly (p= . and . , respectively) increased phosphorylation of camp responsive element binding protein (creb) at ser . these results show for the first time that antagonists of the m r can activate the erk signaling pathway and possibly drive phenotypic change in adult sensory neurons. funded by cihr # mop- . charcot-marie-tooth disease (cmt) is representative of inherited neuropathies affecting an estimated in people. the immense advances in gene discovery gained from next-generation sequencing (ngs) projects have revealed the extent of cmt's genetic heterogeneity, with over loci already identified. this knowledge is rapidly translated into clinical comprehensive gene testing panels, often containing over genes. such a large genomic space will invariantly yield variants of uncertain clinical significance (vus) in nearly any person tested. this rise of the number of vus creates major challenges for genetic counseling. in addition, less individual mutations in already known genes are being published as the academic merit is decreasing, and most such testing now happens in clinical laboratories. we propose to capture more of this data in the cmt field to gain a more complete collection of alleles in cmt genes, ideally in conjunction with detailed phenotypic data. this represents a rational approach to eventually reduce the number of vus. thus, we have created a unique, community-driven variant database for cmt researchers and clinicians. the inherited neuropathy variant browser provides simple, user-friendly access to currently reported cmt variation, including patient-level genotypic and phenotypic information. we have also designed an interactive rating system of genetic variation to assist the community with interpretation of vus. for the initial release, we have collected genetic variation, along with genotypic and phenotypic data when available, from published literature, clinical lab reports, and our in-house database. we highly encourage new submissions of not only observed pathogenic variation, but also variation of unknown significance. the goal is to provide a platform for the cmt community to store, share, and discuss genetic data in order to resolve variation of uncertain significance as a joint-effort. with active participation, we aim to provide the community with a more complete mutational spectrum in cmt genes to assist allelic interpretation and patient diagnosis. neurofilaments are strictly neuron-specific intermediate filaments crucial for maintaining axonal architecture. pathogenic mutations in nefl, which encodes the light chain of neurofilament, cause dominantly and recessively inherited charcot-marie-tooth neuropathy (cmt). the nefl-associated neuropathy can be either axonal (type e) or demyelinating (type f). most pathogenic nefl variants are dominantly inherited missense mutations, which are thought to cause disease by inducing nefl aggregation, leading to the disruption of axonal transport. however, investigation of disease mechanisms caused by the mutations has been complicated by the neuronal specificity of nefl. we identified a homozygous nefl variant c. c>t predicting a nonsense change p.arg * in a patient with early-onset cmt. to elucidate the disease mechanism, we used pluripotent stem cells, reprogrammed from patient's skin fibroblasts, to differentiate patient-specific spinal motor neurons. the motor neurons revealed a near complete loss of nefl mrna, and absence of nefl protein. our results establish that nefl is not essential for the development of human nervous system but its absence causes progressive axonal neuropathy. we currently profile the transcriptomic alterations of the motor neurons lacking nefl using single cell rna sequencing to identify compensatory pathways. friedreich's ataxia (frda) is the most common autosomic recessive ataxia, due to a trinucleotide expansion within the frataxin gene. within the wide phenotype, frda patients present also with an axonal neuropathy whose pathological mechanisms are not completely known. eight patients ( women, mean age . yrs, range - ) with genetically confirmed frda underwent neurophysiological and nerve ultrasound evaluation at four limbs bilaterally. echogenicity and cross-sectional area (csa) of median, ulnar, radial, peroneal, tibial, and sural nerves were recorded. mr neurography and diffusion tensor imaging (dti) analysis were performed in one patient; fractional anisotropy (fa), radial (rd) and axial (ad) diffusivity of median, radial and ulnar nerve were calculated at proximal, intermediate and distal sites. all patients presented with sensory axonal neuropathy. seven patients ( %) presented with increased csa of median and ulnar nerves at arm and axilla. mean median nerve csa at mid-upper arm was . mm (normal values < mm ), mean ulnar nerve csa at mid-upper arm was . mm (normal values < mm ). mean median nerve csa at axilla was . mm (normal values < mm ), mean ulnar nerve csa at axilla was . mm (normal values < mm ). mr neurography (performed in one patient) confirmed diffuse swelling and signal hyperintensity of median and ulnar nerves at the arm and dti analysis showed abnormal values of fa, ad and rd along the whole course of evaluated nerves thus suggesting a wide alteration of nerves structure. frda patients presented with an axonal neuropathy characterized, at ultrasound, by a nerve enlargement strictly limited to mid-upper limbs in all patients, findings that cannot be solely explained by a dying-back axonopathy, as suggested by several authors. neither a dorsal root ganglia neural loss could explain by itself our findings, because a diffuse csa reduction would have been expected. on the whole, these findings represent a peculiar feature in frda, but its pathophysiologic meaning remains unclear. inherited peripheral neuropathies are an important health concern for which there is currently no disease-modifying therapy. dogs are affected by a variety of peripheral neuropathies that are breed-specific, indicating a strong genetic component. the use of in-bred populations, such as pure-bred dogs, is advantageous for genetic dissection of disease. acquired peripheral neuropathy (apn) is an inherited late-onset generalized polyneuropathy with high prevalence in labrador retrievers. the most prominent features of apn, laryngeal paralysis and pelvic limb weakness, are associated with the longest peripheral motor nerves in the dog. the pathologic features of apn are similar to human peripheral neuropathy. our aim is to understand the genetic and pathologic features of apn for development of this condition as a naturally occurring large animal model for human disease. we performed a genome-wide association study (gwas) and short-read high-depth whole genome sequencing (wgs) to investigate the genetic underpinning of apn in the labrador. our gwas data indicates that apn is an autosomal dominant disease. the initial analysis from the wgs study resulted in a potential causal variant with an autosomal dominant pattern; this variant is associated with an axonal gene. the neuropathologic progression and histologic features of apn are poorly defined. using genetic markers from our gwas study, we are able to confidently identify labradors with pre-clinical apn, from which nerve biopsies can be obtained. preliminary analysis of biopsies from labradors suggest apn is an axonopathy. further histologic data from preclinical and symptomatic dogs is being obtained to further define the pathogenesis in this model. mutations in morc lead to an axonal form of neuropathy (cmt z). to date, nine families have been published with mutations in the morc gene, showing that this gene is frequently involved in cmt. while the recent genetic data clearly established the causative role of morc in cmt z, its phenotypic consequences in patients and role in neuronal biology remains to be clarified. therefore, we aim to look for altered genetic and biochemical pathways with a transcriptomic approach in order to investigate the role of morc in hereditary peripheral neuropathy. we have performed transcriptomic analysis using a human gene expression microarray (v x k, agilent technologies), in three hek- t cell lines: control, morc knock-down (kd) and the overexpression of the most common morc mutation, the p.r w (np_ ) (kr). differential gene expression assessment was carried out using limma moderated t-statistics. standard analysis techniques perform one test for each gene. thus, for each gene, a t-test statistic is reported together with its corresponding p-value. in this analysis we have used the conventional multiple testing p-value correction procedures proposed by benjamini hochberg to derive adjusted p-values. the preliminary results reveals that kd shows up-regulated genes involved in transmission of nerve impulse and cilium metabolism, suggesting that morc might act at this level in the peripheral nervous system. otherwise, kr shows a major alteration of main axonal metabolic pathways, including the overexpression of genes related to the generation of neuronal action potential, transport through the axon and its targeting in synapse formation. kr also shows a marked alteration of gene expression related to organization, assembly and cilium movement and with the axonemal dynein complex assembly. this study provides an important step towards understanding the pathomechanism underlying to morc p.r w and its role in cmt z. aifm encodes a mitochondria associated apoptosis inducing factor. mutations in aifm lead to a wide spectrum of neurodegenerative disorders: cowchock syndrome; a combined oxidative phosphorylation deficiency (coxpd ) with severe encephalomyopathy; x-linked deafness with peripheral sensory neuropathy; spondyloepimetaphyseal dysplasia (semd) with mental retardation; and an infantile motor neuron disease. previous studies showed severe defects in mitochondrial metabolism, related to redox function, mitochondrial fragmentation, and respiratory deficiencies. in addition, some mutations impair the protein expression of aifm and cause an increase in caspase-independent apoptosis. by targeted next-generation sequencing, we detected the aifm c. c>t (p.phe ser), in a year-boy. this mutation was confirmed in his year-old affected brother. electromyography and nerve conduction velocities studies revealed an axonal polyneuropathy with exclusive involvement of motor fibers, with an early childhood-onset. both children currently show normal cognitive and cranial nerves functions. the in silico structural modeling of human aifm showed that the mutation of a phenylalanine to serine at position disrupts the hydrophobic interaction between phe and pro , and consequently, it destabilizes an alpha-loop domain. cartoon of protein superposition between two different human aifm structures suggest that a lack of constraints in this region could affect the interaction between - helix and the - -hairpin regions, a very important stage for the functional activity of the aifm protein. patient-derived fibroblasts were used to investigate the pathological effect of the p.phe ser mutation: fibroblasts from patients show a similar mrna but different protein expression of aifm compared to healthy control fibroblasts. however, they have an aberrant morphology, from fibroblastic to polygonal shape, and they are larger than control fibroblast; mitochondria from mutant fibroblasts are markedly fragmented compared to controls; the viability of the patient's fibroblasts is lower, but it does not correlate with an increase in apoptosis. instead, it seems to be caused by an increase in the expression of genes activating the senescent program, like p and p . our study confirms that variable effect of different mutations on the protein function may contribute to the clinical variability observed in aifm patients. funds: isciii (pi / ); fundació per amor a l' art. sango k , takaku s , niimi n , yako h . diabetic neuropathy project, tokyo metropolitan institute of medical science, tokyo, japan. coculture models of neurons and schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained from the primary culture with embryonic or neonatal animals. because it is recognized that some biological properties of both neurons and schwann cells change with maturation and aging, culture systems of adult animal cells appear to mimic peripheral nerve degeneration and regeneration better than those of immature animal cells. we have established spontaneously immortalized schwann cell lines from long-term cultures of adult fischer rat peripheral nerves. one of these cell lines, designated ifrs , has been shown to retain distinct schwann cell phenotypes, such as spindle-shaped morphology with expression of glial cell markers, synthesis and secretion of neurotrophic factors and cytokines, and fundamental ability to myelinate neurites in cocultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed pc cells. our current investigation focuses on the establishment of the coculture system of ifrs cells and nsc- motor neuron-like cells. nsc- cells were seeded at a low density ( x /ml) and maintained for a week in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (bdnf, ng/ml). after overnight exposure to mitomycin c (mmc, micro g/ml), nsc- cells were cocultured with ifrs cells ( x /ml) and maintained in serum-containing medium supplemented with bdnf ( ng/ml), ciliary neurotrophic factor (cntf, ng/ml) and coenzyme q ( micro m). under this culture condition, overgrowth of nsc- cells was prevented and gradual movement of ifrs cells toward the neurites emerging from nsc- cell bodies was observed. double-immunofluorescence staining carried out at day of the coculture showed myelin protein zero-immunoreactive ifrs cells surrounding the beta iii tubulin-immunoreactive neurites. this coculture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g. amyotrophic lateral sclerosis, charcot-marie-tooth diseases and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them. santos pp , torezani gs , maciero l , pagliarini lfd , romão tt , abunahman ms , ferreira is , bittar c , pupe c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. zika virus (zikv) is a flavivirus related to dengue, yellow fever and west nile viruses, and has been recently associated to the occurrence of neurological complications in children and adults. previous studies have linked zikv to the development of guillain-barré syndrome (gbs), myelitis, meningoencephalitis and ophthalmological manifestations in adults. guillain-barré syndrome (gbs) encompasses a spectrum of post-infectious neuropathies characterized by different distributions of weakness and sensory impairment. serum anti-ganglioside antibodies are often found and are related to different clinical patterns. recently we have encountered patients in rio de janeiro, brazil, with distal edema in lower limbs, acute weakness, pain and sensory disturbances during the acute stage of an acute febrile exanthematous illness. the symptoms persist for up to days, with complete resolution afterwards, without specific therapy. blood concentrations of muscle enzymes show normal values, and electromyography and nerve conduction studies (emg/ncs) are usually unremarkable. of note, all cases have had positive rt-pcr for zika virus, indicating an illness that occurred during the viremic phase of this arbovirus infection, and complete recovery within the expected timeframe for the resolution of the systemic viremia. there is a subset of patients who developed acute weakness very early after the initial viral symptoms, with clinical, laboratorial and electrophysiological findings that substantially differ from gbs. we describe three cases with similar features suggestive of an acute infective polyneuritis (aipn). one might hypothesize that zikv might lead to a direct neurotropic injury, significant enough to cause weakness and sensory complaints, but not severe enough to cause permanent damage, resolving in conjunction with decreasing blood viremia. we consider that these patients differ from classical gbs because their illness begins during the acute febrile stage of an infective illness and the clinical course is more rapid leading to complete resolution in a few days. savransky a , mozzoni j , massaro sanchez mp , reisin r , monges ms . department of neurology, hospital de pediatria j.p. garrahan, buenos aires, argentine; department of physical therapy, hospital de pediatria j. p garrahan, buenos aires, argentine; department of neurology, britain hospital, buenos aires, argentine. our objective is to describe a series of children with guillain barré syndrome (gbs) included in the igos protocol. as part of the igos multicenter protocol, pediatric patients meeting gbs diagnostic criteria, who consulted within the first weeks of symptom onset and had parental consent to participate in the study were included. patients were also offered to participate in the extended -year protocol. all patients were evaluated according to the igos protocol. patients were recruited between october and june . twenty-four patients, eight girls, participated. ages ranged from months to years (mean . years). all patients agreed to participate in the extended protocol, which was completed by of them. eight patients completed the -year follow-up and are still under evaluation. five patients were lost to follow-up.twenty-two had the classic variant of gbs and two miller fisher. radicular pain in the back or lower limbs was reported by %. twenty-three patients underwent lumbar puncture and albumin-cytological dissociation was found in . in all cases, csf was stored for proteomic studies. all patients underwent emg showing aidp in , and amsan and aman in each. one patient with aidp developed to cidp. in patients full-spine mri was performed and cauda equina enhancement was found in every case. three patients required ucip, two with invasive and one non-invasive ventilation. all patients were treated with gammaglobulin, with a second dose at weeks in cases with a poor response. all patients who followed the protocol were evaluated with the gbs disability score. median score was at baseline and between and at the -year assessment. we describe a series of children with gbs as a part of an international protocol including patients of different ages. pain was a frequent and early symptom and could be determined despite the young age of our patients. most patients fully recovered. we were invited to participate in igos kids to better assess this age group. saysavath k , somchit v , t. umapathi . mittaphab hospital, vientiane, lao pdr; setthathilath hospital vientiane; national neuroscience institute, singapore, singapore. laos people democratic republic is a country of . million people in south east asia. largely agricultural, its capital and metropolis is vientiane. adult neurological services are concentrated at mittaphab hospital, vientiane serviced by three neurologists. on the average about six cases of guillain-barré syndrome (gbs) are seen per year. it is believed that most patients do not seek medical attention. cases appear to cluster during the rainy season. most patients present late, often at the second week of illness when recovery is unapparent after seeking treatment from traditional medicine doctors and at district hospitals. a substantial number of patients seek treatment at hospitals across the border, in neighboring provinces of thailand. common antecedent symptoms are viral prodrome and diarrhea. miller fisher syndrome appears to be rare, possibly because of the mild deficits that do not prompt patients to seek medical attention. diagnosis is made largely from clinical features and from spinal fluid analysis. nerve conduction studies are not available. patients are often treated with steroids by internists. intravenous immunoglobulin and plasma exchange are not available. common complications include pneumonia, autonomic dysfunction (fluctuating blood pressure), pressure sores and depression. patients who develop respiratory failure are nursed at a twelve-bedded intensive care units. plans are afoot to set up a prospective gbs database, systematically study antecedent infections, including of flaviviruses, and develop low-volume plasma exchange as a feasible therapeutic modality. a gene therapy approach for treating cmt c neuropathy schiza n , markoullis k , richter j , tryfonos c , kagiava a , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory; department of molecular virology; neurology clinics, cyprus institute of neurology and genetics and cyprus school of molecular medicine, nicosia, cyprus. charcot-marie-tooth type c (cmt c) is the most frequent form among recessively inherited demyelinating neuropathies and results from mutations in the sh tc /kiaa gene. sh tc mutations cause loss of function of the sh tc protein suggesting that gene replacement therapy may be useful for treating cmt c. sh tc −/− mice develop all major aspects of the human pathology including early onset progressive peripheral neuropathy with hypo-and demyelination along with decreased motor and sensory nerve conduction velocities, offering a relevant model for testing treatments for cmt c. in order to develop a gene replacement strategy for cmt c, we generated a novel lentiviral vector, lv-mpz-sh tc .myc, to drive expression of the human sh tc cdna under the control of the myelin protein zero (mpz/ p ) promoter specifically in myelinating schwann cells. a c-terminus myc tag was included to facilitate expression analysis. a control vector (mock) was also produced in which the sh tc cdna was replaced by the egfp reporter gene. we first confirmed expression of hsh tc in hela cells transfected with the pcdna -cmv-sh tc .myc vector. immunofluorescence analysis confirmed a strong expression of sh tc specifically at the plasma membrane with additional localization in a dotted pattern intracellularly. for in vivo gene delivery we used both intraneural and intrathecal injections of the lv-mpz-sh tc .myc vector in -week to -month old sh tc −/− mice. expression of virally delivered hsh tc was assessed weeks after injection. immunofluorescence analysis showed hsh tc immunoreactivity in perinuclear schwann cell cytoplasm in sciatic nerve teased fibers of sh tc −/− mice following both intraneural and intrathecal delivery, while lumbar intrathecal gene delivery resulted additionally in expression of hsh tc in the lumbar roots. real time pcr analysis confirmed hsh tc mrna expression in both lumbar roots and sciatic nerves. thus, we have developed a novel lentiviral vector for schwann cell targeted gene delivery to treat cmt c and for testing possible therapeutic effects in the mouse model of the disease. hereditary neuropathies are a group of disorders which are characterised by the systemic impairment of peripheral nerves. more than neuropathies are associated with causative gene defects [ ]. charcot marie tooth (cmt a) neuropathy is the most frequent hereditary neuropathy, triggered by a mutation in the peripheral myelin protein gene (pmp ). cmt a leads to a primary loss of myelin sheath and afterwards to a degeneration of axons [ ] . symptoms appear with the degeneration of axons, whereas demyelination is thought to be largely asymptomatic. for that reason, we investigate in the mechanisms of axonal degeneration. for our analysis, we used purified axoplasma without detectable myelin proteins of the sciatic nerves of weeks old pmp -c mice. in this early stage of the axonal degeneration sarm was significantly increased. the nad + concentration in the axoplasma was dramatically reduced. this correlates to the previous finding that sarm promotes axonal degeneration by cleavage of nad + . additional studies showed an increase of the nad + -dependent axonal protective factor sirt in pmp -c sciatic nerves. nmnat- , known as the active component of the wallerian degeneration slow gene, was unaffected in axoplasma of pmp -c sciatic nerves. summarised, these results indicate that the pathway of sarm , nad + and sirt may play a critical role in axonal degeneration in neuropathy. we report two unrelated czech patients with cmt b , both sporadic cases in the family. patient is a year old man with congenital glaucoma after ophthalmological surgeries. his early motor development was normal. at the age of years parents noted gait problems, first neurological examination was at the age of years, when emg showed diffuse motor and sensory neuropathy with severely decreased ncv ( - m/s). he developed foot deformities (pes cavovarus) and underwent corrective orthopedic surgeries at the age of years. after several dna tests for demyelinating cmt the sfb gene was sanger sequenced and a novel missense mutation p.ile asn was found in homozygous state in the patient and in heterozygous state in both parents. the patient was later tested also by ngs of a panel of all genes to be causal for hereditary neuropathies and no other potentially causal variants were detected. patient is years old man, with normal early motor development. at the age of years parents noted gait problems with distal leg weakness which progressed into distal leg plegia at the age of years. hand weakness was noticed since the age of years. he has severe atrophies of distal muscles of all extremities, is self ambulant. at the age of , emg showed unrecordable responses from nerves of lower limbs and ncv was measurable only on ulnar nerve and was m/s. at the age of years increased intraocular pressure was diagnosed and he uses anti glaucoma eye drops. after many single gene tests for demyelinating cmt, we used ngs of a panel of genes known to be causal for hereditary neuropathies and two novel heterozygous, probably pathogenic variants affecting invariant splice sites were detected: c. - a>g and c. - _ del, both confirmed by sanger sequencing. the first variant is also in the father, but the second is probably de-novo (not detected in parents, despite correct parentity). sekiguchi y , kikuchi si , konno si , sekiguchi m . department of orthopaedic surgery, fukushima medical university school of medicine, fukushima, japan. carpal tunnel syndrome (cts) is the most common entrapment neuropathy. ultrasonography can be used to detect anatomic changes in cts. more recently, it has been shown that doppler ultrasonography can detect increased intraneural blood flow in cts. the purpose of this study was to determine the most suitable finding of pre-and postoperation in cts by ultrasonography. a total of wrists of patients with nerve conduction study (ncs) proven cts were evaluated with ultrasonography. we measured the median nerve's cross-sectional area (csa) and intraneural blood flow of median nerve by ultrasonography. the correlation between these ultrasonographic measurements, ncs severity and duration of clinical cts symptoms was analyzed. the csa (mean, mm ) was no significantly reduction after successful carpal tunnel release. morphologic median nerve changes may persist for a longer period regardless of successful surgery and clinical improvement. however, intraneural blood flow is increasing after successful carpal tunnel release. we conclude that doppler ultrasonography results strongly correlate with post operated cts improvement. hence doppler ultrasonography is a useful method for functional improvement of pre-and post cts operation. senger jl , chan km , olson jl , webber ca . university of alberta, edmonton, canada. the beneficial effects of a preinjury crush conditioning lesion (cl) on peripheral nerve regeneration is well-documented in animal models. no human studies have been attempted to date, given the ethical dilemma of deliberately injuring an intact nerve, and the difficulty in predicting the timing of a nerve injury. recent studies demonstrate that hour of electrical stimulation (es) produces effects similar to cl in neuronal cultures. this, coupled with a surgical environment favoring nerve transfers, in which an intact nerve is deliberately cut to reinnervated a denervated muscle, means that es may be clinically translatable to enhance regeneration. this study hypothesizes that es prior to nerve injury will enhance nerve regeneration. twelve sprague-dawley rats were divided into four groups based on conditioning-type to the mid-common peroneal (cp) nerve: es ( ), crush ( ), sham-es ( ), and naïve ( ). one week following conditioning, they underwent a cut/coaptation of the cp nerve at the sciatic trifurcation. post-cut day , nerves and dorsal root ganglia (drgs) were collected. axonal counts of nerves stained with nf- revealed similar regeneration between es and crush ( . vs. . mm, p= . ) that was superior to sham-stimulation ( . mm) or no-conditioning ( . mm, p< . ). a greater number of axons at the distal tip were present in animals that received either type of conditioning compared to the unconditioned cohorts. drgs were stained with neuronal injury marker growth associated factor- (gap- ), and satellite cell glial cells with glial fibrillary acidic protein (gfap). significant increase in gap- expression at three days was observed in es and crush cohorts compared to sham or naïve (p< . ) cohorts. the satellite glial cells of es and crush conditioning showed a significant increase in gfap expression ( . % and . % respectively) compared to sham ( . %) and naïve ( . %) drgs. by demonstrating similar improvements in axon regeneration, this proof of principle project suggests that es conditioning may produce regenerative outcomes comparable to the classical crush injury model. in turn, this suggests that es may be a promising method for delivering conditioning lesions in clinical trials for conditioning nerves prior to surgical intervention. we report a unique case of newly developed waldenstrom's macroglobulinemia (wm) in a patient with chronic inflammatory demyelinating polyneuropathy (cidp) with antibodies against myelin-associated glycoprotein (mag) and sulfatide who was undergoing treatment with intravenous immunoglobulines (iv-ig). subsequent rituximab infusions did not have a positive impact. patients with wm can develop demyelinating and axonal polyneuropathies and few patients have anti-mag and/or anti-sulfatide antibodies. anti-mag antibodies ( % of wm) are associated with sensorimotor axon loss and demyelination and anti-sulfatide ( % of wm) with sensory axonal loss. rarely, both antibodies can be present, with a more severe clinical phenotype. cidp associated with anti-mag and anti-sulfatide antibodies can represent independent entities, not associated with wm. there are no reports to date of patients with cidp associated with anti-mag and anti-sulfatide antibodies that developed wm during immunomodulatory therapy with iv-ig. in addition, subsequent rituximab infusions after the iv-ig were stopped have not been proven beneficial, as has been previously reported for anti-mag cidp patients. seventy-six year old right-handed gentleman presented with persistent numbness in his left foot, three months following artificial disc placement in his lumbar spine. gradually he developed sensory ataxia. no radicular signs were present on exam or impingement on serial spine mri's. ncs/emg studies were consistent with a cidp variant with severely prolonged distal motor latencies. serum anti-mag and anti-sulfatide antibodies were elevated. chronic therapy with iv-ig was able to partially stabilize the symptoms; however, six years later he newly developed wm. subsequent infusions with rituximab, after iv-ig was stopped, did not improve the clinical picture or the ncs/emg findings. wm can newly develop in an autoimmune setting, such as cidp associated with anti-mag and anti-sulfatide antibodies. in this particular case, there was an ongoing immunomodulatory therapy for our cidp patient, as he had monthly iv-ig infusions. this may reflect a possible induction of pathological b cell clone proliferation during the iv-ig treatment. subsequent rituximab infusions, after the iv-ig was stopped, did not improve the symptoms or the demyelination features on ncs/emg. he continues to be symptomatic despite efforts. charcot-marie-tooth disease (cmt) is an inherited neuropathy without known cure (prevalance: : ). duplication of the gene encoding the peripheral myelin protein of kda (pmp ) underlies the most common subtype cmt a. severely affected cmt patients suffer from sensory and motor symptoms with wheelchair-boundness. the clinical phenotype is highly variable and is determined by the amount of axonal loss, but the molecular mechanisms of the disturbed neuron-glia interaction are poorly understood. risk factors have not been investigated. therefore, cmt-net, a german network funded by the german ministry of education and research (bmbf, bonn, germany) includes interdisciplinary expertise from molecular biology, neurology, neuropathology and human genetics in order to identify genetic and non-genetic risk factors of disease severity of cmt by: (i) examining the mechanisms of the disturbed axon-glia-interaction and neuronal vulnerability, (ii) identification of genetic modifiers and (iii) novel therapeutic targets, (iv) validating outcome measures in children and adults, (v) establishing a biobank and (vi) exploring the disease burden via an internationally harmonised patient registry. cmt-net includes three service structures cmt-net will focus on cmt a, but also includes rarer subforms. we will provide the scientific basis for the development of translational approaches to therapy in patients. our approach bridges cutting edge molecular screening techniques, transgenic animal models of altered axon-glia interactions (fly, chick, mouse, rat), state-of-the-art genomic technologies and human patient trials in order to understand and treat the disease aggravation in cmt. sezer g , tekol y , sezer z , . erciyes university, betül ziya eren genome and stem cell centre, kayseri, turkey; erciyes university, school of medicine, pharmacology department, kayseri, turkey; erciyes university, good clinical practice and research centre, kayseri, turkey. analgesic effects of antidepressant drugs are well known for a long time, however, their systemic side effects limit their usage as an analgesics. venlafaxine is an antidepressant drug that has different structure. our purpose was to investigate whether systemic analgesic effect has been proved drug, venlafaxine, has local peripheral antinociceptive action. we applied vanlafaxine ( μl , , , μg) to male, sprague-dawley rats' paws by intraplantar injection and also by intraperitoneal route ( , , , mg/kg) in formalin test, a model for acute and tonic pain. we also pretreated another groups of rats with mg/kg naloxone (opioid receptor antagonist), mg/kg cpt (adenosine a receptor antagonist) or saline (ip.) before μg/paw venlafaxine injection. to check the effect is local or not, we determined the blood levels of venlafaxine in at different time points after both the local and systemic applications by gc-ms method. datas were expressed as number of flinches and total time for biting/licking of the injected paw over phase ( - min) or phase ( - min) and analyzed using the student's t-test. venlafaxine induced antinociception at , and μg concentrations by the local peripheral application and at , mg/kg doses by the systemic application in formalin test and the effects were comparable. pretreatment with naloxone diminished the effect of venlafaxine in the both phases, however, it was not statistically significant. pretreatment with cpt decreased venlafaxine induced antinociception only in phase . neither naloxone nor cpt changed formalin induced nociceptive behaviors alone. this is the first that determines the peripheral antinociceptive actions of venlafaxine in rat formalin test. with roles of opioid and adenosine a receptors in this action. our results suggest that venlafaxine has local peripheral antinociceptive effect and such an activity may led to trials for to use this drug as a cream-gel formulation for analgesia in clinics in the future. topical application might permit the attainment of higher and more efficacious concentrations in the region of the sensory nerve terminal, with limited systemic side effects. shah a , hoffman em , klein cj , staff np . mayo clinic, rochester, usa. cipn is a common dose-limiting complication for patients with cancer. the long-term disease burden of cipn is compounded by increasing cancer survivorship, yet there are minimal data on long term outcomes following onset of cipn, especially in population-based studies. we utilized the rochester epidemiology project to examine incidence and disease burden of cipn among individuals of olmsted county, minnesota with neurotoxic chemotherapy exposure between and . clinical records were queried for the presence of neuropathic signs, symptoms and icd- diagnostic codes as well as for patient provided information on impairment with activities of daily living and use of pain medications. a total of individuals with incident exposure to neurotoxic chemotherapy agents between and were identified. based on aan criteria for identifying peripheral neuropathy, ( . %) of these individuals were determined to have cipn, while ( . %) controls did not. the median time from incident exposure to reported symptom onset was days (iqr . - ). patients with cipn received a neuropathy icd- diagnosis in merely cases ( . %). median survival following incident chemotherapy exposure among all cases and controls was . years with a significantly longer mean survival in cases with cipn as compared to that of controls ( . years vs. . years, p< . ). in addition to acute effects in cipn, individuals surviving greater than years following exposure to neurotoxic chemotherapy continue to self-report increased symptoms of numbness (or . , % ci . - . ) and pain (or . , % ci . . . ) of the extremities. through utilization of patient provided information, our study was able to collect data on long-term impairment associated with previous history of exposure to neurotoxic chemotherapy. our results are consistent with previous reports of the high incidence of cipn in the first two years following incident exposure. additionally, our results provide evidence of high incidence of cipn independent of individual chemotherapeutic agent used. additionally, our results indicate icd- -cm diagnostic code attribution may dramatically underestimate the magnitude of cipn. increased survival following exposure to neurotoxic chemotherapy and its long-term disease burden necessitates further study of among survivors. the utility of quantitative muscle ultrasound as a marker of disease severity in charcot-marie-tooth (cmt) disease subtypes was investigated. muscle ultrasound was prospectively performed on individual muscles from cmt patients ( cmt a, cmtx , cmt a) and compared to muscles from age and gender-matched controls. muscle ultrasound recorded echogenicity and thickness in representative muscles including first dorsal interosseus (fdi) and tibialis anterior (ta charcot-marie-tooth (cmt) disease is the most frequent inherited peripheral neuropathy, and there is currently no available cure. the most common subtype of cmt, cmt a, is completely associated with duplication of the pmp gene, which encodes peripheral myelin protein of schwann cells. previous studies of cmt a mainly relied on rodent models, and it is not yet clear how pmp overexpression leads to the phenotype in patients. based on induced pluripotent stem cell (ipsc) technology, we herein developed a brand new in vitro cell model of cmt a, called cmt a-hipscs, in the hopes of simulating the developmental progress of the disease and gaining new insights into its pathogenesis. here, we efficiently derived ncscs from cmt a-ipscs and assessed the potential of the isolated cmt a-neural crest stem cells (ncscs) to differentiate into peripheral neurons and schwann cells using defined media. we found that, unlike normal control ncscs, cmt a-ncscs rarely generated schwann cells. instead, cmt a-ncscs produced numerous endoneurial fibroblasts in the schwann cell differentiation system. we further established a pmp -overexpressing ipsc model, and obtained similar results when pmp -ncscs were subjected to schwann cell differentiation. these results suggest that the development of schwann cells in cmt a patients is interrupted by the duplication of pmp . with the exception of the demyelination-remyelination process, developmental disabilities of schwann cells should be considered as an underlying cause of cmt a. shimoi t , yamada t . international university of health and welfare, tochigi, japan, cmt japan, tokyo, japan. charcot-marie-tooth (cmt) disease is the most common hereditary motor and sensory neuropathy. our preliminary report suggests that a certain cmt patient has the recruitment disorder of motor units during muscle fatigue and this disorder may be a factor of "super fatigability" in motor neuropathy patients. if the "super fatigability" occurs, we would expect patients with this characteristic to become slower in recovery from muscle fatigue than patients without this characteristic. in order to verify this hypothesis, we measured characteristic of recovery from muscle fatigue in charcot-marie-tooth patients with electromyographic study. twenty three participants were asked to maintain their % of maximal voluntary isometric contraction (mvc) of elbow flexor until exhaustion as the fatigue exercise. in addition, the participants asked to perform s of their % of mvc at , , , , , , , , , s after the fatigue exercise as recovery tasks. the surface emg (semg) signals of biceps brachii muscle were determined during the exercise and tasks. muscle force, median power frequency (mdpf) and the root mean square of semg amplitude (rms) were used as objective parameters of muscle fatigue. borg scale was used as a subjective parameter of muscle fatigue. six of twenty three participants showed significant decrease of rms during the fatigue exercise. in consideration of this result, we compared alteration of mdpf in recovery task between six participants with decrease of rms (abnormal group) and seventeen participants with increase of rms (normal group). as the result, the abnormal group had at least s as the recovery time from muscle fatigue in contrast with s of normal group. the recovery time from muscle fatigue in subjective parameter was shorter than the time in objective parameters in each group. our data support the "super fatigability" hypothesis. and that hypothesis may induce "hidden muscle fatigue". comparison between complex regional pain syndrome type and based on electrophysiologic, imaging and clinical findings shin jy , moon jy , sung jj . seoul national university hospital, seoul, republic of korea. complex regional pain syndrome (crps) is a constant regional neuropathic pain characterized by various kinds of motor, sensory, and autonomic changes. conventionally, the crps is divided into type i and ii according to the absence and presence of nerve injury. but the pathogenesis of crps is not fully understood yet. and there is still no systematic comparative study between crps type i and ii. we compared between crps type i and ii using multimodal approaches including electrophysiologic, imaging, and clinical findings. the patients ( type i and type ii) diagnosed with crps using the international association for the study of pain (iasp) diagnostic criteria were included. type i and ii were divided by electromyography and nerve conduction study. we obtained clinical information such as continuing pain, allodynia, hyperalgesia, edema, temperature, skin color, sweating, trophic change from patients. all patients were evaluated by bone scan, thermography, quantitative sudomotor axon reflex test (qsart), quantitative somatosensory test (qst). the ratio of qsart and temperature threshold abnormality in type ii was higher compared to type i ( . % vs . %, . % vs . % respectively, p = . and . ), among clinical symptoms, sweating change significantly high in type ii compared to type i ( . % vs . %, p = . ). other electrophysiologic and imaging, clinical findings were not significantly different in both type. in this study, we identified that crps type i and ii are distinguished not only by the nerve injury but also by the sudomotor function, and qsart can serve as a good technique to differentiate between crps type i and ii. it is estimated that there are two distinct pathogenesis in crps. our results may be helpful to diagnose crps correctly and understand the pathogenesis of crps. sjogren syndrome (ss) is an autoimmune inflammatory disorder of exocrine glands resulting in xerophthalmia and xerostomia. ross syndrome is a rare entity characterized by tonic pupil, hyporeflexia, and segmental anhidrosis. we present a -year-old hispanic woman with debilitating sensory and autonomic neuropathies, and persistently elevated anti-ss-a and anti-ss-b antibodies, without the classic sicca complex. she initially developed diarrhea and an ear infection, then felt toe and later leg numbness, which eventually spread to her cheeks and tongue, over few months. four years later, her thumbs and index fingers started tingling. also, she developed orthostatic lightheadedness, tachycardia, segmental hypohidrosis of the right abdomen, and hyperhidrosis of the remaining trunk, intermittent erythema, chronic diarrhea, and a -pound weight loss. her exam demonstrated orthostatic hypotension, bilateral tonic pupils and light-near dissociation, sectoral palsy of the right iris sphincter, stocking-distribution diminished sensation to all modalities, pseudoathetosis, areflexia, dysmetria, intention tremor, romberg, and sensory gait ataxia. mri of neuraxis demonstrated t -weighted hyperintensity in the dorsal spinal cord from c to the lower thoracic level, with mild atrophy. electrodiagnostic testing was consistent with moderate-to-severe, length-dependent, asymmetric, sensory polyganglionopathies. csf showed oligoclonal bands. serology showed elevated antinuclear antibody ( : , reference < : ), ss-a ( , reference < u), ss-b ( , reference < u), and rheumatoid factor ( , reference < iu/ml) titers. the remaining workup was negative for infection (syphilis, hiv, htlv, hepatitis, lyme disease), paraneoplastic syndrome (anti-hu and ganglionic nicotinic acetylcholine receptor antibodies), pyridoxine intoxication, malignancy (chest/abdomen/pelvis ct, breast ultrasound, axillary lymph node flow cytometry, colon and esophagus biopsy), celiac disease, vitamin deficiency, autoimmune disease (anti-aquaporin and anti-gq b antibodies), and adrenoleukodystrophy. the patient received ivig and steroid with some gait improvement and currently takes mycophenolate. midodrine and fludrocortisone resolved her dizziness. this case highlights an important, treatable sensory ganglionopathy and systemic autonomic neuropathy due to sjogren syndrome, and illustrates the overlapping clinical triad of ross syndrome, which may guide future management. siles am , , assylbekova d , , diaz-manera j , , rojas-garcia r , , cortes e , , gallardo e , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, univeristat autònoma de barcelona, barcelona, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. inflammatory neuropathies are a heterogeneous group of peripheral nerve diseases that respond to immune-therapies. chronic inflammatory polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) are two chronic inflammatory neuropathies responding to intravenous immunoglobulins (ivig). b lymphocytes are involved in their pathogenesis. while widely used in clinical practice, ivig's mechanism(s) remain not completely understood. ivig are reported to lead to b-cell anergy and to increase regulatory t-cell function and frequency. regulatory b cells (bregs) are a rare subset of b lymphocytes that suppress immunopathology acting upon several target cells in the immune system. impaired breg yields have been described in a plethora of autoimmune conditions. the presence of regulatory b cells in inflammatory neuropathies and the effect of ivig therapy on their frequencies has not been studied. the aim of this study is to describe the frequencies of bregs in cidp and mmn and the effect of ivig on their frequencies. patients fulfilling diagnostic criteria for cidp or mmn and matching controls were included. pbmcs were obtained by gradient centrifugation before ivig infusion and one week after treatment. b-cells were isolated with negative selection magnetic beads, cultured and activated with the tlr agonist odn and anti-human igg+iga+igm. il- secretion capacity was assessed by flow-cytometry. twenty-eight patients were included of whom where cidp and mmn. of all patients included, received ivig and were suitable for pre and post ivig breg frequency comparisons. breg frequencies did not differ in patients (before ivig treatment) and controls (p= . , mann whitney test, two-tailed). however, the frequencies of bregs significantly increased one-week after treatment with ivig (p= . , wilcoxon matched pairs test, two-tailed). when stratifying by disease subtype, breg frequencies increased in cidp patients after ivig (p= . , wilcoxon matched pairs test, two-tailed) and in mmn (p= . , wilcoxon matched pairs test, two-tailed) although results did not reach statistical significance in mmn. this is the first study that studies the breg frequencies in cidp and the first study that addresses the effect of ivig on breg frequencies. our study provides the proof of principle that bregs could become a biomarker for response to ivig but this would need a larger and prospective study. siles am , , martínez-hernández e , diaz-manera j , , rojas-garcia r , , gallardo e , , illa i , , graus f , querol l , . paraneoplastic neuropathies (pn) are rare, immune-mediated disorders of the peripheral nerve with important prognostic implications. ectopical expression of neural antigens in the tumor leads to the development of onconeural antibodies. several autoantibodies associate to pn, including anti-hu, anti-caspr or anti-cv antibodies but a significant proportion of pn lack identifiable antigens. adhesion molecules that are autoantigens in other neuropathies, like contactin- , are present in several types of tumors. our study proposes a systematic screening of autoantibodies against neural cell-adhesion molecules and neural structures to detect novel antigenic reactivies in pn. thirty-five patients followed in our centre and at the neuroimmunology-multiple sclerosis unit at hospital clínic de barcelona, with pn fulfilling diagnostic criteria of possible (n= ; . %) and definite (n= ; . %) paraneoplastic disease were included. serum samples were obtained and tested by immunocytochemistry against contactin- (cntnt ), neurofascin (nf ) and the cntn /caspr complex. primary cultures of dorsal-root ganglia (drg) and rat schwann cells were incubated with patients' sera to detect antibodies targeting neural structures. ten individuals ( . %) presented with a tumor and a neuropathy involving both sensory and motor symptoms. the remaining patients ( . %) presented with a tumor and a classical sensory neuronopathy without anti-hu or any other onconeuronal antibody. among the latter, ( . %) patients were diagnosed with small-cell lung carcinoma. the rest of the individuals ( . %) associated diverse malignancies. we did not detect any sera reacting against cntn , nf or the cntn /caspr complex. in igg antibody screening experiments, patients ( . %) reacted against drg neurons, of them ( . %) reacting strongly, and patients ( . %) reacted mildly against rat schwann cells. in igm experiments, patients ( . %) reacted slightly against drg neurons and patients ( . %) against rat schwann cells, of them ( . %) featuring strong staining. experiments screening antibodies against motor neurons and immunoprecipitation assays are ongoing. overall, % of patients reacted strongly against either neurons or schwann cells. our study did not detect antibodies against the neural adhesion molecules cntn , nf and the cntn /caspr complex in patients with pn. however, a significant proportion of pn patients harbour antibodies targeting neural structures, which suggests that novel neoplasm-associated antigens remain to be discovered. simmons m , tao f , abreu l , zuchner s , li j . department of neurology, vanderbilt university school of medicine, nashville, tennessee, usa; hussman institute for human genomics, university of miami, miami, florida, usa. objective: despite of a shared genetic mutation of the trisomy of chromosome p (c p ), patients with charcot-marie-tooth type- a (cmt a) present with a high variability of their disease severities. the underlying cause for the variability is still unclear. in this study, we tested a hypothesis whether a second genetic mutation known to damage the nervous system is also present in cmt a patients with early onset and severe phenotypes. methods: from a cohort of patients with cmt a mutation (chromosome p duplication), we identified patients with an early onset (< or = years of age) of the disease. four of the eleven also had dna testing for a panel of known cmt-related genes and sequencing of mitochondrial dna in addition to the dna testing for c p duplication. results: besides the c p duplication, we identified three additional mutations in the four patients with early onset. the mutations were a missense mutation of arg his in mpz gene, an a g mutation in mitochondrial trna for glycin and a homozygous mutation of c p duplication. three of the four had symptomatic onset at birth. one showed symptoms at years of age. conduction velocities were severely reduced in all four patients (from to m/s). interpretation: traditional approaches to identify genetic modifiers, including snp association, assume that those modifiers are clustered in a small region of human genome and shared by the studied patients. however, our study suggests that genetic modifiers in cmt a may be highly diverse and scattered throughout the genome, which could make the conventional approach via the genetic variants association difficult. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr sjogren's syndrome(ss) is a systemic auto-immune disease that apart from exocrine glands may affect any organ. involvement of peripheral nervous system results in wide spectrum of neuropathic manifestations. the aim of our study was to evaluate the clinico-electrophysiological patterns and pathological characteristics of neuropathy in sjogren's syndrome (ss) patients presented to neuromuscular clinic in a tertiary hospital from south india. this is a retrospective study from the departments of neurology, rheumatology and pathology from nizam's institute of medical sciences. twenty one patients with diagnosis of ss and peripheral neuropathy between to were analysed. clinical records, conventional nerve conduction studies, lip and nerve biopsy reports were collected.in patients with ss associated neuropathy,male to female ratio was : . in ( . %) neuropathy was the initial manifestation,while in ( %)exocrinopathy preceded neuropathy. the patterns of neuropathy included mononeuropathy multiplex(mnm) in patients ( %),ganglionopathy in ( %),length dependant, trigeminal, autonomic neuropathy and cidp in ( %)and cranial neuropathy in ( %).eighteen( %) were seropositive..schirmer's test was positive in ( . %).nerve biopsy showed vasculitis in patients, demyelinating and axonopathy in patients each.we conclude that neuropathy is frequently the initial presentation of ss.mnm is the common pattern followed by ganglionopathy. pattern of neuropathy helps in arriving at the diagnosis of ss. confirmation of ss is not by mere serology. schirmer's test and lip biopsy are equally essential for the diagnosis especially in seronegative patients when clinical index of suspicion is high. siskind ce , tesi rocha c . stanford health care, palo alto, ca, usa; stanford university, stanford, ca, usa. here, we report the first case of a human found to have a homozygous, presumed disease causing variant in the arl ip gene, causing charcot marie tooth disease (cmt) with central nervous system findings. the proband was born at term with apgars of and at and minutes. he was found to have iugr and was hospitalized for days for weight gain. he developed respiratory distress during the admission and was intubated. due to inability to extubate, he was transferred to stanford children's hospital, where he remained for three months. noted during admission was hypotonia, areflexia, minimal voluntary movment, sinus tachycardia, and dysautonomia. brain mri found polymicrogyria and cerebral underdevelopment with generally normal-appearing brainstem, with moderate ventriculomegaly. ncs found length dependent polyneuropathy with axonal degeneration. follow up muscle and nerve biopsy found immature muscle and amyelinating neuropathy the patient had normal plasma amino acid, acylcarnitine, lactate, pyruvate, urine organic acid testing, opththalmology exam, newborn screen, and normal array cgh. genetics ordered whole exome sequencing through baylor genetics laboratory (houston, tx, usa), which found a homozygous nonsense variant in arl ip : c. c>t, p.r x. a second child had been identified by baylor with two variants in this gene. that child had hypotonia, respiratory distress and seizures, and a muscle biopsy consistent with sma. the parents of that child chose to withdraw care at months of age. our patient's parents continued with aggressive therapies, including tracheostomy and g-tube for feedings. he had several subsequent hospitalizations for respiratory distress, possible seizure activity, and buldging anterior fontenelle, but now, at two years of age, has made developmental progress and is living at home with his family. he is able to smile, reaching for toys and swatting objects. he has little voluntary movement, and no longer responds to light touch stimuli. overall, this is the first picture of a child affected with a severe amyelinating form of cmt that causes weakness, hypotonia, and possible seizures, with the main concerning feature being the severe respiratory distress that may be life threatening, but can be managed with extreme care. charcot marie tooth (cmt) disease is the most common inherited peripheral neuropathy. patients frequently ask whether pregnancy will affect their cmt, whether cmt will affect their pregnancy, the optimal delivery and whether they or their child will have a higher risk of complications during pregnancy or delivery. so far few studies address these questions. currently no guidelines exist for the management of pregnancy, delivery and postnatal care in cmt patients. the aim of the study is assess the impact of pregnancy on cmt and assess how cmt affects pregnancy, delivery and care of the new born baby. we designed a retrospective questionnaire with expert help from an obstetrician with a special interest in pregnancy in patients with medical conditions. the questionnaire is divided into four parts (prior, during, after pregnancy and delivery) and includes questions on impairment, falls, pain, fatigue and respiratory complications during those periods; type of delivery, possible complications, details of anaesthesia and difficulties looking after the baby in the first months postpartum. so far women ( pregnancies) with cmt and related disorders have answered the questionnaire. % of patients had cmt a, the remaining had various subtypes of cmt and related disorders. patients reported deterioration of cmt symptoms during pregnancy in % of pregnancies with resolution of symptoms after pregnancy in % of pregnancies. of symptoms questioned walking ( %), balance ( %), and hand function ( %) deteriorated the most. there was an increased use of orthoses and walking aids during pregnancy. the majority of women ( %) had natural delivery, % were assisted and % had caesarian sections which was similar to the uk population ( %). no complications with anaesthesia were reported. the survey is currently ongoing. we plan to survey consecutive patients. data acquired from this survey will provide valuable information on current practice and will inform future guidelines and standard of care in charcot marie tooth disease. multifocal motor neuropathy (mmn) is a slowly progressive disorder in adults, characterized by asymmetrical limb weakness, mainly affecting the arms. despite beneficial effect of immunoglobulins, weakness gradually progresses. a major determinant of muscle weakness is the degeneration of affected motor axons. treatments aiming to reduce loss of motor axons require objective tools to quantify such an effect. therefore, we applied the compound muscle action potential (cmap) scan, which is an electrophysiological method that, with increasing transcutaneous stimulus-currents, successively activates all motor units (mus) in a muscle. it captures the contribution of enlarged mus due to reinnervation by the presence of relative large discontinuities in the scan. the aim of the present study was to identify pathophysiological changes of mu-loss and reinnervation in mmn patients by means of the cmap-scan. recordings were obtained from mmn patients. cmap-scan recordings were performed in the median nerve at the wrist where motor responses were recorded from the thenar muscle. we determined the number of largest cmap-scan discontinuities by means of a novel marker, d , where a low number is indicative of mu-loss and enlarged mus. furthermore, we applied the recently developed method of professor hugh bostock for obtaining a mu-number estimate from the cmap-scan. the median peak cmap amplitude was . mv (range . - . mv) and median d was (range - ). in three mmn patients with a normal maximum cmap amplitude (> mv) a reduced d (< ) was found indicative of mu-loss and enlarged mus. furthermore, d and the estimate of mu number were significantly related (r = . , p < . , n = ). the findings suggest that the cmap-scan is a sensitive tool in detecting the underlying pathological changes of reinnervation and mu-loss in mmn, more so than standard maximum cmap amplitude. it is quick and easy to perform and has the potential to be useful for follow-up studies. smith ag , thurgood b , revere c , hauer p , aperghis a , singleton jr . university of utah, salt lake city, utah, usa. corneal confocal microscopy (ccm) directly and quantitatively assesses corneal innervation including nerve fiber length (nfl) and density (nfd). ccm has shown promise as a diagnostic test. we have previously demonstrated that ccm has a diagnostic performance for diabetic neuropathy (dpn) similar to skin biopsy with assessment of intraepidermal nerve fiber density (ienfd) and nerve conduction studies (ncs). the responsiveness of these surrogate measures to dpn progression and their relation to clinically meaningful outcomes has not been well explored. diabetic patients undergoing annual retinopathy examination were recruited. each underwent ccm, ienfd, ncs including sural sensory and peroneal motor responses, the utah early neuropathy score (uens), the norfolk quality of life -diabetic neuropathy (nqol-dn, a validated neuropathy specific qol scale), and a minute walk test ( mwt). with dpn based on symptoms ( %) or signs underwent repeat testing at months and at months. at baseline, nqol-dn correlated with sural sensory amplitude (ssa) (− . , p< . ), peroneal motor conduction velocity (pcv) (− . , p< . ) and ienfd (− . , p< . ). no ccm metric was related to qol. mwt distance correlated with ssa ( . , p< . ), nfl ( . , p< . ) and nfd ( . , p< . ). over months, there was a significant worsening in dpn signs assessed by the uens (increase . +/− . , p< . ). ssa declined . uv (p< . ) and ienfd . fibers/mm (p< . ). there was no change in any ccm metric, pcv or nqol-dn. these findings suggest measures of distal axonal integrity are most sensitive to neuropathy progression, with ienfd having the greatest responsiveness. in contrast, ccm was not responsive to dpn progression. both ncs and ienfd (but not ccm) were significantly correlated with neuropathy-specific qol, whereas ncs and ccm measures correlated with physical functioning. the responsiveness of ienfd and ssa, and their relationship to qol support their selection as endpoints in dpn clinical trials. chronic inflammatory demyelination polyradiculoneuropathy (cidp) affects in , people, and is marked by chronic autoimmune infiltration of peripheral nerves and destruction of the myelin sheath. with current therapies, only % of cidp patients achieve complete remission. to produce more effective, mechanism-based therapies, we study mice with a partial loss of function g w substitution in the autoimmune regulator (aire) gene on the non-obese diabetic (nod) background (nod.aire gw/+ ) that develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. autoimmunity can result from defective immunosuppression. the potent, immunosuppressive cytokine interleukin (il- ) is increased in the peripheral blood mononuclear cells (pbmcs) of active phase cidp patients relative to remission phase patients. further, pbmcs from cidp patients produce il- in response to the myelin protein p . despite these findings, whether il- is important for cidp pathogenesis is not known. thus, we sought to determine the role of il- in sapp. il- was highly upregulated in sciatic nerves of nod.aire gw/+ mice with sapp, suggesting it may play an immunosuppressive role in pathogenesis. however, genetic ablation of il- in nod.aire gw/+ mice lead to a paradoxical delay in disease development. age-matched il- -deficient nod.aire gw/+ mice exhibited no sciatic nerve infiltrate and no reduction in nerve conduction during electrodiagnostic studies. interestingly, the delay in sapp was specific, since the incidences of five other autoimmune manifestations in il- -deficient nod.aire gw/+ mice were unchanged relative to il- -sufficient nod.aire gw/+ controls. importantly, il- -deficient nod.aire gw/+ mice did not have colitis, which is consistent with previous studies of il- deficiency on the nod background. il- is known to perform effector functions in autoimmunity by promoting b cell secretion of immunoglobulins. however, genetic ablation of b cells did not affect neuropathy development in nod.aire gw/+ mice, suggesting b cells are dispensable for pathogenesis and unlikely to mediate the protective effect of il- deficiency. il- -deficient nod.aire gw/+ cd + t cells, which are sufficient to transfer sapp, exhibited increased activation, increased interferon gamma secretion, and preserved nerve-specific t cell activation. these data suggest t cell activation and priming are unperturbed and not the mechanism of protection. in summary, our data showed that il- was paradoxically an effector cytokine in sapp. long exercise test (let) has been used especially in myotonic syndromes and muscle channelopathies. marked decrement in compound muscle action potential (cmap) amplitude after prolonged exercise was previously reported in patients with paramyotonia congenita, hyperkalemic or hypokalemic periodic paralysis. we describe a patient with secondary hypokalemic paralysis who showed abnormal let results. a -year-old man presented with ascending flaccid paralysis which evolved in a hyperacute fashion. the patient became quadriplegic after two hours. initial laboratory evaluation revealed severe hypokalemia, with normal thyroid function. we performed electrodiagnostic studies including long exercise test as proposed by mcmanis et al. nerve conduction study was normal, but marked decrement in cmap amplitude (up to % decrease after minutes) was noted after prolonged exercise. despite oral and intravenous potassium replacement, serum potassium level was not corrected as expected. the unusual clinical course prompted for evaluation of secondary etiologies. abdomen computed tomography scan revealed a . x . cm-sized mass in the left adrenal gland. aldosterone to renin ratio was elevated, suggestive of primary hyperaldosteronism. genetic study for cacna s mutation turned negative. after receiving laparoscopic adrenalectomy, the patient experienced no further attacks, and also was able to stop his antihypertensive medication. let may show abnormal results in condition with reduced membrane excitability, even without true channelopathy. according to international criteria, the diagnosis small fiber neuropathy (sfn) is based on clinical symptoms in combination with a reduced intraepidermal nerve fiber density (ienfd) in skin biopsy and/or abnormal temperature threshold testing (ttt). the sensitivity of skin biopsy is moderate to good, although ienfd is normal in about % of patients with sfn complaints. furthermore, ttt is a widely available diagnostic tool, but lacks specificity. corneal confocal microscopy (ccm) has been described and is used in clinical practice as an objective, non-invasive diagnostic tool to detect small nerve fiber damage in patients with diabetes mellitus. this study examines the applicability of ccm in patients with sfn, and the value of ccm as an additional diagnostic tool in sfn. we will include healthy participants to compare the results with the recently published ccm normative values, and patients referred to the sfn center maastricht with the clinical picture of sfn. corneal nerve fiber density (cnfd), branch density (cnbd), fiber length (cnfl), and the tortuosity coefficient (cnft) will be determined in all participants. the results will be compared with the ienfd and ttt. preliminary results will be presented. southanalinh k , university of health sciences, vientiane capital, lao p.d.r. located in south east asia, lao pdr is a landlocked country with a population of about . million inhabitants. the health indicators are among the lowest in south east asia. the total health caregivers in consisted of , persons corresponding to a ratio of . health workers per inhabitants. the main network for health care service provision remains the public system. its health care facilities consist of four central teaching and referral hospitals; five regional hospitals, including one teaching hospital; provincial hospitals; district hospitals, and about health centers. only one in seven sick people receives modern health care treatment. most people rely on self-medication and/or reliance on self-healing. neurological care is a very new field. knowledge of common neurological disorders among both the lao population and medical staff is only beginning to be spread. there are three neurologists in the country. six neurology residents are currently being trained in a three-year program supported by the association pour la promotion des neuro-sciences au laos (association for the promotion of neuro-sciences in laos) and the asean neurological association. indeed, resources are scarce. in the peripheral nerve diseases domain for example, we have only one electromyography machine that was only temporary used when emg experts from france and from singapore came to teach residents. a significant mismatch between the provision of specialized neurologic services and the needs for them exists, especially in rural areas. also, health insurance is not available for the majority. as a consequence, patients have to bear the costs themselves, which constitutes a limit to the access of available healthcare facilities. neurologic training centers, laboratory facilities and equipments are limited. optimizing available human resources, integrating primary, secondary, and tertiary healthcare tiers and making medical treatment more affordable are need to improve neurologic care in the developing world. in certain low-income countries with limited human and financial resources, it may be difficult for governments to apply some of these recommendations on their own. in these circumstances, it is suggested that countries work with international agencies, nongovernmental organizations or other partners to put their plans into practice. spina e , topa a , iodice r , tozza s , dubbioso r , ruggiero l , santoro l , manganelli f . department of neuroscience, odontostomatological and reproductive sciences, university of naples "federico ii", naples, italy. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a disabling disease and about % of patients may become persistently disabled over time. our aim was identify clinical prognostic factors of long-term disability in a large series of cidp patients. we collected data from cidp patients with definite diagnosis according efns/pns criteria and positive response to first-line therapies (immunoglobulin or corticosteroids) including sex, age of onset, phenotype, disease duration, course of disease (monophasic/relapsing-remitting or chronic progressive) and disability at the time of diagnosis assessed using the modified rankin scale (baseline mrs). all patients had clinical assessment of disability through mrs within the last months (last mrs). ordinal logistic regression model was applied to evaluate the relationship among the clinical parameters and last mrs, considered as ordinal outcome ( - ). anova test for repeated measures was applied to test the overall effects of different course on disability accumulation while t-test was performed to evaluate inter-group differences for parametric variables. we found a significant relationship between last mrs and the course of disease [p< . , z= . , or: . ]. disability accumulation was greater in patients with chronic progressive course than those with monophasic/relapsing-remitting course of disease [p= . ]. moreover, patients with progressive course were older [p= . ]. our data suggest that chronic progressive course of disease may be a major negative prognostic factor for long-term disability in cidp patients. to note that a chronic progressive course of disease is also associated with an older age from the beginning and a more pronounced worsening over the course of disease. sprenger a , lichtenstein t , henning t , lehmann hc . department of neurology, university hospital of cologne, cologne, germany; institute of diagnostic and interventional radiology, university hospital of cologne, cologne, germany; department of neurology, university hospital of cologne, cologne, germany. an unresolved problem in the treatment of inflammatory neuropathies is the lack of valid and reliable diagnostic biomarkers to evaluate axonal damage. we investigated if "diffusion tensor imaging" (dti) and mri t w multi echo dixon imaging are eligible methods to determine proximal nerve injury in chronic inflammatory demyelinating polyneuropathy (cidp). in this prospective observational cohort study the sciatic nerve of cidp patients and age matched healthy controls was investigated. all subjects underwent multimodal mri imaging to determine fractional anisotropy (fa) and muscle fat fraction of the biceps femoris and quadriceps femoris muscle. patients were evaluated by mri, clinical examination and nerve conduction studies at baseline and after six months. the mean fractional anisotropy (fa) value was significantly lower in the sciatic nerve from cidp patients compared to controls. fat fraction of the biceps femoris and quadriceps femoris muscle were significantly higher in cidp patients compared to controls. mri outcome parameters remained unchanged after six months. our study demonstrates the utility of mri imaging to differentiate between "healthy" and functional constricted proximal nerve segments. we postulate that dti and dixon mri might be eligible methods to assess proximal nerve damage in cidp. the presence of peripheral myelin protein (pmp ) has been known for decades, but its functional role was uncovered only recently. recent characterization of pmp -deficient mice revealed a role of pmp in the lipid homeostasis of myelinating schwann cells. in this study, we analyzed the functional impact of pmp on myelination. to decipher the role of pmp , experimental demyelination was performed in myelinating dorsal root ganglia cultures, and in vivo re-myelination was assessed after experimental peripheral nerve damage. we used the myelinating dorsal root ganglia (drg) model in pmp -deficient schwann cell cultures, combined with an established de-and remyelinating protocol in order to analyze myelination in vitro. we also performed experimental nerve crush in pmp -deficient mice. morphometric parameters were defined for the in-vitro experiments and functional parameters such as nerve conduction velocity and the clinical score were additionally measured for the in vivo experiments. structural analyses of the drg cultures revealed fibers expressing myelin basic protein (mbp) and pmp , as well as fibers positive for mbp alone. in contrast to our previous in vivo data, we were also able to detect myelin segments that stained positive for pmp , but were negative for mbp. pmp -deficient drg-cultures demonstrated slightly greater nodal lengths than the control cultures. this trend was significantly augmented after in vitro de-and remyelination, which also resulted in decreased internodal lengths only now, while conserving an intact myelin structure. concomitantly, in vivo nerve crush gives rise to a more severe phenotype in pmp -deficient mice than in wild-type controls. consistent with this, nerve conduction studies showed a delay in remyelination, and analysis of semi-thin sections demonstrated an altered fiber structure in the peripheral nerve biopsies. together, these data suggest that in addition to its role in glial cell lipid homeostasis, pmp also plays a role in remyelination of the injured peripheral nervous system. anti-mag neuropathy remains a difficult diagnosis to treat given its limited therapeutic options. of all interventions, rituximab has emerged as the most effective, although its effect has been with mixed results, especially in patients with advanced axonal loss. lenalidomide is another promising immune modulating therapy, whose effect has been well demonstrated in neuropathy associated with poems (polyneuropathy, endocrinopathy, organomegaly, m-spike protein, and skin changes) syndrome, a condition that has several striking parallels to anti-mag neuropathy. the use of lenalidomide has not been previously described in anti-mag neuropathy. herein, we describe a case of lenalidomide-responsive anti-mag neuropathy in a patient with advanced axonal loss. suichi t , misawa s , sato y , beppu m , sekiguchi y , shibuya k , watanabe k , amino h , kuwabara s . department of neurology, graduate school of medicine, chiba university, chiba, japan; clinical research center, chiba university hospital, chiba, japan. polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes (poems) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscrasia and vegf overproduction. several diagnostic criteria for the disorder have been published, but sensitivity/specificity analyses, and their validation have never been performed. the aim of this study is to establish valid diagnostic criteria for poems syndrome. consecutive poems patients, seen at chiba university hospital since , were screened. of these, we have set a gold standard group of poems syndrome, based on treatment response and exclusion criteria during -year follow-up, and patients was diagnosed as having definite poems syndrome. we also collected patients with cidp (demyelinating neuropathy control) and with multiple myeloma, primary amyloidosis, or mgus (m-protein control). criteria for poems syndrome was defined as having two of the three major criteria (polyneuropathy, m-protein, and elevated serum vegf level) and at least two of the four minor criteria (extravascular volume overload, skin changes, sclerotic bone lesions, and thrombocytosis) which were determined by logistic regression analyses. according to the criteria the sensitivity was %, and the specificity was %. our results indicate that the proposed criteria have an excellent diagnostic accuracy, and are useful in clinical practice, presumably leading to early diagnosis and treatment. intraepidermal electrical stimulation (ies) is a new technique to that assesses the function of a-delta fibers in the epidermis. using this technique, we previously reported that the epidermal pain threshold was two-hold in asymptomatic diabetic patients than in normal subjects (muscle nerve : - , ). subsequently, we reported that the elevated pain threshold negatively correlated with intraepidermal nerve fiver density (jpns : s , ). empirically, it is known that lowering the skin temperature makes it less likely to feel pain. therefore, it is necessary to investigate whether the results of ies are affected by skin temperature. the aim of this study was to investigate the influence of a low skin temperature on pain threshold. we recruited subjects with a mean age of . years. for nociceptive stimulation, we used an ies method with a concentric micro-needle electrode that was developed specifically for the selective stimulation of cutaneous a-delta fibers. we placed the ies electrode onto the extensor digitorum brevis and began stimulation with intensity strong enough for the subject to feel a pricking sensation, then reduced the current in steps of . ma until no sensation was felt. we defined pain threshold as the minimum electrical intensity at which a subject felt a pricking sensation. firstly, we measured pain threshold at skin temperature above degrees celsius. then, we put an ice pack on the extensor digitorum brevis for min to lower the skin temperature, and measured pain threshold at skin temperatures below degrees. mean pain threshold values above degrees and below degrees of skin temperature were . and . ma (p< . ), respectively. our data indicated an elevated pain threshold in epidermis with a low skin temperature. one of most common methods for nociceptive stimulation is painful co laser stimulation. some co laser stimulation studies reported pain threshold increased with a low skin temperature. our result is similar to that of co laser stimulation. pain threshold using ies is very easy and non-invasive technique. it may be useful for the evaluation of small fiber neuropathy. svačina mkr , röth p , bobylev i , sprenger a , zhang g , sheikh ka , lehmann hc . department of neurology, university hospital cologne, cologne, germany; department of neurology, university of texas, houston, usa. intravenous immunoglobulins (ivig) are an effective treatment in guillain-barré-syndrome (gbs). in most patients, the optimal ivig dose and regime is unknown. in serum and ivig preparations, immunoglobulin (ig) g form igg dimers, which are assumed to consist of idiotypic/anti-idiotypic antibody pairs. however, data about kinetics of igg dimer formation in gbs are lacking. to study igg dimer formation, c bl/ mice were injected with ivig and anti-gd b antibody or pbs. blood sera were collected h, h and week post injection. a third cohort received an anti-gd a/gt b antibody and blood was collected h post injection. igg was extracted and subtyped into polymeric, dimeric and monomeric fractions using the Äkta fplc system. dialysed dimeric and monomeric igg fractions were examined for the presence of anti-ganglioside antibodies by anti-ganglioside antibody elisa. further, blood samples from gbs patients were collected before (pre-ivig) and after treatment with ivig (post ivig). serum samples were examined for igg dimers and monomers using the Äkta fplc system. in the mouse model, a maximum peak of igg dimer formation was observed h post injection. in gbs patients' samples, igg serum levels and igg dimer content was significantly higher after treatment with ivig. we demonstrate here the feasibility to assess igg dimer formation in an animal model and in gbs patients' samples after treatment with ivig. h after ivig treatment appears to be the optimal time point to assess igg dimer formation. further studies are warranted to determine the utility of igg dimer formation as surrogate marker for treatment response in gbs. svaren j , moran jj , wu x , gutmann l , shy m . university of wisconsin-madison, madison, wisconsin, usa; university of iowa, iowa city, iowa, usa. development of outcome measures for clinical trials in cmt a is a major challenge given the slowly progressive nature of the disease. outcome measures can be used to measure a) target engagement for a given therapy, as well as b) disease process and c) disease burden. several candidate therapies have been shown to reduce pmp levels in cmt a rodent models and thereby ameliorate the symptoms of pmp overexpression. measuring pmp mrna reduction in human trials has so far been limited to analysis of skin biopsies by qrt-pcr, which did not demonstrate clear elevations of pmp mrna during, nor a reduction following, ascorbic acid trials. the analysis of skin biopsies is hampered by variable amounts of schwann cells (sc) in skin biopsies, as well as the variable amount of pmp in sc as previously established by immuno-em in cmt a skin biopsies (katona et al., ) . therefore, it is important to develop optimal normalization criteria to address the variability inherent in skin biopsy analysis. ideally this will employ normalization to sc-specific genes that are not altered by cmt a status. to optimize normalization, we have performed rna-seq analysis of skin biopsies from patient and control skin biopsy samples. analysis of these data after normalization to read depth indicated that pmp levels were . fold higher in cmt a patient samples compared to control skin biopsies. however, there was significant variability in pmp levels particularly in cmt a samples, which may be due to variable amounts of schwann cells in cmt a skin. using a combination of sc-specific genes for normalization, we were able to reduce the apparent variability and optimize the differential levels between cmt a and control skin biopsy samples. we also identified other sc-specific genes that were apparently induced in cmt a skin biopsies relative to control. these studies provide a new framework for gene expression analysis in skin biopsies, enabling more precise evaluation of pmp levels in clinical trials for cmt a as a measure of target engagement. in addition, the normalization framework may also be applicable to other types of cmt. chronic inflammatory demyelinating polyneuropathy (cidp) is the most common chronic autoimmune neuropathy, with an estimated prevalence of between and per , people. it can cause temporary disability in the affected individuals and may eventually lead to permanent disability or death. cidp is commonly treated with intravenous immunoglobulin (ivig) therapy or corticosteroids. octagam ® % is licensed for cidp in france, while octagam ® % is licensed for cidp in germany and belgium. this analysis presents data from three open, multicenter, non-interventional, single-arm, non-controlled studies of a post-authorisation safety surveillance (pass) program for the subset of patients receiving octagam ® % or % for neurological indications, focusing on patients with cidp. briefly, data from in-and out-patients in austria, france, germany, and uk treated with octagam ® for neurological disorders were collected by physicians and analyzed to assess safety and tolerability of the treatment. of patients included in the three studies, patients ( . %) received octagam ® for neurological indications, of which patients ( . %; mean age . years [range - ]) had cidp. the mean dose of octagam ® per course was . g/kg bw for patients with cidp; for the other neurologic indications, the dose ranged from . (for multiple sclerosis) to . g/kg bw (for guillain-barré syndrome). premedication was not needed in . % of these patients. the development of clinical appearance since last observation (mean: every . months) was assessed for of the cidp patients by their treating physicians. the majority of observations ( . %) assessed the patients as stable and . % showed even an improved clinical appearance. only . % of the observation periods resulted in deteriorations. adverse drug reactions were rare: of the infusions received by patients with neurological disorders, . % of infusions were associated with an adr ( . % of infusions in cidp patients). overall, treatment with octagam ® was effective and well-tolerated in patients with cidp. these results are consistent with data for the overall patient population (including patients with primary and secondary immunodeficiencies, dermatological and other diseases). szepanowski f , szepanowski lp , kleinschnitz c , kieseier bc , stettner m . department of neurology, medical faculty, university duisburg-essen, essen, germany; department of neurology, medical faculty, heinrich-heine-university, düsseldorf, germany. lysophosphatidic acid (lpa) is a pleiotropic signaling lipid that acts as ligand for at least six specific g protein coupled receptors. schwann cells (sc) are known to mainly express the lpa receptor subtype. an emerging body of in vivo evidence has linked lpa with injury induced peripheral nerve demyelination as well as neuropathic pain. however, the molecular mechanism underlying its demyelinating effect has remained largely unclear. myelinated dorsal root ganglia (drg) cultures were treated either with lpa, lpa + am (lpa antagonist) or vehicle. we assessed myelin basic protein, tumor necrosis factor alpha (tnf-alpha) as well as the sc differentiation marker sox by immunocytochemistry. additionally, myelin was investigated by sudan black staining. to better understand the relevance of lpa signaling for demyelination in vivo, we performed sciatic nerve crush in c bl/ mice treated with am at mg/kg in order to study schwann cell expression of tnf-alpha, sox and sox , a marker for sc dedifferentiation, by immunohistochemistry. in drg cultures, lpa caused a significant reduction of myelin as demonstrated by both sudan black staining and immunocytochemical analysis of myelin basic protein. demyelination was paralleled by an upregulation of tnf-alpha as well as downregulation of sox . lpa mediated effects were found to be blocked by addition of the lpa receptor antagonist am . in c bl/ mice, am treatment prior to crush injury increased sox expression in scs in the distal nerve stump while reducing the number of cells expressing sox . these data indicate that lpa may be a critical factor to shift scs towards an injury-associated phenotype and contribute to the onset of wallerian degeneration. szepanowski lp , szepanowski f , kleinschnitz c , stettner m . department of neurology, university hospital essen, essen, germany. glyphosate-based formulations comprise the world's most commonly used herbicides. in non-resistant plants, glyphosate exerts toxic effects most likely via inhibition of aromatic amino acid synthesis by interfering with the shikimate pathway. while glyphosate is the active ingredient, herbicidal formulations contain several adjuvants, including polyethoxlated alkylamines (poeas). although glyphosate has long been considered safe for use in humans and animals, several studies have implicated glyphosate and/or the commonly used adjuvants in cytotoxicity, carcinogenicity and endocrine disruption. furthermore, glyphosate-based herbicide has been reported to mediate neurotoxicity in immature rat hippocampus involving glutamate excitotoxicity. however, it remains unclear whether glyphosate alone or in combination with its adjuvants may have detrimental effects on myelin integrity in the peripheral nervous system. myelinated dorsal root ganglia (drg) cultures were treated over the course of ten days with either pure glyphosate or a glyphosate-based herbicide at concentrations of . %, . % and . %. the concentration of the glyphosate-based herbicide was matched with regard to glyphosate content ( %). controls were treated with equal amounts of vehicle adjusted for the ph. subsequently, cultures were stained with sudan black and myelin content was assessed by determining the number of internodes per neurons. while glyphosate, regardless of its concentration, did not show any effect on myelin content, the glyphosate-based herbicide caused significant demyelination in a concentration-dependent manner. notably, at . %, drg cultures were completely devoid of myelin and appeared severely necrotic. these data raise the possibility that not glyphosate itself, but rather the adjuvants in glyphosate-based herbicide formulations may cause demyelination. the open question whether demyelination is a direct effect of the adjuvants or a consequence of increased cellular glyphosate uptake due to permeabilization warrants further investigation. tan cy , tan mp , yeoh ky , goh kj , shahrizaila n . department of medicine, university of malaya, kuala lumpur, malaysia. in guillain-barré syndrome (gbs), autonomic dysfunction is common and accounts for significant morbidity and mortality. there have been many studies investigating the electrodiagnosis of gbs but few have studied autonomic dysfunction in gbs. the current study comprehensively investigates quantitative autonomic function in patients with gbs and its variant. ten gbs patients were prospectively recruited and the results were compared to age-and gender-matched healthy controls. a series of autonomic function tests including computational (power spectrum analysis of heart rate variability (hrv) and baroreflex sensitivity (brs) at rest) and challenge tests (deep breathing, eyeball compression, active standing, valsalva manoeuvre, isometric exercise and ice-water hand immersion) were performed. parasympathetic function was represented by high frequency (hf) hrv, heart rate responses to deep breathing, eyeball compression, valsalva manoeuvre and active standing. sympathetic function was represented by low frequency (lf) hrv, blood pressure responses to active standing, sustained handgrip and ice-water hand immersion. in the frequency domain analysis of hrv, low frequency (lf: . ± . vs . ± . ; p= . ), high frequency (hf: . ± . vs . ± . ; p= . ) and total power spectral densities (psd: . ± . vs . ± . ; p= . ) were significantly reduced in patients compared to controls. the mean up slope ( . ± . vs . ± . ; p= . ), down slope ( . ± . vs . ± . ; p= . ) and total brs slope ( . ± . vs . ± . ; p= . ) were significantly lower in the gbs group. the diastolic rise in blood pressure upon ice-water hand immersion was significantly lower in gbs group compared to controls ( . ± . vs . ± . ; p= . ). our findings suggest that computation dependent tests (hrv and brs) were sensitive at detecting autonomic dysfunction and baroreceptor reflex insensitivity in gbs patients. in contrast, ice-water hand immersion was the only reliable challenge test making it useful as a bedside measure of autonomic function in gbs patients. sjögren's syndrome (ss) is an autoimmune disease that affects both east and west. nevertheless, we still have limited knowledge of how autoantibodies in ss affects the peripheral nervous system. in this study, we investigated the peripheral neuropathy in ss and sicca complex using the nerve excitability test, to elucidate how peripheral nerves are affected. we have enrolled a total of patients with ss or sicca complex. of these, two patients were excluded due to co-morbid carpal tunnel syndrome. each patient received clinical evaluation, examination for ssa/ssb antibodies titer, the nerve excitability test, conventional thermal quantitative sensory test, and conventional nerve conduction study. compared to normal control subjects, motor nerve excitability test of ss patients with positive ssa or ssb antibodies (n = ) were found to have increased rheobase (p< . ), increased relative refractory period (rrp) (p< . ), increased refractoriness at . ms (p< . ), increased accommodation toward depolarizing current in threshold electrotonus (te) (p< . ), and decreased superexcitability (p< . ). the sensory axonal study in seropositive ss also revealed increased rrp (p< . ), increased refractoriness at . ms (p< . ), and increased accommodation toward hyperpolarizing current in threshold electrotonus (te) (p< . ). meanwhile, in seronegative ss and sicca complex (n = ), we found no significant axonal properties changes. the present study revealed that peripheral nerves are affected differently in seropositive ss and in seronegative ss/sicca complex. in seropositive ss, motor axons tended to be depolarized, and both sensory and motor axons have increased refractoriness. the findings suggested that ssa and ssb antibodies might play a role in the inactivation of transient sodium channels. the effects of the antibodies on transient sodium channels might be the basis of peripheral neuropathies and even cardiac arrhythmias and heart block in ss. charcot-marie-tooth disease type a (cmt a), caused by the pmp duplication on chromosome p . , is the most common subtype of inherited peripheral neuropathies and affects in , individuals worldwide. while sharing the same genetic cause, cmt a patients often present great variability in their phenotypic presentation and disease severity. the cause of the phenotypic variability is largely unclear. in this study, we performed genome-wide association study (gwas) to identify novel genetic modifiers of various phenotypes in cmt a. dna samples from cmt a patients were genotyped on illumina omniexpress platform. after standard quality control, the dataset includes k markers in individuals ( individuals from european ancestry, and individuals from asian ancestry). we focused our analyses on the european population. logistic regression in plink was used to analyze the association between the clinical outcomes and patients' genotypes in an additive model. for cmt neuropathy score (cmtns), the analysis was performed using linear regression in plink, adjusting for patients' age. the analyses yielded several suggestive association signals. an association peak on chromosome was identified in difficulty with eating utensils (lead snp rs , chr : , p= . e- , odds ratio= . ). the peak is located within a non-coding gene linc . hearing loss showed an association peak on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ), located in an intergenic region near the megf gene. in foot plantar flexion, an association signal was identified in the dscam gene on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ). cmtns showed an association signal on chromosome (lead snp rs , chr : , p= . e- , beta= . ), located within an intergenic region close to dffb, c orf , and linc . while these suggestive signals require further validation, our study provides novel insights into the genetic architecture of cmt a. novel genetic modifiers may serve as potential targets for therapeutic interventions in the future. teng a , ohnmar , kalpana p , chai yh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. we present an intriguing diagnostic puzzle, that was eventually cracked serendipitously. a -year-old man was seen for bilateral ptosis. evaluation for myasthenia gravis was negative. nevertheless, a diagnosis of ocular myasthenia gravis was made and he was put on pyridostigmine. he did not respond. the ptosis progressively worsened. he sought a second opinion. at this evaluation, he was noted to have complex ophthalmoplegia without diplopia, bilateral facial weakness and mild bulbar weakness. he had no sensory complaints. the limb examination was remarkable for slightly reduced reflexes, normal strength, and other than increased vibration threshold at the toes, intact sensory examination. repeat serological and electrodiagnostic work-up for myasthenia gravis was negative. a myopathic disorder such as chronic progressive external ophthalmoplegia was considered. serum creatine kinase and lactate were normal. he underwent biceps muscle biopsy which showed increased cox-negative and sdh positive fibers, supporting the then clinical impression of a mitochondrial cytopathy. at this point, he underwent blepharoplasty to improve his vision. routine histological examination of the levator palpebrae muscle showed amyloid deposits. this prompted a review of the earlier biceps biopsy, which revealed amyloid deposits that were not appreciated before. at this point, the significance of the patient and his son's history of lattice corneal dystrophy became apparent. he also reported that his late mother had similar facial appearance as his. the patient's nerve conduction study showed length-dependent sensory axonal polyneuropathy, right carpal tunnel syndrome and bilateral facial neuropathy. he had no definite symptoms of autonomic neuropathy. cardiac evaluation was unremarkable. the final diagnosis of familial gelsolin amyloid polyneuropathy was made. genetic confirmation for the patient and his family is being planned. we highlight the key clinical features of gelsolin neuropathy. the symmetric cranial neuropathy can resemble a muscle or neuromuscular junction disorder and the relative sparing of the cardiac muscle, somatic and autonomic nerves contrasts with transthyretin-related amyloid polyneuropathy. neuropathy is one of the most common long-term complications of diabetes. furthermore, % to % of diabetic neuropathy patients will develop neuropathic pain. the pathophysiology of neuropathic pain in diabetic peripheral neuropathy is complex and not fully understood. a potential mechanism is a change in voltage gated sodium channels, such as nav . . loss of function mutations in this channel cause insensitivity to pain, whereas gain of function mutations have been linked with different pain syndromes including small fiber neuropathy. in a cohort of patients with diabetic peripheral neuropathy we investigated whether mutations in nav . were associated with diabetic neuropathic pain. twelve nav . variants were identified in nine participants all within a cohort of participants with painful diabetic peripheral neuropathy. five of these variants were previously associated with pain disorders: v l, m l; w r, r h, l v. among the other variants two of them met the criteria of potential pathogenicity based on predictive algorithms and were further studied. functional analysis by whole cell patch clamp showed that one of these variants (m t) drastically impairs the inactivation of the channel by shifting the steady-state fast-inactivation towards more depolarizing potentials. there were no phenotypic difference between those participants with pathogenic variants and those participants without pathogenic variants. no rare nav . variants were found in participants with painless diabetic peripheral neuropathy. these observations suggest that mutations in nav . may contribute to painful diabetic peripheral neuropathy. tholance y , rosier c , f bouhour , psimaras d , kuntzer t , taieb g , créange a , delmont e , camdessanché jp , antoine jc . university hospital, saint-etienne, france; university hospital, lyon, france; university hospital, paris, france; university hospital, lausanne, switzerland; university hospital, montpellier, france; university hospital, creteil, france; university hospital, marseille, france. dysimmune sensory neuronopathies (snn) encompass paraneoplastic snn and snn associated with systemic autoimmune diseases such as sjögren syndrome, lupus or inflammatory bowel or rheumatic diseases but also a number of apparently idiopathic cases. biomarker antibodies are well-known in paraneoplastic snn but are lacking in non paraneoplastic cases. from a mono-center retrospective study we identified in anti-fgfr antibody as a potential biomarker of dysimmunity in patients with idiopathic or systemic autoimmune disease associated sensory neuropathy. the identified patients were more frequently women and had a non lenthg dependent neuropathy suggestive of snn. anti-fgfr antibody was the only immunological marker in / of cases at initial work-up although / of patients eventually developed with time systemic autoimmune disease. to confirm the incidence and the clinical pattern of patients with anti-fgfr antibodies we launched a prospective multicenter french study including patients with a sensory neuropathy suspected to be a snn of no paraneoplastic, genetic or metabolic origin. we present here the results on the first included patients compared to healthy blood donors. anti-fgcr antibodies were searched by elisa using the trk intracellular domain of the protein (invitrogen©). we found patients positive for anti-fgfr antibody ( . %). these patients were women and men aged . years as a mean ( - ). the neuropathy was acute and subacute in one patient respectively and progressive in the others. six patients fulfilled the diagnosis criteria of snn and the last one had a sensory neuropathy in the lower limb with abnormal sensory action potentials in the four limbs suggesting snn without reaching the requested criteria. one patient developed uveitis which is a new symptom with anti-fgfr ab. an unclassified dysimmune context was present at the initial work up in patients and one patient developed sjögren syndrome with follow-up. as a whole the clinical pattern of these patients is consistent with that of the initially published series. the lower prevalence of positive sera may be due to more stringent criteria used for elisa but needs to be confirmed on the complete prospective series. peripheral neuropathy research registry (pnrr) neurological assessment was scored using the total neuropathy score clinical version (tnsc), comprising pinprick and vibration sensibility, deep tendon reflexes, strength and patient symptom report. compound sensory action potential (csap) amplitudes were recorded antidromically at the lateral malleolus, stimulating the sural nerve at the mid-calf. of the total sample, % reported lower limb neuropathy, with % of patients reporting 'quite a bit' or 'very much' severity of tingling and numbness in their feet. the average sural csap amplitude was . ± . v and % of patients had sural amplitudes below the lower limit of normal for age. the total tnsc score correlated with the pro fact-gog ntx score (r = −. , p<. ) and sural amplitude (r = −. , p <. ). vibration sensibility correlated with the overall fact-gog ntx score (r =−. , p <. ), and sural amplitude (r = −. , p <. ). sural amplitude correlated with patient reported severity of numbness and tingling in the lower limbs (r =−. , p <. ) but not with the overall fact-gog ntx score. patient reports of neuropathic symptoms in the lower limb correlate with both objective neurophysiological and clinical measures of neuropathy severity. identifying links between objective neurophysiological markers and patient reported outcomes are critical to assess the impact of clinical interventions. tomaselli pj , gouvea sp , nyshyama kfs , nicolau n jr , lourenço cm , marques w jr , . division of neuromuscular diseases, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; neurogenetics, department of neurosciences and behaviour sciences, university of são paulo, ribeirão preto, brazil. mutations in the gab junction beta -protein gene (gjb ) are the second most frequent cause of charcot-marie-tooth disease (cmt), accounting for approximately % of cmt cases worldwide. the gjb codes for connexin protein (cx ). in the peripheral nervous system, the cx is expressed in the schwann cells and allows intercellular traffic of ions and small molecules between opposed cells. we analysed retrospectively detailed clinical and neurophysiological data of five families carrying novel gjb mutation submitted for testing at our neurogenetics laboratory. mutations were identified by bidirectional sanger sequence analysis of gjb coding region. we identified a total of subjects from five different kindreds with novel mutations (p.a v, p.l w, p.l q, p.f s, p.r l). these five novel mutations segregate with phenotype, are located in highly conserved amino acids among gjb and other gab junction protein sequences and among different species, are not present in any public database (exac, dbsnp and genome database), and were not found in normal brazilian controls. in silico analysis, predict these variants to be pathogenic, there was no male-to-male transmission; males were more severely affected than females. four out seven female have subclinical neuropathy and were only identified after clinical and electrophysiological evaluation. the conduction velocities were in the intermediated range in the males patients and higher in the females included in this study. we describe five new pathogenic mutations causing cmtx in a brazilian population and expand the number of causative mutations in the gjb gene. funded pure neural leprosy (pnl) is a slowly progressive, predominantly sensory patchy neuropathy presenting with positive and/or negative sensory symptoms, which are usually followed over time by distal asymmetrical weakness. despite rare, monomelic involvement in leprosy has already been reported. we sought to describe the clinical and electrophysiological patterns of an unusual leprosy neuropathy presentation. clinical data were retrospectively collected from nine patients who had monomelic involvement and were referred for further investigation to the emg lab. seven out nine patients were male. four patients had a brachial plexus like presentation and five have a lumbosacral plexus like presentation. the initial complaint was hypoesthesia in four patients, tingling in two patients and hypoesthesia with tingling in two patients. severe pain was observed in just one patient. all individuals from the group of patients with lumbosacral plexus-like presentation and three with brachial plexus-like presentation had no sensory nerve action potentials (snaps) for all nerves tested in the affected limb with or without motor involvement. one patient with brachial plexus-like presentation had focal slowing of conduction velocity with temporal dispersion of both median and ulnar nerves in the affected limb. one patient with plexus-like presentation had snaps with low amplitude of all nerves in the affect limb. the diagnosis of leprosy was confirmed by nerve biopsy findings, anti-pgl antibody, and positive response to specific treatment. nerve biopsy was performed in four patients, and the bacillus was found in two. the anti-pgl antibody was positive in four patients. plexus mri was performed in two patients and was normal. we found the distribution of motor and sensory symptoms were restricted to on limb in this group of patients. as a typically patchy disorder pnl may affect any nerve, although the reason why damage are restrict to only one limb has to be elucidated. the description of these cases increases the clinical spectrum of leprosy neuropathy. this possibility should be considered in the differential diagnosis of patients with plexopathy from endemic areas after excluding other causes. funded ataxia with oculomotor apraxia type (aoa ) is a very complex disorder characterized by an early-onset progressive cerebellar ataxia with cerebellar atrophy and peripheral neuropathy and it is caused by recessive mutations in the aprataxin gene (aptx ). when the neuropathy is present, it has been described in % of cases as primarily axonal. we describe a case of aoa due compound heterozygous mutations in aptx associated with demyelinating neuropathy. the patient was born from healthy and non-consanguineous parents and presented in the first decade with progressive cerebellar ataxia, multidirectional ophtalmoparesia, oculomotor apraxia, choreiform movements of limbs and peripheral neuropathy. he had normal cognition and stopped walking at age of . blood tests were unremarkable with normal levels of leucocytes, serum proteins, immunoglobulin, cholesterol, vitamin e, and alfa-feto protein. brain mri showed severe cerebellar atrophy. the motor conduction velocity in the upper limbs was slow with preserved amplitudes. the distal latencies and the minimal f wave latencies were prolonged. there was no evidence for superimposed acquired demyelinating neuropathy. direct sequencing of the aptx gene revealed two variants, c. - a>g, p? and c. g>a; p.w x. the first variant is novel and affects a highly conserved acceptor splice site of exon . the other variant is the most common portuguese variant, the nonsense mutation w x is located in exon . parental dna was tested and confirmed the variants were in different alleles. the presence of a demyelinating neuropathy in aoa suggests that phenotypic variability in this condition may be larger than previously considered. at the same time, it increases the differential diagnosis of inherited conditions with cerebellar ataxia and demyelinating neuropathy. finally, this finding opens the functional effects of the aptx gene. funded by: cnpq, fapesp, faepa, pronas (ministry of health). topa a , spina e , iodice r , tozza s , ruggiero l , dubbioso r , esposito m , santoro l , manganelli f . university of naples "federico ii", naples, italy. we report our -year experience of subcutaneous immunoglobulin (scig) in a cohort of patients with chronic inflammatory demyelinating polyneuropathy (cidp) from a tertiary care neuromuscular center. we analyzed data from cidp patients ( males and females, mean age: ± . years; mean age at onset: . ± . years; disease duration: . ± . years) treated with scig and with a follow-up period of months. all patients were previously responders to intravenous immunoglobulin (ivig). eight patients had a typical cidp and five patients had an atypical variant of cidp. five patients switched to weekly maintenance scig therapy (continuous regimen) because of short-lasting response to ivig therapy. eight patients with a longer lasting response to ivig received scig with a pulsed regimen similar to that used for ivig (from to cycles per year); seven of them because of difficulty in hospitalization and one for allergic reaction to ivig. changes in clinical status were assessed over the period of follow-up by using clinical evaluation of muscle strength, modified rankin scale, overall neuropathy scale and inflammatory neuropathy cause and treatmentsum score. in patients we evaluated also six minute walking test, hole-peg-test and meter walking test. all the five patients treated with a continuous regimen of scig remained clinically stable throughout the follow-up period. among the patients receiving pulsed scig treatment, out of ( %) responded to scig similarly to ivig, while three patients ( . %) worsened and needed to be treated again with ivig and the other one ( . %) stopped any therapy. subcutaneously administered immunoglobulin were well tolerated and no patients complained of adverse events. in conclusion, our findings confirm that continuous scig therapy is efficacious in maintaining clinical stability in patients with short-lasting response to ivig. moreover, our data suggest that pulsed therapy with scig may represent an alternative therapeutic option for the treatment of a subset of cidp patients. touvier t , ferri c , mastrangelo r , glimcher l , , wrabetz l , , , d'antonio m . myelin biology unit, division of genetics and cell biology, san raffaele scientific institute, dibit, milan, italy; department of cancer immunology and virology, dana-farber cancer institute, boston, usa; department of medicine, harvard medical school, boston, usa; hunter james kelly research institute, university at buffalo, buffalo, usa; department of biochemistry, university at buffalo, buffalo, usa; neurology, jacobs school of medicine and biomedical sciences, university at buffalo, buffalo, usa. mpz glycoprotein is an abundant product of terminal differentiation in myelinating schwann cells. the mutant mpzs del causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in transgenic mice. mpzs del protein is retained in the endoplasmic reticulum (er) of schwann cells and induces an unfolded protein response (upr) characterized by activation of perk, atf and ire /xbp pathways. we have previously reported that activation of chop and gadd , two downstream mediators of perk, is pathogenetic in mpzs del mice (pennuto, ; d' antonio, ) but the role of the other upr branches remains to be investigated. in this study, we investigated the role of the er stress sensor enzyme ire and of xbp -a transcription factor specifically activated by ire -in mpzs del pathogenesis. we generated a new mouse model with schwann cells-specific ablation of xbp and in parallel we exploited mpzs del dorsal root ganglia (drg) explant cultures in which xbp signaling is modulated by gain/loss of function approaches. we observed that absence of xbp dramatically worsens hypomyelination and electrophysiological/locomotor parameters in young and adult mpzs del neuropathic animals. interestingly we observed that perk, atf and ire -mediated ridd signalings are upregulated in neuropathic animals lacking xbp . this suggests that activation of xbp targets have an essential role in limiting mpzs del toxicity, which cannot be compensated by other stress responses. moreover, we demonstrated in mpzs del drg cultures that inhibition of xbp splicing by u c (cross, ) decreases myelination whereas activation of xbp splicing by quercetin (wiseman, ) slightly ameliorates myelination. altogether, these data demonstrate that xbp pathway has a critical adaptive role in mpzs del neuropathy and suggest that activation of this pathway may be beneficial for cmt b and perhaps for a broad range of neuropathies characterized by upr activation. tozza s , bruzzese d , iodice r , esposito m , dubbioso r , ruggiero l , topa a , spina e , santoro l , manganelli f . department of neuroscience, reproductive sciences and odontostomatology, university of naples "federico ii", naples, italy; department of public health, university federico ii of naples, naples, italy. in cmt a patients, the clinical impairment progressively increases over time and correlates with the axonal loss. evidence has suggested that the decline of physical performance in cmt a patients may reflect a process of normal ageing. the aim of our study was to describe, by a case-control cross-sectional design, the progression of physical impairment with ageing in cmt a patients. we enrolled cmt a patients ( m; range - years) and sex-and age-matched healthy controls. to assess physical performance, all patients and controls underwent -meter walk test ( mwt), -minute walk test ( mwt) and -hole peg test ( hpt) of their dominant (d) and non-dominant (nd) sides. moreover, to assess clinical disability, impairment and quality of life in the cmt a group we used the charcot-marie-tooth neuropathy score (cmtns), the mrc sum score and the short form- (sf- ) questionnaire. the linear regression model was used to evaluate the changes over time of clinical measures in patients and controls. the chow test was used to determine whether the ageing had a different impact on clinical measures for the two groups. physical performance worsened with ageing in both patients and controls, but with a greater slope for cmt a patients [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. the rate of deterioration of physical performance was not different between patients and controls until the th year of age. after the th year of age the rate of deterioration became greater in cmt a group [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. moreover, in cmt a patients also cmtns, mrc sum score and sf- worsened with ageing and with a greater rate of deterioration after the th year of age. our study demonstrates that clinical decline in cmt a patients goes parallel to the normal ageing process until the th year of age, whereupon the clinical deterioration accelerates. tsouni p , devic p , moura b , planque e , bédat-millet al , devaux j , steck aj , delmont e , hottinger af , kuntzer t . dcn, chuv, lausanne, switzerland; centre de référence maladies neuromusculaires, hospices civils de lyon, lyon, france; cabinet médical, epinal, france; département de neurologie, chu de rouen, rouen, france; cnrs, crn m-umr , université aix-marseille, marseille, france; centre de référence maladies neuromusculaires et sla, hôpital la timone, marseille, france. new therapeutic options in immuno-oncology have allowed significant progress in the management of melanomas. treatment usually consists of a combination of two monoclonal antibodies targeting cytotoxic lymphocyte-associated protein and programmed cell death- . as a result, the immunologic barrier protecting tumor cells is overcome allowing an antitumor response. we report cancer patients with immune checkpoint inhibitor-induced neuropathies as a complication of this immunomodulating oncologic treatment. case reports: our index patient developed severe myalgia days after introduction of ipilimumab-nivolumab followed by painful paresthesias of the face and extremities day after the nd cycle of treatment. generalized areflexic quadriparesis (mrc ) with gowers'sign and distal loss of vibratory sensation were found. a -day course of ivig plus corticosteroids (cs) had no effect. three monthly ivig courses were necessary to improve the deficits at months. a survey of sfnp members revealed other patients with similar acute courses but with phenotypes varying from sensorimotor deficits with areflexia and myalgia to purely sensory ataxic forms following immunomodulating treatment. work-up including anti-nodal antibodies were negative in patients. from the other , had abnormal csf and had necrotizing myopathy. detailed repeat ncs demonstrated signs of nerve hyperexcitability and of demyelination or conduction blocks. evolution was slowly favorable following ivig and cs. discussion: our report underscores that atypical acute generalized demyelinating neuropathies are induced by these novel treatments. they may be associated with severe myalgia or other systemic toxic effects. discontinuation of the oncologic treatment depends on severity of symptoms. outcome was slowly favorable following ivig or cs, albeit slower than in the case of primary inflammatory neuropathies, probably given the long half-life of the monoclonal antibodies. gap junctions (gjs) are membrane channels found in most tissues connecting adjacent cells or different cell compartments as in schwann cells. they are involved in electrical connectivity and metabolic homeostasis allowing the passage of small molecules such as ions, second messengers, nucleotides and peptides. an important functional role of peripheral nerve connexins is suggested by their involvement in x-linked inherited neuropathy as well as in acquired neuropathy caused by oxaliplatin. although gjs play a role in electrical connectivity their specific role in the formation of the sciatic nerve compound action potential (cap) remains unclear. the aim of this study was to investigate the role of peripheral nerve connexins in the electrical responses of the mouse sciatic nerve under normal and stress conditions. for this purpose we used sciatic nerves of three different mouse models, the cx knockout (ko), cx ko and the cx /cx double knockout (dko) mice. using our ex vivo model for extracellular recordings we exposed sciatic nerves from different genotypes to three different gj blockers: octanol, -beta-glycyrrhetinic acid (gra) and octanoic acid (oa) and recorded the cap. amplitude and duration of the cap were used as an indication for the effects of the different blockers on the cap formation. all gj blockers caused a gradual decrease of the cap without any changes in the duration of the cap in all genotypes, suggesting progressive disturbance of axonal membrane excitability in the absence of one or two gj proteins. comparison of the three genotypes showed that cx may play a dominant role in the maintenance of the cap formation since nerves from cx ko mice proved to be more sensitive to the gj blockers compared to the cx ko nerves showing a faster decline of the cap amplitude. moreover the effect of gj blockers was similar in cx ko and dko nerves. finally, the effect of gj blockers on the dko nerves implies the presence of another gj protein. in conclusion, our results confirm the direct functional involvement of cx gj channels and cx hemichannels in the cap formation and indicate the existence of at least one more connexin in peripheral nerve. ca( +)-dependent anti-ganglioside antibody in seronegative guillain-barrÉ syndrome uchibori a , gyohda a , chiba a . kyorin university, tokyo, japan. we have reported ca + -dependent igg anti-ganglioside gq b antibodies in gq b-seronegative patients with fisher syndrome and its related disorders (fs-rd). in patients with fs-rd who were gq b-seronegative in conventional assays using phosphate-buffered saline without ca + , % turned seropositive for gq b-related antigens in assays using ca + -added tris-buffered saline. objective: we investigated whether ca + -dependent anti-ganglioside antibodies was present also in ganglioside-seronegative patients with other clinical disease types of guillain-barré syndrome (gbs) other than fs-rd. methods: the subjects were the following: patients with final clinical diagnosis as gbs (acute motor axonal neuropathy [aman], n = , and acute inflammatory demyelinating polyradiculoneuropathy [aidp], n = ), and patients with final clinical diagnosis as sensory ataxic neuropathy (san), n = . all subjects were ganglioside-seronegative in the conventional assays. we assayed serum igg antibodies against various gangliosides (including asialo-gm ) in elisa using tris-buffered saline as a basal buffer in both ca + -added and -non-added conditions. increase of the optical density (od) more than . in ca + -added condition compared with ca + -non-added one was taken significant, i.e. positive for ca + -dependent antibody. results: ca + -dependent antibody was negative in all aman and san patients. in aidp, the antibody titers (ods) against gainac-gd a were significantly increased in patients, and those against asialo-gm were increased in other patients in ca + -added condition. however, the titiers of those ca + -dependent antibodies were all at low level. conclusions: in clinical disease types of gbs other than fs-rd, ca + -dependent igg antibodies against ganglioside were detected in a few patients with aidp, but the positive rate and the antibody titers were low compared with case of gq b-seronegative fs-rd. ca + -dependent antibodies against ganglioside are considered to be more specific for gq b. the aim of the study was to investigate the relationship between median sensory conduction of median nerve and ulnar nerve in patients diagnosed with carpal tunnel syndrome. two hundred and eighty-six hands with carpal tunnel syndrome and hands in control group were investigated. patients were staged clinically and electrophysiologically. diagnosis of carpal tunnel syndrome was made according to the presence of paresthesia, pain in the innervation area of the median nerve, weakness and atrophy in the median nerve innervated muscles, positive phalen and tinel tests. median motor and sensorial nerve conduction study, including first, second, third finger and palm, and ulnar motor and sensorial nerve conduction of fifth finger studies were performed to all patients and control group. the ratio of distal latency and velocity of nerve conduction of first, second, third and palmar branches to fifth finger was calculated. distal latency of first, second, third finger and palm of patients with cts are longer and velocity is more slowly than controls. in addition to these findings, the velocity of fifth finger is also slower and distal latency of this one is longer than healthy subjects. the most sensitive method of classifying the carpal tunnel syndrome as normal, mild and moderate is the ratio of distal latency and velocity of second finger (p< . ). carpal tunnel syndrome is the most common encountered neuropathy. in nerve conduction studies can be used the ratio of distal latency and velocity of second finger as determine the degree of carpal tunnel syndrome. the most surprising finding of this report is the nerve conduction studies of fifth finger. the subclinical susceptibility of fifth finger can be explained by overusing of wrist. ursino g , gemelli c , grandis m , reni l , bellone e , geroldi a , gotta f , mandich p , ferrara m , schenone a dinogmi university of genoa, genoa, italy; irccs-aou san martino hospital genoa, genoa, italy. charcot-marie-tooth (cmt) neuropathy represents a clinically and genetically heterogeneous group of hereditary peripheral neuropathies characterized by chronic motor and sensory impairment. to date mutations in up to genes may cause cmt. the aim of this study is to describe our large population of cmt patients, and, within this, highlight specific phenotypes. the cmt clinic in genova, started in . during the years, patients underwent complete neurological, rehabilitative, neurophysiological examinations and genetic testing. the patients were routinely tested for common genes (as pmp , gjb , mpz, mfn ), while in specific cases we followed the candidate gene approach testing single genes based on the genotype-phenotype correlation. however, the ngs techniques were used when routine genetic testing was negative and a clear genotype-phenotype correlation could not be identified. cases are present to date in our database of cmt patients. in ( . %) patients, in spite of a clinical diagnosis of cmt, a genetic diagnosis is still lacking; ( . %) patients had alternative diagnosis (i.e hereditary spastic paraparesis etc.). instead, in patients ( . %) a defined genetic diagnosis was reached, of them being females ( . %) and males ( . %). among these, except for the more common cmt a, hnpp and cmt x phenotypes, we frequently observed patients affected by cmt b and cmt f. according to most literature data, we observed ( . %) patients with cmt b and patients ( . %) affected by cmt f. at the first visit, the cmt b phenotype was clearly length-dependent: . % patients showed impairment of the lower limbs and saving of the upper limbs; in terms of severity of the neuropathy, the mean cmtns was . and the mean age was . years. similarly, % of patients affected by cmt f, present with the same phenotype; the mean cmtns at the first visit was . and the mean age was . years. in conclusion, based on the experience of the genova cmt clinic, we describe a large population of cmt patients and a specific phenotype in cmt b and f patients, characterized by involvement of the lower limbs and selective sparing of the upper ones, which may help in addressing the diagnostic algorithm. non-freezing cold injury (nfci) develops following sustained exposure to cold temperatures, resulting in tissue cooling but not freezing. this can result in persistent sensory disturbance of the hands and feet including numbness, paraesthesia and chronic pain. both vascular and neurological aetiologies of this pain have been suggested but remain unproven. we prospectively approached patients referred for clinical assessment of chronic pain following non-freezing cold injury between february and november . of patients approached consented to undergo detailed neurological evaluations including: questionnaires to detail pain location and characteristics, structured neurological examination, quantitative sensory testing, nerve conduction studies and skin biopsy for intra-epidermal fibre assessment. of the study participants all had experienced nfci whilst serving in the united kingdom armed services and the majority were of african descent ( . %) and male ( . %). many patients reported multiple exposures to cold. the median time between initial injury and referral was . years. pain was principally localised to the hands and the feet, neuropathic in nature and in all study participants associated with cold hypersensitivity. clinical examination and quantitative sensory testing were consistent with a sensory neuropathy. in all cases large fibre nerve conduction studies were normal. the intra-epidermal nerve fibre density, however, was markedly reduced; ⋅ % of subjects having a count at or below the ⋅ centile of published normative controls. using the neuropathic pain special interest group (neupsig) of the international association for the study of pain (iasp) grading for neuropathic pain % had probable and ⋅ % definite neuropathic pain. chronic non-freezing cold injury is a disabling neuropathic pain disorder due to a sensory neuropathy. why some individuals develop an acute painful sensory neuropathy on sustained cold exposure is not yet known but individuals of african descent appear vulnerable. screening tools, such as the dn questionnaire, and treatment algorithms for neuropathic pain should now be used in the management of these patients. funded by the wellcome trust and the uk ministry of defence guillain-barré syndrome (gbs) is highly heterogeneous regarding clinical presentation, course, electrophysiological subtype and outcome. in part this variety is associated with differences between geographical regions, although this had not been investigated in a single comparative study. one aim of igos is to define the influence of the geographical origin on the heterogeneity of gbs. in igos all gbs patients within weeks of onset can participate, independent of age, variant, severity and treatment. in february , patients were included from countries. the first inclusions in igos were used in this analysis. seventy-five patients ( %) were excluded because of alternative diagnoses (n= , including a-cidp), protocol violations (n= ) or missing data (n= ). of the remaining patients, % were males and % female with a median age of years (iqr - ). at entry % presented with tetraparesis and % with paraparesis. during follow-up % needed mechanical ventilation and % died. of all gbs patients % received treatment ( % ivig, % pe). the remaining % received supportive care only (mild gbs or low social-economic status). antecedent events were reported in % of patients, including upper respiratory tract infection ( %) and gastro-enteritis ( %) as the most frequent events. the pure motor form was the predominant subtype in patients from bangladesh ( %). in europe/americas and asia (without bangladesh) the predominant subtype was the sensorimotor form ( % in europe/americas and % in asia). in asia (without bangladesh) there was a relatively larger proportion of patients with mfs/mfs-overlap syndrome ( %) than in other regions ( - %, p< . ). the proportion of patients able to walk unaided at months after follow-up was % in europe/americas, % in bangladesh and % in asia (without bangladesh). kaplan-meier analysis comparing electrophysiological subtypes of gbs (as reported by neurologist) showed that patients with inexcitable nerves or axonal neuropathy needed more time to regain the ability to walk unaided than the patients with demyelinating or equivocal result (log-rank test, p < . ). these findings demonstrate the extensive geographical differences in gbs. future igos studies will investigate the role of genetic and environmental factors that additionally could explain these differences. van den bergh pyk , attarian s , grapperon am , nicolas g , cassereau j , rajabally ya , delmont e , woodard jl , piéret f and the university of louvain gbs electrodiagnosis study group * . corticosteroids are considered as one of the first line treatments for chronic inflammatory demyelinating polyneuropathy (cidp). different types of corticosteroids are used and there are no comparative studies assessing the improvement rates, remission rates, tolerability and the side effect profiles of these treatment regimens. in addition, there are currently no reliable predictors of favorable treatment response to steroids, which would greatly ease the choice of first line treatment. in this retrospective study we will compare efficacy, tolerability and safety of three different corticosteroid regimens used as first line treatment in three large cidp centers in netherlands, serbia and italy. treatment naïve cidp patients who received either pulsed dexamethasone, pulsed methylprednisolone or daily prednisone will be included in the study. data will be extracted from patient charts. the primary outcome is the percentage of treatment responders at months after start of first treatment, in which treatment response is defined as subjects who improved after treatment and are either without treatment after six months or are still being treated with the first chosen therapy. secondary endpoints include the remission rates and in case of a relapse, the mean duration of remission to relapse; the discontinuation rate within months of treatment due to inefficacy, adverse events or intolerance; and the frequency of adverse events and serious adverse events (sae) during treatment or within month after stopping treatment. furthermore, we will explore the value of previously reported potential predictors of treatment response. results will be presented at the peripheral nerve society meeting . van lieverloo g , , musters a , adrichem m , esveldt r , doorenspleet m , klarenbeek p , van schaik i , de vries n , eftimov f . academic medical center, department of neurology, amsterdam, the netherlands; academic medical center/university of amsterdam, department of clinical rheumatology and immunology, amsterdam, the netherlands. following reports that pathogenic antibodies are present in a minority of patients with chronic demyelinating inflammatory neuropathy (cidp), we studied whether oligoclonal expansions of b-cell clones are present in patients with cidp. recently, we developed a new method for b-cell receptor (bcr) repertoire landscaping based on high throughput sequencing (hts) of rna extracted from blood. bcr repertoire was analyzed in patients with cidp: patients with active disease and starting treatment (group ), patients with stable disease using intravenous immunoglobulin (ivig) treatment in which treatment withdrawal was attempted (group ), and patients in remission (i.e. no treatment in the last months, group ). clinical parameters and sampling was performed at baseline (group , and ), at months after start of treatment (group ), at months or earlier in case of relapse in group and at baseline only in group . most cidp patients had highly expanded bcr clones, regardless of disease activity and response to treatment. however, in group , the most expanded b-cell clones at baseline showed no overlap with the expanded bcr clones after improvement. based on these preliminary data expanded bcr clones are observed in the peripheral blood of most cidp patients, regardless of disease activity (active, stable disease or remission/cure). functional characterization of these expanded clones remains to be performed. van rijs w , fokkink wjr , tio-gillen ap , brem md , jacobs bc , huizinga r . erasmus mc, university medical centre, rotterdam, the netherlands. myeloid cells, including monocytes, macrophages and dendritic cells, are critically involved in the induction of adaptive immune responses, clearance of pathogens and in the initiation of tissue repair. in the guillain-barré syndrome (gbs), macrophages are present in the peripheral nerve, where they phagocytose (damaged) myelin and axons. dendritic cells (dc) are increased in the cerebrospinal fluid of patients with gbs. however, the composition and phenotype of monocytes and dc subsets in the peripheral blood is unclear and it is unknown if these cells can be used as biomarker to monitor disease activity or response to treatment. here we investigated the frequency and phenotype of six myeloid subsets in the peripheral blood mononuclear cells (pbmc) using advanced -color flow cytometry. pbmc were isolated from patients with gbs, before and after immunomodulatory treatment, and age and gender-matched, healthy controls. the frequency of total monocytes, determined as percentage of cd + cells, was increased in gbs patients compared to controls (p< . ). the monocyte population was skewed towards more intermediate (cd +cd +; p< . ) and less non-classical (cd -cd +; p< . ) monocytes. classical (cd +cd -) and intermediate monocytes as well as cd c+ dc expressed significantly higher levels of cd compared to healthy controls. in contrast, the expression of cd and siglec- was significantly higher in the non-classical monocytes of gbs patients compared to controls. no differences were observed in the expression of cd , cd , cd and siglec- . immunomodulatory treatment strongly reduced the frequency of non-classical monocytes and all dc populations in cd + pbmc. the expression of cd , cd c and hla-dr was reduced in classical monocytes after treatment. in addition, siglec- expression was reduced in several monocyte and dc populations after treatment. in summary, our data identify significant changes in the monocyte compartment in gbs. the decrease in non-classical monocytes may suggest that these cells have migrated to peripheral tissues, promoting the differentiation of classical monocytes into intermediate monocytes. further analysis should reveal whether these changes are related to preceding infections, disease severity and response to treatment. in mme. the two mutations were absent from control databases (e.g. exac), affected highly conserved aminoacids and were predicted to have deleterious effects by in silico analysis. unfortunately, both parents were deceased and we were therefore unable to prove that the two mutations reside on separate alleles. mme encodes the metalloprotease neprilysin whose role in peripheral nervous system is still unclear. higuchi and colleagues have described japanese cmt families with late-onset sensory-motor axonal neuropathy and recessive loss-of-function mutations in mme. we report two novel missense mutations in mme in a case of late-onset cmt . we hypothesize an autosomal-recessive mode of inheritance as most likely given the clinical phenotype and the absence of a family history. anti-contactin- (cntn ) antibodies were recently identified in a subgroup of patients with chronic inflammatory demyelinating polyneuropathy (cidp) showing acute/subacute onset of severe sensory-motor neuropathy and poor response to intravenous immunoglobulin (ivig) and corticosteroids. these antibodies belong to the igg isotype and interact with cntn -neurofascin (nf ) complex at paranodes leading to loss of nodal integrity. a -year-old man presented with acute onset of distal weakness in the lower limbs, four limbs paraesthesias and sensory ataxia. at clinical examination ankle swelling was also observed. nerve conduction study showed a demyelinating polyneuropathy and cerebrospinal-fluid examination revealed cyto-albuminologic dissociation. sural nerve biopsy disclosed diffuse loss of myelinated fibres. at routine blood test serum albumin was reduced and proteinuria was gr/ hours, thus leading to the diagnosis of nephrotic syndrome. kidney biopsy showed changes consistent with membranous glomerulonephritis, together with sub-epithelial deposits of immune complexes and complement deposition. treatment with ivig and corticosteroids did not improve neurological status, while membranous glomerulonephritis showed moderate response to ivig. a six-month course of cyclophosphamide was started leading to normalization of renal function and muscle strength and partial improvement of sensory ataxia. the patient did not require any further treatment and after years his condition remains stable. cntn antibodies were tested on a recently collected patient's serum and resulted positive on both elisa and cell-based assay. the patient here reported showed the typical clinical features of anti cntn -associated cidp including older age, acute onset, severe motor impairment and sensory ataxia. the contemporary occurrence of membranous glomerulonephritis was reported in only one other case. contactin- is expressed at low levels in the kidney and a direct damage of anti-cntn antibodies could be hypothesized. alternatively, renal damage might have been secondary to unspecific immune complexes deposition. a good response to anti-cd rituximab was recently reported in patients with cidp associated with anti-cntn and anti-nf antibodies. notwithstanding this single-case observation, our report suggests that also cyclophosphamide may be considered an effective therapy in anti-cntn antibodies-associated cidp and membranous glomerulonephritis, leading to persistent clinical remission. velasco r , , besora s , santos c , sala r , izquierdo c , simó m , gil-gil m , , jiménez l , pardo b , calvo m , palmero r , clapés v , bruna j duloxetine is the only agent demonstrated effective in treating pain related with chemotherapy-induced peripheral neuropathy (cipn). patients with symptomatic cipn treated with duloxetine were retrospectively collected in a single-institution. aim of the study was to evaluate the drug's efficacy and rate of compliance. only patients with cipn with distressing positive symptoms (pain, numbness and/or paresthesia), and non-progressive disease were included. cipn was graded employing the total neuropathy score (tns © ) and national cancer institute-common toxicity criteria. response to duloxetine was assessed with patient global impression of change (pigc) scale ( : no benefit; : excellent response). consecutive first one-hundred cipn patients treated with duloxetine were analyzed. median age was ( - ). , , and received platinum, taxane, bortezomib and vincristine-based regimen, respectively. median tnsc © was ( - ). severity of neuropathy was grade ( %), grade ( %), and grade ( %). sixteen patients were on treatment with other analgesic agents. median time from finishing chemotherapy to duloxetine initiation was months [ - ]. median pigc score was [ - ]. among responders, . % and . % scored low ( - ) and high ( - ) benefit, respectively. fifty-seven ( %) patients discontinued early duloxetine due to intolerable side effects (n= ) the goal of this study was to determine whether predicted fork stalling and template switching (fostes) during mitosis deletes exon in peripheral myelin protein kd (pmp ) and causes a gain-of-function mutation associated with peripheral neuropathy in a family with charcot marie tooth disease type e. two siblings previously reported to have genomic re-arrangements predicted to involve exon of pmp were evaluated clinically and by electrophysiology. skin biopsies from the proband were studied by rt-pcr to determine the effects of the exon re-arrangements on exon mrna expression in myelinating schwann cells. transient transfection studies with wild type and mutant pmp were performed in cos and rt cells to determine the fate of the resultant mutant protein. both affected siblings had a length-dependent demyelinating neuropathy with severely slow nerve conduction velocities (< m/sec). rt-pcr studies of schwann cell rna from one of the siblings demonstrated a complete in frame deletion of pmp exon (pmp delta ). transfection studies demonstrated that pmp delta protein is retained within the endoplasmic reticulum and not transported to the plasma membrane. our results confirm that that fostes mediated genomic rearrangement produced a deletion of exon of pmp , resulting in expression of both pmp mrna and protein lacking this sequence. in addition, we provide direct experimental evidence for endoplasmic reticulum retention of the mutant protein suggesting a gain-of-function mutational mechanism consistent with the observed cmt e in this family. pmp delta is another example of a mutated myelin protein that is misfolded and thus likely to contribute to the pathogenesis of the neuropathy. in poems and cidp, distal limb nerve conduction studies are limited in identifying demyelination and detecting treatment effects in severely affected patients. blink reflex r latency may help to not only identify demyelination but also provide a meaningful treatment outcome measure especially in severely affected patients. poems and cidp patients having undergone routine nerve conductions and blink reflex testing were identified. correlation between r latency, limb nerve conduction studies and neuropathy impairment scores (nis) was calculated with treatment. blink reflexes were performed in patients ( poems, cidp; nis range: - points). overall, r latency prolongation occurred in . % of patients ( . % poems, . % cidp). patients with r prolongation (> ms) had more severely affected nerve conductions in both poems (ulnar cmap . mv vs . mv, p= . ) and cidp (ulnar cmap . mv vs . mv, p< . ). r latency correlated with nis severity in poems better than cidp (r = . vs . , p=< . vs. . ). follow-up nis and r latency evaluations after treatment were available in patients ( poems, cidp). the r latency changes were concordant with the nis changes in % of poems and % of cidp patients. in severely affected patients [ulnar cmap amplitude ≤ . mv ( . %: / )] except for one, all had prolonged r (> ms), allowing for treatment follow up and initial diagnosis. blink reflex r latencies are valuable in defining demyelination in severely affected poems and cidp patients, but also provide a sensitive, early treatment outcome measure among those same severely affected patients. watson dj , martinez c , wallenhorst c , hubsch a , shebl a , simon tl . csl behring llc, king of prussia, usa; institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl behring ag, bern, switzerland; csl behring gmbh, marburg, germany. chronic inflammatory demyelinating polyneuropathy (cidp) rarely occurs in children. clinical trials in pediatric patients have not been performed and there are little data on therapy with intravenous immunoglobulin (ivig). we performed an observational trial to investigate the risk of adverse events of special interest (aesi) with ivig, i.e. hemolytic anemia, aseptic meningitis, acute renal failure, thromboembolic events and anaphylactic reactions. the study cohort was derived from the us premier perspective database and consisted of patients < years with a diagnosis of cidp (icd- cm diagnosis code . ) treated with the ivig privigen ® (csl behring, bern switzerland) between jan and dec . we identified pediatric cidp patients: preschool children (age - years at first treatment for cidp), children ( - years) and adolescents ( - years); females and males; white, one black and two allocated to "other race". six patients had a history of other ivig use for guillain-barré syndrome, one patient for myasthenia gravis and one for immunodeficiency before the diagnosis of cidp. the mean privigen ® dose calculated from the cumulative quantity of privigen ® per treatment episode and the corresponding age-and gender specific median of the us population body weight estimate was . g/kg body weight. the number of recorded treatment episodes per patient ranged from to . using an at-risk period of days for hemolytic anemia, and days for other aesi after each privigen ® administration, no aesi were observed in the patients with cidp with a total of person-days at-risk for ha and person-days at-risk for other aesi. this observational study shows that ivig (privigen ® ) is used for treatment of cidp in pediatric patients with or without concomitant conditions and revealed no particular safety issues in this patient group. hsn- is a peripheral neuropathy most frequently caused by missense mutations in the sptlc or sptlc genes, which code for two subunits of the enzyme serine palmitoyltransferase (spt). spt catalyzes the first and rate limiting step of de novo sphingolipid synthesis. it has been shown that mutations in spt cause a change in enzyme substrate specificity which results in the production of two atypical sphinganines, deoxysphinganine (dsp) and deoxymethylsphinganine (dmsp), rather than the normal enzyme product, sphinganine (sp). levels of deoxysphingolipids are elevated in the blood of hsn- patients and this has been shown to cause the peripheral nerve damage characteristic of the disease, which affects both sensory as well as motor axons. however, the underlying pathomechanism of how deoxysphingolipids damage neurons remains elusive. here, dsp and dmsp-mediated neurotoxicity was examined in primary mouse motor and sensory neurons, by assessing cell survival and neurite outgrowth following exposure to different concentrations of sp, dsp or dmsp. the abnormal enzyme products were found to have a rapid and dose-dependent neurotoxic effect in primary neurons. we also explored the potential mechanisms that underlie deoxysphingolipid neurotoxicity, by characterizing mitochondrial function and changes in calcium handling. we found that mitochondrial dysfunction and calcium handling deficits may be key mediators of abnormal sphingolipid neurotoxicity, in both motor and sensory cell models. specifically, we revealed mitochondrial abnormalities, signs of endoplasmic reticulum stress and dysfunction of store-operated calcium channels. we propose that early deficits in mitochondria and calcium handling may underlie deoxysphingolipid neurotoxicity and thus present potential therapeutic targets for hsn- . months. one patient was excluded because the decrease in hba c was not contemporaneous with weight loss. the mean and median decrease in bmi per month was . and . respectively. the mean and median interval between surgery and decrease in hba c was . and . days respectively. records of these patients were scrutinized and classified as: 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or temporal relationship to decrease in hba c is uncertain; 'unlikely tind': when an alternative explanation exists for symptoms. only one patient was classified as 'possible tind': a -year-old woman who developed neuropathic pain in both lower limbs month after a rapid hba c decline of . % and about months after bariatric surgery. she had no documented autonomic symptoms. our study is limited by small cohort size, retrospective design and reliance on hospital records. nonetheless, it demonstrates that besides nutritional neuropathies, tind should also be considered in dm patients who develop peripheral neuropathy after bariatric surgery. in another study, we found tind is uncommon in a general cohort of diabetic patients. the occurrence of 'possible tind' in only dm patients corroborates earlier data that weight loss may act in tandem to increase the risk of tind. woolums bm , tabuchi m , sung h , sullivan jm , mamah c , yang m , blum id , wu mn , sumner cj , lloyd te . johns hopkins university, baltimore, usa. dominant missense mutations in the gene encoding the cation channel transient receptor vanilloid, family member (trpv ) cause inherited neuropathies including charcot-marie-tooth disease c (cmt c). in vitro, mutations in trpv that cause cmt c cause a gain of trpv channel function and increased intracellular calcium which subsequently leads to cellular toxicity. however, the mechanisms by which cmt c mutations in trpv lead to neuronal dysfunction in vivo remain poorly understood. we generated transgenic drosophila that express either wild-type or a cmt c causing trpv mutant (trpv r c ) to assess the effect of trpv r c on neuron function in vivo. selective expression of trpv r c in drosophila ccap neurons (n ccap ) results in a failure of drosophila wing expansion that is blocked by genetically inactivating the channel pore, demonstrating the requirement of channel function in mediating this phenotype. perforated patch clamp analysis of n ccap reveals that trpv r c causes a calcium-dependent increase in n ccap neuronal excitability. this hyperexcitability is restored to control levels by application of a trpv selective antagonist. high level expression of trpv r c causes synaptic and dendritic degeneration, both of which are rescued genetically by inactivating the channel pore or pharmacologically by feeding larvae a trpv selective antagonist. we conducted a genetic screen in n ccap and found that camkii knockdown potently suppresses the trpv r c mediated wing expansion phenotype and selectively rescues degeneration of synapses but not dendrites. we also find that trpv r c , but not controls, disrupts mitochondrial transport in axons by increasing the number of stationary mitochondria. our data demonstrate that trpv r c elevates neuronal intracellular calcium which disrupts mitochondrial transport and mediates neurodegeneration through compartment-specific calcium-mediated signaling pathways, and supports the further investigation of trpv antagonists as potential therapeutics for the treatment of cmt c. mood disorders, including anxiety and depression, are commonly observed among chronic pain patients with prevalence estimates ranging from to %. comorbidity between mood and chronic pain disorders has been linked to altered limbic regulation of the hypothalamic-pituitary-adrenal (hpa) axis. stress activates the hpa axis and can initiate and/or exacerbate symptoms related to both chronic pain and mood disorders. previous studies from our laboratory have investigated the influence of early life stress on mechanical pain hypersensitivity, visceral hypersensitivity and behavioral evidence of mood disorder later in life. here, we are testing the hypothesis that chronic stress exposure in adulthood can increase somatic and visceral sensitivity and anhedonic behaviors in a mouse strain with a genetic predisposition to anxiety. adult, female a/j mice were exposed to repeated foot shock stress for continuous days and tested for alterations in mechanical sensitivity, sucrose preference, visceromotor response (vmr) during urinary bladder distension, and serum corticosterone levels. mice that underwent shock stress had a significantly decreased mechanical withdrawal threshold in the hind paw compared to their baseline and sham group measurements. sucrose preference was measured prior to shock exposure and throughout the shock paradigm as an indicator of anhedonic behavior. mice that underwent shock stress displayed a trend toward decreased sucrose preference, indicating anhedonia, in comparison to mice in the sham group that did not display anhedonia. mice that underwent shock stress displayed significant increases in vmr during bladder distension compared to sham mice. finally, serum corticosterone levels were significantly higher in the mice that underwent shock stress compared to sham mice, indicating a stress-induced increase in hpa axis output. together these data suggest that chronic stress exposure can induce mechanical allodynia, visceral hypersensitivity, and depression-like behaviors in an anxiety-prone mouse strain. future studies will incorporate gene expression in the hypothalamus, amygdala, and hippocampus, as well as investigation of possible downstream peripheral neuroimmune modulation and neuronal morphology changes. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated neuropathy. as prognostic markers of gbs, modified erasmus gbs outcome score (megos), erasmus gbs respiratory insufficiency score (egris), and Δigg have been reported. however, the proportions of subtypes of gbs are known to be different between the western countries and japan, it remains to be elucidated whether those markers can also be applied to gbs in japan or not. we here investigated retrospectively gbs patients and determined the megos and the egris of those cases. among them, Δigg could be obtained in cases. we evaluated the prognosis using gbs outcome score (functional grade: fg) at months; we called good prognosis when fg at months was less than and poor prognosis when that was or more. as a result, in cases with higher score than at megos on admission cases ( %) had poor prognosis and in cases with higher score than at megos at day of admission cases had poor prognosis. in cases with higher score than on egris cases ( %) needed the mechanical ventilator. patients with good prognosis had higher Δigg(average: mg/dl) than patients with poor prognosis(average: mg/dl). we calculated the cut-off value of Δigg in patients, which was mg/dl. patients with higher Δigg than mg/dl could significantly walk independently at six months (p< . ). patients ( %) had poor prognosis in patients with lower Δigg than mg/dl. ( %) of patients were treated with the single cycle of intravenous immunoglobulin (ivig). other patients ( %) were treated with the combined therapies, such as intra venous methylprednisolone and/or plasmapheresis or the second cycle of ivig in addition to ivig. although there was no difference in prognosis between patients with the single cycle of intravenous immunoglobulin (ivig) and patients with the combined therapies, in the patients who had fg> and megos > on admission, the combined therapies made better prognosis than the single course of ivig (p< . ). we found that megos, egris and Δigg were also available in japan. the efficacy of the combined therapies in severe gbs patients should be investigated in the future large scale prospective studies. yiu em , , , burns j , , , menezes mp , , ryan mm , , and for the paediatric cmt best practice guidelines consortium. royal children's hospital melbourne, melbourne, victoria, australia; murdoch childrens research institute, melbourne, victoria, australia; university of melbourne, melbourne, victoria, australia; university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia. charcot-marie-tooth disease (cmt) often presents during childhood. common symptoms include weakness, limb pain and cramps, foot deformity, and balance impairment. guidelines for the optimal management of common problems experienced by children with cmt do not currently exist. development of these guidelines will provide an evidence base for the management and monitoring of children with cmt. a series of systematic literature reviews utilising the grade (grades of recommendation, assessment, development, and evaluation) approach were conducted to answer pre-specified key clinical questions related to the management of paediatric cmt. these included treatment recommendations for symptoms such as weakness, pain, balance impairment, joint deformity, and impaired upper limb function, and anticipatory monitoring for associated complications such as hip dysplasia. this yielded minimal to no evidence for the pre-specified clinical questions, and evidence-based management recommendations could not be made. consensus-based statements will therefore be formulated via a three-round delphi process, to be conducted in . the delphi panel will consist of local and international medical and allied health professionals who have experience in the management of children with cmt. efficacy of pxt in the treatment of adult patients cmt a (n= ) was shown in a multicenter, randomized, double-blind, placebo-controlled phase ii study (attarian et al. ) . pxt taken x/day, orally, for consecutive months was well tolerated and safe. significant improvement of disability was observed for the highest tested dose, thought indicative for an early, meaningful change in disease course (meta-analysis by mandel et al., ) . this formed the rationale to initiate a multicenter, randomized, double-blind, placebo-controlled pivotal phase iii study (clin-icaltrials.gov: nct ) of pxt in mildly to moderately affected cmt a patients in december . the primary objective is to assess the efficacy of doses of pxt compared to placebo on disability as measured by the mean change from baseline overall neurology limitations scale (onls) score at month and . furthermore, efficacy on the proportion of responders (i.e. improvement of onls), impairment (cmtns-v ), functional tests ( -mwt, qmt, -hpt), electrophysiological parameters (cmap, snap and ncv) and quality of life (eq- d, vas) are secondary endpoints. pursuant this study, patients will be eligible for a -month extension study, in which pxt assigned patients will continue with the previously assigned dose, whereas placebo patients will be randomized to one of the two pxt doses. the study is conducted in investigational sites in countries (eu, canada and us). in december patient randomization was completed (n= ). the screen failure rate was %, as expected ( patients were screened). the independent dsmb recommended to continue the study as planned following a safety analysis on the first patients treated for > months. preliminary baseline characteristics are based on patients (data not cleaned). the study population had a mean age of . ± . years (range - ; male . %) of which . % had a confirmed genetic diagnosis of cmt a. the mean cmtns-v was . ± . and the mean motor nerve conduction of the ulnar nerve was . ± . m/s. ten patients withdrew from the study, due to adverse events unrelated to study treatment. the last patient completing the study is expected in march . zhang g , ghosh p , lin j , ghauri a , sheikh ka . department of neurology, university of texas health science center at houston, houston, tx, usa. assessment of epidermal nerve fiber density and its structure integrity is critical for the diagnosis and evaluating the effectiveness of potential therapies in small fiber neuropathies. currently, skin biopsies, at multiple sites, are most commonly used to assess these diseases. these studies are expensive and time consuming due to cumbersome processing and quantification techniques and serial biopsies over time are often not feasible due to costs and patient acceptance. moreover, vast majority of normative data for skin biopsies in humans are available only for few distal sites and a significant proportion of patients with small fiber neuropathies have focal or regional symptoms not involving the commonly biopsied sites in the leg. live imaging could overcome these limitations and provide a noninvasive real time assessment of epidermal nerve fibers all over the body. we previously found that anti-ganglioside antibody (aga) is an effective neuronal delivery vector for transport of various cargos, such as fluorescent dyes, to peripheral nerves. in the current proof of concept study, we examined whether non-invasive multiphoton microscopy can be used to probe/image the epidermal nerve fibers in living animals after systemic and/or local delivery of fluorescently conjugated aga. we found that the individual nerve endings in skin and cornea are distinctly labeled, and visualized under two-photon microscope. the epidermal nerve fiber labelling by fluorescent-tagged aga was further validated using transgenic mice selectively expressing yellow fluorescent protein in their nervous system. in-vivo multiphoton imaging provide a tool with potential for dynamic longitudinal evaluation of small fiber neuropathies, including nerve degeneration and regeneration, without tissue removal. thus, the use of multiphoton microscopy in conjunction with fluorescently labeled aga as neuronal vector can have many research and clinical applications, such as labeling and live visualization of epidermal nerve fibers to assess small sensory nerve fibers in health and disease. zhou y , tavori h , lee s , al salihi m , fazio s , notterpek l . mcknight brain institute, university of florida, gainesville, florida, usa; knight cardiovascular institute, oregon health and science university, portland, oregon, usa. the majority of hereditary neuropathies are due to abnormalities in peripheral myelin protein (pmp ), whose genetic variants include increased expression (gene duplication), haploinsufficiency (gene deletion), or point mutations. phenotypic heterogeneity in clinical presentation is common among hereditary neuropathy patients even within the same family, the cause of which has not been determined. to investigate the role of pmp in the pathogenesis of the neuropathies we have generated and characterized pmp null (pmp −/− ) mice (amici et al., ) whose peripheral nerves show alterations in lipid metabolism (lee et al., ) . to examine the molecular changes underlying these abnormalities we determined the expression of cholesterol synthesis (srebp pathway), and cholesterol uptake, transport and efflux genes (lxr pathway). in affected nerves, we found the cholesterol synthesis pathway inhibited, while the lxr pathway, and particularly apoe and abca , upregulated at the mrna and protein levels. since pmp is expressed at low levels in the liver, the central organ for the regulation of cholesterol in the body, we studied liver tissue form pmp −/− mice. liver from pmp −/− mice showed significant hepatomegaly, clear features of microvesicular steatosis, as well as marked increase in lxr pathway genes and proteins (abca and apoe), as compared to wt. ultrastructural studies identified lipid droplets and significantly enlarged mitochondria in the liver of male pmp −/− mice, which is not due to mitochondria fusion, as the levels of mfn and remained similar to wt. as disturbed hepatic cholesterol homeostasis induces the activation of kupffer and stellate cells, we determined the extent of inflammation in nerve and liver tissues from pmp −/− mice with leukocyte (cd b) and macrophage markers (cd ). in nerve sections, we detected an increase in the number of cd b+ cells, which was confirmed by western blots. in the liver of pmp −/− mice we found a significant increase in cd -reactive kupffer cells and elevated levels of tnf-alpha. the severe dysregulation of cholesterol metabolism in nerve and liver, including neuroand hepatic inflammation in the absence of pmp − suggest that dysregulated cholesterol metabolism and inflammation may act as a disease modifier in pmp -dependent neuropathies. our aim was to ascertain frequency of and risk factors for tee, arterial (ate) and venous thromboembolic events (vte) in neuromuscular patients receiving regular ivig. we performed a retrospective case-note review of inflammatory neuropathy patients receiving regular ivig treatment. we collected the following data over a month study period this analysis suggests tee incidence is higher in ivig treated patients than comparable population-based rates. examination of tee occurrence in age and vascular risk factor matched ivig-treated and ivig-naïve individuals is required to appreciate the excess risk associated with ivig treatment. référence des maladies neuromusculaires et la sla, hôpital de la timone ha) and whether temporal dispersion (td) parameters are helpful. fifty-eight patients diagnosed according to asbury and cornblath ( ) were prospectively included between january and september . edx and were performed - (mean= ) and - days (mean= ) after disease onset, respectively. there were no differences in classification consistency between ho and ha at edx (p= . ) and edx (p= . ), but more patients were classified as aman when comparing ra with ho and ha at edx (p< . ). at edx , ra classified more patients as equivocal with ho (p< . ) and as aman with ha (p< . ) ) or edx (ho, fe p= ; ra, fe p= . ; ra with td, fe p= . ). gm , gd a, gd b and gq b igg antibodies were tested (willison at edx , only ho showed maybe more antibodies with aman compared to aidp (fe p= . , phi=− . ) and with aman compared to equivocal cases since correlation with factors associated with axonal gbs, in casu rcf and antibodies, is far from exclusive, the usefulness of edx subtype classification using specific criteria sets, remains doubtful. the frequency of rcf indicates that nodal/paranodal alterations may represent the main pathophysiology in more gbs patients than currently thought ruiz m , lessi f , cacciavillani m , riva m , salvalaggio a , campagnolo m , briani c . neurology, department of neuroscience, university of padua, padua, italy; hematology and clinical immunology unit, department of medicine, university of padua, padua, italy; cemes, data medica group, padua, italy.arsenic trioxide (ato) is highly effective in treatment of acute promyelocytic leukemia (apl). it is licensed in italy for treatment of relapsed-refractory apl and for first line chemotherapy in low risk patients. ato most frequent side effects are well described, but less is known on ato induced. we describe apl patients who were treated with all-trans retinoic acid (atra)/ato as first line therapy. the characteristics of ato induced neuropathy was prospectically analyzed by neurological evaluation using both the total neuropathy score, clinical version (tnsc) (a validated scale for chemotherapy induced peripheral neuropathy) and neurophysiological assessment. patients have been evaluated at baseline, at the end of the induction phase, at the end of ato/atra treatment and year after discontinuation of treatment. baseline neurophysiology was performed at the end of induction phase. both patients were men, respectively and -yr-old. none of the patients had previous history of neuropathy. baseline tnsc was (no clinical signs of neuropathy) in both patients. neurophysiological evaluation performed after the end of induction cycle did not reveal signs of peripheral neuropathy in both patients. patient received mg of ato during induction, total , mg. patient received mg of ato during induction, total , . both patients developed leg numbness during consolidation cycles and patient also hand numbness. tnsc at the end of therapy was in patient and in patient . neurophysiology at the end of therapy detected signs of sensitive axonal neuropathy in both patients. they received full doses of ato consolidation ( . mg/kg/day for days/week, on alternate months for total months and tretinoin weeks on weeks off). during the first year of follow-up both tnsc and neurophysiology year after the end of consolidation cycle were consistent with full recovery. our patients developed sensory axonal neuropathy during ato therapy, that clinically manifested during consolidation cycles and improved up to complete recovery during follow-up. published case reports show that outcomes may not be as good as in our patients. a multicenter prospective study evaluating the characteristics of ato-induced neuropathy in apl is ongoing. ruiz m , campagnolo m , salvalaggio a , cacciavillani m , taioli f , fabrizi gm , briani c . department of neuroscience, neurology unit, university of padua, padua, italy; data medica group, emg unit, cemes, padua, italy;mutations in the mitochondrial copper-binding protein sco , cytochrome c oxidase assembly protein, have been reported in several cases of fatal infantile cardioencephalomyopathy with cox deficiency. we identified compound heterozygous variants in sco in two unrelated patients with isolated length dependent axonal sensorimotor polyneuropathy of variable clinical severity (axonal autosomal recessive charcot-marie-tooth disease type , cmt ) by whole exome sequencing. although peripheral neuropathy has been described as a secondary feature in a few cases of fatal infantile cardioencephalomyopathy, the disease onset, clinical phenotype and survival in our patients differ significantly from the previously described cases. our patients developed predominantly motor neuropathy; moreover, they are still alive and they have not developed cardiomyopathy, which is the main phenotype and cause of death at early infancy in reported patients. both of our patients harbor mutations adjacent or near the conserved copper-binding motif (cxxxc), including the common reported pathogenic variant e k and the novel change d g. in addition, each patient carries a second mutation located in the same loop region of the protein, p t and r q. western blots from fibroblasts from these cmt patients showed reduced levels of cox , a subunit of complex iv, indicating cox deficiency. our findings demonstrate that cmt can be the predominant phenotype associated with sco mutations, pointing to a broader phenotypic heterogeneity. the mechanism linking mitochondrial respiratory chain dysfunction to isolated axonal loss of variable severity remains to be elucidated. the muscarinic acetylcholine (ach) type receptor (m r) is a metabotropic g protein-coupled receptor expressed by adult sensory neurons. cholinergic signaling through muscarinic receptors can modulate axonal plasticity in invertebrates and lower vertebrates. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) m r antagonists elevate neurite outgrowth and protect from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt, et al., ) . furthermore, we demonstrated that excessive cholinergic signaling due to m r overexpression caused a significant reduction in neurite outgrowth (calcutt, et al., ) . the mechanism of m r-antagonist driven neurite outgrowth remains poorly understood, however, we have proposed that ach constrains axonal outgrowth via m r activation. cholinergic signaling is mediated via recruitment of trimeric g proteins, of which g alpha- and g alpha- regulate cytoskeleton dynamics by control of tubulin polymerization. activated gtp-bound g-alpha proteins destabilize microtubules by increasing the intrinsic gtpase activity of tubulin. we have therefore tested the hypothesis that cholinergic signaling regulates neurite outgrowth via modulation of g protein mobilization and the dynamics of the tubulin cytoskeleton. we found that over-expression of m r in adult sensory neurons induced dissolution of the tubulin cytoskeleton in distal neurites. g alpha- expression in adult sensory neurons was significantly higher (p< . , -fold) than g alpha- . subsequent knockdown the peripheral neuropathy research registry (pnrr) is a multicenter collaborative research project sponsored by the foundation for peripheral neuropathy to advance the science in distal symmetrical polyneuropathies (dsp). the registry was designed to prospectively characterize clinical phenotype and natural history of patients with dsp and obtain biofluids to identify new causes and genetic modifiers of dsp with careful genotype/phenotype correlations, and to develop biomarkers. the enrollment in the registry is still ongoing but an interim analysis was carried at the end of december . eligible study participants were years or older with a diagnosis of idiopathic, diabetic, chemotherapy-or hiv-induced peripheral neuropathy. they were examined by a physician at one of the six consortium members (johns hopkins university school of medicine, icahn school of medicine at mount sinai medical center, beth israel deaconess medical center, northwestern university medical center, university of utah medical center and kansas university medical center). the collected data set included ( ) a detailed questionnaire, discussing their symptoms, medical history and family history, ( ) a standardized neurological examination form, ( ) electrodiagnostic evaluation and ( ) diagnostic laboratory testing. blood samples (whole blood, plasma and serum) were collected for future biomarker and genotyping evaluations. at the end of , complete data sets and blood samples were collected from patients. % had diabetic pn, % had hiv-associated pn, % had chemotherapy induced pn and % were diagnosed with idiopathic pn. detailed analysis of clinical presentation, examination findings and diagnostic investigations will be discussed at the presentation. standardized phenotyping with linked bio-specimen banking will help establish the minimum data set required for neuropathy diagnosis and support genotype-phenotype correlations with next generation sequencing technologies and development of novel biomarkers. pnrr data will improve our understanding of disease mechanisms paving the way for new therapeutic discoveries in painful and non-painful neuropathies. chemotherapy-induced peripheral neuropathy (cipn) is a major side effect of treatment, typically presenting as a sensory neuropathy. symptoms include pain, paraesthesia, and numbness in the extremities, resulting in functional impairment. increasingly, patient reported outcomes (pros) are utilized to accurately examine the impact of cipn symptoms on patient function. however, the links between objective neurological assessment and pro measures remain ill defined. this study aimed to identify links between neurophysiological measures and pros in patients treated with neurotoxic chemotherapy. assessments were conducted in patients (f= , mean age . ± . years) who had completed neurotoxic chemotherapy on average . ± months previously (platinum-based n= , taxane-based n= or taxane/platinum combination therapy n= ). patients reported the presence and severity of neuropathic symptoms via the fact-gog ntx , a validated patient questionnaire. clinical the most important neurological side effect of a large number of anti-cancer drugs is a painful peripheral neuropathy. mainly chemotherapeutics that interfere with microtubules, including the plant derived vinca-alkaloids such as vincristine, are well known to cause chemotherapy-induced peripheral neuropathies (cipn). to date, few treatments are available and focus on symptom alleviation and pain reduction rather than on preventing the neuropathy all together. for the first time, we highlight the potential of specific histone deacetylase (hdac ) inhibitors as a preventive therapy for cipn, using novel rodent models for vincristine-induced peripheral neuropathies (vipn), characterized by a sensory axonopathy. one reason so few therapies are available, is because the exact pathophysiological mechanisms are poorly understood. mounting evidence proposes axonal transport, a pathway frequently disturbed in neurological disorders, as a major player in the pathophysiology of vipn. proper axonal transport requires dynamic microtubules which are highly modulated by post-translational modifications. since vincristine interferes with the polymerization of microtubules, we reason disturbances in microtubule dynamics, and by extension axonal transport, could contribute to vipn. we illustrate that increasing acetylation of -tubulin after hdac -inhibition, can restore vincristine-induced defects in axonal transport in cultured dorsal root ganglion neurons. also in vivo, -tubulin acetylation was restored in the saphenous nerve and dorsal root ganglia, two sensory tissues that are affected by vincristine. ultimately, this correlates to a reduced severity of the neurological symptoms, both on the electrophysiological and on the behavioral level. moreover, we discovered that hdac -inhibition was not only protective against neurotoxicity, but also reduced tumor progression in a mouse model for acute lymphoblastic leukemia. taken together, our results show that hdac -inhibition is an ideal strategy to prevent vipn with beneficial effects both on the neurotoxicity as well as on tumor growth.approximately two-thirds of cidp subjects need long-term corticosteroids or intravenous immunoglobulins (ivig), with ivig being slightly preferred based on safety profiles. subcutaneous ig (scig) is an alternative option for ig delivery but has not previously been investigated in a large-scale clinical trial in cidp.we performed a randomized, double-blind trial in cidp investigating . and . g/kg weekly doses of scig igpro (hizentra ® , csl behring) versus placebo in subjects for maintenance treatment. ivig-dependent adults with definite or probable cidp according to efns/pns criteria were eligible. the primary outcome was the percentage of subjects with a cidp relapse ( -point deterioration on adjusted incat disability score) or who were withdrawn for any other reason during the -week scig-treatment period. multiple secondary endpoints were assessed. superiority of at least one igpro dose over placebo was tested one-sided using the cochran-armitage trend test for the primary outcome and the jonckheere-terpstra tests for secondary outcomes.the primary outcome occurred in % of high-dose scig, % of low-dose scig, and % of placebo subjects (p < . ); cidp relapse occurred in % of high-dose scig, % of low-dose scig and % of placebo subjects (p < . ), respectively. both scig doses were superior to placebo (low-dose vs placebo p = . ; high dose vs placebo p < . ). median incat score, mrc sum score, and grip strength remained stable in scig subjects. high-dose scig prevented the r-ods decline seen with low-dose scig and placebo (p < . ). all placebo subjects deteriorated on measures of strength and disability.causally related adverse events occurred in ( %) subjects ( % placebo, % low dose, and % high dose).scig igpro was efficacious and safe as maintenance treatment. mutations in metalloendopeptidase (mme) gene have been associated with autosomal-recessive late-onset charcot-marie-tooth type- (cmt ). to date, all patients have had at least one truncating mutation, either in homozygosity or in trans with a missense mutation. more recently, loss-of-function and missense heterozygous mutations were also identified in autosomal-dominant cmt. we report the case of a previously healthy caucasian woman, born to healthy unrelated parents, who presented at the age of thirty-nine with numbness and cold sensation in the lower limbs. subsequently she developed progressive gait disturbance and impaired hand dexterity. her homozygous twin presented at the same age with similar symptoms. the family history was otherwise uneventful, in particular neither neuropathy nor dementia were described. neurological examination at the age of fifty-three revealed a steppage gait, distal upper and lower limb atrophy and weakness, distal sensory loss and bilateral pes cavus. deep tendon reflexes were normal in the upper limbs and absent in the lower limbs. nerve conduction studies revealed an axonal sensory and motor neuropathy. a sural nerve biopsy revealed a reduction in myelinated nerve fibers and active axonal degeneration. targeted sanger sequencing of mpz , gjb , gdap , nefl, fkrp, bscl , hspb and mfn were negative. sureselect focused exome sequencing was therefore performed and identified two missense heterozygous mutations [c. g>a,p.c y;c. t>c,p.y h] or lack of efficacy (n= ). most frequently reported adverse events were cognitive ( %), gastrointestinal ( %) and genitourinary ( %). discontinuation due to perception of lack of efficacy was more frequently reported by men ( % vs % p= . ). women presented higher punctuations on pigc scale compared with men ( . ± . vs . ± . , p= . ). pigc scores were significantly higher in patients receiving taxane ( . ± . ) than platinum ( . ± . ) agents (p= . ). no significant differences according severity of neuropathy neither type of chemotherapy were observed in drop-out and retention rates. patients with long-lasting cipn (> months) reported lower pigc scores ( . ± . vs . ± . , p= . ) and higher frequency of suspension due to adverse events ( % vs %, p= . ) and less rate of continuation of duloxetine ( % vs %, p= . ). more than one-third of patients with disturbing cipn discontinued duloxetine prematurely due to intolerable side-effects. low tolerability, male gender and long-lasting cipn may limit duloxetine usefulness in the treatment of symptomatic cipn. verboon c , jacobs bc , and the igos consortium. department of neurology, erasmus medical centre, rotterdam, the netherlands; department of immunology, erasmus medical centre, rotterdam, the netherlands.the efficacy of intravenous immunoglobulin (ivig) in guillain-barré syndrome (gbs) has only been demonstrated in severely affected patients who are unable to walk independently. although there is no proof that ivig is effective in milder forms of gbs, some neurologists are treating these patients with ivig considering that even milder forms of gbs may result in poor recovery, residual deficits, fatigue or pain.we determined the effectiveness of a single course of ivig ( g/kg in - days) in relatively mild forms of gbs in the ongoing observational international gbs outcome study (igos). the gbs disability score, mrc sum score and patient reported outcome measures (prom) were compared at and weeks. ordinal logistic regression analysis was used to determine the effect of ivig on the gbs disability score, adjusted for previously identified prognostic factors.data were analyzed from the first patients enrolled in igos by december , including patients with mild gbs at entry, of which patients ( %) were treated with supportive care, while patients ( %) received ivig (start ivig after onset of symptoms in days, median , iqr - ). at baseline, patients in the ivig treated group compared to the untreated group less frequently had pure motor gbs ( % versus %, p< . ) and axonal damage or unresponsive nerves ( % versus %, p= . ), but a worse gbs disability scores at nadir (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ). at weeks, the median mrc sum scores and prom were not significantly different between treated and untreated patients. however, more patients in the ivig group showed complete recovery of muscle strength at weeks than patients in the control group ( % versus %) (p= . ) and more frequently showed full neurological recovery on the gbs disability scale ( % versus %, p= . ). additional results will be presented at the conference.based on the results of this interim analysis in observational data, we conclude that patients with a relatively mild form of gbs may benefit from a single course of ivig. despite treatment with intravenous immunoglobulin (ivig), many patients with guillain-barré syndrome (gbs) recover insufficiently. we primarily aimed to determine whether a second ivig course ( g/kg in - days) in patients with a poor prognosis improves outcome on the gbs disability scale after weeks. we included patients from the prospective, observational international gbs outcome study (igos) treated with ivig and who had a poor prognosis on the modified erasmus gbs outcome score (megos). of patients enrolled in igos, patients were eligible; patients ( %) were treated with one ivig course (control group); patients ( %) received an 'early' second ivig course ( - weeks after start first course) and patients ( %) a 'late' second ivig course (within - weeks). one week after study entry, patients receiving an 'early' or 'late' second ivig course had significantly worse gbs disability scores and mrc sum scores than controls, implying the need for adjustment of baseline characteristics. the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' group, and . ( % ci . - . ) for the 'late' group, suggesting worse outcomes with a second course of ivig compared to controls. at months, patients ( %) in the 'early' second ivig group, patients ( %) in the control group and only ( %) in the 'late' second ivig group were able to walk unaided (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' second ivig group and only . ( % ci . - . ) for the 'late' second ivig group. in gbs patients with a poor prognosis, we did not find a beneficial effect of a second course of ivig after weeks follow-up. our results suggest that an 'early' administered second ivig course might improve outcome at weeks. given the limitations of this observational study, a randomized controlled trial with a larger number of gbs patients with a poor prognosis being treated early in the disease course is needed to confirm or refute these results. vidal c , bhatt n , agudelo c , mahapatra a , saporta ma . department of neurology, university of miami miller school of medicine, miami, usa.multifocal motor neuropathy (mmn) is an inflammatory demyelinating chronic neuropathy characterized by progressive asymmetric weakness in the distribution of two or more peripheral nerves, without objective sensory loss or upper motor neuron signs. the cardinal neurophysiological finding is conduction block outside the usual sites of nerve compression. supportive clinical criteria include high titers of anti-gm antibodies, good response to ivig, increased cerebrospinal fluid (csf) protein (< g/dl) and magnetic resonance imaging (mri) with diffuse swelling of the brachial plexus. we report a case of a year old woman with a two month history of progressive muscular weakness which began in the right upper extremity, described as difficulty in gripping objects, writing and typing on the computer, followed by progressive lower extremity weakness with difficulty rising from a chair and left foot drop. she also complained of pain in her right trapezius and scapular area. she denied any tingling or numbness. on neurological examination there was right scapular winging and asymmetric weakness predominantly involving the right upper and left lower extremities. reflexes were absent throughout. lumbar puncture revealed albuminocytological dissociation (wbc and protein . g/dl). ncs revealed slow conduction velocities for both ulnar and median nerves and a median nerve conduction block at the forearm segment. sensory nerve action potentials were normal for all tested nerves. needle emg revealed acute denervation of right periscapular muscles. brachial plexus mri showed symmetric bilateral thickening from trunks to peripheral nerves. anti-gm igg/igm were negative. two days after lumbar puncture and the beginning of ivig, patient presented binocular diplopia on extreme left lateral gaze. brain mri with and without contrast showed no intracranial pathologies. after five days of ivig, patient was discharged with significant clinical improvement and recovery of the right scapular winging. diplopia improved a week after discharge. we report an atypical multifocal motor neuropathy case. although this patient fulfills all clinical criteria for mmn, we report some features usually not found in mmn such as scapular winging and a mild and transient left vi nerve palsy. mmn should be included in the differential diagnosis of scapular winging. vlckova e , , raputova j , , srotova i , , sommer c , Üçeyler n , birklein f , rebhorn c , rittner hl , kovalova e , , nekvapilova e , , belobradkova j , olsovsky j , weber p , dusek l , jarkovsky j , bednarik j , . despite many studies addressing biomarkers for pain in diabetic polyneuropathy (dpn), little is known about why it affects only a certain proportion of dpn patients. the somatosensory system plays a key role in the pathophysiology of neuropathic pain (neup) and subgroups with different sensory profiles might respond differently to pain treatment. we aimed to characterize sensory phenotypes of patients with painful and painless diabetic neuropathy and to assess demographic, clinical, metabolic, electrophysiological and psychological parameters related to the presence of neup in a large cohort of well-defined dpn subjects.this observational cross-sectional multi-centre cohort study (performed as part of the ncrnapain eu consortium) of subjects with dpn (non-painful, ndpn, n= ; painful, pdpn n= ) associated with diabetes mellitus of type and (median age years, range - years; women) comprised detailed history taking, neurological examination, laboratory tests, quantitative sensory testing, nerve conduction studies, neuropathy severity scores, and neuropsychological questionnaires. all parameters were analysed with regard to the presence and severity of neup. the presence and severity of neup were positively correlated with severity of neuropathy and thermal hyposensitivity (p< . ). a minority of pdpn patients ( . %) had a sensory profile indicating thermal hypersensitivity; this was associated with less severe neuropathy and better response to pain therapy. the presence of neup was also associated with female gender (p< . ) and with higher cognitive appraisal of pain as assessed by the pain catastrophizing scale (p< . ), while parameters related to diabetes (duration, hba c, microangiopathy) showed no influence on neup presence and severity. this study confirms the necessity of comprehensive dpn phenotyping and underlines the importance of the severity of neuropathy that should be taken into account in the stratification of patients with pdpn for analgesic treatment and drug trials.people with charcot marie tooth disease (cmt) experience slowly progressive muscle weakness, sensory loss and musculoskeletal changes over time. this leads to disability and risk of comorbidities due to inactivity. exercise is important to maintain general health but may also help to improve symptoms of cmt. we conducted a randomised controlled crossover trial of aerobic exercise to ascertain the effect of training on fitness levels, muscle strength, function and general well-being. in addition, we monitored the safety of training and feasibility of participation in this type of exercise in local community gyms. motivation, confidence and barriers to exercise were explored using qualitative interviews. the recruitment target was people. in total people with cmt a were approached to participate and were unable to commit to the trial or did not meet the study criteria on initial screening. thirty-one people underwent more detailed screening but three failed to meet the study criteria, five people withdrew before starting and three withdrew part way through the study. the data for the people who started the study were analysed using a random effects model. there was a % participation level in the training and it was well tolerated with no increases in pain or blood serum creatine kinase. an increase in vo peak (ml/min/kg) was observed in the cmt group with (pre training: n= , . ± . , % ci . to . ; post training: n= , . ± . , % ci . to . ; pre control: n= , . ± . % ci . to . ; post control: n= , . ± . , % ci . to . ) . there was wide between subject variation leading to a small overall effect size with cohen d of . ( % ci:- . to . ). a tentative regression model showed no effect of group or time point. there were no major changes in other measures of impairment, function or patient reported outcome measures. this pilot study showed that a community based model of training had a small effect on cardiopulmonary fitness, and was well tolerated with good participation. ankle-foot orthoses (afo) are commonly prescribed for children with charcot-marie-tooth disease (cmt) to manage foot drop, however the type and severity of functional impairment results in gait deviations that might require alternate orthotic designs. the aim of this study was to identify d gait patterns of children with cmt based on severity of functional weakness (based on heel walk, toe walk and foot drop items during gait using the cmt pediatric scale) to inform a design pipeline for d printed orthoses. d gait data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model in children with cmt ( male; ± . yrs, ± . cm, ± . kg), of various cmt types: cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx and compared to typically developing children ( male; . ± . yrs, ± . cm, ± . kg). data were subdivided into three groups denoting increasing severity of dorsiflexion and plantarflexion weakness: no difficulty heel or toe walking (cmt nd ), difficulty heel walking (cmt dh ), difficulty toe and heel walking (cmt dth ). the cmt nd group showed a near-normal gait pattern. the only significant differences noted at the ankle were reduced peak dorsiflexion in stance (p< . ), indicating that an orthotic intervention may not be required. in addition to reduced peak dorsiflexion, the cmt dh group demonstrated significantly reduced dorsiflexion in swing (foot-drop) and a reduced dorsiflexor moment in loading response (p< . ). this suggest, the cmt dh group would require a flexible afo to allow activation of the plantarflexors during push-off, prevent foot-drop and restore a heel rocker in loading response. in contrast, the cmt dth group presented with significantly delayed and increased peak dorsiflexion in stance and reduced plantarflexion and power at push-off (p< . ). they also had significantly increased mean knee extensor moment (p< . ) revealing early signs of 'crouch gait'. therefore, the cmt dth group would require a rigid afo to limit the amount of dorsiflexion and assist movement of the ground reaction force anterior to the knee during stance. three distinct gait patterns at the ankle were identified in children with cmt, indicating patient-specific orthotic design pathways to target specific functional impairment. wojciechowski e , , chang a , cheng t , , little d , , menezes mp , , hogan s , burns j , . university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia.children with cmt are often prescribed ankle-foot orthoses (afo) to manage lower limb impairment such as foot drop and foot deformities. they are handmade by plaster cast followed by thermoplastic moulding. this traditional approach provides limited design options, can be costly, with long outpatient wait times. d printing, also known as additive manufacturing, has the potential to transform the way orthoses are prescribed, designed and manufactured. the aim of this review was to evaluate the evidence of d printing afos compared to traditional manufacturing methods, for children with cmt. there are currently no studies evaluating the application of d printing afos for children with cmt. however, a small, but emerging evidence base exists for d printed afo's in adults from studies including healthy participants and populations with rheumatoid arthritis, post-polio syndrome, foot drop and ankle weakness secondary to injury. samples sizes ranged from - participants for studies related to in-shoe orthoses and from - for studies related to afos. the methods of d printing included sterolithography, selective laser sintering and fused deposition modelling using materials such as nylon , nylon , polylactide, polycarbonate and abs. d printed afos were comparable to traditional manufactured orthoses in terms of patient-perceived comfort, temporal-spatial parameters, plantar pressure measurements and d gait analysis. however, the effects of long-term usage and durability of d printed afos has not been investigated. d printing orthoses have potential advantages including increased design possibilities, improved productivity, higher compliance, and reduced labour needs. disadvantages include redesigning clinical pathways, limited evidence base for clinical effectiveness, limited biocompatible materials, occupational safety considerations and a high level of expertise required for software operation and fabrication of devices. further research is required to determine the feasibility of d printed afos for children with cmt, and the most appropriate and effective printing pathway, materials to improve health outcomes of affected patients. wong shj , koh sj , lee bjh , chng ysk , pawa c , subramaniam t , cheng ksa , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore; yong loo lin school of medicine, national university singapore, singapore; khoo teck puat hospital, singapore.treatment-induced neuropathy of diabetes mellitus (dm) (tind) is a complication of rapid glycaemic control. individuals present with neuropathic pain and autonomic dysfunction within weeks of improvement in glucose control. the use of both insulin and oral hypoglycaemic agents has been associated with tind. its severity is determined by the rate and quantum of hba c decline. other predisposing factors include anorexia and weight loss. we studied the incidence of tind in dm patients who have undergone massive weight loss and hba c decline after bariatric surgery. we screened electronic records of patients ( dm, non-dm) who underwent bariatric surgery between and . dm patients fulfilled the tind hba c criteria of a decrease of ≥ % over months or ≥ % over key: cord- - v k gi authors: bagnasco, annamaria; zanini, milko; hayter, mark; catania, gianluca; sasso, loredana title: covid —a message from italy to the global nursing community date: - - journal: j adv nurs doi: . /jan. sha: doc_id: cord_uid: v k gi during these difficult times, it is not easy to learn all the nursing lessons from the covid- epidemic in italy. it is not easy because - at the time of writing - italian nurses are in the middle of this emergency that shows no sign of diminishing. whatever is said today can change completely after only hours. as a global community we have only known about this virus for a few months, but it has invaded lives, hospitals and homes, subverting habits, practices, and protocols. some of the lessons learned will emerge later - after reflection and retrospective analysis. however, some things are now so evident that sharing them now is vital to help prepare those who are getting ready to face this emergency. dent that sharing them now is vital to help prepare those who are getting ready to face this emergency. the first lesson is the vital importance of personal protective equipment (ppe)-both in terms of amount and suitability. in italy the lack of suitable ppe, in particular, appropriate masks-as the ordinary surgical masks are of no use-has played a key role in spreading the infection among health workers. this lack of appropriate protection for those working on the front lines translates into a drastic daily loss of health professionals. it has been estimated that one tenth of those in italy who are covid- positive are physicians and nurses, but this could be underestimated due to the presence of infected professionals who are without symptoms (anelli et al., ; sorbello et al., ) . it is to their enormous credit that many nurses continue to provide care conscious that the minimum levels of protection cannot be guaranteed. with the global pandemic now accelerating in areas of the world yet to see italian levels of infection, it is vital that ppe equipment is procured and delivered to the covid- front-line critical care environments. protecting staff-as well as being an ethical duty of healthcare providers-is also essential to prevent reductions in skilled staff due to illness when they are needed more than ever. time is precious in this pandemic-italy did not see it coming-many other areas of the world can. the importance of ppe for staff cannot be emphasized enough. learn also from italian nurses' experiences of the harm long-term use of ppe: facial lesions and sores produced by the pressure and sweat caused by masks and goggles worn far beyond the usual time frame in normal clinical practice. further research will be needed on this-with the manufacturers of ppe being involved. but in the immediate situation healthcare providers can advise on self-care for those staff having to wear ppe for protracted periods (suen et al., ) . to help limit face lesions caused by the pressure of masks and goggles, the italian national institute of health (istituto superiore di sanità) organized online courses for health professionals to help them deal with this issue, among others related to keeping safe against covid- . more ergonomic masks, goggles and ppe will need to be available in the future. another very challenging issue, especially at the beginning of the outbreak, has been the antigen or viral testing of front-line staff, unfortunately in most cases this was not possible due to the very rapid spread of covid- infections, because there were not enough testing kits available and places where these analyses could be conducted (paterlini, ) . we also recognize that the lack of antibody testing resulted in an inability to tell who had had the infection but now had immunity-and could therefore safely return to work. more recently, some italian regions are starting to conduct tests on all front-line staff and on the entire population, but it has taken several precious weeks of time to implement this. so, this is another important lesson for the global community. an additional emotional burden facing nurses is the fear of introducing the virus into their own homes and exposing family members to covid- . it is important that nurses and other health professionals are trained in the correct procedures to manage uniforms and other belongings to minimize such a risk. if possible, staff should be encouraged and supported to use alternative accommodation to reduce the risk of family transmission. policy makers need to ensure they provide appropriate logistic and financial support to help with this course of action. this also creates a sense of isolation for healthcare workers who are already highly stressed. it highlights that this epidemic is wreaking a huge emotional toll on all healthcare professionals in italy. the long-term support needs of these staff can be planned later-but the importance of trying to provide some psychological support for staff-including the opportunity to speak about their experiences and fears-if only briefly-should be an important part of the acute response to covid- . front-line covid- care giving is exhausting-especially over long hours-at some point replacement staff will be needed to enable others to take some rest and restore their energy. many of these replacements may be returning to practice or unfamiliar with critical care environments-the importance of training and providing ppe for these staff should not be neglected due to the urgency of the need to plug gaps in the care teams. to healthcare providers and policy makers in areas at the start of their covid- epidemic, our message is to plan for the replacement of staff in critical care areasthink about how this will be done, how they can be prepared and how you plan to recall recently retired nurses back to the hospitals. the peer review history for this article is available at https://publo ns.com/publo n/ . /jan. and encouragement, to ensure that such choices are solidly rooted in noble values. one last but very important lesson from the epidemic is the need to plan for the possibility of caring for patients in their own homes. we have learnt that hospitalization is not necessary for everyone and can even be harmful. home care may be a more viable option. moreover, this would enable to reduce hospital stay and facilitate the fast discharge of recovering patients, thus increasing the availability of beds and other hospital resources. however, to take care of patients at home community nurses and general practitioners must have all the equipment and instruments they need in order to be able to do their job properly. therefore, it is important to educate large numbers of primary and community-based health professionals who, with all the necessary equipment and means, and in collaboration with general practitioners, can take care of patients directly in their own homes. it is also vital that this also included ensuring the infection control measures are in place to protect others living in the same dwelling. to conclude, is the year of the nurse, celebrating the bicentenary of the birth of our colleague florence nightingale, but it will surely be remembered also as the year of the covid- pan- we thank dr giuseppe aleo, phd and lecturer of scientific english from the department of health sciences of the university of genoa for translating this editorial into english. covid- : over italian doctors and scientists call for more testing società italiana di anestesia analgesia rianimazione e terapia intensiva (siaarti) airway research group, and the european airway management society comparing mask fit and usability of traditional and nanofibre n filtering facepiece respirators before and after nursing procedures