key: cord-0699475-5zcw9owm
authors: Gagliardi, Stella; Emanuele Poloni, Tino; Pandini, Cecilia; Garofalo, Maria; Dragoni, Francesca; Medici, Valentina; Davin, Annalisa; Damiana Visonà, Silvia; Moretti, Matteo; Sproviero, Daisy; Pansarasa, Orietta; Guaita, Antonio; Ceroni, Mauro; Tronconi, Livio; Cereda, Cristina
title: Detection of SARS-CoV-2 genome and whole transcriptome sequencing in Frontal Cortex of COVID-19 patients
date: 2021-05-19
journal: Brain Behav Immun
DOI: 10.1016/j.bbi.2021.05.012
sha: e93b4fe278a5e20bcc9c8f92dde815f928bf6e28
doc_id: 699475
cord_uid: 5zcw9owm

SARS-Cov-2 infection is frequently associated with Nervous System manifestations. However, it is not clear how SARS-CoV-2 can cause neurological dysfunctions and which molecular processes are affected in the brain. In this work, we examined the frontal cortex tissue of patients who died of COVID-19 for the presence of SARS-CoV-2, comparing qRT-PCR with ddPCR. We also investigated the transcriptomic profile of frontal cortex from COVID-19 patients and matched controls by RNA-seq analysis to characterize the transcriptional signature. Our data showed that SARS-CoV-2 could be detected by ddPCR in 8 (88%) of 9 examined samples while by qRT-PCR in one case only (11%). Transcriptomic analysis revealed that 11 genes (10 mRNAs and 1 lncRNA) were differential expressed when frontal cortex of COVID-19 patients were compared to controls. These genes fall into categories including hypoxia, hemoglobin-stabilizing protein, hydrogen peroxide processes. This work demonstrated that the quantity of viral RNA in frontal cortex is minimal and it can be detected only with a very sensitive method (ddPCR). Thus, it is likely that SARS-CoV-2 does not actively infect and replicate in the brain; its topography within encephalic structures remains uncertain. Moreover, COVID-19 may have a role on brain gene expression, since we observed an important downregulation of genes associated to hypoxia inducting factor system (HIF) that may inhibit the capacity of defense system during infection and oxigen deprivation, showing that hypoxia, well known multi organ condition associated to COVID-19, also marked the brain.

COVID-19, a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS- infection evolved in the last year into a global pandemic (Zou et al., 2020) , is primary characterized by respiratory symptoms. The most common symptoms are fever, cough, fatigue, shortness of breath, but some COVID-19 patients evolve to acute respiratory distress syndrome and/or multi organ failure Guan et al., 2020) . COVID-19 patients experience moderate to severe hypoxemia that can contribute to multiple organ dysfunction, especially affecting the kidney, heart and central nervous system (CNS). Indeed, neurological, and cardiovascular effects have been described (Helms et al., 2020; Wichmann et al., 2020) . COVID-19 is particularly detrimental in patients with multiple comorbidities (e. g. cardiovascular diseases, diabetes, obesity, pulmonary disorders, and cancer). The cytokine storm induced by SARS-CoV-2 may be uncontrolled and might cause a wide range of symptoms, including several neurological manifestations. Neurological phenomena, observed during and after the acute COVID-19 phase, have now been described in several studies reporting both CNS symptoms (psychomotor retardation, dizziness, confusion, delirium, ataxia, encephalitis, stroke, seizures) and vegetative/peripheral manifestations (vomiting, hypotension, severe asthenia, myalgia, neuralgia, headache, olfactory, and gustatory dysfunction, Guillain-Barré syndrome) (Ahmad et al., 2020; Baig et al., 2020; ; Mao et al., 2020; Zito et al., 2020; Zubair et al., 2020) .

Neurological manifestations caused by COVID-19 may be due to immune-mediated mechanisms or direct viral invasion of CNS. Particularly, SARS-CoV-2 may enter the CNS through different routes: 1) the trans-synaptic route, particularly intranasal to the olfactory bulbs and the basal frontal lobes; 2) the endothelial-astrocytic route by crossing the blood brain barrier or through transport by infected leukocytes (Trojan horse mechanism) (Baig al., 2020; Zubair et al., 2020; ) . Available autopsy data from brains of COVID-19 patients show unspecific findings of brain congestion, oedema, neuronal damage, and inflammatory infiltrates with features of encephalitis and meningitis. Such findings appear attributable more to hypoxic and immune-mediated phenomena rather than to virus-induced lesions; they do not clearly establish the presence of SARS-CoV-2 in the brain and its role in neuronal damage remains unclear ( Matschke et al., 2020; Solomon et al., 2020; Von Weyhern et al., 2020) . Viral protein detection is very scant in CNS and limited to isolate neurons in the medulla oblongata. On the other hand, few papers have been published about the presence of viral RNA in the human brain and no correlation has been found between severity of neurological clinical signs and the presence of viral protein or RNA in the human brain of COVID-19 patients (Frank, 2020) . Nonetheless, in a recent series, RNA was detected in about half of the cases (Matschke et al., 2020) , but the consequences of brain infection in terms of molecular alteration have yet to be revealed.

In this paper, we investigated the presence of SARS-CoV-2 in the brain of elderly patients died of COVID-19 and the possible effect of SARS-CoV-2 infection on transcriptomic profile in frontal cortex comparing COVID-19 patients and matched controls by RNA-seq analysis. Fronto-basal cortex was chosen from the assumption that SARS-CoV-2 may invade the brain through the olfactory pathway, as recently reported (Politi et al., 2020) .

Autoptic human brain samples were provided by Unit of Legal Medicine and Forensic Sciences, Institute of Legal Medicine of University of Pavia and the Abbiategrasso Brain Bank (ABB) of Golgi Cenci Foundation (Milan, Italy). The ABB autopsy and sampling protocol COVID-19 cases. All the bodies were stored at 4 degrees Celsius until the time of the autopsy. The preservation of the body was adequate in all 9 COVID-19 cases considered for the study and none were excluded. A reconstruction of the patients' clinical history was carried out through a retrospective evaluation of medical charts by a forensic medical doctor and a neurologist with expertise in neuropathology, who carried out also the neuropathological evaluation. COVID-19 cases (5 women and 4 men; median age 83 years), were clinically defined for the presence or absence of dementia, and for the presence or absence of comorbidities including cardiovascular disease, diabetes, obesity, pulmonary disorders and cancer (Table 1) . Frontal lobe samples from 8 non-COVID-19 cases provided by ABB were used for comparison. They were selected with age and clinical characteristics similar to those of the COVID-19 group. The control ABB cases differ from the COVID-19 cases for the post mortem-time which was much shorter (from 2 to 16 hours) ( Table 1 ). The PMD was necessarily different because the ABB protocol implies an autopsy performed no later than 30 hours after death . Instead, the COVID-19 autopsies followed the course of the forensic roles. After the brain removal, one slice of anterior frontal lobe was fresh cut into about 10 mm thickness and frozen at -80°C. An adjacent slice was fixed in formalin, included in paraffin and cut into 8 μm thick serial sections. Tissue samples for this study included cortical gray matter and subcortical white matter. The sections were stained with Hematoxylin and Eosin and Luxol Fast Blue (LFB) to evaluate vascular, architectural and structural tissue abnormalities, inflammatory infiltrates, and myelin loss. All the cases were studied for the presence of underlying AD pathology. For this purpose, reactions for TAU (AT8) and beta-amyloid (4G8) were performed on additional sections including frontal, temporal and parieto-occipital lobes, and hippocampi. The inflammatory infiltrates were characterized through antibodies against microglia (CD68) and lymphocytes (CD3, CD20). Anti-SARS-CoV-2 antibodies were used to detect viral inclusions. 

For the detection of SARS-CoV-2, the One-Step RT-ddPCR Advanced Kit for Probes (BioRad, Richmond, CA) was used. For each 20 μl reaction, 5 μl of Supermix, 2 μl of Reverse transcriptase, 1 μl of 300 mM DTT, 1 μl of Probe (1:40), 1 μl of both Forward and Reverse primers (1:10) and 4

μl of H2O were provided and 5 μl of starting RNA was added. The oligonucleotides and probes used in RT-ddPCR were the same used in RT-qPCR. For the droplet generation in RT-ddPCR, 20

μl of reaction volume was transferred to 8-channel disposable droplet generation cartridge and 70 μl of droplet generation oil for probe was added in adjacent oil wells. The cartridge was placed into a QX200 droplet generator (BioRad, Richmond, CA), which makes the partition of each sample into droplets. The droplets were carefully transferred to a semi-skirted 96-well PCR plate (BioRad, Richmond, CA), sealed using the PX1 PCR Plate Sealer (BioRad, Richmond, CA) for PCR in 2720

Thermal Cycler (Applied Biosystems, USA). After PCR of targets presented in the droplets, the QX200 droplet reader (BioRad, Richmond, CA) was used to analyze each droplet individually, which counts positive and negative droplets to establish absolute quantification of samples (concentration).

The QuantaSoft 1.6 software was used to view fluorescence data in 1D amplitude, concentration data, copy number data, events (number of positive, negative or total droplet counts). The multi-well threshold tool was used in all the wells according to results of specificity assay in negative samples to discriminate between positive and negative droplets. The software automatically reported the copy number of each sample. (GRCh37.p13) as a reference, using the "stranded" option. Differential expression analysis for mRNA was performed using R package EBSeq (Leng et al., 2013) . This tool was selected because of its superior performance in identifying isoforms differential expression (Carrara et al., 2015) .

Differential expression analysis for long non-coding RNAs (lncRNAs) was performed with the R package DESeq.2 (Love et al., 2014). Coding and non coding genes were considered differentially expressed and retained for further analysis with |log2(disease sample/healthy control)| ≥ 1 and a FDR ≤ 0.1. We imposed minimum |Log2FC| of 1 and a FDR lower than 0.1 as thresholds to differentially expressed genes. This choice is motivated by the decision to maximize the sensitivity of this analysis, in order to perform a massive screening and identify candidate genes to be validated with real-time analysis (Gagliardi et al., 2018; Zucca et al., 2019) .

RNA sequencing data is available in GEO repository (GSE164332).

Gene enrichment analysis was performed on coding genes (Subramanian et al., 2005) . We 

PCR oligonucleotide for genes pairs were selected spanning introns to optimize amplification from mRNA templates and avoiding nonspecific amplification products, using NCBI's Primer-BLAST or online Primer 3.0. Moreover, primers were designed in specific regions that do not overlap with Antisense sequences (primers upon request). Total cDNAs were prepared using iScript™ cDNA Synthesis Kit (BioRad, Richmond, CA). qPCR reactions were performed with SYBR Green SuperMix (BioRad, Richmond, CA), using 1 μL of cDNA template (or water control). Cycle threshold (Ct) values normalized against those determined for GAPDH. Fold-expression differences relative to healthy controls were determined using the 2ΔΔCt method. Significance of gene expression changes relative to controls was analysed using one-way ANOVA (Kruskal-Wallis) and

the Dunns post-test for all possible test pairings using Prism GraphPad 5.02 software (GraphPad Software, San Diego, CA).

In all COVID-19 cases, the cortical cytoarchitecture was deeply altered, due to severe hypoxia, agonal changes and postmortem artifacts. Hematoxylin-Eosin staining showed widespread edema with marked perineuronal space dilatation and neuronal loss in cortical gray matter, particularly in the supragranular layers ( Figure 1A ). No fresh vascular lesions were found but some COVID-19

cases had pre-existing small vessel disease (SVD). Moderate to severe SVD was present in cases 2, 4 and 8, with perivascular space dilatation, arteriolosclerosis ( Figure 1B) , and myelin loss ( Figure   1C ). Moderate to severe SVD was also present in non-COVID cases (cases 11, 14, 15 and 16).

Nobody showed a clear picture of vasculitis but COVID-19 cases frequently had perivascular infiltrates that represented the main inflammatory feature in subjects without dementia and with a low amount of viral RNA ( Figure 1D ). On the other hand, COVID-19 cases with dementia and higher load of viral RNA presented multifocal inflammatory infiltrates and nodules, mainly in cortical gray matter ( Figures 1E) . The nodules were composed almost entirely by CD68-positive microglial cells ( Figure 1F ), as activation of innate immunity. Lymphocytes were very rare and viral inclusions were detected in very few cells of the lower brainstem (data not shown). Definite AD diagnosis was made in COVID-19 cases 1, 4, 6, 7, 8 ( Figure 1G-K) , and in non-COVID cases 10, 11, 13, 14, and 15. Despite the difference in post-mortem delay between the COVID-19 cases and the ABB controls, they appear to be comparable on a histological level and there is adequate immunohistochemical antigenic detection also in COVID-19 brains. 

RNA extracted from frontal cortex of both COVID-19 patients and controls have been analyzed for SARS-CoV-2 nucleocapsid gene 1 (nCoV_N1) and human Ribonuclease P (hRP), as controls of RNA extraction. The analysis of viral RNA detection by qPCR was positive for viral RNA (nCoV_N1) only in one patient while in all samples the expression of hRP gene was present, demonstrating that the RNA was correctly extracted ( Supplementary table 1 (S1). SARS-CoV-2 detection in frontal cortex. 1A. Ct values obtain by qPCR for SARS-CoV-2 target sequences N1 and for human gene RNase P (RP). 1B. Quantification of viral RNA copy number by ddPCR indicating RNA copy concentration for µl and for reaction (20 µl), the number of accepted droplets as confirmed of well performed experiment and the number of droplets positive and negative to SARS-CoV-2 detection.

In COVID-19 patients, RNA-seq data reported 11 differentially expressed genes (DEGs), 10 were coding RNAs while 1 was a lncRNA. Concerning coding genes, 4 out of 10 have been found downregulated while 6 out of 10 up-regulated. Heat-map separately representing the expression levels of all dysregulated mRNAs and lncRNA in COVID-19 and control subjects is represented in Figure   2A . Here, different expression profiles in COVID-19 and controls can be visibly distinguished. We also performed a Principal Component Analysis (PCA) shown in Figure 2B , where COVID-19 patients' samples are clearly separated from controls cluster. The volcano plot shows the most significant DEGs, validated by qPCR, in COVID-19 patients that confirm the different degree of alteration in the population (Figure 3) . We considered the low number of DEGs in COVID-19 patients an interesting datum. The most deregulated genes are upregulated mRNA coding for hemoglobin subunits (HBA, and HBB) and down-regulated genes associated to hypoxia and infalmmation (SLC14A1, HIF3A and RGS5). This data also correlate with neuropathological findings that showed inflammatory infiltrates in COVID-19 patients' tissue. 

BioPlanet2019 pathway, GO and KEGG enrichment analyses for DEGs were performed to identify the most important pathways and molecular features associated to DEGs.

The most relevant data concern pathway analysis that showed that DEGs are associated to SARS coronavirus protease and Alpha hemoglobin stabilizing protein (AHSP) pathway (Figure 4) . The GO biological processes enriched terms for response to oxidative stress, oxygen and gas transport and hydrogen peroxide catabolic and metabolic process, underline the significant involvement of respiratory aspect in COVID-19 phenotype ( Figure 5 ). 

SARS-CoV-2 infection can cause severe pulmonary disease and complications with possible multiorgan consequences involving CNS (Renu et al., 2020) . The most frequent neurological manifestation of COVID-19 in patients with dementia is a non-specific encephalopathy with behavioral changes, such as psychomotor retardation and delirium, typical of several infectiveinflammatory diseases . These clinical manifestations may be due to nonspecific pathological phenomena that can be attributed to inflammatory state, hypoxia, and sepsis, possibly superimposed on pre-existing neurodegenerative or vascular pathologies. At gross neuropathological examination, we could not find fresh macroscopic vascular lesions. All COVID-19 brains show congestion, oedema, and neuronal damage. These alterations are not SARS-CoV-2specific but related to hypoxic-agonic and postmortem phenomena. Also, other pathological findings such as AD pathology and SVD are clearly pre-existent conditions and cannot be considered virus-specific,. On the other hand, we observed perivascular inflammatory infiltrates and nodules that are, probably, attributable to SARS-CoV-2. The inflammatory infiltrates consist essentially of cells of the innate immunity (monocyte-macrophage-microglia series) with very rare lymphocytes. Although the small number of cases does not allow us to draw firm conclusions, it is interesting to note that the inflammatory load appears greater in cases with dementia and with the greatest amount of viral RNA.

For a deeper understanding of the pathogenic processes induced by SARS-CoV-2 in the CNS, the neuropathological data should be supplemented with biochemical data. Therefore, detecting the presence of the virus in CNS, and the biochemical alterations induced by the virus, is crucial for the interpretation of the brain pathophysiology of COVID-19. Viral SARS-CoV-2 infection causes alterations in transcriptome of affected cells and tissues, as lung and peripheral blood mononuclear cells (PBMCs) (Islam et al., 2020; Yang et al., 2020; Xiong et al., 2020) . Starting from the evidence that transcriptomic profile may be altered by SARS-CoV-2 infection and considering previous data (Asadi-Pooya et al., 2020; Baig et al., 2020; Koralnik et al., 2020) about a CNS involvement, we performed RNA-sequencing of frontal basal cortex to characterize the gene expression profile of COVID-19 patients. First, we checked for the presence of viral RNA demonstrating that SARS-CoV-2 RNA was detectable by qRT-PCR only in one sample, while using a more sensitive method, ddPCR, it was appreciable in almost all COVID-19 samples (88%). Interestingly, literature reports that in COVID-19 brain tissue (not specifically in frontal cortex) viral RNA is detectable in about 40-50% of samples or fewer (Matschke et al., 2020; Solomon et al., 2020) . In this work, we showed that detection of virus in brain tissue was increasable to 90% through ddPCR (Suo et al., 2020) .

Indeed, it should be considered that the time elapsed between the acute infection and the death is relevant for virus clearance and, probably, affects the presence of SARS-CoV-2 inside the brain.

Thus, it is necessary to use an extremely sensitive method. In any case, the low amount of viral RNA detected does not allow to distinguish whether the virus is located in brain parenchyma or in brain blood vessels. However, these data, together with the absence of a specific neuropathologic picture of encephalitis, testify to the absence of viral replicative activity within the brain.

About transcriptome profiling results, we found that 11 genes (10 mRNAs and 1 lncRNA) were differential expressed when frontal cortex of COVID-19 patients was compared to controls. These genes fall into categories including hypoxia, hemoglobin stabilizing protein, hydrogen peroxide processes, which all play important roles in SARS-CoV-2 infection (Cavezzi et al., 2020; Jahani et al., 2020; Thomas et al., 2020) . Interesting genes HIF3A, SLC14A1 and RGS5 have been found down-regulated. HIF3A belongs to hypoxia-inducible factor family genes; they are transcription factors that respond to decrease in cellular environment oxygen or hypoxia (Smith et al., 2008; Wilkins et al., 2016) . Inhibition of HIF-1 and dysregulation in Akt/mTOR/HIF-1 has been found in SARS-CoV-2 infected cells (Appelberg et al., 2020) and it has been shown that inhibition of HIF-1 can promote replication of influenza A virus and severe inflammation mediated via promotion of autophagy . Also increasing HIF-1α level promotes the defense capacity of macrophages in cases of infection (Imtiyaz et al., 2010) . Our data showed a reduction of HIF suggesting a possible inhibition of defense capacity . In fact, HIF pathway usually plays a protective role after injury and promotes cell recovery that in COVID-19 patients is not possible due to HIF down-regulation (Corrado et al., 2020) . Previous work reported that the HIF response is variable depending upon the type of hypoxic stress (Mandic et al., 2018) . Particularly, HIF was induced by chronic or intermittent hypoxia (Powel et al., 2008) . Our COVID-19 cases had a severe acute hypoxia due pulmonary failure, which was not corrected through mechanical ventilation. As highlighted by the neuropathological picture, this peculiar condition caused an extreme hypoxic suffering of the brain, which, probably, induced a down-regulation of the HIF response, thus creating a vicious circle that might be important in COVID-19 pathogenesis.

Moreover, SLC2A1 has been described as part of HIF-1 signaling (Appelberg et al., 2020; Dengler et al., 2014) , and also RGS5, involved in endothelial apoptosis, is regulated by HIF (Jin et al., 2009 ).

About up-regulated genes, the most relevant data concerned hemoglobin subunits (HBB, A1 and A2) increase. Hb up-regulation may be linked to downregulation of mTOR activity, suggesting that the overexpression of Hb may act as response to mTOR downregulation (Codrich et al., 2017) .

Moreover, Hb overexpression may be a protective system against oxidative stress. In fact, it has been demonstrated that Hb overexpression may be neuroprotective (Amri et al., 2017) .

About lncRNA CTB-36O1.7, an uncharacterized gene, it has been found associated to Multiple Sclerosis in brain (Chiricosta et al., 2020) . Although its role is still unknown, this finding may confirm a role of lncRNA CTB-36O1.7 in inflammation and in the regulation of microglia. The neuropathological picture observed in our COVID-19 cases is characterized by microglial activation, in the absence of clear signs of encephalitis due to viral replication. Thus, our data corroborates the involvement of lncRNA CTB-36O1.7 in the activation of microglia and innate immunity rather than in adaptive immunity linked to an active infection.

In conclusion, our data confirm the presence of viral RNA at very low amount in frontal cortex tissues. As a matter of fact, COVID-19 detection is variable; therefore a sensitive method is required to reveal the viral RNA.

This work demonstrates that the quantity of viral RNA is minimal, thus, it is likely that SARS-CoV-2 does not actively infect and replicate in the brain. Also, this work showed that hypoxia, a wellknown condition associated to COVID-19 infection, is a multi-organ feature (lung, kidney, and hearth) that marks even the CNS, and specifically brain cortex. Our data showed an important deregulation of hypoxia inducting factor system that inhibits defense system from infection. Our data also suggested an activation of protective mechanism by Hb in response to oxidative stress and deficit in mTOR system. Furthermore, as demonstrated by the neuropathological picture and the upregulation of lncRNA CTB-36O1.7, the activation of microglia seems to have an important role in the pathogenesis of the neurological manifestations of COVID-19. Therefore, the topography and intensity of microglial activation should be investigated further, and comparison studies with non-SARS-CoV-2 cases should be conducted. Supplementary table 1 (S1). SARS-CoV-2 detection in frontal cortex. 1A. Ct values obtain by qPCR for SARS-CoV-2 target sequences N1 and for human gene RNase P (RP). 1B. Quantification of viral RNA copy number by ddPCR indicating RNA copy concentration for µl and for reaction (20 µl), the number of accepted droplets as confirmed of well performed experiment and the number of droplets positive and negative to SARS-CoV-2 detection. 

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We thank the Abbiategrasso Brain Bank (ABB) donors and the COVID-19 patients who donated the noblest organ of their body.We also would like to thank Dr. Antonio Traversi and Prysmian Group for the support.

Project was funded by the Italian Ministry of Health (Ricerca Corrente 2020)