Oral Microbiota in Children Who Started Antiretroviral Therapy at Young Ages

Background: Infancy is an important developmental period when the human microbiome is shaped. Given links between young age at antiretroviral treatment (ART) initiation and smaller persisting viral reservoirs, we hypothesized that earlier ART initiation may leave distinct microbial signatures in the oral cavity detectable in children living with HIV (CLWH). Method(s): Oral swab samples were collected from 477 CLWH and 123 children without HIV at two sites in Johannesburg, South Africa. CLWH had started ART < 2 years of age with 60% starting < 6 months of age. Most were wellcontrolled on ART at a median of 10 years of age when the swab was collected. Controls were age-matched and recruited from the same communities. Sequencing of the V4 amplicon of the 16S rRNA gene was done using established protocols. DADA2, decontam, and phyloseq were used for sequence inference, contaminant removal, and subsequent analyses. All p-values were adjusted for multiple testing using Benjamini-Hochberg false discovery rate method. Statistical analyses were performed with R. Result(s): CLWH had lower alpha diversity than uninfected children (Shannon index p< 0.0001). Genus-level abundances of Granulicatella, Streptococcus and Gemella were greater and Neisseria and Haemophilus were less abundant among CLWH compared to uninfected children. Associations were strongest among boys. There was no evidence of attenuation of associations with earlier ART initiation. In fact, decreased bacterial diversity and differences in taxa abundances in CLWH versus controls were consistent regardless of whether ART was started before or after 6 months of age. Shifts in genus-level taxa abundances relative to uninfected controls were most marked in children on regimens containing lopinavir/ritonavir;with few shifts seen if on regimens containing efavirenz. Conclusion(s): A distinct profile of less diverse oral bacterial taxa was observed in school-age CLWH on ART versus uninfected age-matched children suggesting persisting interference of HIV and its treatments on microbiota in the mouth. Any effects of earlier ART initiation were not detectable at this age. Studies of treated adults with HIV have observed similar shifts in taxa abundances. Oral microbiota have been linked to salivary cytokine levels with associations between Granulicatella and IL-8 and Neisseria and IL-6. Declines in Neisseria abundances in oral samples have been associated with more severe outcomes in influenza and COVID-19.

BASELINE AND TEMPORAL DIFFERENCES IN PROTEOMICS FOLLOWING SARS-CoV-2 INFECTION IN PWH

Background: COVID-19 may be more severe in persons with HIV (PWH). However, underlying biological mechanisms associated with the development of COVID-19 and its clinical severity among antiretroviral therapy (ART) treated PWH are largely unknown. Therefore, we wished to evaluate temporal changes in plasma proteins following SARS-CoV-2 infection and identify pre-infection proteomic markers associated with future COVID-19. Method(s): We analyzed the data of clinical, antibody-confirmed COVID-19 ARTtreated PWH from the global Randomized Trial to Prevent Vascular Events in HIV (REPRIEVE). Individuals were matched on geographic region, age, and sample timing to antibody-negative controls. For cases and controls, pre-COVID-19 pandemic specimens were obtained prior to January 2020 to assess temporal changes and baseline differences in protein expression in relationship to COVID-19 severity, using mixed effects models adjusted for false-discovery rate. Result(s): We compared 257 unique plasma proteins (Olink Proteomics) in 94 COVID-19 antibody-confirmed clinical cases and 113 matched antibody-negative controls, excluding COVID-19 vaccinated participants (median age 50 years, 73% male). 40% of cases were characterized as mild;60% moderate to severe. Median time from COVID-19 infection to follow-up sampling was 4 months. Temporal changes in protein expression differed based on COVID-19 disease severity. Among moderate to severe cases vs. controls, NOS3 increased, whereas ANG, CASP-8, CD5, GZMH, GZMB, ITGB2, and KLRD1 decreased. Higher baseline circulating concentrations of granzymes A, B and H (GZMA, GZMB and GZMH) were associated with the future development of moderate-severe COVID-19 in PWH and were related to immune function, including CD4, CD8 and the CD4/ CD8 ratio. Conclusion(s): We identified temporal changes in novel proteins in closely linked inflammatory, immune, and fibrotic pathways which may relate to COVID-19-related morbidity among ART-treated PWH. Further, we identified key granzyme proteins, serine proteases expressed by cytotoxic T lymphocytes and NK cells in response to foreign antigens, associated with future COVID-19 in PWH. Our results provide unique insights into the biological susceptibility and responses to COVID-19 infection in PWH. (Figure Presented).

RNA-sequencing unravels unique host-response to infection with SARS-CoV-2

Introduction/Aim: As the causative agent of COVID-19, SARS-CoV-2 remains a global cause for concern. Compared to other highly pathogenic coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 exhibits stronger transmissibility but less lethality, indicating that SARS-CoV-2 displays unique characteristics, despite the partial genomic proximity. Thus, we aim to employ RNA sequencing to define transcriptional differences in epithelial responses following infection with SARS-CoV-2 compared to pathogenic SARS-CoV and MERS-CoV, and low pathogenic HCoV-229E. Method(s): Primary human bronchial epithelial cells (PBEC) were differentiated for 6 weeks at the air-liquid interface (ALI) before parallel infection by the 4 different coronaviruses (n = 4). After infection following apical application of coronaviruses at low dose (MOI 0.1), cells were harvested for bulk RNA sequencing. Gene were considered significant with a fold change (FC) > 2 and false discovery rate of FDR < 0.05. Inhibitor experiments were conducted on CALU-3 cells using DIM-C-pPhOH 10 muM (NR4A1 antagonist), Sp600125 10 muM (JNK inhibitor), T-5224 10 muM (AP-1 transcription factor inhibitor) and Cytosporone B (CsB 5 muM;NR4A1 agonist) preincubated for 1 h with these compounds and subsequently infected with SARS-CoV-2 or MERS-CoV (MOI of 1). Samples were collect 24 h later for PCR. Result(s): PCR and RNA-Seq demonstrated that all tested coronaviruses efficiently infected ALI-PBEC and replicated over 72 h (p < 0.05). RNA sequencing analysis revealed that infection with SARS-CoV, MERS-CoV and HCoV-229E resulted in largely similar transcriptional responses by the epithelial cells. However, whereas infection with these viruses was accompanied by an increased expression of genes associated with JNK/AP-1 signalling, including FOS, FOSB and NR4A1 (FC > 1, FDR < 0.05), no such increase was observed following SARS-CoV-2 infection. Further, we found that an NR4A1 antagonist reduced viral replication of MERS and SARs-CoV-2 100-fold in Calu-3 cells. Conclusion(s): In conclusion, these data suggest that SARS-CoV-2-infected ALI-PBEC exhibit a unique transcriptional response compared to other coronaviruses, which might relate to the pathogenicity of the virus.

Persistence of inflammatory and vascular damage mediators 5 months after acute COVID-19

Background: In the acute phase, patients with severe COVID-19 exhibit pulmonary inflammation and vascular injury, as well as an exaggerated cytokine response. Aim(s): To describe the inflammatory cytokine and vascular injury mediator profiles in patients previously hospitalised with COVID-19, and to compare these profiles with those in healthy controls and in patients recovering from severe sepsis of other aetiology. Method(s): Plasma levels of 28 different cytokine, chemokine, angiogenic and vascular injury markers were measured by MSD V-PLEX multiplex assays in 49 post-COVID patients 5.0+/-1.9 (mean+/-SD) months after hospitalisation with COVID-19 pneumonia, 11 post-sepsis patients (5.4+/-2.9 months after hospitalised non-COVID sepsis) and 18 healthy controls. Kruskal-Wallis or ANOVA were used to compare groups;false discovery rate correction (Benjamini Hochberg) allowed for multiple testing. Result(s): In the post-COVID group, IL-6, TNFalpha, SAA, MCP1, Tie-2, Flt1, PIGF and CRP were significantly elevated, whereas IL-7 and bFGF were significantly depressed. The differences in TNFalpha, SAA, MCP1, Tie-2, Flt1, IL-7 and bFGF appeared unique to the post-COVID group, but increased IL-6, PIGF and CRP levels were also seen in postsepsis patients compared with controls. In post-COVID patients we found strong negative spearman correlations between each of IL-6 (r=-0.51) and CRP (r=-0.57) with TLCO %predicted (p<0.001) and positive correlations with post-recovery CT abnormality scores: IL-6 (r=0.28) and CRP (r=0.46), p<0.05. Conclusion(s): A unique signature of inflammatory and vascular damage markers is seen months after acute COVID19 infection. Further research is needed to determine their pathophysiological significance.

Proteomic profiling of severe lung diffusion impairment in the recovery stage of COVID-19-induced ARDS

Background: Around 80% of patients who developed COVID-19-driven ARDS present lung ailment. There is a lack of knowledge of the mechanisms that mediate the pulmonary outcomes. Aim(s): To characterize the factors linked to diffusion impairment in survivors of severe COVID-19. Method(s): Prospective cohort study including 87 COVID-19-induced ARDS survivors. A complete pulmonary evaluation was performed 3 months after hospital discharge. 364 proteins were quantified using the proximity extension assay (PEA). Partial least square-discriminant analysis (PLS-DA) and random forest (RF) were used for multivariable analyses. Result(s): Moderate to severe diffusion impairment (DLCO<60% predicted) was observed in the 30% of the cohort. 15 proteins were differentially detected [false discovery rate (FDR)<0.05] in the univariate analysis. Pleiotrophin showed the highest differences (fold change=2.22 and FDR=0.001). In continuous analysis, proteins were inversely and independently associated with DLCO, and in some cases showed a robust dose-response relationship. PLS-DA and RF identified proteomic profiles related to the severity of diffusion capacity. Clusters identified were enriched in mediators of cell proliferation and differentiation, tissue remodeling, angiogenesis, coagulation, inflammation, immune response and fibrosis. Proteins are expressed in immune and non-immune lung cells. Conclusion(s): In survivors of COVID-19-driven ARDS, lung dysfunction is linked to plasma factors involved in injury and repair mechanisms. The host proteomic profile provides a novel understanding of post-acute sequelae and may be source of therapeutic strategies and biomarkers.

Exploring Platelet Proteins Altered in Severe COVID-19 Using Gene Ontology (GO) Pathways

Background: SARS-CoV- 2 infection causing COVID-19 is associated with a high incidence of thrombotic complications, a phenomenon in which platelets play an important contributory role. COVID-19 is associated with alterations in platelet function, including increased platelet-neutrophil aggregates and impaired integrin alphaIIbbeta3 activation. The mechanisms underlying such effects remain unclear. Aim(s): In this study, we aimed to identify changes in the platelet proteome in patients with COVID-19 and explore implicated biological pathways. Method(s): Patients hospitalized with COVID-19 (N = 7) and healthy controls (N = 6) were recruited between October 2020 and February 2021. Proteomics analysis was performed on washed platelets using Tandem Mass Tag Mass Spectrometry (TMT-MS). Alterations in platelet proteins were analyzed using Gene Ontology (GO) pathways. Result(s): A total of 5773 proteins were quantified in COVID-19 patients and controls. There was differential expression of 858 (15%) proteins between patients with severe COVID-19 infection and controls (false discovery rate p < 0.05). Pathway analysis revealed expression changes in gene products associated with regulation of platelet activation (GO:0010543), with reduced expression of platelet kinases (PRKCA, PRKCD, PRKCQ, LYN, SYK and JAK2), glycoproteins (GP5 and GP9) and PLEK, known positive regulators of integrin activation. There was increased expression of alpha-and dense-granule lumen gene products (GO:0031093 and GO:0031089), including protease inhibitors SERPINA3, SERPING1 and SERPINF2 Conclusion(s): Our results demonstrate diverse changes in signaling and secretion pathways important to platelet activity in patients hospitalized with COVID-19. Increased abundance of platelet granule proteins is in keeping with published plasma proteome descriptions, suggesting platelet uptake of plasma proteins. Platelet granule proteins involved in the regulation of hemostasis may contribute to the increased risk of thrombosis seen in this patient cohort and point towards potential therapeutic targets for COVID-19 related thrombotic complications.

Higher Proinflammatory Cytokines Are Associated with Increased Antibody Titer After a Third Dose of SARS-CoV-2 Vaccine in Solid Organ Transplant Recipients

Purpose: Solid organ transplant recipients (SOTRs) are at increased risk for severe COVID-19 and exhibit lower antibody responses to SARS-CoV-2 vaccines. This study aimed to determine if pre-vaccination cytokine levels are associated with antibody response to SARS-CoV-2 vaccination. Method(s): A cross-sectional study was performed among 58 SOTRs before and after two-dose mRNA vaccine series, 35 additional SOTRs before and after a third vaccine dose, with comparison to 16 healthy controls (HCs). Anti-spike antibody was assessed using the IgG Euroimmun ELISA. Electrochemiluminescence detectionbased multiplexed sandwich immunoassays were used to quantify plasma cytokine and chemokine concentrations (n=20 analytes). Concentrations between SOTRs and HCs, stratified by ultimate antibody response to the vaccine, were compared using Wilcoxon-rank-sum test with false discovery rates (FDR) computed to correct for multiple comparisons. Result(s): In the study population, 100% of HCs, 59% of SOTRs after two doses and 63% of SOTRs after three doses had a detectable antibody response. Multiple baseline cytokines were elevated in SOTRs versus HCs. There was no significant difference in cytokine levels between SOTRs with high vs low-titer antibodies after two doses of vaccine. However, as compared to poor antibody responders, SOTRs who went on to develop a high-titer antibody response to a third dose of vaccine had significantly higher pre-third dose levels of several innate immune cytokines including IL-17, IL-2Ra, IL-6, IP-10, MIP-1alpha, and TNF-alpha (FDR <0.05). Conclusion(s): A specific inflammatory profile or immune state may identify which SOTRs are likely to develop stronger sero-response and possible protection after a third dose of SARS-CoV-2 vaccine.

COVID-19 IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN TREATMENT-FREE REMISSION: DISEASE SEVERITY AND IMPACT ON TFR STATUS

Background: Severe SARS-CoV-2 infections associated with high mortality rates are reported in a higher percentage of patients (pts) with hematologic malignancies compared to general population. In chronic myeloid leukemia (CML), pts with uncontrolled disease have a higher mortality risk. The impact of SARS-CoV-2 infection on CML pts in treatment-free remission (TFR) has not been studied so far. In particular, as immune control of residual disease may be important for TFR, the concern is that the infection could induce loss of TFR. Aims: To evaluate the outcome of SARS-CoV-2 infection in CML pts in TFR and assess any impact on maintenance of TFR. Methods: From March 2020 to December 2021, the CANDID study organized by the international CML Foundation has collected data on COVID-19 positive CML pts worldwide. Details on the registry were presented recently (Pagano ASH 2021). For this sub-analysis on pts in TFR additional information were collected including;molecular remission status (BCR::ABL1 ratios) before, during and after SARS-CoV-2 infection covering at least 6 months. For molecular analyses, BCR::ABL1 ratios were classified according to Cross et al (Leukemia 2015). In addition, ratios of 0% without indication of sensitivity were allocated as MR4 i.e. 0.01%IS. PCR outlier results were identified using the ROUT method by nonlinear regression with a maximum false discovery rate (FDR) of 1% (Motulsky et al 2006). Time to molecular relapse (MR) was measured from the date of COVID-19 diagnosis to the date of MR defined as loss of major molecular remission (MMR, BCR::ABL1 >0.1%IS) or the date of last molecular test. Molecular relapse-free survival (MRFS) and overall survival (OS) were estimated with the Kaplan-Meier method. The statistical difference between groups was performed using log-rank test. Results: By December 2021, 1050 COVID-19 positive CML pts were registered. 95 pts were in TFR at the time point of SARS-CoV-2 infection of which 89 (93.68%) recovered and 6 deceased (6.32%). Median age of TFR pts was 57 years, male were 51 (53.68%). Median time from CML diagnosis to reporting date was 13 years (range 3.7-27.0 years). TFR duration was 2.83 years in median (range 0.5 months - 10.1 years) including 19 pts with a duration < 1 year. From the 89 recovered TFR pts, 74 pts completed the 6-month follow up (83%), a further 6 pts with molecular follow-up of 3-5 months after COVID-19 diagnosis were still in TFR, 9 pts were lost to follow-up. Of 74 pts with complete reports, 69 pts remained in TFR (93%) and 5 pts lost TFR. For 71 pts, PCR results were obtained before, during and after infection. With the ROUT method 10 pts demonstrated outlier PCR tests, 61 pts demonstrated stable PCR results. There was no statistically significant difference in PCR results before and during/after infection (p>0.2). MRFS for these 71 pts 15 months after COVID-19 diagnosis was 86%. Probability of TFR loss was higher in pts with a TFR duration < 6 months compared to pts with TFR duration >6 months (27% vs 10%, Fig 1A). Additionally, there were no statistically differences in hospitalization rate (16% vs 23%, p=0.12) and severity of COVID-19 symptoms (12.6% vs 12%, p=0.87) comparing TFR and TKI treated pts. OS of COVID-19 positive TFR pts did not differ from COVID-19 positive pts on TKI therapy (HR 1.1, CI 0.47-2.54) (Fig 1B). Summary/Conclusion: In this sub-analysis of the CANDID study, CML pts in TFR had similar severity and survival to CML pts who were on TKI therapy and there was no evidence of an increased risk of TFR loss after SARS-CoV-2 infection.

Monocytosis in the acute phase of SARS-CoV-2 infection predicts the presence of anosognosia for cognitive deficits in the chronic phase.

Reduced awareness of neuropsychological disorders (i.e., anosognosia) is a striking symptom of post-COVID-19 condition. Some leukocyte markers in the acute phase may predict the presence of anosognosia in the chronic phase, but they have not yet been identified. This study aimed to determine whether patients with anosognosia for their memory deficits in the chronic phase presented specific leukocyte distribution in the acute phase, and if so, whether these leukocyte levels might be predictive of anosognosia. First, we compared the acute immunological data (i.e., white blood cell differentiation count) of 20 patients who displayed anosognosia 6-9 months after being infected with SARS-CoV-2 (230.25 ± 46.65 days) versus 41 patients infected with SARS-Cov-2 who did not develop anosognosia. Second, we performed an ROC analysis to evaluate the predictive value of the leukocyte markers that emerged from this comparison. Blood circulating monocytes (%) in the acute phase of SARS-CoV-2 infection were associated with long-term post-COVID-19 anosognosia. A monocyte percentage of 7.35% of the total number of leukocytes at admission seemed to predict the presence of chronic anosognosia 6-9 months after infection.

THE COVID-19 PANDEMIC AND MENTAL HEALTH: EFFECTS OF SOCIAL SUPPORT AND SELF-REGULATION

Social support has been widely associated with various morbidity and mortality. How does social support availability help youths cope with the global pandemic of Covid-19 and maintain their mental well-being? Utilizing data from a UK national birth cohort, namely the Millennium Cohort Study, this research investigated the joint role of social support availability and self-regulation against such major life stressors, inclusding G × E mechanisms. Results from 4095 cohort members (399 males (47.20%) and 2602 females (63.54%), others refused to report) suggested that social support availability at the outburst of Covid-19 pandemic, as well as age 7 emotional self-regulation (rated by cohort members' parents) contributes to youths' better mental health (viz., mental well-being and non-specific psychological distress) shortly after the outburst of Covid-19 (;B;s > 0.072, ps < .018). Most importantly, age 7 cognitive self-regulation and social support availability jointly predicted better their well-being 4 months later after the local outbursts of pandemic (for mental well-being, B = 0.309, p = .017, 95% CI = [0.056, 0.562];and for non-specific psychological distress, B =-0.299, p = .043, 95% CI = [-0.587,-0.011]). Johnson-Neyman plots (false discovery rate limited) suggested that it was those high but not low in cognitive self-regulation that benefited more from the perceived social support availability. Within the ranges of significance (81.19% for mental well-being and 80.94% for non-specific psychological well-being), social support availability positively predicted mental health and such effect increased gradually as the increase of age 7 cognitive self-regulation. Findings filled in the research gap such that social support and self-regulation have been investigated separately as two coping mechanisms, by revealing that self-regulation (i.e., internal resources) determines the utility of social support availability (i.e., external resources). Findings here inspired new research questions for the field, such as whether relevant developmental trajectories of self-regulation and social support might intertwin to cast on health trajectories, and whether these processes are subject to potential G × E interactions such as the exposure to childhood adversity or relevant genetic risks. Models to examine these hypotheses will be discussed.

Microarray-Based Transcriptome Analysis of Peripheral Blood Mononuclear Cells in Lung Cancer Patients

: Lung cancer is the most commonly occurring cancer in men worldwide. To search for new biological markers of this pathology, the transcriptome of the blood mononuclear cells from patients and healthy donors (residents of Kemerovo oblast, Russia) was studied using SurePrint G3 Human Gene Expression microarray technology. A total of 288 differentially expressed genes were identified, including 108 up-regulated genes and 180 down-regulated genes. Functional enrichment analysis using the WebGestalt resource and different databases (Gene Ontology, KEGG, and Reactome) indicated changes in the expression profiles of genes involved in the processes of immune response, protein synthesis, cell cycle control, and apoptosis. Analysis of protein–protein interactions using the STRING algorithm made it possible to identify functional clusters of gene products with different expression levels.

Alterations in the Covid-19 - Associated Proteome Relative to Length of Hospital Stay

RATIONALE: The proteomic responses of hospitalized patients with SARS Co-V-2 infection may provide insight into risk, time course, and mechanisms associated with this infection. We used a high throughput proteomic platform to examine proteins that were differentially expressed relative to the length of hospital stay (LOS). METHOD:26 patients, hospitalized with SARS CoV-2 infection (mean age 48 yrs, 44% women) had blood samples obtained within 72 hours of admission. Initial plasma samples were analyzed from patients who were hospitalized for < 3 days (n=6), < 7days (n=12) and > 7 days (n=8) of LOS and compared to healthy controls (HC, n=8). Samples were analyzed with the modified aptamer-based array (SomaScan) that measures more than 7,000 human proteins representing different molecular pathways and gene families. Differentially regulated proteins with > 1.5 fold change and a false discovery rate of 5% were analyzed using the Ingenuity Pathway Analysis (IPA). Unique protein categories associated with LOS were assessed. RESULT: Compared to HC, differentially expressed proteins were detected among the 3 groups: 461 at < 3 days, 1,635 proteins at < 7 days and 1,738 proteins in >7 days. 407 proteins were common among all hospitalized COVID 19 individuals independent of LOS and 12, 250 and 361 proteins were uniquely present at < 3 days, < 7 days and > 7 days respectively compared to HC. The table below demonstrates the top highly enriched canonical pathway, molecular function and upstream regulator of differentially expressed proteins. The temporal sequence of these protein networks varied with LOS. Representative examples include early responses;platelet membrane glycoprotein GP6 signaling pathway that involves the FcR gamma-chain and the Src kinases linked to platelet aggregation, signaling involved in T cell receptor-mediated IL-2 production (TEC kinase). Less than 7 days include diacylglycerol associated with T cell activation, carnitine palmitoyltransferase associated with mitochondrial beta-oxidation of long chain fatty acids. CXCR4 a receptor for stromal -cell derived factor 1 and associated with COVID-19 prognosis. Late responses after 7 days include pathways involved in remodeling of epithelial adherens junctions. CONCLUSIONS : A high throughput proteomic approach provides insight into the dynamic regulation of protein pathways associated with the progression of SARS-Co-V2 infection. This may provide additional insight into risk and mechanisms associated with outcomes in COVID. (Table Presented).

Neurological impact of COVID-19: Sub-acute brain microstructural alterations in self-isolated individuals

Objective: This study investigates the effects of COVID-19 on brain microstructure among those recently recovering from COVID-19 through self isolation. Background: Microstructural differences have previously been detected in comparisons of COVID-19 patients with controls, particularly in regions related to the olfactory system. The olfactory system is connected with the caudate, putamen, thalamus, precuneus, and cingulate regions. Design/Methods: Here we report diffusion magnetic resonance imaging (3 T Siemens MRI) findings from 40 patients (mean age: 43.7, 68% female) who self-isolated after testing positive for COVID (COV+), and 14 COVID negative (COV-) subjects (mean age: 43, 64% female) who had flu-like symptoms. This is part of the Canadian-based NeuroCOVID-19 study. Fractional anisotropy (FA), mean diffusivity (MD), mode of anisotropy (MO), free water fraction (F), tissue-specific FA (FAt) and tissue-specific MD (MDt) were obtained using data with b=700 and 1400 (DIPY free-water model). Regions of interest in the grey matter and white matter were delineated using FreeSurfer. Differences between groups were assessed using an analysis of variance (ANOVA), the Kruskal-Wallis Test and the Mann-Whitney Test, corrected for false-discovery rate of 0.05. Effect size (Cohen's d) was also computed (d>0.45 deemed large effect). Results: In the COV+ group, all three tests revealed decreased FA and FAt in the insula, and increased MD in the parstriangularis cortex. Increased FA and FAt in the cuneus (along with decreased MD) was also found. MD was reduced in COV+ in the temporal and supramarginal areas. MO was lower in COV+ in the insula and amygdala regions. Conclusions: In patients, higher MD with lower FA and MO suggest increased extracellular fluids, while lower MD with decreased FA and MO may suggest necrotic debris built up following inflammation. The cuneus and insula are involved in visual and taste processing, respectively. This study highlights the need to study neurological effects of COVID-19.

“Long-hauler ” impact of COVID-19 on brain microstructure: a 3-month follow-up study

Objective: This study investigates the chronic effects of COVID-19 on brain microstructure. Background: Microstructural differences have previously been detected in comparisons of COVID-19 patients with controls, particularly in the insula, cuneus, inferior temporal and anterior cingulate regions. Design/Methods: Here we report diffusion magnetic resonance imaging (3 T Siemens MRI) findings from 20 participants (mean age: 45.3, 55% female), both immediately after recovery and at a 3-month follow-up. Fractional anisotropy (FA), mean diffusivity (MD), mode of diffusivity (MO), free water fraction (F), tissue-specific FA (FAt) and tissue-specific MD (MDt) were obtained using DTI data with b=700 and 1400 (DIPY free-water model). Regions of interest in the grey matter and white matter were delineated using FreeSurfer. To assess differences between baseline and follow-up, a paired t-test, the Wilcoxon Test and Friedman Test were performed, corrected for false-discovery rate of 0.05. Effect size (Cohen's d) was also computed (d>0.45 deemed large effect). Results: All three tests revealed decreased F in the hippocampus and decreased MD in the parahippocampal region of the WM at follow-up. In the GM, F was increased in the medial orbitofrontal region. In the WM, MD was increased in the paracentral region and MDt was increased in the parahippocampal and lateral orbitofrontal regions. Conclusions: These results suggest that microstructural abnormalities persist following recovery. Increased extracellular fluid (i.e. F and MD) in the frontal lobe suggest spreading of COVID-19 impact, while decreased F and MD in the hippocampal region suggest debris accumulation as part of the inflammatory process. None of the regions affected in sub-acute COVID-19 appear to fully recover within three months.

Null-free False Discovery Rate Control Using Decoy Permutations.

The traditional approaches to false discovery rate (FDR) control in multiple hypothesis testing are usually based on the null distribution of a test statistic. However, all types of null distributions, including the theoretical, permutation-based and empirical ones, have some inherent drawbacks. For example, the theoretical null might fail because of improper assumptions on the sample distribution. Here, we propose a null distribution-free approach to FDR control for multiple hypothesis testing in the case-control study. This approach, named target-decoy procedure, simply builds on the ordering of tests by some statistic or score, the null distribution of which is not required to be known. Competitive decoy tests are constructed from permutations of original samples and are used to estimate the false target discoveries. We prove that this approach controls the FDR when the score function is symmetric and the scores are independent between different tests. Simulation demonstrates that it is more stable and powerful than two popular traditional approaches, even in the existence of dependency. Evaluation is also made on two real datasets, including an arabidopsis genomics dataset and a COVID-19 proteomics dataset.

A 'Multiomic' Approach of Saliva Metabolomics, Microbiota, and Serum Biomarkers to Assess the Need of Hospitalization in Coronavirus Disease 2019.

BACKGROUND AND AIMS: The SARS-CoV-2 pandemic has overwhelmed the treatment capacity of the health care systems during the highest viral diffusion rate. Patients reaching the emergency department had to be either hospitalized (inpatients) or discharged (outpatients). Still, the decision was taken based on the individual assessment of the actual clinical condition, without specific biomarkers to predict future improvement or deterioration, and discharged patients often returned to the hospital for aggravation of their condition. Here, we have developed a new combined approach of omics to identify factors that could distinguish coronavirus disease 19 (COVID-19) inpatients from outpatients. METHODS: Saliva and blood samples were collected over the course of two observational cohort studies. By using machine learning approaches, we compared salivary metabolome of 50 COVID-19 patients with that of 270 healthy individuals having previously been exposed or not to SARS-CoV-2. We then correlated the salivary metabolites that allowed separating COVID-19 inpatients from outpatients with serum biomarkers and salivary microbiota taxa differentially represented in the two groups of patients. RESULTS: We identified nine salivary metabolites that allowed assessing the need of hospitalization. When combined with serum biomarkers, just two salivary metabolites (myo-inositol and 2-pyrrolidineacetic acid) and one serum protein, chitinase 3-like-1 (CHI3L1), were sufficient to separate inpatients from outpatients completely and correlated with modulated microbiota taxa. In particular, we found Corynebacterium 1 to be overrepresented in inpatients, whereas Actinomycetaceae F0332, Candidatus Saccharimonas, and Haemophilus were all underrepresented in the hospitalized population. CONCLUSION: This is a proof of concept that a combined omic analysis can be used to stratify patients independently from COVID-19.

Pulmonary endothelial nedd9 and the prothrombotic pathophenotype of acute respiratory distress syndrome due to sars-cov-2 infection

Background: The pathobiology of in situ pulmonary thrombosis in acute respiratory distress syndrome (ARDS) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is incompletely characterized. In human pulmonary artery endothelial cells (HPAECs), hypoxia upregulates expression of a pro-thrombotic NEDD9 peptide (N9 ) on the extracellular plasma membrane surface. We hypothesized that increased pulmonary endothelial N9 is a novel feature of the SARS-CoV-2 pathophenotype. Methods: Paraffin-embedded autopsy lung specimens were acquired from patients with ARDS due to SARS-CoV-2 infection (n=13), ARDS of other causes (n=10), and non-disease controls (n=5). Immunofluorescence characterized expression of N9 , fibrin, and TCF12, a putative binding target of SARS-CoV-2 and known transcriptional regulator of NEDD9. We performed RNA-Seq on mRNA isolated from control HPAECs treated with normoxia or hypoxia (0.2% O2 ) for 24 hr. Immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) profiled protein-protein interactions involving N9 relevant to thrombus stabilization. Results: Compared to non-SARS-CoV-2-ARDS lungs, pulmonary endothelial N9 expression and N9-fibrin colocalization was increased by 174% (P<0.002) and 212% (P<0.001) in SARS-CoV-2-ARDS, respectively. Compared to normoxia, hypoxia increased TCF12 mRNA quantity significantly in HPAECs in vitro [+1.19-fold, P=0.001;false discovery rate (FDR)=0.005]. Pulmonary endothelial nuclear TCF12 expression was also increased by 370% in SARS-CoV-2-ARDS vs. controls. In HPAEC plasma membranes, IP-LC-MS identified a novel protein-protein interaction between NEDD9 and the β3 subunit of the αvβ3 integrin, which regulates fibrin anchoring to endothelial cells. Conclusions: Compared to non-SARS-CoV-2-ARDS, SARS-CoV-2-ARDS is associated with increased pulmonary endothelial N9 expression and N9-fibrin colocalization in microthrombi in situ. Increased hypoxia signaling or SARS-CoV-2-mediated regulation of TCF12 are potential mechanisms by which to explain these findings. Identifying N9 in the pulmonary microthrombi of SARS-CoV-2 lungs may have important pathobiological and, potentially, therapeutic implications for ARDS patients.

Limited negative effects of the COVID-19 pandemic on mental health measures of Ghanaian university students.

BACKGROUND: Stress and mental health outcomes are negatively correlated among university students throughout the world. Reports of differences in stress perception by gender exist, but there is limited data on students from sub-Saharan African countries. This study describes the burden of perceived and financial stress; characterizes mood and degree of anxiety symptoms; examines stress coping mechanisms, including resilience and repetitive negative thinking (RNT); and explores how students at a Ghanaian university believed the COVID-19 pandemic affected these measures. METHODS: Students (n = 129) were recruited from the Kwame Nkrumah University of Science and Technology, Kumasi, Ghana from October 2020 - January 2021. Validated surveys were used. Participants were asked "Are your answers to the questions affected by the COVID-19 pandemic?" RESULTS: No differences in mean scores were observed between genders. For female students, financial stress was positively associated with RNT (p = 0.009), negative mood (p = 0.002), and anxiety (p < 0.001). Males were more likely to report decreased stress during the pandemic (p = 0.002), but there was no difference in mental health outcomes by perceived stress (PS) change category among males. Effects of the pandemic on mental health outcomes were mixed, but substantial proportions of students reported improvements or no change in financial stress, mood, anxiety, and RNT. LIMITATIONS: Students from one university particiapted in this cross-sectional survey. CONCLUSIONS: This study adds to the understanding of how higher education students are experiencing stress and are coping with the uncertainties of the COVID-19 pandemic in Ghana.

MICROBIAL COLONIZATION SUPRESSES COLONIC ACE2 EXPRESSION

Background: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has been responsible for the global pandemic and disease known as COVID-19. Clinical studies have found >50% of COVID-19 patients report gastrointestinal (GI) symptoms, with some studies suggesting longer viral clearance in patients with GI symptoms. SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor for viral attachment and intracellular entry, which is expressed in the intestine, making the GI tract a potential route of infection. Aim: To determine the effect of microbial colonization on colonic ACE2 expression using a humanized mouse model. Methods: Human stool was collected from healthy volunteers and individuals with irritable bowel syndrome, a common gastrointestinal disorder, and fecal slurries were prepared. 4-6 week old female Swiss-Webster mice (N=6/group) were gavaged with fecal slurries and maintained in sterile ex-isolator cages for 6 weeks. Posthumanization stool was collected along with proximal colonic tissue. Shotgun metagenomics was done on mouse pellets. RNAseq on tissue performed at a depth of 20 million reads/ sample using NovaSeq 6000. MAP-Rseq workflow using the mm10 genome was done to identify differentially expressed genes. Associations of ACE2 expression with α –diversity between microbiome from healthy and irritable bowel syndrome humanized mice was done using linear modeling with compositional associations assessed using PERMANOVA (BrayCurtis distance). To identify taxa associated with ACE2 expression, a permutation-based approach using the F-statistic of a linear model was used with false discovery rate (FDR) to correct for multiple testing. Results: Humanized mice demonstrated significantly lower colonic ACE2 expression compared to the germ-free mice (333.4 ± 191.1 vs. 1914 4 ± 309.9, FDR<0.001). However, ACE2 expression was similar post-humanization across all mouse groups regardless of stool used for humanization, despite decreased diversity in stool from irritable bowel syndrome patients and compositional differences from healthy volunteer stool. No associations between microbiome α-diversity (Shannon p=0.825, observed p= 0.400, InvSimpson p=0.512), β-diversity (p=0.568) or individual taxa were seen with ACE2 expression. Conclusions: Commensal microbial colonization significantly suppresses colonic ACE2 expression. However, in this pilot study, mice colonized with dysbiotic and healthy microbial communities had similar ACE2 expression. Future studies will have to explore the role of commensal microbes on gastrointestinal expression of ACE2 which may in turn reflect predisposition for infection or intestinal involvement with SARS-CoV-2. Supported by DK103911, DK120745.

Multiplicity Eludes Peer Review: The Case of COVID-19 Research.

Multiplicity arises when data analysis involves multiple simultaneous inferences, increasing the chance of spurious findings. It is a widespread problem frequently ignored by researchers. In this paper, we perform an exploratory analysis of the Web of Science database for COVID-19 observational studies. We examined 100 top-cited COVID-19 peer-reviewed articles based on p-values, including up to 7100 simultaneous tests, with 50% including >34 tests, and 20% > 100 tests. We found that the larger the number of tests performed, the larger the number of significant results (r = 0.87, p < 10-6). The number of p-values in the abstracts was not related to the number of p-values in the papers. However, the highly significant results (p < 0.001) in the abstracts were strongly correlated (r = 0.61, p < 10-6) with the number of p < 0.001 significances in the papers. Furthermore, the abstracts included a higher proportion of significant results (0.91 vs. 0.50), and 80% reported only significant results. Only one reviewed paper addressed multiplicity-induced type I error inflation, pointing to potentially spurious results bypassing the peer-review process. We conclude the need to pay special attention to the increased chance of false discoveries in observational studies, including non-replicated striking discoveries with a potentially large social impact. We propose some easy-to-implement measures to assess and limit the effects of multiplicity.