Covid-19: Is the placenta a safe space

Problem: Several large studies have demonstrated that COVID-19 pregnant individuals are at a significant risk for severe disease and adverse pregnancy outcomes. The mechanisms underlying these phenomena remain to be elucidated and are the focus of our project. Although fetal and placental infection is rare, placental abnormalities and adverse pregnancy outcomes associated with placental dysfunction in COVID-19 cases have been widely reported. In particular, placental thrombosis and lesions consistent with maternal vascular malperfusion (MVM) of the placenta are common in individuals with COVID-19. Since thrombotic complications have been associated with COVID-19, it is not surprising that pregnant individuals with COVID- 19 are at risk for placental thrombosis. Method of Study: Placentas were evaluated histologically. Extracellular vesicles were isolated by serial centrifugation. Result(s): Adverse pregnancy outcomes associated with these placental lesions, including hypertensive disorders of pregnancy (gestational hypertension and preeclampsia), small for gestational age (SGA, birthweight < 10th percentile for gestational age), and preterm birth (PTB, < 37 weeks) are significantly increased among pregnant individuals with COVID-19. Placental infection with SARSCoV- 2 is uncommon, but multiple inflammatory and metabolic factors are likely to affect the placenta, including circulating extracellular vesicles (EVs) derived from various organs that have been associated with COVID-19 pathology and disease severity.We have analyzed over 500 placentas from COVID-19 pregnancies and found marked changes in placental morphology, characterized by abnormal maternal and fetal vessels, intervillous thrombi, and fibrin deposition, even in the face of mild or asymptomatic disease. We detected increased levels of small EVs in maternal serum from COVID-19 cases compared to controls and increased levels of mitochondrial DNA in EVs from COVID-19 cases. In in vitro experiments, we found increased oxidative stress in uterine endothelial cells and primary trophoblasts. Syncytialization of trophoblast cells following exposure to EVs from pregnant COVID-19 patients was markedly reduced. RNAseq of trophoblast cells exposed to EVs from pregnant COVID-19 patients revealed disruption of multiple pathways related to mitochondria function, oxidative stress, coagulation defects, and inflammation. Timing of infection during pregnancy (first, second, and third trimester) altered EV size distribution, cargo content, and functional consequences of trophoblast EV exposure. Conclusion(s): Our studies show that COVID-19 infection during pregnancy has profound effects on placenta morphology and function. It remains to be determined what the long-term consequences are on the offspring.

Pericytes may facilitate SARS-CoV-2 entry into the nervous system

Objective: Although the neurological and olfactory symptoms of coronavirus disease 2019 have been identified, the neurotropic properties of the causative virus, severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), remain unknown. We sought to identify the susceptible cell types and potential routes of SARS-CoV-2 entry into the central nervous system, olfactory system, and respiratory system. Method(s): We collected single-cell RNA data from normal brain and nasal epithelium specimens, along with bronchial, tracheal, and lung specimens in public datasets. The susceptible cell types that express SARS-CoV-2 entry genes were identified using single-cell RNA sequencing and the expression of the key genes at protein levels was verified by immunohistochemistry. We compared the coexpression patterns of the entry receptor angiotensin-converting enzyme 2 (ACE2) and the spike protein priming enzyme transmembrane serine protease (TMPRSS)/cathepsin L among the specimens. Result(s): The SARS-CoV-2 entry receptor ACE2 and the spike protein priming enzyme TMPRSS/cathepsin L were coexpressed by pericytes in brain tissue;this coexpression was confirmed by immunohistochemistry. In the nasal epithelium, ciliated cells and sustentacular cells exhibited strong coexpression of ACE2 and TMPRSS. Neurons and glia in the brain and nasal epithelium did not exhibit coexpression of ACE2 and TMPRSS. However, coexpression was present in ciliated cells, vascular smooth muscle cells, and fibroblasts in tracheal tissue;ciliated cells and goblet cells in bronchial tissue;and alveolar epithelium type 1 cells, AT2 cells, and ciliated cells in lung tissue. Conclusion(s): Neurological symptoms in patients with coronavirus disease 2019 could be associated with SARS-CoV-2 invasion across the blood-brain barrier via pericytes. Additionally, SARS-CoV-2-induced olfactory disorders could be the result of localized cell damage in the nasal epithelium.Copyright © Wolters Kluwer Health, Inc. All rights reserved.

Transcriptomic analysis in patients with SARS-CoV-2 virus infection. Identification of the association of risk/ protective profiles against the disease

Background/Objectives: SARS-CoV2 causes the COVID-19 disease, capable of producing a severe acute respiratory syndrome. Several clinical variables and genetic variants have been related to a worse prognosis. The aim of this study is to measure if difference in the gene expression are associated with COVID-19 severity. Method(s): We performed RNA-seq Transcriptome in RNA extracted from lymphoblastoid cell line in 20 patients who require hospitalization (10 from the intensive care unit) in a GeneStudio S5 Plus Sequencer (Ion Torrent Technology). FASTQ files were obtained and trimmed using BBtools, BBduk for cutting, filtering and masking the data, and Dedupe for the elimination of duplicates. Mapping and counting matrix was done in bash using the Salmon program. Differential expression analysis and subsequent functional enrichment was performed using Rstudio (DESeq2, ClusterProfiler, GO and KEGG). Result(s): We observed that 2042 differentially expressed genes (1996 overexpressed, LFC>0 and 406 underexpressed, LFC<0) were obtained between patients who require hospitalization versus those in the intensive care unit. We found some genes previously SARS-CoV-2 associated (PGLYRP1, HDAC9 and FUT4). Furthermore, genes involved in the activity of the immune system and in inflammatory processes showed significant differences between cohorts (ABCF1 (LFC = -25.14, padj = 1.05e-13), ABHD16A (LFC = 25.00, padj = 1.05e-13) and IER3 (LFC = -24.45, padj = 2.43e-13)). Conclusion(s): We described differential expression in genes of the immune system and inflammatory processes that might be have a role in the risk of develop severe symptoms of COVID-19, including admission in the intensive care unit. This results should be validated by additional functional studies.

Gene expression profiling reveals a role of immune system and inflammation in innate and stress-induced anxiety-like behavior.

Anxiety is an evolutionarily conserved response that is essential for survival. Pathological anxiety, however, is a maladaptive response to nonthreatening situations and greatly affects quality of life. The recent COVID-19 pandemic has increased the prevalence of anxiety symptoms and highlighted the urge to identify the molecular events that initiate pathological anxiety. To this aim, we investigated the extent of similarity of brain region-specific gene expression patterns associated with innate and stress-induced anxiety-like behavior. We compared the cortico-frontal (FCx) and hippocampal (Hpc) gene expression patterns of five inbred mouse strains with high or low levels of innate anxiety-like behavior with gene expression patterns of mice subjected to chronic social defeat stress. We found significantly large overlap of the Hpc but small overlap of the FCx gene expression patterns in innate and stress-induced anxiety, that however, converged onto common inflammation and immune system canonical pathways. Comparing the gene expression data with drug-gene interaction datasets revealed drug candidates, including medrysone, simvastatin, captopril, and sulpiride, that produced gene expression changes opposite to those observed in innate or stress-induced anxiety-like behavior. Together, our data provide a comprehensive overview of FCx and Hpc gene expression differences between innate and stress-induced anxiety and support the role of inflammation and immune system in anxiety-like behavior.

Computational identification of differentially-expressed genes as suggested novel COVID-19 biomarkers: A bioinformatics analysis of expression profiles.

COVID-19 was declared a pandemic in March 2020, and since then, it has not stopped spreading like wildfire in almost every corner of the world, despite the many efforts made to stem its spread. SARS-CoV-2 has one of the biggest genomes among RNA viruses and presents unique characteristics that differentiate it from other coronaviruses, making it even more challenging to find a cure or vaccine that is efficient enough. This work aims, using RNA sequencing (RNA-Seq) data, to evaluate whether the expression of specific human genes in the host can vary in different grades of disease severity and to determine the molecular origins of the differences in response to SARS-CoV-2 infection in different patients. In addition to quantifying gene expression, data coming from RNA-Seq allow for the discovery of new transcripts, the identification of alternative splicing events, the detection of allele-specific expression, and the detection of post-transcriptional alterations. For this reason, we performed differential expression analysis on different expression profiles of COVID-19 patients, using RNA-Seq data coming from NCBI public repository, and we obtained the lists of all differentially expressed genes (DEGs) emerging from 7 experimental conditions. We performed a Gene Set Enrichment Analysis (GSEA) on these genes to find possible correlations between DEGs and known disease phenotypes. We mainly focused on DEGs coming out from the analysis of the contrasts involving severe conditions to infer any possible relation between a worsening of the clinical picture and an over-representation of specific genes. Based on the obtained results, this study indicates a small group of genes that result up-regulated in the severe form of the disease. EXOSC5, MESD, REXO2, and TRMT2A genes are not differentially expressed or not present in the other conditions, being for that reason, good biomarkers candidates for the severe form of COVID-19 disease. The use of specific over-expressed genes, whether up-regulated or down-regulated, which have an individual role in each different condition of COVID-19 as a biomarker, can assist in early diagnosis.

Co-exposure to urban particulate matter and aircraft noise adversely impacts the cerebro-pulmonary-cardiovascular axis in mice

Worldwide, up to 8.8 million excess deaths/year have been attributed to air pollution, mainly due to the exposure to fine particulate matter (PM). Traffic-related noise is an additional contributor to global mortality and morbidity. Both health risk factors substantially contribute to cardiovascular, metabolic and neuropsychiatric sequelae. Studies on the combined exposure are rare and urgently needed because of frequent co-occurrence of both risk factors in urban and industrial settings. To study the synergistic effects of PM and noise, we used an exposure system equipped with aerosol generator and loud-speakers, where C57BL/6 mice were acutely exposed for 3d to either ambient PM (NIST particles) and/or noise (aircraft landing and take-off events). The combination of both stressors caused endothelial dysfunction, increased blood pressure, oxidative stress and inflammation. An additive impairment of endothelial function was observed in isolated aortic rings and even more pronounced in cerebral and retinal arterioles. The increase in oxidative stress and inflammation markers together with RNA sequencing data indicate that noise particularly affects the brain and PM particularly affects the lungs. Noise also increased levels of circulating stress hormones adrenaline and noradrenaline, while PM increased levels of circulating cytokines CD68 and MCP-1. The combination of both stressors has additive adverse effects on the cardiovascular system that are based on PM-induced systemic inflammation and noise-triggered stress hormone signaling. We demonstrate an additive upregulation of ACE-2 in the lung, suggesting that there may be an increased vulnerability to COVID-19 infection. The data warrant further mechanistic studies to characterize the propagation of primary target tissue damage (lung, brain) to remote organs such as aorta and heart by combined noise and PM exposure.Copyright © 2023

Molecular genetics and genomics of the ABO blood group system

The A and B oligosaccharide antigens of the ABO blood group system are produced from the common precursor, H substance, by enzymatic reactions catalyzed by A and B glycosyltransferases (AT and BT) encoded by functional A and B alleles at the ABO genetic locus, respectively. In 1990, my research team cloned human A, B, and O allelic cDNAs. We then demonstrated this central dogma of ABO and opened a new era of molecular genetics. We identified four amino acid substitutions between AT and BT and inactivating mutations in the O alleles, clarifying the allelic basis of ABO. We became the first to achieve successful ABO genotyping, discriminating between AA and AO genotypes and between BB and BO, which was impossible using immunohematological/serological methods. We also identified mutations in several subgroup alleles and also in the cis-AB and B(A) alleles that specify the expression of the A and B antigens by single alleles. Later, other scientists interested in the ABO system characterized many additional ABO alleles. However, the situation has changed drastically in the last decade, due to rapid advances in next-generation sequencing (NGS) technology, which has allowed the sequencing of several thousand genes and even the entire genome in individual experiments. Genome sequencing has revealed not only the exome but also transcription/translation regulatory elements. RNA sequencing determines which genes and spliced transcripts are expressed. Because more than 500,000 human genomes have been sequenced and deposited in sequence databases, bioinformaticians can retrieve and analyze this data without generating it. Now, in this era of genomics, we can harness the vast sequence information to unravel the molecular mechanisms responsible for important biological phenomena associated with the ABO polymorphism. Two examples are presented in this review: the delineation of the ABO gene evolution in a variety of species and the association of single nucleotide variant (SNV) sites in the ABO gene with diseases and biological parameters through genome-wide association studies (GWAS).Copyright © Annals of Blood. All rights reserved.

Blood gene expression predicts intensive care unit admission in hospitalised patients with COVID-19.

Background: The COVID-19 pandemic has created pressure on healthcare systems worldwide. Tools that can stratify individuals according to prognosis could allow for more efficient allocation of healthcare resources and thus improved patient outcomes. It is currently unclear if blood gene expression signatures derived from patients at the point of admission to hospital could provide useful prognostic information. Methods: Gene expression of whole blood obtained at the point of admission from a cohort of 78 patients hospitalised with COVID-19 during the first wave was measured by high resolution RNA sequencing. Gene signatures predictive of admission to Intensive Care Unit were identified and tested using machine learning and topological data analysis, TopMD. Results: The best gene expression signature predictive of ICU admission was defined using topological data analysis with an accuracy: 0.72 and ROC AUC: 0.76. The gene signature was primarily based on differentially activated pathways controlling epidermal growth factor receptor (EGFR) presentation, Peroxisome proliferator-activated receptor alpha (PPAR-α) signalling and Transforming growth factor beta (TGF-ß) signalling. Conclusions: Gene expression signatures from blood taken at the point of admission to hospital predicted ICU admission of treatment naïve patients with COVID-19.

cGAS-STING PATHWAY LIMITS SARS-CoV-2 REPLICATION IN ACE2+ AIRWAY CELL LINES

Background: We previously screened 10 human lung and upper airway cell lines expressing variable levels of endogenous ACE2/TMPRSS2. We found that H522 human lung adenocarcinoma cells supported SARS-CoV-2 replication independent of ACE2, whereas the ACE2 positive cell lines were not permissive to infection. Type I/III interferons (IFNs) potently restrict SARS-CoV-2 replication through the actions of hundreds of interferon-stimulated genes (ISGs) that are upregulated upon IFN signaling. Here we report that a number of ACE2 positive airway cell lines are unable to support SARS-CoV-2 replication due to basal activation of the cGAS-STING DNA sensing pathway and subsequent upregulation of IFNs and ISGs which restrict SARS-CoV-2 replication. Method(s): SARS-CoV-2 WT strain 2019-nCoV/USA-WA1/2020 viral replication was detected through analysis of cell associated RNA. RNA sequencing was used to study the basal level of genes in the type-I IFN pathway in the 10 cell lines, which was further validated by western blotting and qRT-PCR. A panel of 5 cell lines, with varying expression levels of ACE2 and TMPRSS2, were pre-treated with Ruxolitinib, a JAK1/2 inhibitor. A siRNA-mediated screen was used to determine the molecular basis of basally high expression of ISGs in cell lines. CRISPR knockout of IFN-alpha receptor and cGAS-STING pathway components was conducted in parallel Results: Here we show that higher basal levels of IFN pathway activity underlie the inability of ACE2+ cell lines to support virus replication. Importantly, this IFN-induced block can be overcome by chemical inhibition and genetic disruption of the IFN signaling pathway or by ACE2 overexpression, suggesting that one or more saturable ISGs underlie the lack of permissivity of these cells. Ruxolitinib treatment increased SARS-CoV-2 RNA levels by nearly 3 logs in OE21 and SCC25. Furthermore, the baseline activation of the STING-cGAS pathway accounts for the high ISG levels and genetic disruption of the cGAS-STING pathway enhances levels by nearly 2 and 3 logs of virus replication in the two separate ACE2+ cell line models respectively. Conclusion(s): Our findings demonstrate that cGAS-STING-dependent activation of IFN-mediated innate immunity underlies the inability of ACE2+ airway cell lines to support SARS-CoV-2 replication. Our study highlights that in addition to ACE2, basal activation of cGAS-STING pathway, IFNs and ISGs may play a key role in defining SARS-CoV-2 cellular tropism and may explain the complex SARS-CoV- 2 pathogenesis in vivo.

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.

TARGETING THE HOST-VIRUS INTERFACE TO BLOCK SARS-CoV-2 ASSEMBLY IN AIRWAY CELLS

Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.

Predicting COVID-19 Severity Integrating RNA-Seq Data Using Machine Learning Techniques

A fundamental challenge in the fight against COVID-19 is the development of reliable and accurate tools to predict disease progression in a patient. This information can be extremely useful in distinguishing hospitalized patients at higher risk for needing UCI from patients with low severity. How SARS-CoV-2 infection will evolve is still unclear. Method(s): A novel pipeline was developed that can integrate RNA-Seq data from different databases to obtain a genetic biomarker COVID-19 severity index using an artificial intelligence algorithm. Our pipeline ensures robustness through multiple cross-validation processes in different steps. Result(s): CD93, RPS24, PSCA, and CD300E were identified as COVID-19 severity gene signatures. Furthermore, using the obtained gene signature, an effective multi-class classifier capable of discrimi-nating between control, outpatient, inpatient, and ICU COVID-19 patients was optimized, achieving an accuracy of 97.5%. Conclusion(s): In summary, during this research, a new intelligent pipeline was implemented to develop a specific gene signature that can detect the severity of patients suffering COVID-19. Our approach to clinical decision support systems achieved excellent results, even when processing unseen samples. Our system can be of great clinical utility for the strategy of planning, organizing and managing human and material resources, as well as for automatically classifying the severity of patients affected by COVID-19.Copyright © 2023 Bentham Science Publishers.

Application of single-cell RNA sequencing on human testicular samples: a comprehensive review.

So far there has been no comprehensive review using systematic literature search strategies to show the application of single-cell RNA sequencing (scRNA-seq) in the human testis of the whole life cycle (from embryos to aging males). Here, we summarized the application of scRNA-seq analyses on various human testicular biological samples. A systematic search was conducted in PubMed and Gene Expression Omnibus (GEO), focusing on English researches published after 2009. Articles related to GEO data-series were also retrieved in PubMed or BioRxiv. 81 full-length studies were finally included in the review. ScRNA-seq has been widely used on different human testicular samples with various library strategies, and new cell subtypes such as State 0 spermatogonial stem cells (SSC) and stage_a/b/c Sertoli cells (SC) were identified. For the development of normal testes, scRNA-seq-based evidence showed dynamic transcriptional changes of both germ cells and somatic cells from embryos to adults. And dysregulated metabolic signaling or hedgehog signaling were revealed by scRNA-seq in aged SC or Leydig cells (LC), respectively. For infertile males, scRNA-seq studies revealed profound changes of testes, such as the increased proportion of immature SC/LC of Klinefelter syndrome, the somatic immaturity and altered germline autophagy of patients with non-obstructive azoospermia, and the repressed differentiation of SSC in trans-females receiving testosterone inhibition therapy. Besides, the re-analyzing of public scRNA-seq data made further discoveries such as the potential vulnerability of testicular SARS-CoV-2 infection, and both evolutionary conservatism and divergence among species. ScRNA-seq analyses would unveil mechanisms of testes' development and changes so as to help developing novel treatments for male infertility.

Single cell meta-analysis of EndMT and EMT state in COVID-19.

COVID-19 prognoses suggests that a proportion of patients develop fibrosis, but there is no evidence to indicate whether patients have progression of mesenchymal transition (MT) in the lungs. The role of MT during the COVID-19 pandemic remains poorly understood. Using single-cell RNA sequencing, we profiled the transcriptomes of cells from the lungs of healthy individuals (n = 45), COVID-19 patients (n = 58), and idiopathic pulmonary fibrosis (IPF) patients (n = 64) human lungs to map the entire MT change. This analysis enabled us to map all high-resolution matrix-producing cells and identify distinct subpopulations of endothelial cells (ECs) and epithelial cells as the primary cellular sources of MT clusters during COVID-19. For the first time, we have identied early and late subgroups of endothelial mesenchymal transition (EndMT) and epithelial-mesenchymal transition (EMT) using analysis of public databases for single-cell sequencing. We assessed epithelial subgroups by age, smoking status, and gender, and the data suggest that the proportional changes in EMT in COVID-19 are statistically significant. Further enumeration of early and late EMT suggests a correlation between invasive genes and COVID-19. Finally, EndMT is upregulated in COVID-19 patients and enriched for more inflammatory cytokines. Further, by classifying EndMT as early or late stages, we found that early EndMT was positively correlated with entry factors but this was not true for late EndMT. Exploring the MT state of may help to mitigate the fibrosis impact of SARS-CoV-2 infection.

Single-cell sequencing of PBMC characterizes the altered transcriptomic landscape of classical monocytes in BNT162b2-induced myocarditis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been the most dangerous threat to public health worldwide for the last few years, which led to the development of the novel mRNA vaccine (BNT162b2). However, BNT162b2 vaccination is known to be associated with myocarditis. Here, as an attempt to determine the pathogenesis of the disease and to develop biomarkers to determine whether subjects likely proceed to myocarditis after vaccination, we conducted a time series analysis of peripheral blood mononuclear cells of a patient with BNT162b2-induced myocarditis. Single-cell RNA sequence analysis identified monocytes as the cell clusters with the most dynamic changes. To identify distinct gene expression signatures, we compared monocytes of BNT162b2-induced myocarditis with monocytes under various conditions, including SARS-CoV-2 infection, BNT162b2 vaccination, and Kawasaki disease, a disease similar to myocarditis. Representative changes in the transcriptomic profile of classical monocytes include the upregulation of genes related to fatty acid metabolism and downregulation of transcription factor AP-1 activity. This study provides, for the first time, the importance of classical monocytes in the pathogenesis of myocarditis following BNT162b2 vaccination and presents the possibility that vaccination affects monocytes, further inducing their differentiation and infiltration into the heart.

Effect of polycyclic aromatic hydrocarbons on skin pigmentation

Despite COVID-19 lockdowns, gradual restarting of the global economy has rapidly increased air quality index (AQI) values. With over 99 % of the world population living in areas exceeding air quality guidelines, air pollution is more so a threat to our health. Within particulate matter, a major air pollutant, lies polycyclic aromatic hydrocarbons (PAH). While recent studies explore the link between air pollution and pigmentation disorders, the molecular mechanisms responsible for this alteration remain largely unknown. To challenge our hypothesis that exposure to PAH leads to an increase in abnormal pigmentation, we have utilized in vitro and in vivo assays. In vitro, novel 2D and 3D co-culture assays were developed to analyze pigment production, transfer and total melanin content in human keratinocytes and melanocytes. Following that, bulk RNA-sequencing was also carried out on isolated melanocytes post co-culture to possibly elucidate the mechanism behind this phenomenon. In vivo, a mouse model bearing epidermal melanin was generated to investigate the effect of PAH exposure. Notably, our initial studies have indicated a significant increase in melanin production, transfer and total melanin content when exposed to PAH. From our transcriptome analysis, we have also pinpointed to several genes which have been differentially expressed, most significant being CYP1A1. This prompted us to look further into the AhR signaling pathway. Interestingly, we did not see an increase in classic melanogenesis genes, but instead genes which are usually associated with senescence-associated secretory phenotype (SASP). This hints to a possible alternative pathway leading to an eventual increase in melanin production. We believe that our findings highlight potential approaches for novel therapeutics the treatment of skin pigmentation disorders triggered by air pollution.Copyright © 2023

Transcriptome in Human Mycoses

Mycoses are infectious diseases caused by fungi, which incidence has increased in recent decades due to the increasing number of immunocompromised patients and improved diagnostic tests. As eukaryotes, fungi share many similarities with human cells, making it difficult to design drugs without side effects. Commercially available drugs act on a limited number of targets and have been reported fungal resistance to commonly used antifungal drugs. Therefore, elucidating the pathogenesis of fungal infections, the fungal strategies to overcome the hostile environment of the host, and the action of antifungal drugs is essential for developing new therapeutic approaches and diagnostic tests. Large-scale transcriptional analyses using microarrays and RNA sequencing (RNA-seq), combined with improvements in molecular biology techniques, have improved the study of fungal pathogenicity. Such techniques have provided insights into the infective process by identifying molecular strategies used by the host and pathogen during the course of human mycoses. This chapter will explore the latest discoveries regarding the transcriptome of major human fungal pathogens. Further we will highlight genes essential for host–pathogen interactions, immune response, invasion, infection, antifungal drug response, and resistance. Finally, we will discuss their importance to the discovery of new molecular targets for antifungal drugs. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2014, 2022.

Possible Biological Mechanisms Underlying the Association between COVID-19 Severity and HLA-C*04:01

A strong link between COVID-19 severity and HLAC* 04:01 allele has been replicated in several Caucasian populations including Armenians. The results have led to an idea that HLA-C*04:01 may affect the immune response via three biological mechanisms: (i) disruption of the HLA-C mediated protection harnessing natural killer cells (NK);(ii) causing NK hypo-responsiveness through KIR2DL1;or (iii) over-activation and exhaustion of CTL and NK cells by stimulating functional KIR2DS4. To test those hypotheses, we re-analyzed HLA-genotypes and RNA-sequencing data of Overmyer et al. [Cell Systems 2021;12:23-40]. An ordinal regression of patients' status (i.e., non-COVID vs. COVIDnon- ICU vs. COVID-ICU) against HLA-C has corroborated the increase in the disease severity with increasing HLA-C*04:01 dosage (p< 0.003). DESeq2 analyses of the transcriptome (16444 loci) within COVID subset mapped 3586 down-regulated and 4031 up-regulated loci to the disease severity at FDR p<0.05. The results of enrichment analyses of those 7617 genes indicated aberrations in processes, such as T cell activation, inflammatory response, positive regulation of both NK-mediated cytotoxicity and interferon-gamma production. However, only 563 down- and 341 up-regulated loci had nominally associated with the HLA-C*04:01 carriage, reflecting its genetic association with severe symptoms. Using GTEx data and rs5010528 as proxy for HLAC* 04:01 (R2 = 0.97, 1kG EUR cohort), we found that HLA-C*04:01 was associated with multiple tissue (e.g., lung, heart and blood) RNA expressional and splicing changes in >10 protein-coding loci situated close to HLA-C. The ontology analysis of the loci implicated HLA-C*04:01 in altering antigen processing and presentation of endogenous peptide antigen via HLA class I via ER pathway (FDR p<0.0001), protection from NKmediated cytotoxicity (p<0.004), and innate immune response to other organisms (p<0.009). The work was supported by the Science Committee of RA (grant E17).

Constructing custom-made radiotranscriptomic signatures from CT angiograms: An application in COVID-19 vascular inflammation

Background: Advances in computational methodologies have enabled processing of large datasets originating from imaging studies. However, most imaging biomarkers suffer from a lack of direct links with underlying biology, as they are only observationally correlated with pathophysiology. Purpose(s): To develop and validate a novel AI-assisted image analysis platform, by applying quantitative radiotranscriptomics that quantifies cytokinedriven vascular inflammation from routine CT angiograms (CTA) performed as part of clinical care in COVID-19. Method(s): We used this platform to train the radiotranscriptomic signature C19-RS, derived from the perivascular space around the aorta and the internal mammary artery in routine chest CTAs, to best describe cytokinedriven vascular inflammation, defined using transcriptomic profiles from RNA sequencing data from human arterial biopsies (A). This signature was validated externally in 358 clinically indicated CT pulmonary angiograms from patients with or without COVID-19 from 3 different geographical regions. Result(s): First, 22 patients who had a CTA before the pandemic underwent repeat CTA <6 months post COVID-19 infection (B). Compared with 22 controls (matched for age, gender, and BMI) C19-RS was increased only in the COVID-19 group (C). Next, C19-RS was calculated in a cohort of 331 patients hospitalised during the pandemic, and was higher in COVID-19 positives (adjusted OR=2.97 [95% CI: 1.43-6.27], p=0.004, D). C19-RS had prognostic value for in-hospital mortality in COVID-19, with HR=3.31 ([95% CI: 1.49-7.33], p=0.003) and 2.58 ([95% CI: 1.10-6.05], p=0.028) in two testing cohorts respectively (E, F), adjusted for clinical factors and biochemical biomarkers of inflammation and myocardial injury. The corrected HR for in-hospital mortality was 8.24 [95% CI: 2.16-31.36], p=0.002 for those who received no treatment with dexamethasone, but only 2.27 [95% CI: 0.69-7.55], p=0.18 in those who received dexamethasone subsequently to the C19-RS based image analysis, suggesting that vascular inflammation may have been a therapeutic target of dexamethasone in COVID-19. Finally, C19-RS was strongly associated (r=0.61, p=0.0003) with a whole blood transcriptional module representing dysregulation of coagulation and platelet aggregation pathways. Conclusion(s): We present the first proof of concept study that combines transcriptomics with radiomics to provide a platform for the development of machine learning derived radiotranscriptomics analysis of routine clinical CT scans for the development of non-invasive imaging biomarkers. Application in COVID-19 produced C19-RS, a marker of cytokine-driven inflammation driving systemic activation of coagulation, that predicts inhospital mortality and identifies people who will have better response to anti-inflammatory treatments, allowing targeted therapy. This AI-assisted image analysis platform may have applications across a wide range of vascular diseases, from infections to autoimmune diseases.

In silico identification of potential miRNAs -mRNA inflammatory networks implicated in the pathogenesis of COVID-19

COVID-19 has been found to affect the expression profile of several mRNAs and miRNAs, leading to dysregulation of a number of signaling pathways, particularly those related to inflammatory responses. In the current study, a systematic biology procedure was used for the analysis of high-throughput expression data from blood specimens of COVID-19 and healthy individuals. Differentially expressed miRNAs in blood specimens of COVID-19 vs. healthy specimens were then identified to construct and analyze miRNA-mRNA networks and predict key miRNAs and genes in inflammatory pathways. Our results showed that 171 miRNAs were expressed as outliers in box plot and located in the critical areas according to our statistical analysis. Among them, 8 miRNAs, namely miR-1275, miR-4429, miR-4489, miR-6721-5p, miR-5010-5p, miR-7110-5p, miR-6804-5p and miR-6881-3p were found to affect expression of key genes in NF-KB, JAK/STAT and MAPK signaling pathways implicated in COVID-19 pathogenesis. In addition, our results predicted that 25 genes involved in above-mentioned inflammatory pathways were targeted not only by these 8 miRNAs but also by other obtained miRNAs (163 miRNAs). The results of the current in silico study represent candidate targets for further studies in COVID-19.Copyright © 2023 Elsevier B.V.