ABSTRACT
Background: COVID-19 causes significant morbidity and mortality, albeit with considerable heterogeneity among affected individuals. It remains unclear which host factors determine disease severity and survival. Given the propensity of clonal hematopoiesis (CH) to promote inflammation in healthy individuals, we investigated its effect on COVID-19 outcomes. Method(s): We performed a multi-omics interrogation of the genome, epigenome, transcriptome, and proteome of peripheral blood mononuclear cells from COVID-19 patients (n=227). We obtained clinical data, laboratory studies, and survival outcomes. We determined CH status and TET2-related DNA methylation. We performed single-cell proteogenomics to understand clonal composition in relation to cell phenotype. We interrogated single-cell gene expression in isolation and in conjunction with DNA accessibility. We integrated these multi-omics data to understand the effect of CH on clonal composition, gene expression, methylation of cis-regulatory elements, and lineage commitment in COVID-19 patients. We performed shRNA knockdowns to validate the effect of one candidate transcription factor in myeloid cell lines. Result(s): The presence of CH was strongly associated with COVID-19 severity and all-cause mortality, independent of age (HR 3.48, 95% CI 1.45-8.36, p=0.005). Differential methylation of promoters and enhancers was prevalent in TET2-mutant, but not DNMT3A-mutant CH. TET2- mutant CH was associated with enhanced classical/intermediate monocytosis and single-cell proteogenomics confirmed an enrichment of TET2 mutations in these cell types. We identified celltype specific gene expression changes associated with TET2 mutations in 102,072 single cells (n=34). Single-cell RNA-seq confirmed the skewing of hematopoiesis towards classical and intermediate monocytes and demonstrated the downregulation of EGR1 (a transcription factor important for monocyte differentiation) along with up-regulation of the lncRNA MALAT1 in monocytes. Combined scRNA-/scATAC-seq in 43,160 single cells (n=18) confirmed the skewing of hematopoiesis and up-regulation of MALAT1 in monocytes along with decreased accessibility of EGR1 motifs in known cis-regulatory elements. Using myeloid cell lines for functional validation, shRNA knockdowns of EGR1 confirmed the up-regulation of MALAT1 (in comparison to wildtype controls). Conclusion(s): CH is an independent prognostic factor in COVID-19 and skews hematopoiesis towards monocytosis. TET2-mutant CH is characterized by differential methylation and accessibility of enhancers binding myeloid transcriptions factors including EGR1. The ensuing loss of EGR1 expression in monocytes causes MALAT1 overexpression, a factor known to promote monocyte differentiation and inflammation. These data provide a mechanistic insight to the adverse prognostic impact of CH in COVID-19.
ABSTRACT
The proceedings contain 96 papers. The topics discussed include: practical pharmacotherapy for opioid use disorder in the age of fentanyl;can COVID-19 cause acute psychosis in pediatric patients? a case report;a survey of bullying experiences in a child and adolescent psychiatric clinic population;acute emergence of suicidal thoughts following Lemborexant initiation: an adverse reaction case report;assessing the unmet clinical need and opportunity for digital therapeutic intervention in schizophrenia: perspective from people with schizophrenia;rapid antidepressant effects and MADRS item improvements with AXS-05 (DEXTROMETHORPHAN-BUPROPION), an oral NMDA receptor antagonist in major depressive disorder: results from two randomized double-blind, controlled trials;targeting lncRNA NEAT1 impedes Alzheimers disease progression via MicroRNA-193a mediated CREB/BDNF and NRF2/NQO1 pathways;and impact of AXS-05 (DEXTROMETHORPHAN-BUPROPION), an Oral NMDA receptor antagonist, on Anhedonic symptoms in major depressive disorder.
ABSTRACT
Background: Infection with SARS-CoV-2 triggers reprogramming through global transcriptomic changes that drive the development of Coronavirus disease 2019 (COVID-19). Although the expression and functions of proteincoding transcripts have been widely studied in SARS-CoV-2 infection, most of the transcriptome consists of non-protein-coding RNAs (ncRNAs). Long noncoding RNAs (lncRNAs), which constitute a large proportion of the transcriptome, regulate immune responses and play prominent roles in health and disease. However, the impact of lncRNAs on SARS-CoV-2 infection is poorly understood. Our study will provide fundamental insights into the role of lncRNAs in SARS-CoV-2 infection. Method(s): We hypothesized that SARS-CoV-2-induced lncRNAs are critical regulators of viral replication and immune response. To test our hypothesis, we identified lncRNAs with significant differential expression in SARS-CoV-2 infected vs. uninfected cells across two cell types (A549-hACE2 and Calu) from published transcriptome data. We silenced the expression of the top lncRNA Bre- AS1 (BA) a human lung epithelial cell model (A549 cells stably expressing hACE2 and hTMPRSS2, A549AT) using lncRNA-specific ASO (lncsi) or negative control (NC) and compared viral replication in lncsi vs. NC cells. BA-silencing (BA-si) increased SARS-CoV-2 replication. and inhibited the expression of antiviral interferon-stimulated genes (ISG). (Tyr 705) pSTAT3 forms suppressor molecular complexes (pSTAT3-pSTAT1 or pSTAT3-PLSCR2) that inhibit ISG transcription. Using molecular methods such as gene-silencing, immunoprecipitation, western blot, and measuring promoter activity, we further show that Bre-AS1 inhibits the phosphorylation of STAT3 and enhances ISG transcription. Result(s): Our data show that cellular lncRNA, Bre-AS1 enhances antiviral interferon-stimulated genes (ISG) expression and inhibits replication of SARSCoV- 2. Our data show that Bre-AS1 inhibits the (Tyr705) phosphorylation of STAT3 that forms ISG repressor complexes (pSTAT3-pSTAT1 or pSTAT3-PLSCR2) and thus enhances ISG transcription. Conclusion(s): Cellular lncRNA Bre-AS1 enhances expression of antiviral interferon-stimulated genes and inhibits the replication of SARS-CoV-2. Our data show that cellular lncRNAs could play significant roles in immune response and viral propagation. Thus, unraveling the mechanisms of lncRNA-mediated regulation of virus replication and immune response may lead to identifying new, highly selective therapeutic targets Bre-AS1 inhibits STAT3 phosphorylation and enhances ISG transcription.
ABSTRACT
Introduction Entry factors angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) facilitate Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) entry into the host cells. Despite SARS-CoV-2s preference for respiratory system, extra-pulmonary organ involvement has been suggested. Recent studies report that SARS-CoV-2 leads to direct hepatic impairment in COVID-19 patients, necessitating further investigations about hepatic involvement. ACE2 and TMPRSS2 are expressed in primary human hepatocytes (PHH), suggesting a possible susceptibility to SARS-CoV-2. Despite this, data on infection and factors modulating functional regulation of SARS-CoV-2 infection in PHH are lacking. MicroRNAs (miRNAs) are approximately 22 nucleotide-long non-coding RNAs that have been shown to regulate various cellular processes including virus-host interactions. We aimed to study the susceptibility of PHH to SARS-CoV-2 and to evaluate the potential of miRNAs in modulating viral infection. Materials and methods We investigated the role of miRNAs to regulate SARS-CoV-2 infection in PHH in vitro. To strengthen our fndings, we analysed liver autopsies from COVID-19 patients. Results We demonstrate that PHH can be readily infected with SARS-CoV-2, resulting in robust replication and sustained host responses as indicated by the upregulation of several interferon-stimulated genes. In silico analyses unravelled miR-200c-3p, miR-429 and miR-141-3p as candidate miRNAs targeting ACE2 and, let-7c-5p targeting TMPRSS2. Expression of these miRNAs reduced SARS-CoV-2 infection in PHH. Furthermore, expression of several endogenous miRNAs was altered upon SARS-CoV-2 infection in PHH and human liver autopsies. Conclusion Our results show that PHH are susceptible towards SARS-CoV-2 and cellular miRNAs can diminish SARS-CoV-2 viral burden.
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The proceedings contain 429 papers. The topics discussed include: a short peptide encoded by long non-coding RNA small nucleolar RNA host gene 6 promotes cell migration and epithelial-mesenchymal transition by activating transforming growth factor-beta/SMAD signaling pathway in human endometrial cells;a short peptide encoded by long non-coding rna small nucleolar rna host gene 6 promotes cell migration and epithelial-mesenchymal transition by activating transforming growth factor-beta/smad signaling pathway in human endometrial cells;compatible cut-off values for luteinizing hormone and the luteinizing hormone/follicle-stimulating hormone ratio in diagnostic criteria of the Japan society of obstetrics and gynecology for polycystic ovary syndrome;intracytoplasmic sperm injection cycle success in patients under 35 years old with diminished ovarian reserve plus severe male factor;assisted reproductive technology and neonatal intensive care unit: a retrospective observational study from a single center;the value of clinical symptoms, the neutrophil-to-lymphocyte ratio, and ultrasonographic features in predicting adnexal torsion: a case-control study;construction of a diagnostic classifier for cervical intraepithelial neoplasia and cervical cancer based on xgboost feature selection and random forest model;and impact of the COVID-19 pandemic on surgery for benign diseases in gynecology: a nationwide survey by the japan society of obstetrics and gynecology.
ABSTRACT
Bulk transcriptomic analyses of virus-cell interactions are commonly performed on mixed populations of infected and uninfected bystander cells and may thus lead to inaccurate interpretations. Moreover, they generally focus on the expression of the coding genome and not on the total transcriptome, which is largely composed of long non-coding RNAs (lncRNAs). We performed polyA+ and whole transcriptome analysis of lung epithelial A549 cells infected with SARS-CoV-2, which were sorted based on the expression of the viral protein Spike (S). To increase the sequencing depth and ameliorate the robustness of the analysis, the samples were depleted of viral transcripts, which constituted up to 85% of total reads in the S-positive cells. Results highlighted a high number of down-regulated genes upon infection, an indication of massive host transcription shutdown. We also noticed an increased level of intronic reads in infected cells as compared to control cells, suggesting a defect in mRNA splicing. Among the upregulated coding genes in S-positive cells, we recovered candidates previously identified by analysis performed on non-sorted cells, such as CXCL8 and CCL20, but also novel candidates, including IL- 32, ITGAM and the down-regulated FEN1. Comparison of mRNA abundances of few coding, non-coding and unannotated genes between non-sorted cells and sorted ones confirmed the accuracy of the approach. We also observed that S-negative bystander cells and mock-infected control cells exhibited very similar transcriptomic profiles. This was not due to a lack of communication between infected and bystander cells: transcripts related to inflammatory cytokines underwent normal splicing and maturation in infected cells, with concomitant high levels of protein secretion. The observed lack of major transcriptomic changes in the bystander population can be linked to the insufficient interferon response in infected cells. Finally, we explored the functional implications of genes selectively upregulated in the infected subpopulation and observed previously uncharacterized proviral activities of lncRNA ADIRFAS1 and endogenous Interleukin 32 (IL-32), making them attractive targets for putative therapeutic strategies. Thus, analyzing the whole transcriptome of pure populations of infected lung cells allowed the accurate identification of cellular functions that are directly affected by infection and recovery of coding and non-coding genes relevant for SARS-CoV-2 replication.