Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2, TMPRSS2, and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta-analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2, TMPRSS2, and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2(+)TMPRSS2(+) cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross-talk. Amongst these are IL6, its receptor and co-receptor, IL1R, TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis

Temporal and Spatial Dynamics of Peroxynitrite-Induced Oxidative Damage after Spinal Cord Contusion Injury

The reactive nitrogen species peroxynitrite (PN) has been suggested to be an important mediator of the secondary oxidative damage that occurs following acute spinal cord injury (SCI). The PN decomposition products nitrogen dioxide (•NO2), hydroxyl radical (•OH), and carbonate radical (•CO3) are highly reactive with cellular lipids and proteins. In this immunohistochemical study, we examined the temporal (3, 24, and 72 h, and 1 and 2 weeks) and spatial relationships of PN-mediated oxidative damage in the contusion-injured rat thoracic spinal cord (IH device, 200 kdyn, T10) using 3-nitrotyrosine (3-NT), a marker for protein nitration by PN-derived •NO2 and 4-hydroxynonenal (4-HNE), an indicator of lipid peroxidation (LP) initiated by any of the PN radicals. Minimal 3-NT or 4-HNE immunostaining was seen in sham, non-injured spinal cords. In contrast, both markers showed a substantial increase at 3 h post-injury at the epicenter, that extended throughout the gray matter and into the surrounding white matter. At 24 and 72 h, the oxidative damage expanded circumferentially to involve all but a small rim of white matter tissue at the injury site, and longitudinally as much as 6–9 mm in the rostral and caudal directions. The staining was observed in neuronal soma, axons, and microvessels. At all time points except 3 h, there was no significant difference in the mean rostral or caudal extent of 3-NT and 4-HNE staining. By 1, and more so at 2 weeks, the longitudinal extent of the oxidative damage staining was greatly decreased. The spatial and temporal overlap of 3-NT and 4-HNE staining supports the concept that PN is involved in both damage produced by lipid peroxidation and protein nitration, and that antioxidant agents that target PN or PN-derived radicals should be effective neuroprotectants for acute SCI if administered during the first post-injury hours

Reference-based cell type matching of in situ image-based spatial transcriptomics data on primary visual cortex of mouse brain

With the advent of multiplex fluorescence in situ hybridization (FISH) and in situ RNA sequencing technologies, spatial transcriptomics analysis is advancing rapidly, providing spatial location and gene expression information about cells in tissue sections at single cell resolution. Cell type classification of these spatially-resolved cells can be inferred by matching the spatial transcriptomics data to reference atlases derived from single cell RNA-sequencing (scRNA-seq) in which cell types are defined by differences in their gene expression profiles. However, robust cell type matching of the spatially-resolved cells to reference scRNA-seq atlases is challenging due to the intrinsic differences in resolution between the spatial and scRNA-seq data. In this study, we systematically evaluated six computational algorithms for cell type matching across four image-based spatial transcriptomics experimental protocols (MERFISH, smFISH, BaristaSeq, and ExSeq) conducted on the same mouse primary visual cortex (VISp) brain region. We find that many cells are assigned as the same type by multiple cell type matching algorithms and are present in spatial patterns previously reported from scRNA-seq studies in VISp. Furthermore, by combining the results of individual matching strategies into consensus cell type assignments, we see even greater alignment with biological expectations. We present two ensemble meta-analysis strategies used in this study and share the consensus cell type matching results in the Cytosplore Viewer (https://viewer.cytosplore.org) for interactive visualization and data exploration. The consensus matching can also guide spatial data analysis using SSAM, allowing segmentation-free cell type assignment.Radiolog