Single cell sequencing reveals cellular landscape alterations in the airway mucosa of patients with pulmonary long COVID.

To elucidate the important cellular and molecular drivers of pulmonary long COVID, we generated a single-cell transcriptomic map of the airway mucosa using bronchial brushings from patients with long COVID who reported persistent pulmonary symptoms.Adults with and without long COVID were recruited from the general community in Greater Vancouver, Canada. The cohort was divided into those with pulmonary long COVID (PLC), which was defined as persons with new or worsening respiratory symptoms following at least one year from their initial acute SARS-CoV-2 infection (N=9); and control subjects defined as SARS-CoV-2 infected persons whose acute respiratory symptoms had fully resolved or individuals who had no history of acute COVID-19 (N=9). These participants underwent bronchoscopy from which a single cell suspension was created from bronchial brush samples and then sequenced.A total of 56 906 cells were recovered for the downstream analysis, with 34 840 cells belonging to the PLC group, which strikingly showed a unique cluster of neutrophils in the PLC group (p<0.05). Ingenuity Pathway Analysis revealed that the neutrophil degranulation pathway was enriched across epithelial cell clusters. Differential gene expression analysis between the PLC and control groups demonstrated upregulation of inflammatory chemokines and epithelial barrier dysfunction across epithelial cell clusters, as well as over-expression of mucin genes across secretory cell clusters.In conclusion, a single-cell transcriptomic landscape of the small airways suggest that neutrophils may play a significant role in mediating the chronic small airway inflammation driving pulmonary symptoms of long COVID.

The cell-type-specific spatial organization of the anterior thalamic nuclei of the mouse brain.

Understanding the cell-type composition and spatial organization of brain regions is crucial for interpreting brain computation and function. In the thalamus, the anterior thalamic nuclei (ATN) are involved in a wide variety of functions, yet the cell-type composition of the ATN remains unmapped at a single-cell and spatial resolution. Combining single-cell RNA sequencing, spatial transcriptomics, and multiplexed fluorescent in situ hybridization, we identify three discrete excitatory cell-type clusters that correspond to the known nuclei of the ATN and uncover marker genes, molecular pathways, and putative functions of these cell types. We further illustrate graded spatial variation along the dorsomedial-ventrolateral axis for all individual nuclei of the ATN and additionally demonstrate that the anteroventral nucleus exhibits spatially covarying protein products and long-range inputs. Collectively, our study reveals discrete and continuous cell-type organizational principles of the ATN, which will help to guide and interpret experiments on ATN computation and function.

Single-cell RNA sequencing of bronchoscopy specimens: development of a rapid minimal handling protocol.

Single-cell RNA sequencing (scRNA-seq) is an important tool for understanding disease pathophysiology, including airway diseases. Currently, the majority of scRNA-seq studies in airway diseases have used invasive methods (airway biopsy, surgical resection), which carry inherent risks and thus present a major limitation to scRNA-seq investigation of airway pathobiology. Bronchial brushing, where the airway mucosa is sampled using a cytological brush, is a viable, less invasive method of obtaining airway cells for scRNA-seq. Here we describe the development of a rapid and minimal handling protocol for preparing single-cell suspensions from bronchial brush specimens for scRNA-seq. Our optimized protocol maximizes cell recovery and cell quality and facilitates large-scale profiling of the airway transcriptome at single-cell resolution.

Sharp cell-type-identity changes differentiate the retrosplenial cortex from the neocortex.

The laminae of the neocortex are fundamental processing layers of the mammalian brain. Notably, such laminae are believed to be relatively stereotyped across short spatial scales such that shared laminae between nearby brain regions exhibit similar constituent cells. Here, we consider a potential exception to this rule by studying the retrosplenial cortex (RSC), a brain region known for sharp cytoarchitectonic differences across its granular-dysgranular border. Using a variety of transcriptomics techniques, we identify, spatially map, and interpret the excitatory cell-type landscape of the mouse RSC. In doing so, we uncover that RSC gene expression and cell types change sharply at the granular-dysgranular border. Additionally, supposedly homologous laminae between the RSC and the neocortex are effectively wholly distinct in their cell-type composition. In collection, the RSC exhibits a variety of intrinsic cell-type specializations and embodies an organizational principle wherein cell-type identities can vary sharply within and between brain regions.

Apoptotic and proliferative defects characterize ocular development in a microphthalmic BMP model.

PURPOSE: Vision is critically dependent on ocular size, which is regulated by environmental and genetic factors. Mutation of human Growth and Differentiation Factor 6 (GDF6) or zebrafish gdf6a results in a spectrum of small eye phenotypes (microphthalmia, anophthalmia, and coloboma). However, current models do not explain their etiology fully. As such, analyses of apoptosis and cell cycle regulation were undertaken in a zebrafish gdf6a mutant. METHODS: Microarray analysis was performed at 2 days after fertilization to uncover novel gdf6a-dependent cell cycle regulators. Altered expression of Gdf6a targets was confirmed by in situ hybridization, and resulting changes in cell proliferation were assessed by phosphohistone H3 immunohistochemistry. Analysis of apoptosis was evaluated through activated Caspase 3 immunohistochemistry and chemical inhibitors of cell death. RESULTS: Reduced numbers of retinal progenitor cells are observed at 24 hours post fertilization (hpf), resulting in microphthalmic eyes in gdf6a(-/-) embryos. At 28 hpf, a wave of apoptosis occurs; however, apoptosis inhibition does not rescue eye size, indicating a limited contribution. Mutants display altered proliferation and expression levels of cell cycle regulators, including members of the forkhead box i (foxi) transcription factor family expressed in the ciliary marginal zone. Notably, inhibition of foxi2 in gdf6a(-/-) embryos further reduces eye size. CONCLUSIONS: These data support a model whereby the gdf6a(-/-)-induced microphthalmia is based on early regulation of retinal progenitor cell number, and later by regulation of proliferation in the ciliary marginal zone. Foxi genes represent downstream effectors of Gdf6a function in the CMZ required for eye size determination.