Single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cellular variability by capturing gene expression profiles of individual cells. The importance of cell-to-cell variability in determining and shaping cell function has been widely appreciated. Nevertheless, differential expression (DE) analysis remains a cornerstone method in analytical practice. Current computational analyses overlook the rich information encoded by variability within the single-cell gene expression data by focusing exclusively on mean expression. To offer a deeper understanding of cellular systems, there is a need for approaches to assess data variability rather than just the mean. Here we present spline-DV, a statistical framework for differential variability (DV) analysis using scRNA-seq data. The spline-DV method identifies genes exhibiting significantly increased or decreased expression variability among cells derived from two experimental conditions. Case studies show that DV genes identified using spline-DV are representative and functionally relevant to tested cellular conditions, including obesity, fibrosis, and cancer.

Gene expression is a dynamic and stochastic process characterized by transcriptional bursting followed by periods of silence. Single-cell RNA sequencing (scRNA-seq) is a powerful tool to measure transcriptional bursting and silencing at the individual cell level. In this study, we introduce the single-cell Stochastic Gene Silencing (scSGS) method, which leverages the natural variability in single-cell gene expression to decipher gene function. For a target gene g under investigation, scSGS classifies cells into transcriptionally active (g + ) and silenced (g-) samples. It then compares these cell samples to identify differentially expressed genes, referred to as SGS-responsive genes, which are used to infer the function of the target gene g. Analysis of real data demonstrates that scSGS can reveal regulatory relationships up- and downstream of target genes, circumventing the survivorship bias that often affects gene knockout and perturbation studies. scSGS thus offers an efficient approach for gene function prediction, with significant potential to reduce the use of genetically modified animals in gene function research.

Epicardial adipose tissue (EAT) regulates lipid metabolism and immune cell recruitment in coronary arteries. Increased EAT contributes to coronary artery disease (CAD), but exercise prevents CAD. We hypothesized that exercise, irrespective of CAD presence, would produce EAT with increased M2 macrophages and up-regulation of anti-inflammatory cytokine transcripts. Female Yucatan pigs (n=7) were sedentary or exercised, and the left circumflex coronary artery was occluded or remained non-occluded (2x2 design). Bulk and single-nuclei transcriptomic sequencing performed on EAT identified immune, endothelial, smooth muscle, adipocytes, adipocyte progenitor cells (APSCs), and neuronal cells, with adipocytes and APSCs predominant. Non-occluded (N) sedentary (Sed) EAT had the most M1 macrophages and CD8+ T cells. Sed EAT had the most cells expressing tumor necrosis factor (TNF) superfamily genes. Exercise (Ex) upregulated peroxisome proliferator-activated receptor ( PPAR) gamma ( G) expression and enriched PPAR signaling, which suppresses activation, in macrophages and T cells, particularly in occluded (O) Ex EAT. By contrast, N_Ex EAT had few CD8+ T cells with low PPARG expression. Adipocytes and immune cells in O_Sed EAT had the most communication via growth factors and adhesion molecules. Exercise mitigates EAT inflammation via modulation of immune cell subpopulations, decreased TNF superfamily, and increased PPARG gene expression, and decreased communication between adipocytes and immune cells. However, the effect of exercise on the EAT immune environment is modulated by coronary artery occlusion status. Future studies of the impact of exercise and coronary artery occlusion on EAT would benefit from using a progressive nutritionally induced model of CAD.

Coronary artery disease (CAD) is a leading cause of death in women. Although exercise mitigates CAD, the mechanisms by which exercise impacts epicardial adipose tissue (EAT) are unknown. We hypothesized that exercise promotes an anti-inflammatory microenvironment in EAT from female pigs. Yucatan pigs (n=7) were assigned to sedentary (Sed) or exercise (Ex) treatments and coronary arteries were occluded (O) with an ameroid to mimic CAD or remained non-occluded (N). EAT was collected for bulk and single nucleus transcriptomic sequencing (snRNA-seq). Exercise upregulated G-protein coupled receptor, S100 family, and FAK pathways and downregulated the coagulation pathway. Exercise increased the interaction between immune, endothelial, and mesenchymal cells in the insulin-like growth factor pathway and between endothelial and other cell types in the platelet endothelial cell adhesion molecule 1 pathway. Sub- clustering revealed nine cell types in EAT with fibroblast and macrophage populations predominant in O-Ex EAT and T cell population predominant in N-Ex EAT. Coronary occlusion impacted the largest number of genes in T and endothelial cells. Genes related to fatty acid metabolism were the most highly upregulated in non-immune cells from O-Ex EAT. Sub-clustering of endothelial cells revealed that N-Ex EAT separated from other treatments. In conclusion, aerobic exercise increased interaction amongst immune and mesenchymal and endothelial cells in female EAT. Exercise was minimally effective at reversing alterations in gene expression in endothelial and mesenchymal cells in EAT surrounding occluded arteries. These findings lay the foundation for future work focused on the impact of exercise on cell types in EAT. Significance Statement Coronary artery disease (CAD) is a leading cause of death in women. However, the role of epicardial adipose tissue (EAT) in the development of CAD in females and how exercise, which is recommended to slow CAD progression, impacts EAT are unknown. The effect of aerobic exercise on gene expression in EAT was investigated with RNA-sequencing, revealing significant alterations in fatty acid processing and immunoregulatory processes. This study provides valuable insights into the molecular and cellular changes induced in EAT by exercise in the context of chronic ischemic heart disease in females. These findings bolster current understanding of the impact of aerobic exercise on cardiac health in females and provide a foundation for future research in the field of exercise science.

HIV-associated neurological complications remain a major concern in people living with HIV (PLWH), even under effective viral suppression with combination antiretroviral therapy (cART), underscoring unresolved mechanisms driving HIV-related neurodegeneration. To address this gap, we used HIV-1-infected human cerebral organoids (hCOs) containing microglia as a physiologically relevant three-dimensional model of the central nervous system (CNS) and validated key findings in simian immunodeficiency virus (SIV)-infected, cART-treated rhesus macaque model. Integrating single-nucleus RNA sequencing with ATAC sequencing, we identified cell-type-specific alterations in dual innate immune-driven inflammatory signaling pathways across neural and glial populations. Microglia were preferentially infected and activated, initiating IL-17A-mediated cascades involving IFNγ, STAT1, CEBPB, and NFkB pathways, which converged with cGAS-STING and Notch-NEURL1 signaling, both associated with neuroinflammation and synaptic dysfunction. cART suppressed viral replication, reduced IL-17A-driven neuroinflammation, improved mitochondrial function, and restored synaptic gene expression. Collectively, these findings identify IL-17A, STAT1, CEBPB, and cGAS-STING as key molecular drivers of HIV-associated neuroinflammation and establish a novel mechanistic link between IL-17A signaling and microglial remodeling, providing a translational framework for the development of CNS-targeted therapies in PLWH.

Bis-indole-derived compounds including 1,1-bis(3′-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) analogs bind both orphan nuclear receptors 4A1 (NR4A1) and NR4A2, and DIM-3,5 compounds act as dual receptor inverse agonists and inhibit both NR4A1- and NR4A2-regulated responses. Chromatin immunoprecipitation assays show that β1-integrin and the methyltransferase gene G9a are regulated by both NR4A1 and NR4A2 acting as cofactors for Sp1- and Sp4-dependent gene expression. DIM-3,5 treatment results in the loss of one or more of these nuclear factors from the β1-integrin and G9a promoters. Single-cell and RNAseq analyses show that both receptors regulate common (<10%) and unique genes in SW480 colon cancer cells; however, functional enrichment analysis of the differentially expressed genes converges to several common pathways and gene ontology terms.