Search Results (1,778)

Background: Cardiovascular complications stemming from diabetes pose a grave threat to patients’ survival. Both type 1 diabetes (T1D) and type 2 diabetes (T2D) significantly increase the risk of heart failure, yet no reports have clarified whether there are differences in the pathway alterations involved in these two conditions. Investigating the heterogeneity of the cardiac remodeling between these two types of diabetes is conducive to reducing the incidence of cardiovascular events in diabetic patients in clinical practice. Methods: T1D and T2D models were established in adult mice, and the hearts were collected for RNA sequencing. Differential expression analysis (DEA) was performed. Integrating functional enrichment analyses, we probed into gene and pathway heterogeneity. Subsequently, we compared single-cell RNA sequencing (scRNA-seq) data of hearts from T1D and T2D mice, focusing on three cell populations (endothelial cells, macrophages, and fibroblasts) to identify gene and pathway differences. Finally, we evaluated shared genes and common signaling pathway changes across these three cell populations in both diabetes types. Results: We have successfully established T1D and T2D models in mice. Compared with shared genes, the two types of diabetes had more consistent pathway changes. Further scRNA-seq analysis identified endothelial cells, macrophages, and fibroblasts as significantly associated with the diabetic phenotype. In shared pathway, endothelial cells were significantly enriched in pathways related to endothelial proliferation and angiogenesis; macrophages were enriched in immune response pathways; and fibroblasts were enriched in pathways involving fibrosis, cell proliferation, and apoptosis. In endothelial cells, inflammatory response and fatty acid metabolism pathways were predominantly enriched in T1D, while energy metabolism pathways were dominant in T2D. In macrophages, antiviral immune pathways were specifically enriched in T1D, whereas macrophages in T2D were additionally implicated in the regulation of cardiomyocyte function. In fibroblasts, immune-related pathways were characteristically enriched in T1D, while cell respiration and energy supply pathways were prominent in T2D. Common functional enrichment pathways across the three cell types in both diabetes types mainly involved innate immune responses and cardiac morphogenesis, with the proportion of shared pathways being significantly higher than that of shared genes. Conclusions: This study, by combining RNA sequencing and scRNA-seq, revealed that cardiac pathologies induced by T1D and T2D exhibit a higher degree of consistent pathway changes compared to shared gene changes. Interventions targeting these common pathways may hold greater value in preventing and treating diabetic cardiomyopathy. Full article

Taste dysfunction, or dysgeusia, is a frequent symptom associated with infections and systemic diseases, yet its cellular and molecular basis remains poorly understood. The COVID-19 pandemic provided an opportunity to study dysgeusia as a “natural experiment” due to its high prevalence in those with acute and long COVID (LC). We investigated salivary epithelial cells (SECs) using single-cell RNA sequencing to elucidate molecular changes underlying taste dysfunction in LC. Functional enrichment analysis of SEC transcriptomes from individuals with bitter taste dysfunction (LC-D) revealed downregulation of genes involved in cytoskeletal dynamics and taste cell–nerve synapse assembly. Further, specific Type II and III taste receptor genes, critical for bitter taste perception, were reduced. Microbial defense markers such as Toll-like receptors TLR2 and TLR4 were also downregulated, suggesting chronic inflammation. These findings support a model of sustained dysregulated epithelial turnover due to impaired taste in LC-D. Saliva-based single-cell approaches offer promising tools for future diagnostics and mechanistic studies of taste systems. Full article

Background/Objectives: Milk somatic cells reflect the cellular composition and functional state of the lactating mammary gland and represent a valuable, non-invasive source for transcriptomic studies. Single-cell RNA sequencing (scRNA-seq) enables cell-type-resolved analysis of bovine milk; however, sequencing depth strongly influences the detection of lowly expressed genes and the resolution of transcriptional cell states. The aim of this study was to further characterise the single-cell transcriptome of bovine milk somatic cells, with particular emphasis on high-resolution gene expression profiling and cellular heterogeneity. Methods: Milk somatic cells were isolated from two healthy Holstein Friesian cows in mid-lactation and profiled using a droplet-based scRNA-seq platform. Newly generated high-depth datasets were integrated with two previously published bovine milk scRNA-seq datasets using an identical bioinformatics pipeline. Data integration, clustering and cell-type annotation were performed using the Seurat framework, and transcription factor expression was evaluated across datasets with different sequencing depths. Results: Single-cell transcriptomic analysis revealed a diverse cellular landscape in bovine milk, comprising epithelial, progenitor, and immune cell populations. Unsupervised clustering identified 21 transcriptionally distinct clusters, including multiple CD8⁺ T-cell subpopulations, monocytes, neutrophils, mast cells, and B cells, as well as luminal epithelial and luminal progenitor cells. While overall cell-type composition was comparable across datasets, deeply sequenced samples exhibited higher transcriptomic complexity and enabled refined resolution of immune and epithelial subpopulations. Deeper sequencing facilitated the detection of low-abundance transcription factors that were not observed in lower-depth datasets. Among these, NANOG was detected exclusively in deeply sequenced samples, suggesting the presence of rare transcriptional states associated with cellular plasticity. Conclusions: This study expands the single-cell transcriptomic landscape of bovine milk somatic cells and demonstrates the importance of sequencing depth for resolving functional cellular heterogeneity. The results highlight milk as a powerful, non-invasive source for investigating mammary gland biology and cellular plasticity during lactation. Full article

Pulmonary large-cell neuroendocrine carcinoma (LCNEC) is a rare lung malignancy characterized by an aggressive clinical course and an unfavorable prognosis. Next-generation sequencing (NGS) has revealed that LCNECs exhibit molecular features resembling either small-cell lung carcinoma (SCLC-like LCNEC) or non-small cell lung carcinoma (NSCLC-like LCNEC). This study aimed to characterize the incidence of actionable gene variants in a retrospective cohort of LCNEC patients using a targeted NGS approach. Microscopic diagnosis was established according to the 2021 World Health Organization (WHO) classification using a standard immunohistochemical (IHC) panel. In total, 216 LCNEC tumor samples were analyzed for molecular variants in 17 genes using the RNA-based Archer FusionPlex Lung NGS assay (Integrated DNA Technologies, USA) and the MiSeq platform (Illumina, USA)—an algorithm utilized for routine NSCLC diagnosis. Overall, 46 variants were identified in 46/216 (21.3%) tumor samples, with 28/216 (13%) LCNECs harboring at least one actionable molecular variant potentially targetable by registered or investigational agents. KRAS variants (5%; including G12C at 2%) and PIK3CA variants (5%) were the most prevalent, followed by RET single-nucleotide variants (3%), uncommon EGFR variants (1%), and BRAF class II and III variants (<1%). Notably, no classical EGFR exon 18–21 mutations nor ALK, FGFR1/2/3, or ROS1 alterations (mutations or fusions) were detected, despite the technical capability of the assay to identify such variants. A novel in-frame gene fusion (TMEM79::NTRK1) was identified in a single tumor sample (0.5%). Our results confirm that LCNECs harbor potentially targetable alterations in KRAS, PIK3CA, RET, BRAF, and NTRK1, albeit at lower frequencies than those typically observed in NSCLC. Full article

Background: The progression of idiopathic pulmonary fibrosis (IPF) involves distal airway remodeling and bronchiolization; however, the mechanisms driving these changes, particularly the contributions of epithelial stem cells, are not fully understood. K13⁺ hillock cells, normally quiescent in proximal airways, were examined for their potential contribution to IPF pathogenesis. Methods: Spatial immunofluorescence was used to profile K13 expression along the airway axes in IPF and control lungs. Multiplex staining complemented by ex vivo culture assays was used to test expression stability. Single-cell RNA-sequencing (scRNA-seq) data were re-analyzed to identify cell subclusters and pathway enrichments. Meanwhile, cell–cell communication was inferred by using CellChat. Results: K13 was ectopically upregulated in IPF honeycomb cysts, triggering a proximal-like pseudostratified phenotype. This shift was marked by surges in K13⁺ regionally overlapping expression patterns (K5⁺, ~9%; CC10⁺, ~53%; ACE-TUB⁺, ~44%; MUC5AC⁺, ~23%) and a decline in SOX2 expression (~95% to ~64%), with ~70% of residual SOX2^low cells exhibiting elevated K13. Accompanying the expansion of K13⁺ subclusters (basal: 1.8% to 41.5%; club: 10.7% to 31.5%), it was observed that the profibrotic markers (K17, S100A2, LGALS7, IGFBP6) and ontologies related to RNA processing, stress response, and senescence were also enriched. These subclusters also amplified pro-fibrotic signaling (e.g., TGF-β, SEMA3, and GALECTIN-9) associated with epithelial subtypes and HAS1^high fibroblasts. Conclusions: Here, we demonstrate that K13⁺ cell activation is a pivotal event, driving the dysregulated proximalization of distal airways in IPF through fate reprogramming and epithelial-mesenchymal crosstalk. Thus, elucidating these K13-mediated fate dynamics provides a critical framework for understanding IPF pathogenesis. Full article

Ionizing radiation (IR) causes severe vascular damage, yet the dynamic functional states and regulatory mechanisms of vascular endothelial cells (VECs) after irradiation remain poorly understood. To elucidate the underlying processes, we analyzed single-cell RNA sequencing data from mouse dorsal skin collected at multiple post-irradiation (p.i.) time points using trajectory inference, pathway enrichment, transcription factor activity inference, and cell–cell communication analyses. Our results showed that VECs exhibited marked temporal dynamics after irradiation, transitioning from early-stage stress responses to middle-stage angiogenic remodeling and late-stage restoration of homeostasis. A transient Gpihbp1⁺ capillary endothelial subpopulation (capVEC2) emerged predominantly during the middle stage (2–3 days p.i.) and was enriched for angiogenesis- and migration-related programs. Enhanced Sp1 regulatory activity was associated with its pro-angiogenic phenotype. At 2 days p.i., capVEC2 engaged in pro-angiogenic and pro-repair signaling with keratinocytes, whereas by 3 days p.i. these interactions shifted toward immune surveillance and tissue homeostasis, accompanied by increased pro-inflammatory and pro-apoptotic signaling and a decline in capVEC2 abundance. Collectively, our findings identify a radiation-induced, transient functional endothelial subpopulation that is associated with vascular–epidermal communication during skin repair post irradiation. Full article

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense desmoplastic stroma that actively drives malignant progression. However, the specific contributions of E3 ubiquitin ligases within the cancer-associated fibroblast (CAFs) compartment to the PDAC landscape remain largely elusive. Methods: Pancreatic tissue samples were collected from the First Affiliated Hospital of Harbin Medical University. Gene expression was analyzed by RT-PCR, and single-cell RNA sequencing (scRNA-seq) data were integrated for cell subtype identification. Kaplan-Meier survival analysis assessed gene expression and survival. Pseudotime analysis and CellChat evaluated fibroblast transitions and intercellular communication. Cell lines were transfected with RBX1 siRNAs, and protein levels were measured by Western blotting. Proliferation was assessed using colony formation and EdU staining. Statistical analyses were performed using R (v4.4) and GraphPad Prism 8.0. Results: Thirteen E3 ubiquitin ligases were significantly upregulated in PDAC and correlated with unfavorable clinical outcomes. Among these, RBX1 was identified as a candidate preferentially expressed in CAF populations and strongly associated with poor prognosis. Single-cell transcriptomic profiling and pseudotime analysis further revealed that RBX1-positive CAFs were predominantly involved in extracellular matrix remodeling and pro-tumorigenic pathways. Functional assays demonstrated that silencing RBX1 markedly inhibited PAAD cell proliferation and tumor growth both in vitro and in xenograft models. Mechanistically, RBX1 was found to upregulate Tenascin C (TNC) expression, while ectopic overexpression of TNC partially rescued the growth suppression induced by RBX1 knockdown. Conclusions: Our findings suggest that RBX1 facilitates PDAC progression through a CAF-related mechanism and TNC regulation, positioning RBX1 as a potential therapeutic target for PDAC intervention. Full article

Respiratory syncytial virus (RSV), a member of the genus Orthopneumovirus, is an etiological agent in infant lower respiratory tract infections (LRTIs) producing substantial global morbidity. Here, secretoglobin (Scgb1a1)-derived progenitors play a primary role in triggering innate, inflammatory, and cell state transitions in response to RSV LRTIs. Whether RSV activation of innate signaling in this epithelial sentinel population leads to chronic airway disease is unknown. To understand the role of innate signaling in Scgb1a1-derived progenitors, a model of RSV post-viral disease (PVLD) was developed and studied in the presence or absence of RelA conditional knockout (CKO). Single-cell RNA sequencing (scRNA-seq) studies showed that RSV-PVLD induced a transition of atypical, differentiation-intermediate, alveolar type 2 (aAT2) cells characterized by tumor protein 63 (TRP63), aquaporin 3 (AQP3), and Itgβ4 expression, as well as changes in PDGFRβ mesenchyme. A single-cell trajectory analysis and lineage-tracing experiments using Scgb1a1 CreER^TM X mTmG mice demonstrated that the Scgb1a1+ populations were precursors to the aAT2 population. Mechanistically, we found that the formation of the aAT2 population was prevented by RelA CKO. A differential gene expression analysis revealed that RSV-PVLD coordinately upregulates nuclear receptor subfamily 1 group D (Nr1d1/2), clock and basic helix-loop-helix ARNT-like 1 (Bmal) genes both in the aAT2 cell and in its Pdgfrα+ mesenchymal niche in a RelA-dependent manner. A systematic analysis of intercellular epithelial–mesenchymal communication in the scRNA-seq data showed that the clock-dysregulated epithelial–mesenchymal niche produces aberrant ANGPTL4 expression. ANGPTL4 upregulation was confirmed by the measurement of both its mRNA and protein. Moreover, ANGPTL4 is biologically active in the BALF of RSV-PVLD mice, inhibiting lipoprotein lipase activity. We conclude that RSV-PVLD is mediated, at least in part, by RelA signaling in Scgb1a1-derived epithelial progenitors, dysregulating ANGPTL4 signaling in an epithelial–mesenchymal niche, resulting in persistence of atypical alveolar epithelial cells with dysregulated of clock gene expression. Full article

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by genetic heterogeneity. Post-transcriptional regulation—particularly alternative polyadenylation (APA)—plays a critical role in the pathogenesis of ASD. APA controls mRNA stability, translational efficiency, and subcellular localization through modulating the length of the 3′ untranslated region of mRNA. APA profiling can uncover functionally relevant post-transcriptional alterations often missed by conventional gene expression analyses. However, current ASD analyses still largely rely on differential gene expression or individual APA event detection, which ignores the collective explanatory power of ASD risk genes or co-dysregulated functional gene modules within specific cell types. In this study, we present an integrative computational framework that combines matrix factorization and machine learning to identify ASD-associated gene modules driven by APA and to predict cell-type-specific ASD-related cells. Applied to human brain single-nucleus RNA sequencing (snRNA-seq) data, our approach systematically uncovers APA regulatory patterns that are specific to cell type, brain region, and sex in ASD. The identified APA modules are significantly enriched in pathways related to synaptic function, neurodevelopment, and immune response, with the strongest signals observed in excitatory neurons of the prefrontal cortex. Using APA genes from these modules as features, we built a classification model that effectively distinguishes ASD cells from normal cells. Moreover, we found that integrating APA with gene expression—two complementary modalities—substantially improves prediction accuracy, underscoring APA as an independent and biologically informative regulatory layer. Our work delineates a high-resolution APA regulatory landscape in ASD, offering novel insights and potential therapeutic avenues beyond transcriptional abundance. Full article

The immunosuppressive nature of porcine reproductive and respiratory syndrome virus (PRRSV) remains the central obstacle to its effective control. Conventional microRNA (miRNA)-based antiviral approaches are limited by their modest potency and the high risk of viral escape. Here, we rationally designed an engineered miRNA carrying a 5′-triphosphate (5′-PPP) terminus that integrates RIG-I-driven innate immune activation and sequence-specific gene silencing within a single molecule. In vitro-transcribed 5′-PPP miRNAs are efficiently recognized by the pattern-recognition receptor RIG-I, triggering a robust type I interferon response that counteracts PRRSV-induced immunosuppression. In MARC-145 cells, one such construct, 5′-PPP BZL-sRNA-20, potently inhibited PRRSV replication through the synergistic action of immune activation and gene silencing. However, in porcine alveolar macrophages (PAMs)—the natural host cells for PRRSV—the antiviral effect depended primarily on 5′-PPP-induced interferon responses, with the targeting sequence providing limited or context-dependent benefits. Dual-luciferase assays confirmed that the gene-silencing activity depends on 5′-PPP modification, which enhances the stability of BZL-sRNA-20. This bifunctional strategy establishes an “immune activation plus targeting” paradigm by simultaneously acting as a RIG-I ligand that triggers broad antiviral responses and specifically cleaves viral RNA via direct base-pairing to conserved regions of the PRRSV genome. These findings reveal the potential of engineered 5′-PPP miRNAs as immunomodulatory antiviral agents, while highlighting that the contribution of RNAi targeting varies depending on the cellular context. Full article

As a typical C3-C4 intermediate plant, cassava (Manihot esculenta Crantz) exhibits high photosynthetic efficiency and low photorespiration. NADP-malic enzyme (NADP-ME) is a key enzyme in the C4 photosynthetic pathway that provides elevated ${\mathrm{CO}}_2$ concentrations for Rubisco. However, research on NADP-ME in C3-C4 intermediate species remains limited. In this study, we identified four NADP-ME genes in the cassava genome, with segmental duplication serving as the primary driving force for gene evolution. Cis-acting element analysis indicated potential roles of MeNADP-ME genes in environmental adaptation, stress responses, and growth regulation. Expression profiling using bulk RNA sequencing and single-cell RNA sequencing revealed distinct expression patterns in different tissues and cell subsets. Comparative analysis with Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) NADP-ME families demonstrated that MeNADP-ME3 exhibits bundle sheath cell-specific expression analogous to ZmchlC4NADP-ME in maize. Notably, photosynthetic genes and plasmodesmata (PD)-related genes exhibited high co-expression within mesophyll subcluster 13 and bundle sheath cells, providing molecular evidence for a limited C4 photosynthetic pathway in cassava. Protein–protein interaction predictions implicated MeNADP-ME3 in photosynthetic carbon metabolism and photorespiration regulation. Furthermore, qRT-PCR revealed significant responsiveness of MeNADP-ME3 to various abiotic stresses, and confocal imaging confirmed its chloroplast localization. Functional validation demonstrated that Arabidopsis overexpressing MeNADP-ME3 exhibited 30–120% enhanced antioxidant enzyme activities (SOD, POD, CAT) and 20–32% reduced oxidative damage markers (MDA, ${\mathrm{H}}_2{\mathrm{O}}_2$) under drought and salt stresses. These findings reveal the evolutionary trajectory of NADP-ME genes in C3-C4 intermediate species and provide genetic resources for developing stress-tolerant cassava cultivars. Full article

The human cerebral cortex is organized into distinct area-specific regions along the rostral–caudal axis, yet current human brain organoid models incompletely recapitulate this regional diversity. Here, we establish an area-specific brain organoid platform by leveraging transcription factors (TFs) identified through re-analysis of in vivo human cortical transcriptomic datasets. Publicly available single-cell RNA sequencing datasets from human developing cortex were re-analyzed to identify differentially expressed genes associated with rostral and caudal cortical identities. From this analysis, we identified SP9 (rostral-enriched) and DMRTA2 (caudal-enriched) as candidate TFs governing regional specification. To model cortical area identity, these TFs were overexpressed in an inducible manner during human cerebral organoid (hCO) generation. Overexpression of SP9 resulted in hCOs exhibiting rostral cortical characteristics, whereas DMRTA2 overexpression promoted caudal cortical features. The resulting hCOs showed distinct regional identities, reflected by differential expression of area-specific markers. In addition, these regional identities were accompanied by distinct functional phenotypes, as calcium imaging revealed divergent patterns of spontaneous neural activity between rostral and caudal hCOs. Altogether, our findings demonstrate that overexpression of TFs enables the controlled generation of area-specific hCOs. This approach provides a scalable and reproducible platform for studying human cortical regionalization and offers a framework for investigating region-specific mechanisms underlying neurodevelopmental and neurological disorders. Full article

Background/Objectives: Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to sustain proliferation, survive under metabolic stress, and develop therapeutic resistance. While oncogenic signaling pathways regulating cancer metabolism have been extensively studied, increasing evidence indicates that non-coding RNAs (ncRNAs) play essential roles in coordinating metabolic adaptation. This review aims to synthesize current knowledge on long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) as important but relatively less characterized regulators of cancer metabolic adaptation and discuss their potential as biomarkers and therapeutic targets. Methods: We analyzed their roles across multiple types of cancer, prioritizing studies that integrate ncRNA profiling with metabolomics and mechanistic investigations, with particular attention to their diagnostic, prognostic, and predictive value. Results: LncRNAs and circRNAs regulate major metabolic pathways, including glycolysis, mitochondrial function, glutaminolysis, lipid metabolism, and redox balance. They act through transcriptional and epigenetic mechanisms, protein scaffolding, peptide encoding, and miRNA sponging, frequently converging on key regulators such as HIF-1α, c-Myc, p53, AMPK, and mTOR. However, many reported associations remain largely correlative, with limited integration of quantitative metabolic flux analyses and insufficient validation in physiologically relevant models. Conclusions: Although lncRNAs and circRNAs constitute an important context-dependent regulatory layer linking oncogenic signaling to metabolic reprogramming, future studies should combine ncRNA perturbation with stable isotope tracing, fluxomics, spatial metabolomics, long-read sequencing, and single-cell approaches to define causal and spatially resolved metabolic functions. Such integrative strategies may improve biomarker development and support ncRNA-informed, metabolism-oriented therapeutic interventions. Full article

The role of platelet-derived growth factor D (PDGFD) in mesenchymal cells is well-established, but its specific function in skeletal muscle generation remains unknown. This study reveals for the first time PDGFD’s dual regulatory role in myogenesis: it acts both as a “guardian” maintaining the myoblast pool and as an “initiator” driving myogenic differentiation. Through single-cell RNA sequencing analysis of skeletal muscle from Jiangquan Black pigs, we identified PDGFD as a common candidate gene for both muscle and fat development. In the C2C12 cell model, PDGFD knockdown significantly inhibited cell proliferation and promoted apoptosis, while overexpression enhanced viability and inhibited apoptosis, indicating its critical role in maintaining myoprogenic precursor cell homeostasis. Further studies revealed that PDGFD interference downregulated key myogenic differentiation markers MyoD and MyoG, inhibiting differentiation. Its expression peaked during mid-differentiation (D5), suggesting temporal regulation of differentiation. Interestingly, although PDGFD primarily acts through the PI3K/Akt pathway downstream of PDGFR-β, PDGFD knockdown did not show significant synergistic effects with PI3K/Akt pathway activation in inhibiting differentiation. This suggests PDGFD may specifically regulate myogenic differentiation via an independent or parallel signaling axis. This study not only expands the known functions of PDGFD in muscle biology but also provides new insights into the mechanisms by which growth factors coordinate cell fate decisions. Full article

Myocardial ischemia/reperfusion (MI/R) injury affects heart attack outcomes. Endothelial cells dysfunction immediately after MI/R, but the key molecules and how to block them remain unclear. We combined single-cell atlas analysis, AI simulation, and experimental single-cell RNA sequencing data from mouse MI/R; we did quality control, cell annotation, hdWGCNA, and differential gene screening to identify endothelial genes. We constructed a protein network with STRING, predicted structure with AlphaFold3, and used AutoDock for molecular docking to find potential drugs. Virtual knockout simulations were used to check gene deletion effects. The compound andrographolide (AG) was tested in in vitro and in vivo MI/R models by measuring cell viability, inflammation, pathway activity, infarct size, and cardiac function. Single-cell analysis showed that S100 calcium binding protein A8 (S100A8) is an important element in vascular inflammation. It promotes inflammation by interacting indirectly with Cluster of differentiation 14 (CD14). Molecular docking showed that AG binds stably to S100A8. In vitro, AG reduced endothelial injury and blocked the IL-17 pathway. In vivo, AG reduced infarct size, improved cardiac function, and lowered S100A8 and IL-17 pathway proteins. Using single-cell analysis, AI, and experiments, we showed that S100A8 is related to MI/R injury. Andrographolide protects microvasculature via the S100A8 pathway, offering a promising treatment approach and new insights into heart injury mechanisms. Full article