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SnRNA-seq reveals the ameliorative effects of optimized Xueyu Jingshen formula on high altitude cerebral edema by modulating energy metabolism, inflammation and BBB integrity.
BACKGROUND: High-altitude cerebral edema (HACE) is a type of lethal neurological emergency, whose underlying pathogenic mechanisms and core therapeutic targets remain largely unelucidated to date. Optimized Xueyu Jingshen formula (OXJF) has been shown to exert definite clinical efficacy in the intervention of HACE, whereas the underlying molecular mechanisms remain to be systematically and deeply explored. PURPOSE: This work leverages single-nucleus RNA sequencing (snRNA-seq) to elucidate pathological rewiring of cell-type-specific interactions in HACE, and to uncover molecular targets of OXJF, thereby laying the groundwork for novel clinical therapeutic strategies. METHODS: We firstly conducted a qualitative analysis of the chemical components of OXJF using UHLPC-Q-TOF-MS. We comprehensively evaluated the pharmacodynamic effects of OXJF firstly by detecting the behavioral changes, brain water content, BBB permeability and pathological damage of HACE mice, combined with the analysis of cerebral microvascular blood flow imaging. Afterwards, we constructed a cerebral cell atlas of HACE mice by using snRNA-seq and achieved systematic identification of various cell types. The proportion of each cell type was quantified and verified through multiplex immunofluorescence. AUCell was employed to ascertain the activity of gene modules, thereby identifying cell population exhibiting specific functional states or transcriptional signatures. Differential genes and signaling pathways enrichment analyses were employed to elucidate vital biomarkers and pathways. To clarify the differentiation patterns of cell subtypes, we introduced pseudotime analysis for relevant investigations. CellChat was performed to decipher cell-cell communication networks based on ligand-receptor pairs analysis. Ultimately, we verified whether mitochondrial Complex I was the pivotal target mediating cellular responses to hypoxic stimulation and OXJF intervention in C8-D1A, HT22, BV2 and MO3.13 cell lines. RESULTS: A total of 163 chemical components, mainly including flavonoids, organic acids and terpenoids, were successfully identified. Pharmacodynamic experiments certified that OXJF could largely improve cognitive and motor functions, and reduce BWC in HACE mice. Meanwhile, by upregulating the expression of tight junction proteins, OXJF reversed high-altitude-induced increases in BBB permeability, and effectively alleviated cerebral microvascular hemodynamic disorders as well as pathological damage in HACE mice. 92,616 annotated nuclei were mainly identified as astrocyte, excitatory and inhibitory neurons, microglia, oligodendrocyte precursor cells and oligodendrocytes. Cell proportion analysis and multiplex immunofluorescence staining verified that no significant alterations in the proportions of the aforementioned cell populations were observed in both HACE mice and OXJF-treated mice. Gene modules associated with energy metabolism, inflammation and BBB integrity exhibited high AUCell scores, implying their potential involvement as underlying mechanisms through which OXJF ameliorated HACE. Furthermore, key regulatory components within the above modules were identified including Hif1a, Vhl, Ikbkb, Rela, Tlr4, Tnfrsf11a, Irak1, Ndufb8, Nfe2l2, Ndufs4, Vegfa, Pdgfa and Gja1 genes. Cell trajectory analysis indicated that diverse cell types manifested marked heterogeneity in response to HACE and OXJF intervention. Further CellChat analysis unveiled widespread ligand-receptor interactions across cell population, and these interactions were mapped to central signaling molecules including ADGRB, EPHB, PTN, UNC5, CLDN, CypA, JAM, SLIT and GAP. In vitro cellular assays had confirmed that OXJF suppressed reactive oxygen species production and mitochondrial permeability transition pore opening via upregulating the expression of mitochondrial Complex Ⅰ subunits NDUFA9, NDUFS4 and NDUFB8. This effect drove a noteworthy increase in ATP content, which in turn rescued energy supply in C8-D1A, HT22, BV2 and MO3.13 cell lines. CONCLUSION: This study provides and defines the first systematic snRNA-seq profiles under OXJF-treated HACE mice. The potential mechanisms of OXJF in treating HACE were related to maintaining energy metabolism, inflammation and BBB homeostasis.
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Extracellular cancer‑associated fibroblasts: A novel subgroup in the cervical cancer microenvironment that exhibits tumor‑promoting roles and prognosis biomarker functions.
Tumor invasion and metastasis are the processes that primarily cause adverse outcomes in patients with cervical cancer. Cancer-associated fibroblasts (CAFs), which participate in cancer progression and metastasis, are novel targets for the treatment of tumors. The present study aimed to assess the heterogeneity of CAFs in the cervical cancer microenvironment through single-cell RNA sequencing. After collecting five cervical cancer samples and obtaining the CAF-associated gene sets, the CAFs in the cervical cancer microenvironment were divided into myofibroblastic CAFs and extracellular (ec)CAFs. The ecCAFs appeared with more robust pro-tumorigenic effects than myCAFs according to enrichment analysis. Subsequently, through combining the ecCAF hub genes and bulk gene expression data for cervical cancer obtained from The Cancer Genome Atlas and Gene Ontology databases, univariate Cox regression and least absolute shrinkage and selection operator analyses were performed to establish a CAF-associated risk signature for patients with cancer. The established risk signature demonstrated a stable and strong prognostic capability in both the training and validation cohorts. Subsequently, the association between the risk signature and clinical data was evaluated, and a nomogram to facilitate clinical application was established. The risk score was demonstrated to be associated with both the tumor immune microenvironment and the therapeutic responses. Moreover, the signature also has predictive value for the prognosis of head and neck squamous cell carcinoma, and bladder urothelial carcinoma, which were also associated with human papillomavirus infection. In conclusion, the present study assessed the heterogeneity of CAFs in the cervical cancer microenvironment, and a subgroup of CAFs that may be closely associated with tumor progression was defined. Moreover, a signature based on the hub genes of ecCAFs was shown to have biomarker functionality in terms of predicting survival rates, and therefore this CAF subgroup may become a therapeutic target for cervical cancer in the future.
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Decoding the epithelial-stromal interactome in allergic rhinitis through single-cell multiomics integration.
BACKGROUND: Allergic rhinitis (AR) is a prevalent chronic condition, yet the cellular and molecular changes associated with its pathogenesis remain incompletely understood. OBJECTIVE: We sought to construct a comprehensive cellular atlas of the nasal mucosa in AR and nonallergic rhinitis and elucidate disease-associated transcriptional and epigenetic alterations. METHODS: We performed single-cell RNA sequencing and single-cell ATAC sequencing on nasal mucosa samples from 39 subjects (25 AR, 14 nonallergic rhinitis). Differentially expressed gene analysis, differentially accessible peaks analysis, cell-cell communication, trajectory inference, and gene regulatory network reconstruction were applied. A deep learning framework was developed to integrate multiomics data for disease prediction. RESULTS: We profiled 1,024,146 cells, constructing a comprehensive nasal mucosa atlas. The AR epithelium exhibited aberrant differentiation with suppressed maturation of basal and club cells, while fibroblasts displayed inflammatory activation and matrix remodeling signatures. Epithelial-stromal cross talk was enhanced in the AR group. Cell subset-specific epigenetic alterations were also observed. Single-cell multiomics for AR integrative analysis, or scMARIA, can simultaneously predict AR risk and clinically relevant disease parameters, and prioritize putative cell type-specific regulatory linkages. CONCLUSION: This multiomics study establishes a comprehensive molecular framework of the nasal mucosa, revealing dysregulated epithelial-stromal interactions and gene regulatory networks that are correlated with AR status.
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Clinical characterization of EFHD2 (swiprosin-1) in Glioma-associated macrophages and its role in regulation of immunosuppression.
Glioblastoma has been extensively studied due to its high mortality and short survival. The evolution mechanism of tumor-associated macrophages (TAMs) to Glioma-associated microglia and macrophages (GAMs) in the tumor microenvironment (TME) remains to be elucidated. The tumor cell-to-cell interaction patterns have not been well defined yet. The EF-Hand Domain Family Member D2 (EFHD2) has been reported to be differentially expressed as an immunomodulatory molecule in a variety of cancers. But large-scale clinical data from multiple ethnic communities have not been used to investigate the role of EFHD2 in glioma. RNA-seq data from 313 or 657 glioma patients from the Chinese Glioma Genome Atlas (CGGA) database and 603 glioma patients from the Cancer Genome Atlas (TCGA) database were analyzed retrospectively. Cell localization was performed using single-cell sequencing data from the CGGA database and the GSE131928 dataset. Mouse glioma cell lines and primary macrophages isolated from Efhd2 knockout mice were co-cultured to validate the immunomodulatory effects of EFHD2 on macrophages and the remodeling of TME of glioblastoma. EFHD2 is enriched in high-grade gliomas, isocitrate dehydrogenase wild-type, and 1p/19q non-co-deficient gliomas. It is a potential biomarker of glioma-proneuronal subtypes and an independent prognostic factor for overall survival in patients with malignant glioblastoma. EFHD2 regulates the monocyte-macrophage system function and positively correlates with immunosuppressive checkpoints. Further experimental data demonstrates that Efhd2 influences the polarization state of GAMs and inhibits the secretion of TGF-β1. In vitro experiments have revealed that macrophages lacking Efhd2 suppress the vitality of two glioma cell lines and decelerate the growth of glioma xenografts. In conclusion, EFHD2 promises to be a key target for TME-related immunotherapy.
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Bioinformatics analysis of aging-related genes in thoracic aortic aneurysm and dissection.
OBJECTIVE: Thoracic aortic aneurysm and dissection (TAAD) is a cardiovascular disease with a high mortality rate. Aging is an important risk factor for TAAD. This study explored the relationship between aging and TAAD and investigated the underlying mechanisms, which may contribute to the diagnosis and treatment of TAAD. METHODS: Human aging genes were obtained from the Aging Atlas official website. Various datasets were downloaded from the GEO database:the human TAAD dataset GSE52093 were used for screening differentially expressed genes (DEGs); GSE137869, GSE102397 and GSE153434 were used as validation sets, and GSE9106 was used for diagnostic prediction of receiver operating characteristic (ROC) curves. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), and protein-protein interaction (PPI) network analysis were used to screen differentially co-expressed genes from human aging genes and TAAD. Using five methods of the cytoHubba plugin in Cytoscape software (Degree, Closeness, EPC, MNC, Radiality), hub genes were identified from the differentially co-expressed genes. Single-cell RNA sequencing was used to verify the expression levels of hubgenes in different cell types of aortic tissue. ROC curves were used to further screen for diagnostic genes. RESULTS: A total of 70 differentially co-expressed genes were screened from human aging genes and DEGs in human TAAD dataset GSE52093. GO enrichment analysis revealed that the DEGs played a major role in regulating DNA metabolism and damaged DNA binding. KEGG enrichment analysis revealed enrichment in the longevity regulating pathway, cellular senescence, and HIF-1 signaling pathway. GSEA indicated that the DEGs were concentrated in the cell cycle and aging-related p53 signaling pathway. The five identified hubgenes were MYC, IL6, HIF1A, ESR1, and PTGS2. Single-cell sequencing of the aging rat aorta showed that hubgenes were expressed differently in different types of cells in aortic tissue. Among these five hubgenes, HIF1A and PTGS2 were validated in the aging dataset GSE102397; MYC, HIF1A and ESR1 were validated in the TAAD dataset GSE153434. The combined area under the diagnostic ROC curve (AUC) values for the five hub genes were >0.7 in the testing and training sets of the dataset GSE9106. The combined AUC values of MYC and ESR1 were equal to the combin ed AUC values of the five hub genes. CONCLUSION: The HIF-1 signaling pathway may play an important role in TAAD and aging. MYC and ESR1 may have diagnostic value for aging-related TAAD.
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A predefined sulphur-handling transcriptional programme is associated with colorectal cancer molecular subtypes.
BACKGROUND: Colorectal cancer (CRC) exhibits substantial metabolic and transcriptomic heterogeneity, but the relationship between sulphur-handling transcriptional programmes and consensus molecular subtypes (CMS) remains incompletely resolved. METHODS: We analysed a predefined 14-gene sulphur-handling panel across The Cancer Genome Atlas colon and rectal adenocarcinoma cohorts (TCGA-COAD/READ) and six independent HG-U133 validation cohorts (n = 1,337 tumours). Analytical workflows integrated differential expression comparing tumours with adjacent-normal mucosa, CMS association modelling, random-effects meta-analysis, adjustment for three orthogonal tumour-purity estimators (ESTIMATE, consensus purity estimates, and ABSOLUTE), reference-based single-cell deconvolution with independent cross-atlas verification, and direction-aware therapeutic triage. RESULTS: Colorectal tumours demonstrated branch-specific transcriptional alterations, marked by coordinated downregulation of sulphur oxidation and clearance genes (SQOR, ETHE1, TST) and of the cysteine-catabolism genes CDO1 and CTH, and selective upregulation of SLC7A11 and AHCY, whereas CBS was not significantly altered. The composite programme score differed across CMS subtypes and was consistently lower in mesenchymal CMS4 tumours than in non-CMS4 tumours across all discovery and validation series. This association remained statistically detectable after adjustment for three global tumour-purity metrics. The programme score showed no statistically detectable independent association with overall, recurrence-free, or disease-free survival. Single-cell deconvolution supported expression across epithelial, stromal, and immune compartments. Direction-aware therapeutic triage prioritised SLC7A11/xCT inhibition as an exploratory candidate for preclinical testing. CONCLUSION: Sulphur-handling transcriptional architecture is reproducibly linked to CRC molecular taxonomy and is not fully explained by measured tumour purity variation, providing a computational framework for future functional and compartment-resolved investigation of sulphur metabolism in stratified CRC populations.
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GeoMx and RNAscope: A Comparative Assessment of Their Utility for Spatial mRNA Expression Profiling in Formalin-Fixed Breast Cancer Tissue.
mRNA expression analysis in formalin-fixed tissue is essential for many biomarker and tumor microenvironment studies. NanoString GeoMx Digital Spatial Profiler is a recent technique that offers high-plex spatial transcriptomics (up to 18,000 genes), while RNAscope (1-4 plex) is a well-established mRNA in situ hybridization method. This study compares quantitative expression estimates obtained by GeoMX and RNAscope. Serial sections from two TMAs containing mammary cancers, including triple-negative (n = 48) and varying ER/HER2 status (n = 45), were analyzed using GeoMx Cancer Transcriptomics Atlas (CTA) covering approximately 1,800 genes and RNAscope probes for GATA3, SOX10, and PD-L1 mRNAs. Expression was quantified as counts/cell (GeoMx, geometric mean of five probes per gene) and average dots/cell (RNAscope, QuPath). Positivity was defined as above the limit of quantification (GeoMx) or > 0.1 dots/cell (RNAscope). High correlation was observed for GATA3 (R = 0.87) and SOX10 (R = 0.77) between methods. PD-L1 expression was high in only one core, precluding correlation analysis. RNAscope demonstrated higher sensitivity and a broader dynamic range than GeoMx. In conclusion, GeoMx CTA and RNAscope exhibit a strong correlation. GeoMx enabled highly multiplexed gene expression analysis, whereas RNAscope provided better sensitivity and single-cell resolution. The choice of method should be guided by study objectives.
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Hymeglusin Enhances the Pro-Apoptotic Effects of Venetoclax in Acute Myeloid Leukemia.
Venetoclax is used for the priority treatment of elderly patients with acute myeloid leukemia (AML). Resistance or intolerance to venetoclax offsets its clinical benefits in some patients. Combination strategies with other drugs are promising alternatives to overcome the current complications associated with venetoclax use. Hymeglusin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMGCS1), regulates the mevalonate pathway, which is vital for AML growth and chemosensitivity. The effects of the combination of venetoclax and hymeglusin on AML were explored in this study. The correlations between HMGCS1 and apoptosis-related genes were analyzed using the Gene Expression Profiling Interactive Analysis 2 and The Cancer Genome Atlas databases. Apoptosis and cell viability were detected in HL-60 and KG-1 cells after treatment with gradient concentrations of venetoclax or hymeglusin. The transcriptomic profiles of HL-60 and KG-1 cells were compared via RNA-Seq analysis. The effects of venetoclax and hymeglusin on apoptosis were validated in primary cells. The results showed that HMGCS1 expression was closely associated with apoptosis-related genes based on the data from large clinical databases. B cell lymphoma (BCL)-2 expression was elevated in AML and negatively associated with overall survival. Hymeglusin decreased BCL2 expression levels in HL-60 and KG-1 cells. Venetoclax and hymeglusin inhibited cell viability in both cell lines, but induced apoptosis in HL-60 cells. This discrepancy in sensitivity to hymeglusin may be attributed to the positive increase in the expression levels of HMGCS1 and multiple upregulated pro-leukemia genes in KG-1 cells. Combination treatment with venetoclax and hymeglusin significantly increased the apoptotic rates compared to single-agent treatment in both AML cell lines and primary AML cells. Furthermore, the combination strategy did not result in remarkably enhanced toxicity in normal mononuclear cells. Collectively, hymeglusin enhanced the effects of venetoclax on apoptosis. This combination strategy showed enhanced antileukemic activity with acceptable toxicity in AML.
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Primary nasal viral infection rewires the tissue-scale memory response.
The nasal mucosa is frequently the initial site of respiratory viral infection, replication, and transmission. Recent work has started to clarify the independent responses of epithelial, myeloid, and lymphoid cells to viral infection in the nasal mucosa, but their spatiotemporal coordination and relative contributions remain unclear. Furthermore, understanding whether and how primary infection shapes tissue-scale memory responses to secondary challenge is critical for the rational design of nasal-targeting therapeutics and vaccines. Here, we generated a single-cell RNA-sequencing (scRNA-seq) atlas of the murine nasal mucosa sampling three distinct regions before and during primary and secondary influenza infection. Primary infection was largely restricted to respiratory mucosa and induced stepwise changes in cell type, subset, and state composition over time. Type I Interferon (IFN)-responsive neutrophils appeared 2 days post infection (dpi) and preceded transient IFN-responsive/cycling epithelial cell responses 5 dpi, which coincided with broader antiviral monocyte and NK cell accumulation. By 8 dpi, monocyte-derived macrophages (MDMs) expressing Cxcl9 and Cxcl16 arose alongside effector cytotoxic CD8 and Ifng-expressing CD4 T cells. Following viral clearance (14 dpi), rare, previously undescribed Krt13+ nasal immune-interacting floor epithelial (KNIIFE) cells expressing multiple genes with immune communication potential increased concurrently with tissue-resident memory T (TRM)-like cells and early IgG+/IgA+ plasmablasts. Proportionality analysis coupled with cell-cell communication inference, alongside validation by in situ microscopy, underscored the CXCL16-CXCR6 signaling axis between MDMs and effector CD8 T cells 8dpi and KNIIFE cells and TRM cells 14 dpi. Secondary influenza challenge with a homologous or heterologous strain administered 60 dpi induced an accelerated and coordinated myeloid and lymphoid response without epithelial proliferation, illustrating how tissue-scale memory to natural infection engages both myeloid and lymphoid cells to reduce epithelial regenerative burden. Together, this atlas serves as a reference for viral infection in the upper respiratory tract and highlights the efficacy of local coordinated memory responses upon rechallenge.
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A single-cell atlas of the Culex tarsalis midgut during West Nile virus infection.
The mosquito midgut functions as a key interface between pathogen and vector. However, studies of midgut physiology and virus infection dynamics are scarce, and in Culex tarsalis-an extremely efficient vector of West Nile virus (WNV)-nonexistent. We performed single-cell RNA sequencing on Cx. tarsalis midguts, defined multiple cell types, and determined whether specific cell types are more permissive to WNV infection. We identified 20 cell states comprising 8 distinct cell types, consistent with existing descriptions of Drosophila and Aedes aegypti midgut physiology. Most midgut cell populations were permissive to WNV infection. However, there were higher levels of WNV RNA (vRNA) in enteroendocrine cells (EE), suggesting enhanced replication in this population. In contrast, proliferating intestinal stem cells (ISC) had the lowest levels of vRNA, a finding consistent with studies suggesting ISC proliferation in the midgut is involved in infection control. ISCs were also found to have a strong transcriptional response to WNV infection; genes involved in ribosome structure and biogenesis, and translation were significantly downregulated in WNV-infected ISC populations. Notably, we did not detect significant WNV-infection induced upregulation of canonical mosquito antiviral immune genes (e.g., AGO2, R2D2, etc.) at the whole-midgut level. Rather, we observed a significant positive correlation between immune gene expression levels and vRNA load in individual cells, suggesting that within midgut cells, high levels of vRNA may trigger antiviral responses. Our findings establish a Cx. tarsalis midgut cell atlas, and provide insight into midgut infection dynamics of WNV by characterizing cell-type specific enhancement/restriction of, and immune response to, infection at the single-cell level.