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Page 142 of 492-
Single-Nucleus Transcriptional Profiling Revealed Cell Diversity and Albino Mutation Mechanism in the Skin of Channa argus.
Body color is the most prominent phenotypic trait in animals. To investigate the molecular regulatory mechanisms underlying skin pigmentation and body color in Channa argus, single-nucleus RNA sequencing technology was employed to analyze cell diversity and functional changes in the skin of normal and albino C. argus. Three pigment-related cell types, seven immune-related cell types, and nine other skin-related structural and functional cell types were identified. The skin of albino C. argus, which appears white to the naked eye, contains numerous melanocytes and iridophores with reflective silver properties. Compared to normal C. argus, melanocytes in albino individuals contained fewer melanin granules, while iridophores exhibited increased chromogenic substances. Melanocyte-specific genes-kitlg, myo5a, and scarb1-were significantly downregulated in albino melanocytes (p < 0.05). Conversely, iridophore-specific genes alk, pnp, and gpnmb were significantly upregulated in albino skin, whereas mlph was significantly downregulated (p < 0.05). Weighted gene co-expression network analysis revealed that scarb1 was associated with the melanocyte module, alk was identified as a core gene, and pnp was linked to the iridophore module. Functionally, scarb1 is involved in pigment transport, pnp in purine synthesis, and alk is essential for iridophore development. Therefore, scarb1, pnp, and alk may be correlated to albinism in C. argus. Overall, this study constructed a single-cell transcriptional atlas of C. argus skin, providing valuable reference data for further research into the regulatory mechanisms governing body color formation and maintenance in this species.
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Deciphering the cellular landscape and genetic underpinnings of fiber diameter determined by dermal papilla cells in fine-wool sheep.
Wool fiber diameter (FD) is a key economic trait in fine-wool sheep, and its growth process is closely associated with the development and cyclical growth of hair follicles (HFs). Therefore, a comprehensive understanding of the molecular mechanisms regulating HF development is of significant importance. In this study, single-cell RNA sequencing (scRNA-seq) was performed on skin tissues from six half-sibling fine-wool sheep reared under identical conditions but exhibiting different fiber diameters, successfully constructing a transcriptional atlas of the HF microenvironment. A total of 59,732 high-quality cells were captured and systematically annotated into 14 distinct cell types using known marker genes. Each cell type exhibited unique gene expression profiles. Analysis of cell proportions across the three experimental groups revealed that the ultra-fine group had a higher proportion of dermal papilla cells (DPCs), hair follicle stem cells (HFSC), and outer root sheath cells (ORS) compared to the other two groups. Cell-cell communication analysis identified multiple ligand-receptor pairs involved in HF regulation, suggesting that DPCs may play an important role in modulating HF growth and development. Pseudotime trajectory analysis reconstructed the developmental dynamics of the epidermal lineage. Furthermore, we identified that DPCs may exist in three distinct states to perform their physiological functions. To elucidate the molecular mechanisms underlying FD, we identified and analyzed differentially expressed genes across cell types in the ultra-fine, medium-fine, and fine wool groups, and performed cellular-level validation of CRABP1, which was significantly upregulated in DPCs, revealing that overexpression of this gene significantly upregulated PCNA and CTNNB1, suppressed the expression of SFRP2 and BMP2, and subsequently promoted the proliferation of DPCs. This study successfully constructed a single-cell atlas of skin tissues from one-year-old fine-wool sheep, revealing the potential important role of DPCs in HF development and uncovering potential molecular mechanisms of FD. These findings provide new insights into the biology of HF development in sheep and offer valuable references for molecular-targeted precision breeding of ultra-fine wool sheep.
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A single-cell transcriptomic atlas of immune cells in Wilson disease identifies copper-specific immune regulation.
Wilson disease (WD) is caused by mutations of the copper-transporting gene, ATP7B, leading to abnormal copper metabolism. A better characterization of WD is essential in understanding the effects of excess copper and how it disrupts immune regulation and hematopoietic development. Furthermore, the exploration of the relationship between copper-mediated proliferation or cuproptosis and immune regulation is critical for developing new immune therapies. Therefore, we performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) to develop an atlas of the immune landscape. Cells were clustered into several immune subsets, and cuproptosis-associated genes were assessed. Differential expression analysis was performed to identify WD-specific signatures by comparing transcriptome profiles of patients with WD with HDs. Excess copper impaired immune homeostasis and hematopoietic development. Then, we developed a map of the immune landscape of patients with WD. Excess copper is involved in the metabolic reprogramming of immune cells, such as glycolysis in CD14+ monocytes. We found that the antigen processing-related pathway is dysregulated in immune cells of patients with WD. Our study revealed that abnormal copper concentration influences the expression of HLA-I and HLA-II molecules. It is noteworthy that a high concentration of intracellular copper differs significantly from the high concentration of extracellular copper. We have also identified a gene set of neurologic abnormalities, which were dysregulated in PBMCs of patients with WD. We also observed abnormal expression of cuproptosis-associated genes in proliferating or malignant cells, providing new insights into the application of cuproptosis in cancer treatment.
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Integrated single-cell and bulk RNA sequencing analyses reveal a prognostic signature of cancer-associated fibroblasts in head and neck squamous cell carcinoma.
Objectives: To identify a prognosis-related subtype of cancer-associated fibroblasts (CAFs) in head and neck squamous cell carcinoma (HNSCC) and comprehend its contributions to molecular characteristics, immune characteristics, and their potential benefits in immunotherapy and chemotherapy for HNSCC. Materials and Methods: We performed single-cell RNA sequencing (scRNA-seq) analysis of CAFs from the samples of HNSCC patients derived from Gene Expression Omnibus (GEO), to identify the prognosis-related subtype of CAFs. CAFs were clustered into five subtypes, and a prognosis-related subtype was identified. Univariate and multivariate cox regression analyses were performed on the cohort selected from The Cancer Genome Atlas (TCGA) to determine signature construction, which was validated in GSE65858 and GSE42743. A prognostic signature based on 4 genes was constructed, which were derived from prognosis-related CAFs. The molecular characteristics, immune characteristics as well as the predicted chemosensitivity and immunotherapeutic response in the signature-defined subgroups were analyzed subsequently. Results: The patients with higher CAF scores correlated with poor survival outcomes. Additionally, a high CAF score correlated with lower infiltration levels of many immune cells including M1 macrophages, CD8+ T cells, follicular T helper cells, monocytes, and naïve B cells. High CAF score also demonstrated different enrichment pathways, mutation genes and copy number variated genes. Furthermore, patients with high CAF scores showed lower sensitivity for chemotherapy and immunotherapy than those with low CAF scores. Conclusion: The results of our study indicate the potential of the CAF signature as a biomarker for the prognosis of HNSCC patients. Furthermore, the signature could be a prospective therapeutic target in HNSCC.
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Single-Cell Profiling Uncovers the Roles of Endometrial Fibrosis and Microenvironmental Changes in Adenomyosis.
PURPOSE: Adenomyosis (AM) is a common benign uterine disorder that has deleterious effects on women's health. However, the pathogenesis of AM is not clearly understood. We aimed to investigate the pathophysiological changes and molecular mechanism in AM. METHODS: Single-cell RNA sequencing (scRNA-seq) was employed to construct a transcriptomic atlas of various cell subsets from the ectopic endometrium (EC) and eutopic endometrium (EM) of one AM patient and evaluate differential expression. The Cell Ranger software pipeline (version 4.0.0) was applied to conduct sample demultiplexing, barcode processing and mapping reads to the reference genome (human GRCh38). Different cell types were classified with markers with the "FindAllMarkers" function, and differential gene expression analysis was performed with Seurat software in R. The findings were confirmed by Reverse Transcription Real-Time PCR using samples from three AM patients. RESULTS: We identified nine cell types: endothelial cells, epithelial cells, myoepithelial cells, smooth muscle cells, fibroblasts, lymphocytes, mast cells, macrophages and unknown cells. A number of differentially expressed genes, including CLO4A1, MMP1, TPM2 and CXCL8, were identified from all cell types. Functional enrichment showed that aberrant gene expression in fibroblasts and immune cells was related to fibrosis-associated terms, such as extracellular matrix dysregulation, focal adhesion and the PI3K-Akt signaling pathway. We also identified fibroblast subtypes and determined a potential developmental trajectory related to AM. In addition, we identified increased cell-cell communication patterns in EC, highlighting the imbalanced microenvironment in AM progression. CONCLUSION: Our results support the theory of endometrial-myometrial interface disruption for AM, and repeated tissue injury and repair could lead to increased fibrosis in the endometrium. Therefore, the present study reveals the association between fibrosis, the microenvironment, and AM pathogenesis. This study provides insight into the molecular mechanisms regulating AM progression.
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Single cell transcriptional analysis of human adenoids identifies molecular features of airway microfold cells.
The nasal, oropharyngeal, and bronchial mucosa are primary contact points for airborne pathogens like Mycobacterium tuberculosis (Mtb), SARS-CoV-2, and influenza virus. While mucosal surfaces can function as both entry points and barriers to infection, mucosa-associated lymphoid tissues (MALT) facilitate early immune responses to mucosal antigens. MALT contains a variety of specialized epithelial cells, including a rare cell type called a microfold cell (M cell) that functions to transport apical antigens to basolateral antigen-presenting cells, a crucial step in the initiation of mucosal immunity. M cells have been extensively characterized in the gastrointestinal (GI) tract in murine and human models. However, the precise development and functions of human airway M cells are unknown. Here, using single-nucleus RNA sequencing (snRNA-seq), we generated an atlas of cells from the human adenoid and identified 26 unique cell types representing basal, club, hillock, and hematopoietic lineages, defined their developmental trajectories, and determined cell-cell relationships. Using trajectory analysis, we found that human airway M cells develop from progenitor club cells and express a gene signature distinct from intestinal M cells. Surprisingly, we also identified a heretofore unknown epithelial cell type demonstrating a robust interferon-stimulated gene signature. Our analysis of human adenoid cells enhances our understanding of mucosal immune responses and the role of M cells in airway immunity. This work also provides a resource for understanding early interactions of pathogens with airway mucosa and a platform for development of mucosal vaccines.
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Human Pancreatic Cancer Single-Cell Atlas Reveals Association of CXCL10+ Fibroblasts and Basal Subtype Tumor Cells.
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) patients with tumors enriched for the basal-like molecular subtype exhibit enhanced resistance to standard-of-care treatments and have significantly worse overall survival compared with patients with classic subtype-enriched tumors. It is important to develop genomic resources, enabling identification of novel putative targets in a statistically rigorous manner. EXPERIMENTAL DESIGN: We compiled a single-cell RNA sequencing (scRNA-seq) atlas of the human pancreas with 229 patient samples aggregated from publicly available raw data. We mapped cell type-specific scRNA-seq gene signatures in bulk RNA-seq (n = 744) and spatial transcriptomics (ST; n = 22) and performed validation using multiplex immunostaining. RESULTS: Analysis of tumor cells from our scRNA-seq atlas revealed nine distinct populations, two of which aligned with the basal subtype, correlating with worse overall survival in bulk RNA-seq. Deconvolution identified one of the basal populations to be the predominant tumor subtype in nondissociated ST tissues and in vitro tumor cell and patient-derived organoid lines. We discovered a novel enrichment and spatial association of CXCL10+ cancer-associated fibroblasts with basal tumor cells. We identified that besides immune cells, ductal cells also express CXCR3, the receptor for CXCL10, suggesting a relationship between these cell types in the PDAC tumor microenvironment. CONCLUSIONS: We show that our scRNA-seq atlas (700,000 cells), integrated with ST data, has increased statistical power and is a powerful resource, allowing for expansion of current subtyping paradigms in PDAC. We uncovered a novel signaling niche marked by CXCL10+ cancer-associated fibroblasts and basal tumor cells that could be explored for future targeted therapies.
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Genotype-phenotype profile of global ASPH-associated ectopia lentis and clinical findings from a Chinese cohort.
BACKGROUND: Traboulsi syndrome is an under-recognized syndromic form of ectopia lentis (EL) caused by the aspartate beta-Hydroxylase (ASPH) variant. The genotype-phenotype profile of ASPH-associated disease is poorly understood due to the rarity of the condition. METHODS: We conducted targeted next-generation sequencing and bioinformatics analysis to identify potentially pathogenic ASPH variants in the cohort. Furthermore, we characterized the expression pattern of ASPH and major components of the zonules using single-cell RNA-sequencing (scRNA-seq) and evaluated the genotype-phenotype correlations by combining our data and those from the literature. RESULTS: We identified a novel missense variant c.2075G > A (p.G692D) and a recurrent nonsense variant c.1126C > G (p.R376*) of ASPH in two pedigrees from a Chinese cohort of EL. Both probands were 5-year-old boys with canonical facial dysmorphisms and bilateral anteriorly-dislocated lenses. Other ocular comorbidities included microspherophakia, shallow anterior chamber, and narrow chamber angel. No cardiac involvements or filtering blebs were identified. The single-cell expression atlas of ciliary epithelium demonstrated the coexpression of ASPH with FBN1, FBN2, and LTBP2 in the non-pigmented ciliary epithelium cells. Furthermore, molecular modeling simulation of p.G692D revealed increased affinity to the cb EGF-like domain and a subsequent destabilized calcium-binding motif. The genotype-phenotype analysis demonstrated that patients with cardiac involvements all harbored biallelic truncation variants. CONCLUSIONS: The data from this study provide new insights into the genotype-phenotype profile of ASPH-associated disease and implicate the potential role of ASPH in the pathogenesis of EL.
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A cell atlas of the human fallopian tube throughout the menstrual cycle and menopause.
The fallopian tube undergoes extensive molecular changes during the menstrual cycle and menopause. We use single-cell RNA and ATAC sequencing to construct a comprehensive cell atlas of healthy human fallopian tubes during the menstrual cycle and menopause. Our scRNA-seq comparison of 85,107 pre- and 46,111 post-menopausal fallopian tube cells reveals substantial shifts in cell type frequencies, gene expression, transcription factor activity, and cell-to-cell communications during menopause and menstrual cycle. Menstrual cycle dependent hormonal changes regulate distinct molecular states in fallopian tube secretory epithelial cells. Postmenopausal fallopian tubes show high chromatin accessibility in transcription factors associated with aging such as Jun, Fos, and BACH1/2, while hormone receptors were generally downregulated, a small proportion of secretory epithelial cells had high expression of ESR2, IGF1R, and LEPR. While a pre-menopausal secretory epithelial gene cluster is enriched in the immunoreactive molecular subtype, a subset of genes expressed in post-menopausal secretory epithelial cells show enrichment in the mesenchymal molecular type of high-grade serous ovarian cancer.
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Gene-Length Bias and Compartment Non-Identifiability in Cell-Type Deconvolution of Sheep Skin: A Prospectively Specified and Timestamped Diagnostic Re-Analysis of Wool-Density Transcriptomes in Hetian Sheep.
Bulk skin transcriptomes cannot distinguish cell-intrinsic gene regulation from shifts in tissue-type composition, and computational deconvolution is widely applied to resolve this limitation. In this study, we performed deconvolution on published Hetian sheep skin RNA-seq data from three high-wool-density and three low-wool-density animals, using the available ovine skin single-cell reference. All hypotheses and analytical workflows were prospectively specified and timestamped prior to estimating cell-type fractions. Our work identifies two major methodological pitfalls for livestock tissue deconvolution. First, gene-length bias distorts deconvolution outputs derived from mixed bulk and 3' single-cell references. After bias correction, the estimated hair-follicle epithelial fraction changed substantially. This bias cannot be uncovered by standard benchmark workflows, highlighting a hidden failure mode for deconvolution. Second, limited signature separability within the reference prevents reliable discrimination between related epidermal compartments. This non-identifiability strongly disturbs apparent compartment-level effects. When analysed at the granularity the reference reliably supports, we found no compositional difference between high- and low-wool-density groups for the epithelial hypothesis. The dermal hypothesis could not be meaningfully evaluated due to incompatible reference compartment merging requirements. Group-level differential expression remained robust despite this null compositional outcome. We recovered a large set of differentially expressed genes consistent with the original study, including most qPCR-validated candidates. Adjusting for cell-type composition altered part of this gene list, as expected given negligible group-level compositional divergence. Co-expression modules correlated more strongly with inter-individual compositional variation than with wool-density phenotype. This indicates compositional heterogeneity shapes transcriptome co-expression even without measurable mean differences across experimental groups. To avoid misleading biological inference, we propose a set of practical diagnostic checks for deconvolution when developmental or limited single-cell atlases are used for livestock tissues. Collectively, these transcriptomic patterns support cell-state-driven regulation rather than detectable mean shifts in tissue compartment composition.