JOURNAL/nrgr/04.03/01300535-202512000-00030/figure1/v/2025-01-31T122243Z/r/image-tiff Studies have shown that vascular dysfunction is closely related to the pathogenesis of Alzheimer's disease. The middle temporal gyrus region of the brain is susceptible to pronounced impairment in Alzheimer's disease. Identification of the molecules involved in vascular aberrance of the middle temporal gyrus would support elucidation of the mechanisms underlying Alzheimer's disease and discovery of novel targets for intervention. We carried out single-cell transcriptomic analysis of the middle temporal gyrus in the brains of patients with Alzheimer's disease and healthy controls, revealing obvious changes in vascular function. CellChat analysis of intercellular communication in the middle temporal gyrus showed that the number of cell interactions in this region was decreased in Alzheimer's disease patients, with altered intercellular communication of endothelial cells and pericytes being the most prominent. Differentially expressed genes were also identified. Using the CellChat results, AUCell evaluation of the pathway activity of specific cells showed that the obvious changes in vascular function in the middle temporal gyrus in Alzheimer's disease were directly related to changes in the vascular endothelial growth factor (VEGF)A-VEGF receptor (VEGFR) 2 pathway. AUCell analysis identified subtypes of endothelial cells and pericytes directly related to VEGFA-VEGFR2 pathway activity. Two subtypes of middle temporal gyrus cells showed significant alteration in AD: endothelial cells with high expression of Erb-B2 receptor tyrosine kinase 4 (ERBB4 high ) and pericytes with high expression of angiopoietin-like 4 (ANGPTL4 high ). Finally, combining bulk RNA sequencing data and two machine learning algorithms (least absolute shrinkage and selection operator and random forest), four characteristic Alzheimer's disease feature genes were identified: somatostatin ( SST ), protein tyrosine phosphatase non-receptor type 3 ( PTPN3 ), glutinase ( GL3 ), and tropomyosin 3 ( PTM3 ). These genes were downregulated in the middle temporal gyrus of patients with Alzheimer's disease and may be used to target the VEGF pathway. Alzheimer's disease mouse models demonstrated consistent altered expression of these genes in the middle temporal gyrus. In conclusion, this study detected changes in intercellular communication between endothelial cells and pericytes in the middle temporal gyrus and identified four novel feature genes related to middle temporal gyrus and vascular functioning in patients with Alzheimer's disease. These findings contribute to a deeper understanding of the molecular mechanisms underlying Alzheimer's disease and present novel treatment targets.

Despite significant progress in diagnostic and therapeutic modalities, lung adenocarcinoma (LUAD) still exhibits a high recurrence risk and a low 5-year survival rate. Reliable prognostic signatures are imperative for risk stratification in LUAD patients. This study encompassed 2740 patients from 23 LUAD cohorts, including one single-cell RNA sequencing (scRNA-seq) dataset, five bulk RNA-seq datasets, and 17 microarray datasets. Using scRNA-seq dataset, we defined a group of epithelial-specific transcription factors significantly over-represented in the epithelial-to-mesenchymal transition (EMT) gene set (enrichment ratio [ER] = 5.80, Fisher's exact test p < .001), and the corresponding target genes were significantly enriched in the cancer driver gene set (ER = 2.74, p < .001), indicating of their crucial roles in the EMT process and tumor progression. We constructed a single-cell gene pairs (scGPS) signature, composed of 3521 gene pairs derived from the epithelial cell-specific transcription factor regulatory network, to predict overall survival (OS) of LUAD. High-risk patients identified by scGPS in the discovery cohort exhibited significantly worse OS compared to low-risk patients (Hazard ratio [HR] = 1.78, 95% CI: 1.29-2.46, log-rank p = 1.80 × 10-4). The scGPS outperformed other established gene signatures and demonstrated robust prognostic stratification across various independent datasets, including microarray data and even early-stage LUAD patients. It remained an independent prognostic factor after adjusting for clinical and pathologic factors. In addition, combining scGPS with tumor stage further enhanced prognostic accuracy compared to using stage alone. The scGPS signature offers individualized prognosis estimations, showing significant potential for practical application in clinical settings.

Malignant melanoma is a highly aggressive tumor originating from the pigment producing cells, the melanocytes. It accounts for the majority of skin cancer related deaths worldwide. This is often due to the development of therapy resistance or tumor dormancy, eventually resulting in tumor relapse by yet undefined mechanisms. Tumor dormancy is thought to be mediated by the cellular microenvironment and models taking this factor into account are urgently needed. We 3D bioprinted melanoma cells in the hydrogels Cellink Bioink (CIB) or Matrigel (MG), each as a substitute of the extracellular matrix, and, thereby, induced a quiescent or a proliferative phenotype of the melanoma cell lines, respectively. RNA-Seq with subsequent comprehensive bioinformatical and molecular analyses assigned CIB-cultured cells to a predominantly mesenchymal and Matrigel-cultured cells to a more mitotic phenotype, emphasizing the CIB model as a suitable platform for the investigation of dormancy under consideration of the microenvironment. Melanoma cells in CIB 3D culture reflect a quiescent and migratory active cell state e.g. by revealing significant downregulation of genes associated with replication and cell cycle progression in this setting. Using this model system, we identified the mechanosensory gene FHL2 as one early sensor of changes in the ECM and suggest a FHL2-p21/AP-1 axis contributing to the dormant phenotype of melanoma cells in CIB.

In recent years, RNA transcriptome sequencing technology has been continuously evolving, ranging from single-cell transcriptomics to spatial transcriptomics. Although these technologies are all based on RNA sequencing, each sequencing technology has its own unique characteristics, and there is an urgent need to develop an algorithmic toolkit that integrates both sequencing techniques. To address this, we have developed OmniClust, a toolkit based on single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics data. OmniClust employs deep learning algorithms for feature learning and clustering of spatial transcriptomics data, while utilizing machine learning algorithms for clustering scRNA-seq data. OmniClust was tested on 12 spatial transcriptomics benchmark datasets, demonstrating high clustering accuracy across multiple clustering evaluation metrics. It was also evaluated on four scRNA-seq benchmark datasets, achieving high clustering accuracy based on various clustering evaluation metrics. Furthermore, we applied OmniClust to downstream analyses of spatial transcriptomics and single-cell RNA breast cancer data, showcasing its potential to uncover and interpret the biological significance of cancer transcriptome data. In summary, OmniClust is a clustering tool designed for both single-cell transcriptomics and spatial transcriptomics data, demonstrating outstanding performance.

Regulated cell death (RCD), a genetically controlled process mediated by specialized molecular pathways (commonly termed programmed cell death), plays pivotal roles in diverse pathophysiological processes. However, the landscape and functional implications of RCD subtypes in chronic rhinosinusitis with nasal polyps (CRSwNP) remain poorly characterized. This study aimed to systematically investigate the involvement of RCD mechanisms in the pathogenesis and progression of CRSwNP.

Transcriptomic datasets (GSE136825, GSE23552, GSE198950, GSE196169, GSE156285) related to CRSwNP were retrieved from the Gene Expression Omnibus (GEO) database. A comprehensive panel of 18 RCD-associated gene sets was compiled through a systematic literature review. Gene set variation analysis (GSVA) was employed to profile RCD activation patterns in CRSwNP. Integrative bioinformatics approaches including weighted gene co-expression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) regression were implemented to identify hub RCD-related genes and construct a cell death index (CDI). Single-cell RNA sequencing (scRNA-seq) data were analyzed to map RCD dynamics across cellular subpopulations. Clinical validation was performed using qRT-PCR quantification of key genes in nasal polyp/inferior turbinate tissues, with the concurrent assessment of symptom severity via visual analogue scale (VAS) scores.

GSVA revealed significant upregulation of 8 RCD subtypes in CRSwNP: apoptosis, ferroptosis, necroptosis, entotic cell death, lysosome-dependent cell death, NETosis, immunogenic cell death, and anoikis. Pathway enrichment analysis demonstrated that RCD-related differentially expressed genes were predominantly involved in epithelial-mesenchymal transition (EMT) and immune-inflammatory regulation. Furthermore, the WGCNA algorithm and LASSO analysis identified 8 key cell death genes (PTHLH, GRINA, S100A9, SCG2, HMOX1, RNF183, TYROBP, SEMA7A), which were utilized to construct the cell death-related index (CDI). In training and validation cohorts, the CDI was significantly elevated in CRSwNP compared to control and exhibited high diagnostic performance, with elevated scores correlating with enhanced immune cell infiltration. Single-cell resolution analysis uncovered cell type-specific RCD activation patterns. Clinical validation confirmed significantly higher expression of S100A9, PTHLH, and HMOX1 in eosinophilic versus non-eosinophilic polyps. Notably, expression levels of PTHLH, S100A9, HMOX1, GRINA, and TYROBP showed strong positive correlations with VAS scores.

Our investigation delineates an RCD activation signature in CRSwNP pathogenesis, characterized by 8 key cell death modalities and their regulatory genes. The novel CDI exhibits promising diagnostic potential, while mechanistic insights suggest RCD pathways may drive disease progression through EMT potentiation and inflammatory cascade amplification. These findings provide a framework for developing RCD-targeted therapeutic strategies in CRSwNP.

Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by the gradual loss of neurons and the accumulation of amyloid plaques and neurofibrillary tangles. Despite advancements in the understanding of AD's pathophysiology, the cellular organization and interactions in the prefrontal cortex (PFC) remain elusive. Eight single-cell RNA sequencing (scRNA-seq) datasets from both normal controls and individuals with AD were harmonized. Stringent preprocessing protocols were implemented to uphold dataset integrity. Unsupervised clustering and annotation revealed 22 distinct cell clusters corresponding to 19 unique cell types. The spatial architecture of the PFC region was constructed using the CARD tool. Further analyses encompassed trajectory examination of Oligodendrocyte subtypes, evaluation of regulon activity scores, and spot clustering within white matter regions (WM). Differential expression analysis and functional enrichment assays unveiled molecular signatures linked to AD progression and were validated using microarray data sourced from neurodegenerative disorder patients. Our investigation employs scRNA-seq and spatial transcriptomics to uncover the cellular atlas and spatial architecture of the human PFC in AD. Moreover, our results indicate that Oligodendrocytes are more prevalent in AD patients, showcasing diverse subtypes and spatial organization within WM regions. Each subtype appears to be associated with distinct biological processes and transcriptional regulators, shedding light on their involvement in AD pathology. Notably, the Oligodendrocyte_C6 subtype is linked to neurological damage in AD patients, characterized by heightened expression of genes involved in cell-cell connections, cell membrane stability, and myelination. Additionally, 12 target genes regulated by NFIA were identified, which are upregulated in AD patients and associated with disease progression. Elevated PLXDC2 expression in peripheral blood was also identified, suggesting its potential as a non-invasive biomarker for early AD detection. Our study provides novel insights into the role of Oligodendrocytes in AD and highlights the potential of PLXDC2 as a blood biomarker for non-invasive diagnosis and monitoring of AD patients.

Smooth muscle cells within the tumor microenvironment play a crucial role in cancer progression. However, their involvement in the local invasion of head and neck squamous cell carcinoma remains poorly understood. In this research, we aim to investigate the role of smooth muscle cells-mediated cell interactions in facilitating the local invasion of head and neck squamous cell carcinoma.

Single-cell sequencing data from the public databases GSE164690 and GSE181919 were utilized to identify a specific smooth muscle cells cluster. Smooth muscle cells were isolated from tumor microenvironment of head and neck squamous cell carcinoma. PHLDA1 expression in smooth muscle cells was assessed through immunofluorescence staining. The role of THBS1 was investigated through in vitro studies.

PHLDA1-positive smooth muscle cells were significantly enriched in head and neck squamous cell carcinoma. PHLDA1 promoted the expression of THBS1 in smooth muscle cells. In vitro, THBS1 facilitated head and neck squamous cell carcinoma migration and invasion through SDC1 receptor.

PHLDA1-positive smooth muscle cells play a critical role in head and neck squamous cell carcinoma invasion through THBS1. Targeting PHLDA1-positive smooth muscle cells or THBS1 may offer a promising therapeutic approach for head and neck squamous cell carcinoma treatment.

Patients with liver metastatic colorectal cancer (mCRC) have a poor prognosis and are the leading cause of death in colorectal cancer (CRC) patients, but the mechanisms associated with CRC metastasis have not been fully elucidated. In this study, we obtained data from the Gene Expression Omnibus database and characterized the single-cell profiles of CRC, mCRC and healthy samples at single-cell resolution, and explored the cells that influence CRC metastasis. We find that AQP1+ CRC identified as highly malignant tumor cells exhibited proliferative and metastatic characteristics. Immunosuppressive properties are present in the tumor microenvironment (TME), while NOTCH3+ Fib is identified to play a facilitating role in the metastatic colonization of CRC. Importantly, we reveal that tumor-associated macrophages (TAM) characterized by SPP1-specific high expression may be involved in TME remodeling through intercellular communication. Specifically, SPP1+ TAM mediates the generation of Fib-derived extracellular vesicle through the APOE-LRP1 axis, which in turn delivers tumor growth-promoting factors in the TME. This study deepens the understanding of the mechanism of TME in mCRC and lays the scientific foundation for the development of therapeutic regimens for mCRC patients.

Brainstem stroke accounts only 7-10 % of all ischemic stroke while it had more morbidity and mortality. As the predominant cellular component of nerve tracts, oligodendrocytes might provide some neuroprotection against ischemic injury in the context of brainstem stroke, but the underlying mechanism remains unclear.

A mouse model of brainstem stroke was established, and single-cell RNA sequencing and spatial transcriptomic sequencing analysis were performed to elucidate the phenotype of oligodendrocytes within this context.

Loss of oligodendrocytes led to neurological impairment following brainstem stroke, and subsequent proliferation of oligodendrocytes was observed. We identified a subcluster of Pros1+ oligodendrocytes, designated OLG8 cells. These cells increased in number after brainstem stroke and were enriched around the peri-infarct zone. OLG8 cells were derived from oligodendrocyte progenitor cells, and this process was found to be regulated by Myo1e. We found that OLG8 cells protected interneurons. Notably, the overexpression of Myo1e within OLG8 cells led to a marked reduction in infarct volume while simultaneously improving the recovery of neurological function.

In conclusion, we identified a novel cell subcluster, OLG8 cells, in the context of brainstem stroke, and found that overexpression of Myo1e alleviated ischemic injury by facilitating the differentiation of OLG8 cells. Our study provided insight into the mechanism of brainstem stroke.

Nuclear technology has been extensively used in various fields, increasing the possibility of radiation exposure to humans. Radiation exposure outcomes may be classified as whole-body irradiation or local irradiation. Clinically, local irradiation refers to the exposure of a relatively limited portion of the body, with injury confined to the directly exposed tissues. However, locally irradiated tissues can trigger systemic reactions through the release of inflammatory factors or damage to blood cells at the irradiated site. The circulating population of peripheral blood mononuclear cells (PBMCs), a component of normal tissue, is particularly sensitive to ionizing radiation. The present study applied single-cell RNA sequencing (scRNA-Seq) to profile PBMCs from one irradiated patient and 10 healthy controls matched for sex and age. In total, 6,447 and 7,892 cells were collected for analysis from the PBMCs of the irradiated patient on the 113rd and 631st days post radiation, respectively, whereas 9,101 cells were obtained from 10 healthy controls. Following scRNA-Seq, five cell types were annotated via representative markers, revealing distinct cell types whose proportions changed markedly in the irradiated patient. Trajectory analysis indicated that the dysregulation of multiple signaling pathways was associated with radiation exposure. Furthermore, single-cell regulatory network inference and clustering analysis revealed gene regulatory networks and suggested the involvement of several signaling pathways, such as those related to viral infection, in the context of radiation exposure. The present study elucidated the dynamic landscape of human blood immune responses to ionizing radiation and provides evidence of its therapeutic potential for treating radiation injury.

The striatum, a critical component of the basal ganglia, is essential for motor control, cognitive processing, and emotional regulation. Medium spiny neurons (MSNs) are the primary neuronal population in the striatum, classified into D1 and D2 subtypes. The transcription factor Pou3f1 has been hypothesized to play a crucial role in the development of pyramidal neurons. Recently, a comprehensive analysis of the human embryonic scRNA-seq dataset predicted and emphasized the bridging function of POU3F1 between striatal progenitor cells and immature neurons, though this finding lacked genetic validation. In this study, we found that Pou3f1 expression was significantly reduced after Six3 deletion. However, Pou3f1 deletion does not significantly affect the number or subtype composition of MSNs, nor the proliferation and differentiation of progenitor cells, in our Pou3f1 conditional knockout (cko) mice, challenging the in silico predictions based on human data. These results suggest that Pou3f1 is not required for the specification, generation, or differentiation of MSNs, though its potential involvement in other aspects of striatal development cannot be entirely ruled out.

The enteric nervous system (ENS), which is composed of neurons and glia, regulates intestinal motility. Hirschsprung disease (HSCR) results from defects in ENS formation; however, although neuronal aspects have been studied extensively, enteric glia remain disregarded. This study aimed to explore enteric glia diversity in health and disease.

Full-thickness intestinal resection material from pediatric controls and patients with HSCR was collected, dissociated, and enriched for the ENS population through fluorescence-activated cell sorting. Single-cell RNA sequencing was performed to uncover the transcriptomic diversity of the ENS in controls and HSCR patients, as well as in wild-type and ret mutant zebrafish. Immunofluorescence and fluorescence in situ hybridization confirmed the presence of distinct subtypes.

Two major enteric glial classes emerged in the pediatric intestine: Schwann-like enteric glia, which are reminiscent of Schwann cells, and enteric glia expressing classical glial markers. Comparative analysis with previously published datasets confirmed our classification and revealed that although classical enteric glia are predominant prenatally, Schwann-like enteric glia become more abundant postnatally. In HSCR, ganglionic segments mirrored controls and aganglionic segments featured only Schwann-like enteric glia. Leveraging the regenerative potential of Schwann cells, we explored therapeutic options using a ret mutant zebrafish. Prucalopride, a serotonin-receptor (5-HT) agonist, induced neurogenesis partially rescuing the HSCR phenotype in ret+/- mutants.

Two major enteric glial classes were identified in the pediatric intestine, highlighting the significant postnatal contribution of Schwann-like enteric glia to glial heterogeneity. Crucially, these glial subtypes persist in aganglionic segments of patients with HSCR, offering a new target for their treatment using 5-HT agonists.

There is a recent dramatic increase in research on thoracic aortic diseases that includes aneurysms, dissections, and rupture. Experimental studies predominantly use mice in which aortopathy is induced by chemical interventions, genetic manipulations, or both. Many parameters should be deliberated in experimental design in concert with multiple considerations when providing dimensional data and characterization of aortic tissues. The purpose of this review is to provide recommendations on guidance in (1) the selection of a mouse model and experimental conditions for the study, (2) parameters for standardizing detection and measurements of aortic diseases, (3) meaningful interpretation of characteristics of diseased aortic tissue, and (4) reporting standards that include rigor and transparency.

Circulating monocytes largely contribute to macrophage buildup in atheromata, which is crucial for clearing subendothelial LDLs (low-density lipoproteins) and dead cells; however, the transitional trajectory from monocytes to macrophages in atherosclerotic plaques and the underlying regulatory mechanism remain unclear. Moreover, the role of alternative polyadenylation, a posttranscriptional regulator of cell fate, in monocyte/macrophage fate decisions during atherogenesis is not entirely understood.

To identify monocyte/macrophage subtypes in atherosclerotic lesions and the effect of alternative polyadenylation on these subtypes and atherogenesis, single-cell RNA sequencing, 3'-end sequencing, flow cytometric, and histopathologic analyses were performed on plaques obtained from Apoe-/- mouse arteries with or without myeloid deletion of Srsf3 (serine/arginine-rich splicing factor 3). Cell fractionation, polysome profiling, L-azidohomoalanine metabolic labeling assay, and metabolomic profiling were conducted to disclose the underlying mechanisms. Reprogramming of widespread alternative polyadenylation patterns was estimated in human plaques via bulk RNA sequencing.

We identified a subset of lesional cells in a monocyte-to-macrophage transitional state, which exhibited high expression of chemokines in mice. Srsf3 deletion caused a maturation delay of these transitional cells and phagocytic impairment of lesional macrophages, aggravating atherosclerosis. Mechanistically, Srsf3 deficiency shortened 3' untranslated regions of mitochondria-associated Aars2 (alanyl-tRNA synthetase 2), disrupting its translation. The resultant impairment of protein synthesis in mitochondria led to mitochondrial dysfunction with declined NAD+ (nicotinamide adenine dinucleotide, oxidized form) levels, activation of the integrated stress response, and metabolic reprogramming in macrophages. Administering an NAD+ precursor nicotinamide mononucleotide or the integrated stress response inhibitor partially restored Srsf3-deficient macrophage maturation, and nicotinamide mononucleotide treatment mitigated the proatherosclerotic effects of Srsf3 deficiency. Consistently, Srsf3 downregulation, global 3' untranslated region shortening, and accumulation of these transitional macrophages were associated with atherosclerosis progression in humans.

Our study reveals that Srsf3-dependent generation of long 3' untranslated region is required for efficient mitochondrial translation, which promotes mature phagocytic macrophage formation, thereby playing a protective role in atherosclerosis.

Head and neck squamous cell carcinoma (HNSCC) treatment faces significant clinical challenges. Lactate metabolism plays a crucial role in the initiation of many cancers and the tumor microenvironment (TME). However, the prognostic significance of lactate metabolism-related genes (LMRGs) and the role of TME in HNSCC require further elucidation.

We built a prognostic multigene signature with LMRGs and systematically correlated the risk signature with immunological characteristics and immunotherapy efficacy. Next, a series of single-cell sequencing analyses were used to characterize lactate metabolism in TME. Finally, single-cell sequencing analysis, immunofluorescence analyses, and a series of in vitro experiments were used to explore the role of PYGL in HNSCC. Potential drugs targeting PYGL were screened using AutoDock 4.2.

A prognostic multigene signature based on LMRGs was developed, which effectively stratified patients into high- and low-risk groups, with significant differences in overall survival (OS) and progression-free survival (PFS). Patients in the low-risk group exhibited reduced lactate metabolism, higher CD8 + T cell infiltration, and improved response to immunotherapy. Single-cell sequencing revealed that tumor cells had the most active lactate metabolism compared to other cells in the TME. PYGL, identified as the most critical prognostic gene, was highly expressed in tumor-associated macrophages and played a role in inhibiting M1 macrophage polarization. Knockdown of PYGL led to reduced lactate levels, and its expression was inversely correlated with CD8 + T cell infiltration. Furthermore, PYGL was involved in copper-dependent cell death, highlighting its potential as a therapeutic target. Drug screening identified elesclomol, which showed promising results in PYGL-knockdown cells.

The study established a robust LMRGs-based prognostic model that not only predicts patient survival but also correlates with the immune microenvironment in HNSCC. PYGL emerged as a key biomarker with significant implications for both prognosis and therapeutic intervention. Its role in regulating lactate metabolism and immune suppression suggests that targeting PYGL could enhance the efficacy of immunotherapies. This research provides a foundation for future clinical strategies aimed at improving outcomes in HNSCC by modulating the tumor's metabolic and immune landscapes.

Hepatoblastoma, the most aggressive childhood liver tumor, poses significant challenges due to limited knowledge of its pathogenesis, particularly in poorly differentiated advanced tumors where the prognosis is dismal. Single-cell sequencing provides an in-depth exploration at the single-cell level and offers a deep understanding of tumor heterogeneity. Herein, single-cell transcriptomics analysis is used to identify a unique malignant-hepatoblast (HB)-like cell subpopulation as the possible origin of poorly differentiated hepatoblastoma. These cells are associated with an unfavorable clinical prognosis in hepatoblastoma patients. The malignant-HB-like cell subpopulation generated insulin-like growth factor 2 (IGF2) to sustain stem-like features by promoting abnormal cholesterol accumulation via SREBF2. IGF2 also stimulated fibroblast 2 to secrete collagen 1, intensifying tumor malignancy via the collagen 1/integrin α1 signaling pathway. This suggests that targeting malignant HB-like cells by inhibiting IGF2-induced pathways can lead to promising treatments for hepatoblastoma. Additionally, serum IGF2 levels may serve as a diagnostic biomarker for advanced hepatoblastoma. In summary, these findings provide valuable insight into the genesis and malignancy of hepatoblastoma and a foundation for more effective diagnostic tools and therapeutic strategies for this challenging disease.

Soil salinization, exacerbated by environmental deterioration and improper cultivation, is a major challenge for sustainable agriculture. The root is the primary organ in plants to perceive and respond to salt stress. Utilizing single-cell sequencing, we have created the first single-cell transcriptional and chromatin accessibility landscape for normal and salt-stressed root tips in non-heading Chinese cabbage (NHCC). Our study reveals that salt stress disrupts the normal differentiation of root hairs, leaving many in an undifferentiated state and preventing stress response gene expression. Inter-species analyses show that both salt and osmotic stresses inhibit root hair differentiation and elongation similarly, resulting in fewer, malfunctioning root hairs. We found that high salinity affects root hair iron transport. Salt stress-responsive genes, cell type-specific transcriptional regulatory networks, and trajectory curves are linked to iron transport. Specifically, the expression of BcIRT2, a metal transporter gene, is influenced by salt stress. Silencing BcIRT2 causes chlorotic leaves and increases salt sensitivity, reducing iron content in NHCC roots. Our findings offer significant insights into plant salt stress responses and provide valuable information for breeding salt-tolerant NHCC and other crops.

Glutamine metabolism is a hallmark of cancer metabolism. This study aims to perform a comprehensive and systematic single-cell profile of glutamine metabolism in premalignant and malignant gastric lesions. We use single-cell transcriptomics data from chronic atrophic gastritis (CAG) and early gastric cancer (EGC) lesions and investigate glutamine metabolism features at the single-cell level. Experiments are implemented to validate the expression and biological role of ERO1LB in gastric cancer (GC). A single-cell atlas based on 22511 cells from premalignant and early-malignant gastric lesions is established. Among these cells, epithelial cells constitute the dominant cell population in both CAG and EGC lesions. The activity of glutamine metabolism is higher in epithelial cells from EGC lesions than in those from CAG lesions. Among the epithelial cell subpopulations, glutamine metabolism is more active in the epithelial cell subpopulation cluster_4 in EGCs than in CAG lesions. As a key marker gene of this subpopulation, ERO1LB is experimentally proven to be overexpressed in human GC tissue lesions. In both in vitro and in vivo experiments, overexpression of ERO1LB in GC cells increases glutamine metabolism, facilitates cell growth and migration and prevents cell apoptosis, and vice versa. This study provides insight into the cellular heterogeneityof glutamine metabolism within the gastric mucosa in premalignant and malignant gastric lesions and identifies ERO1LB as a key orchestrator of glutamine metabolism, which may help to identify markers for GC prevention and contribute to our understanding of GC pathogenesis.

Cancer-associated fibroblasts (CAFs) display significant functional and molecular heterogeneity within the tumor microenvironment, playing diverse roles in cancer progression. Employing single-cell RNA sequencing data of colorectal cancer (CRC), we identified a subset of matrix CAFs (mCAFs) as a critical subtype that secretes THBS2, a molecule linked to advanced cancer stages and poor prognosis. Spatial transcriptomics and multiplex immunohistochemistry revealed clear spatial colocalization between THBS2-producing mCAFs and tumor cells. Mechanically, CAF-secreted THBS2 binds to CD47 on tumor cells, triggering the MAPK/ERK5 signaling pathway, which enhances tumor progression. The tumor-promoting role of THBS2 was further validated using fibroblast-specific THBS2 knockout mice, patient-derived organoids, and xenografts. Moreover, the transcription factor CREB3L1 was identified as a regulator of the transformation of normal fibroblasts into THBS2-producing mCAFs. These findings underscore the pivotal role of THBS2 in CRC progression and highlight the therapeutic potential of targeting the THBS2-CD47 axis and CREB3L1 in CRC.

Leber hereditary optic neuropathy (LHON) is a paradigm for mitochondrial retinopathy due to mitochondrial DNA (mtDNA) mutations. However, the mechanism underlying retinal cell-specific effects of LHON-linked mtDNA mutations remains poorly understood, and there has been no effective treatment or cure for this disorder. Using a mouse model bearing an LHON-linked ND6P25L mutation, we demonstrated that the mutation caused retinal cell-specific deficiencies, especially in retinal ganglion cells (RGCs), rods, and Müller cells. Single-cell RNA sequencing revealed cell-specific dysregulation of oxidative phosphorylation and visual signaling pathways in the mutant retina. Strikingly, ND6 mutation-induced dysfunctions caused abnormal vitamin A (VA) metabolism essential for visual function. VA supplementation remarkably alleviated retinal deficiencies, including reduced fundus lesion and retinal thickness and increased numbers of RGCs, photoreceptors, and Müller cell neurites. The restoration of visual functions with VA treatment were further evidenced by correcting dysregulations of phototransduction cascade and neurotransmitter transmission and restoring electrophysiological properties. Interestingly, VA supplementation markedly rescued the abnormal mitochondrial morphologies and functions in the mutant retina. These findings provide insight into retina-specific pathophysiology of mitochondrial retinopathy arising from VA deficiency and mitochondrial dysfunction induced by mtDNA mutation and a step toward therapeutic intervention for LHON and other mitochondrial retinopathies.

Blood cancers are generally more common in males, and the prevalence of most mutations that drive clonal hematopoiesis and myeloid malignancies is higher in males. In contrast, hematopoietic DNMT3A mutations are more common in females. Among ∼450,000 participants in the UK Biobank, the prevalence of DNMT3A mutations and copy-number abnormalities is higher in females than males. In a murine model, Dnmt3a-mutant hematopoietic stem cells (HSCs) from unperturbed female mice had increased stemness gene expression compared to male and wild-type (WT) mice. Estrogen regulates HSCs, and we found that Dnmt3a mutations maintain stemness in the setting of estrogen-induced proliferative stress. Dnmt3a-mutant myeloid cells outcompeted WT cells under chronic estrogen treatment, an effect that was dependent on cell-intrinsic estrogen receptor alpha activity. Our studies indicate that estrogen might contribute to the female predominance of DNMT3A-mutant clonal hematopoiesis.

The lymphoid cycle serves as a sentinel of the immune response, yet the cell subtypes and immune properties within lymph fluid remain unclear. This study describes a comprehensive characterization of immune cells in rat lymph fluid using single-cell RNA sequencing, identifying a unique subset of CD4+ T cells (CD4_Icos) that suppresses inflammation in early sepsis. Trajectory analysis reveals that CD4+Icos+ T cells can differentiate into regulatory T cells (Tregs). Transferring CD4+Icos+ T cells alleviates CLP-induced organ injury, while CD4+ Icos-knockout (KO) mice show reduced Treg numbers, increased inflammation, and higher mortality. Further experiments identify Npas2 as an Icos-specific transcription factor regulating Icos expression and promoting the differentiation of CD4+Icos+ T cells. Clinical data show a negative correlation between ICOS expression in CD4+ T cells and clinical outcomes in septic patients. These findings highlight the protective role of CD4+ T cells in modulating immune responses and mitigating sepsis progression.

Chronic itch is a devastating clinical condition, and its central mechanisms remain poorly understood. We reported that spinal cannabinoid receptor type 2 (CB2R) activation exerts antipruritic effects and that itch escalates in mice lacking Cnr2 in mouse models of dermatitis and psoriasis. In the spinal cord, CB2R is mainly expressed in microglia, and microglial ablation or inhibition attenuated chronic itch, suggesting that microglial activation contributes to chronic itch. Particularly, conditional Cnr2 deletion in microglia also exacerbated chronic itch in mice. Single-cell RNA sequencing and molecular mechanistic studies suggest that CB2R activation reprogrammed microglia by inducing anti-inflammatory suppressor of cytokine signaling 3 (SOCS3) and reducing itch-related p38 and signal transducer and activator of transcription 1 (STAT1) phosphorylation. Finally, CB2R activation suppressed neuronal excitability and synaptic transmission in gastrin-releasing peptide (GRP)/GRP receptor (GRPR) interneurons and ascending projection neurons by inhibiting microglia-derived cytokines. These findings demonstrate that microglial activation contributes to chronic itch, while CB2R activation in microglia alleviates chronic itch via neuro-immune interactions.

The development of early human tooth primordia is not well understood. Here, we linked single-cell RNA sequencing, spatial transcriptomics, and secretome analysis to characterize human fetal tooth development over time. A spatiotemporal atlas of human tooth development at multiple levels was mapped, identifying previously uncharacterized epithelial subpopulations with distinct gene expression profiles and spatial localization. Dynamic changes in epithelial-mesenchymal interactions across developmental stages were characterized. Secretome analysis confirmed the extensive paracrine signaling from the epithelial to mesenchymal compartments and uncovered signaling factors produced by dental epithelium (DE) that regulate mesenchymal cell fate and differentiation. Integration of these datasets highlighted the crucial role of the DE in orchestrating tooth morphogenesis. Our multi-omics approach not only provides unprecedented insights into the cellular and molecular mechanisms of ectoderm-derived tissue development but also serves as a valuable resource, which is publicly available online, for future studies on human tooth regeneration and related diseases.

Human brain development requires generating diverse cell types, a process explored by single-cell transcriptomics. Through parallel meta-analyses of the human cortex in development (seven datasets) and adulthood (16 datasets), we generated over 500 gene co-expression networks that can describe mechanisms of cortical development, centering on peak stages of neurogenesis. These meta-modules show dynamic cell subtype specificities throughout cortical development, with several developmental meta-modules displaying spatiotemporal expression patterns that allude to potential roles in cell fate specification. We validated the expression of these modules in primary human cortical tissues. These include meta-module 20, a module elevated in FEZF2+ deep layer neurons that includes TSHZ3, a transcription factor associated with neurodevelopmental disorders. Human cortical chimeroid experiments validated that both FEZF2 and TSHZ3 are required to drive module 20 activity and deep layer neuron specification but through distinct modalities. These studies demonstrate how meta-atlases can engender further mechanistic analyses of cortical fate specification.

We present hypergraph variational autoencoder (HyperG-VAE), a Bayesian deep generative model that leverages hypergraph representation to model single-cell RNA sequencing (scRNA-seq) data. The model features a cell encoder with a structural equation model to account for cellular heterogeneity and construct gene regulatory networks (GRNs) alongside a gene encoder using hypergraph self-attention to identify gene modules. The synergistic optimization of encoders via a decoder improves GRN inference, single-cell clustering, and data visualization, as validated by benchmarks. HyperG-VAE effectively uncovers gene regulation patterns and demonstrates robustness in downstream analyses, as shown in B cell development data from bone marrow. Gene set enrichment analysis of overlapping genes in predicted GRNs confirms the gene encoder's role in refining GRN inference. Offering an efficient solution for scRNA-seq analysis and GRN construction, HyperG-VAE also holds the potential for extending GRN modeling to temporal and multimodal single-cell omics.

Identifying cell types and states remains a time-consuming, error-prone challenge for spatial biology. While deep learning increasingly plays a role, it is difficult to generalize due to variability at the level of cells, neighborhoods, and niches in health and disease. To address this, we develop TACIT, an unsupervised algorithm for cell annotation using predefined signatures that operates without training data. TACIT uses unbiased thresholding to distinguish positive cells from background, focusing on relevant markers to identify ambiguous cells in multiomic assays. Using five datasets (5,000,000 cells; 51 cell types) from three niches (brain, intestine, gland), TACIT outperforms existing unsupervised methods in accuracy and scalability. Integrating TACIT-identified cell types reveals new phenotypes in two inflammatory gland diseases. Finally, using combined spatial transcriptomics and proteomics, we discover under- and overrepresented immune cell types and states in regions of interest, suggesting multimodality is essential for translating spatial biology to clinical applications.

There is a sex bias in the incidence and progression of many kidney diseases. To better understand such sexual dimorphism, we integrated data from six platforms, characterizing 76 kidney samples from 68 mice at six developmental and adult time points, creating a molecular atlas of the mouse kidney across the lifespan for both sexes. We show that proximal tubules have the most sex-biased differentially expressed genes emerging after 3 weeks of age and are associated with hormonal regulations. We reveal potential mechanisms involving both direct and indirect regulation by androgens and estrogens. Spatial profiling identifies distinct sex-biased spatial patterns in the cortex and outer stripe of the outer medulla. Additionally, older mice exhibit more aging-related gene alterations in loops of Henle, proximal tubules and collecting ducts in a sex-dependent manner. Our results enhance the understanding of spatially resolved gene expression and hormone regulation underlying kidney sexual dimorphism across the lifespan.

As part of the innate immune system, NK cells contribute to optimizing cancer immunotherapy strategies and are becoming a focal point in cancer research. However, limited research has been conducted to further investigate changes in NK cell subsets and their critical genes following ibrutinib treatment in CLL patients.

Peripheral blood samples from patients clinically and pathologically diagnosed with monoclonal B-cell lymphocytosis (MBL), newly diagnosed with CLL (ND-CLL), postibrutinib-treated patients who achieved a complete response (CR) or partial response (PR), and those with Richter's syndrome (RS) were collected. Single-cell transcriptome sequencing was performed, followed by pseudotemporal analysis and functional enrichment to characterize the NK cell subsets. Mendelian randomization analysis and colocalization analysis were employed to identify key genes. Multiple algorithms were used for immune infiltration analysis, and drug sensitivity analysis was conducted to pinpoint potential therapeutic agents.

Three distinct NK cell subsets were identified: CD56bright_NK cells, CD56dim_NK cells, and a highly cytotoxic CLL_NK subset. The core genes of the CLL_NK subset were elucidated through Mendelian randomization and colocalization analyses. A cell subset-specific novel index (CNI) was constructed based on these core genes and was shown to be capable of predicting responses to immunotherapy. Oncopredictive algorithms and molecular docking screenings further identified semaxanib and ulixertinib as potential therapeutic candidates for CLL.

The CLL_NK subset plays a crucial role in the development and progression of CLL. The CNI, derived from its key genes, holds promise as a predictor of immune therapeutic responses, highlighting the significance of CLL_NK subset dynamics and their genetic underpinnings in CLL management.

Dysregulation of fibroblast function is closely associated with the occurrence of heart failure after myocardial infarction (post-MI HF). Myocardial fibrosis is a detrimental consequence of aberrant fibroblast activation and extracellular matrix deposition following myocardial infarction (MI). However, the heterogeneity of fibroblasts in normal cardiac tissue and heart failure tissue remains to be further investigated. We discovered that the abundance of FGF7+MGST1+ fibroblasts were down-regulated in post-MI HF according to scRNA-seq analysis. Key gene characteristics of FGF7+MGST1+ fibroblasts were uncovered through both differential expression analysis and hdWGCNA pipeline. Pseudotime analysis revealed that FGF7+MGST1+ fibroblasts were gradually decreased with the occurrence of heart failure. Cell-cell communication analysis indicated an enhanced secretory ability in FGF7+MGST1+ fibroblasts compared to other fibroblasts. Utilizing machine learning algorithms, we identified 17 feature genes of this cell population. A deep learning model capable of predicting heart failure was successfully built based on these feature genes and immune infiltration levels of post-MI HF. FGF7 was highly related to cardioprotective pathway terms, including "PI3K/AKT pathway" and "protein secretion". Parallelly, mendelian randomization analysis was adopted to better understand the causal relationships between feature genes and post-MI HF. Results indicated that MGST1 was causally associated with heart failure, consistent with single cell data. And the post-MI HF mouse model was constructed and qRT-PCR assays supported that both FGF7 and MGST1 were largely down-regulated in myocardial infarction area than other cardiac tissues. These findings provide new insights into the roles of FGF7+MGST1+ fibroblasts in post MI HF.

Integrating biological data with in silico modeling offers the transformative potential to develop virtual human models, or "digital twins." These models hold immense promise for deepening our understanding of diseases and uncovering new therapeutic strategies. This approach is especially valuable for diseases lacking reliable models. Here we review current modelling efforts in of human lung development, highlighting the role of interdisciplinary collaboration and key advances toward a digital lung twin.

Diffuse intrinsic pontine glioma (DIPG), a lethal pediatric cancer driven by H3K27M oncohistones, exhibits aberrant epigenetic regulation and stem-like cell states. Here, we uncover an axis involving H3.3K27M oncohistones, CREB5/ID1, which sustains the stem-like state of DIPG cells, promoting malignancy. We demonstrate that CREB5 mediates elevated ID1 levels in the H3.3K27M/ACVR1WT subtype, promoting tumor growth; while BMP signaling regulates this process in the H3.1K27M/ACVR1MUT subtype. Furthermore, we reveal that H3.3K27M directly enhances CREB5 expression by reshaping the H3K27me3 landscape at the CREB5 locus, particularly at super-enhancer regions. Additionally, we elucidate the collaboration between CREB5 and BRG1, the SWI/SNF chromatin remodeling complex catalytic subunit, in driving oncogenic transcriptional changes in H3.3K27M DIPG. Intriguingly, disrupting CREB5 super-enhancers with ABBV-075 significantly reduces its expression and inhibits H3.3K27M DIPG tumor growth. Combined treatment with ABBV-075 and a BRG1 inhibitor presents a promising therapeutic strategy for clinical translation in H3.3K27M DIPG treatment.

In vitro fertilization efficiency is limited in part because a fraction of retrieved oocytes fails to fertilize. Accurately evaluating their quality could significantly improve in vitro fertilization efficiency, which would require better understanding how their maturation may be disrupted. Here, we quantitatively investigate the interplay between superovulation and aging in mouse oocytes and their paired granulosa cells using a newly adapted experimental methodology. We test the hypothesis that superovulation disrupts oocyte maturation, revealing the key intercellular communication pathways dysregulated at the transcriptional level by forced hormonal stimulation. We further demonstrate that granulosa cell transcriptional markers can prospectively predict an associated oocyte's early developmental potential. By using naturally ovulated old mice as a non-stimulated reference, we show that aging and superovulation dysregulate similar genes and interact with each other. By comparing mice and human transcriptional responses of granulosa cells, we find that age-related dysregulation of hormonal responses and cell cycle pathways are shared, though substantial divergence exists in other pathways.