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The complex pathology of mild traumatic brain injury (mTBI) is a main contributor to the difficulties in achieving a successful therapeutic regimen. Thyroxine (T4) administration has been shown to prevent the cognitive impairments induced by mTBI in mice but the mechanism is poorly understood. To understand the underlying mechanism, we carried out a single cell transcriptomic study to investigate the spatiotemporal effects of T4 on individual cell types in the hippocampus and frontal cortex at three post-injury stages in a mouse model of mTBI. We found that T4 treatment altered the proportions and transcriptomes of numerous cell types across tissues and timepoints, particularly oligodendrocytes, astrocytes, and microglia, which are crucial for injury repair. T4 also reversed the expression of mTBI-affected genes such as Ttr, mt-Rnr2, Ggn12, Malat1, Gnaq, and Myo3a, as well as numerous pathways such as cell/energy/iron metabolism, immune response, nervous system, and cytoskeleton-related pathways. Cell-type specific network modeling revealed that T4 mitigated select mTBI-perturbed dynamic shifts in subnetworks related to cell cycle, stress response, and RNA processing in oligodendrocytes. Cross cell-type ligand-receptor networks revealed the roles of App, Hmgb1, Fn1, and Tnf in mTBI, with the latter two ligands having been previously identified as TBI network hubs. mTBI and/or T4 signature genes were enriched for human genome-wide association study (GWAS) candidate genes for cognitive, psychiatric and neurodegenerative disorders related to mTBI. Our systems-level single cell analysis elucidated the temporal and spatial dynamic reprogramming of cell-type specific genes, pathways, and networks, as well as cell-cell communications as the mechanisms through which T4 mitigates cognitive dysfunction induced by mTBI.

BACKGROUND: Acute lung allograft dysfunction (ALAD) is an imprecise syndrome denoting concern for the onset of chronic lung allograft dysfunction (CLAD). Mechanistic biomarkers are needed that stratify risk of ALAD progression to CLAD. We hypothesized that single cell investigation of bronchoalveolar lavage (BAL) cells at the time of ALAD would identify immune cells linked to progressive graft dysfunction. METHODS: We prospectively collected BAL from consenting lung transplant recipients for single cell RNA sequencing. ALAD was defined by a ≥10% decrease in FEV1 not caused by infection or acute rejection and samples were matched to BAL from recipients with stable lung function. We examined cell compositional and transcriptional differences across control, ALAD with decline, and ALAD with recovery groups. We also assessed cell-cell communication. RESULTS: BAL was assessed for 17 ALAD cases with subsequent decline (ALAD declined), 13 ALAD cases that resolved (ALAD recovered), and 15 cases with stable lung function. We observed broad differences in frequencies of the 26 unique cell populations across groups (p = 0.02). A CD8 T cell (p = 0.04) and a macrophage cluster (p = 0.01) best identified ALAD declined from the ALAD recovered and stable groups. This macrophage cluster was distinguished by an anti-inflammatory signature and the CD8 T cell cluster resembled a Tissue Resident Memory subset. Anti-inflammatory macrophages signaled to activated CD8 T cells via class I HLA, fibronectin, and galectin pathways (p

Objective To evaluate the efficacy of Tiaochang Xiaoliu Granules in preventing the recurrence of colorectal adenoma (CRA) and to conduct a further pharmacological analysis. Methods A single-center, double-blind, randomized controlled trial was conducted in this study. Patients who underwent resection for CRA were randomly divided into two groups: one group received the treatment with Tiaochang Xiaoliu Granules, while the other group received the treatment with placebo Granules for a duration of six months. One year later, colonoscopy results were reviewed, with the primary outcome being the CRA detection rate at follow-up. Additionally, ultra-high-performance liquid chromatography coupled with quadrupole-exactive orbitrap mass spectrometry was employed to identify the active ingredients in Tiaochang Xiaoliu Granules. Core target screening and functional enrichment were performed using R software, and single-cell RNA sequencing (scRNA-seq)data were analyzed for cell annotation and communication pathways to identify key effector and signaling pathways. Results The randomized controlled trial revealed a one-year adenoma recurrence rate of 12.0% in the Tiaochang Xiaoliu Granules group significantly lower than that observed in the placebo group (37.1%), with statistically significant difference between the two groups. Pharmacological analyses identified core genes associated with CRA recurrence prevention, including AKT1, TP53, TNF, IL6, CASP3, VEGFA, INSR, IL1B, MAPK3, and EGFR. Further analysis of single-cell sequencing data suggested that the VISFATIN pathway plays a crucial role in preventing CRA recurrence, with the primary cellular communication occurring through the NAMPT-INSR pathway, involved in transmitting signals from embryonic stem cells to epithelial cells and endothelial cells. This process may be regulated by endothelial-mesenchymal transition (EndMT) and lipid metabolism. Conclusion Tiaochang Xiaoliu Granules significantly reduce the one-year recurrence rate following CRA surgery. The mechanism may involve regulation of the VISFATIN pathway, influencing EndMT and lipid metabolism to mitigate CRA recurrence and progression. These findings support the clinical application of Tiaochang Xiaoliu Granules in preventing CRA. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is caused by altered maturation and differentiation of myeloid blasts, as well as transcriptional/epigenetic alterations, all leading to excessive proliferation of malignant blood cells in the bone marrow. Tumor heterogeneity due to the acquisition of new somatic alterations leads to a high rate of resistance to current therapies or reduces the efficacy of hematopoietic stem cell transplantation (HSCT), thus increasing the risk of relapse and mortality. Single-cell RNA sequencing (scRNA-seq) will enable the classification of AML and guide treatment approaches by profiling patients with different facets of the same disease, stratifying risk, and identifying new potential therapeutic targets at the time of diagnosis or after treatment. ScRNA-seq allows the identification of quiescent stem-like cells, and leukemia stem cells responsible for resistance to therapeutic approaches and relapse after treatment. This method also introduces the factors and mechanisms that enhance the efficacy of the HSCT process. Generated data of the transcriptional profile of the AML could even allow the development of cancer vaccines and CAR T-cell therapies while saving valuable time and alleviating dangerous side effects of chemotherapy and HSCT in vivo. However, scRNA-seq applications face various challenges such as a large amount of data for high-dimensional analysis, technical noise, batch effects, and finding small biological patterns, which could be improved in combination with artificial intelligence models. Highlights: Tumor heterogeneity and drug resistance reduce therapeutic efficacy in AML treatment. ScRNA-seq improves the accuracy and quality of HSCT, resulting in a durable antitumor response. ScRNA-seq introduces neoantigens, biomarkers and cell subtypes that may contribute to reversing resistance to standard therapies in AML. Combination approaches using scRNA-seq and AI may provide effective AML management in the future. [ABSTRACT FROM AUTHOR]

Marek's disease (MD), a T cell lymphoma disease in chickens, is caused by the Marek's disease virus (MDV) found ubiquitously in the poultry industry. Genetically resistant Line 63 (L6) and susceptible Line 72 (L7) chickens have been instrumental to research on avian immune system response to MDV infection. In this study we characterized molecular signatures unique to splenic immune cell types across different genetic backgrounds 6 days after infection. Using three populations, L6, L7, and an F1 cross between L6xL7, we evaluated the immune cell transcriptome of responding cell types using single cell RNA sequencing. Several MDV genes were found expressed mainly in cytotoxic T cells while ICP4 and MEQ MDV genes were expressed across infected cell types. Using the F1 we quantified allele specific expression (ASE) of biallelic SNPs and found biased expression of parental alleles specific to immune cell subtypes. We identified 22 SNPs with ASE in response to MDV infection mapped to gene rich regions surrounding 59 genes of critical importance for chromatin remodeling and transcriptional regulation. Histone deacetylase genes (HDAC1 and HDAC8) had increased expression of L6 alleles, while small nuclear RNA genes (SNORA68 and SNORA72) expressed higher levels of L7 alleles with infection in T cell subsets. SNPs with ASE also mapped genes important for an adequate immune response including GNLY (cytotoxic activity) and PDIA3 (component of MHC class I peptide loading complex), and genes known to promote viral replication (MCM5 and EIF3M). These results show that functional variants associated with susceptibility to MD may have a bigger impact in subsets of immune cell types, and by characterizing the transcriptomes of these subtypes we can unravel molecular signatures specific to MD genomic resistance. [ABSTRACT FROM AUTHOR]

Background: Tumor microenvironment (TME), particularly immune cell infiltration, programmed cell death (PCD) and stress, has increasingly become a focal point in colorectal cancer (CRC) treatment. Uncovering the intricate crosstalk between these factors can enhance our understanding of CRC, guide therapeutic strategies, and improve patient prognosis. Methods: We constructed an immune-related cell death and stress (ICDS) prognostic model utilizing machine learning methodologies. Furthermore, we performed enrichment analyses and deconvolution algorithms to elucidate the complex interactions between immune cell infiltration and the processes of PCD and stress within a substantial array of transcriptomic data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus data base (GEO) related to CRC. Single-cell sequencing and biochemical experiments were used to validate the interaction between the model genes and programmed cell death in tumor cells. Results: The ICDS prognostic model exhibited robust predictive performance in seven independent cohorts, revealing an inverse correlation between model scores and patient prognosis. Meanwhile, the ICDS index was positively correlated with clinical stage. Model analysis indicated that patient subgroups with low ICDS index exhibited heightened immune activation features and elevated activity in PCD and stress pathways. Single-cell analysis further revealed that macrophages were the central drivers of immune characteristics underlying prognostic differences within the ICDS prognostic model. Pseudotime analysis and cellular experiments indicated that the model gene GAL3ST4 promotes the transition of macrophages toward an M2 pro-tumor phenotype. Furthermore, cell communication analysis and experimental validation revealed that the cuproptosis in tumor cells suppress GAL3ST4 expression, thereby inhibiting M2-like macrophage polarization. Conclusion: In summary, we constructed the ICDS prognostic model and uncovered the mechanism by which tumor cells downregulate GAL3ST4 expression via cuproptosis to inhibit M2-like macrophage polarization, providing new targets and biomarkers for CRC treatment and prognosis evaluation. [ABSTRACT FROM AUTHOR]

Macrophages play important roles in metabolic dysfunction-associated steatohepatitis (MASH), an advanced and inflammatory stage of metabolic dysfunction-associated steatotic liver disease (MASLD). In humans and mice, the cellular heterogeneity and diverse function of hepatic macrophages in MASH have been investigated by single cell RNA sequencing (scRNA-seq). However, little is known about their roles in rats. Here, we collected liver tissues at the postnatal week 16, when our previously characterized Lep∆I14/∆I14 rats developed MASH phenotypes. By scRNA-seq, we found an increase in the number of macrophages and endothelial cells and a decrease in that of NK and B cells. Hepatic macrophages in rats underwent a unique M1 to M2 transition without expression of the classical markers such as Arg1 and Nos2, except for Cd163. Lipid-associated macrophages (LAMs) were increased, which could be detected by the antibody against Cd63. In the microenvironment, macrophages had an increased number of interactions with hepatocytes, myofibroblasts, T cells, neutrophils, and dendritic cells, while their interaction strengths remained unchanged. Finally, the macrophage migration inhibitory factor (MIF) pathway was identified as the top upregulated cell-communication pathway in MASH. In conclusion, we dissected hepatic macrophage dynamics during MASH at single cell resolution and provided fundamental tools for the investigation of MASH in rat models. [ABSTRACT FROM AUTHOR]

Background: Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Single-cell RNA sequencing (scRNA-seq) provides gene expression profiles at the single-cell level. Hence, we evaluated gene expression in the peripheral blood of patients with COPD. Methods: Peripheral blood samples from seven healthy controls and eight patients with COPD were obtained in this study. The 10X Genomics Chromium Instrument and cDNA synthesis kit were utilized to generate a barcoded cDNA library for single cell RNA-sequencing. We compared the scRNA-seq data between the COPD and control groups using computational analysis. Functional analyses were performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Results: scRNA-seq was used to analyze the transcriptome of peripheral blood mononuclear cells from seven normal controls and eight patients with COPD. We found an increased number of monocyte/macrophages in the COPD group compared to the normal control group. Among the differentially expressed genes (DEGs) in monocyte/macrophages, we identified 15 upregulated genes (EGR1, NR4A1, CCL3, CXCL8, PTGS2, CD83, BCL2A1, SGK1, IL1B, BTG2, NFKBIZ, DUSP2, MAFB, PLAUR and CCL3L1) and 7 downregulated genes (FOLR3, RPS4Y1, HLA-DRB5, NAMPT, CD52, TMEM176A and TMEM176B) in the COPD group compared to the normal control group. Conclusions: Using scRNA-seq, we found differences in cell type distribution, especially in monocyte/ macrophages. Several upregulated and downregulated genes were found in the monocyte/macrophages of the COPD group. [ABSTRACT FROM AUTHOR]

Neurotropic alphaviruses such as Venezuelan equine encephalitis virus (VEEV) are critical human pathogens that continually expand to naïve populations and for which there are no licensed vaccines or therapeutics. VEEV is highly infectious via the aerosol route and is a recognized weaponizable biothreat that causes neurological disease in humans. The neuropathology of VEEV has been attributed to an inflammatory immune response in the brain yet the underlying mechanisms and specific immune cell populations involved are not fully elucidated. This study uses single-cell RNA sequencing to produce a comprehensive transcriptional profile of immune cells isolated from the brain over a time course of infection in a mouse model of VEEV. Analyses reveal differentially activated subpopulations of microglia, including a distinct type I interferon-expressing subpopulation. This is followed by the sequential infiltration of myeloid cells and cytotoxic lymphocytes, also comprising subpopulations with unique transcriptional signatures. We identify a subpopulation of myeloid cells that form a distinct localization pattern in the hippocampal region whereas lymphocytes are widely distributed, indicating differential modes of recruitment, including that to specific regions of the brain. Altogether, this study provides a high-resolution analysis of the immune response to VEEV in the brain and highlights potential avenues of investigation for therapeutics that target neuroinflammation in the brain. [ABSTRACT FROM AUTHOR]

Abstract Background Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Single-cell RNA sequencing (scRNA-seq) provides gene expression profiles at the single-cell level. Hence, we evaluated gene expression in the peripheral blood of patients with COPD. Methods Peripheral blood samples from seven healthy controls and eight patients with COPD were obtained in this study. The 10X Genomics Chromium Instrument and cDNA synthesis kit were utilized to generate a barcoded cDNA library for single cell RNA-sequencing. We compared the scRNA-seq data between the COPD and control groups using computational analysis. Functional analyses were performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Results scRNA-seq was used to analyze the transcriptome of peripheral blood mononuclear cells from seven normal controls and eight patients with COPD. We found an increased number of monocyte/macrophages in the COPD group compared to the normal control group. Among the differentially expressed genes (DEGs) in monocyte/macrophages, we identified 15 upregulated genes (EGR1, NR4A1, CCL3, CXCL8, PTGS2, CD83, BCL2A1, SGK1, IL1B, BTG2, NFKBIZ, DUSP2, MAFB, PLAUR and CCL3L1) and 7 downregulated genes (FOLR3, RPS4Y1, HLA-DRB5, NAMPT, CD52, TMEM176A and TMEM176B) in the COPD group compared to the normal control group. Conclusions Using scRNA-seq, we found differences in cell type distribution, especially in monocyte/ macrophages. Several upregulated and downregulated genes were found in the monocyte/macrophages of the COPD group.

BackgroundMany approaches have been developed to overcome technical noise in single cell RNA-sequencing (scRNAseq). As researchers dig deeper into data-looking for rare cell types, subtleties of cell states, and details of gene regulatory networks-there is a growing need for algorithms with controllable accuracy and fewer ad hoc parameters and thresholds. Impeding this goal is the fact that an appropriate null distribution for scRNAseq cannot simply be extracted from data in which ground truth about biological variation is unknown (i.e., usually).ResultsWe approach this problem analytically, assuming that scRNAseq data reflect only cell heterogeneity (what we seek to characterize), transcriptional noise (temporal fluctuations randomly distributed across cells), and sampling error (i.e., Poisson noise). We analyze scRNAseq data without normalization-a step that skews distributions, particularly for sparse data-and calculate p values associated with key statistics. We develop an improved method for selecting features for cell clustering and identifying gene-gene correlations, both positive and negative. Using simulated data, we show that this method, which we call BigSur (Basic Informatics and Gene Statistics from Unnormalized Reads), captures even weak yet significant correlation structures in scRNAseq data. Applying BigSur to data from a clonal human melanoma cell line, we identify thousands of correlations that, when clustered without supervision into gene communities, align with known cellular components and biological processes, and highlight potentially novel cell biological relationships.ConclusionsNew insights into functionally relevant gene regulatory networks can be obtained using a statistically grounded approach to the identification of gene-gene correlations.

Juntao Chen,1,2,* Chunjiao Lu,2,* Wenjie Xie,1,2 Xiaoqian Cao,1 Jiannan Zhang,1 Juanjuan Luo,2 Juan Li1 1Key Laboratory of Bioresources and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, People’s Republic of China; 2Engineering Research Center of Key Technique for Biotherapy of Guangdong Province, Shantou University Medical College, Shantou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Juan Li, Key Laboratory of Bioresources and Eco-environment of Ministry of Education, College of Life Science, Sichuan University, No. 24 South Section 1, Yihuan Road, Chengdu, 610065, People’s Republic of China, Email lijuanscuhk@163.com Juanjuan Luo, Engineering Research Center of Key Technique for Biotherapy of Guangdong Province, Shantou University Medical College, 22 Xinling Road, Shantou, 515041, People’s Republic of China, Email 15jjluo1@stu.edu.cnIntroduction: The proliferation of nanoplastics (NPs) has emerged as a significant environmental concern due to their extensive use, raising concerns about potential adverse effects on human health. However, the exact impacts of NPs on the early development of hematopoietic organs remain poorly understood.Methods: This investigation utilized fluorescence microscopy to observe the effects of various NP concentrations on the caudal vein plexus (CVP) development in zebrafish embryos. Subsequent RNA sequencing (RNA-seq) identified genes related to CVP deformities and hematopoietic stem/progenitor cells (HSPCs) in zebrafish embryos exposed to NPs. Additionally, single cell RNA sequencing (scRNA-seq) analysis identified genes associated with the development of CVP and HSPCs. RT-qPCR assessed changes in expression of these genes in zebrafish embryos exposed to different NP concentrations.Results: The impact of NPs on zebrafish embryos was investigated, revealing significant reductions in survival and hatching rates and decreases in body length alongside increased heart rates. Exposure to NPs at 8 mg/L severely impaired zebrafish CVP development. RNA-seq revealed that NPs exposure altered the activity of oxidative enzymes, hydrolases, and the extracellular matrix in zebrafish embryos. Treatment with 10 μM NAC effectively rescued the CVP defects induced by NPs. Additionally, scRNA-seq identified genes associated with EC and HSPC development, and subsequent RT-qPCR validation confirmed significant expression changes in these genes.Conclusion: The results of this study suggest that NPs induce oxidative stress in vascular ECs and HSPCs, which mediates CVP damage and impairs hematopoiesis in zebrafish embryos.Keywords: nanoplastics, caudal vein plexus, oxidative stress, endothelial cells, hematopoietic stem/progenitor cells, zebrafish, single cell RNA sequencing

Xiangnan Zhou,1,2,* Jingyuan Ning,3,* Rui Cai,2 Jiayi Liu,2 Haoyu Yang,4 Yanping Bai1 1Department of Dermatology, China-Japan Friendship Hospital, National Center for Integrative Medicine, Beijing, 100029, People’s Republic of China; 2Beijing University of Chinese Medicine, China-Japan Friendship Clinical School of Medicine, Beijing, 100029, People’s Republic of China; 3State Key Laboratory of Medical Molecular Biology & Department of Medical Genetics, Institute of Basic Medical Sciences & School of Basic Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100005, People’s Republic of China; 4Department of Dermatology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, 100010, People’s Republic of China*These authors contributed equally to this work: Xiangnan Zhou, Jingyuan NingCorrespondence: Yanping Bai, Department of Dermatology, China-Japan Friendship Hospital, Beijing, 100029, People’s Republic of China, Email yanpbcjfh@163.comBackground: Psoriasis represents a persistent, immune-driven inflammatory condition affecting the skin, characterized by a lack of well-established biologic treatments without adverse events. Consequently, the identification of novel targets and therapeutic agents remains a pressing priority in the field of psoriasis research.Methods: We collected single-cell RNA sequencing (scRNA-seq) datasets and inferred T cell differentiation trajectories through pseudotime analysis. Bulk transcriptome and scRNA-seq data were integrated to identify differentially expressed genes (DEGs). Machine learning was employed to screen candidate genes. Correlation analysis was used to predict the interactions between cells expressing insulin-induced gene 1 (INSIG1) and other immune cells. Finally, drug docking was performed on INSIG1, and the expression levels of INSIG1 in psoriasis were verified through clinical and in vivo experiments, and further in vivo experiments established the efficacy of tetrandrine in the treatment of psoriasis.Results: T cells were initially categorized into seven states, with differentially expressed genes in T cells (TDEGs) identified and their functions and signaling pathways. INSIG1 emerged as a characteristic gene for psoriasis and was found to be downregulated in psoriasis and potentially negatively associated with T cells, influencing psoriasis fatty acid metabolism, as inferred from enrichment and immunoinfiltration analyses. In the cellular communication network, cells expressing INSIG1 exhibited close interactions with other immune cells through multiple signaling channels. Furthermore, drug sensitivity showed that tetrandrine stably binds to INSIG1, could be a potential therapeutic agent for psoriasis.Conclusion: INSIG1 emerges as a specific candidate gene potentially regulating the fatty acid metabolism of patients with psoriasis. In addition, tetrandrine shows promise as a potential treatment for the condition.Keywords: psoriasis, single-cell RNA sequencing, machine learning, INSIG1, pseudotime analysis

Abstract Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by an insidious onset. Despite the emphasis on motor symptom-based diagnosis, there remains an unmet clinical need for effective diagnostic approaches during the prodromal phase of PD. Recent advances in single-cell RNA sequencing (scRNA-seq) and bulk transcriptomic analyses of PD patients open avenues for identifying potential diagnostic biomarkers. A comprehensive cell trajectory analysis was conducted using scRNA-seq datasets to identify gene expressions associated with the cellular transition from healthy to PD-associated states. Integration of scRNA-seq datasets with weighted gene co-expression network analysis (WGCNA) allowed extraction of pyroptosis-associated differentially expressed genes (PDEGs). Using LASSO logistic regression, support vector machine recursive feature elimination (SVM-RFE) and random forest methods, we developed a diagnostic model centred on PDEGs. In addition, immunoinfiltration, inflammatory signalling pathways and intercellular communication were detected by scRNA-seq analyses. In PD patients, the number of cells including metencephalic-like cells, excitatory neurons, inhibitory neurons and MHB-like cells was significantly reduced, whereas the proportion of astrocytes and microglia, immunoinfiltration and inflammatory signalling pathways were upregulated compared to healthy individuals. Using scRNA-seq and WGCNA analyses, two pyroptosis-related diagnostic genes, POLR2K and TIMM8B, were identified and a diagnostic model based on them was constructed, which showed promising performance upon validation. This study established a pyroptosis-related diagnostic model for PD through the analyses of scRNA-seq combined with bulk transcriptome data, which improved the understanding of the role of PDEGs in PD and provided new insights into the diagnostic strategies for this neurodegenerative disease.

Key points Gut hormones control intestinal function, metabolism and appetite, and have been harnessed therapeutically to treat type 2 diabetes and obesity. Our understanding of the enteroendocrine axis arises largely from animal studies, but intestinal organoid models make it possible to identify, genetically modify and purify human enteroendocrine cells (EECs). This study aimed to map human EECs using single‐cell RNA sequencing. Organoids derived from human duodenum and ileum were genetically modified using CRISPR‐Cas9 to express the fluorescent protein Venus driven by the chromogranin‐A promoter. Fluorescent cells from CHGA‐Venus organoids were purified by flow cytometry and analysed by 10X single‐cell RNA sequencing. Cluster analysis separated EEC populations, allowing an examination of differentially expressed hormones, nutrient‐sensing machinery, transcription factors and exocytotic machinery. Bile acid receptor GPBAR1 was most highly expressed in L‐cells (producing glucagon‐like peptide 1 and peptide YY), long‐chain fatty acid receptor FFAR1 was highest in I‐cells (cholecystokinin), K‐cells (glucose‐dependent insulinotropic polypeptide) and L‐cells, short‐chain fatty acid receptor FFAR2 was highest in ileal L‐cells and enterochromaffin cells, olfactory receptor OR51E1 was notably expressed in ileal enterochromaffin cells, and the glucose‐sensing sodium glucose cotransporter SLC5A1 was highly and differentially expressed in K‐ and L‐cells, reflecting their known responsiveness to ingested glucose. The organoid EEC atlas was merged with published data from human intestine and organoids, with good overlap between enteroendocrine datasets. Understanding the similarities and differences between human EEC types will facilitate the development of drugs targeting the enteroendocrine axis for the treatment of conditions such as diabetes, obesity and intestinal disorders. Gut hormones regulate intestinal function, nutrient homeostasis and metabolism and form the basis of the new classes of drugs for obesity and diabetes. As enteroendocrine cells (EECs) comprise only ∼1% of the intestinal epithelium, they are under‐represented in current single‐cell atlases. To identify, compare and characterise human EECs we generated chromogranin‐A labelled organoids from duodenal and ileal biopsies by CRISPR‐Cas9. Fluorescent chromogranin‐A positive EECs were purified and analysed by single‐cell RNA sequencing, revealing predominant cell clusters producing different gut hormones. Cell clusters exhibited differential expression of nutrient‐sensing machinery including bile acid receptors, long‐ and short‐chain fatty acid receptors and glucose transporters. Organoid‐derived EECs mapped well onto data from native intestinal cell populations, extending coverage of EECs. [ABSTRACT FROM AUTHOR]

Background: Undifferentiated pleomorphic sarcoma (UPS) is a highly malignant mesenchymal tumor that ranks as one of the most common types of soft tissue sarcoma. Even though chemotherapy increases the 5-year survival rate in UPS, high tumor heterogeneity frequently leads to chemotherapy resistance and consequently to recurrences. In this study, we characterized the cell composition and the transcriptional profile of UPS with resistance to chemotherapy at the single cell resolution. Methods: A 58-year-old woman was diagnosed with a 13.6 × 9.3 × 6.0 cm multi-nodular tumor with heterogeneous cysto-solid structure at the level of the distal metadiaphysis of the left thigh during magnetic resonance tomography. Morphological and immunohistochemical analysis led to the diagnosis of high-grade (G3) UPS. Neoadjuvant chemotherapy, surgery (negative resection margins), and adjuvant chemotherapy were conducted, but tumor recurrence developed. The UPS sample was used to perform single-cell RNA sequencing by chromium-fixed RNA profiling. Results: Four subpopulations of tumor cells and seven subpopulations of tumor microenvironment (TME) have been identified in UPS. The expression of chemoresistance genes has been detected, including KLF4 (doxorubicin and ifosfamide), ULK1, LUM, GPNMB, and CAVIN1 (doxorubicin), and AHNAK2 (gemcitabine) in tumor cells and ETS1 (gemcitabine) in TME. Conclusions: This study provides the first description of the single-cell transcriptome of UPS with resistance to two lines of chemotherapy, showcasing the gene expression in subpopulations of tumor cells and TME, which may be potential markers for personalized cancer therapy. [ABSTRACT FROM AUTHOR]

Lymph node metastasis, the initial step in distant metastasis, represents a primary contributor to mortality in patients diagnosed with oral squamous cell carcinoma (OSCC). However, the underlying mechanisms of lymph node metastasis in OSCC remain incompletely understood. Here, the transcriptomes of 56 383 single cells derived from paired tissues of six OSCC patients are analyzed. This study founds that CXCR4+ epithelial cells, identified as highly malignant disseminated tumor cells (DTCs), exhibited a propensity for lymph node metastasis. Importantly, a distinct subset of tumor‐associated macrophages (TAMs) characterized by exclusive expression of phosphoprotein 1 (SPP1) is discovered. These TAMs may remodel the metastatic lymph node microenvironment by potentially activating fibroblasts and promoting T cell exhaustion through SPP1‐CD44 and CD155‐CD226 ligand‐receptor interactions, thereby facilitating colonization and proliferation of disseminated tumor cells. The research advanced the mechanistic understanding of metastatic tumor microenvironment (TME) and provided a foundation for the development of personalized treatments for OSCC patients with metastasis. [ABSTRACT FROM AUTHOR]

Backgrounds: Gastric cancer (GC) remains a global health threat due to frequent treatment failures caused by primary or acquired resistance. Although cancer-associated fibroblasts (CAFs) have been implicated in this process, it is still unclear which specific subtype(s) of CAFs hinder T-cell infiltration and promote resistance to immunotherapy. Methods: We analyzed the GC fibroblast atlas in detail by combining 63,955 single cells from 14 scRNA-seq datasets. We also performed RNA-seq data in a local GC cohort and examined 13 bulk RNA-seq datasets to understand the biological and clinical roles of different CAF subsets. Additionally, we conducted in vitro experiments to study the role of specific proteins in GC development. Results: We identified a total of 17 fibroblast subsets in gastric cancer, nine of which did not fit into the existing CAFs classification. These subsets exhibited significant heterogeneity in distribution and biological characteristics (metabolism, cell-cell interactions, differentiation state), as well as clinical functions such as prognosis and response to immunotherapy. In particular, cluster 6 stood out for its high expression of MFAP5, CFD, and PI16; it was found to be negatively associated with both overall survival and response to immunotherapy in GC. This association was linked to an immunosuppressive microenvironment characterized by an increase in M2 macrophages but higher levels of T cell dysfunction and exclusion—a feature shared by tumors expressing MFAP5. Furthermore, the addition of human recombinant MFAP5 promoted proliferation and migration of HGC-27 cells by inducing the MFAP5/NOTCH1/HEY1 signaling pathway. Conclusion: We introduce a high-resolution GC fibroblast atlas. The 17 identified fibroblast clusters provide valuable opportunities for gaining deeper biological insights into the relationship between fibroblasts and GC development. Particularly, cluster 6 and its specific marker MFAP5 could serve as prognostic factors in GC and form a foundation for personalized therapeutic combinations to address primary resistance to ICIs. [ABSTRACT FROM AUTHOR]

In developed economies, the growing number of older individuals is a pressing issue. As a result, research progress into ageing has emphasized the significance of staying healthy in one's later years. Stem cells have a fundamental role to play in fostering diverse cell types and necessary processes for tissue repair and regeneration. Stem cells experience the effects of ageing over time, which is caused by their functional deterioration. Changes to stem cells, their niches and signals from other tissues they interact with are crucial factors in the ageing of stem cells. Progress in single-cell RNA sequencing (scRNA-seq) technology has greatly advanced stem cell research. This review examines the mechanisms of stem cell ageing, its impact on health and investigates the potential of stem cell therapy, with a special emphasis on the skin. [ABSTRACT FROM AUTHOR]

Background: Tracheal adenoid cystic carcinoma (ACC) is a slow growing yet aggressive malignancy with high rates of local recurrence as well as distant metastasis. Tracheal ACC exhibit a low mutation burden along with high mutational diversity, and generally do not respond well to chemotherapeutics. Methods: We present a rare case of primary tracheal ACC initially presenting with nonspecific cervicalgia and globus sensation that was ultimately treated with tracheal resection followed by chemoradiation. Immune profiling of intratumoral T‐cell receptor (TCR) repertoire was subsequently performed using single cell RNA sequencing (scRNAseq). Results: We describe a rare case of primary tracheal adenoid cystic carcinoma highlighting several management principles as well as providing new insights into intratumor T cell populations. Conclusions: Primary tracheal ACC is most commonly treated with surgical resection followed by adjuvant therapy. Further characterization of the tumor immune microenvironment is necessary to better understand ACC disease biology and to identify potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Cleft lip and/or primary palate (CL/P) represent a prevalent congenital malformation, the aetiology of which is highly intricate. Although it is generally accepted that the condition arises from failed fusion between the upper lip and primary palate, the precise mechanism underlying this fusion process remains enigmatic. In this study, we utilized transposase‐accessible chromatin sequencing (scATAC‐seq) and single‐cell RNA sequencing (scRNA‐seq) to interrogate lambdoidal junction tissue derived from C57BL/6J mouse embryos at critical stages of embryogenesis (10.5, 11.5 and 12.5 embryonic days). We successfully identified distinct subgroups of mesenchymal and ectodermal cells involved in the fusion process and characterized their unique transcriptional profiles. Furthermore, we conducted cell differentiation trajectory analysis, revealing a dynamic repertoire of genes that are sequentially activated or repressed during pseudotime, facilitating the transition of relevant cell types. Additionally, we employed scATAC data to identify key genes associated with the fusion process and demonstrated differential chromatin accessibility across major cell types. Finally, we constructed a dynamic intercellular communication network and predicted upstream transcriptional regulators of critical genes involved in important signalling pathways. Our findings provide a valuable resource for future studies on upper lip and primary palate development, as well as congenital defects. [ABSTRACT FROM AUTHOR]

The monkeypox (MPXV) outbreak in 2022 is more prevalent among individuals with human immunodeficiency virus (HIV). While it is plausible that HIV-induced immunosuppression could result in a more severe progression, the exact mechanisms remain undetermined. To better understand the immunopathology of MPXV in patients with and without HIV infection, we employed single-cell RNA sequencing (scRNA-seq) to analyze peripheral blood mononuclear cells (PBMCs) from 6 patients hospitalized for MPXV, 3 of whom had HIV infection (HIV antibody positive & HIV RNA level below the detection limit), and 3 patients only infected with MPXV (HIV-). We map the peripheral immune response in both the acute phase and the recovery period, showing the reconfiguration of peripheral immune cell phenotypes in acute stage compared with recovery stage of 6 patients, characterized by disturbed cell subsets and intense cell interactions mediated by monocytes and neutrophils. Importantly, besides different Mono/DC dynamic between HIV + and HIV- patients, HIV + patients showed decreased NK cells subsets and expansion of some CD8 T cell subsets, we also found obviously dysregulated gene expression in B cells, thus proposing mechanism underlying serious condition underlying HIV + patients. In conclusion, our findings provide a comprehensive cell atlas of MPXV patients, shed light on the mechanisms underlying the severe disease progression and longer recovery time in HIV + individuals.

Abstract Background Many approaches have been developed to overcome technical noise in single cell RNA-sequencing (scRNAseq). As researchers dig deeper into data—looking for rare cell types, subtleties of cell states, and details of gene regulatory networks—there is a growing need for algorithms with controllable accuracy and fewer ad hoc parameters and thresholds. Impeding this goal is the fact that an appropriate null distribution for scRNAseq cannot simply be extracted from data in which ground truth about biological variation is unknown (i.e., usually). Results We approach this problem analytically, assuming that scRNAseq data reflect only cell heterogeneity (what we seek to characterize), transcriptional noise (temporal fluctuations randomly distributed across cells), and sampling error (i.e., Poisson noise). We analyze scRNAseq data without normalization—a step that skews distributions, particularly for sparse data—and calculate p values associated with key statistics. We develop an improved method for selecting features for cell clustering and identifying gene–gene correlations, both positive and negative. Using simulated data, we show that this method, which we call BigSur (Basic Informatics and Gene Statistics from Unnormalized Reads), captures even weak yet significant correlation structures in scRNAseq data. Applying BigSur to data from a clonal human melanoma cell line, we identify thousands of correlations that, when clustered without supervision into gene communities, align with known cellular components and biological processes, and highlight potentially novel cell biological relationships. Conclusions New insights into functionally relevant gene regulatory networks can be obtained using a statistically grounded approach to the identification of gene–gene correlations.

Abstract Background At present, immunotherapy has become a powerful treatment for advanced gastric cancer (AGC), but not all patients can benefit from it. According to the latest research, the impact of B cell subpopulations on the immune microenvironment of gastric cancer (GC) is unknown. Exploring whether the interaction between B cells and tumor cells in GC affects the effectiveness of immunotherapy has attracted our interest. Methods This study involved the re-analysis of single-cell RNA (scRNA) and spatial transcriptomics (ST) data from publicly available datasets. The focus was on investigating the subpopulations and differentiation trajectories of B cells in the gastric cancer (GC) tumor immune microenvironment (TIME). Spatial transcriptomics (ST) and multiple immunofluorescence (mIF) revealed a clear co-localization pattern between B cells and tumor cells. Multiple immunotherapy datasets were collected to identify unique immunotherapy biomarkers. The unique immunotherapeutic potential of targeting CCL28 was validated through a mouse gastric cancer model. In addition, flow cytometry revealed changes in the tumor immune microenvironment targeting CCL28. Results The re-analysis of ST data from multiple cancer types revealed a co-localization pattern between B cells and tumor cells. A significant number of IgA plasma cells were identified in the GC TIME. Five different tumor-infiltrating B cell subpopulations and two unique B cell differentiation trajectories were characterized, along with seven GC-related states. By analyzing the communication between GC cells and B cells, it was further discovered that tumor cells can influence and recruit plasma cells through CCL28-CCR10 signaling. Additionally, there was a crosstalk between GC cells and B cells. Finally, we identified the LAMA/CD44 signaling axis as a potential prognostic marker for immunotherapy through a large amount of immunotherapy data. We also validated through various animal tumor models that targeting CCL28 can significantly promote CD8+T cell infiltration and function in the TME by regulating B cell and plasma cell functions, and has the ability to synergize immunotherapy. Conclusion The co-localization and crosstalk between GC cells and B cells significantly affect the efficacy of immunotherapy, and inhibiting the CCL28-CCR10 signal axis is a potential immunotherapy target for GC. Meanwhile, LAMA/CD44 pair may be a potential adverse indicator for immunotherapy and tumor prognosis.

Abstract Background Metabolite-associated cell communications play critical roles in maintaining human biological function. However, most existing tools and resources focus only on ligand-receptor interaction pairs where both partners are proteinaceous, neglecting other non-protein molecules. To address this gap, we introduce the MRCLinkdb database and algorithm, which aggregates and organizes data related to non-protein L-R interactions in cell-cell communication, providing a valuable resource for predicting intercellular communication based on metabolite-related ligand-receptor interactions. Results Here, we manually curated the metabolite-ligand-receptor (ML-R) interactions from the literature and known databases, ultimately collecting over 790 human and 670 mouse ML-R interactions. Additionally, we compiled information on over 1900 enzymes and 260 transporter entries associated with these metabolites. We developed Metabolite-Receptor based Cell Link Database (MRCLinkdb) to store these ML-R interactions data. Meanwhile, the platform also offers extensive information for presenting ML-R interactions, including fundamental metabolite information and the overall expression landscape of metabolite-associated gene sets (such as receptor, enzymes, and transporter proteins) based on single-cell transcriptomics sequencing (covering 35 human and 26 mouse tissues, 52 human and 44 mouse cell types) and bulk RNA-seq/microarray data (encompassing 62 human and 39 mouse tissues). Furthermore, MRCLinkdb introduces a web server dedicated to the analysis of intercellular communication based on ML-R interactions. MRCLinkdb is freely available at https://www.cellknowledge.com.cn/mrclinkdb/ . Conclusions In addition to supplementing ligand-receptor databases, MRCLinkdb may provide new perspectives for decoding the intercellular communication and advancing related prediction tools based on ML-R interactions.

Abstract Cuproptosis is a novel type to regulate cell death with copper-dependent manner, and has been reported to involve in the occurrence and development of various malignant tumors. However, the association between cuproptosis and the tumor microenvironment (TME) of clear cell renal cell carcinoma (ccRCC) remained unclear. To address this question, we integrated the single cell RNA sequencing (scRNA-seq) datasets of ccRCC across different stages, systematically examined the distinctive expression patterns of cuproptosis-related genes (CRGs) within the TME of ccRCC, and explored the crucial signatures using the spatial transcriptome sequencing (ST-seq) dataset. The cuproptosis activities reduced in cancer tissues along with the ccRCC development, and recovered after therapy. We identified HILPDA+ ccRCC1 subtype, characterized with hypoxia, as cuproptosis susceptible cells associated with a better prognosis. The main co-expression modules of HILPDA+ ccRCC1 subtype highlighted the role in anion transport, response to oxygen species and PD-L1-PD-1 pathway. Furthermore, the immunosuppressive cells might interact with HILPDA+ ccRCC1 subtype via HAVCR2-LGALS9, C3-C3AR1, HLA-A-CD8B and HLA-C-CD8A axises to shape the cuproptosis-related TME landscape. In summary, we anticipate that this study will offer valuable insights and potential strategies of cuproptosis for therapy of ccRCC. Graphical Abstract

Background: Clear cell renal cell carcinoma (ccRCC) poses substantial treatment challenges, especially in advanced stages where the efficacy of immune checkpoint blockade (ICB) therapy varies significantly. Elevated expression of the oncogene TUBA1C has been correlated with poor prognosis in various cancers, however, its role in ccRCC is unclear, especially concerning ICB resistance. Methods: Single-cell analysis was used to examine gene expression variations in malignant cells post-ICB therapy. This included investigating TUBA1C expression across different ICB response groups and its relationship with CD274. A general module of action was identified through pan-cancer and pan-tissue analysis. TUBA1C expression and its association with clinical characteristics and prognosis was further validated. Multiple algorithms were employed to explore immune cell infiltration levels, and the DepMap database was utilized to assess gene dependency and mutation status in kidney cancer cell lines. The in silico knockout of TUBA1C was performed using deep learning model, complemented by immunohistochemical assays, clinical cohort and functional assays validations. Results: TUBA1C expression is elevated in malignant cells following ICB therapy and is correlated with ICB resistance in ccRCC. High TUBA1C expression activates PI3K/AKT pathway and is associated with increased infiltration of regulatory T cells and myeloid-derived suppressor cells, which contributes to an immunosuppressive microenvironment in ccRCC. Patients with high TUBA1C expression exhibit a greater tumor mutation burden and increased genetic variation, which causes a worse prognosis. Additionally, TUBA1C dependency and its effects were evident in kidney cancer cell lines, where mutations conferred resistance to anti-PD-L1 therapy. In silico knockout analyses indicated that treatment targeting TUBA1C shifted malignant cells to a state responsive to ICB therapy. Immunohistochemistry, RT-qPCR and clinical cohort validation further confirmed that TUBA1C expression was upregulated and contributed to poorer outcome in ccRCC. Finaly, wound healing and CCK-8 assays demonstrated the potent oncogenic function of TUBA1C. Conclusions: TUBA1C is a pivotal regulator in ccRCC, affecting both disease progression and the effectiveness of ICB therapy by fostering an immunosuppressive microenvironment mediated by the PI3K/AKT pathway. Additionally, TUBA1C holds promise, both as a prognostic biomarker and a therapeutic target, for enhancing responsiveness to ICB. [ABSTRACT FROM AUTHOR]

Traumatic spinal cord injury (tSCI) is a severe injury to the central nervous system that is categorized into primary and secondary injuries. Among them, the local microenvironmental imbalance in the spinal cord caused by secondary spinal cord injury includes accumulation of cytokines and chemokines, reduced angiogenesis, dysregulation of cellular energy metabolism, and dysfunction of immune cells at the site of injury, which severely impedes neurological recovery from spinal cord injury (SCI). In recent years, single-cell techniques have revealed the heterogeneity of multiple immune cells at the genomic, transcriptomic, proteomic, and metabolomic levels after tSCI, further deepening our understanding of the mechanisms underlying tSCI. However, spatial information about the tSCI microenvironment, such as cell location and cell cell interactions, is lost in these approaches. The application of spatial multiomics technology can solve this problem by combining the data obtained from immunohistochemistry and multiparametric analysis to reveal the changes in the microenvironment at different times of secondary injury after SCI. In this review, we systematically review the progress of spatial multi-omics techniques in the study of the microenvironment after SCI, including changes in the immune microenvironment and discuss potential future therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Inflammatory Bowel Diseases (IBDs), encompassing Ulcerative Colitis (UC) and Crohn's Disease (CD), are chronic, recurrent inflammatory conditions of the gastrointestinal tract. The microRNA (miRNA) -mRNA regulatory network is pivotal in the initiation and progression of IBDs. Although individual studies provide valuable insights into miRNA mechanisms in IBDs, they often have limited scope due to constraints in population diversity, sample size, sequencing platform variability, batch effects, and potential researcher bias. Our study aimed to construct comprehensive miRNA-mRNA regulatory networks and determine the cellular sources and functions of key miRNAs in IBD pathogenesis. Methods: To minimize potential bias from individual studies, we utilized a text mining-based approach on published scientific literature from PubMed and PMC databases to identify miRNAs and mRNAs associated with IBDs and their subtypes. We constructed miRNA-mRNA regulatory networks by integrating both predicted and experimentally validated results from DIANA, Targetscan, PicTar, Miranda, miRDB, and miRTarBase (all of which are databases for miRNA target annotation). The functions of miRNAs were determined through gene enrichment analysis of their target mRNAs. Additionally, we used two large-scale single-cell RNA sequencing datasets to identify the cellular sources of miRNAs and the association of their expression levels with clinical status, molecular and functional alternation in CD and UC. Results: Our analysis systematically summarized IBD-related genes using textmining methodologies. We constructed three comprehensive miRNA-mRNA regulatory networks specific to IBD, CD, and UC. Through cross-analysis with two large-scale scRNA-seq datasets, we determined the cellular sources of the identified miRNAs. Despite originating from different cell types, hsa-miR-142, hsa-miR-145, and hsa-miR-146a were common to both CD and UC. Notably, hsa-miR-145 was identified as myofibroblast-specific in both CD and UC. Furthermore, we found that higher tissue repair and enhanced glucose and lipid metabolism were associated with hsa-miR-145 in myofibroblasts in both CD and UC contexts. Conclusion: This comprehensive approach revealed common and distinct miRNA-mRNA regulatory networks in CD and UC, identified cell-specific miRNA expressions (notably hsa-miR-145 in myofibroblasts), and linked miRNA expression to functional alterations in IBD. These findings not only enhance our understanding of IBD pathogenesis but also offer promising diagnostic biomarkers and therapeutic targets for clinical practice in managing IBDs. [ABSTRACT FROM AUTHOR]

The dorsal aorta (DA) is the first major blood vessel to develop in the embryonic cardiovascular system. Its formation is governed by a coordinated process involving the migration, specification, and arrangement of angioblasts into arterial and venous lineages, a process conserved across species. Although vascular endothelial growth factor a (VEGF-A) is known to drive DA specification and formation, the kinases involved in this process remain ambiguous. Thus, we investigated the role of protein kinase B (Akt) in zebrafish by generating a quadruple mutant (aktΔ/Δ), in which expression and activity of all Akt genes – akt1, -2, -3a and -3b – are strongly decreased. Live imaging of developing aktΔ/Δ DA uncovers early arteriovenous malformations. Single-cell RNA-sequencing analysis of aktΔ/Δ endothelial cells corroborates the impairment of arterial, yet not venous, cell specification. Notably, endothelial specific expression of ligand-independent activation of Notch or constitutively active Akt1 were sufficient to re-establish normal arterial specification in aktΔ/Δ. The Akt loss-of-function mutant unveils that Akt kinase can act upstream of Notch in arterial endothelial cells, and is involved in proper embryonic artery specification. This sheds light on cardiovascular development, revealing a mechanism behind congenital malformations. [ABSTRACT FROM AUTHOR]

Background: At present, immunotherapy has become a powerful treatment for advanced gastric cancer (AGC), but not all patients can benefit from it. According to the latest research, the impact of B cell subpopulations on the immune microenvironment of gastric cancer (GC) is unknown. Exploring whether the interaction between B cells and tumor cells in GC affects the effectiveness of immunotherapy has attracted our interest. Methods: This study involved the re-analysis of single-cell RNA (scRNA) and spatial transcriptomics (ST) data from publicly available datasets. The focus was on investigating the subpopulations and differentiation trajectories of B cells in the gastric cancer (GC) tumor immune microenvironment (TIME). Spatial transcriptomics (ST) and multiple immunofluorescence (mIF) revealed a clear co-localization pattern between B cells and tumor cells. Multiple immunotherapy datasets were collected to identify unique immunotherapy biomarkers. The unique immunotherapeutic potential of targeting CCL28 was validated through a mouse gastric cancer model. In addition, flow cytometry revealed changes in the tumor immune microenvironment targeting CCL28. Results: The re-analysis of ST data from multiple cancer types revealed a co-localization pattern between B cells and tumor cells. A significant number of IgA plasma cells were identified in the GC TIME. Five different tumor-infiltrating B cell subpopulations and two unique B cell differentiation trajectories were characterized, along with seven GC-related states. By analyzing the communication between GC cells and B cells, it was further discovered that tumor cells can influence and recruit plasma cells through CCL28-CCR10 signaling. Additionally, there was a crosstalk between GC cells and B cells. Finally, we identified the LAMA/CD44 signaling axis as a potential prognostic marker for immunotherapy through a large amount of immunotherapy data. We also validated through various animal tumor models that targeting CCL28 can significantly promote CD8+T cell infiltration and function in the TME by regulating B cell and plasma cell functions, and has the ability to synergize immunotherapy. Conclusion: The co-localization and crosstalk between GC cells and B cells significantly affect the efficacy of immunotherapy, and inhibiting the CCL28-CCR10 signal axis is a potential immunotherapy target for GC. Meanwhile, LAMA/CD44 pair may be a potential adverse indicator for immunotherapy and tumor prognosis. [ABSTRACT FROM AUTHOR]

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a serious pulmonary vascular disease characterized by residual thrombi in the pulmonary arteries and distal pulmonary microvascular remodeling. The pathogenesis of CTEPH remains unclear, but many factors such as inflammation, immunity, coagulation and angiogenesis may be involved. Monocytes are important immune cells that can differentiate into macrophages and dendritic cells and play an important role in thrombus formation. However, the distribution, gene expression profile and differentiation trajectory of monocyte subsets in CTEPH patients have not been systematically studied. This study aims to reveal the characteristics and functions of monocytes in CTEPH patients using single-cell sequencing technology, and to provide new insights for the diagnosis and treatment of CTEPH. Methods: Single-cell RNA sequencing (scRNA-seq) were performed to analyze the transcriptomic features of peripheral blood mononuclear cells (PBMCs) from healthy controls, CTEPH patients and the tissues from CTEPH patients after the pulmonary endarterectomy (PEA). We established a CTEPH rat model with chronic pulmonary embolism caused by repeated injection of autologous thrombi through a central venous catheter, and used flow cytometry to detect the proportion changes of monocyte subsets in CTEPH patients and CTEPH rat model. We also observed the infiltration degree of macrophage subsets in thrombus tissue and their differentiation relationship with peripheral blood monocyte subsets by immunofluorescence staining. Results: The results showed that the monocyte subsets in peripheral blood of CTEPH patients changed significantly, especially the proportion of CD16+ monocyte subset increased. This monocyte subset had unique functional features at the transcriptomic level, involving processes such as cell adhesion, T cell activation, coagulation response and platelet activation, which may play an important role in pulmonary artery thrombus formation and pulmonary artery intimal remodeling. In addition, we also found that the macrophage subsets in pulmonary endarterectomy tissue of CTEPH patients showed pro-inflammatory and lipid metabolism reprogramming features, which may be related to the persistence and insolubility of pulmonary artery thrombi and the development of pulmonary hypertension. Finally, we also observed that CD16+ monocyte subset in peripheral blood of CTEPH patients may be recruited to pulmonary artery intimal tissue and differentiate into macrophage subset with high expression of IL-1β, participating in disease progression. Conclusion: CD16+ monocytes subset had significant gene expression changes in CTEPH patients, related to platelet activation, coagulation response and inflammatory response. And we also found that these cells could migrate to the thrombus and differentiate into macrophages with high expression of IL-1b involved in CTEPH disease progression. We believe that CD16+ monocytes are important participants in CTEPH and potential therapeutic targets. [ABSTRACT FROM AUTHOR]