ABSTRACT:Background:After ischemic stroke,neutrophils hyperactivate,increasing in number and worsening inflammation,causing neural damage.Prior scRNA-seq showed Lrg1 modulates cells subsentence to cerebral ischemiarep...ABSTRACT:Background:After ischemic stroke,neutrophils hyperactivate,increasing in number and worsening inflammation,causing neural damage.Prior scRNA-seq showed Lrg1 modulates cells subsentence to cerebral ischemiareperfusion injury,but its mechanism in regulating neutrophil accumulation/differentiation post-injury is unclear.Methods:Lrg1 knockout impact on neutrophil accumulation was assessed via immunofluorescence and western blot.Three-dimensional reconstruction of immunofluorescent staining analyzed cell-cell interactions among neutrophils and microglia.scRNA-seq of WT and Lrg1^(-/-)mice from GSE245386 and GSE279462 was conducted.Each group conducted oxidative phosphorylation scoring via Gene Set Enrichment Analysis(GSEA),while Metascape was employed to perform GO and KEGG enrichment analyses for elucidating functional mechanisms.CellChat exhibited cell-cell communication.Furthermore,alterations in microglial phagocytic activity were evaluated by immunostaining for CD68,a well-established marker of phagolysosomal activity in phagocytic cells.Brain energy metabolism was evaluated via glutamate dehydrogenase activity and ATP levels with ELISA,and enzyme expression was analyzed by immunofluorescence and western blot.Results:Lrg1 knockout decreased neutrophil accumulation and NET formation in mice.3D immunofluorescence reconstruction confirmed neutrophil co-localization with endothelial cells/microglia.scRNA-seq revealed that the oxidative phosphorylation score was significantly higher in the MCAO/R+WT group compared to both the Sham-operated+WT and Lrg1^(-/-)groups.Notably,the oxidative phosphorylation score was further elevated in the MCAO/R+Lrg1^(-/-)group.Immunostaining showed that Lrg1 knockout elevated CD68+lysosome expression post-MCAO/R,with TMEM119 colocalizing with these lysosomes.MCAO/R raised CD68 expression in ischemic brains,an effect further intensified by Lrg1 knockout.KEGG analysis linked differential genes to oxidative phosphorylation pathways.Validation in MCAO/R vs.sham groups revealed increased ROS production and reduced expression of complex enzymes I-V(NDUFB8,SDHB,UQCRC1,MTCO2,ATP5A1).Lrg1 intervention increased enzyme expression.Immunofluorescence and western blot in brain tissue showed similar patterns in microglia and enzymes I-V.Conclusions:Lrg1 knockout significantly enhances microglial phagocytic activity towards neutrophils subsequent to cerebral ischemia-reperfusion injury,through its regulatory effect on the oxidative phosphorylation pathway.This finding accentuates Lrg1 as a highly potential therapeutic target for intervening in and modulating post-ischemic inflammatory responses.展开更多
Gut microbes exhibit complex interactions with their hosts and shape an organism's immune system throughout its lifespan.As the largest secondary lymphoid organ,the spleen has a wide range of immunological functio...Gut microbes exhibit complex interactions with their hosts and shape an organism's immune system throughout its lifespan.As the largest secondary lymphoid organ,the spleen has a wide range of immunological functions.To explore the role of microbiota in regulating and shaping the spleen,we employ scRNA-seq and Stereo-seq technologies based on germ-free(GF)mice to detect differences in tissue size,anatomical structure,cell types,functions,and spatial molecular characteristics.We identify 18 cell types,9 subtypes of T cells,and 7 subtypes of B cells.Gene differential expression analysis reveals that the absence of microorganisms results in alterations in erythropoiesis within the red pulp region and congenital immune deficiency in the white pulp region.Stereo-seq results demonstrate a clear hierarchy of immune cells in the spleen,including marginal zone(MZ)macrophages,MZ B cells,follicular B cells and T cells,distributed in a well-defined pattern from outside to inside.However,this hierarchical structure is disturbed in GF mice.Ccr7 and Cxcl13 chemokines are specifically expressed in the spatial locations of T cells and B cells,respectively.We speculate that the microbiota may mediate the structural composition or partitioning of spleen immune cells by modulating the expression levels of chemokines.展开更多
The process of lymphatic metastasis was proved to be associated with podoplanin-expressing macrophages in breast cancer(BC).This study aimed to investigate the role of the M2 phenotype of tumor-associated macrophages ...The process of lymphatic metastasis was proved to be associated with podoplanin-expressing macrophages in breast cancer(BC).This study aimed to investigate the role of the M2 phenotype of tumor-associated macrophages and mine the key M2 macrophages-related genes for lymph node metastasis in BC.We downloaded the GSE158399 dataset from the Gene Expression Omnibus(GEO)database,which includes transcriptomic profiles of individual cells from primary tumors,negative lymph nodes(NLNs),and positive lymph nodes(PLNs)of breast cancer patients.The cell subsets were identified by clustering analysis after quality control of the scRNA-seq using Seurat.The activation and migration capability of M2 macrophages were evaluated with R package“GSVA”.The key M2 macrophages-related genes were screened from the differential expressed genes(DEGs)and M2 macrophages activation and migration gene sets collected from MSigDB database.Our analysis identified three main cell types in primary tumors,NLNs,and PLNs:basal cells,luminal cells,and immune cell subsets.The further cell type classification of immune cell subsets indicated M2 macrophages accumulation in NLs and PLs.The GSVA enrichment scores for activation and migration capability were increased significantly in M2 macrophages from primary tumors than NLNs and PLNs(pvalue<0.001).Seven M2 macrophages activation-related and 15 M2 macrophages migration-related genes were significantly up-regulated in primary tumors than NLNs and PLNs.The proportion and GSVA enrichment scores for activation and migration of M2 macrophages may be potential markers for lymph node metastasis in breast cancer.Our study demonstrated that twenty-two up-regulated mRNA may be possible therapeutic targets for lymph node metastasis in breast cancer.展开更多
Single-cell RNA-sequencing(scRNA-seq)is a rapidly increasing research area in biomed-ical signal processing.However,the high complexity of single-cell data makes efficient and accurate analysis difficult.To improve th...Single-cell RNA-sequencing(scRNA-seq)is a rapidly increasing research area in biomed-ical signal processing.However,the high complexity of single-cell data makes efficient and accurate analysis difficult.To improve the performance of single-cell RNA data processing,two single-cell features calculation method and corresponding dual-input neural network structures are proposed.In this feature extraction and fusion scheme,the features at the cluster level are extracted by hier-archical clustering and differential gene analysis,and the features at the cell level are extracted by the calculation of gene frequency and cross cell frequency.Our experiments on COVID-19 data demonstrate that the combined use of these two feature achieves great results and high robustness for classification tasks.展开更多
The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis.The single-cell RNA sequencing(scRNA-seq)analysis of the testis was performed to identify genes upr...The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis.The single-cell RNA sequencing(scRNA-seq)analysis of the testis was performed to identify genes upregulated in spermatogonia.Using scRNAseq analysis,we identified the spermatogonia upregulated gene origin recognition complex subunit 6(Orc6),which is involved in DNA replication and cell cycle regulation;its protein expression in the human and mouse testis was detected by western blot and immunofluorescence.To explore the potential function of Orc6 in spermatogonia,the C18-4 cell line was transfected with control or Orc6 siRNA.Subsequently,5-ethynyl-2-deoxyuridine(EdU)and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assays,flow cytometry,and western blot were used to evaluate its effects on proliferation and apoptosis.It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells.Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated(Wnt)/β-catenin signaling.Western blot revealed that the expression ofβ-catenin protein and its phosphorylation(Ser675)were significantly decreased when silencing the expression of ORC6.Our findings indicated that Orc6 was upregulated in spermatogonia,whereby it regulated proliferation and apoptosis by activating Wnt/β-catenin signaling.展开更多
Background:Colorectal cancer(CRC)is a highly heterogeneous malignant tumor that significantly impacts clinical diagnosis and treatment.Single-cell RNA sequencing is an innovative method for exploring tumor heterogenei...Background:Colorectal cancer(CRC)is a highly heterogeneous malignant tumor that significantly impacts clinical diagnosis and treatment.Single-cell RNA sequencing is an innovative method for exploring tumor heterogeneity and understanding its role at cellular and genetic levels.Method:The colorectal cancer Single-cell RNA sequencing data were analysed on the immune.RNA-seq data in bulk form was utilized to assess the major genes of the immune cell subsets linked to CRC.We conducted an analysis of the abundance of immune cells in the microenvironment of CRC,and also performed weighted gene co-expression network analysis.Gene set enrichment analysis helped perform two analytical procedures of subtype groups.Furthermore,Least absolute shrinkage and selection operator regression was employed to analyse and screen for a gene signature.Finally,quantitative PCR Was performed to detect the expression levels of signature genes in CRC.Results:The Single-cell RNA sequencing(GSE146771)dataset was integrated to obtain 9 cell clusters.The Single-sample gene set enrichment analysis showed that the related gene expression of T-cell subsets of different functional statuses could vary greatly between patients with GSE146771.Immune cell analysis of TCGA-CRC indicated an improved overall survival rate for patients with elevated Th2 cell abundance.Five-gene signature(Risk Score=-0.205×CDC25C-0.231×GSTCD-0.010×KPNA2-0.002×KIF15-0.171×ORC1)was developed by weighted correlation network analysis,and lasso Cox regression.Then,the risk prediction efficacy of the signature was validated in four GSE datasets.Furthermore,the expression of five genes was reduced in CRC tissue by quantitative PCR.Conclusion:Five-gene signature based on CRC heterogeneity was developed as a prognosis predictor,which can serve as a potential treatment target.展开更多
In recent years,advancements in single-cell and spatial transcriptomics,which are highly regarded developments in the current era,particularly the emerging integration of single-cell and spatiotemporal transcriptomics...In recent years,advancements in single-cell and spatial transcriptomics,which are highly regarded developments in the current era,particularly the emerging integration of single-cell and spatiotemporal transcriptomics,have enabled a detailed molecular comprehension of the complex regulation of cell fate.The insights obtained from these methodologies are anticipated to significantly contribute to the development of personalized medicine.Currently,single-cell technology is less frequently utilized for prostate cancer compared with other types of tumors.Start-ing from the perspective of RNA sequencing technology,this review outlined the signifcance of single-cell RNA sequencing(scRNA-seq)in prostate cancer research,encompassing preclinical medicine and clinical applications.We summarize the differences between mouse and human prostate cancer as revealed by scRNA-seq studies,as well as a combination of multi-omics methods involving scRNA-seq to highlight the key molecular targets for the diagnosis,treatment,and drug resistance characteristics of prostate cancer.These studies are expected to provide novel insights for the development of immunotherapy and other innovative treatment strategies for castration-resistant prostate cancer.Furthermore,we explore the potential clinical applications stemming from other single-cell technologies in this review,paving the way for future research in precision medicine.展开更多
Objective:The key molecular events signifying the Helicobacter pylori-induced gastric carcinogenesis process are largely unknown.Methods:Bulk tissue-proteomics profiling were leveraged across multi-stage gastric lesio...Objective:The key molecular events signifying the Helicobacter pylori-induced gastric carcinogenesis process are largely unknown.Methods:Bulk tissue-proteomics profiling were leveraged across multi-stage gastric lesions from Linqu(n=166)and Beijing sets(n=99)and single-cell transcriptomic profiling(n=18)to decipher key molecular signatures of H.pylori-related gastric lesion progression and gastric cancer(GC)development.The association of key proteins association with gastric lesion progression and GC development were prospectively studied building on follow-up of the Linqu set and UK Biobank(n=48,529).Results:Concordant proteomics signatures associated with H.pylori infection and gastric carcinogenesis(ρ=0.784,correlation P=1.80×10^(−36))were identified.RNA expression of genes encoding 13 up-and 15 down-regulated key proteins displayed trending alterations in the transition from normal gastric epithelium to intestinal metaplasia,then to malignant cells.A 15-tissue protein panel integrating these signatures demonstrated potential for targeting individuals at high risk for progressing to gastric neoplasia(OR=7.22,95%CI:1.31-39.72 for the high-score group).A 4-circulating protein panel may be used as non-invasive markers predicting the risk of GC development(hazard ratio=3.73,95%confidence interval:1.63-8.54,high-risk vs.low-risk populations,area under the curve=0.75).Conclusions:Concordant proteomics signatures associated with H.pylori infection and gastric carcinogenesis were unveiled with potential as biomarkers for targeted prevention strategies.展开更多
Adolescent depression is increasingly recognized as a serious mental health disorder with distinct clinical and molecular features.Using single-nucleus RNA sequencing,we identified cell-specific transcriptomic changes...Adolescent depression is increasingly recognized as a serious mental health disorder with distinct clinical and molecular features.Using single-nucleus RNA sequencing,we identified cell-specific transcriptomic changes in the nucleus accumbens(NAc),particularly in astrocytes,of adolescent macaques exhibiting depressive-like behaviors.The level of diacylglycerol kinase beta was significantly reduced in neurons and glial cells of depressed macaques,while FKBP5 levels increased in glial cells.Disruption of GABAergic synapses and disruption of D-glutamine and D-glutamate metabolism were linked to depressive phenotypes in medium spiny neurons(MSNs)and subtypes of astrocytes.Communication pathways between astrocytes and D1/D2-MSNs were also disrupted,involving factors like bone morphogenetic protein-6 and Erb-B2 receptor tyrosine kinase-4.Bulk transcriptomic and proteomic analyses corroborated these findings,and FKBP5 upregulation was confirmed by qRT-PCR,western blotting,and immunofluorescence in the NAc of rats and macaques with chronic unpredictable mild stress.Our results highlight the specific roles of different cell types in adolescent depression in the NAc,offering potential targets for new antidepressant therapies.展开更多
Background Thermogenic adipose tissue,both beige and brown,experiences whitening as animals are exposed to warmth and age,but the potential mechanisms are not fully understood.In this study,we employed singlenucleus R...Background Thermogenic adipose tissue,both beige and brown,experiences whitening as animals are exposed to warmth and age,but the potential mechanisms are not fully understood.In this study,we employed singlenucleus RNA-seq to construct a cell atlas during whitening progression and identified the characteristics of thermogenic adipocytes.Results Our histological studies and bulk transcriptome gene expression analysis confirmed that both perirenal and omental adipose tissues(pAT and oAT)exhibited progressive whitening in goats.Compared to the classic brown adipocytes in mice,goat thermogenic adipocytes were more closely related in gene expression patterns to human beige adipocytes,which was also confirmed by adipocyte type-and lineage-specific marker expression analysis.Furthermore,trajectory analysis revealed beige-and white-like adipocytes deriving from a common origin,coexisting and undergoing the transdifferentiation.In addition,differences in gene expression profiles and cell communication patterns(e.g.,FGF and CALCR signaling)between oAT and pAT suggested a lower thermogenic capacity of oAT than that of pAT.Conclusions We constructed a cell atlas of goat pAT and oAT and descripted the characteristics of thermogenic adipocytes during whitening progression.Altogether,our results make a significant contribution to the molecular and cellular mechanisms behind the whitening of thermogenic adipocytes,and providing new insights into obesity prevention in humans and cold adaptation in animals.展开更多
Although the spatial characteristics within the tumor microenvironment of lung adenocarcinoma(LUAD)have been identified,the mechanisms by which these factors promote LUAD progression and immune evasion remain unclear....Although the spatial characteristics within the tumor microenvironment of lung adenocarcinoma(LUAD)have been identified,the mechanisms by which these factors promote LUAD progression and immune evasion remain unclear.Using spatial transcriptomics and single-cell RNA-sequencing data from multi-regional LUAD biopsies consisting of tumor core,tumor edge,and normal area,we sought to delineate the spatial heterogeneity and driving factors of cell colocalization.Two cancer cell sub-clusters(Cancer_c1 and Cancer_c2),associated with LUAD initiation and metastasis,respectively,exhibit distinct spatial distributions and immune cell colocalizations.In particular,Cancer_c1,enriched within the tumor core,could directly interact with B cells or indirectly recruit B cells through macrophages.Conversely,Cancer_c2 enriched within the tumor edge exhibits colocalization with CD8^(+)T cells.Collectively,our work elucidates the spatial distribution of cancer cell subtypes and their interaction with immune cells in the core and edge of LUAD,providing insights for developing therapeutic strategies for cancer intervention.展开更多
Orthopedic conditions have emerged as global health concerns,impacting approximately 1.7 billion individuals worldwide.However,the limited understanding of the underlying pathological processes at the cellular and mol...Orthopedic conditions have emerged as global health concerns,impacting approximately 1.7 billion individuals worldwide.However,the limited understanding of the underlying pathological processes at the cellular and molecular level has hindered the development of comprehensive treatment options for these disorders.The advent of single-cell RNA sequencing(scRNA-seq)technology has revolutionized biomedical research by enabling detailed examination of cellular and molecular diversity.Nevertheless,investigating mechanisms at the single-cell level in highly mineralized skeletal tissue poses technical challenges.In this comprehensive review,we present a streamlined approach to obtaining high-quality single cells from skeletal tissue and provide an overview of existing scRNA-seq technologies employed in skeletal studies along with practical bioinformatic analysis pipelines.By utilizing these methodologies,crucial insights into the developmental dynamics,maintenance of homeostasis,and pathological processes involved in spine,joint,bone,muscle,and tendon disorders have been uncovered.Specifically focusing on the joint diseases of degenerative disc disease,osteoarthritis,and rheumatoid arthritis using scRNA-seq has provided novel insights and a more nuanced comprehension.These findings have paved the way for discovering novel therapeutic targets that offer potential benefits to patients suffering from diverse skeletal disorders.展开更多
Recent data suggest that vascular endothelial growth factor receptor inhibitor(VEGFRi)can enhance the anti-tumor activity of the anti-programmed cell death-1(anti-PD-1)antibody in colorectal cancer(CRC)with microsatel...Recent data suggest that vascular endothelial growth factor receptor inhibitor(VEGFRi)can enhance the anti-tumor activity of the anti-programmed cell death-1(anti-PD-1)antibody in colorectal cancer(CRC)with microsatellite stability(MSS).However,the comparison between this combination and standard third-line VEGFRi treatment is not performed,and reliable biomarkers are still lacking.We retrospectively enrolled MSS CRC patients receiving anti-PD-1 antibody plus VEGFRi(combination group,n=54)or VEGFRi alone(VEGFRi group,n=32),and their efficacy and safety were evaluated.We additionally examined the immune characteristics of the MSS CRC tumor microenvironment(TME)through single-cell and spatial transcriptomic data,and an MSS CRC immune cell-related signature(MCICRS)that can be used to predict the clinical outcomes of MSS CRC patients receiving immunotherapy was developed and validated in our in-house cohort.Compared with VEGFRi alone,the combination of anti-PD-1 antibody and VEGFRi exhibited a prolonged survival benefit(median progression-free survival:4.4 vs.2.0 months,P=0.0024;median overall survival:10.2 vs.5.2 months,P=0.0038)and a similar adverse event incidence.Through single-cell and spatial transcriptomic analysis,we determined ten MSS CRC-enriched immune cell types and their spatial distribution,including naive CD4+T,regulatory CD4+T,CD4+Th17,exhausted CD8+T,cytotoxic CD8+T,proliferated CD8+T,natural killer(NK)cells,plasma,and classical and intermediate monocytes.Based on a systemic meta-analysis and ten machine learning algorithms,we obtained MCICRS,an independent risk factor for the prognosis of MSS CRC patients.Further analyses demonstrated that the low-MCICRS group presented a higher immune cell infiltration and immune-related pathway activation,and hence a significant relation with the superior efficacy of pan-cancer immunotherapy.More importantly,the predictive value of MCICRS in MSS CRC patients receiving immunotherapy was also validated with an in-house cohort.Anti-PD-1 antibody combined with VEGFRi presented an improved clinical benefit in MSS CRC with manageable toxicity.MCICRS could serve as a robust and promising tool to predict clinical outcomes for individual MSS CRC patients receiving immunotherapy.展开更多
基金supported by the Foundation Project:National Natural Science Foundation of China(Nos.:82460249,82100417,81760094)The Foundation of Jiangxi Provincial Department of Science and Technology Outstanding Youth Fund Project(20242BAB23080).
文摘ABSTRACT:Background:After ischemic stroke,neutrophils hyperactivate,increasing in number and worsening inflammation,causing neural damage.Prior scRNA-seq showed Lrg1 modulates cells subsentence to cerebral ischemiareperfusion injury,but its mechanism in regulating neutrophil accumulation/differentiation post-injury is unclear.Methods:Lrg1 knockout impact on neutrophil accumulation was assessed via immunofluorescence and western blot.Three-dimensional reconstruction of immunofluorescent staining analyzed cell-cell interactions among neutrophils and microglia.scRNA-seq of WT and Lrg1^(-/-)mice from GSE245386 and GSE279462 was conducted.Each group conducted oxidative phosphorylation scoring via Gene Set Enrichment Analysis(GSEA),while Metascape was employed to perform GO and KEGG enrichment analyses for elucidating functional mechanisms.CellChat exhibited cell-cell communication.Furthermore,alterations in microglial phagocytic activity were evaluated by immunostaining for CD68,a well-established marker of phagolysosomal activity in phagocytic cells.Brain energy metabolism was evaluated via glutamate dehydrogenase activity and ATP levels with ELISA,and enzyme expression was analyzed by immunofluorescence and western blot.Results:Lrg1 knockout decreased neutrophil accumulation and NET formation in mice.3D immunofluorescence reconstruction confirmed neutrophil co-localization with endothelial cells/microglia.scRNA-seq revealed that the oxidative phosphorylation score was significantly higher in the MCAO/R+WT group compared to both the Sham-operated+WT and Lrg1^(-/-)groups.Notably,the oxidative phosphorylation score was further elevated in the MCAO/R+Lrg1^(-/-)group.Immunostaining showed that Lrg1 knockout elevated CD68+lysosome expression post-MCAO/R,with TMEM119 colocalizing with these lysosomes.MCAO/R raised CD68 expression in ischemic brains,an effect further intensified by Lrg1 knockout.KEGG analysis linked differential genes to oxidative phosphorylation pathways.Validation in MCAO/R vs.sham groups revealed increased ROS production and reduced expression of complex enzymes I-V(NDUFB8,SDHB,UQCRC1,MTCO2,ATP5A1).Lrg1 intervention increased enzyme expression.Immunofluorescence and western blot in brain tissue showed similar patterns in microglia and enzymes I-V.Conclusions:Lrg1 knockout significantly enhances microglial phagocytic activity towards neutrophils subsequent to cerebral ischemia-reperfusion injury,through its regulatory effect on the oxidative phosphorylation pathway.This finding accentuates Lrg1 as a highly potential therapeutic target for intervening in and modulating post-ischemic inflammatory responses.
基金funded by the National Natural Science Foundation of China(81700436)the Science Technology and Innovation Committee of Shenzhen Municipality,China(SGDX20190919142801722)。
文摘Gut microbes exhibit complex interactions with their hosts and shape an organism's immune system throughout its lifespan.As the largest secondary lymphoid organ,the spleen has a wide range of immunological functions.To explore the role of microbiota in regulating and shaping the spleen,we employ scRNA-seq and Stereo-seq technologies based on germ-free(GF)mice to detect differences in tissue size,anatomical structure,cell types,functions,and spatial molecular characteristics.We identify 18 cell types,9 subtypes of T cells,and 7 subtypes of B cells.Gene differential expression analysis reveals that the absence of microorganisms results in alterations in erythropoiesis within the red pulp region and congenital immune deficiency in the white pulp region.Stereo-seq results demonstrate a clear hierarchy of immune cells in the spleen,including marginal zone(MZ)macrophages,MZ B cells,follicular B cells and T cells,distributed in a well-defined pattern from outside to inside.However,this hierarchical structure is disturbed in GF mice.Ccr7 and Cxcl13 chemokines are specifically expressed in the spatial locations of T cells and B cells,respectively.We speculate that the microbiota may mediate the structural composition or partitioning of spleen immune cells by modulating the expression levels of chemokines.
文摘The process of lymphatic metastasis was proved to be associated with podoplanin-expressing macrophages in breast cancer(BC).This study aimed to investigate the role of the M2 phenotype of tumor-associated macrophages and mine the key M2 macrophages-related genes for lymph node metastasis in BC.We downloaded the GSE158399 dataset from the Gene Expression Omnibus(GEO)database,which includes transcriptomic profiles of individual cells from primary tumors,negative lymph nodes(NLNs),and positive lymph nodes(PLNs)of breast cancer patients.The cell subsets were identified by clustering analysis after quality control of the scRNA-seq using Seurat.The activation and migration capability of M2 macrophages were evaluated with R package“GSVA”.The key M2 macrophages-related genes were screened from the differential expressed genes(DEGs)and M2 macrophages activation and migration gene sets collected from MSigDB database.Our analysis identified three main cell types in primary tumors,NLNs,and PLNs:basal cells,luminal cells,and immune cell subsets.The further cell type classification of immune cell subsets indicated M2 macrophages accumulation in NLs and PLs.The GSVA enrichment scores for activation and migration capability were increased significantly in M2 macrophages from primary tumors than NLNs and PLNs(pvalue<0.001).Seven M2 macrophages activation-related and 15 M2 macrophages migration-related genes were significantly up-regulated in primary tumors than NLNs and PLNs.The proportion and GSVA enrichment scores for activation and migration of M2 macrophages may be potential markers for lymph node metastasis in breast cancer.Our study demonstrated that twenty-two up-regulated mRNA may be possible therapeutic targets for lymph node metastasis in breast cancer.
文摘Single-cell RNA-sequencing(scRNA-seq)is a rapidly increasing research area in biomed-ical signal processing.However,the high complexity of single-cell data makes efficient and accurate analysis difficult.To improve the performance of single-cell RNA data processing,two single-cell features calculation method and corresponding dual-input neural network structures are proposed.In this feature extraction and fusion scheme,the features at the cluster level are extracted by hier-archical clustering and differential gene analysis,and the features at the cell level are extracted by the calculation of gene frequency and cross cell frequency.Our experiments on COVID-19 data demonstrate that the combined use of these two feature achieves great results and high robustness for classification tasks.
基金This work was supported by the National Key Research and Development Program of China(No.2022YFC2702700)the National Natural Science Foundation of China(No.82171597)Clinical Research Plan of Shanghai Hospital Development Center(No.SHDC2020CR3077B).
文摘The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis.The single-cell RNA sequencing(scRNA-seq)analysis of the testis was performed to identify genes upregulated in spermatogonia.Using scRNAseq analysis,we identified the spermatogonia upregulated gene origin recognition complex subunit 6(Orc6),which is involved in DNA replication and cell cycle regulation;its protein expression in the human and mouse testis was detected by western blot and immunofluorescence.To explore the potential function of Orc6 in spermatogonia,the C18-4 cell line was transfected with control or Orc6 siRNA.Subsequently,5-ethynyl-2-deoxyuridine(EdU)and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assays,flow cytometry,and western blot were used to evaluate its effects on proliferation and apoptosis.It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells.Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated(Wnt)/β-catenin signaling.Western blot revealed that the expression ofβ-catenin protein and its phosphorylation(Ser675)were significantly decreased when silencing the expression of ORC6.Our findings indicated that Orc6 was upregulated in spermatogonia,whereby it regulated proliferation and apoptosis by activating Wnt/β-catenin signaling.
基金supported by the Guangzhou Science and Technology Plan Project(No.202201010786&2023A04J1129)the Basic Research Project of Guangzhou Municipal School(Hospital),(No.202201020483)+4 种基金the Guangdong Second Provincial General Hospital(No.3DA2021015)Doctoral workstation foundation of Guangdong Second Provincial General Hospital(2021BSGZ018)the science foundation of Guangdong Second Provincial General Hospital(TJGC-2021007)Guangdong Medical Scientific Research(grant No.B2023038)National Natural Science Foundation of China(No.82302640).
文摘Background:Colorectal cancer(CRC)is a highly heterogeneous malignant tumor that significantly impacts clinical diagnosis and treatment.Single-cell RNA sequencing is an innovative method for exploring tumor heterogeneity and understanding its role at cellular and genetic levels.Method:The colorectal cancer Single-cell RNA sequencing data were analysed on the immune.RNA-seq data in bulk form was utilized to assess the major genes of the immune cell subsets linked to CRC.We conducted an analysis of the abundance of immune cells in the microenvironment of CRC,and also performed weighted gene co-expression network analysis.Gene set enrichment analysis helped perform two analytical procedures of subtype groups.Furthermore,Least absolute shrinkage and selection operator regression was employed to analyse and screen for a gene signature.Finally,quantitative PCR Was performed to detect the expression levels of signature genes in CRC.Results:The Single-cell RNA sequencing(GSE146771)dataset was integrated to obtain 9 cell clusters.The Single-sample gene set enrichment analysis showed that the related gene expression of T-cell subsets of different functional statuses could vary greatly between patients with GSE146771.Immune cell analysis of TCGA-CRC indicated an improved overall survival rate for patients with elevated Th2 cell abundance.Five-gene signature(Risk Score=-0.205×CDC25C-0.231×GSTCD-0.010×KPNA2-0.002×KIF15-0.171×ORC1)was developed by weighted correlation network analysis,and lasso Cox regression.Then,the risk prediction efficacy of the signature was validated in four GSE datasets.Furthermore,the expression of five genes was reduced in CRC tissue by quantitative PCR.Conclusion:Five-gene signature based on CRC heterogeneity was developed as a prognosis predictor,which can serve as a potential treatment target.
基金Chinese Scholarship Council(202206240086)National Natural Science Foundation of China(81974099,82170785,81974098,82170784)+4 种基金National Key Research and Development Program of China(2021YFC2009303)programs from Science and Technology Department of Sichuan Province(2021YFH0172)Young Investigator Award of Sichuan University 2017(2017SCU04A17)Technology Innovation Research and Development Project of Chengdu Science and Technology Bureau(2019-YF05-00296-SN)Sichuan University-Panzhihua science and technology cooperation special fund(2020CDPZH-4).
文摘In recent years,advancements in single-cell and spatial transcriptomics,which are highly regarded developments in the current era,particularly the emerging integration of single-cell and spatiotemporal transcriptomics,have enabled a detailed molecular comprehension of the complex regulation of cell fate.The insights obtained from these methodologies are anticipated to significantly contribute to the development of personalized medicine.Currently,single-cell technology is less frequently utilized for prostate cancer compared with other types of tumors.Start-ing from the perspective of RNA sequencing technology,this review outlined the signifcance of single-cell RNA sequencing(scRNA-seq)in prostate cancer research,encompassing preclinical medicine and clinical applications.We summarize the differences between mouse and human prostate cancer as revealed by scRNA-seq studies,as well as a combination of multi-omics methods involving scRNA-seq to highlight the key molecular targets for the diagnosis,treatment,and drug resistance characteristics of prostate cancer.These studies are expected to provide novel insights for the development of immunotherapy and other innovative treatment strategies for castration-resistant prostate cancer.Furthermore,we explore the potential clinical applications stemming from other single-cell technologies in this review,paving the way for future research in precision medicine.
基金funded by the National Natural Science Foundation of China(Grant No.82273704)Noncommunicable Chronic Diseases-National Science and Technology Major Project(Grant Nos.2023ZD0501400-2023ZD0501402)+3 种基金Beijing Hospitals Authority’s Ascent Plan(Grant No.DFL20241102)Beijing Hospitals Authority Clinical Medicine Development of Special Funding Support(Grant No.ZLRK202325)Peking University Medicine Fund for World’s Leading Discipline or Discipline Cluster Development(Grant No.BMU2022XKQ004)the Science Foundation of Peking University Cancer Hospital(Grant Nos.BJCH2024BJ02,XKFZ2410,and 2022-27).
文摘Objective:The key molecular events signifying the Helicobacter pylori-induced gastric carcinogenesis process are largely unknown.Methods:Bulk tissue-proteomics profiling were leveraged across multi-stage gastric lesions from Linqu(n=166)and Beijing sets(n=99)and single-cell transcriptomic profiling(n=18)to decipher key molecular signatures of H.pylori-related gastric lesion progression and gastric cancer(GC)development.The association of key proteins association with gastric lesion progression and GC development were prospectively studied building on follow-up of the Linqu set and UK Biobank(n=48,529).Results:Concordant proteomics signatures associated with H.pylori infection and gastric carcinogenesis(ρ=0.784,correlation P=1.80×10^(−36))were identified.RNA expression of genes encoding 13 up-and 15 down-regulated key proteins displayed trending alterations in the transition from normal gastric epithelium to intestinal metaplasia,then to malignant cells.A 15-tissue protein panel integrating these signatures demonstrated potential for targeting individuals at high risk for progressing to gastric neoplasia(OR=7.22,95%CI:1.31-39.72 for the high-score group).A 4-circulating protein panel may be used as non-invasive markers predicting the risk of GC development(hazard ratio=3.73,95%confidence interval:1.63-8.54,high-risk vs.low-risk populations,area under the curve=0.75).Conclusions:Concordant proteomics signatures associated with H.pylori infection and gastric carcinogenesis were unveiled with potential as biomarkers for targeted prevention strategies.
基金supported by STI2030-Major Projects(2022ZD0212900)the Joint Project of Chongqing Municipal Science and Technology Bureau and Chongqing Health Commission(2023CCXM003)+4 种基金the National Key Research and Development Program of China(2017YFA0505700)the National Natural Science Foundation of China(82271565 and 82301714)the China Postdoctoral Science Foundation(2023TQ0398,GZB20230916,2023MD734124)the Natural Science Foundation of Chongqing,China(CSTB2023NSCQ-BHX0106)the Postdoctoral Innovation Talents Support Program of Chongqing,China(2208013341918508).
文摘Adolescent depression is increasingly recognized as a serious mental health disorder with distinct clinical and molecular features.Using single-nucleus RNA sequencing,we identified cell-specific transcriptomic changes in the nucleus accumbens(NAc),particularly in astrocytes,of adolescent macaques exhibiting depressive-like behaviors.The level of diacylglycerol kinase beta was significantly reduced in neurons and glial cells of depressed macaques,while FKBP5 levels increased in glial cells.Disruption of GABAergic synapses and disruption of D-glutamine and D-glutamate metabolism were linked to depressive phenotypes in medium spiny neurons(MSNs)and subtypes of astrocytes.Communication pathways between astrocytes and D1/D2-MSNs were also disrupted,involving factors like bone morphogenetic protein-6 and Erb-B2 receptor tyrosine kinase-4.Bulk transcriptomic and proteomic analyses corroborated these findings,and FKBP5 upregulation was confirmed by qRT-PCR,western blotting,and immunofluorescence in the NAc of rats and macaques with chronic unpredictable mild stress.Our results highlight the specific roles of different cell types in adolescent depression in the NAc,offering potential targets for new antidepressant therapies.
基金supported by the National Key Research and Development Programme of China(grant number 2021YFF1001000)the National Natural Science Foundation of China(grant number 32170627)+1 种基金the Postdoctoral Innovative Talents Support Program of China(grant number BX20200282)the Natural Science Foundation of Sichuan Province(grant number 23NSFSC1804).
文摘Background Thermogenic adipose tissue,both beige and brown,experiences whitening as animals are exposed to warmth and age,but the potential mechanisms are not fully understood.In this study,we employed singlenucleus RNA-seq to construct a cell atlas during whitening progression and identified the characteristics of thermogenic adipocytes.Results Our histological studies and bulk transcriptome gene expression analysis confirmed that both perirenal and omental adipose tissues(pAT and oAT)exhibited progressive whitening in goats.Compared to the classic brown adipocytes in mice,goat thermogenic adipocytes were more closely related in gene expression patterns to human beige adipocytes,which was also confirmed by adipocyte type-and lineage-specific marker expression analysis.Furthermore,trajectory analysis revealed beige-and white-like adipocytes deriving from a common origin,coexisting and undergoing the transdifferentiation.In addition,differences in gene expression profiles and cell communication patterns(e.g.,FGF and CALCR signaling)between oAT and pAT suggested a lower thermogenic capacity of oAT than that of pAT.Conclusions We constructed a cell atlas of goat pAT and oAT and descripted the characteristics of thermogenic adipocytes during whitening progression.Altogether,our results make a significant contribution to the molecular and cellular mechanisms behind the whitening of thermogenic adipocytes,and providing new insights into obesity prevention in humans and cold adaptation in animals.
基金supported by the National Natural Science Foundation of China(82002432 to J.W.,82302068 to M.Z.,and 32300568 to T.W.)the Natural Science Foundation of Shandong Province(ZR2024MH159 to Y.Z.,ZR2020QH179 to J.W.,ZR2022QH057 to M.Z.,and ZR2021QH005 to T.W.)the China Postdoctoral Science Foundation(2024M752006 to S.M.)。
文摘Although the spatial characteristics within the tumor microenvironment of lung adenocarcinoma(LUAD)have been identified,the mechanisms by which these factors promote LUAD progression and immune evasion remain unclear.Using spatial transcriptomics and single-cell RNA-sequencing data from multi-regional LUAD biopsies consisting of tumor core,tumor edge,and normal area,we sought to delineate the spatial heterogeneity and driving factors of cell colocalization.Two cancer cell sub-clusters(Cancer_c1 and Cancer_c2),associated with LUAD initiation and metastasis,respectively,exhibit distinct spatial distributions and immune cell colocalizations.In particular,Cancer_c1,enriched within the tumor core,could directly interact with B cells or indirectly recruit B cells through macrophages.Conversely,Cancer_c2 enriched within the tumor edge exhibits colocalization with CD8^(+)T cells.Collectively,our work elucidates the spatial distribution of cancer cell subtypes and their interaction with immune cells in the core and edge of LUAD,providing insights for developing therapeutic strategies for cancer intervention.
基金National Key Research and Development Program of China(2022YFA1103202)National Natural Science Foundation of China(82272507,32270887,and 32200654)+6 种基金Natural Science Foundation of Chongqing(CSTB2023NSCQ-ZDJO008)Postdoctoral Innovative Talent Support Program(BX20220397)Independent Research Project of State Key Laboratory of Trauma and Chemical Poisoning(SFLKF202201)Project for Enhancing Innovation of Army Medical University(2023X1839)Talent Innovation Training Program at the Army Medical Center(ZXZYTSYS09)General Hospital of Western Theater Command Research Project(2021-XZYG-B10)University Grants Committee,Research Grants Council of Hong Kong,China(14113723,N_CUHK472/22,C7030-18G,T13-402/17-N,and AoE/M-402/20)。
文摘Orthopedic conditions have emerged as global health concerns,impacting approximately 1.7 billion individuals worldwide.However,the limited understanding of the underlying pathological processes at the cellular and molecular level has hindered the development of comprehensive treatment options for these disorders.The advent of single-cell RNA sequencing(scRNA-seq)technology has revolutionized biomedical research by enabling detailed examination of cellular and molecular diversity.Nevertheless,investigating mechanisms at the single-cell level in highly mineralized skeletal tissue poses technical challenges.In this comprehensive review,we present a streamlined approach to obtaining high-quality single cells from skeletal tissue and provide an overview of existing scRNA-seq technologies employed in skeletal studies along with practical bioinformatic analysis pipelines.By utilizing these methodologies,crucial insights into the developmental dynamics,maintenance of homeostasis,and pathological processes involved in spine,joint,bone,muscle,and tendon disorders have been uncovered.Specifically focusing on the joint diseases of degenerative disc disease,osteoarthritis,and rheumatoid arthritis using scRNA-seq has provided novel insights and a more nuanced comprehension.These findings have paved the way for discovering novel therapeutic targets that offer potential benefits to patients suffering from diverse skeletal disorders.
基金supported by the National Natural Science Foundation of China(No.81972012).
文摘Recent data suggest that vascular endothelial growth factor receptor inhibitor(VEGFRi)can enhance the anti-tumor activity of the anti-programmed cell death-1(anti-PD-1)antibody in colorectal cancer(CRC)with microsatellite stability(MSS).However,the comparison between this combination and standard third-line VEGFRi treatment is not performed,and reliable biomarkers are still lacking.We retrospectively enrolled MSS CRC patients receiving anti-PD-1 antibody plus VEGFRi(combination group,n=54)or VEGFRi alone(VEGFRi group,n=32),and their efficacy and safety were evaluated.We additionally examined the immune characteristics of the MSS CRC tumor microenvironment(TME)through single-cell and spatial transcriptomic data,and an MSS CRC immune cell-related signature(MCICRS)that can be used to predict the clinical outcomes of MSS CRC patients receiving immunotherapy was developed and validated in our in-house cohort.Compared with VEGFRi alone,the combination of anti-PD-1 antibody and VEGFRi exhibited a prolonged survival benefit(median progression-free survival:4.4 vs.2.0 months,P=0.0024;median overall survival:10.2 vs.5.2 months,P=0.0038)and a similar adverse event incidence.Through single-cell and spatial transcriptomic analysis,we determined ten MSS CRC-enriched immune cell types and their spatial distribution,including naive CD4+T,regulatory CD4+T,CD4+Th17,exhausted CD8+T,cytotoxic CD8+T,proliferated CD8+T,natural killer(NK)cells,plasma,and classical and intermediate monocytes.Based on a systemic meta-analysis and ten machine learning algorithms,we obtained MCICRS,an independent risk factor for the prognosis of MSS CRC patients.Further analyses demonstrated that the low-MCICRS group presented a higher immune cell infiltration and immune-related pathway activation,and hence a significant relation with the superior efficacy of pan-cancer immunotherapy.More importantly,the predictive value of MCICRS in MSS CRC patients receiving immunotherapy was also validated with an in-house cohort.Anti-PD-1 antibody combined with VEGFRi presented an improved clinical benefit in MSS CRC with manageable toxicity.MCICRS could serve as a robust and promising tool to predict clinical outcomes for individual MSS CRC patients receiving immunotherapy.
