Landscape of four different stages of human gastric cancer revealed by single-cell sequencing

BACKGROUND Gastric cancer(GC)poses a substantial risk to human health due to its high prevalence and mortality rates.Nevertheless,current therapeutic strategies remain insufficient.Single-cell RNA sequencing(scRNA-seq)offers the potential to provide comprehensive insights into GC pathogenesis.AIM To explore the distribution and dynamic changes of cell populations in the GC tumor microenvironment using scRNA-seq techniques.METHODS Cancerous tissues and paracancerous tissues were obtained from patients diagnosed with GC at various stages(I,II,III,and IV).Single-cell suspensions were prepared and analyzed using scRNA-seq to examine transcriptome profiles and cell-cell interactions.Additionally,quantitative real-time polymerase chain reaction(qRT-PCR)and flow cytometry were applied for measuring the expression of cluster of differentiation(CD)2,CD3D,CD3E,cytokeratin 19,cytokeratin 8,and epithelial cell adhesion molecules.RESULTS Transcriptome data from 73645 single cells across eight tissues of four patients were categorized into 25 distinct cell clusters,representing 10 different cell types.Variations were observed in these cell type distribution.The adjacent epithelial cells in stages II and III exhibited a degenerative trend.Additionally,the quantity of CD4 T cells and CD8 T cells were evidently elevated in cancerous tissues.Interaction analysis displayed a remarkable increase in interaction between B cells and other mast cells in stages II,III,and IV of GC.These findings were further validated through qRT-PCR and flow cytometry,demonstrating elevated T cells and declined epithelial cells within the cancerous tissues.CONCLUSION This study provides a comprehensive analysis of cell dynamics across GC stages,highlighting key interactions within the tumor microenvironment.These findings offer valuable insights for developing novel therapeutic strategies.

Identification and validation of a pyroptosis-related prognostic model for colorectal cancer based on bulk and single-cell RNA sequencing data

BACKGROUND Pyroptosis impacts the development of malignant tumors,yet its role in colorectal cancer(CRC)prognosis remains uncertain.AIM To assess the prognostic significance of pyroptosis-related genes and their association with CRC immune infiltration.METHODS Gene expression data were obtained from The Cancer Genome Atlas(TCGA)and single-cell RNA sequencing dataset GSE178341 from the Gene Expression Omnibus(GEO).Pyroptosis-related gene expression in cell clusters was analyzed,and enrichment analysis was conducted.A pyroptosis-related risk model was developed using the LASSO regression algorithm,with prediction accuracy assessed through K-M and receiver operating characteristic analyses.A nomo-gram predicting survival was created,and the correlation between the risk model and immune infiltration was analyzed using CIBERSORTx calculations.Finally,the differential expression of the 8 prognostic genes between CRC and normal samples was verified by analyzing TCGA-COADREAD data from the UCSC database.RESULTS An effective pyroptosis-related risk model was constructed using 8 genes-CHMP2B,SDHB,BST2,UBE2D2,GJA1,AIM2,PDCD6IP,and SEZ6L2(P<0.05).Seven of these genes exhibited differential expression between CRC and normal samples based on TCGA database analysis(P<0.05).Patients with higher risk scores demonstrated increased death risk and reduced overall survival(P<0.05).Significant differences in immune infiltration were observed between low-and high-risk groups,correlating with pyroptosis-related gene expression.CONCLUSION We developed a pyroptosis-related prognostic model for CRC,affirming its correlation with immune infiltration.This model may prove useful for CRC prognostic evaluation.

Single-cell transcriptome sequencing reveals the mechanism regulating rice plumule development

Seed plumules comprise multiple developing tissues and are key sites for above-ground plant organ morphogenesis.Here,the spatial expression of genes in developing rice seed plumules was characterized by single-cell transcriptome sequencing in Zhongjiazao 17,a popular Chinese indica rice cultivar.Of 15 cell clusters,13 were assigned to cell types using marker genes and cluster-specific genes.Marker genes of multiple cell types were expressed in several clusters,suggesting a complex developmental system.Some genes for signaling by phytohormones such as abscisic acid were highly expressed in specific clusters.Various cis-elements in the promoters of genes specifically expressed in cell clusters were calculated,and some key hormone-related motifs were frequent in certain clusters.Spatial expression patterns of genes involved in rapid seed germination,seedling growth,and development were identified.These findings enhanced our understanding of cellular diversity and specialization within plumules of rice,a monocotyledonous model crop.

Applications of single-cell RNA sequencing in spermatogenesis and molecular evolution

Spermatogenic cell heterogeneity is determined by the complex process of spermatogenesis differentiation.However,effectively revealing the regulatory mechanisms underlying mammalian spermatogenic cell development and differentiation via traditional methods is difficult.Advances in technology have led to the emergence of many single-cell transcriptome sequencing protocols,which have partially addressed these challenges.In this review,we detail the principles of 10x Genomics technology and summarize the methods for downstream analysis of single-cell transcriptome sequencing data.Furthermore,we explore the role of single-cell transcriptome sequencing in revealing the heterogeneity of testicular ecological niche cells,delineating the establishment and disruption of testicular immune homeostasis during human spermatogenesis,investigating abnormal spermatogenesis in humans,and,ultimately,elucidating the molecular evolution of mammalian spermatogenesis.

Revolutionizing stem cell research:unbiased insights through single-cell sequencing

Stem cells have shown great application potential in wound repair,tissue regeneration,and disease treatment.Therefore,a full understanding of stem cells and their related regulatory mechanisms in disease treatment is conducive to improving the therapeutic effect of stem cells.However,thus far,there are still many unsolved mysteries in thefield of stem cells due to technical limitations,which hinder the in-depth exploration of stem cells and their wide clinical application.Single-cell sequencing(SCS)has provided very powerful and unbiased insights into cell gene expression profiles at the single-cell level,bringing exciting results to the stem cellfield.At present,SCS has been widely applied in thefield of stem cells,covering various aspects,including lineage tracing the development of stem cells,identifying new stem cell types,exploring cellular heterogeneity,and identifying internal functional subpopulations.In this paper,we focus on the latest research progress and discuss the application of SCS technology in stem cells.

Food nutrition and toxicology targeting on specific organs in the era of single-cell sequencing

Due to the complex natures of dietary food components,it is difficult to elucidate how the compounds affect host health.Dietary food often selectively presents its mechanism of action on different cell types,and participates in the modulation of targeted cells and their microenvironments within organs.However,the limitations of traditional in vitro assays or in vivo animal experiments cannot comprehensively examine cellular heterogeneity and the tissue-biased influences.Single-cell RNA sequencing(sc RNA-seq)has emerged as an indispensable methodology to decompose tissues into different cell types for the demonstration of transcriptional profiles of individual cells.Sc RNA-seq applications has been summarized on three typical organs(brain,liver,kidney),and two representative immune-and tumor related health problems.The everincreasing role of sc RNA-seq in dietary food research with further improvement can provide sub-cellular information and the coupling between other cellular modalities.In this review,we propose utilizing sc RNAseq to more effectively capture the subtle and complex effects of food chemicals,and how they may lead to health problems at single-cell resolution.This novel technique will be valuable to elucidate the underlying mechanism of both the health benefits of food nutrients and the detrimental consequences food toxicants at the cellular level.

Single-cell sequencing technology in diabetic wound healing:New insights into the progenitors-based repair strategies

Diabetes mellitus(DM),an increasingly prevalent chronic metabolic disease,is characterised by prolonged hyperglycaemia,which leads to long-term health consequences.Although much effort has been put into understanding the pathogenesis of diabetic wounds,the underlying mechanisms remain unclear.The advent of single-cell RNA sequencing(scRNAseq)has revolutionised biological research by enabling the identification of novel cell types,the discovery of cellular markers,the analysis of gene expression patterns and the prediction of develop-mental trajectories.This powerful tool allows for an in-depth exploration of pathogenesis at the cellular and molecular levels.In this editorial,we focus on progenitor-based repair strategies for diabetic wound healing as revealed by scRNAseq and highlight the biological behaviour of various healing-related cells and the alteration of signalling pathways in the process of diabetic wound healing.ScRNAseq could not only deepen our understanding of the complex biology of diabetic wounds but also identify and validate new targets for inter-vention,offering hope for improved patient outcomes in the management of this challenging complication of DM.

Single-cell RNA sequencing analysis of the retina under acute high intraocular pressure

High intraocular pressure causes retinal ganglion cell injury in primary and secondary glaucoma diseases,yet the molecular landscape characteristics of retinal cells under high intraocular pressure remain unknown.Rat models of acute hypertension ocular pressure were established by injection of cross-linked hyaluronic acid hydrogel(Healaflow■).Single-cell RNA sequencing was then used to describe the cellular composition and molecular profile of the retina following high intraocular pressure.Our results identified a total of 12 cell types,namely retinal pigment epithelial cells,rod-photoreceptor cells,bipolar cells,Müller cells,microglia,cone-photoreceptor cells,retinal ganglion cells,endothelial cells,retinal progenitor cells,oligodendrocytes,pericytes,and fibroblasts.The single-cell RNA sequencing analysis of the retina under acute high intraocular pressure revealed obvious changes in the proportions of various retinal cells,with ganglion cells decreased by 23%.Hematoxylin and eosin staining and TUNEL staining confirmed the damage to retinal ganglion cells under high intraocular pressure.We extracted data from retinal ganglion cells and analyzed the retinal ganglion cell cluster with the most distinct expression.We found upregulation of the B3gat2 gene,which is associated with neuronal migration and adhesion,and downregulation of the Tsc22d gene,which participates in inhibition of inflammation.This study is the first to reveal molecular changes and intercellular interactions in the retina under high intraocular pressure.These data contribute to understanding of the molecular mechanism of retinal injury induced by high intraocular pressure and will benefit the development of novel therapies.

Action of circulating and infiltrating B cells in the immune microenvironment of colorectal cancer by single-cell sequencing analysis

BACKGROUND The complexity of the immune microenvironment has an impact on the treatment of colorectal cancer(CRC),one of the most prevalent malignancies worldwide.In this study,multi-omics and single-cell sequencing techniques were used to investigate the mechanism of action of circulating and infiltrating B cells in CRC.By revealing the heterogeneity and functional differences of B cells in cancer immunity,we aim to deepen our understanding of immune regulation and provide a scientific basis for the development of more effective cancer treatment strategies.AIM To explore the role of circulating and infiltrating B cell subsets in the immune microenvironment of CRC,explore the potential driving mechanism of B cell development,analyze the interaction between B cells and other immune cells in the immune microenvironment and the functions of communication molecules,and search for possible regulatory pathways to promote the anti-tumor effects of B cells.METHODS A total of 69 paracancer(normal),tumor and peripheral blood samples were collected from 23 patients with CRC from The Cancer Genome Atlas database(https://portal.gdc.cancer.gov/).After the immune cells were sorted by multicolor flow cytometry,the single cell transcriptome and B cell receptor group library were sequenced using the 10X Genomics platform,and the data were analyzed using bioinformatics tools such as Seurat.The differences in the number and function of B cell infiltration between tumor and normal tissue,the interaction between B cell subsets and T cells and myeloid cell subsets,and the transcription factor regulatory network of B cell subsets were explored and analyzed.RESULTS Compared with normal tissue,the infiltrating number of CD20+B cell subsets in tumor tissue increased significantly.Among them,germinal center B cells(GCB)played the most prominent role,with positive clone expansion and heavy chain mutation level increasing,and the trend of differentiation into memory B cells increased.However,the number of plasma cells in the tumor microenvironment decreased significantly,and the plasma cells secreting IgA antibodies decreased most obviously.In addition,compared with the immune microenvironment of normal tissues,GCB cells in tumor tissues became more closely connected with other immune cells such as T cells,and communication molecules that positively regulate immune function were significantly enriched.CONCLUSION The role of GCB in CRC tumor microenvironment is greatly enhanced,and its affinity to tumor antigen is enhanced by its significantly increased heavy chain mutation level.Meanwhile,GCB has enhanced its association with immune cells in the microenvironment,which plays a positive anti-tumor effect.

Progress and challenges of cerebrovascular endothelial cells research promoted by single-cell transcriptome sequencing technology

Single-cell transcriptome sequencing has been a rapidly developing and powerful biological tool in recent years,and it plays a vital role in describing tissue development,cell heterogeneity,stress response,etc.Cerebrovascular disease is one of the leading causes affecting human health in the world.Thus,it is important to understand the characteristics of cerebrovascular structure,function,and environmental response.Notably,single-cell transcriptome sequencing provides deeper insights into cerebrovascular research in health and disease states.This article will briefly introduce the basic structure and function of cerebrovascular endothelial cells(ECs),summarize the current research and new findings on cerebrovascular ECs at the single-cell transcriptome level,and discuss the challenges in this field.

Transcriptome and single-cell profiling of the mechanism of diabetic kidney disease

BACKGROUND The NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome may play an important role in diabetic kidney disease(DKD).However,the exact link remains unclear.AIM To investigate the role of the NLRP3 inflammasome in DKD.METHODS Using datasets from the Gene Expression Omnibus database,30 NLRP3 inflammasome-related genes were identified.Differentially expressed genes were selected using differential expression analysis,whereas intersecting genes were selected based on overlapping differentially expressed genes and NLRP3 inflammasome-related genes.Subsequently,three machine learning algorithms were used to screen genes,and biomarkers were identified by overlapping the genes from the three algorithms.Potential biomarkers were validated by western blotting in a db/db mouse model of diabetes.RESULTS Two biomarkers,sirtuin 2(SIRT2)and caspase 1(CASP1),involved in the Leishmania infection pathway were identified.Both biomarkers were expressed in endothelial cells.Pseudo-temporal analysis based on endothelial cells showed that DKD mostly occurs during the mid-differentiation stage.Western blotting results showed that CASP1 expression was higher in the DKD group than in the control group(P<0.05),and SIRT2 content decreased(P<0.05).CONCLUSION SIRT2 and CASP1 provide a potential theoretical basis for DKD treatment.

Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing

Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.

Variation of microbiological and small molecule metabolite profiles of Nuodeng ham during ripening by high-throughput sequencing and GC-TOF-MS

The internal microbial diversity and small molecular metabolites of Nuodeng ham in different processing years(the first,second and third year sample)were analyzed by high-throughput sequencing technology and gas chromatography-time of flight mass spectrography(GC-TOF-MS)to study the effects of microorganisms and small molecular metabolites on the quality of ham in different processing years.The results showed that the dominant bacteria phyla of Nuodeng ham in different processing years were Proteobacteria and Firmicutes,the dominant fungi phyla were Ascomycota and Basidiomycota,while Staphylococcus and Aspergillus were the dominant bacteria and fungi of Nuodeng ham,respectively.Totally,252 kinds of small molecular metabolites were identified from Nuodeng ham in different processing years,and 12 different metabolites were screened through multivariate statistical analysis.Further metabolic pathway analysis showed that 23 metabolic pathways were related to ham fermentation,of which 8 metabolic pathways had significant effects on ham fermentation(Impact>0.01,P<0.05).The content of L-proline,phenyllactic acid,L-lysine,carnosine,taurine,D-proline,betaine and creatine were significantly positively correlated with the relative abundance of Staphylococcus and Serratia,but negatively correlated with the relative abundance of Halomonas,Aspergillus and Yamadazyma.

Single-cell RNA sequencing of meiocytes and microspores reveals the involvement of the Rf4 gene in redox homeostasis of CMS-C maize

Normal microsporogenesis is determined by both nuclear and mitochondrial genes. In maize C-type cytoplasmic male sterility, it is unclear how the development of meiocytes and microspores is affected by the mitochondrial sterility gene and the nuclear restorer gene. In this study, we sequenced the transcriptomes of single meiocytes(tetrad stage) and early mononucleate microspores from sterile and restorer lines. The numbers of expressed genes varied in individual cells and fewer than half of the expressed genes were common to the same cell types. Four comparisons revealed 3379 differentially expressed genes(DEGs), with 277 putatively associated with mitochondria, 226 encoding transcription factors,and 467 possibly targeted by RF4. KEGG analysis indicated that the DEGs in the two lines at the tetrad stage were involved predominantly in carbon metabolism and in amino acid biosynthesis and metabolism, whereas the DEGs during the transition from the tetrad stage to the early mononucleate stage were associated mostly with regulation of protein metabolism, fatty acid metabolism, and anatomical structure morphogenesis. Thus, meiocyte and microspore development was affected by the surrounding cells and the restorer gene, and the restorer gene helped restore the redox homeostasis of microspores and the normal cellular reconstruction during the transition.

Characteristics of alveolar macrophages in bronchioalveolar lavage fluids from active tuberculosis patients identified by single-cell RNA sequencing

Tuberculosis(TB),is an infectious disease caused by Mycobacterium tuberculosis(M.tuberculosis),and presents with high morbidity and mortality.Alveolar macrophages play an important role in TB pathogenesis although there is heterogeneity and functional plasticity.This study aimed to show the characteristics of alveolar macrophages from bronchioalveolar lavage fluid(BALF)in active TB patients.Single-cell RNA sequencing(scRNA-seq)was performed on BALF cells from three patients with active TB and additional scRNA-seq data from three healthy adults were established as controls.Transcriptional profiles were analyzed and compared by differential gene expression and functional enrichment analysis.We applied pseudo-temporal trajectory analysis to investigate correlations and heterogeneity within alveolar macrophage subclusters.Alveolar macrophages from active TB patients at the single-cell resolution are described.We found that TB patients have higher cellular percentages in five macrophage subclusters.Alveolar macrophage subclusters with increased percentages were involved in inflammatory signaling pathways as well as the basic macrophage functions.The TB-increased alveolar macrophage subclusters might be derived from M1-like polarization state,before switching to an M2-like polarization state with the development of M.tuberculosis infection.Cell-cell communications of alveolar macrophages also increased and enhanced in active TB patients.Overall,our study demonstrated the characteristics of alveolar macrophages from BALF in active TB patients by using scRNA-seq.

Advances in single-cell sequencing:insights from organ transplantation

Single-cell RNA sequencing(scRNA-seq)is a comprehensive technical tool to analyze intracellular and intercellular interaction data by whole transcriptional profile analysis.Here,we describe the application in biomedical research,focusing on the immune system during organ transplantation and rejection.Unlike conventional transcriptome analysis,this method provides a full map of multiple cell populations in one specific tissue and presents a dynamic and transient unbiased method to explore the progression of allograft dysfunction,starting from the stress response to final graft failure.This promising sequencing technology remarkably improves individualized organ rejection treatment by identifying decisive cellular subgroups and cell-specific interactions.

Recent advances of single-cell RNA sequencing technology in mesenchymal stem cell research

Mesenchymal stem cells(MSCs)are multipotent stromal cells with great potential for clinical applications.However,little is known about their cell heterogeneity at a single-cell resolution,which severely impedes the development of MSC therapy.In this review,we focus on advances in the identification of novel surface markers and functional subpopulations of MSCs made by single-cell RNA sequencing and discuss their participation in the pathophysiology of stem cells and related diseases.The challenges and future directions of single-cell RNA sequencing in MSCs are also addressed in this review.

Panoramic comparison between NK cells in healthy and cancerous liver through single-cell RNA sequencing

Objective:NK cells play crucial roles in the immune defense mechanisms against viral infections and transformed cells.However,the developmental progression,transcriptomic landscape,and functional subtypes of liver NK cells are not well defined.Hepatocellular carcinoma(HCC)accounts for approximately 80%of primary liver cancer worldwide,yet the biological characteristics of NK cells in the HCC environment are unclear.Therefore,we aimed to determine these cells’roles in tumorigenesis and prognosis.Methods:We compared the single-cell RNA sequencing profiles of NK cells purified from blood(n=1),healthy liver tissues(n=3),HCC tumor tissues(n=4),and peritumor liver tissues(n=1)to identify NK cell subsets.Furthermore,we performed bioinformatics analysis by using The Cancer Genome Atlas(TCGA)data to identify prognostic biomarkers simultaneously overexpressed in the blood and tumor tissues of patients with HCC.Results:Transcriptomic analysis revealed 5 NK cell subsets(L1-NK-CD56bright,L2-NK-CD56dim,L3-NK-HLA,L4-LrNK-FCGR3A,and L5-LrNK-XCL1)in the healthy liver tissues.However,the transitional L3 subset and the CXCR6+CD16+L4 subset with strong anti-tumor activity were absent in the HCC and peritumor liver tissues.Furthermore,4 common prognosis-associated genes(RHOB,TALDO1,HLA-DPA1,and TKT)were significantly overexpressed in the paired tumor tissue and blood.Conclusions:Our study revealed 5 specific subsets of NK cells in healthy human liver tissues.However,only 3 of the 5 NK cell subsets were present in HCC and peritumor tissues.The cytotoxic NK cell subsets were absent in HCC tissues.Furthermore,we identified 4 potential non-invasive prognostic biomarkers in patients with HCC.

Investigating intratumour heterogeneity by single-cell sequencing

Intratumour heterogeneity is a longstanding field of focus for both researchers and clinicians. It refers to the diversity amongst cells within the same tumour. Two major hypotheses have attempted to explain the existence of intratumour heterogeneity： （i） the clonal evolution （CE） theory and （ii） the cancer stem cell （CSC） model. CE theory emphasizes the evolutionary biological characteristics of the tumour, underscoring the initiation and progression of the disease. In contrast, the CSC model focuses on stem cell differentiation into distinct functions in order to stabilize the tumour microenvironment. Here we consider single-cell sequencing （SCS） as a newly developed technique for application to the investigation of intratumour heterogeneity and assess its relevance within research and clinical environments. Early detection of rare tumour cells, monitoring of circulating tumour cells （CTCs） and control of the occurrence of drug resistance are imoortant goals in early diagnosis, prognosis prediction and individualized medicine.

Single-cell RNA sequencing reveals classical monocytes are the major precursors of rat osteoclasts

To dissect which subset of bone marrow monocyte is the major precursor of osteoclast,3-month-old rat bone marrow was obtained for single-cell RNA sequencing.A total of 6091 cells were acquired for detailed analysis,with a median number of 1206 genes detected per cell and 17,959 genes detected in total.A total of 19 cell clusters were recognized,with the main lineages identified as B cells,Granulocytes,Monocytes,T cells,Erythrocytes and Macrophages.Monocytes were further divided into classical monocytes and non-classical monocytes.Compared with non-classical monocytes,classical monocytes highly expressed osteoclast differentiation related genes Mitf,Spi1,Fos and Csf1r.Additionally,biological processes of classical monocytes were related to osteoclast differentiation.qPCR revealed differentially expressed genes of classical monocytes played a role in osteoclast differentiation.In conclusion,classical monocytes were identified as the main precursors of osteoclasts in rats,and may contribute to osteoclast differentiation by regulating S100a4,S100a6,S100a10,Fn1,Vcan and Bcl2a1.The results of this study contribute to the understanding of the origin of osteoclasts and may provide potential biomarkers for early diagnosis of bone metabolic diseases,as well as molecular and cellular targets for clinical intervention in bone metabolic diseases.