AB040.Single-cell transcriptomics identifies cell-specific signatures of pathological angiogenesis

Background:To treat vascular proliferative diseases,anti-VEGF therapies have shown systemic adverse effects attributable to the lack of selectivity between pathological and physiological angiogenesis.Thus,identifying the molecular mechanisms that are only specific to pathological cell types is crucial to develop better precision medicine.Methods:Here,we used different cell type enrichment approaches combined with single-cell RNA sequencing to define the transcriptomic changes within each retinal cell types in a mouse model of ischemic retinopathy.This retinal model develops pathological neovascularization(NV)in response to local hypoxia following oxygen-induced vessel obliteration(P7 to P12).The NV phenotype is characterized by the progressive appearance of vascular tufts resulting from misguided,abnormal proliferation of endothelial cells that we monitored at 3 consecutive time points-P12,P14 and P17(peak of NV).Results:By following the dynamic response to hypoxia,our experimental design reveals how pathological angiogenesis is specifically associated with significant metabolic adaptations in different subtypes of endothelial cells(i.e.,Tips vs Stalk cells).We also identify a pathological subtype of glial cells over-expressing VEGFA and pro-inflammatory IL-1 receptor subunits.This subtype of activated glial cells was targeted using selective IL1R antagonist treatment which reduced glial activation,inflammation,NV and promotes physiological angiogenesis,therefore improving tissue regeneration.Conclusions:Our results illustrate how analyzing cell type heterogeneity in tissues developing pathological angiogenesis allows establishing better targeting therapies to restore vascular integrity.

Single-cell transcriptomics reveals T-cell heterogeneity and immunomodulatory role of CD4^(+) T native cells in Candida albicans infection

Objective:Candida albicans is a common fungal pathogen that triggers complex host defense mechanisms,including coordinated innate and adaptive immune responses,to neutralize invading fungi effectively.Exploring the immune microenvironment has the potential to inform the development of therapeutic strategies for fungal infections.Methods:The study analyzed individual immune cell profiles in peripheral blood mononuclear cells from Candida albicans-infected mice and healthy control mice using single-cell transcriptomics,fluorescence quantitative PCR,and Western blotting.We investigated intergroup differences in the dynamics of immune cell subpopulation infiltration,pathway enrichment,and differentiation during Candida albicans infection.Results:Our findings indicate that infiltration of CD4^(+)naive cells,regulatory T(Treg)cells,and Microtubules(MT)-associated cells increased after infection,along with impaired T cell activity.Notably,CD4^(+) T cells and plasma cells were enhanced after infection,suggesting that antibody production is dependent on T cells.In addition,we screened 6 hub genes,transcription factor forkhead box protein 3(Foxp3),cytotoxic T-lymphocyte associated protein 4(CTLA4),Interleukin 2 Receptor Subunit Beta(Il2rb),Cd28,C-C Motif Chemokine Ligand 5(Ccl5),and Cd27 for alterations associated with CD4^(+) T cell differentiation.Conclusions:These results provide a comprehensive immunological landscape of the mechanisms of Candida albicans infection and greatly advance our understanding of adaptive immunity in fungal infections.

Single-cell transcriptomics reveals tumor landscape in ovarian carcinosarcoma

Objective:The present study used single-cell RNA sequencing(scRNA-seq)to characterize the cellular composition of ovarian carcinosarcoma(OCS)and identify its molecular characteristics.Methods:scRNA-seq was performed in resected primary OCS for an in-depth analysis of tumor cells and the tumor microenvironment.Immunohistochemistry staining was used for validation.The scRNA-seq data of OCS were compared with those of high-grade serous ovarian carcinoma(HGSOC)tumors and other OCS tumors.Results:Both malignant epithelial and malignant mesenchymal cells were observed in the OCS patient of this study.We identified four epithelial cell subclusters with different biological roles.Among them,epithelial subcluster 4 presented high levels of breast cancer type 1 susceptibility protein homolog(BRCA1)and DNA topoisomerase 2-α(TOP2A)expression and was related to drug resistance and cell cycle.We analyzed the interaction between epithelial and mesenchymal cells and found that fibroblast growth factor(FGF)and pleiotrophin(PTN)signalings were the main pathways contributing to communication between these cells.Moreover,we compared the malignant epithelial and mesenchymal cells of this OCS tumor with our previous published HGSOC scRNA-seq data and OCS data.All the epithelial subclusters in the OCS tumor could be found in the HGSOC samples.Notably,the mesenchymal subcluster C14 exhibited specific expression patterns in the OCS tumor,characterized by elevated expression of cytochrome P450 family 24 subfamily A member 1(CYP24A1),collagen type XXIIIα1 chain(COL23A1),cholecystokinin(CCK),bone morphogenetic protein 7(BMP7),PTN,Wnt inhibitory factor 1(WIF1),and insulin-like growth factor 2(IGF2).Moreover,this subcluster showed distinct characteristics when compared with both another previously published OCS tumor and normal ovarian tissue.Conclusions:This study provides the single-cell transcriptomics signature of human OCS,which constitutes a new resource for elucidating OCS diversity.

DVsc:An Automated Framework for Efficiently Detecting Viral Infection from Single-cell Transcriptomics Data

Single-cell RNA sequencing(scRNA-seq)has emerged as a valuable tool for studying cellular heterogeneity in various fields,particularly in virological research.By studying the viral and cellular transcriptomes,the dynamics of viral infection can be investigated at a single-cell resolution.However,limited studies have been conducted to investigate whether RNA transcripts from clinical samples contain substantial amounts of viral RNAs,and a specific computational framework for efficiently detecting viral reads based on scRNA-seq data has not been developed.Hence,we introduce DVsc,an open-source framework for precise quantitative analysis of viral infection from single-cell transcriptomics data.When applied to approximately 200 diverse clinical samples that were infected by more than 10 different viruses,DVsc demonstrated high accuracy in systematically detecting viral infection across a wide array of cell types.This innovative bioinformatics pipeline could be crucial for addressing the potential effects of surreptitiously invading viruses on certain illnesses,as well as for designing novel medicines to target viruses in specific host cell subsets and evaluating the efficacy of treatment.DVsc supports the FASTQ format as an input and is compatible with multiple single-cell sequencing platforms.Moreover,it could also be applied to sequences from bulk RNA sequencing data.DVsc is available at http://62.234.32.33:5000/DVsc.

Single-cell transcriptomic atlas of goat ovarian aging

Background The ovaries are one of the first organs that undergo degenerative changes earlier in the aging process,and ovarian aging is shown by a decrease in the number and quality of oocytes.However,little is known about the molecular mechanisms of female age-related fertility decline in different types of ovarian cells during aging,especially in goats.Therefore,the aim of this study was to reveal the mechanisms driving ovarian aging in goats at single-cell resolution.Results For the first time,we surveyed the single-cell transcriptomic landscape of over 27,000 ovarian cells from newborn,young and aging goats,and identified nine ovarian cell types with distinct gene-expression signatures.Functional enrichment analysis showed that ovarian cell types were involved in their own unique biological processes,such as Wnt beta-catenin signalling was enriched in germ cells,whereas ovarian steroidogenesis was enriched in granulosa cells(GCs).Further analysis showed that ovarian aging was linked to GCs-specific changes in the antioxidant system,oxidative phosphorylation,and apoptosis.Subsequently,we identified a series of dynamic genes,such as AMH,CRABP2,THBS1 and TIMP1,which determined the fate of GCs.Additionally,FOXO1,SOX4,and HIF1A were identified as significant regulons that instructed the differentiation of GCs in a distinct manner during ovarian aging.Conclusions This study revealed a comprehensive aging-associated transcriptomic atlas characterizing the cell typespecific mechanisms during ovarian aging at the single-cell level and offers new diagnostic biomarkers and potential therapeutic targets for age-related goat ovarian diseases.

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.

Comprehensive integration of single-cell transcriptomic data illuminates the regulatory network architecture of plant cell fate specification

Plant morphogenesis relies on precise gene expression programs at the proper time and position which is orchestrated by transcription factors(TFs)in intricate regulatory networks in a cell-type specific manner.Here we introduced a comprehensive single-cell transcriptomic atlas of Arabidopsis seedlings.This atlas is the result of meticulous integration of 63 previously published scRNA-seq datasets,addressing batch effects and conserving biological variance.This integration spans a broad spectrum of tissues,including both below-and above-ground parts.Utilizing a rigorous approach for cell type annotation,we identified 47 distinct cell types or states,largely expanding our current view of plant cell compositions.We systematically constructed cell-type specific gene regulatory networks and uncovered key regulators that act in a coordinated manner to control cell-type specific gene expression.Taken together,our study not only offers extensive plant cell atlas exploration that serves as a valuable resource,but also provides molecular insights into gene-regulatory programs that varies from different cell types.

Single-cell transcriptomics of immune cells in lymph nodes reveals their composition and alterations in functional dynamics during the early stages of bubonic plague

Bubonic plague caused by Yersinia pestis is highly infectious and often fatal.Characterization of the host immune response and its subsequent suppression by Y.pestis is critical to understanding the pathogenesis of Y.pestis.Here,we utilized single-cell RNA sequencing to systematically profile the transcriptomes of immune cells in draining lymph nodes(d LNs)during the early stage of Y.pestis infection.Dendritic cells responded to Y.pestis within 2 h post-infection(hpi),followed by the activation of macrophages/monocytes(Mφs/Mons)and recruitment of polymorphonuclear neutrophils(PMNs)to d LNs at 24 hpi.Analysis of cell-to-cell communication suggests that PMNs may be recruited to lymph nodes following the secretion of CCL9 by Mφs/Mons stimulated through CCR1-CCL9 interaction.Significant functional suppression of all the three innate immune cell types occurred during the early stage of infection.In summary,we present a dynamic immune landscape,at single-cell resolution,of murine d LNs involved in the response to Y.pestis infection,which may facilitate the understanding of the plague pathogenesis of during the early stage of infection.

Decoding the molecular landscape of keloids:new insights from single-cell transcriptomics

Keloids are a fibrotic disease caused by an excessive accumulation of extracellular matrix in the dermis;they have neoplasia-like properties of aggressive growth and high posttreatment recurrence rates.Therefore,it is imperative to gain additional insight into the pathobiology of keloid formation.Single-cell RNA sequencing(scRNA-seq)technology has brought data-driven innovation to understanding the pathogenesis of keloids by breaking the limitations of traditional sequencing technologies to resolve cell composition and to distinguish functional cell subtypes at an unprecedented resolution.The present review aims to cover the application of scRNAseq technology in keloids and its exploratory findings,including the depiction of the cellular landscape of keloids,fibroblast heterogeneity,the lineage development of Schwann cells and the mesenchymal-activation phenomenon of endothelial cells.Furthermore,scRNA-seq records the transcriptional profiles of fibroblasts and immune cells in a more refined manner,and this gene expression information provides excellent material for inferring intercellular communication networks and lays an important theoretical foundation for future studies.

Single-cell and spatial omics:exploring hypothalamic heterogeneity

Elucidating the complex dynamic cellular organization in the hypothalamus is critical for understanding its role in coordinating fundamental body functions. Over the past decade, single-cell and spatial omics technologies have significantly evolved, overcoming initial technical challenges in capturing and analyzing individual cells. These high-throughput omics technologies now offer a remarkable opportunity to comprehend the complex spatiotemporal patterns of transcriptional diversity and cell-type characteristics across the entire hypothalamus. Current single-cell and single-nucleus RNA sequencing methods comprehensively quantify gene expression by exploring distinct phenotypes across various subregions of the hypothalamus. However, single-cell/single-nucleus RNA sequencing requires isolating the cell/nuclei from the tissue, potentially resulting in the loss of spatial information concerning neuronal networks. Spatial transcriptomics methods, by bypassing the cell dissociation, can elucidate the intricate spatial organization of neural networks through their imaging and sequencing technologies. In this review, we highlight the applicative value of single-cell and spatial transcriptomics in exploring the complex molecular-genetic diversity of hypothalamic cell types, driven by recent high-throughput achievements.

The early life immune dynamics and cellular drivers at single-cell resolution in lamb forestomachs and abomasum

Background Four-chambered stomach including the forestomachs(rumen,reticulum,and omasum)and abomasum allows ruminants convert plant fiber into high-quality animal products.The early development of this four-chambered stomach is crucial for the health and well-being of young ruminants,especially the immune development.However,the dynamics of immune development are poorly understood.Results We investigated the early gene expression patterns across the four-chambered stomach in Hu sheep,at 5,10,15,and 25 days of age.We found that forestomachs share similar gene expression patterns,all four stomachs underwent widespread activation of both innate and adaptive immune responses from d 5 to 25,whereas the metabolic function were significantly downregulated with age.We constructed a cell landscape of the four-chambered stomach using single-cell sequencing.Integrating transcriptomic and single-cell transcriptomic analyses revealed that the immune-associated module hub genes were highly expressed in T cells,monocytes and macrophages,as well as the defense-associated module hub genes were highly expressed in endothelial cells in the four-stomach tissues.Moreover,the non-immune cells such as epithelial cells play key roles in immune maturation.Cell communication analysis predicted that in addition to immune cells,non-immune cells recruit immune cells through macrophage migration inhibitory factor signaling in the forestomachs.Conclusions Our results demonstrate that the immune and defense responses of four stomachs are quickly developing with age in lamb's early life.We also identified the gene expression patterns and functional cells associated with immune development.Additionally,we identified some key receptors and signaling involved in immune regulation.These results help to understand the early life immune development at single-cell resolution,which has implications to develop nutritional manipulation and health management strategies based on specific targets including key receptors and signaling pathways.

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.

Spatial transcriptomics:recent developments and insights in respiratory research

The respiratory system's complex cellular heterogeneity presents unique challenges to researchers in this field.Although bulk RNA sequencing and single-cell RNA sequencing(scRNA-seq)have provided insights into cell types and heterogeneity in the respiratory system,the relevant specific spatial localization and cellular interactions have not been clearly elucidated.Spatial transcriptomics(ST)has filled this gap and has been widely used in respiratory studies.This review focuses on the latest iterative technology of ST in recent years,summarizing how ST can be applied to the physiological and pathological processes of the respiratory system,with emphasis on the lungs.Finally,the current challenges and potential development directions are proposed,including high-throughput full-length transcriptome,integration of multi-omics,temporal and spatial omics,bioinformatics analysis,etc.These viewpoints are expected to advance the study of systematic mechanisms,including respiratory studies.

When blood development meets single-cell transcriptomics

Blood cells arise during embryonic development by three temporally distinct waves.Belonging to the third wave,hematopoietic stem cells(HSCs)are generated from hemogenic endothelium via endothelial-to-hematopoietic transition in mid-gestational embryos.Recently,studies combined with single-cell transcriptomics have provided massive new insights into the molecular evolutions and the underlying mechanisms of distinct waves of hematopoietic specification.In this review,we discuss the current single-cell profiling techniques,the most recent novel findings involved in the generation of distinct waves of blood cells,especially the HSCs,using single-cell transcriptional profiling combined with functional evaluations,and the perspectives to use the accumulating huge singlecell transcriptional data sets to study developmental hematopoiesis.

Integrated spatial metabolomics and transcriptomics decipher the hepatoprotection mechanisms of wedelolactone and demethylwedelolactone on non-alcoholic fatty liver disease

Eclipta prostrata L.has been used in traditional medicine and known for its liver-protective properties for centuries.Wedelolactone(WEL)and demethylwedelolactone(DWEL)are the major coumarins found in E.prostrata L.However,the comprehensive characterization of these two compounds on non-alcoholic fatty liver disease(NAFLD)still remains to be explored.Utilizing a well-established zebrafish model of thioacetamide(TAA)-induced liver injury,the present study sought to investigate the impacts and mechanisms of WEL and DWEL on NAFLD through integrative spatial metabolomics with liver-specific transcriptomics analysis.Our results showed that WEL and DWEL significantly improved liver function and reduced the accumulation of fat in the liver.The biodistributions and metabolism of these two compounds in whole-body zebrafish were successfully mapped,and the discriminatory endogenous metabolites reversely regulated by WEL and DWEL treatments were also characterized.Based on spatial metabolomics and transcriptomics,we identified that steroid biosynthesis and fatty acid metabolism are mainly involved in the hepatoprotective effects of WEL instead of DWEL.Our study unveils the distinct mechanism of WEL and DWEL in ameliorating NAFLD,and presents a“multi-omics”platform of spatial metabolomics and liver-specific transcriptomics to develop highly effective compounds for further improved therapy.

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.

Single-cell profiling of the pig cecum at various developmental stages

The gastrointestinal tract is essential for food digestion,nutrient absorption,waste elimination,and microbial defense.Single-cell transcriptome profiling of the intestinal tract has greatly enriched our understanding of cellular diversity,functional heterogeneity,and their importance in intestinal tract development and disease.Although such profiling has been extensively conducted in humans and mice,the single-cell gene expression landscape of the pig cecum remains unexplored.Here,single-cell RNA sequencing was performed on 45572 cells obtained from seven cecal samples in pigs at four different developmental stages(days(D)30,42,150,and 730).Analysis revealed 12 major cell types and 38 subtypes,as well as their distinctive genes,transcription factors,and regulons,many of which were conserved in humans.An increase in the relative proportions of CD8^(+)T and Granzyme A(low expression)natural killer T cells(GZMA^(low)NKT)cells and a decrease in the relative proportions of epithelial stem cells,Tregs,RHEX^(+)T cells,and plasmacytoid dendritic cells(pDCs)were noted across the developmental stages.Moreover,the post-weaning period exhibited an up-regulation in mitochondrial genes,COX2 and ND2,as well as genes involved in immune activation in multiple cell types.Cell-cell crosstalk analysis indicated that IBP6^(+)fibroblasts were the main signal senders at D30,whereas IBP6^(−)fibroblasts assumed this role at the other stages.NKT cells established interactions with epithelial cells and IBP6^(+)fibroblasts in the D730 cecum through mediation of GZMA-F2RL1/F2RL2 pairs.This study provides valuable insights into cellular heterogeneity and function in the pig cecum at different development stages.

Single-cell transcriptome analysis reveals the regulatory effects of artesunate on splenic immune cells in polymicrobial sepsis

Sepsis is characterized by a severe and life-threatening host immune response to polymicrobial infection accompanied by organ dysfunction.Studies on the therapeutic effect and mechanism of immunomodulatory drugs on the sepsis-induced hyperinflammatory or immunosuppression states of various immune cells remain limited.This study aimed to investigate the protective effects and underlying mechanism of artesunate(ART)on the splenic microenvironment of cecal ligation and puncture-induced sepsis model mice using single-cell RNA sequencing(scRNA-seq)and experimental validations.The scRNA-seq analysis revealed that ART inhibited the activation of pro-inflammatory macrophages recruited during sepsis.ART could restore neutrophils’chemotaxis and immune function in the septic spleen.It inhibited the activation of T regulatory cells but promoted the cytotoxic function of natural killer cells during sepsis.ART also promoted the differentiation and activity of splenic B cells in mice with sepsis.These results indicated that ART could alleviate the inflammatory and/or immunosuppressive states of various immune cells involved in sepsis to balance the immune homeostasis within the host.Overall,this study provided a comprehensive investigation of the regulatory effect of ART on the splenic microenvironment in sepsis,thus contributing to the application of ART as adjunctive therapy for the clinical treatment of sepsis.

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.

Integrated Transcriptomics and Metabolomics Analysis for the Mechanism Underlying White-to-Pink Petal Color Transition in Hibiscus mutabilis Flowers

Cotton rose(Hibiscus mutabilis)is a well-known ornamental plant that produces large flowers of vibrant colors.However,metabolites in H.mutabilis flowers with vibrant color have not been fully understood.By performing a combined analysis of metabolomics and transcriptomics data,we here explored mechanisms for the production of primary active compounds in this plant.Multivariate statistics unveiled differences in flavonoid metabolism between white and pink flowers,with pink flowers exhibiting a greater flavonoid abundance.The white-to-pink transition of cotton rose flowers may be attributed to pelargonidin-3-O-glucoside formation.On examining the expression of genes related to the structure of flavonoids,pink flowers were found to have a higher number of upregulated genes than white flowers,which resulted in higher flavonoid accumulation in the pink flowers.These results underscore the potential applications and development value of cotton rose flowers.Our study provides relevant insights into the regulation of key active components and the theoretical basis for the efficient utilization of cotton rose flowers.