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 nucleus/cell RNA-seq of the chicken hypothalamicpituitaryovarian axis offers new insights into the molecular regulatory mechanisms of ovarian development

The hypothalamic-pituitary-ovarian(HPO)axis represents a central neuroendocrine network essential for reproductive function.Despite its critical role,the intrinsic heterogeneity within the HPO axis across vertebrates and the complex intercellular interactions remain poorly defined.This study provides the first comprehensive,unbiased,cell type-specific molecular profiling of all three components of the HPO axis in adult Lohmann layers and Liangshan Yanying chickens.Within the hypothalamus,pituitary,and ovary,seven,12,and 13 distinct cell types were identified,respectively.Results indicated that the pituitary adenylate cyclase activating polypeptide(PACAP),follicle-stimulating hormone(FSH),and prolactin(PRL)signaling pathways may modulate the synthesis and secretion of gonadotropin-releasing hormone(GnRH),FSH,and luteinizing hormone(LH)within the hypothalamus and pituitary.In the ovary,interactions between granulosa cells and oocytes involved the KIT,CD99,LIFR,FN1,and ANGPTL signaling pathways,which collectively regulate follicular maturation.The SEMA4 signaling pathway emerged as a critical mediator across all three tissues of the HPO axis.Additionally,gene expression analysis revealed that relaxin 3(RLN3),gastrin-releasing peptide(GRP),and cocaine-and amphetamine regulated transcripts(CART,also known as CARTPT)may function as novel endocrine hormones,influencing the HPO axis through autocrine,paracrine,and endocrine pathways.Comparative analyses between Lohmann layers and Liangshan Yanying chickens demonstrated higher expression levels of GRP,RLN3,CARTPT,LHCGR,FSHR,and GRPR in the ovaries of Lohmann layers,potentially contributing to their superior reproductive performance.In conclusion,this study provides a detailed molecular characterization of the HPO axis,offering novel insights into the regulatory mechanisms underlying reproductive biology.

Single-cell transcriptomic dissection of the cellular and molecular events underlying the triclosan-induced liver fibrosis in mice

Background: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage.Methods: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing(scRNA-seq) was then carried out on TCS-or mock-treated mice livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor(TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the cellular and molecular events after TCS exposure. To verify the TCS-induced liver fibrosis,the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red stainings. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies.Results: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control groups profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells(HSCs)were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition,other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interactionmediated cellular communication in promoting liver fibrosis.Conclusions: TCS modulates the cellular activities and fates of several specific cell types(including hepatocytes, HSCs,endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis.Overall, we provide the first comprehensive single-cell atlas of mice livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

Application of single-cell omics in inflammatory bowel disease

Over the past decade,the advent of single cell RNA-sequencing has revolutionized the approach in cellular transcriptomics research.The current technology offers an unbiased platform to understand how genotype correlates to phenotype.Single-cell omics applications in gastrointestinal(GI)research namely inflammatory bowel disease(IBD)has become popular in the last few years with multiple publications as single-cell omics techniques can be applied directly to the target organ,the GI tract at the tissue level.Through examination of mucosal tissue and peripheral blood in IBD,the recent boom in single cell research has identified a myriad of key immune players from enterocytes to tissue resident memory T cells,and explored functional heterogeneity within cellular subsets previously unreported.As we begin to unravel the complex mucosal immune system in states of health and disease like IBD,the power of exploration through single-cell omics can change our approach to translational research.As novel techniques evolve through multiplexing single-cell omics and spatial transcriptomics come to the forefront,we can begin to fully comprehend the disease IBD and better design targets of treatment.In addition,hopefully these techniques can ultimately begin to identify biomarkers of therapeutic response and answer clinically relevant questions in how to tailor individual therapy to patients through personalized medicine.

Single-cell RNA sequencing in cornea research:Insights into limbal stem cells and their niche regulation

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye,which acts as a protective barrier and is critical for clear and stable vision.Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells(LSCs),a cell population that resides at the limbus in a highly regulated niche.Dysfunction of LSCs or their niche can cause limbal stem cell deficiency,a disease that is manifested by failed epithelial wound healing or even blindness.Nevertheless,compared to stem cells in other tissues,little is known about the LSCs and their niche.With the advent of single-cell RNA sequencing,our understanding of LSC characteristics and their microenvironment has grown considerably.In this review,we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology,including the heterogeneity of the LSC population,novel LSC markers and regulation of the LSC niche,which will provide a reference for clinical issues such as corneal epithelial wound healing,ocular surface reconstruction and interventions for related diseases.

Tunable microfluidic chip for single-cell deformation study

Microfluidic phenotyping methods have been of vital importance for cellular characterization,especially for evaluating single cells.In order to study the deformability of a single cell,we devised and tested a tunable microfluidic chip-based method.A pneumatic polymer polydimethylsiloxane(PDMS)membrane was designed and fabricated abutting a single-cell trapping structure,so the cell could be squeezed controllably in a lateral direction.Cell contour changes under increasing pressure were recorded,enabling the deformation degree of different types of single cell to be analyzed and compared using computer vision.This provides a new perspective for studying mechanical properties of cells at the single cell level.

System analysis based on the T cell exhaustion‑related genes identifies CD38 as a novel therapy target for ovarian cancer

Ovarian cancer(OV)is highly heterogeneous tumor with a very poor prognosis.Studies increasingly show that T cell exhaustion is prognostically relevant in OV.The aim of this study was to dissect the heterogeneity of T cell subclusters in OV through single cell transcriptomic analysis.The single RNA-sequencing(scRNA-seq)data of five OV patients were analyzed,and six major cell clusters were identified after threshold screening.Further clustering of T cell-associated clusters revealed four subtypes.Pathways related to oxidative phosphorylation,G2M checkpoint,JAK-STAT and MAPK signaling were significantly activated,while the p53 pathway was inhibited in the CD8+exhausted T cells.The standard marker genes of CD8+T cell exhaustion were screened to develop a T-cell related gene score(TRS)based on random forest plots in TCGA cohort.The patients with low TRS have better prognosis compared to the patients with high TRS in both TCGA and GEO.In addition,most genes included in the TRS showed significant differences in expression levels between the high-and low-risk groups.Immune cell infiltration was analyzed using the MCPcounter and xCell algorithms,which revealed significant differences between the two risk groups,indicating that the different prognoses may stem from the respective immune landscapes.In addition,CD38 knockdown in OV cell lines increased apoptosis and inhibited invasion in vitro.Finally,we performed a drug sensitivity analysis and identified six potential drug candidates for OV.To summarize,we identified the heterogeneity and clinical significance of T cell exhaustion in OV and built a superior prognostic model based on T cell exhaustion genes,which can contribute to the development of more precise and effective therapies.

Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development

BACKGROUND Accumulating evidence suggests that the maxillary process,to which cranial crest cells migrate,is essential to tooth development.Emerging studies indicate that Cd271 plays an essential role in odontogenesis.However,the underlying mechanisms have yet to be elucidated.AIM To establish the functionally heterogeneous population in the maxillary process,elucidate the effects of Cd271 deficiency on gene expression differences.METHODS p75NTR knockout(Cd271-/-)mice(from American Jackson laboratory)were used to collect the maxillofacial process tissue of p75NTR knockout mice,and the wildtype maxillofacial process of the same pregnant mouse wild was used as control.After single cell suspension,the cDNA was prepared by loading the single cell suspension into the 10x Genomics Chromium system to be sequenced by NovaSeq6000 sequencing system.Finally,the sequencing data in Fastq format were obtained.The FastQC software is used to evaluate the quality of data and CellRanger analyzed the data.The gene expression matrix is read by R software,and Seurat is used to control and standardize the data,reduce the dimension and cluster.We search for marker genes for subgroup annotation by consulting literature and database;explore the effect of p75NTR knockout on mesenchymal stem cells(MSCs)gene expression and cell proportion by cell subgrouping,differential gene analysis,enrichment analysis and protein-protein interaction network analysis;understand the interaction between MSCs cells and the differentiation trajectory and gene change characteristics of p75NTR knockout MSCs by cell communication analysis and pseudo-time analysis.Last we verified the findings single cell sequencing in vitro.RESULTS We identified 21 cell clusters,and we re-clustered these into three subclusters.Importantly,we revealed the cell–cell communication networks between clusters.We clarified that Cd271 was significantly associated with the regulation of mineralization.CONCLUSION This study provides comprehensive mechanistic insights into the maxillary-process-derived MSCs and demonstrates that Cd271 is significantly associated with the odontogenesis in mesenchymal populations.

Observing single cells in whole organs with optical imaging

Cells are the basic unit of human organs that are not fully understood.The revolutionary advancements of optical imaging alowed us to observe single cells in whole organs,revealing the complicated composition of cells with spatial information.Therefore,in this review,we revisit the principles of optical contrast related to those biomolecules and the optical techniques that transform optical contrast into detectable optical signals.Then,we describe optical imaging to achieve threedimensional spatial discrimination for biological tisutes.Due to the milky appearance of tissues,the spatial information burred deep in the whole organ.Fortunately,strategies developed in the last decade could circumvent this issue and lead us into a new era of investigation of the cells with their original spatial information.

A SARS-CoV-2 neutralizing antibody discovery by single cell sequencing and molecular modeling

Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.

Glycogen metabolism-mediated intercellular communication in the tumor microenvironment influences liver cancer prognosis

Glycogen metabolism plays a key role in the development of hepatoellular carcinoma(HCC),but the function of glycogen metabolism genes in the tumor microenvironment(TME)is still to be elucidated.Single cell RNA-seq data were obtained from ten HCC tumor samples totaling 64,545 cells and 65 glycogen metabolism genes were analyzed bya nonnegative matrix factorization(NMF).The prognosis and immune response of new glycogen TME cell dusters were predicted by using HCC and immunotherapy cohorts from public databases.HOC single cell analysis was divided into fibroblasts,NT T cells,macrophages,endothelial clls,and B cells,which were separately divided into new cell clusters by glycogen metabolism gene annotation.Pseudo temporal trajectory analysis demonstrated the temporal differentiation trajectory of different glycogen subtype cell dusters.Cellular communication analysis revealed extensive interactions between endothelial cells with glycogen metabolizing TME cell.related subtypes and diferent glycogen subtype cell clusters.SCENIC analysis of transcription factors upstream of TME cell clusters with different glycogen metabolism.In addition,TME cell dusters of glycogen metabolism were found to be enriched in expression in CAF subtypes,CD8 depleted,M1,and M2 types.Bulk seq analysis showed the prognostic signifcance of glycogen metabolism.mediated TME cell dusters in HCC,while a significant immune response was found in the immunotherapy cohort in patients treated with immune checkpoint blockade(ICB),especially for CAFs,T cells,and macrophages In summary,our study reveals for the first time that glycogen metabolism mediates intercellular communication in the hepatocellular carcinoma microenvironment while elucidating the anti-tumor mechanisms and immune prognostic responses of different subtypes of cell dusters.

Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

The Superiority of Like-rocket Immunoelectrophoresis Using in Single Cell Gel Electrophoresis Assay

[Objective] The like-rocket immunoelectrophoresis was used to explore a new feasible electrophoresis method for single cell gel electrophoresis assay （comet assay）.[Method] The like-rocket immunoelectrophoresis was used for single cell gel electrophoresis assay to detect DNA damage at single cell level,then it was compared with traditional electrophoresis method to analyze its advantage and disadvantages.[Result] Under cell DNA undamaged state,the results of two electrophoresis methods were consistent.When cell DNA was damaged,the comet tail divergence of some cells under traditional electrophoresis method were drifted,however,the comet tail image of like-rocket immunoelectrophoresis was concentrated and not shifted.[Conclusion] The like-rocket immunoelectrophoresis had some advantages.

Single-cell analyses of circulating tumor cells

Circulating tumor cells(CTCs) are a population of tumor cells mediating metastasis, which results in most of the cancer related deaths. The number of CTCs in the peripheral blood of patients is rare, and many platforms have been launched for detection and enrichment of CTCs. Enumeration of CTCs has already been used as a prognosis marker predicting the survival rate of cancer patients. Yet CTCs should be more potential. Studies on CTCs at single cell level may help revealing the underlying mechanism of tumorigenesis and metastasis. Though far from developed, this area of study holds much promise in providing new clinical application and deep understanding towards metastasis and cancer development.

Analysis of Amino Acids in a Single Human Red Blood Cell by Capillary Zone Electrophoresis with Intracellular NDA- derivatization and Electrochemical Detection

A novel method for determination of amino acids in individual human red blood cells has been developed. In this method, the derivatization reagents (NDA and CN-) are introduced into living cells by electroporation. After completion of derivatization, the amino acids in a single cell is determined by capillary zone electrophoresis with end-column amperometric detection.

Successful Nitrogen Doping of 1.3 GHz Single Cell Superconducting Radio-Frequency Cavities

A high intrinsic quality factor （Q0） of a superconducting radio-frequency cavity is beneficial to reducing the oper- ation costs of superconducting accelerators. Nitrogen doping （N-doping） has been demonstrated as a aseful way to improve Q0 of the superconducting cavity in recent years. N-doping researches with 1.3 GHz single cell cavities are carried out at Peking University and the preliminary results are promising. Our recipe is slightly different from other laboratories. After 250μm polishing, high pressure rinsing and 3 h high temperature annealing, the cavities are nitrogen doped at 2.7-4.0Pa for 20rain and then followed by 15μm electropolishing. Vertical test results show that Q0 of a 1.3 GHz single cell cavity made of large grain niobium has increased to 4 ×10 10 at 2.0K and medium gradient.

Studies on Single Cell Culture in vitro in Wheat——The variation of grain protein content and its fractions from regenerated plants

On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11531770%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -2069% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE774 increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

Microfluidic-based single cell trapping using a combination of stagnation point flow and physical barrier

Single cell trapping in vitro by microfluidic device is an emerging approach for the study of the relationship between single cells and their dynamic biochemical microenvironments. In this paper, a hydrodynamic-based microfluidic device for single cell trapping is designed using a combination of stagnation point flow and physical barrier.The microfluidic device overcomes the weakness of the traditional ones, which have been only based upon either stagnation point flows or physical barriers, and can conveniently load dynamic biochemical signals to the trapped cell. In addition, it can connect with a programmable syringe pump and a microscope to constitute an integrated experimental system.It is experimentally verified that the microfluidic system can trap single cells in vitro even under flow disturbance and conveniently load biochemical signals to the trapped cell. The designed micro-device would provide a simple yet effective experimental platform for further study of the interactions between single cells and their microenvironments.

Detection of Sperm DNA Damage in Workers Exposed to Benzene by Modified Single Cell Gel Electrophoresis

Objective To assess the effect of benzene on sperm DNA damage ;Methods Twenty-seven benzene-exposed workers were selected as exposed group and 35 normal sperm donors as control group. Air concentration of benzene series in workshop was determined by gas chromatography. As an internal exposure dose of benzene, the concentration of trans, trans-muconic acid （ttMA） was determined by high performance liquid chromatography. DNA was detected by modified single cell gel electrophoresis （SCGE）. Results The air concentrations of benzene, toluene and xylene at the workplace were 86.49±2.83 mg/m^3, 97.20±3.52 mg/m^3 and 97.45± 2.10 mg/m^3, respectively. Urinary ttMA in exposed group （1.040 ± 0.617 mg/L） was significantly higher than that of control group （0.819 ± 0.157 mg/L）. The percentage of head DNA, determined by modified SCGE method, significantly decreased in the exposed group （n=13, 70.18% ± 7.36%） compared with the control （n=16, 90.62% ± 2.94%）（P〈0.001）. Conclusion The modified SCGE method can be used to investigate the damage of sperm DNA. As genotoxin and reprotoxins, benzene had direct effect on the germ cells during the spermatogenesiss.

COVID-19 Related Research by Data Mining in Single Cell Transcriptome Profiles

The outbreak of coronavirus disease 2019(COVID-2019)has drawn public attention all over the world.As a newly emerging area,single cell sequencing also exerts its power in the battle over the epidemic.In this review,the up-to-date knowledge of COVID-19 and its receptor is summarized,followed by a collection of the mining of single cell transcriptome profiling data for the information in aspects of the vulnerable cell types in humans and the potential mechanisms of the disease.