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.

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.

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.

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.

Single cell sequencing: technique, application,and future development

The progression of next generation sequencing is continuously changing the landscape of genomic, tran- scriptomic, and epigenomic studies. Particularly, advances in single cell manipulation and amplification techniques bring sequencing technology to the single-cell level. Single cell genome sequencing allows us to study tumor evolu- tion, gamete genesis, somatic mosaicism at genome-wide level; single cell transcriptome sequencing unveils the dynamic gene expression during early embryonic devel- opment, differentiation and reprogramming; single cell methylome sequencing is just taking off and shows great potential in cancer and stem cell studies. Lots of attempts are still being made in other dimensions of sequencing. The increasing need for single cell sequencing requires the future techniques with the following features： （1） high accuracy and fidelity; （2） able to perform multiple omics analyses in one cell; （3） high degree of automation and standardized pipeline. These progresses and improvements will lower the barrier for single cell sequencing to enter ordinary laboratories. The wide application of single cell sequencing techniques will substantially change biomedi- cal research in future.

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.

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.

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.

FSPAM:A Feature Construction Method to Identifying Cell Populations in ScRNA-seq Data

The emergence of single-cell RNA-sequencing(scRNA-seq)technology has introduced new information about the structure of cells,diseases,and their associated biological factors.One of the main uses of scRNA-seq is identifying cell populations,which sometimes leads to the detection of rare cell populations.However,the new method is still in its infancy and with its advantages comes computational challenges that are just beginning to address.An important tool in the analysis is dimensionality reduction,which transforms high dimensional data into a meaningful reduced subspace.The technique allows noise removal,visualization and compression of high-dimensional data.This paper presents a new dimensionality reduction approach where,during an unsupervised multistage process,a feature set including high valuable markers is created which can facilitate the isolation of cell populations.Our proposed method,called fusion of the Spearman and Pearson affinity matrices(FSPAM),is based on a graph-based Gaussian kernel.Use of the graph theory can be effective to overcome the challenge of the nonlinear relations between cellular markers in scRNA-seq data.Furthermore,with a proper fusion of the Pearson and Spearman correlation coefficient criteria,it extracts a set of the most important features in a new space.In fact,the FSPAM aggregates the various aspects of cell-to-cell similarity derived from the Pearson and Spearman metrics,and reveals new aspects of cell-to-cell similarity,which can be used to extract new features.The results of the identification of cell populations via k-means++clustering method based on the features extracted from the FSPAM and different datasets of scRNA-seq suggested that the proposed method,regardless of the characteristics that govern each dataset,enjoys greater accuracy and better quality compared to previous methods.

Insights into the genetic architecture of congenital heart disease from animal modeling

Congenital heart disease(CHD)is observed in up to 1%of live births and is one of the leading causes of mortality from birth defects.While hundreds of genes have been implicated in the genetic etiology of CHD,their role in CHD pathogenesis is still poorly understood.This is largely a reflection of the sporadic nature of CHD,as well as its variable expressivity and incomplete penetrance.We reviewed the monogenic causes and evidence for oligogenic etiology of CHD,as well as the role of de novo mutations,common variants,and genetic modifiers.For further mechanistic insight,we leveraged single-cell data across species to investigate the cellular expression characteristics of genes implicated in CHD in developing human and mouse embryonic hearts.Understanding the genetic etiology of CHD may enable the application of precision medicine and prenatal diagnosis,thereby facilitating early intervention to improve outcomes for patients with CHD.

Single cell metabolic phenome and genome via the ramanome technology platform:Precision medicine of infectious diseases at the ultimate precision?

Due to the limitations of existing approaches,a rapid,sensitive,accurate,comprehensive,and generally applicable strategy to diagnose and treat bacterial and fungal infections remains a major challenge.Here,based on the ramanome technology platform,we propose a culture‐free,one cell resolution,phenome‐genome‐combined strategy called single‐cell identification,viability and vitality tests and source tracking(SCIVVS).For each cell directly extracted from a clinical specimen,the fingerprint region of the D2O‐probed single cell Raman spectrum(SCRS)enables species‐level identification based on a reference SCRS database of pathogen species,whereas the C‐D band accurately quantifies viability,metabolic vitality,phenotypic susceptibility to antimicrobials,and their intercellular heterogeneity.Moreover,to source track a cell,Raman‐activated cell sorting followed by sequencing or cultivation proceeds,producinging an indexed,high coverage genome assembly or a pure culture from precisely one pathogenic cell.Finally,an integrated SCIVVS workflow that features automated profiling and sorting of metabolic and morphological phenomes can complete the entire process in only a few hours.Because it resolves heterogeneity for both the metabolic phenome and genome,targets functions,can be automated,and is orders‐of‐magnitude faster while cost‐effective,SCIVVS is a new technological and data framework to diagnose and treat bacterial and fungal infections in various clinical and disease control settings.

Explore potential biomarkers to provide a theoretical basis for the treatment of liver fibrosis with pirfenidone

Background:Hepatic fibrosis is a common chronic liver disease in clinic,the purpose of our study is to explore potential biomarkers to provide a theoretical basis for the treatment of liver fibrosis with pirfenidone.Methods:We downloaded a gene-sequencing dataset and a single-cell dataset from the GEO database and pirfenidone target genes from three different databases.First,we performed GO,KEGG,and DO analysis on pirfenidone target genes.Then,we grouped the liver tissue sequencing data(GSE162694)in the sequencing data set(N-F0 group and F1-F4 group)and performed gene expression differential analysis on these two groups,weighted gene co-expression network analysis and gene Enrichment analysis.Finally,we intersected the significantly upregulated genes in the F1-F4 group with the pirfenidone target genes and performed PPI network analysis.In order to further explore the expression of both pirfenidone drug target genes and liver fibrosis disease genes(PDLFG)in different immune cells of liver tissue,we used the CD45+cell data in the GSE136103 data set for further analysis.Results:A subnetwork consisting of CDC42,HNF4A,BHLHE40,CCDC71L,NR1H3,TNF,MGLL,GPT,SCD and PLIN1 was screened out,and by analysis,we finally identified the SCD as PDLFG.In single-cell sequencing analysis,we found that SCD was highly expressed in M2-polarized macrophages.Conclusion:SCD may be an important target protein to inhibit the progression of liver fibrosis.

Dysregulated N6-methyladenosine modification in peripheral immune cells contributes to the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis(ALS)is a progressive neurogenerative disorder with uncertain origins.Emerging evidence implicates N6-methyladenosine(m6A)modification in ALS pathogenesis.Methylated RNA immunoprecipitation sequencing(MeRIP-seq)and liquid chromatography–mass spectrometry were utilized for m6A profiling in peripheral immune cells and serum proteome analysis,respectively,in patients with ALS(n=16)and controls(n=6).The single-cell transcriptomic dataset(GSE174332)of primary motor cortex was further analyzed to illuminate the biological implications of differentially methylated genes and cell communication changes.Analysis of peripheral immune cells revealed extensive RNA hypermethylation,highlighting candidate genes with differential m6A modification and expression,including C-X3-C motif chemokine receptor 1(CX3CR1).In RAW264.7 macrophages,disrupted CX3CR1 signaling affected chemotaxis,potentially influencing immune cell migration in ALS.Serum proteome analysis demonstrated the role of dysregulated immune cell migration in ALS.Cell type-specific expression variations of these genes in the central nervous system(CNS),particularly microglia,were observed.Intercellular communication between neurons and glial cells was selectively altered in ALS CNS.This integrated approach underscores m6A dysregulation in immune cells as a potential ALS contributor.

Integrated identification of growth pattern and taxon of bacterium in gut microbiota via confocal fluorescence imaging‐oriented single‐cell sequencing

Despite the fast progress in our understanding of the complex functions of gut microbiota,it is still challenging to directly investigate the in vivo microbial activities and processes on an individual cell basis.To gain knowledge of the indigenous growth/division patterns of the diverse mouse gut bacteria with a relatively high throughput,here,we propose an integrative strategy,which combines the use of fluorescent probe labeling,confocal imaging with single‐cell sorting,and sequencing.Mouse gut bacteria sequentially labeled by two fluorescent D‐amino acid probes in vivo were first imaged by confocal microscopy to visualize their growth patterns,which can be unveiled by the distribution of the two fluorescence signals on each bacterium.Bacterial cells of interest on the imaging slide were then sorted using a laser ejection equipment,and the collected cells were then sequenced individually to identify their taxa.Our strategy allows integrated acquirement of the growth pattern knowledge of a variety of gut bacteria and their genomic information on a single‐cell basis,which should also have great potential in studying many other complex bacterial systems.

Single-cell transcriptome reveals core cell populations and androgen-RXFP2 axis involved in deer antler full regeneration

Deer antlers constitute a unique mammalian model for the study of both organ formation in postnatal life and annual full regeneration.Previous studies revealed that these events are achieved through the proliferation and differentiation of antlerogenic periosteum(AP)cells and pedicle periosteum(PP)cells,respectively.As the cells resident in the AP and the PP possess stem cell attributes,both antler generation and regeneration are stem cell-based processes.However,the cell composition of each tissue type and molecular events underlying antler development remain poorly characterized.Here,we took the approach of single-cell RNA sequencing(scRNA-Seq)and identified eight cell types(mainly THY1^(+)cells,progenitor cells,and osteochondroblasts)and three core subclusters of the THY1^(+)cells(SC2,SC3,and SC4).Endothelial and mural cells each are heterogeneous at transcriptional level.It was the proliferation of progenitor,mural,and endothelial cells in the activated antler-lineage-specific tissues that drove the rapid formation of the antler.We detected the differences in the initial differentiation process between antler generation and regeneration using pseudotime trajectory analysis.These may be due to the difference in the degree of stemness of the AP-THY1+and PP-THY1^(+)cells.We further found that androgen-RXFP2 axis may be involved in triggering initial antler full regeneration.Fully deciphering the cell composition for these antler tissue types will open up new avenues for elucidating the mechanism underlying antler full renewal in specific and regenerative medicine in general.

VASC: Dimension Reduction and Visualization of Single-cell RNA-seq Data by Deep Variational Autoencoder

Single-cell RNA sequencing(scRNA-seq) is a powerful technique to analyze the transcriptomic heterogeneities at the single cell level. It is an important step for studying cell subpopulations and lineages, with an effective low-dimensional representation and visualization of the original scRNA-Seq data. At the single cell level, the transcriptional fluctuations are much larger than the average of a cell population, and the low amount of RNA transcripts will increase the rate of technical dropout events. Therefore, scRNA-seq data are much noisier than traditional bulk RNA-seq data. In this study, we proposed the deep variational autoencoder for scRNA-seq data(VASC), a deep multi-layer generative model, for the unsupervised dimension reduction and visualization of scRNA-seq data. VASC can explicitly model the dropout events and find the nonlinear hierarchical feature representations of the original data. Tested on over 20 datasets, VASC shows superior performances in most cases and exhibits broader dataset compatibility compared to four state-of-the-art dimension reduction and visualization methods. In addition, VASC provides better representations for very rare cell populations in the 2D visualization. As a case study, VASC successfully re-establishes the cell dynamics in pre-implantation embryos and identifies several candidate marker genes associated with early embryo development. Moreover, VASC also performs well on a 10× Genomics dataset with more cells and higher dropout rate.

Longitudinal single-cell RNA-seq of hESCs-derived retinal organoids

Human retina development involves multiple well-studied signaling pathways that promote the genesis of a wide arrange of different cell types in a complex architectural structure.Human embryonic stem cells(hESCs)-derived retinal organoids could recapitulate the human retinal development.We performed single-cell RNA-seq of retinal organoids from 5 time points(D36,D66,D96,D126,D186)and identified 9 distinct populations of cells.In addition,we analyzed the molecular characteristics of each main population and followed them from genesis to maturity by pseudotime analysis and characterized the cell-cell interactions between different cell types.Interestingly,we identified insulin receptor(INSR)as a specifically expressed receptor involved in the genesis of photoreceptors,and pleiothropin(PTN)-protein tyrosine phosphatase receptor type Z1(PTPRZ1)as a mediator of a previously unknown interaction between Müller and retinal progenitor cells.Taken together,these findings provide a rich transcriptome-based lineage map for studying human retinal development and modeling developmental disorders in retinal organoids.

Advancement in research on genes associated with fetal congenital heart disease(CHD)and diagnostic testing methods

Introduction:Congenital heart disease(CHD)is one of the most common congenital malformations,and is a polygenic disease related to some major genes and involved in environmental factors.With the progress of science and technology,the progress was both in the studies of genetic patterns and testing methods.Understanding how each gene participates in normal and pathological anatomy is an important goal of CHD research.We reviewed the development of testing methods and CHD-related genes,to provide some enlightenment for the CHD prenatal diagnosis and hope to realize the intervention and treatment on the gene level of CHD in the future.

Emerging approaches and technologies in transplantation:the potential game changers

Newly emerging technologies are rapidly changing conventional approaches to organ transplantation.In the modern era,the key challenges to transplantation include(1)how to best individualize and possibly eliminate the need for life-long immunosuppression and(2)how to expand the donor pool suitable for human transplantation.This article aims to provide readers with an updated review of three new technologies that address these challenges.First,single-cell RNA sequencing technology is rapidly evolving and has recently been employed in settings related to transplantation.The new sequencing data indicate an unprecedented cellular heterogeneity within organ transplants,as well as exciting new molecular signatures involved in alloimmune responses.Second,sophisticated nanotechnology platforms provide a means of therapeutically delivering immune modulating reagents to promote transplant tolerance.Tolerogenic nanoparticles with regulatory molecules and donor antigens are capable of targeting host immune responses with tremendous precision,which,in some cases,results in donor-specific tolerance.Third,CRISPR/Cas9 gene editing technology has the potential to precisely remove immunogenic molecules while inserting desirable regulatory molecules.This technology is particularly useful in generating genetically modified pigs for xenotransplantation to solve the issue of the shortage of human organs.Collectively,these new technologies are positioning the transplant community for major breakthroughs that will significantly advance transplant medicine.

Organoid research on human early development and beyond

The organoid field has been developing rapidly during the last decade.Organoids for human pre-,peri-and post-implantation development have opened an avenue to study these biological processes in vitro,which have been hampered by lack of accessible research models for long term.The technologies of four fields,single cell omics sequencing,genome editing and lineage tracing,micro-fluidics and tissue engineering,have fueled the rapid development of the organoid field.In this review,we will discuss the organoid research on human early development as well as future directions of the organoid field combining with other powerful technologies.