Project description:Single cell technologies provide an unprecedented opportunity to explore the heterogeneity in a biological process at the level of single cells. One major challenge in analyzing single cell data is to identify cell subpopulations, stable cell states, and cells in transition between states. To elucidate the transition mechanisms in cell fate dynamics, it is highly desirable to quantitatively characterize cellular states and intermediate states. Here, we present scRCMF, an unsupervised method that identifies stable cell states and transition cells by adopting a nonlinear optimization model that infers the latent substructures from a gene-cell matrix. We incorporate a random coefficient matrix-based regularization into the standard nonnegative matrix decomposition model to improve the reliability and stability of estimating latent substructures. To quantify the transition capability of each cell, we propose two new measures: single-cell transition entropy (scEntropy) and transition probability (scTP). When applied to two simulated and three published scRNA-seq datasets, scRCMF not only successfully captures multiple subpopulations and transition processes in large-scale data, but also identifies transition states and some known marker genes associated with cell state transitions and subpopulations. Furthermore, the quantity scEntropy is found to be significantly higher for transition cells than other cellular states during the global differentiation, and the scTP predicts the "fate decisions" of transition cells within the transition. The present study provides new insights into transition events during differentiation and development.
Project description:Neisseria meningitidis secretes a protease that specifically cleaves the hinge region of immunoglobulin A1 (IgA1), releasing the effector (Fc) domain of IgA1 from the antigen binding (Fab) determinants. Theoretically, the remaining Fab fragments can block pathogen receptors or toxins and still provide protection. Here, we describe binding of V-gene-matched human IgA1 and IgA2 to PorA of strain H44/76. On live meningococci, efficient cleavage of IgA1, but not cleavage of IgA2, was observed, and up to approximately 80% of the IgA1 Fc tails were lost from the meningococcal surface within 30 min. No cleavage of IgA1 was found on an isogenic H44/76 strain lacking IgA1 protease. Furthermore, our data indicate that PorA-bound IgA1 is masked by the serogroup B polysaccharide capsule, rendering the IgA1 less accessible to degradation by secreted IgA1 protease present in the bacterial surroundings. Experiments with protein synthesis inhibitors showed that de novo production of IgA1 protease was responsible for cleavage of PorA-bound IgA1 on encapsulated bacteria. Finally, our data suggest that cleavage of IgA1 by IgA1 protease releases a significant proportion of Fab fragments from the bacterium, probably as a result of their reduced avidity compared to that of whole antibodies.
Project description:Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igalpha and Igbeta of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating order of magnitude increases in BCR surface expression. Surface Ig levels correlated with transgenic Igalpha expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS).
Project description:The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases of Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis had an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence in S. mitis that proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors.
Project description:Vaccines are among the most effective public health tools for combating certain infectious diseases such as influenza. The role of the humoral immune system in vaccine-induced protection is widely appreciated; however, our understanding of how antibody specificities relate to B cell function remains limited due to the complexity of polyclonal antibody responses. To address this, we developed the Spec-seq framework, which allows for simultaneous monoclonal antibody (mAb) characterization and transcriptional profiling from the same single cell. Here, we present the first application of the Spec-seq framework, which we applied to human plasmablasts after influenza vaccination in order to characterize transcriptional differences governed by B cell receptor (BCR) isotype and vaccine reactivity. Our analysis did not find evidence of long-term transcriptional specialization between plasmablasts of different isotypes. However, we did find enhanced transcriptional similarity between clonally related B cells, as well as distinct transcriptional signatures ascribed by BCR vaccine recognition. These data suggest IgG and IgA vaccine-positive plasmablasts are largely similar, whereas IgA vaccine-negative cells appear to be transcriptionally distinct from conventional, terminally differentiated, antigen-induced peripheral blood plasmablasts.
Project description:IntroductionComplexins (CPLXs), initially identified in neuronal presynaptic terminals, are cytoplasmic proteins that interact with the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex to regulate the fusion of vesicles to the plasma membrane. Although much is known about CPLX function in neuronal synaptic vesicle exocytosis, their distribution and role in immune cells are still unclear. In this study, we investigated CPLX2 knockout (KO) mice to reveal the role of CPLXs in exocytosis of lymphocytes.MethodsWe examined the expression of CPLXs and SNAREs in lymphocytes. To study the effect of CPLXs on the immune system in vivo, we analyzed the immune phenotype of CPLX2 KO mice. Furthermore, antibodies secretion from the peritoneal cavity, spleen, and bone marrow cells of wild-type (WT) and CPLX2 KO mice were determined.ResultsCPLX2 was detected in B cells but not in T cells, while other CPLXs and SNAREs were expressed at a similar level in both B and T cells. To clarify the function of CPLX2 in B lymphocytes, serum concentrations of immunoglobulin G (IgG), IgA, IgM, and IgE were measured in WT and CPLX2 KO mice using enzyme-linked immunosorbent assay. The level of IgM, which mainly consists of natural antibodies, was higher in KO mice than that in WT mice, while the levels of other antibodies were similar in both types of mice. Additionally, we found that spontaneous secretion of IgM and IgG1 was enhanced from the splenic antibody-secreting cells (ASCs) of CPLX2 KO mice.ConclusionOur data suggest that CPLX2 inhibits spontaneous secretion of IgM and IgG1 from splenic ASCs. This study provides new insight into the mechanism of antibody secretion of ASCs.
Project description:Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches.
Project description:BackgroundCiliates are single-celled microbial eukaryotes that diverged from other eukaryotic lineages more than a billion years ago. The long evolutionary timespan of ciliates has led to enormous genetic and phenotypic changes, contributing significantly to their high level of diversity. Recent analyses based on molecular data have revealed numerous cases of cryptic species complexes in different ciliate lineages, demonstrating the need for a robust approach to delimit species boundaries and elucidate phylogenetic relationships. Species of the genus Spirostomum are difficult to identify due to the lack of distinctive morphological characters. Previous molecular studies have focused on only a few loci, namely the nuclear ribosomal RNA genes, alpha-tubulin, and mitochondrial CO1, suggesting the presence of several cryptic Spirostomum species. In this study, we increased taxon sampling and obtained single-cell transcriptomes of 25 Spirostomum specimens (representing six morphospecies) sampled from South Korea and the USA. We evaluated the utility of the transcriptomic data by constructing species trees using concatenation and coalescent-based methods. In addition, we used neighbor-net network analysis to visualize and quantify potential phylogenetic conflicts within the concatenated dataset. Furthermore, coalescent-based species delimitation was performed with transcriptomic data to define the species boundaries within the genus Spirostomum.ResultsPhylogenomic analysis of 37 Spirostomum specimens (25 newly generated transcriptomes and 12 from GenBank) and 265 protein-coding genes provides robust insight into the evolutionary relationships among Spirostomum species. Our results confirm that species with moniliform and compact macronucleus each form a distinct monophyletic lineage, with the compact macronucleus likely representing the ancestral state, while the moniliform macronucleus being a derived trait. Furthermore, our analyses suggest that ancestral polymorphism and rapid radiation may have shaped the genetic diversity and evolutionary history of Spirostomum, and the S. minus-like appearance represents the ancestral state of the species with a moniliform macronucleus. Therefore, the S. minus-like species share ancestral morphological traits and cannot be morphologically delimited. The multispecies coalescent (MSC) model suggests that two cryptic species from each of S. minus, S. ambiguum, S. subtilis, S. teres, and S. aff. minus represent distinct lineages within the genus Spirostomum. We also provide a workflow for reconstructing nuclear ribosomal RNA gene sequences from transcriptome sequences using a read mapping approach, and compare different mapping methods to reconstruct reliable contigs.ConclusionOur sampling of closely related Spirostomum populations and comprehensive single-cell RNA sequencing (scRNA-seq) data allowed us to reveal the hidden crypticity of species within the genus Spirostomum and to resolve and provide much stronger support than hitherto to the phylogeny of this model ciliate genus.
Project description:Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level.
Project description:Beta cell failure and apoptosis following islet inflammation have been associated with autoimmune type 1 diabetes pathogenesis. As conveyors of biological active material, extracellular vesicles (EV) act as mediators in communication with immune effectors fostering the idea that EV from inflamed beta cells may contribute to autoimmunity. Evidence accumulates that beta exosomes promote diabetogenic responses, but relative contributions of larger vesicles as well as variations in the composition of the beta cell's vesiculome due to environmental changes have not been explored yet. Here, we made side-by-side comparisons of the phenotype and function of apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of MIN6 beta cells exposed to inflammatory, hypoxic or genotoxic stressors. Under normal conditions, large vesicles represent 93% of the volume, but only 2% of the number of the vesicles. Our data reveal a consistently higher release of AB and sEV and to a lesser extent of MV, exclusively under inflammatory conditions commensurate with a 4-fold increase in the total volume of the vesiculome and enhanced export of immune-stimulatory material including the autoantigen insulin, microRNA, and cytokines. Whilst inflammation does not change the concentration of insulin inside the EV, specific Toll-like receptor-binding microRNA sequences preferentially partition into sEV. Exposure to inflammatory stress engenders drastic increases in the expression of monocyte chemoattractant protein 1 in all EV and of interleukin-27 solely in AB suggesting selective sorting toward EV subspecies. Functional in vitro assays in mouse dendritic cells and macrophages reveal further differences in the aptitude of EV to modulate expression of cytokines and maturation markers. These findings highlight the different quantitative and qualitative imprints of environmental changes in subpopulations of beta EV that may contribute to the spread of inflammation and sustained immune cell recruitment at the inception of the (auto-) immune response.