B cell receptor repertoire analysis in primary Sjogren's syndrome salivary glands identifies repertoire features associated with clinical activity.

BACKGROUND: Primary Sjogren's syndrome (pSS) is a complex autoimmune disease featuring damage to salivary and lacrimal glands, with the possibility of manifestations across multiple organs. Antibody-producing B cells have long been appreciated to play a significant role in pSS pathogenesis, with a number of autoreactive antibody species having been identified to be elevated in pSS patients. While several studies have attempted to characterize the BCR repertoires of peripheral blood B cells in pSS patients, much remains unknown about the repertoire characteristics of gland-infiltrating B cells. METHODS: Through paired scRNAseq and scBCRseq, we profiled the BCR repertoires of both infiltrating and circulating B cells in a small cohort of patients. We further utilize receptor reconstruction analyses to further investigate repertoire characteristics in a wider cohort of pSS patients previously profiled through RNAseq. RESULTS: Via integrated BCR and transcriptome analysis of B cell clones, we generate a trajectory progression pattern for infiltrated memory B cells in pSS. We observe significant differences in BCR repertoires between the peripheral blood and labial gland B cells of pSS patients in terms of relative expansion, isotype usage, and BCR clustering. We further observe significant decreases in IgA2 isotype usage among pSS patient labial and parotid gland B cells these analyses relative to controls as well as a positive correlation between kappa/lambda light chain usage and clinical disease activity. CONCLUSIONS: Through BCR repertoire analysis of pSS patient salivary glands, we identify a number of novel repertoire characteristics that may serve as useful indicators of clinical disease and disease activity. By collecting these BCR repertoires into an accessible database, we hope to also enable comparative analysis of patient repertoires in pSS and potentially other autoimmune disorders.

Screening single-cell trajectories via continuity assessments for cell transition potential.

Advances in single-cell sequencing and data analysis have made it possible to infer biological trajectories spanning heterogeneous cell populations based on transcriptome variation. These trajectories yield a wealth of novel insights into dynamic processes such as development and differentiation. However, trajectory analysis relies on an assumption of trajectory continuity, and experimental limitations preclude some real-world scenarios from meeting this condition. The current lack of assessment metrics makes it difficult to ascertain if/when a given trajectory deviates from continuity, and what impact such a divergence would have on inference accuracy is unclear. By analyzing simulated breaks introduced into in silico and real single-cell data, we found that discontinuity caused precipitous drops in the accuracy of trajectory inference. We then generate a simple scoring algorithm for assessing trajectory continuity, and found that continuity assessments in real-world cases of intestinal stem cell development and CD8 + T cells differentiation efficiently identifies trajectories consistent with empirical knowledge. This assessment approach can also be used in cases where a priori knowledge is lacking to screen a pool of inferred lineages for their adherence to presumed continuity, and serve as a means for weighing higher likelihood trajectories for validation via empirical studies, as exemplified by our case studies in psoriatic arthritis and acute kidney injury. This tool is freely available through github at qingshanni/scEGRET.

Single-cell Clonal Tracing of Glandular and Circulating T cells identifies a population of CD9+CD8+T cells in primary Sjogren's Syndrome.

Primary Sjogren's syndrome (pSS) is a complex chronic autoimmune disease in which local tissue damage in exocrine glands are combined with broader systemic involvement across the body in tissues including the skin. These combined manifestations negatively impact patient health and quality of life. While studies have previously reported differences in immune cell composition in the peripheral blood of pSS patients relative to healthy controls, a detailed immune cell landscape of the damaged exocrine glands of these patients remains lacking. Through single-cell transcriptomics and repertoire sequencing of immune cells in paired peripheral blood samples and salivary gland biopsies, we present here a preliminary picture of adaptive immune response in pSS. We characterize a number of points of divergence between circulating and glandular immune responses that have been hitherto underappreciated, and identify a novel population of CD8+CD9+ cells with tissue-residential properties that are highly enriched in the salivary glands of pSS patients. Through comparative analyses with other sequencing data, we also observe a potential connection between these cells and the tissue-resident memory cells found in cutaneous vasculitis lesions. Together, these results indicate a potential role for CD8+CD9+ cells in mediating glandular and systemic effects associated with pSS and other autoimmune disorders.

Highly diastereoselective synthesis of benzothiazolo[3,2-a]pyridines via [4 + 2] annulation reaction of 2-vinylbenzothiazoles and azlactones.

An efficient AgOTf-catalyzed [4 + 2] annulation reaction of 2-vinylbenzothiazoles and azlactones was successfully performed under mild reaction conditions. With this approach, a series of novel benzothiazolo[3,2-a]pyridine derivatives was readily obtained in good to excellent yields (68-96%), with high diastereoselectivities and tolerating quite a broad scope of substituents. By using chiral phosphoric acid catalyst, the desired products were obtained in high enantioselectivities, up to -94%. This methodology provides a rapid and useful method for constructing fused benzothiazole derivatives.

TIPS: trajectory inference of pathway significance through pseudotime comparison for functional assessment of single-cell RNAseq data.

Recent advances in bioinformatics analyses have led to the development of novel tools enabling the capture and trajectory mapping of single-cell RNA sequencing (scRNAseq) data. However, there is a lack of methods to assess the contributions of biological pathways and transcription factors to an overall developmental trajectory mapped from scRNAseq data. In this manuscript, we present a simplified approach for trajectory inference of pathway significance (TIPS) that leverages existing knowledgebases of functional pathways and other gene lists to provide further mechanistic insights into a biological process. TIPS identifies key pathways which contribute to a process of interest, as well as the individual genes that best reflect these changes. TIPS also provides insight into the relative timing of pathway changes, as well as a suite of visualizations to enable simplified data interpretation of scRNAseq libraries generated using a wide range of techniques. The TIPS package can be run through either a web server or downloaded as a user-friendly GUI run in R, and may serve as a useful tool to help biologists perform deeper functional analyses and visualization of their single-cell data.

Mapping the spatial distribution of T cells in repertoire dimension.

T cells mediate adaptive immunity in diverse anatomic compartments through recognition of specific antigens via unique T cell receptor (TCR) structures. However, little is known about the spatial distribution of an organism's TCR repertoire. Here, using high-throughput TCR sequencing (TCRseq), we investigated the TCR repertoires of sixteen tissues in healthy C57B/L6 mice. We found that TCR repertoires generally classified into three categories (lymph nodes, non-lymph node tissues and small intestine) based on sequence similarity. Clonal distribution and diversity analyses showed that small intestine compartment had a more skewed repertoire as compared to lymph nodes and non-lymph node tissues. However, analysis of TRBV and TRBJ gene usage across tissue compartments, as well as comparison of CDR3 length distributions, showed no significant tissue-dependent differences. Interestingly, analysis of clonotype sharing between mice showed that although non-redundant public clonotypes were found more easily in lymph nodes, small intestinal CD4 + T cells harbored more abundant public clonotypes. These findings under healthy physiological conditions offer an important reference dataset, which may contribute to our ability to better manipulate T cell responses against infection and vaccination.

Conversion of effector CD4+ T cells to a CD8+ MHC II-recognizing lineage.

CD4+ and CD8+ T cells are dichotomous lineages in adaptive immunity. While conventionally viewed as distinct fates that are fixed after thymic development, accumulating evidence indicates that these two populations can exhibit significant lineage plasticity, particularly upon TCR-mediated activation. We define a novel CD4-CD8αß+ MHC II-recognizing population generated by lineage conversion from effector CD4+ T cells. CD4-CD8αß+ effector T cells downregulated the expression of T helper cell-associated costimulatory molecules and increased the expression of cytotoxic T lymphocyte-associated cytotoxic molecules. This shift in functional potential corresponded with a CD8+-lineage skewed transcriptional profile. TCRß repertoire sequencing and in vivo genetic lineage tracing in acutely infected wild-type mice demonstrated that CD4-CD8αß+ effector T cells arise from fundamental lineage reprogramming of bona fide effector CD4+ T cells. Impairing autophagy via functional deletion of the initiating kinase Vps34 or the downstream enzyme Atg7 enhanced the generation of this cell population. These findings suggest that effector CD4+ T cells can exhibit a previously unreported degree of skewing towards the CD8+ T cell lineage, which may point towards a novel direction for HIV vaccine design.

MiR-26a targets EphA2 to resist intracellular Listeria monocytogenes in macrophages.

At infection sites, macrophages are sentinels that resist and destroy various pathogens, through direct phagocytosis. In macrophages, microRNAs play a variety of crucial roles, the most striking of which is the regulation of the ability of the host cell to resist infection. However, the underlying mechanisms associated with the anti-infection effects mediated by microRNAs remain largely unknown. Here, we demonstrated that miR-26a is downregulated during infection by Listeria monocytogenes (Lm). In miR-26a overexpressing mice, the Lm bacterial burden of liver and spleen decreased significantly within 72 h of infection, compared with that in control mice. Subsequently, RNA sequencing (RNA-seq) data suggested that miR-26a may attenuate the survival of Lm by targeting the Ephrin receptor tyrosine kinase A2 (EphA2). The knockdown of EphA2 in RAW264.7 macrophage cells resulted in decreased intracellular Lm burden. Taken together, these findings validate EphA2 as a target of miR-26a and provide a mechanism through which Lm may survive within macrophages by altering host miRNA expression.

VisTCR: An Interactive Software for T Cell Repertoire Sequencing Data Analysis.

Recent progress in high throughput sequencing technologies has provided an opportunity to probe T cell receptor (TCR) repertoire, bringing about an explosion of TCR sequencing data and analysis tools. For easier and more heuristic analysis TCR sequencing data, we developed a client-based HTML program (VisTCR). It has a data storage module and a data analysis module that integrate multiple cutting-edge analysis algorithms in a hierarchical fashion. Researchers can group and re-group samples for different analysis purposes by customized "Experiment Design File." Moreover, the VisTCR provides a user-friendly interactive interface, by all the TCR analysis methods and visualization results can be accessed and saved as tables or graphs in the process of analysis. The source code is freely available at https://github.com/qingshanni/VisTCR.

TCR repertoire and CDR3 motif analyses depict the role of αß T cells in Ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory disease with worldwide high prevalence. Although AS is strongly associated with HLA-B27 MHC-I antigen presentation, the role played by αß T cells in AS remains elusive. METHODS: Utilizing TCRß repertoire sequencing and bioinformatics tools developed in house, we analyzed overall TCR repertoire structures and antigen-recognizing CDR3 motifs in AS patients with different disease activities. FINDINGS: We found that disease progression is associated with both CD4+ and CD8+ T cell oligo-clonal expansion, which suggests that αß T cell activation may mediate AS disease progression. By developing a bioinformatics platform to dissect antigen-specific responses, we discovered a cell population consisting of both CD4+ and CD8+ T cells expressing identical TCRs, herein termed CD4/8 T cells. CD4/8 clonotypes were highly enriched in the spondyloarthritic joint fluid of patients, and their expansion correlated with the activity of disease. INTERPRETATION: These results provide evidence on the T cell clone side to reveal the potential role of CD4/8 T cells in the etiology of AS development.

Comparative analysis and characterization of Enterobacteria phage SSL-2009a and 'HK578likevirus' bacteriophages.

Enterobacteria phage SSL-2009a is a virulent bacteriophage with strong and abroad lytic ability against lots of engineering E. coli strains. In this study, we re-sequenced its whole genome and made a detail analysis on its genomic and proteomic characteristics according to the updated genomic sequence. The genome of SSL-2009a is a circular double-stranded DNA of 44,899 base pairs in length, with a 54.67% G + C content. A total of 67 open reading frames were predicted as protein coding sequences, 24 of which encode products highly homologous to known phage proteins. There are 10 promoters and 22 terminators identified in the genome of SSL-2009a, but no tRNA is found. SSL-2009a belongs to the 'HK578likevirus' genus of Siphoviridae. Comparative analyses indicated that other twelve phages share high homology with SSL-2009a at nucleotide and amino acid levels and also should be clustered into the same genus. In-depth analysis was performed to reveal the genomic, proteomic, and morphological features of these 'HK578likevirus' phages, which may promote our understanding of Enterobacteria phage SSL-2009a and the 'HK578likevirus' genus, even the biodiversity and evolution of bacteriophages.

Rab32-related antimicrobial pathway is involved in the progression of dextran sodium sulfate-induced colitis.

Inflammatory bowel disease (IBD) is a multifactorial disease involving defective immune responses against invasive microbiota. Genes associated with innate immune responses to microbes have been highlighted in the pathogenesis of IBD. To determine the role of Rab32 in the pathogenesis of IBD, we administered dextran sodium sulfate (DSS) to CD11c+ cell-specific Rab32 knockout (CD11c-Cre+Rab32f/f) mice to induce colitis. Rab32 deficiency in CD11c+ cells resulted in more severe disease progression and increased mortality. Histopathological analysis showed extensive damage to the colon mucosa in DSS-treated CD11c-Cre+Rab32f/f mice, including more severe damage to the epithelial layer and crypts, as well as more inflammatory cell infiltration. The pro-inflammatory cytokines IL1A, IL1B, IL6, and CSF3 and chemokines CXCL1 and CXCL2 were significantly increased, and the frequency of CD11b+Ly6G+ neutrophils was higher in CD11c-Cre+Rab32f/f colitis mice. Furthermore, CD11c+ cells deficient for Rab32 exhibited a significant increase in bacterial translocation in inflamed colon tissue. The present data demonstrate that Rab32 knockout in CD11c+ cells aggravates the development of DSS-induced colitis and suggest that the Rab32-related antimicrobial pathway is involved in the pathogenesis of IBD.

Transcriptomic and Metabolomic Analysis Revealed Multifaceted Effects of Phage Protein Gp70.1 on Pseudomonas aeruginosa.

The impact of phage infection on the host cell is severe. In order to take over the cellular machinery, some phage proteins were produced to shut off the host biosynthesis early in the phage infection. The discovery and identification of these phage-derived inhibitors have a significant prospect of application in antibacterial treatment. This work presented a phage protein, gp70.1, with non-specific inhibitory effects on Pseudomonas aeruginosa and Escherichia coli. Gp70.1 was encoded by early gene - orf 70.1 from P. aeruginosa phage PaP3. The P. aeruginosa with a plasmid encoding gp70.1 showed with delayed growth and had the appearance of a small colony. The combination of multifaceted analysis including microarray-based transcriptomic analysis, RT-qPCR, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics and phenotype experiments were performed to investigate the effects of gp70.1 on P. aeruginosa. A total of 178 genes of P. aeruginosa mainly involved in extracellular function and metabolism were differentially expressed in the presence of gp70.1 at three examined time points. Furthermore, our results indicated that gp70.1 had an extensive impact on the extracellular phenotype of P. aeruginosa, such as motility, pyocyanin, extracellular protease, polysaccharide, and cellulase. For the metabolism of P. aeruginosa, the main effect of gp70.1 was the reduction of amino acid consumption. Finally, the RNA polymerase sigma factor RpoS was identified as a potential cellular target of gp70.1. Gp70.1 was the first bacterial inhibitor identified from Pseudomonas aeruginosa phage PaP3. It was also the first phage protein that interacted with the global regulator RpoS of bacteria. Our results indicated the potential value of gp70.1 in antibacterial applications. This study preliminarily revealed the biological function of gp70.1 and provided a reference for the study of other phage genes sharing similarities with orf70.1.

Glimpse of natural selection of long-lived T-cell clones in healthy life.

Homeostatic maintenance of T cells with broad clonal diversity is influenced by both continuing output of young T cells from the thymus and ongoing turnover of preexisting clones in the periphery. In the absence of infection, self and commensal antigens are thought to play important roles in selection and homeostatic maintenance of the T-cell pool. Most naïve T cells are short-lived due to lack of antigen encounter, whereas antigen-experienced T cells may survive and persist as long-lived clones. Thus far, little is known about the homeostasis, antigenic specificity, and clonal diversity of long-lived T-cell clones in peripheral lymphoid organs under healthy living conditions. To identify long-lived T-cell clones in mice, we designed a lineage-tracing method to label a wave of T cells produced in the thymus of young mice. After aging the mice for 1.5 y, we found that lineage-tracked T cells consisted of primarily memory-like T cells and T regulatory cells. T-cell receptor repertoire analysis revealed that the lineage-tracked CD4 memory-like T cells and T regulatory cells exhibited age-dependent enrichment of shared clonotypes. Furthermore, these shared clonotypes were found across different mice maintained in the same housing condition. These findings suggest that nonrandom and shared antigens are involved in controlling selection, retention, and immune tolerance of long-lived T-cell clones under healthy living conditions.

Analysis of the Rab GTPase Interactome in Dendritic Cells Reveals Anti-microbial Functions of the Rab32 Complex in Bacterial Containment.

Dendritic cells (DCs) orchestrate complex membrane trafficking through an interconnected transportation network linked together by Rab GTPases. Through a tandem affinity purification strategy and mass spectrometry, we depicted an interactomic landscape of major members of the mammalian Rab GTPase family. When complemented with imaging tools, this proteomic analysis provided a global view of intracellular membrane organization. Driven by this analysis, we investigated dynamic changes to the Rab32 subnetwork in DCs induced by L. monocytogenes infection and uncovered an essential role of this subnetwork in controlling the intracellular proliferation of L. monocytogenes. Mechanistically, Rab32 formed a persistent complex with two interacting proteins, PHB and PHB2, to encompass bacteria both during early phagosome formation and after L. monocytogenes escaped the original containment vacuole. Collectively, we have provided a functional compartmentalization overview and an organizational framework of intracellular Rab-mediated vesicle trafficking that can serve as a resource for future investigations.

Association of CD8(+) T lymphocyte repertoire spreading with the severity of DRESS syndrome.

T-cell receptor (TCR)-mediated cross-recognition is a major mechanism in the pathogenesis of drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome. However, the characteristics of the TCR repertoire and the clinical significance of repertoire reformation throughout the course of DRESS are unknown. Here, we isolated CD4(+) and CD8(+) T-cells from peripheral blood of 8 DRESS patients at 10-day intervals and, sequenced CDR3-regions of the TCRB chain by high-throughput sequencing to analyze the dynamic reformation in the T-cell repertoire hierarchy. Compared with healthy donors, T-cell expanded in peripheral repertoires from DRESS patient. The extent of fluctuation of dominant CD8(+) T-cell clones, but not of CD4(+) counterparts, correlated positively with the clinical severity and helped classify the enrolled subjects into "fluctuant" and "flat" repertoire groups. The anti-herpesvirus response, which was measured using anti-EBV/HHV antibodies, and the proportion of the homologous CD8(+) EBV-specific clonotypes, in the "fluctuant" group was substantial higher than that in the "flat" group. Furthermore, autoimmune sequelae were observed in a cured "fluctuant" patient. Collectively, the clinical relevance of the fluctuant CD8(+) T-cell repertoires supports the notion that herpes virus-mediated continuously de novo priming of newly pathogenic CD8(+) T-cell clones is an alternate mechanism responsible for the pathogenicity of DRESS.

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity.

BACKGROUND: Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome. RESULTS: After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51 N-6-methyladenines and 152 N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5α as a host bacterium to construct a shotgun library. CONCLUSIONS: This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.

Modulation, bioinformatic screening, and assessment of small molecular peptides targeting the vascular endothelial growth factor receptor.

Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) are important factors in tumor growth and metastasis. Molecular probes or drugs designed to target VEGF/VEGFR interactions are crucial in tumor molecular imaging and targeted therapy. Bioinformatic methods enable molecular design based on the structure of bio-macromolecules and their interactions. This study was aimed to identify tumor-targeting small-molecule peptides with high affinity for VEGFR using bioinformatics screening. The VEGFR extracellular immunoglobulin-like modules Ig1-Ig3 were used as the target to systematically alter the primary peptide sequence of VEGF125-136. Molecular docking and surface functional group interaction methods were combined in an in silico screen for polypeptides, which in theory, would have higher affinities for VEGFR. In vitro receptor competition binding assays were used to assess the affinity of the putative VEGFR-binding polypeptides. Rhodamine-conjugated peptides were used to label and visualize peptide-binding sites on A549 cells. Using bioinformatic screening, we identified 20 polypeptides with potentially higher affinity for VEGFR. The polypeptides were capable of inhibiting the binding of (125)I-VEGF to VEGFR in a dose-dependent manner. The IC50 values of QKRKRKKSRKKH and RKRKRKKSRYIVLS (80 and 185 nmol/L, respectively) were significantly lower than that of VEGF125-136 (464 nmol/L); thus, the affinity of these peptides for VEGFR was 6- and 2.5-fold higher, respectively, than that of VEGF125-136. Rhodamine labeling of A549 cells revealed peptide binding mainly on the plasma membrane and in the cytoplasm. Bioinformatic approaches hold promise for the development of molecular imaging probes. Using this approach, we designed two peptides that showed higher affinity toward VEGFR. These polypeptides may be used as molecular probes or drugs targeting VEGFR, which can be utilized in molecular imaging and targeted therapy of certain tumors.

Accurate discrimination of outer membrane proteins using secondary structure element alignment and support vector machine.

Outer membrane proteins (OMPs) play critical roles in many cellular processes and discriminating OMPs from other types of proteins is very important for OMPs identification in bacterial genomic proteins. In this study, a method SSEA_SVM is developed using secondary structure element alignment and support vector machine. Moreover, a novel kernel function is designed to utilize secondary structure information in the support vector machine classifier. A benchmark dataset, which consists of 208 OMPs, 673 globular proteins, and 206 α-helical membrane proteins, is used to evaluate the performance of SSEA_SVM. A high accuracy of 97.7% with 0.926 MCC is achieved while SSEA_SVM is applied to discriminating OMPs and non-OMPs. In comparison with existing methods in the literature, SSEA_SVM is also highly competitive. We suggest that SSEA_SVM is a much more promising method to identify OMPs in genomic proteins. A web server that implements SSEA_SVM is freely available at http://bioinfo.tmmu.edu.cn/SSEA_SVM/.