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Background Single cell RNA sequencing provides unprecedented opportunity to simultaneously explore the transcriptomic and immune receptor diversity of T and B cells. However, there are limited tools available that simultaneously analyse large multi-omics datasets integrated with metadata such as patient and clinical information. Results We developed VDJView, which permits the simultaneous or independent analysis and visualisation of gene expression, immune receptors, and clinical metadata of both T and B cells. This tool is implemented as an easy-to-use R shiny web-application, which integrates numerous gene expression and TCR analysis tools, and accepts data from plate-based sorted or high-throughput single cell platforms. We utilised VDJView to analyse several 10X scRNA-seq datasets, including a recent dataset of 150,000 CD8 + T cells with available gene expression, TCR sequences, quantification of 15 surface proteins, and 44 antigen specificities (across viruses, cancer, and self-antigens). We performed quality control, filtering of tetramer non-specific cells, clustering, random sampling and hypothesis testing to discover antigen specific gene signatures which were associated with immune cell differentiation states and clonal expansion across the pathogen specific T cells. We also analysed 563 single cells (plate-based sorted) obtained from 11 subjects, revealing clonally expanded T and B cells across primary cancer tissues and metastatic lymph-node. These immune cells clustered with distinct gene signatures according to the breast cancer molecular subtype. VDJView has been tested in lab meetings and peer-to-peer discussions, showing effective data generation and discussion without the need to consult bioinformaticians. Conclusions VDJView enables researchers without profound bioinformatics skills to analyse immune scRNA-seq data, integrating and visualising this with clonality and metadata profiles, thus accelerating the process of hypothesis testing, data interpretation and discovery of cellular heterogeneity. VDJView is freely available at https://bitbucket.org/kirbyvisp/vdjview .

T cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood samples from a unique cohort of individuals with primary infection using single-cell multi-omics to identify the functions and phenotypes of HCV-specific CD8 + T cells. Early elevated IFN-γ response against the transmitted virus is associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome, we find heterogeneous subsets of progenitors of exhaustion, based on the level of PD-1 expression and loss of AP-1 transcription factors. Intra-clonal analysis shows distinct trajectories with multiple fates and evolutionary plasticity of precursor cells. These findings challenge the current paradigm on the contribution of CD8 + T cells to HCV disease outcome and provide data for future studies on T cell differentiation in human infections. The early immune response following exposure to HCV is not fully explored. Here the authors use single cell analysis and immune profiling to relate the infection sequence and immune response to early HCV infection showing that exhausted phenotypes of T cells arise early post infection.

The emergence of single cell RNA sequencing technologies has opened a vast number of strategies to look at various aspects of immunity to infectious diseases, especially into the adaptive immune system. Here, we applied scRNA-seq to study rare antigen-specific memory B cells in Hepatitis C and COVID-19 disease. Firstly, a tool for the reconstruction of single T cell and B cell receptor (TCR and BCR) sequencing method was developed that can obtain full-length repertoire from short read sequencing of T and B cell at single cell resolution. This tool, VDJPuzzle was able to report the detection of V(D)J genes, isotypes, somatic hypermutations, membrane vs secreted exon isoforms. VDJPuzzle showed highly accurate detection of V(D)J when benchmarked against available tools together. The utility of this tool to explore infection dynamics associated between BCR and disease outcome was then explored for two diseases: Hepatitis C (HCV) and COVID19. In the HCV study, properties associated with rare HCV-specific memory B cells and varied HCV infection outcome in primary infection and reinfection were examined. In the primary infection cohort, a higher maturation of memory B cells and a reduced level of aromatic residues was observed in individuals that developed chronic infection. In the reinfection cohort, no association of BCR recall was observed, however transcriptomic differences were observed prior to re-infection across clearers and chronics where, clearers had a pre-existing enrichment of genes associated with the TNF-α signaling via NF-kB pathway. There was also a population of memory B cells with increased CD95 surface expression that showed migration of these cells towards effector long-lived plasma cells showing higher expression of ZBTB32, TBX21, FAS, ITGAX, etc. In the COVID-19 study, we successfully extracted rare SARS-CoV-2 specific memory B cells from mild/moderate and severely infected subjects, at 1 month and 4 months convalescence. Interestingly, the VDJPuzzle tool observed a higher percentage of dual kappa-lambda chains in SARS-CoV-2-specific B cells. The presence of unique phenotypes was also identified in SARSCoV-2-specific memory B cells from subjects with severe disease. In particular, there was an increase in two populations defined as ‘actBC1’ (CD80hiTNFAIP3hi) and ‘actBC2’ (CD11chiCD95hi) which we hypothesized might be associated with increased longevity of the memory B cell response. The severe patients also maintained a higher level of activation genes associated with TNF-α signaling via NF-kB pathway, which may relate to the ‘cytokine storm’ that is often observed in severe SARS-CoV-2 infected individuals. Whereas, in mild/moderate infected individuals a decline in activated genes was observed over time. In summary, this thesis took advantage of unique sets of scRNA-seq techniques to identify the causes associated with HCV-specific memory B cells towards the clearance and chronic infection outcome in primary and reinfection cohort. Furthermore, we looked at the potential mechanistic insights associated with SARS-CoV-2-specific memory B cell response towards how transcriptome contributes towards the long-term immunity in a mild/moderate or severe infected individuals up to 4 months at convalescence.

Coronavirus pandemic has brought forth the urgency of providing affordable health care to everyone. Generic medicines are often one-fourth to one-tenth of the cost of the branded drugs, and so offer a remarkable opportunity to significantly lower the health-care expenditure. However, the argument for promoting generic medicines is indisputable, we have to think about the other enabling conditions which are necessary for a successful health policy on encouraging generics without causing unintended adverse repercussions. This paper attempts to answer such questions by considering the motivations of the various stakeholders of the broader health services ecosystem in India and undertaking a systematic analysis of the winners and losers from such a policy. We argue that generic prescription will not be successful without prior improvement in the state capacity for quality control of drug manufacturing; rise in awareness among the doctors, patients, and pharmacists; improved trust in the medical systems; and innovative demand-side interventions.

A significant number of esophageal atresia and tracheoesophageal fistula patients have long gaps and a high propensity to leak. Anastomotic leak in esophageal atresia is associated with a significant morbidity and mortality. In a prospective randomized trial, we analyzed the risk factors leading to anastomotic dehiscence and studied the effect of pleural wrap as an additional vascular cover around the esophageal anastomosis. Forty patients were divided into two groups A and B randomly. In 20 patients of group A, pleural wrap was utilized for covering the anastomosis and in 20 patients of group B, no such wrap was utilized. Both the groups were comparable regarding age, sex, weight, gap length, tension at anastomosis and the hospital stay. The overall leak rate was 25% (10/40) in both the groups. The leak rate was not significantly different in two groups whenever a gap length was less than 2 cm or more than 3 cm. However, for a gap length of 2-3 cm, the leak rate in group A was 18% (2/11) and in group B was 50% (4/8) (P = 0.05). Thirty percent (3/10) of patients, whose anastomosis was under tension, leaked in group A as compared to 75% (6/8) in group B patients (P = 0.001). Use of pleural wrap was associated with less anastomotic dehiscence in patients with moderate gap esophageal atresia (2-3 cm) especially when the anastomosis was under tension.

The degradation of hydrocarbons plays an important role in the eco-balancing of petroleum products, pesticides and other toxic products in the environment. The degradation of hydrocarbons by microbes such as Geobacillus thermodenitrificans , Burkhulderia , Gordonia sp. and Acinetobacter sp. has been studied intensively in the literature. The present study focused on the in silico protein engineering of alkane monooxygenase (ladA)—a protein involved in the alkane degradation pathway. We demonstrated the improvement in substrate binding energy with engineered ladA in Burkholderia thailandensis MSMB121. We identified an ortholog of ladA monooxygenase found in B. thailandensis MSMB121, and showed it to be an enzyme involved in an alkane degradation pathway studied extensively in Geobacillus thermodenitrificans . Homology modeling of the three-dimensional structure of ladA was performed with a crystal structure (protein databank ID: 3B9N) as a template in MODELLER 9v11, and further validated using PROCHECK, VERIFY-3D and WHATIF tools. Specific amino acids were substituted in the region corresponding to amino acids 305–370 of ladA protein, resulting in an enhancement of binding energy in different alkane chain molecules as compared to wild protein structures in the docking experiments. The substrate binding energy with the protein was calculated using Vina (Implemented in VEGAZZ). Molecular dynamics simulations were performed to study the dynamics of different alkane chain molecules inside the binding pockets of wild and mutated ladA. Here, we hypothesize an improvement in binding energies and accessibility of substrates towards engineered ladA enzyme, which could be further facilitated for wet laboratory-based experiments for validation of the alkane degradation pathway in this organism. This abstract shows homology based 3-D structure of alkane monooxygenase in Burkholderia thailandensis MSMB121, it was further validated using Ramachandaran plot, Whatif and verify-3D. Both wild and mutated alkane monooxygenase were docked with long chain alkane molecules (c16-c36) using Vina. Finally, a plot for RMSD of molecular dynamics simulations was generated using GROMACS showing fluctutations in wild and mutated protein structures

T-cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood samples from a unique cohort of subjects with primary infection using single cell multi-omics to identify the functions and phenotypes of HCV-specific CD8+ T cells. Early elevated IFN-γ response against the transmitted virus was associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome we discovered progenitors of early-exhaustion with intermediate expression of PD-1. Intra clonal analysis revealed distinct trajectories with multiple fates suggesting evolutionary plasticity of precursor cells. These findings challenge current paradigm on the contribution of CD8+ T cells to HCV disease outcome and provide data for future studies on T-cell differentiation in human infections. Competing Interest Statement The authors have declared no competing interest.

This was a prospective randomized study of 40 patients of EA and TEF in two groups of 20 each. Group A patients were provided with an additional pleural cover around the esophageal anastomosis whereas in group B no such cover was given. Mean age at presentation in group A was 1.85 days as compared to 1.75 days in group B. In patients who presented early, the mortality was less however the leak rate was not affected. Male to female ratio in group A was 5.6; 1 and 2.3:1 in group B. Survival rate in the male babies was slightly higher. Mean birth weight in group A was 2.43 kg as compared to 2.35 kg in group B. Babies with more than 2.5 kg birth weight in both the groups had better survival rates. However, no such relation was found with the leak rate. Mean length of gap between the two esophageal ends in group A was 2.85 cm and in group B was 2.5 cm. Leak rate in patients with intermediate gap length was significantly less where pleural wrap was applied as compared to group B patients in whom no such cover was given. Associated anomalies were found in 37.5 % of the patients. Thirteen out of 40 patients had been fed one or more number of times before hospitalization and it did not influence the mortality or morbidity. Polyhydramnios was seen in 12% of our patients. Fifty percent of patients in group A had anastomotic tension as compared to 40% of patients in group B and leak rate was significantly higher in group B if there was tension on the anastomosis (30% vs 75%). Lower pouch mobilization was done in 40% of group A patients and 35% of group B patients. However, there was no significant difference in leak rate in group A or B patients undergoing lower pouch mobilization. Post operative ventilation was required in 15 patients in group A and 14 patients in group B. Increased duration of ventilation was associated with increased mortality and higher leak rates. Anastomotic leak was 20% in group A and 30% in group B. Mean hospital stay was comparable in both the groups. Seventy five percent of group A patients went home as compared to 80% of patients in group B.

Background Single cell RNA sequencing provides unprecedented opportunity to simultaneously explore the transcriptomic and immune receptor diversity of T and B cells. However, there are limited tools available that simultaneously analyse large multi-omics datasets integrated with metadata such as patient and clinical information.Results We developed VDJView, which permits the simultaneous or independent analysis and visualisation of gene expression, immune receptors, and clinical metadata of both T and B cells. This tool is implemented as an easy-to-use R shiny web-application, which integrates numerous gene expression and TCR analysis tools, and accepts data from plate-based sorted or high-throughput single cell platforms. We utilised VDJView to analyse several 10X scRNA-seq datasets, including a recent dataset of 150,000 CD8+ T cells with available gene expression, TCR sequences, quantification of 15 surface proteins, and 44 antigen specificities (across viruses, cancer, and self-antigens). We performed quality control, filtering of tetramer non-specific cells, clustering, random sampling and hypothesis testing to discover antigen specific gene signatures which were associated with immune cell differentiation states and clonal expansion across the pathogen specific T cells. We also analysed 563 single cells (plate-based sorted) obtained from 11 subjects, revealing clonally expanded T and B cells across primary cancer tissues and metastatic lymph-node. These immune cells clustered with distinct gene signatures according to the breast cancer molecular subtype. VDJView has been tested in lab meetings and peer-to-peer discussions, showing effective data generation and discussion without the need to consult bioinformaticians.Conclusions VDJView enables researchers without profound bioinformatics skills to analyse immune scRNA-seq data, integrating and visualising this with clonality and metadata profiles, thus accelerating the process of hypothesis testing, data interpretation and discovery of cellular heterogeneity. VDJView is freely available at https://bitbucket.org/kirbyvisp/vdjview .

Background: Single cell RNA sequencing provides unprecedented opportunity to simultaneously explore the transcriptomic and immune receptor diversity of T and B cells. However, there are limited tools available that simultaneously analyse large multi-omics datasets integrated with metadata such as patient and clinical information. Results: We developed VDJView, which permits the simultaneous or independent analysis and visualisation of gene expression, immune receptors, and clinical metadata of both T and B cells. This tool is implemented as an easy-to-use R shiny web-application, which integrates numerous gene expression and TCR analysis tools, and accepts data from plate-based sorted or high-throughput single cell platforms. We utilised VDJView to analyse several 10X scRNA-seq datasets, including a recent dataset of 150,000 CD8+ T cells with available gene expression, TCR sequences, quantification of 15 surface proteins, and 44 antigen specificities (across viruses, cancer, and self-antigens). We performed quality control, filtering of tetramer non-specific cells, clustering, random sampling and hypothesis testing to discover antigen specific gene signatures which were associated with immune cell differentiation states and clonal expansion across the pathogen specific T cells. We also analysed 563 single cells (plate-based sorted) obtained from 11 subjects, revealing clonally expanded T and B cells across primary cancer tissues and metastatic lymph-node. These immune cells clustered with distinct gene signatures according to the breast cancer molecular subtype. VDJView has been tested in lab meetings and peer-to-peer discussions, showing effective data generation and discussion without the need to consult bioinformaticians. Conclusions: VDJView enables researchers without profound bioinformatics skills to analyse immune scRNA-seq data, integrating and visualising this with clonality and metadata profiles, thus accelerating the process of hypothesis testing, data interpretation and discovery of cellular heterogeneity. VDJView is freely available at https://bitbucket.org/kirbyvisp/vdjview. Footnotes * The revised paper solely documents VDJView, detailing added features, and presents analysis on a 10X dataset of over 150,000 CD8+ T cells with TotalSeq and dextramer staining. * https://bitbucket.org/kirbyvisp/vdjview