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Biology subjects

Singh, K. P.

Publications and source records attributed to Singh, K. P..

13 recordsLinked to original sources

The ZNF512B-NuRD complex co-ordinates a neural-specific gene expression program

The nucleosome remodelling and deacetylase complex (NuRD) plays a key role in chromatin regulation and a wide range of biological processes including development, haemopoiesis, immunity and neurogenesis. Its interaction with tissue-enriched and sequence-specific transcription factors (TFs) leads to distinct functional outputs in the given tissue by targeting a specific set of genes. However, how NuRD dynamically and specifically regulates gene expression in a tissue-specific manner is poorly understood. Here, we refine an N-terminal specific NuRD interaction motif which enables direct engagement with many transcriptional regulatory proteins. Using a series of structural modelling and biochemical techniques, we show that ZNF512B, a poorly characterised neuronal-expressed zinc finger protein, directly binds to the RBBP4 subunit of the NuRD complex. Subsequent knockdown of ZNF512B results in the downregulation of several neural-related molecular pathways suggesting that ZNF512B may play a regulatory role during neurogenesis. We also show that in NTERA-2 neural cells, the expression of ZNF512B is necessary for cell growth and survival, and is markedly enhanced during neural progenitor cell (NPC) differentiation. In summary, our data suggest that ZNF512B might regulate neural-specific transcriptional programs via engagement with the NuRD complex.

molecular biology↗

Unleashing the Immune Arsenal: Development of Broad-spectrum Multiepitope Bluetongue Vaccine Targeting Conserved T Cell Epitopes of Structural Proteins

Bluetongue (BT) is a severe arboviral disease affecting sheep, cows, and other wild ruminants, caused by the Bluetongue virus (BTV). The virus has evolved into over 32 serotypes, rendering existing vaccines less effective. While the structural proteins of this virus represent promising targets for vaccine development, they unfortunately exhibit high amino acid polymorphism and are laden with numerous inhibitory epitopes. Structural proteins such as VP1 and VP7 are highly conserved and may contain epitopes capable of triggering cross-reactive cell-mediated immunity (CMI). In this study, we identified highly conserved MHC-I and -II-restricted T cell epitopes within VP1, VP5, and VP7 BTV proteins and developed an effective in silico-immuno-informatics-based broad-spectrum BT multiepitope vaccine for bovine and laboratory mouse systems. The conserved epitopes utilized in the vaccines are highly antigenic, non-allergenic, non-toxic, and capable of inducing IFN-{gamma} (only CD4+ T cell epitopes). Both mouse and bovine vaccines were tethered with Toll-like receptor (TLR)-4-agonist adjuvants, beta-defensin 2, and the 50s ribosomal unit to stimulate innate immunity for CMI development. Protein-protein docking analysis revealed strong binding affinities, while extensive 100-nanosecond molecular dynamics simulations indicated stable complexes between the vaccine structures and TLR4. Vaccination simulation studies demonstrated their ability to trigger proinflammatory responses. Therefore, these novel vaccine designs necessitate further exploration through wet lab experiments to evaluate their immunogenicity, safety, and effectiveness for practical deployment in livestock.

immunology↗

The integration of network biology and pharmacophore modeling suggests repurposing Clindamycin as an inhibitor of pyroptosis via Caspase-1 blockage in tumor-associated macrophages

BackgroundUveal melanoma (UM) is a highly malignant intraocular tumor with a poor prognosis and response to therapy, including immune checkpoint inhibitors (ICIs), after the onset of liver metastasis. The metastatic microenvironment contains high levels of tumor-associated macrophages (TAMs) that correlate positively with a worse patient prognosis. We hypothesized that one could increase the efficacy of ICIs in UM metastases by immunomodulating UM-associated macrophages. MethodsTo identify potential targets for the immunomodulation, we created a network-based representation of the biology of TAMs and employed (bulk and single-cell) differential gene expression analysis to obtain a regulatory core of UM macrophages-associated genes. We utilized selected targets for pharmacophore-based virtual screening against a library of FDA-approved chemical compounds, followed by refined flexible docking analysis. Finally, we ranked the interactions and selected one novel drug-target combination for in vitro validation. ResultsBased on the generated TAM-specific interaction network (3863 nodes, 9073 edges), we derived a UM macrophages-associated regulatory core (74 nodes, 286 edges). From the regulatory core genes, we selected eight potential targets for pharmacophore-based virtual screening (YBX1, GSTP1, NLRP3, ISG15, MYC, PTGS2, NFKB1, CASP1). Of 266 drug-target interactions screened, we identified the interaction between the antibiotic Clindamycin and Caspase-1 as a priority for experimental validation. Our in vitro validation experiments showed that Clindamycin specifically interferes with activated Caspase-1 and inhibits the secretion of IL-1{beta}, IL-18, and lactate dehydrogenase (LDH) in macrophages after stimulation. Our results suggest that repurposed Clindamycin could reduce pyroptosis in TAMs, a pro-inflammatory form of programmed immune cell death favouring tumor progression. ConclusionWe were able to predict a novel Clindamycin-Caspase-1 interaction that effectively blocks Caspase-1-mediated inflammasome activity and pyroptosis in vitro in macrophages. This interaction is a promising clinical immunomodulator of the tumor microenvironment for improving ICI responsivenss. This work demonstrates the power of combining network-based transcriptomic analysis with pharmacophore-guided screening for de novo drug-target repurposing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/576201v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@310a9aorg.highwire.dtl.DTLVardef@1af0dcaorg.highwire.dtl.DTLVardef@1b272aborg.highwire.dtl.DTLVardef@86572b_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

Immuno-informatics Study Identifies Conserved T Cell Epitopes in Non-structural Proteins of Bluetongue Virus Serotypes: Formulation of Computationally Optimized Next-Generation Broad-spectrum Multiepitope Vaccine

Bluetongue (BT) is a significant arboviral disease affecting sheep, cattle, goats, and wild ruminants, posing serious economic challenges to livestock industry. Control efforts have been hampered by the existence of over 32 distinct BT virus (BTV) serotypes and the absence of broad-spectrum vaccines. Some key non-structural proteins of BTV, including NS1, NS2, and NS3, exhibit notable amino acid sequence conservation. Our findings reveal that mouse MHC class I (MHC-I) CD8+ T cell epitopes are highly conserved in NS1 and NS3, while MHC-II epitopes are prevalent in all the three non-structural NS 1-3 proteins. Similarly, both class I and II Bovine Leukocyte antigen-restricted CD8+ and CD4+ T cell epitopes are conserved within NS1, NS2, and NS3 proteins. To construct in silico broad-spectrum vaccine, we subsequently screened these conserved epitopes based on antigenicity, allergenicity, toxicity, and solubility. Modeling and Refinement of the 3D structure models of vaccine constructs were achieved using protein modeling web servers. Our analysis revealed promising epitopes that exhibit strong binding affinities with low energies against two TLR receptors (TLR3 and TLR4). To ensure atomic-level stability, we evaluated the docking complexes of epitopes and receptors through all-atom molecular dynamics simulations (MDS). Encouragingly, our 100 nanoseconds MDS showed stable complexes with minimal RMSF values. Our study offers valuable insights into these conserved T cell epitopes as promising candidates for a broad-spectrum BT vaccine. We therefore encourage for their evaluation in animal models and natural hosts to assess their immunogenicity, safety, and efficacy for field use in the livestock.

bioinformatics↗

Regenerative Signatures in Bronchioalveolar Lavage of Acute Respiratory Distress Syndrome

BackgroundIn patients with severe acute respiratory distress syndrome (ARDS) associated with sepsis, lung recovery is considerably delayed, and mortality is much high. More insight into the process of lung regeneration in ARDS patients is needed. Exosomes are important cargos for intercellular communication by serving as autocrine and/or paracrine. Cutting-edge exomics (exosomal proteomics) makes it possible to study the mechanisms of re-alveolarization in ARDS lungs. AimsThis study aimed to identify potential regenerative niches by characterizing differentially expressed proteins in the exosomes of bronchioalveolar lavage (BAL) in ARDS patients. MethodsWe purified exosomes from BAL samples collected from ARDS patients by NIH-supported ALTA and SPIROMICS trials. The abundance of exosomal proteins/peptides was quantified using liquid chromatography-mass spectrometry (LC-MS). Differentially expressed exosomal proteins between healthy controls and ARDS patients were profiled for functional annotations, cell origins, signaling pathways, networks, and clinical correlations. ResultsOur results show that more exosomal proteins were identified in the lungs of late-stage ARDS patients. Immune cells and lung epithelial stem cells were major contributors to BAL exosomes in addition to those from other organs. We enriched a wide range of functions, stem cell signals, growth factors, and immune niches in both mild and severe patients. The differentially expressed proteins that we identified were associated with key clinical variables. The severity-associated differences in protein-protein interaction, RNA crosstalk, and epigenetic network were observed between mild and severe groups. Moreover, alveolar type 2 epithelial cells could serve as both exosome donors and recipients via autocrine and paracrine mechanisms. ConclusionsThis study identifies novel exosomal proteins associated with diverse functions, signaling pathways, and cell origins in ARDS lavage samples. These differentiated proteins may serve as regenerative niches for re-alveolarization in injured lungs.

pathology↗

Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India

Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed using an Indian field strain (LSDV/India/2019/Ranchi). The virus was attenuated by continuous passage (P=50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n=10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune response and exhibited complete protection upon virulent LSDV challenge. We observed a minimum Neethling response (0.018% animals; 5 out of 26940 animals) of the vaccine in field trials among 26940 animals. There was no significant reduction in the milk yield in lactating animals (n=10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n=2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.

microbiology↗

Whole-Genome Sequence of African Swine Fever Virus isolate from India provides insights into diversity and evolution

African swine fever (ASF) was first reported in 1921, and since then has posed a major threat to the world pig industry and still remains a major challenge as there is no vaccine or therapy available. In May 2020, the first incidence of ASF was recorded in India, followed by a number of outbreaks in the north eastern part of India. In this study, we report the first whole genome of an Indian isolate of ASF virus (ASF/IND/20/CAD/543) using next generation sequencing and compared with the other ASFV complete genome. On phylogenetic analysis, the virus was assigned to genotype II on the basis of p72 genotyping. However, the whole genome based phylogeny distinguished it from other genotype II isolates of clade 1.1.1. This study adds to our understanding of ASFVs genetic diversity and molecular evolution.

microbiology↗

Systemic infection of SARS-CoV-2 in free ranging Leopard (Panthera pardus fusca) in India

We report patho-morphological and virological characterization of SARS-CoV-2 in naturally infected, free ranging Indian Leopard (Panthera pardus fusca). Whole genome sequence analysis confirmed infection of Delta variant of SARS-CoV-2, possibly spill over from humans, but the case was detected when infection level had dropped significantly in human population. This report underlines the need for intensive screening of wild animals for keeping track of the virus evolution and development of carrier status of SARS-CoV-2 among wildlife species.

microbiology↗

Heterogeneity versus the COVID-19 Pandemic

In this paper, heterogeneity is formally defined, and its properties are explored. We define and distinguish observable versus non-observable heterogeneity. It is proposed that heterogeneity among the vulnerable is a significant factor in the contagion impact of COVID-19, as demonstrated with incidence rates on a Diamond Princess Cruise ship in February 2020. Given the nature of the disease, its heterogeneity and human social norms, pre-voyage and post-voyage quick testing procedures may become the new standard for cruise ship passengers and crew. The technological advances in testing available today would facilitate more humanistic treatment as compared to more archaic quarantine and isolation practices for all onboard ship. With quick testing, identification of those infected and thus not allowed to embark on a cruise or quarantining those disembarking and other mitigation strategies, the popular cruise adventure could be available safely again. Whatever the procedures implemented, the methodological purpose of this study should add valuable insight in the modeling of disease and specifically, the COVID-19 virus.

scientific communication and education↗

Predicting COVID-19 cases with unknown homogeneous or heterogeneous resistance to infectivity

This article constructs a restricted infection rate inverse binomial-based approach to predict COVID-19 cases after a family gathering. The traditional inverse binomial (IB) model is unqualified to match the reality of COVID-19, because the data contradicts the models requirement that variance should be greater than expected value. A refined version of the IB model is a necessity to predict COVID-19 cases after family gatherings. Our refined version of an IB model is more appropriate and versatile, as it accommodates all potential data scenarios: equal, lesser, or greater variance than expected value. Application of the approach is based on a restricted infectivity rate and methodology on Fan et al.s COVID-19 data, which exhibits two clusters of infectivity. Cluster 1 has a smaller number of primary cases and exhibits larger variance than the expected cases with a negative correlation of 28%, implying that the number of secondary cases is lesser when the number of primary cases increases and vice versa. The traditional inverse binomial (IB) model is appropriate for Cluster 1. The probability of contracting COVID-19 is estimated to be 0.13 among the primary, but is 0.75 among the secondary in Cluster 1, with a wider gap. Conversely, Cluster 2, exhibits smaller variance than the expected cases with a correlation of 79%, implying the number of primary and secondary cases increase or decrease together. Cluster 2 disqualifies the traditional IB model and demands its refined version. Probability of contracting COVID-19 is estimated to be 0.74 among the primary, but is 0.72 among the secondary in Cluster 2, with a narrower gap. The models ability to estimate the communitys health system memory for future policies to be developed is an asset of this approach. The current hazard level to be infected with COVID-19 among the primary and secondary groups are estimable and interpretable. Author SummaryCurrent statistical models are not able to accurately predict disease infection spread in the COVID-19 pandemic. We have applied a widely-used inverse binomial method to predict rates of infection after small gatherings, going from primary (original) cases to secondary (later) cases after family gatherings or social events, using the data from the Wuhan and Gansu provinces in China, where the virus first spread. The advantages of the proposed approach include that the models ability to estimate the communitys health system memory for future policies to be developed, as such policies might reduce COVIDs spread if not its control. In our approach, as demonstrated, the current hazard level of becoming infected with COVID-19 and the odds of contracting COVID-19 among the primary in comparison to the secondary groups are estimable and interpretable. We hope the proposed approach will be used in future epidemics.

scientific communication and education↗

Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

Visceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen-/-). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen-/- against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen-/- did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen-/--immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen-/- immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-{gamma} and TNF-, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen-/- which showed equal protection as laboratory-grade LmCen-/- parasites in hamsters. Importantly, GLP-grade LmCen-/- parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

immunology↗

A Web-resource for Nutrient Use Efficiency related Genes, QTLs, and microRNA in important cereals and model plants

Cereals are the key contributors to global food security. Genes involved in uptake (transport), assimilation and utilization of macro- and micro-nutrients are responsible for their content in grain and straw. Although many cereal genomic databases are available, currently there is no cohesive web-resource of manually curated nutrient use efficiency (NtUE) related genes and QTLs, etc. In this study, we present a web-resource containing information on NtUE related genes/QTLs and the corresponding available microRNAs for some of these genes in four major cereal crops [wheat (Triticum aestivum), rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare)], two alien species (Triticum urartu and Aegilops tauschii) related to wheat, and two model species including Brachypodium distachyon and Arabidopsis thaliana. Gene annotations integrated in the current web-resource were collected from the existing databases and the available literature. The primary goal of developing this web-resource is to provide descriptions of the NtUE related genes and their functional annotation. MicroRNA targeting some of the NtUE related genes and the quantitative trait loci (QTLs) for NtUE related traits are also included. The available information in the web-resource should help the users to readily search the desired information.\n\nWeb-resource URLhttp://bioclues.org/NtUE/

bioinformatics↗

Genome-Wide Mining, Characterization and Development of miRNA-SSRs in Arabidopsis thaliana

Simple Sequence Repeats (SSRs), also known as microsatellites are short tandem repeats of DNA sequences that are 1-6 bp long. In plants, SSRs serve as a source of important class of molecular markers because of their hypervariabile and co-dominant nature, making them useful both for the genetic studies and marker-assisted breeding. The SSRs are widespread throughout the genome of an organism, so that a large number of SSR datasets are available, most of them from either protein-coding regions or untranslated regions. It is only recently, that their occurrence within microRNAs (miRNA) genes has received attention. As is widely known, miRNA themselves are a class of non-coding RNAs (ncRNAs) with varying length of 19-22 nucleotides (nts), which play an important role in regulating gene expression in plants under different biotic and abiotic stresses. In this communication, we describe the results of a study, where miRNA-SSRs in full length pre-miRNA sequences of Arabidopsis thaliana were mined. The sequences were retrieved by annotations available at EnsemblPlants using BatchPrimer3 server with miRNA-SSR flanking primers found to be well distributed. Our analysis shows that miRNA-SSRs are relatively rare in protein-coding regions but abundant in non-coding region. All the observed 147 di-, tri-, tetra-, penta- and hexanucleotide SSRs were located in non-coding regions of all the 5 chromosomes of A. thaliana. While we confirm that miRNA-SSRs were commonly spread across the full length pre-miRNAs, we envisage that such studies would allow us to identify newly discovered markers for breeding studies.

bioinformatics↗