Biology subjects

Yadav, A.

Publications and source records attributed to Yadav, A..

79 records · Page 5Linked to original sources

bioRxiv2020

A Sequence-Pair-Classification-Based Method For Detecting And Correcting Under-Clustered Gene Families

Gene families are groups of genes that have descended from a common ancestral gene present in the species under study. Current, widely used gene family building algorithms can produce family clusters that may be fragmented or missing true family sequences (under-clustering). Here we present a classification method based on sequence pairs that, first, inspects given families for under-clustering and then predicts the missing sequences for the families using family-specific alignment score cutoffs. We have tested this method on a set of curated, gold-standard ("true") families from the Yeast Gene Order Browser (YGOB) database, including 20 yeast species, as well as a test set of intentionally under-clustered ("deficient") families derived from the YGOB families. For 83% of the modified yeast families, our pair-classification method was able to reliably detect under-clustering in "deficient" families that were missing 20% of sequences relative to the full/" true" families. We also attempted to predict back the missing sequences using the family-specific alignment score cutoffs obtained during the detection phase. In the case of "pure" under-clustered families (under-clustered families with no "wrong"/unrelated sequences), for 78% of families the prediction precision and recall was [≥]0.75, with mean precision = 0.928 and mean recall = 0.859. For "impure" under-clustered families, (under-clustered families containing closest sequences from outside the family, in addition to missing true family sequences), the prediction precision and recall was [≥]0.75 for 63% of families with mean precision = 0.790 and mean recall = 0.869. To check if our method can detect and correct incomplete families obtained using existing family building methods, we attempted to correct 374 under-clustered yeast families produced using the OrthoFinder tool. We were able to predict missing sequences for at least 19 yeast families with mean precision of 0.9 and mean recall of 0.65. We also analyzed 14,663 legume families built using the OrthoFinder program, with 14 legume species. We were able to identify 1,665 OrthoFinder families that were missing one or more sequences - sequences which were previously un-clustered or clustered into unusually small families. Further, using a simple merging strategy, we were able to merge 2,216 small families into 933 under-clustered families using the predicted missing sequences. Out of the 933 merged families, we could confirm correct mergings in at least 534 families using the maximum-likelihood phylogenies of the merged families. We also provide recommendations on different types of family-specific alignment score cutoffs that can be used for predicting the missing sequences based on the "purity" of under-clustered families and the chosen precision and recall for prediction. Finally, we provide the containerized version of the pair-classification method that can be applied on any given set of gene families.

bioinformatics↗

bioRxiv2019

DNA replication protein Cdc45 directly interacts with PCNA via its PIP box in Leishmania donovani and the Cdc45 PIP box is essential for cell survival

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, interacting with each other directly in vitro also. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival in this organism also. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins have been identified to associate with PCNA in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it is not confined to trypanosomatids.\n\nAuthor SummaryLeishmaniases are manifested in three forms: cutaneous, sub-cutaneous and visceral. The prevalent form in the Indian subcontinent is visceral Leishmaniasis (VL), which is fatal if not treated on time. While there are drugs for treatment, the hunt for additional drugs continues due to emerging drug resistance patterns. The parasite is transmitted by the bite of the sandfly, whereupon it establishes itself within cells of the host immune system (macrophages) and reproduces by binary fission. The replication of its genome is essential for parasite survival. Eukaryotic DNA replication is generally conserved across species. This study targets Cdc45, a protein that helps unwind the DNA double helix to enable copying of the two strands into two daughter strands. The new chains of DNA are synthesized by DNA polymerases, and a trimeric protein, proliferating cell nuclear antigen (PCNA), helps clamp the polymerases onto the template. In this study we find Cdc45 to interact with PCNA, and have identified the motif in Cdc45 via which it does so. Our results suggest this interaction is seen in some other eukaryotes as well. Based on the results of our experiments we propose that Cdc45 may help moor PCNA-polymerase complexes to template DNA.

molecular biology↗

bioRxiv2019

An alanine permease mutation or exposure to D-cycloserine re-sensitizes MRSA to beta-lactam antibiotics

Prolonging the clinical effectiveness of {beta}-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted D-cycloserine (DCS) transporter gene cycA re-sensitized MRSA to {beta}-lactam antibiotics. The cycA mutation also resulted in hyper-susceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and D-alanine ligase required for D-alanine incorporation into cell wall peptidoglycan (PG). Alanine transport was impaired in the cycA mutant and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal D-ala-D-ala and reduced PG crosslinking, prompting us to investigate synergism between {beta}-lactams and DCS. DCS re-sensitised MRSA to {beta}-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteraemia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to {beta}-lactam antibiotics.

microbiology↗

bioRxiv2019

A reference map of the human protein interactome

Global insights into cellular organization and function require comprehensive understanding of interactome networks. Similar to how a reference genome sequence revolutionized human genetics, a reference map of the human interactome network is critical to fully understand genotype-phenotype relationships. Here we present the first human \"all-by-all\" binary reference interactome map, or \"HuRI\". With ~53,000 high-quality protein-protein interactions (PPIs), HuRI is approximately four times larger than the information curated from small-scale studies available in the literature. Integrating HuRI with genome, transcriptome and proteome data enables the study of cellular function within essentially any physiological or pathological cellular context. We demonstrate the use of HuRI in identifying specific subcellular roles of PPIs and protein function modulation via splicing during brain development. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms underlying tissue-specific phenotypes of Mendelian diseases. HuRI thus represents an unprecedented, systematic reference linking genomic variation to phenotypic outcomes.

systems biology↗

bioRxiv2019

Whole exome sequencing and characterization of coding variation in 49,960 individuals in the UK Biobank

The UK Biobank is a prospective study of 502,543 individuals, combining extensive phenotypic and genotypic data with streamlined access for researchers around the world. Here we describe the first tranche of large-scale exome sequence data for 49,960 study participants, revealing approximately 4 million coding variants (of which ~98.4% have frequency < 1%). The data includes 231,631 predicted loss of function variants, a >10-fold increase compared to imputed sequence for the same participants. Nearly all genes (>97%) had [≥]1 predicted loss of function carrier, and most genes (>69%) had [≥]10 loss of function carriers. We illustrate the power of characterizing loss of function variation in this large population through association analyses across 1,741 phenotypes. In addition to replicating a range of established associations, we discover novel loss of function variants with large effects on disease traits, including PIEZO1 on varicose veins, COL6A1 on corneal resistance, MEPE on bone density, and IQGAP2 and GMPR on blood cell traits. We further demonstrate the value of exome sequencing by surveying the prevalence of pathogenic variants of clinical significance in this population, finding that 2% of the population has a medically actionable variant. Additionally, we leverage the phenotypic data to characterize the relationship between rare BRCA1 and BRCA2 pathogenic variants and cancer risk. Exomes from the first 49,960 participants are now made accessible to the scientific community and highlight the promise offered by genomic sequencing in large-scale population-based studies.

genomics↗

bioRxiv2019

Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

The Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) overexpression represents ~10-25% of the prostate cancer (PCa) cases associated with shorter recurrence-free survival and poor prognosis. Nonetheless, androgen-deprivation therapy (ADT) remains the mainstay treatment for locally advanced and metastatic PCa patients. However, majority of these individuals eventually progress to castration-resistant stage, and a subset of these patients develop ADT-induced neuroendocrine PCa. Despite adverse effects of ADT, possible role of androgen signaling in SPINK1-mediated prostate oncogenesis remains unexplored. Here, we show that androgen receptor (AR) and its corepressor, the RE1-silencing transcription factor (REST), occupy SPINK1 promoter and functions as a direct transcriptional repressor of SPINK1, thus blocking AR signaling via ADT relieves its repression, leading to SPINK1 upregulation. In agreement, an inverse association between SPINK1 levels and AR expression was observed across multiple PCa cohorts, and in neuroendocrine differentiated cells. While, lineage reprogramming factor SOX2 in turn binds to SPINK1 promoter leading to its transactivation in androgen-deprived conditions with concomitant increase in neuroendocrine markers. Additionally, we also confirm the role of SPINK1 in epithelial-mesenchymal transition, drug resistance, stemness and cellular plasticity. Moreover, we show that Casein Kinase 1 inhibitor stabilizes the REST levels, which in cooperation with AR, conjures transcriptional repression of SPINK1 expression, and impedes SPINK1-mediated oncogenesis. Collectively, our findings provide a plausible explanation to the paradoxical clinical outcomes of ADT, possibly due to increased SPINK1 levels. This study highlights the need to take a well-informed decision prior to ADT and develop alternative therapeutic strategies for castrate-resistant PCa patients.

cancer biology↗

bioRxiv2018

Polycomb Group protein EZH2-mediated transcriptional repression of microRNA-338/-421 drives SPINK1-positive prostate cancer

PurposeSerine Peptidase Inhibitor, Kazal type-1 (SPINK1) overexpression defines the second most recurrent and aggressive prostate cancer (PCa) subtype. However, the underlying molecular mechanism and pathobiology of SPINK1 in PCa remains largely unknown.\n\nExperimental DesignMicroRNA-prediction tools were employed to examine the SPINK1-3UTR for miRNAs binding. Luciferase reporter assays were performed to confirm the SPINK1-3UTR binding of shortlisted miR-338-5p/miR-421. Further, miR-338-5p/-421 overexpressing cancer cells (SPINK1-positive) were evaluated for oncogenic properties using cell-based functional assays and mice xenograft model. Global gene expression profiling was performed to unravel the biological pathways altered by miR-338-5p/-421. Immunohistochemistry and RNA in-situ hybridization was carried-out on PCa patients tissue microarray for SPINK1 and EZH2 expression respectively. Chromatin immunoprecipitation assay was performed to examine EZH2 occupancy on the miR-338-5p/-421 regulatory regions. Bisulfite sequencing and methylated DNA-immunoprecipitation was performed on PCa cell lines and patients specimens.\n\nResultsWe established a critical role of miRNA-338-5p/-421 in post-transcriptional regulation of SPINK1. Ectopic expression of miRNA-338-5p/-421 in SPINK1-positive PCa cells abrogate oncogenic properties including cell-cycle progression, stemness and drug resistance, and show reduced tumor burden and distant metastases in mice model. Importantly, we show SPINK1-positive PCa patients exhibit increased EZH2 expression, suggesting its role in miRNA-338-5p/-421 epigenetic silencing. Furthermore, presence of CpG dinucleotide DNA methylation marks on the regulatory regions of miR-338-5p/-421 in SPINK1-positive PCa cells and patients specimens confirms epigenetic silencing.\n\nConclusionOur findings revealed that miRNA-338-5p/-421 are epigenetically silenced in SPINK1-positive PCa, while restoring the expression of these miRNAs using epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis.\n\nTRANSLATIONAL IMPACTWe establish a regulatory model involving the functional interplay between SPINK1, miRNA-338-5p/miRNA-421 and EZH2, thereby, revealing hitherto unknown mechanism of SPINK1 up-regulation in SPINK1-positive subtype. Our findings provide a strong rationale for the development of potential therapeutic strategies for SPINK1-positive malignancies. We demonstrate that restoring miRNA-338-5p/miRNA-421 expression using epigenetic drugs including DNMTs inhibitors in combination with HDACs or HKMTs inhibitors or miRNA synthetic mimics in SPINK1-positive prostate cancer abrogate SPINK1-mediated oncogenicity. The major findings of this study will not only advance the prostate cancer field, but will also be valuable for treatment and disease management of other SPINK1-positive malignancies.

cancer biology↗