Biology subjects

Joshi, M.

Publications and source records attributed to Joshi, M..

33 records · Page 2Linked to original sources

bioRxiv2022

RNASeq as a tool to understand dysregulation of potential biomarkers in HNSC

With its rising fatality rates, oral cancer is one of the most concerning public health issues. To reduce disease-related mortality and morbidity, advancements in screening and detection are critical. Finding specific biomarkers is one of the most successful approaches for screening, diagnosing, and staging this dreadful disease. In this study differentially expressed genes associated with oral cancer were analyzed using RNASeq to find the potential biomarkers. Functional enrichment of upregulated genes found that 253 genes were present in the plasma membrane. Three clusters were formed using KMean Clustering from the PPI networks, and highly connected hub genes were identified from each cluster. Eventually, expression and survival analyses of hub genes were performed using The Cancer Genome Atlas (TCGA) database targeting Head and Neck Squamous Cell Carcinoma. Among those genes, expression levels of eight genes SLC2A1, ITGA6, LAMC2, COL1A2, COL1A1, TNC, THY1, and CD276 have significantly changed in Head and Neck Squamous cell carcinoma. There are reports that suggest these genes were significantly dysregulated in Oral Squamous cell carcinoma and can be explored further as potential biomarkers for margin clearance.

cancer biology↗

bioRxiv2022

Comprehensive analysis using DNA metabarcoding, PCR, and HPLC unveils the adulteration in Brahmi herbal products

BackgroundThe herbal products market is expanding and creating a bottleneck for raw materials. Hence, economically motivated adulteration has a high prevalence. DNA barcoding and species-specific PCR assays are now revolutionising the molecular identification of herbal products and are included in a number of pharmacopoeias for the identification of raw materials. High-throughput sequencing with barcoding advances toward metabarcoding, which enables the identification of unintentionally or intentionally unlabelled plant material present in herbal products. Brahmi is one of the most commercially significant and nootropic botanicals, with great controversy over the terms "Brahmi" being used to describe both Bacopa monneri (BM) and Centella asiatica (CA) species. PurposeThis study evaluates DNA-based methods for Brahmi herbal products with the traditional HPLC-based analytical approach in order to assess their effectiveness. MethodsWe employed a species-specific PCR assay, DNA metabarcoding using rbcL minibarcode, and HPLC to detect the presence of the Brahmi (either BM or CA) in eighteen market samples. All the methods have been validated using in-house blended formulations. ResultsComprehensive analysis of all three methods revealed the presence of 22.2%, 55.6%, and 50.0% of Brahmi by PCR assay, DNA metabarcoding, and HPLC, respectively, in Brahmi market formulations, whereas blended formulations only exhibited targeted plant species with all three methods. ConclusionSpecies-specific PCR can be used as a cost-effective and rapid method to detect the presence of the Brahmi, while in high-throughput methods, DNA metabarcoding can be used to detect the presence of widespread adulterated botanicals, and further, bioactive compounds could be detected by HPLC. These results emphasise the need for quality control of the marketed Brahmi herbal products as well as the implementation of all methodologies in accordance with fit for purpose.

molecular biology↗

bioRxiv2022

Dual mode of PGRP-LE-dependent NF-κB pathway activation in bacteria infected guts

Interactions between prokaryotes and eukaryotes require a dialogue between MAMPs and PRRs. In Drosophila, bacterial peptidoglycan is detected by PGRP receptors. While the components of the signaling cascades activated upon PGN/PGRP interactions are well characterized, little is known about the subcellular events that translate these early signaling steps into target gene transcription. Using a Drosophila enteric infection model, we show that gut-associated bacteria can induce the formation of intracellular PGRP-LE aggregates which colocalized with the early endosome marker Rab5. Combining microscopic and RNA-seq analysis, we demonstrate that RNAi inactivation of the endocytosis pathway in the Drosophila gut affects the expression of essential regulators of the NF-{kappa}B response leading not only to a disruption of the immune response locally in the gut but also at the systemic level. This work sheds new light on the involvement of the endocytosis pathway in the control of the gut response to intestinal bacterial infection Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=181 HEIGHT=200 SRC="FIGDIR/small/490610v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1ed549eorg.highwire.dtl.DTLVardef@4e5315org.highwire.dtl.DTLVardef@30b1eaorg.highwire.dtl.DTLVardef@f08401_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Delimiting Continuity: Comparison of Target Enrichment and ddRAD for Delineating Admixing Parapatric Melitaea Butterflies

Parapatrically distributed taxa pose a challenge for species delimitation due to the presence of gene flow and inherent arbitrariness of exactly defining the species boundaries in such systems. We tackled the problem of species delimitation in a parapatric species pair of Melitaea butterflies using two popular genomic methods - double digest restriction-site associated DNA sequencing (ddRAD) and target enrichment. The former is mainly applied at shallow phylogenetic scales and the latter at both deep and shallow scales. Although both of these methods have been adequately utilised for species delimitation purposes, there are only a handful of studies that have compared these two genomic approaches in the same study system. We applied phylogenetic, population genetic and species delimitation methods and compared the results obtained from the two approaches. Using a recently developed target enrichment probe kit, we were able to capture 1,743 loci with a low amount of missing data and compared these with already available ddRAD data from a previous study on the same set of specimens. We recovered consistent phylogenetic relationships across the datasets, both demonstrating the presence of a genetically distinct Balkan lineage and paraphyly of Melitaea athalia with respect to Melitaea celadussa. The same relationships were also found in a species tree analysis of the target enrichment dataset using ASTRAL. Population genetic STRUCTURE analyses supported the presence of two species when using ddRAD data, but three species when using target enrichment, while the Bayes factor delimitation analysis found both two and three species scenarios equally decisive in both datasets. From the geographic distribution of genomic admixture, we confirm the patterns observed by a previous study that used ddRAD data. As the results obtained from both methods were largely congruent, we discuss some practical considerations and benefits of target enrichment over RAD sequencing. We conclude that the choice of method of genomic data collection does not influence the results of phylogenetic analyses at alpha taxonomic level, given a sufficient number of loci. Finally, we recommend a solution for delineating species in parapatric scenarios by proposing that parapatric taxa be consistently classified as subspecies or complete species, but not both, to promote taxonomic stability.

evolutionary biology↗

bioRxiv2022

De novo transcriptome of Taverniera cuneifolia (Roth) Ali.

Taverniera cuneifolia has been described as a potent substitute of Licorice in India. It has been used as an expectorant, anti-inflammatory, anti-ulcer, wound healing, blood purifier etc. Glycyrrhizin is one of the most useful bioactive sesquiterpenoid present in this plant. The present study aim to carry out transcriptome analysis in root tissue of Taverniera cuneifolia to identify specific functional genes involved in the biosynthesis of secondary metabolites. The root transcriptome sequencing of Taverniera cuneifolia resulted in a total of ~7.29 Gb of raw data and generated 55,991,233 raw reads. The high quality reads were de novo assembled by Trinity assembler followed through CD-HIT resulted into 35,590 "Unigene" transcripts with an average size of 419 bp. The unigenes were analyzed using BLAST2GO resulted in 27,884 (78.35%) transcript with blast hits, 22,510 (63.25%) transcript with mapping and 21,066 (59.19%) transcript with annotation. Functional annotation was carried out using NCBIs non-redundant and Uniprot databases resulted in the identification of 21,066 (59.19%) annotated transcripts and GO assigned to 24751 (69.54%) transcripts. The gene ontology result shows maximum sequences match with Biological Processes (48%), Molecular Function (27%) and Cellular components (23%). A total of 289 metabolic enriched pathways were identified, which included pathways like Sesquiterpenoid and triterpenoid pathway which were involved in synthesis of secondary metabolite Glycyrrhizin biosynthesis. The enzymes, squalene monooxygenase, farnesyl-diphosphate farnesyltransferase, beta amyrin synthase, beta-amyrin 24-hydroxylase, were identified by functional annotation of transcriptome data. There were several other pathways like terpenoid backbone biosynthesis, steroid biosynthesis, Carotenoid biosynthesis, Flavonoids biosynthesis etc. which have been reported first time from this plant. Transcription factors were predicted by comparison with Plant Transcription Factor Database, and 1557 trancripts belonging to 85 trancription factor families were identified. This transcriptome analysis provided an important resource for future genomic studies in Taverniera cuneifolia, therefore representing basis in further investigation of the plant. SignificanceLicorice (Glycyrrhiza glabra roots) is used as traditional Chinese herbal medicines in majority of formulations. Licorice is also used in Industries like food, herbal and cosmetics etc. due to its high demand in the market it is imported from foreign countries and is not available locally of superior quality (Liu et al., 2015). In India, Taverniera cuneifolia has been described as a potent substitute of Licorice, it has been quoted in ancient books like Charak Samhita during the Nigandu period (Kamboj, 2000) and Barda dungar ni Vanaspati ane upyog (Thaker 1910). It has been used as an expectorant, anti-inflammatory, anti-ulcer, wound healing, blood purifier etc. Transcriptomic studies will assist in understanding the basic molecular structure, function and organization of information within the genome of Taverniera cuniefolia. This study will help us to identify the key metabolites their expressions and genes responsible for their production.

genomics↗

bioRxiv2021

Comparative analysis of two NGS platforms and different databases for analysis of AMR genes

The use of antibiotics in human medicine and livestock production has contributed to the widespread occurrence of antimicrobial resistance (AMR). Recognizing the relevance of AMR to human and livestock health, it is important to assess the occurrence of genetic determinants of resistance in medical, veterinary, and public health settings in order to understand risks of transmission and treatment failure. Advances in Next Generation Sequencing (NGS) technologies have had a significant impact on research in microbial genetics and microbiome analyses. Now, strategies for high throughput sequencing from panels of PCR amplicons representing known AMR genes offer opportunities for targeted characterization of complex microbial populations. Aim of the present study was to compare the Illumina MiSeq and Ion Torrent S5 Plus sequencing platforms for use with the Ion AmpliSeq AMR Research Panel in a veterinary/public health setting. All samples were processed in parallel for the two sequencing technologies, subsequently following a common bioinformatics workflow to define the occurrence and abundance of AMR gene sequences. Regardless of sequencing platform, the results were closely comparable with minor differences. The Comprehensive Antibiotic Resistance Database (CARD), QIAGEN Microbial Insight - Antimicrobial Resistance (QMI-AR), Antimicrobial resistance database (AR), and CARD-CLC databases were compared for analysis, with the most genes identified using CARD. Drawing on these results we describe an end-to-end workflow for AMR gene analysis using NGS.

bioinformatics↗

bioRxiv2021

Trisomy 21 increases microtubules and disrupts centriolar satellite localization

Trisomy 21, the cause of Down syndrome, causes a 0.5-fold protein increase of the chromosome 21-resident gene Pericentrin (PCNT) and reduces primary cilia formation and signaling. Here we investigate the mechanisms by which PCNT imbalances disrupt cilia. Using isogenic RPE-1 cells with increased chromosome 21 dosage, we find PCNT protein accumulates around the centrosome as a pericentrosomal cluster of enlarged cytoplasmic puncta that localize along and at MT ends. Cytoplasmic PCNT puncta impact the intracellular MT trafficking network required for primary cilia, as the PCNT puncta sequester cargo peripheral to centrosomes in what we call pericentrosomal crowding. The centriolar satellite proteins, PCM1, CEP131 and CEP290, important for ciliogenesis, accumulate at sites of enlarged PCNT puncta in trisomy 21 cells. Reducing PCNT when chromosome 21 ploidy is elevated is sufficient to decrease PCNT puncta, reestablish a normal density of MTs around the centrosome, restore ciliogenesis to wild type levels and decrease pericentrosomal crowding. A transient reduction in MTs also decreases pericentrosomal crowding and partially rescues ciliogenesis in trisomy 21 cells, indicating that increased PCNT leads to defects in the microtubule network deleterious to normal centriolar satellite distribution. We propose that chromosome 21 aneuploidy disrupts MT-dependent intracellular trafficking required for primary cilia. TOCMcCurdy et al explore why elevated Pericentrin in trisomy 21 negatively impacts primary cilia formation and signaling. They find that elevated Pericentrin produces more pericentrosomal puncta that associates with and increases microtubules. Changes to Pericentrin and microtubules mislocalizes centriolar satellites in a pericentrosomal crowd.

cell biology↗

bioRxiv2021

The M1 aminopeptidase NPEPPS is a novel regulator of cisplatin sensitivity

There is an unmet need to improve efficacy of platinum-based cancer chemotherapy. Using multi-omic assessment of cisplatin-responsive and -resistant human bladder cancer cell lines and whole-genome CRISPR screens, we identified Puromycin-Sensitive Aminopeptidase, NPEPPS, as a novel driver of cisplatin resistance. NPEPPS depletion sensitizes resistant bladder cancer cells to cisplatin in vitro and in vivo. Conversely, overexpression of NPEPPS in sensitive cells increased cisplatin resistance. We show that NPEPPS affects treatment response by regulating intracellular cisplatin concentrations. Patient-derived organoids (PDOs) generated from bladder cancer samples before and after cisplatin-based treatment, and from patients who did not receive cisplatin, were evaluated for sensitivity to cisplatin and they were found to be concordant with clinical response. In PDOs, shRNA depletion or pharmacologic inhibition of NPEPPS led to increased cisplatin sensitivity, while NPEPPS overexpression had the opposite effect. Our data present NPEPPS as a novel and druggable driver of cisplatin resistance by regulating intracellular cisplatin concentrations, along with providing the preclinical data to support clinical trials combining NPEPPS inhibition with cisplatin.

cancer biology↗

bioRxiv2020

Conformational plasticity and dynamic interactions of the N-terminal domain of the chemokine receptor CXCR1

Dynamic interactions between G protein-coupled receptors (GPCRs) and their cognate protein partners at the membrane interface control several cellular signaling pathways. An important example is the association of CXC chemokine receptor 1 (CXCR1) with its cognate chemokine, interleukin-8 (IL8 or CXCL8) that regulates neutrophil-mediated immune responses. Although the N-terminal domain of the receptor is known to confer ligand selectivity, the conformational dynamics of this intrinsically disordered region of CXCR1 in particular, and chemokine receptors in general, remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations that were validated by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo-receptor N-terminal region is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we validated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies. Our results indicate that caution should be exercised when chemical shift perturbation is used as a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces. Author summaryHow cells communicate with the outside environment is intricately controlled and regulated by a large family of receptors on the cell membrane (G protein-coupled receptors or GPCRs) that respond to external signals (termed ligands). Chemokine receptors belong to this GPCR family and regulate immune responses. We analyze here the first step of binding of a representative chemokine receptor (CXCR1) with its natural ligand, interleukin 8 (IL8) by an extensive set of molecular dynamics simulations. Our work complements previous mutational and NMR experiments which lack molecular-level resolution. We show that in the inactive state, one of the extracellular domains of the CXCR1 receptor, namely the N-terminal domain, is highly flexible and like a "shape-shifter" can exist in multiple conformational states. However, when IL8 binds, the N-terminal domain undergoes a conformational freezing, and acquires a C-shaped "claw-like" structure. The complex between the receptor and IL8 is still quite dynamic as this C-shaped N-terminal domain forms an extensive but slippery interface with the ligand. We further validated these results by quantitative comparison with NMR and mutagenesis studies. Our work helps clarify the inherent disorder in N-terminal domains of chemokine receptors and demonstrates how this domain can acquire functionally important conformational states in dynamic protein-protein interfaces.

biophysics↗

bioRxiv2020

In-Silico analysis reveals lower transcription efficiency of C241T variant of SARS-CoV-2 with host replication factors MADP1 and hnRNP-1

Novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has claimed more than 1.5 million lives worldwide and counting. As per the GISAID database, the genomics of SARS-CoV2 is extensively studied with more than 500 genome submissions per day. Out of several hotspot mutations within the SARS-CoV-2 genome, researchers have focused a lot on missense variants but the least work is done on the UTRs. One of the most frequent 5 UTR variants in the SARS-CoV-2 genome is the C241T with a global frequency of more than 0.9. In the present study, the effect of the C241T mutation has been studied with respect to change in RNA structure and its interaction with the host replication factors MADP1 Zinc finger CCHC-type and RNA-binding motif 1 (hnRNP1). The results obtained from molecular docking and molecular dynamics simulation indicated weaker interaction of C241T mutant stem loops with host transcription factor MADP1 indicating reduced replication efficiency. The results are also correlated with increased recovery rates and decreased death rates of global SARS-CoV-2 cases.

bioinformatics↗

bioRxiv2020

HERC1 regulates breast cancer cells migration and invasion

Tumor cell migration and invasion into adjacent tissues is one of the hallmarks of cancer and the first step towards secondary tumors formation, which represents the leading cause of cancer-related deaths. This process is considered an unmet clinical need in the treatment of this disease, particularly in breast cancers characterized by high aggressiveness and metastatic potential. In order to identify and characterize genes with novel functions as regulators of tumor cell migration and invasion, we performed a genetic loss-of-function screen using a shRNA library directed against the Ubiquitin Proteasome System (UPS) in a highly invasive breast cancer derived cell line. Among the candidates, we validated HERC1 as a gene regulating cell migration and invasion. Furthermore, using animal models, our results indicated that HERC1 silencing affects primary tumor growth and lung colonization. Finally, we conducted an in-silico analysis using publicly available protein expression data and observed an inverse correlation between HERC1 expression levels and breast cancer patients overall survival. Altogether, our findings demonstrate that HERC1 might represent a novel therapeutic target for the development or improvement of breast cancer treatment.

cancer biology↗

bioRxiv2020

USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

Tumor cell dissemination in cancer patients is associated with a significant reduction in their survival and quality of life. The ubiquitination pathway plays a fundamental role in the maintenance of protein homeostasis both in normal and stressed conditions and its dysregulation has been associated with malignant transformation and invasive potential of tumor cells, thus highlighting its value as a potential therapeutic target. In order to identify novel molecular targets of tumor cell migration and invasion we performed a genetic screen with an shRNA library against ubiquitination pathway-related genes. To this end, we set up a protocol to specifically enrich positive migration regulator candidates. We identified the deubiquitinase USP19 and demonstrated that its silencing reduces the migratory and invasive potential of highly invasive breast cancer cell lines. We extended our investigation in vivo and confirmed that mice injected with USP19 depleted cells display increased tumor-free survival, as well as a delay in the onset of the tumor formation and a significant reduction in the appearance of metastatic foci, indicating that tumor cell invasion and dissemination is impaired. In contrast, overexpression of USP19 increased cell invasiveness both in vitro and in vivo, further validating our findings. More importantly, we demonstrated that USP19 catalytic activity is important for the control of tumor cell migration and invasion, and that its molecular mechanism of action involves LRP6, a Wnt co-receptor. Finally, we showed that USP19 overexpression is a surrogate prognostic marker of distant relapse in patients with early breast cancer. Altogether, these findings demonstrate that USP19 might represent a novel therapeutic target in breast cancer.

cancer biology↗

bioRxiv2020

Identification of unique mutations in SARS-CoV-2 strains isolated from India suggests its attenuated pathotype

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first reported in Wuhan, China in November 2019 has developed into a pandemic since March 2020, causing substantial human casualties and economic losses. Studies on SARS-CoV-2 are being carried out at an unprecedented rate to tackle this threat. Genomics studies, in particular, are indispensable to elucidate the dynamic nature of the RNA genome of SARS-CoV-2. RNA viruses are marked by their unique ability to undergo high rates of mutation in their genome, much more frequently than their hosts, which diversifies their strengths qualifying them to elude host immune response and amplify drug resistance. In this study, we sequenced and analyzed the genomic information of the SARS-CoV-2 isolates from two infected Indian patients and explored the possible implications of point mutations in its biology. In addition to multiple point mutations, we found a remarkable similarity between relatively common mutations of 36-nucleotide deletion in ORF8 of SARS-CoV-2. Our results corroborate with the earlier reported 29-nucleotide deletion in SARS, which was frequent during the early stage of human-to-human transmission. The results will be useful to understand the biology of SARS-CoV-2 and itsattenuation for vaccine development.

microbiology↗

bioRxiv2020

Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in Myc-driven Medulloblastoma

Myc-driven Medulloblastoma remains a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. Myc gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in Myc-amplified medulloblastoma we performed a CRISPR-Cas9 essentiality screen targeting 1140 genes annotated as the druggable genome. CDK7 was identified as a mediator of medulloblastoma tumorigenesis. Using covalent inhibitors and genetic depletion of CDK7 we observe the cessation of tumor growth in xenograft mouse models and increase in apoptotic mechanisms. The results are attributed to repression of a core set of Myc-driven transcriptional programs mediating DNA repair. We further establish that blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage and extended survival and tumor latency in medulloblastoma xenograft mouse models. Our studies establish a mechanism for selective inhibition of Myc-driven MB by CDK7 inhibition combined with radiation as a viable therapeutic strategy for Myc-amplified medulloblastoma.

cancer biology↗

bioRxiv2019

Metabolic control of immune-competency by odors in Drosophila

Drosophila blood-progenitor cells generate an inflammatory cell-type termed lamellocyte, in response to parasitic wasp-infections. In this study we show that olfaction primes lamellocyte potential. Specifically, larval odor-detection mediated release of systemic {gamma}-aminobutyric acid (GABA) from neurosecretory cells, is detected and internalized by blood progenitor-cells. GABA catabolism through the GABA-shunt pathway prevents Sima (HIF) protein degradation. Sima is necessary and sufficient for lamellocyte induction. However, limited systemic GABA availability during development restricts blood-progenitor Sima levels and consequently their lamellocyte potential. Preconditioning Drosophila larvae in odor environments mimicking parasitoid-threatened conditions raises systemic GABA and blood-progenitor Sima levels. As a result, infection responses in these animals are rapid and efficient. Overall, this study explores the importance of sensory control of myeloid-immunity and unravels the adaptive influence of environmental odor-experience on myeloid-metabolism and priming innate-immune potential.

developmental biology↗