Biology subjects

Seshadri, V.

Publications and source records attributed to Seshadri, V..

5 recordsLinked to original sources

bioRxiv2025

Mon1-Rab7 axis is essential for transport, localization and anchoring of oskar mRNA

Asymmetric localization of oskar mRNA to the posterior of the oocyte is a complex process driven by autonomous and non-cell autonomous mechanisms. The former includes Oskar protein that reinforces localization and anchoring of its mRNA through activation of endocytosis and regulation of actin cytoskeleton; the latter includes signals from the posterior follicle cells (PFCs) that regulates microtubule orientation for polarized transport. Here we identify Monensin Sensitivity 1 (Mon1), as a novel factor regulating anterior-posterior (A-P) patterning. Mon1 is an evolutionarily conserved activator of Rab7-a key regulator of the endo-lysosomal pathway. Embryos lacking maternal mon1 (mon1m) show mislocalized oskar and bicoid mRNAs leading to loss of patterning and lethality. In the mutant oocyte Staufen appears clumpy and the levels of Oskar protein and Par-1 is significantly reduced. Abnormal actin rings are seen in the ooplasm. Driving expression of mon1 in the germline rescues these phenotypes and restores viability. In contrast, expression in the PFC predominantly rescues the Par-1 phenotype with a modest effect on viability. We demonstrate that oskar mRNA interacts with Rab7 suggesting possible role for the Mon1-Rab7 axis in the transport of oskar. We show that Mon1 in the PFCs, regulates PIP2 levels to influence accumulation of Par-1 in the oocyte. We propose that Mon1 regulates oskar localization in two distinct ways: cell autonomously in the germline by regulating Rab7, and non-cell autonomously through the PFCs by regulating accumulation of Par-1. Summary statementMon1, an established Rab converter, has roles in embryonic axial patterning, modulating transport, localisation and anchoring of posteriorly localised mRNA during oocyte maturation. Mon1 influence is both cell autonomous, from within the oocyte and non-cell autonomous, through posterior follicle cells.

developmental biology↗

bioRxiv2024

Single-cell fluorescence imaging reveals heterogeneity in senescence biomarkers and identifies rapamycin-responsive sub-populations

Cellular senescence is a state of irreversible cell cycle arrest accompanied by a distinctive inflammatory secretory profile known as the senescence-associated secretory phenotype (SASP). While various biomarkers, such as senescence-associated beta-galactosidase (SA-{beta}gal), EdU incorporation, P21, and P16, are used to identify senescent cells, no single biomarker universally defines cellular senescence, and current methods often fail to address heterogeneity in biomarker expression levels. This study leverages single-cell fluorescence imaging to assess multiple senescence markers including SA-{beta}gal enzymatic activity, P21 and IL-6 expression, and nuclear and cell area, in chemotherapy-induced (mitomycin C) and oxidative stress-induced (D-galactose) senescence models in human fibroblasts. Our findings reveal significant heterogeneity in SA-{beta}gal activity and distinct sub-populations within senescent cells. Nuclear and cell area measurements emerged as robust indicators of cellular senescence, displaying similar variability across individual cells. Importantly, we identified specific nuclear area sub-populations that strongly correlate with IL-6 expression levels, demonstrating a relationship between the heterogeneous expression of senescence biomarkers and the SASP. To address this heterogeneity, we introduced an induction threshold method to more accurately quantify the percentage of cells expressing senescence biomarkers. Furthermore, in both senescence models, we observed that rapamycin, a well-known senomorphic agent, selectively targets specific biomarker-expressing sub-populations. This study underscores the value of assessing cellular heterogeneity in senescence research and provides an improved approach for analysing senescence markers in diverse cellular contexts.

cell biology↗

bioRxiv2022

Prolonged exposure to insulin causes epigenetic alteration leading to insulin resistance

Glucose homeostasis is maintained by insulin. It has been observed that hyperinsulinemia precedes insulin resistance and Type 2 diabetes. Insulin resistance is caused by multiple factors including genetic and diet. The molecular mechanism underlying insulin resistance (IR) is not completely understood. Using Glut4 and insulin receptor-expressing CHO cells we had previously shown that prolonged exposure of these cells to insulin in the absence of high levels of glucose led to insulin resistance in the cells. In the present study, we have shown that the underlying cause for the impaired GLUT4 trafficking is the defective PI3K/AKT pathway. This insulin resistance is likely due to epigenetic alterations as it is stable and can be maintained for several generations even when insulin is not provided, and epigenetic modifiers can reverse the insulin resistance. We extended these studies to liver cell line (BRL-3A) and show that these cells also develop impaired insulin signaling upon exposure to insulin in the absence of high levels of glucose. Transcriptomic analysis of the insulin-sensitive and -resistance cells uncover altered signaling networks involved in chromatin remodelling, Rho GTPases, and ubiquitination. Pathway analysis reveals the role of demethylase Kdm5b and lysine methyltransferase (Kmt2a and Kmt2e) in the development of insulin resistance. It is also observed that trimethylation of histone H3 at lysine 4 (H3K4me3) is increased in insulin resistance cellular models. We further showed that mice injected with low doses of insulin when fasting develop insulin resistance with impaired glucose tolerance and increased HOMA-IR index. Altogether, these findings suggest dysregulated synthesis of insulin in the absence of glucose stimulus could lead to epigenetic alterations that may lead to insulin resistance. Summary StatementInsulin stimulation in the absence of glucose leads to insulin resistance. We have developed a cell and mouse model of insulin resistance in this study to characterise the molecular signalling involved in insulin resistance and early onset of type 2 diabetes. The transcriptomic analysis provides new insights on epi-transcriptomic regulation in insulin resistance.

molecular biology↗

bioRxiv2021

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

molecular biology↗

bioRxiv2020

Phylogenomic analysis of SARS-CoV-2 genomes from western India reveals unique linked mutations

India has become the third worst-hit nation by the COVID-19 pandemic caused by the SARS-CoV-2 virus. Here, we investigated the molecular, phylogenomic, and evolutionary dynamics of SARS-CoV-2 in western India, the most affected region of the country. A total of 90 genomes were sequenced. Four nucleotide variants, namely C241T, C3037T, C14408T (Pro4715Leu), and A23403G (Asp614Gly), located at 5UTR, Orf1a, Orf1b, and Spike protein regions of the genome, respectively, were predominant and ubiquitous (90%). Phylogenetic analysis of the genomes revealed four distinct clusters, formed owing to different variants. The major cluster (cluster 4) is distinguished by mutations C313T, C5700A, G28881A are unique patterns and observed in 45% of samples. We thus report a newly emerging pattern of linked mutations. The predominance of these linked mutations suggests that they are likely a part of the viral fitness landscape. A novel and distinct pattern of mutations in the viral strains of each of the districts was observed. The Satara district viral strains showed mutations primarily at the 3' end of the genome, while Nashik district viral strains displayed mutations at the 5' end of the genome. Characterization of Pune strains showed that a novel variant has overtaken the other strains. Examination of the frequency of three mutations i.e., C313T, C5700A, G28881A in symptomatic versus asymptomatic patients indicated an increased occurrence in symptomatic cases, which is more prominent in females. The age-wise specific pattern of mutation is observed. Mutations C18877T, G20326A, G24794T, G25563T, G26152T, and C26735T are found in more than 30% study samples in the age group of 10-25. Intriguingly, these mutations are not detected in the higher age range 61-80. These findings portray the prevalence of unique linked mutations in SARS-CoV-2 in western India and their prevalence in symptomatic patients. ImportanceElucidation of the SARS-CoV-2 mutational landscape within a specific geographical location, and its relationship with age and symptoms, is essential to understand its local transmission dynamics and control. Here we present the first comprehensive study on genome and mutation pattern analysis of SARS-CoV-2 from the western part of India, the worst affected region by the pandemic. Our analysis revealed three unique linked mutations, which are prevalent in most of the sequences studied. These may serve as a molecular marker to track the spread of this viral variant to different places.

genomics↗