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Srivastava, T.

Publications and source records attributed to Srivastava, T..

12 recordsLinked to original sources
bioRxiv2026

Jamestown Canyon virus rapidly adapts to mosquito cells through multiple M segment mutations

Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus with an unusually broad host and vector range. Despite increasing mosquito-to-human spillover, the viral determinants governing host adaptation remain poorly defined. We examined changes in JCV replication during serial passage in mosquito cells and sought to link adaptive changes in viral fitness to specific genetic mutations. In mosquito-derived C6/36 cells, JCV exhibited a distinct "lag-burst" phenotype in which viral replication remained nearly undetectable for 10 days before abruptly increasing. Strikingly, following reinfection of fresh C6/36 cells, JCV that had been passaged once in mosquito cells exhibited immediate, robust replication with no detectable lag phase. Sequencing before and after passage identified multiple M segment mutations associated with enhanced replication. Using a plasmid-based reverse genetics system, individual mutations were introduced into recombinant JCV and evaluated for their effects on replication in mosquito cells. All tested mutations independently enhanced replication efficiency, demonstrating that adaptation can arise through multiple independent genetic pathways. However, no individual mutation fully reproduced the phenotype acquired naturally through mosquito cell passage. Together, these findings demonstrate that JCV rapidly adapts to mosquito cells under minimal selective pressure and highlight the potential for emergence of increasingly well-adapted viral variants.

microbiology↗
bioRxiv2026

ULK1 drives NDP52-mediated selective autophagic degradation of MHC-I to promote immune evasion in HPV-positive head and neck cancer

Antigen presentation by major histocompatibility complex class I (MHC-I) is critical for tumor cell killing by CD8+ T cells. Accordingly, tumor cells downregulate MHC-I expression through multiple mechanisms, thereby evading the immune response. Importantly, lower levels of MHC-I are associated with poor responses to immune checkpoint inhibitor therapy. Our recent study has shown that the human papillomavirus (HPV) oncoproteins induce MHC-I protein ubiquitination by membrane-associated Ring-CH-type finger 8 (MARCHF8) in HPV-positive head and neck cancer (HPV+ HNC). However, the mechanism by which ubiquitinated MHC-I is degraded remains elusive. By performing genome-wide CRISPR screens, we identified components of the ULK1 and PIK3C3 complexes for autophagy initiation complexes among the top negative regulators of cell-surface MHC-I expression in HPV+ HNC cells. We show that MHC-I is recruited from the ER to autophagosomes by the cargo receptor NDP52, decreasing MHC-I levels. Further, inhibiting the initiation or nucleation steps of autophagy before autophagosome formation is critical for restoring MHC-I levels on the cell surface. Finally, genetic inhibition of autophagy initiation suppresses HPV+ HNC tumor growth in vivo and enhances the CD8+ T cell-mediated antitumor response. Our findings suggest that autophagic degradation of MARCHF8-ubiquitinated MHC-I is a key immune evasion mechanism in HPV+ HNC.

cancer biology↗
bioRxiv2024

Withaferin A downregulates NDRG1 to overcome hypoxia mediated EMT and chemoresistance in lung adenocarcinoma and glioma cells

One of the important hurdles to be overcome in the management of cancers is the acquired resistance to therapy. There is a crucial need to not only understand molecular pathways underpinning resistance to therapy but to find better and more effective therapeutic avenues to overcome such acquired resistance. Tumor microenvironmental conditions such as hypoxia have long been known to result in chemoresistance in various cancers that become unresponsive to standard-of-care drugs such as cisplatin. Although many new compounds are screened for their anticancer properties, the recalcitrant tumor microenviroenment has not been given due importance in many potential drugs. In this study, we examine the efficacy of Withaferin A (WA), a bioactive compound isolated from Withania somnifera, in overcoming hypoxia-induced metabolic adaptation, chemoresistance, and epithelial-to-mesenchymal transition (EMT) in both lung adenocarcinoma and glioma cells. Our results reveal that WA significantly inhibits hypoxia-induced increased migration, EMT, and metabolic reprogramming, leading to enhanced cytotoxicity in otherwise chemoresistant cancer cells. WA treatment suppressed the expression of markers of EMT, glucose metabolism, and bonafide hypoxia markers, particularly NDRG1 thereby inducing cell death in these cancer cells. WA showed significant cytotoxic effects in chemoresistant lung adenocarcinoma and glioma cells, both alone or in combination with cisplatin, highlighting its potential as a therapeutic agent for overcoming chemoresistance in these cancers. Our results provide new insights into the anti-cancer mechanisms of WA especially under hypoxic conditions, and support its further investigation as a promising adjunctive therapy for the treatment against hypoxia-induced chemoresistance in cancers.

cancer biology↗
bioRxiv2024

Mild neonatal hypoxia disrupts adult hippocampal learning and memory and is associated with CK2-mediated dysregulation of synaptic calcium-activated potassium channel KCNN2

ObjectiveAlthough nearly half of preterm survivors display persistent neurobehavioral dysfunction including memory impairment without overt gray matter injury, the underlying mechanisms of neuronal or glial dysfunction, and their relationship to commonly observed cerebral white matter injury are unclear. We developed a mouse model to test the hypothesis that mild hypoxia during preterm equivalence is sufficient to persistently disrupt hippocampal neuronal maturation related to adult cellular mechanisms of learning and memory. Methods: Neonatal (P2) mice were exposed to mild hypoxia (8%O2) for 30 min and evaluated for acute injury responses or survived until adulthood for assessment of learning and memory and hippocampal neurodevelopment. ResultsNeonatal mild hypoxia resulted in clinically relevant oxygen desaturation and tachycardia without bradycardia and was not accompanied by cerebral gray or white matter injury. Neonatal hypoxia exposure was sufficient to cause hippocampal learning and memory deficits and abnormal maturation of CA1 neurons that persisted into adulthood. This was accompanied by reduced hippocampal CA3-CA1 synaptic strength and LTP and reduced synaptic activity of calcium-sensitive SK2 channels, key regulators of spike timing dependent neuroplasticity, including LTP. Structural illumination microscopy revealed reduced synaptic density, but intact SK2 localization at the synapse. Persistent loss of SK2 activity was mediated by altered casein kinase 2 (CK2) signaling. InterpretationClinically relevant mild hypoxic exposure in the neonatal mouse is sufficient to produce morphometric and functional disturbances in hippocampal neuronal maturation independently of white matter injury. Additionally, we describe a novel persistent mechanism of potassium channel dysregulation after neonatal hypoxia. Collectively our findings suggest an unexplored explanation for the broad spectrum of neurobehavioral, cognitive and learning disabilities that paradoxically persist into adulthood without overt gray matter injury after preterm birth.

neuroscience↗
bioRxiv2023

Huntingtin Interacting Protein-1 expression is regulated via HIF2 axis in Lung Adenocarcinoma

Non-Small Cell Lung Cancer (NSCLC) patients are diagnosed late when the disease has metastasized. Kras is a prevalent mutation in NSCLC besides EGFR and TP53 and targeted therapies against this have been challenging. We have explored deregulation of an endocytic adapter protein, Huntingtin Interacting Protein-1(HIP1) and its relevance in a Kras mutant lung adenocarcinoma cell line as a model system. HIP1 RNA expression is observed to be significantly reduced in high-grade and metastatic lung cancer patients as compared to low-grade tumours and this correlates with poor survival. HIP1 depletion followed by global proteome profiling in A549 cells identified metabolic pathways to be majorly upregulated, followed by RNA transport and surveillance, amongst others. HIP1 depletion also significantly increased anchorage independent growth and invasion of these cells. However, the EMT markers did not follow the canonical regulation. We observed E-Cadherin and Vimentin induction, which is suggestive of collective migration. Additionally, we observed a hypoxic microenvironment to induce HIP1 expression, mediated by Hypoxia Inducible Factor 2 (HIF2), suggesting that a HIF2-HIP1 axis can cause tumour suppression and needs further exploration.

cancer biology↗
bioRxiv2023

The tetrapeptide sequence of IL-1β regulates its recruitment and activation by inflammatory caspases

The mammalian innate immune system uses germline-encoded cytosolic pattern-recognition receptors (PRRs) to detect intracellular danger signals. At least six of these PRRs are known to form multiprotein complexes called inflammasomes which activate cysteine proteases known as caspases. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines such as IL-1{beta} and IL-18, as well as the pore forming protein, gasdermin D (GSDMD), to induce pyroptotic cell death. In contrast, non-canonical inflammasomes, caspases-4/-5 (CASP4/5) in humans and caspase-11 (CASP11) in mice, are activated by intracellular LPS to cleave GSDMD, but their role in direct processing of inflammatory cytokines has not been established. Here we show that active CASP4/5 directly cleave IL-18 to generate the active species. Surprisingly, we also discovered that CASP4/5/11 cleave IL-1{beta} at D27 to generate a 27 kDa fragment that is predicted to be inactive and cannot signal to the IL-1 receptor. Mechanistically, we discovered that the sequence identity of the P4-P1 tetrapeptide sequence adjacent to the caspase cleavage site (D116) regulates the recruitment and processing of IL-1{beta} by inflammatory caspases to generate the bioactive species. Thus, we have identified new substrates of the non-canonical inflammasomes and reveal key mechanistic details regulating inflammation.

immunology↗
bioRxiv2021

Interaction of hypoxia and nicotine acetylcholine receptor signaling network reveals a novel mechanism for lung adenocarcinoma progression in never-smokers

High incident of lung cancer among never smokers and their disease pathogenesis is an unexplained phenomenon. We have analyzed 1727 lung cancer patient data to understand the impact of smoking on overall survival of lung cancer patients and have observed a difference of only 47 days between smokers and never smokers in adenocarcinoma patients suggesting that the disease is equally fatal in never-smokers irrespective of gender. In this study, we have investigated the possible collaboration between the nAChR and hypoxia signaling pathway to elucidate a mechanism of disease progression in never-smokers. We report a previously unidentified increase in both acetylcholine and nAChR-7 levels in non small cell lung cancer cells in hypoxia. Similar increase in ubiquitously expressed nAChR-7 transcripts was also observed in other cancer lines. A direct binding of HIF-1 with the hypoxia response element (HRE) present at -48 position preceding the transcriptional start site in nAChR-7 promoter region was established. Significantly, the increased acetylcholine levels in hypoxia drove a feedback loop via modulation of PI3K/AKT pathway to stabilize HIF-1 in hypoxia. Further, Bungarotoxin, an antagonist of nAChR-7 significantly reversed hypoxia mediated metastasis and induction of HIF-1 in these cells. Our study gives a plausible explanation for the equally worse prognosis of lung adenocarcinoma in never-smokers wherein the nAChR signaling is enhanced in hypoxia by acetylcholine, in the absence of nicotine.

cancer biology↗
bioRxiv2020

Genotypic and antigenic study of SARS-CoV-2 from an Indian isolate.

Coronaviruses (CoVs) are one of the largest groups of positive-sense RNA virus families within the Nidovirales order, which are further classified into four genera: alpha, beta, gamma, and delta. Coronaviruses have an extensive range of natural hosts and are known to be responsible for a broad spectrum of diseases in multiple species. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) that has unleashed a global threat to public health and the economy. Coronaviruses are extensively present in birds and mammals, with horseshoe bats (Rhinolophus affinis), being the reservoir for the ongoing SARS-CoV-2 that seems to have resulted from a zoonotic spillover to the human host, causing respiratory infections, lung injury and Acute Respiratory Distress Syndrome(ARDS). About six coronavirus serotypes are linked with the disease in humans, namely HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, SARS-CoV-2, and MERS-CoV. SARS-CoV-2 is the seventh CoV to infect humans. We analyzed the genome sequence of CoV-2 from isolates derived from China as well from India and encountered minute variations in their sequence. A cladogram analysis revealed the predominant strain circulating in India belongs to the A2a clad. We took one such strain (MT012098) and performed a rigorous in-silico genotypic and antigenic analysis to identify its relatedness to other strains. Further, we also performed a detailed prediction for B and T cell epitopes using BepiPred 2.0 server and NetCTL 1.2 server (DTU Bioinformatics), respectively. We hope this information may assist in an effective vaccine designing program against SARS-CoV-2.

biochemistry↗
bioRxiv2020

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death

Phosphatidylinositol 3-phosphate (PI(3)P) levels in Plasmodium falciparum correlate with tolerance to cellular stresses caused by artemisinin, a first-line malaria treatment, and environmental factors. However, the functional role of PI(3)P in the Plasmodium stress response and a possible mechanism of protection were unknown. Here, we used multiple chemical probes including PI3K inhibitors and known antimalarial drugs to examine the importance of PI(3)P under thermal conditions that recapitulate malaria fever. Live cell microscopy using both chemical and genetic reporters revealed that PI(3)P stabilizes the acidic and proteolytic digestive vacuole (DV) under heat stress. We demonstrate that heat-induced DV destabilization in PI(3)P-deficient P. falciparum precedes cell death and is reversible after withdrawal of the stress condition and the PI3K inhibitor. These phenotypes are not observed with an inactive structural analog of the PI3K inhibitor. A chemoproteomic and biochemical approach identified PfHsp70-1 as a parasite PI(3)P-binding protein. Targeting PfHsp70-1 with a small molecule inhibitor phenocopied PI(3)P-deficient parasites under heat shock. Furthermore, tunable knockdown of PfHsp70-1 showed that PfHsp70-1 downregulation causes DV destabilization and hypersensitizes parasites to heat shock and PI3K inhibitors. Our findings underscore a mechanistic link between PI(3)P and PfHsp70-1, and present a novel PI(3)P function in stabilizing the DV compartment during heat stress.

microbiology↗
bioRxiv2019

RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

The apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease that poses a significant global health burden. Plasmodium spp. initiate infection of the human host by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood when compared to other Plasmodium life stages, which has hindered our ability to target these parasites for disease prevention. We conducted an extensive RNA-seq analysis throughout the Plasmodium berghei LS, covering as early as 2 hours post infection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi, and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using hierarchical clustering along with co-expression analysis, we identified clusters functionally enriched for important liver-stage processes such as interactions with the host cell and redox homeostasis. Furthermore, some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding motifs. This finding presents potential LS targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our work presents a window into the previously undescribed transcriptome of Plasmodium upon host hepatocyte infection to enable a comprehensive view of the parasites LS. These findings also provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. berghei to human-infective Plasmodium parasites. IMPORTANCEThe LS of Plasmodium infection is an asymptomatic yet necessary stage for producing blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle can prevent clinical malaria, but relatively less is known about the parasites biology at this stage. Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) have not been accessible before due to the technical challenges associated with liver-stage infections. Our data now provides insights into these early parasite fluxes that may facilitate establishment of infection, transformation and replication in the liver.

microbiology↗
bioRxiv2018

Redefining Nephrotic Syndrome in Molecular Terms: Outcome-associated molecular clusters and patient stratification with noninvasive surrogate biomarkers

A tissue transcriptome driven classification of nephrotic syndrome patients identified a high risk group of patients with TNF activation and established a non-invasive marker panel for pathway activity assessment paving the way towards precision medicine trials in NS.\n\nAbstractNephrotic syndrome from primary glomerular diseases can lead to chronic kidney disease (CKD) and/or end-stage renal disease (ESRD). Conventional diagnoses using a combination of clinical presentation and descriptive biopsy information do not accurately predict risk for progression in patients with nephrotic syndrome, which complicates disease management. To address this challenge, a transcriptome-driven approach was used to classify patients with minimal change disease and focal segmental glomerulosclerosis in the Nephrotic Syndrome Study Network (NEPTUNE). Transcriptome-based classification revealed a group of patients at risk for disease progression. High risk patients had a transcriptome profile consistent with TNF activation. Non-invasive urine biomarkers TIMP1 and CCL2 (MCP1), which are causally downstream of TNF, accurately predicted TNF activation in the NEPTUNE cohort setting the stage for patient stratification approaches and precision medicine in kidney disease.

systems biology↗