Biology subjects

Choudhary, V.

Publications and source records attributed to Choudhary, V..

13 recordsLinked to original sources

bioRxiv2026

Dengue virus-induced thrombocytopenia is mediated by dysregulation of the RUNX1-MYH9 axis

Dengue virus (DENV) infection causes an acute febrile illness that is usually self-limiting but can become fatal. Thrombocytopenia, or a decrease in circulating platelets, is a major clinical feature of dengue and is strongly associated with the progression to dengue hemorrhagic fever and dengue shock syndrome. In this report, MYH9, a cytoskeletal protein involved in platelets maturation and release, was found to be down-regulated in DENV-infected Huh7 cell lines. The expression of RUNX1, an important transcription factor involved in megakaryopoiesis, was also decreased following DENV-infection. In-silico analysis identified three potential RUNX1 transcription-factor binding site in the promoter-region of MYH9 gene. We therefore investigated whether RUNX1 regulates MYH9 expression during DENV-infection. Luciferase-assay and ChIP assays showed that DENV-infection affect the binding of RUNX1 to the MYH9 promoter, providing insight into the molecular basis underlying the reduced expression of MYH9 expression. Furthermore, the megakaryocyte differentiation markers GATA1, ITGB3, and MYH9 were upregulated in PMA-induced K562-cells. However, DENV infection downregulated these genes and increased the expression of apoptosis marker gene CYCS. These findings suggest that the dengue affects megakaryopoiesis through multiple mechanisms, potentially reducing platelets production while also promoting platelets destruction. Additionally, Fluorescence and electron microscopy revealed that functional defects of MYH9 may affects megakaryopoiesis biogenesis. Overall, this study provides insights into the molecular mechanisms contributing to thrombocytopenia during DENV infection.

molecular biology↗

bioRxiv2026

Conformational activation of GSK3β by an environmental toxicant suppresses hedgehog signalling

Primary cilium-dependent hedgehog signalling is essential for embryonic development, tissue patterning, and organ homeostasis, and its dysfunction causes ciliopathies, a clinically diverse spectrum of developmental and reproductive disorders. Whether environmental chemicals can phenocopy genetic ciliopathies by directly targeting ciliary kinase machinery has remained unknown. Here we show that endosulfan, a banned organochlorine pesticide linked to congenital and reproductive defects, suppresses hedgehog signalling by driving proteolytic processing of GLI transcription factors into repressor forms. Excluding ciliary receptor trafficking, cAMP signalling, and GLI-DNA binding, we identify PKA and GSK3{beta} as direct endosulfan targets: endosulfan allosterically fine-tunes PKA activity and, to our knowledge, is the first reported small-molecule activator of GSK3{beta}, stabilising its active conformation, a profile distinct from all known inhibitors. We further identify Cetn3 and Cep250 as novel GLI-regulated genes required for centriole cohesion, both of which are repressed upon endosulfan exposure, linking this kinase axis to the reproductive defects reported in exposed human populations and animal models. These findings establish a chemical-biological axis through which an environmental toxicant hijacks core kinase signalling to phenocopy a genetic ciliopathy.

cell biology↗

bioRxiv2026

Usherin in the pineal gland: altered sleep in zebrafish models of Usher syndrome type 2a

Usher syndrome type 2A (USH2a), the most common form of hereditary deaf-blindness, is frequently accompanied by fatigue and poor sleep quality. As these sleep problems occur independently of visual decline, it is hypothesized that the USH2A-encoded protein usherin regulates sleep and circadian rhythmicity via an extra-retinal mechanism. Ush2a knockout zebrafish models were utilized to investigate this hypothesis. Immunohistochemical analysis demonstrated usherin localisation in pineal gland photoreceptor cells in wild-type larvae, alongside the USH2 complex proteins Adgrv1 and Whrna. Cross-species transcriptomic and proteomic analyses confirmed USH2A expression in all mammalian pineal gland tissues studied. Circadian clock gene expression was measured over 24 h and showed preserved oscillatory patterns in wild-type and mutant zebrafish. Ex vivo superfusion of pineal glands revealed sustained circadian melatonin release with comparable phase and period to controls, although potential differences in absolute melatonin levels could not be excluded. Despite intact clock gene expression and melatonin release in ush2a mutants, behavioural classification over 24-h recordings revealed altered sleep-wake behaviour: ush2a mutants displayed elevated daytime sleep and significantly prolonged and more variable sleep latency. The dissociation between intact molecular rhythms and abnormal sleep behaviour likely implicates that usherin plays a role in sleep-wake regulation independent of the circadian pacemaker and melatonin synthesis. These findings suggest a novel role of usherin in the pineal gland and establish a mechanistic link between usherin dysfunction and sleep disturbances, providing a biological basis for the fatigue and sleep problems reported in USH2a patients.

animal behavior and cognition↗

bioRxiv2025

Satb1 integrates cohesin mediated genome organization and transcriptional regulation during T cell development

Three-dimensional (3D) genome folding, which is highly cell type-specific, plays a crucial role in orchestrating spatiotemporal gene expression. Although factors such as CTCF have been extensively studied in the hierarchical regulation of 3D chromatin organization, the mechanisms driving dynamic genome folding during T cell fate transitions remain incompletely defined. In this study, we reveal that Satb1, a chromatin organizer enriched in the T cell lineage, co-occupies genomic regions with the cohesin complex and Ctcf in double-positive (DP) thymocytes, where chromatin interactions are notably increased. We show that Satb1 physically interacts with the cohesin subunit Smc1a, and its deletion results in aberrant Smc1a binding and reduced chromatin contacts at sites co-occupied by Satb1 and cohesin. In both DP and immature CD4 single-positive (SP) T cells, Satb1 is essential for proper T cell activation and cytokine signaling. At the Cd3 locus, Satb1 and cohesin collaboratively regulate gene expression, with Satb1 loss leading to disrupted Smc1a occupancy and compromised chromatin interactions. Furthermore, Satb1 shows in vitro properties consistent with liquid-liquid phase separation, and disease-associated mutations impair these properties. Together, our findings uncover a molecular mechanism in which Satb1 facilitates chromatin looping through direct interaction with the cohesin complex and its ability to form nuclear condensates, thereby governing transcriptional regulation during T cell development.

immunology↗

bioRxiv2025

Lipid Droplets are active translation depots that respond to infection

Lipid droplets (LDs) are increasingly recognized as dynamic organelles with roles extending beyond lipid storage. While the core LD proteome across cells reveals minimal overlap, several proteins with "housekeeping" functions such as ribosomal proteins and translation factors have largely been ignored as contaminants. Here, we provide biochemical and microscopy-based evidence of ribosome components and the ribosome to be present in association with the LD. By developing a modified method based on puromycylation, we demonstrate that active ribosomes are localised in close-proximity of LDs, demonstrating that LDs serve as sites of active protein translation. By using Mycobacterium tuberculosis (Mtb) infection as a model, we show that LD-associated translation is regulated under infection conditions. Although the specific mRNAs translated on LDs remain to be identified, these findings reveal LDs as critical platforms for protein synthesis and provide insights into how the LD proteome is established. This work highlights the multifaceted role of LDs in cellular homeostasis and host-pathogen interactions. IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports wil be available in ful to the handling editor. C_LIO_LIThe identity of the referees wil NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee wil be confidentia ly disclosed to any a filiate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons o fice can be contacted directly at: office@reviewcommons.org

cell biology↗

bioRxiv2025

Characterization of Woodsmoke Generated in the Air Pollution Exposure Lab and Comparison to Diesel Exhaust

To address the increasing concern of woodsmoke (WS) and better understand its effects on human health, a woodsmoke generation system was built in the Air Pollution Exposure Laboratory to facilitate future controlled human exposure studies. Two different woodsmoke conditions, flaming (WSFL) and smoldering (WSSM), were generated, and PM2.5 concentrations of approximately 500 g/m3 were achieved. The woodsmoke produced using the system was characterized in this study and compared with diesel exhaust (DE) generated and collected at the same facility. Within the gas phase generated by the pollutants, WS showed slight increases in CO and CO2 compared to filtered air (FA), while DE contained significantly higher levels of NOx, CO2, and total volatile organic compounds compared to FA. The WS aerosols were comprised of approximately 98% organics, 0.6% NH4, 0.9% NO3, and 0.2% SO4. Among the organic species, the CHO1 and CHOgt1 families encompassed around 60%, which was higher than the fraction of oxygenated families in DE aerosols. Moreover, the WS aerosols had higher concentrations of Cd compared to the DE aerosols. Greater oxidative potentials were also observed for WSFL and WSSM compared to DE, with DTT consumption rates normalized to the PM mass being 0.0099 and 0.0090 nmol/min/g, respectively. The difference in the compositions and properties of WS and DE suggests that it is critical to conduct further studies on how these pollutants can affect health differently.

pharmacology and toxicology↗

bioRxiv2024

Low RT-based Genome Editing Fidelity in Mouse Hepatocytes: Challenges and Solutions

Abstract/SummaryIntegrase-mediated Programmable Genomic Integration (I-PGI) uses a Cas9 nickase (nCas9) with a reverse transcriptase (RT), to write a large serine integrase (LSI) target site (attB/P, here called "beacon") in a programmed location. Co-delivery of the LSI and a DNA template containing the cognate recognition site results in precise integration of the template in a specific genomic location. While we were able to achieve high-fidelity beacon placement in a range of primate cycling and non-dividing cells, when translating our technology into an in vivo rodent model (liver) we surprisingly observed very low beacon fidelity, with the vast majority of beacons being unsuitable for integration. This phenomenon was independent of mouse strain, but was specific to non-dividing cells, as a cycling mouse hepatocyte cell line (Hepa1-6) demonstrated very high levels of fidelity. To address this issue we utilized neonatal mice, which have a much higher proportion of proliferating hepatocytes than adult mice. This resulted in a significant increase in the placement of high-fidelity beacons, and achieved functional gene expression after I-PGI in a therapeutically relevant target site. In an alternate approach, we engineered transgenic mice with intact beacons placed in specific genomic locations, allowing us to optimize integrase and DNA template dosing and kinetics. In summary, we have identified a previously undescribed challenge when using RT-based editing to write long sequences (~40 bp) in non-dividing rodent hepatocytes. This phenomenon was specific to rodents and was not observed in primate dividing or non-dividing cells. This previously unidentified challenge using RTs will limit the use of I-PGI in mouse models, however here we describe two methods that address this issue.

bioengineering↗

bioRxiv2024

Substrate Trapping in Polyketide Synthase Thioesterase Domains: Structural Basis for Macrolactone Formation

Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the termina PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TEDAP) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TEDAP, the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 [A] from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide new insights to facilitate TE engineering and optimization.

biochemistry↗

bioRxiv2024

Real-time visualization reveals Mycobacterium tuberculosis ESAT-6 disrupts phagosome via fibril-mediated vesiculation

Mycobacterium tuberculosis (Mtb) evades host defense by hijacking and rupturing the phagosome, enabling it to escape to the host cytosol for its survival. ESAT-6, a secreted virulence protein of Mtb, is known to be critical for phagosome rupture. However, the mechanism of ESAT-6-mediated disruption of the phagosomal membrane remains unknown. Using in vitro reconstitution and numerical simulations, we discover that ESAT-6 polymerization remodels and vesiculates phagosomal membrane. In contrast to the pore formation triggered by a bilayer-spanning conformation, we find that the binding of ESAT-6 to the phagosomal membrane is shallow. Such shallow insertion leads to membrane shape transition leading to tubular and bud-like deformations on the membrane in a concentration-dependent manner, facilitated by the reduction in membrane tension and compressibility modulus. Strikingly, our observations suggest that ESAT-6 polymerizes in bulk and on the membrane, both in vitro and in macrophage. Numerical simulations demonstrate that growing fibrils generate both radial and tangential forces causing local remodeling and shape transition of the membrane. Using micropipette aspiration, we quantitatively show that ESAT-6 bound tensed membrane undergoes local changes in membrane curvature and lipid phase separation, also facilitated by the direct contact of the bacteria inside the phagosome. Nonetheless, the vesiculation of the buds is primarily driven by the forces exerted by the polymerization of ESAT-6. Such ESAT-6 mediated vesiculation induces apoptosis and host cell death in a concentration and time-dependent manner that promotes infection. Overall, the findings provide mechanistic insights into the long-standing question of phagosome disruption by Mtb for its escape.

biophysics↗

bioRxiv2021

An unconventional GABAergic circuit differently controls pyramidal neuron activity in two visual cortical areas via endocannabinoids

Perisomatic inhibition of neocortical pyramidal neurons (PNs) coordinates cortical network activity during sensory processing, and it has been mainly attributed to parvalbumin-expressing basket cells (BCs). However, cannabinoid receptor type 1 (CB1)-expressing interneurons also inhibit the perisomatic region of PNs but the connectivity and function of these elusive - yet prominent - neocortical GABAergic cells is unknown. We found that the connectivity pattern of CB1-positive BCs strongly differs between primary and high-order cortical visual areas. Moreover, persistently active CB1 signaling suppresses GABA release from CB1 BCs in the medial secondary visual cortex (V2M), but not in the primary (V1) visual area. Accordingly, in vivo, tonic CB1 signaling is responsible for higher but less coordinated PN activity in V2M than in V1. Our results indicate a differential CB1-mediated mechanism controlling PN activity, and suggest an alternative connectivity schemes of a specific GABAergic circuit in different cortical areas

neuroscience↗

bioRxiv2020

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER). Relatively little is known about how sites of LD formation are selected, and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)-synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER-LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG synthases and additional LD biogeneiss factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG. We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.

cell biology↗

bioRxiv2020

Alterations of specific inhibitory circuits of the prefrontal cortex underlie abnormal network activity in a mouse model of Down syndrome

Down syndrome (DS) results in various degrees of cognitive deficits. In DS mouse models, recovery of behavioral and neurophysiological deficits using GABAAR antagonists led to hypothesize an excessive activity of inhibitory circuits in this condition. Nonetheless, whether over-inhibition is present in DS and whether this is due to specific alterations of distinct GABAergic circuits is unknown. In the prefrontal cortex of Ts65Dn mice (a well-established DS model), we found that the dendritic synaptic inhibitory loop formed by somatostatin-positive Martinotti cells (MCs) and pyramidal neurons (PNs) was strongly enhanced, with no alteration of their excitability. Conversely, perisomatic inhibition from parvalbumin-positive (PV) interneurons was unaltered, but PV cells of DS mice lost their classical fast-spiking phenotype and exhibited increased excitability. These microcircuit alterations resulted in reduced pyramidal-neuron firing and increased phase locking to cognitive-relevant network oscillations in vivo. These results define important synaptic and circuit mechanisms underlying of cognitive dysfunctions in DS.

neuroscience↗

bioRxiv2017

Lipid droplet and peroxisome biogenesis occur at the same ER subdomains.

Nascent lipid droplet (LD) formation occurs in the ER membrane1-4. It is not known whether LD biogenesis occurs stochastically in the ER or at subdomains with unique protein and lipid composition. We previously identified ER subdomains in S. cerevisiae that contain Pex30, a reticulon-like ER-resident membrane protein5. There are ~25 Pex30-containing puncta in the ER per cell. These sites are regions where preperoxisomal vesicles (PPVs) are generated5. Here we show that Pex30 subdomains are also the location where most nascent LDs form. Mature LDs remain associated with Pex30 subdomains and the same Pex30 subdomain can simultaneously associate with a LD and a PPV. Pex30 subdomains become highly enriched in diacylglycerol (DAG) during LD biogenesis, indicating they have a unique lipid composition. We find that in higher eukaryotes multiple C2 domain containing transmembrane protein (MCTP2) is the functional homologue of Pex30; MCTP2 resides in ER subdomains where most nascent LD biogenesis occurs and that are often associated with peroxisomes. Together, these findings indicate that most LDs and PPVs form and remain associated with conserved ER subdomains and suggest a link between LD and peroxisome biogenesis.

cell biology↗