Biology subjects

Bajpai, A.

Publications and source records attributed to Bajpai, A..

13 recordsLinked to original sources

bioRxiv2026

A multi-electrode array and immunofluorescence workflow to characterize impacts of HHV-6 infection in induced pluripotent stems cells differentiated to mixed neuronal endpoints

Roseoloviruses, notably human herpesviruses 6A and 6B (HHV-6A and HHV-6B), are neurotropic viruses implicated as agents in neurological disorders, including: epilepsy, multiple sclerosis, and chronic fatigue syndrome. However, the effects of roseolovirus infection on neuronal signaling and network activity are not characterized. This is, in part, due to the complexities of monitoring electrical activity in individual neurons and across neuronal connections during viral infection. This protocol describes a human induced pluripotent stem cell (iPSC)-derived neuronal culture system that employs multi-electrode array (MEA) recordings to study neurophysiological changes during viral infection. Two culture platforms are described: (a) NGN2-induced forebrain neuronal cultures; and, (b) progenitor cell-derived neuron-astrocyte mixed cultures. Cell composition in cultures is validated by immunofluorescence staining using neuronal, glial, and viral markers. Functional activity is assessed using extracellular recordings detected via the MEA2100 system. Mean firing rate, inter-spike interval, single-electrode bursting, and network burst activity are characterized between roseolovirus-infected versus uninfected (control) states. Pharmacological treatment with bicuculline, gabazine, and nicotine in conjunction with immunofluorescence is used to confirm functional responsiveness of defined neuronal neurotransmitter chemotypes. Here, we show that HHV-6A infection changes neuronal firing compared to uninfected controls. The methods/workflows described provide a strategy to study how virus infection alters neuronal excitability and may be adapted to compare the effects of different viruses on nerve cell function, infection time courses, different multiplicities of infection (MOI), and therapeutic interventions.

microbiology↗

bioRxiv2026

SARS-CoV-2 PLpro Drives Epithelial Barrier Disruption Across Drosophila and Mammalian Epithelia

Severe SARS-CoV-2 infection manifests as a systemic disorder characterized by catastrophic loss of epithelial homeostasis and dysregulated host inflammation. However, which specific viral factors initiate this tissue damage in intact, living organisms remains unresolved. Using a comprehensive in vivo screen of all 25 SARS-CoV-2 nonstructural proteins (NSPs) and accessory factors (ORFs) in the Drosophila wing imaginal epithelium, here we have identified NSP5 (Main protease, Mpro) and NSP3--specifically its papain-like protease (PLpro) domain--as individual triggers of tissue injury. Focusing further on PLpro, we demonstrate that its expression initiates a self-amplifying host cell stress signaling circuit driven by metabolic overdrive, oxidative stress, and hyperactivation of Akt, JNK, and JAK-STAT signaling. However, this pathogenic loop is fragile; genetic suppression of any of the hyperactivated signaling nodes collapses this network, thereby restoring epithelial homeostasis. This organ-intrinsic, PLpro-induced epithelial vulnerability is also seen in the adult midgut and larval respiratory system. Notably, in the larval respiratory system, PLpro drives compartment-specific pathologies: it induces fibrosis-like remodeling in terminally differentiated squamous tracheal tubes, while catastrophically depleting the progenitor-competent tracheoblasts and air sac precursors (ASP). Both these defects are reversed by the genetic knockdown of PLpro-induced JAK-STAT signaling. Finally, we show that PLpro expression in mammalian MDCK epithelia similarly triggers junctional remodeling and inflammatory stress signaling. Our findings position PLpro as a central driver of COVID-19 pathogenesis through a self-amplifying epithelial stress circuit that compromises epithelial homeostasis across organs and species.

cell biology↗

bioRxiv2025

Systematic Comparative Analysis of Pathway Databases from a User-Perspective

Pathway Databases are essential particularly in the post-genomic era as they provide organized information about biological pathways and processes. However, there are too many pathway databases with varying features. These variations, stemming from multiple factors, create challenges for users in making objective selections among the databases. Hence, there is a need to systematically compare these databases from a users perspective. Indeed, there is a need to compile all such databases first. We first compiled pathway databases through a thorough literature search and shortlisted those suitable for human pathway studies. We designed a query set consisting of genes, pathways, and conditions and performed a quantitative comparison of the output from these shortlisted databases. Furthermore, to assess update frequency among the pathway databases, we curated potential novel genes involved in the MAPK pathway and compared them with the information available in pathway databases. The quantitative comparison of pathway databases showed that PathDIP yielded the highest number of pathways when queried with gene and condition names, followed by other databases. However, when queried for pathway names, Reactome yielded the highest number of pathways, followed by PathDIP, KEGG, and others. The results also support the notion that most scientists may have been making very arbitrary choice of pathway databases.

bioinformatics↗

bioRxiv2023

In silico and in vitro studies suggest epigallocatechin gallate (EGCG), a polyphenol in green tea, can bind and modulate the aggregation and cytotoxicity of the full-length TDP-43 protein implicated in TDP-43 proteinopathies.

Misfolding and aggregation of TDP-43 protein are implicated in several proteinopathies like ALS and FTLD. Extracellular TDP-43 is also proposed to propagate in a prion-like pathogenic manner to the neighbouring cells. Here, using turbidity and sedimentation assay, we show that a polyphenol in green tea, epigallocatechin gallate (EGCG), can inhibit the in vitro aggregation of the full-length TDP-43 protein. Furthermore, Alexa-Fluor-labelled TDP-43 protein failed to show aggregates in the presence of EGCG in fluorescence microscopy. Also, AFM imaging revealed that EGCG co-incubation with TDP-43 allows formation of only small oligomers in contrast to the larger TDP-43 aggregates formed otherwise. A physical binding of EGCG with TDP-43 was observed using triphenyl tetrazolium chloride (TTC) staining and isothermal titration calorimetry (ITC). ITC also revealed a high-affinity binding site for EGCG on TDP-43 with a Kd value of 7.8 {micro}M and a binding free energy of -6.9 kcal/mol. Furthermore, in silico molecular docking and molecular dynamic simulation (MDS) studies using different available structures of the N-terminal, RRM1-2 and C-terminal domains of TDP-43, predicted a preferable and stable binding of EGCG to the structure of the aggregation prone C-terminal domain (CTD) (PDB ID:7KWZ). Also, EGCG complexed with CTD of TDP-43 yielded a negative {Delta}G value of -20.29 kcal/mol using MM-PBSA analysis of the MDS data thereby further suggesting a stable complex formation. Also, in MDS, EGCG interacted with the amino acids Phe-313 and Ala-341 of TDP-43, which were previously projected to be important for the recruitment of monomers for the amyloid formation by CTD, thereby suggesting a possible mechanism of EGCGs inhibition of the TDP-43 aggregation. Notably, while the in vitro-made aggregates of full-length TDP-43 caused mild cytotoxicity to the HEK293 cells, the small oligomers of TDP-43 formed in presence of EGCG did not. In totality, EGCG can in vitro interact with TDP-43 and inhibit its aggregation, possibly via interaction with the amyloidogenic domain, thereby preventing it from assuming cytotoxic conformations. As EGCG is a natural molecule, it could be relevant to the therapeutic quest against the TDP-43 proteinopathies.

biochemistry↗

bioRxiv2023

Loss of Myeloid Cell-Specific β2-Adrenergic Receptor Expression Ameliorates Cardiac Function and Remodeling after Acute Ischemia

Myeloid cells, including neutrophils, monocytes and macrophages, accumulate quickly after ischemic injury in the heart where they play integral roles in the regulation of inflammation and repair. We previously reported that deletion of {beta}2-adrenergic receptor ({beta}2AR) in all cells of hematopoietic origin resulted in generalized disruption of immune cell responsiveness to injury, but with unknown impact on myeloid cells specifically. To investigate this, we crossed floxed {beta}2AR (F/F) mice with myeloid cell-expressing Cre (LysM-Cre) mice to generate myeloid cell-specific {beta}2AR knockout mice (LB2) and subjected them to myocardial infarction (MI). Via echocardiography and immunohistochemical analyses, LB2 mice displayed better cardiac function and less fibrotic remodeling after MI than the control lines. Despite similar accumulation of myeloid cell subsets in the heart at 1-day post-MI, LB2 mice displayed reduced numbers of Nu by 4 days post-MI, suggesting LB2 hearts have enhanced capacity for Nu efferocytosis. Indeed, bone marrow-derived macrophage (BMDM)-mediated efferocytosis of Nu was enhanced in LB2-versus F/F-derived cells in vitro. Mechanistically, several pro-efferocytosis-related genes were increased in LB2 myeloid cells, with annexin A1 (Anxa1) in particular elevated in several myeloid cell types following MI. Accordingly, shRNA-mediated knockdown of Anxa1 in LB2 bone marrow prior to transplantation into irradiated LB2 mice reduced Mac-induced Nu efferocytosis in vitro and prevented the ameliorative effects of myeloid cell-specific {beta}2AR deletion on cardiac function and fibrosis following MI in vivo. Altogether, our data reveal a previously unrecognized role for {beta}2AR in the regulation of myeloid cell-dependent efferocytosis in the heart following injury.

immunology↗

bioRxiv2023

Aging-associated Decline in Vascular Smooth Muscle Cell Mechanosensation is Mediated by Piezo1 Channel

Aging of the vasculature is associated with detrimental changes in vascular smooth muscle cell (VSMC) mechanosensitivity to extrinsic forces in their surrounding microenvironment. However, how chronological aging alters VSMCs ability to sense and adapt to mechanical perturbations remains unexplored. Here, we show defective VSMC mechanosensation in aging measured with ultrasound tweezers-based micromechanical system, force instantaneous frequency spectrum and transcriptome analyses. The mechanobiological study reveals that aged VSMCs adapt a relatively inert solid-like state with altered actin cytoskeletal integrity, resulting in an impairment in their mechanosensitivity and dynamic mechanoresponse to mechanical perturbations. The aging-associated decline in mechanosensation behaviors is mediated by hyperactivity of Piezo1-dependent calcium signaling. Inhibition of Piezo1 alleviates vascular aging and partially restores the loss in dynamic contractile properties in aged cells. Altogether, our study reveals the novel signaling pathway underlying aging-associated aberrant mechanosensation in VSMC and identifies Piezo1 as a potential therapeutic mechanobiological target to alleviate vascular aging.

bioengineering↗

bioRxiv2022

Sex- and GABAergic-modulated neuronal subnetwork assemblies in the developing somatosensory cortex

Neuronal subnetworks, also known as ensembles, are functional units formed by interconnected neurons for information processing and encoding in the adult brain. Our study investigates the establishment of neuronal subnetworks in the mouse primary somatosensory (S1) cortex from postnatal days (P)11 to P21 using in vivo two-photon calcium imaging. We found that at P11, neuronal activity was highly synchronized but became sparser by P21. Clustering analyses revealed that while the number of subnetworks remained constant, their activity patterns became more distinct, with increased coherence, independent of cortical layer or sex. Furthermore, the coherence of neuronal activity within individual subnetworks significantly increased when synchrony frequencies were reduced by augmenting GABAergic activity at P15/16, a period when the neuronal subnetworks were still maturing. Together, these findings indicate the early formation of subnetworks and underscore the pivotal roles of GABAergic inputs in modulating S1 neuronal subnetworks.

neuroscience↗

bioRxiv2022

Human ERG Oncoprotein Represses Chip/LDB1 LIM-Domain Binding Gene in Drosophila

ERG oncoprotein, a master transcription factor, targets diverse arrays of genes in different cancers. Identifying oncogenically relevant ones from these ERG targets, however, is challenging. Here we show that heterologous ERG disrupts a LIM-homeodomain (LIM-HD) complex, Chip-Tailup, in Drosophila. In the posterior thorax (notum) primordium, ERG-induced upregulation of E(z)/EZH2 trimethylates histones in Chip promoter. A consequent loss of the Chip-Tailup complex releases repression of N-Wg signaling in the notum, inducing de novo wings and, alternatively, carcinogenesis of ERG-expressing notal cells displaying loss of Lgl tumor suppressor. ERG-induced developmental or oncogenic fallouts are abrogated upon gain of Chip, N, or E(z) loss, besides Wg ligand sequestration. ERG-positive prostate cancer (PCa) cells, too, display suppression of mammalian homolog of Drosophila Chip, LIM Domain Binding1, LDB1. Deep homology in gene regulatory networks, like that of Chip-Tup complex, thus help prioritize identification of functionally relevant targets of human oncoproteins in Drosophila. HighlightsO_LIHuman ERG suppresses Chip, a LIM-domain binding, LDB gene in Drosophila via E(z) C_LIO_LIERG-mediated Chip loss induces ectopic Wg morphogen signaling in the notum primordium C_LIO_LIChip gain suppresses ERG-induced Wg morphogen and tumor progression in lgl clones C_LIO_LIERG-positive human PCa cell lines show downregulation of a Chip homolog, LDB1 C_LI In briefMammalian ERG oncoprotein displays a diverse and perplexing range of targets in different cancers. By driving ERG in Drosophila developing appendages, Bharti et al. reveal its repression of a LIM-domain coding gene, Chip/LDB1. ERG-positive prostate cancer cells, too, display Chip/LDB1 repression. Deep homology across phylogeny thus helps uncover oncoprotein targets.

developmental biology↗

bioRxiv2021

Stress-responsive Long Non-coding RNA, hsrω, is a genetic modifier of JNK-dependent Intrinsic Tumor Suppression in Drosophila

Host genetics is known to influence cancer susceptibility. However, the specific candidate genes and molecular mechanisms that confer resistance remain poorly understood. Here, we demonstrate the power of haploinsufficiency screen to uncover host genetic regulators of cancer in Drosophila and identify the long noncoding RNA (lncRNA) hsr{omega}, a structural component of nuclear biomolecular condensate known as omega speckles, as a key host cancer susceptibility locus. Loss of hsr{omega} disrupts proteostasis and cell fitness, while its haploinsufficiency accelerates epithelial tumor progression driven by loss of the Lethal giant larvae (Lgl) tumor suppressor. Further validating the breadth of this screening strategy, we independently identified Drosophila STING (innate immunity) and Keap1 (oxidative stress defense) as genetic modifiers of cancer. Moreover, in humans, copy number variations (CNV) in these genes and Sat III (a functional human homolog of hsr{omega}) correlate with poor cancer prognosis, thereby revealing conserved stress pathways as potential host genetic susceptibility regulators.

cancer biology↗

bioRxiv2021

A Serpin27A-dependent Toll Signaling Underlies Host Genetics of Cancer Resistance in Drosophila

Cancer resistance varies amongst individuals, although its host genetic underpinnings remain largely elusive. Remissions of sarcomas were first reported following repeated injections of patients with mixtures of killed bacteria--Coleys toxins--a phenomenon, which was subsequently causally traced to induction of innate immunity. Here we reveal remission of Drosophila epithelial neoplasms by genetically triggered host innate immunity via Toll signaling. These neoplasms display capacities to receive and, in rare instances, induce Toll signaling. A tumor-induced and progressive Toll signaling, however, did not culminate in tumor suppression. By contrast, Drosophila hosts heterozygous for spn27A1 mutation, which constitutively produce activated Toll ligand, SpzAct, displayed comprehensive tumor remission via Toll-induced, NF-{kappa}B-mediated, tumor cell death. Our results reveal a novel node of host genetic cancer resistance via serpin-dependent Toll signaling.

cancer biology↗

bioRxiv2020

A Drosophila model of oral peptide therapeutics for adult intestinal stem cell tumors

The proto-oncogene YAP /Yki, a transcription co-factor of the Hippo pathway, has been linked to many cancers. YAP interacts with DNA-binding TEAD/Sd proteins to regulate expression of its transcriptional targets. Disruption of YAP-TEAD therefore offers a potential therapeutic strategy. The mammalian Vestigial Like (VGLL) protein, specifically its TONDU domain, has been shown to competitively inhibit YAP-TEAD interaction and a TONDU peptide can suppress YAP-induced cancer. As TONDU could potentially be developed into a therapeutic peptide for multiple cancers, we evaluated its efficacy in Yki-driven adult Intestinal Stem Cell (ISC) tumors in Drosophila. We show that oral uptake of the TONDU peptide is highly effective at inhibiting Yki-driven gut tumors by suppressing YAP-TEAD interaction. Comparative proteomics of early and late stage Yki-driven ISC tumors revealed enrichment of a number of proteins, including members of the integrin signaling pathway, such as Talin, Vinculin and Paxillin. These, in turn displayed a decrease in their levels in TONDU-peptide treated tumors. Further, we show that Sd binds to the regulatory region of integrin-coding gene, mew, which codes for PS1, a key integrin of the ISCs. In support to a possible role of integrins in Yki-driven ISC tumors, we show that genetic downregulation of mew arrests Yki-driven ISC proliferation, reminiscent of the effects of TONDU peptide. Altogether, our findings present a novel platform for screening therapeutic peptides and provide insights into tumor suppression mechanisms. SIGNIFICANCE STATEMENTDiscovering novel strategies to inhibit oncogene activity is a priority in cancer biology. As signaling pathways are widely conserved between mammals and Drosophila, these questions can be effectively addressed in this model organism. Here, we show that progression of Drosophila Intestinal Stem Cell (ISC) tumors induced by gain of an oncogenic form of the transcription co-factor Yki can be suppressed by feeding a peptide corresponding to the conserved TONDU domain of Vestigial (Vg), which blocks binding of Yki to the Sd transcription factor. Further, we show that down regulation of the integrin signaling pathway is causally linked to TONDU-peptide-mediated ISC tumor suppression. Our findings reveal that Drosophila can be successfully used to screen peptides for their therapeutic applications.

cancer biology↗

bioRxiv2018

Single cell variations in expression of codominant alleles A and B on RBC of AB blood group individuals

One of the key questions in biology is whether all cells of a "cell type" have more or less the same phenotype, especially with relation to non-imprinted autosomal loci. Recent studies point to differential allelic expression of autosomal genes being a prevalent phenomenon responsible to confer phenotypic variability at individual cell level. However, most studies have been carried out in actively transcribing cells. Here we display cellular mosaicism arising from differential allelic expression for the cell surface glycoprotein in the enucleated RBCs. We studied the expression of the A and B histo-blood group antigens encoded by the co-dominant alleles in individual RBCs using immunofluorescence. We assessed the relative levels of the co-dominant alleles IA and IB in 2512 RBC from 24 individuals with AB blood group using Cy3- and FITC- tagged antibodies. Quantification of individual fluorescence intensities from each cell and test of their normal distribution revealed that contrary to the general belief that all RBC in AB individuals express both antigens in comparable amounts, they segregated into 4 groups: showing normal distribution for both antigens, either antigen, and neither antigen; the deviation from normal distribution could not be correlated to maternal/paternal origin, thus appear to be stochastic. Surprisingly, very few people showed any correlation between the amounts of these two antigens on RBC. In fact, the ratio of antigen A to B in the entire set of samples spanned over 5 orders of magnitude. This variability in amount of the antigens A and/or B, combined with a lack of correlation between the amounts of these two antigens resulted in unique staining patterns for RBC, generating widespread mosaicism in the RBC population of AB blood group individuals.

genetics↗

bioRxiv2018

Phenylhydrazones are active against non-replicating Mycobacterium tuberculosis

There is an urgent need for the development of shorter, simpler and more tolerable drugs to treat antibiotic tolerant populations of Mycobacterium tuberculosis. We previously identified a series of phenylhydrazones (PHY) active against M. tuberculosis. We selected six representative compounds for further analysis. All compounds were active against non-replicating M. tuberculosis, with two compounds demonstrating greater activity under hypoxic conditions than aerobic culture. Compounds had bactericidal activity with MBC/MIC of <4 and demonstrated an inoculum-dependent effect against aerobically replicating bacteria. Bacterial kill kinetics demonstrated a faster rate of kill against non-replicating bacilli generated by nutrient starvation. Compounds had limited activity against other bacterial species. In conclusion, we have demonstrated that the PHY compounds have some attractive properties in terms of their anti-tubercular activity.

microbiology↗