Biology subjects

Verma, J.

Publications and source records attributed to Verma, J..

14 recordsLinked to original sources

bioRxiv2026

FAST-MaP: Chemical Mapping of RNA Structures Using Primer-less Sequencing

RNA structure governs the function of non-coding RNAs and influences mRNA stability and translation, making testing of models of experimental RNA structure relevant to fields spanning structural biology, virology, and molecular therapeutics. Here we present FAST-MaP (Fast and Accessible Sequencing Technology for Mutational Profiling), a protocol that enables per-nucleotide RNA structure characterization using standard molecular biology equipment and requiring no sequencing infrastructure or bioinformatics expertise. RNA is chemically modified with orthogonal probes (2A3 and DMS), reverse-transcribed, and PCR-amplified to produce dsDNA amplicons that are submitted directly to a commercial primer-less sequencing service. Returned FASTQ files are processed through a freely accessible web server to generate normalized reactivity profiles within minutes. The complete protocol, from DNA template to structural data, can be completed in approximately one week. We illustrate the workflow on a 659-nucleotide RNA, demonstrating how to test structure preservation across buffers, and how to test specific secondary and tertiary structure predictions of the RNA from computational modeling or cryo-electron microscopy. The protocol requires only standard molecular-biology skills and does not require sequencing or bioinformatics expertise.

biochemistry↗

bioRxiv2026

Molecular Determinants of Allosteric Inhibitor Affinity and Selectivity in PDE5

Allosteric inhibitors targeting unique regulatory pockets provide an opportunity for selective modulation of proteins within the phosphodiesterase (PDE) family. In this work, we investigate the molecular determinants for binding of an evodiamine derivative (EVO) to PDE5 and the structural basis for differential recognition of this inhibitor in PDE6 isoforms. We integrated structural modeling, all-atom molecular dynamics (MD) simulations, and extensive alchemical free-energy calculations to quantify residue-level contributions to inhibitor binding in PDE5 through systematic alanine scanning and PDE6-derived mutations. Thermodynamic analysis based on free energy identified key residues that govern EVO binding through a network of hydrogen bonds and hydrophobic contacts. Specifically, we examined structural changes associated with allosteric pocket variations in PDE5, identified key structural elements, and evaluated the role of chemical substituents of EVO in allosteric inhibition. Moreover, PDE6-derived substitutions revealed isoform specific effects, with PDE6-rod substitutions resulting in stronger destabilization than PDE6-cone substitutions. Together, these results define the energetic and structural basis for allosteric inhibition of PDE5 and provide mechanistic insights for the rational design of selective PDE inhibitors. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712116v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@5934b4org.highwire.dtl.DTLVardef@9346d7org.highwire.dtl.DTLVardef@10fcd8corg.highwire.dtl.DTLVardef@7fc5c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Scalable single-cell metagenomic analysis with Bascet and Zorn

Single-cell metagenomic sequencing (scMetaG) can provide maximum-resolution insights into complex microbial communities. However, existing bioinformatic tools are not equipped to handle the massive amounts of data generated by novel high-throughput scMetaG methods. Here, we present a bioinformatic toolkit for complete, end-to-end scMetaG analysis: (i) Bascet, a command-line suite designed to scale to massive scMetaG datasets ([≥]1 million cells); (ii) Zorn, an R package/workflow manager that enables reproducible scMetaG data analysis, exploration, and visualization (http://zorn.henlab.org/). Enabled by recent advances in droplet microfluidics, we use Bascet and Zorn to develop and optimize a high-throughput scMetaG method on a ten-species mock community. To showcase their utility on a real-world sample, we use Bascet and Zorn to characterize a human saliva sample, generating single-amplified genomes (SAGs) from >10k prokaryotic cells. Overall, Bascet and Zorn enable reproducible scMetaG analysis, allowing users to query microbiomes at unprecedented resolution and scale.

microbiology↗

bioRxiv2025

Biofabrication of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves with their anticancer, antibacterial, and antioxidant activities.

In this study, we describe the cytotoxic and antibacterial capabilities of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves. Here, we used an environmentally friendly method to synthesize GNPs and then characterized them using techniques such as transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible spectroscopy, XRD, and Fourier transform infrared spectroscopy. The Surface Plasmon Resonance of GNPs was identified by an absorption peak at 533 nm in the UV-visible spectroscopic studies. The biosynthesized GNPs had an average size of 83 nm and were spherical in shape, according to TEM examination. Its interesting to note that the biosynthesized GNPs exhibited antibacterial action against a variety of bacterial isolates, both Gram-positive and negative. By using an MTT assay, growth inhibition and the cytotoxic effect of the synthesized GNPs against Daltons lymphoma cell lines were also evaluated.

plant biology↗

bioRxiv2025

Conformational Dynamics in Insulin Receptor Kinase Reveals a Type III Allosteric Pocket

Allosteric modulation of kinases is a promising approach for pharmacological intervention, particularly for designing selective kinase modulators. However, the structural information on allosteric pockets remains limited for most kinases in the human kinome. In this work, we present comprehensive characterization of a type III allosteric pocket in the insulin receptor kinase (IRK) by uncovering its structural features and conformational dynamics. Specifically, we used microsecond-scale atomistic molecular dynamics (MD) simulations to investigate apo and inhibitor-bound IRK structures. Our findings suggest that the type III allosteric site is a "back pocket" in IRK, sandwiched between the N-terminal and the C-terminal lobes, beneath the C-helix and the {beta} - sheets of the N-terminal lobe. It has a hydrophobic cleft composed of both aliphatic and aromatic non-polar residues and a charge center to facilitate electrostatic interactions. We explored the binding of an experimentally known IRK inhibitor to the newly discovered allosteric pocket. Our results indicate that the C-helix adopts an "out" conformation stabilized by the inhibitor which promotes an inactive conformation of the kinase. Furthermore, we observed a helical intermediate formation in the activation loop and a stable "DFG-out" conformation in both inhibitor-bound and unbound states. Our results also suggest that the residue M1051 in IRK functions as a gatekeeper residue, essential for maintaining the structural integrity of the C-helix and regulating the binding of the allosteric inhibitor. Our findings are relevant for developing allosteric IRK modulators and informing therapeutic strategies targeting proteins in the insulin receptor family.

biophysics↗

bioRxiv2024

Conserved ancillary residues situated proximally to the VIM-2 active site affect its metallo β-lactamase activity

Verona-integron-metallo-{beta}-lactamase (VIM-2) is one of the most widespread class B {beta}-lactamase, responsible for {beta}-lactam resistance. Although active-site residues help in metal binding, the residues nearing the active-site possess functional importance. Here, to decipher the role of such residues in the activity and stability of VIM-2, the residues E146, D182, N210, S207, and D213 were selected through in-silico analyses and substituted with alanine using site-directed mutagenesis. The effects of substitution mutations were assessed by comparing the changes in the {beta}-lactam susceptibility pattern of E. coli host cells expressing VIM-2 and its mutated proteins. VIM-2_N210A enhanced the susceptibility of the host by [~]4-8 folds against penicillins and cephalosporins while the expression of VIM-2_D182A radically increased the susceptibility of the host. However, expression of VIM-2_E146A reduced the susceptibility of the host by 2-fold. Further, proteins were purified to homogeneity, and VIM_N210A and VIM_D182A displayed reduced thermal stability than VIM-2. Moreover, in vitro catalytic efficiencies of VIM-2_D182A were drastically reduced against all the {beta}-lactams tested whereas the same were moderately reduced for VIM-2_N210A. Conversely, the catalytic efficiency was marginally altered for VIM_E146A. Overall, we infer that both N210A and D182A substitutions negatively affect the performance of VIM-2 by influencing substrate specificity and stability, respectively.

microbiology↗

bioRxiv2024

(p)ppGpp and DksA play crucial role in reducing the efficacy of b-lactam antibiotics by modulating bacterial membrane permeability

The key signaling molecules in the bacterial stress sensing pathway, the alarmone (p)ppGpp and transcription factor DksA, help in survival during nutritional deprivation and exposure to xenobiotics by modulating cellular metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked with the functions of three proteins: RelA, SpoT, and RelV. At threshold or elevated concentrations of (p)ppGpp, the level of cellular metabolites and proteins in the presence and absence of DksA in V. cholerae and other bacteria has not yet been comprehensively studied. We engineered the genome of V. cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp biosynthetic enzymes. We observed a higher sensitivity of the (p)ppGpp0{Delta}dksA V. cholerae mutant to different -lactam antibiotics compared to the wild-type (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the cell membrane and peptidoglycan biosynthesis pathways are significantly altered in the (p)ppGpp0, {Delta}dksA, and (p)ppGpp0{Delta}dksA V. cholerae strains. Further, the mutant strains displayed enhanced inner and outer membrane permeability in comparison to the WT strains. These results directly correlate with the tolerance and survival of V. cholerae to -lactam antibiotics. These findings may help in the development of adjuvants for -lactam antibiotics by inhibiting the functions of stringent response modulators. ImportanceThe (p)ppGpp biosynthetic pathway is widely conserved in bacteria. Intracellular levels of (p)ppGpp and the transcription factor DksA play crucial roles in bacterial multiplication and viability in the presence of antibiotics and/or other xenobiotics. The present findings have shown that (p)ppGpp and DksA significantly reduces the efficacy of -lactam and other antibiotics by modulating the availability of peptidoglycan and cell membrane-associated metabolites by reducing membrane permeability. Nevertheless, the whole-cell proteome analysis of (p)ppGpp0, {Delta}dksA, and (p)ppGpp0{Delta}dksA strains identified the biosynthetic pathways and associated enzymes that are directly modulated by the stringent response effector molecules. Thus, the (p)ppGpp metabolic pathways and DksA could be a potential target for increasing the efficacy of antibiotics and developing antibiotic adjuvants.

microbiology↗

bioRxiv2024

PAN deadenylase is required for mitosis in response to microtubule stress in yeast and humans

HIGHLIGHTSO_LIPAN2-PAN3 catalytic activity is required for mitotic robustness under MT stress C_LIO_LICatalytic-dead Pan2-D1020A phenocopies pan2{Delta}: spindle defects and G2/M arrest C_LIO_LILoss of Pan2 catalytic activity amplifies nocodazole-induced changes in CLB1 and CLN3 mRNA C_LIO_LIPAN2 depletion in human cells produces multipolar spindles under MT stress C_LI IN BRIEFVerma et al. show that PAN2-PAN3 deadenylase catalytic activity is selectively required for mitotic fidelity under microtubule stress. Yeast pan2{Delta} cells exhibit spindle defects, G2/M arrest, and altered CLB1 and CLN3 mRNA levels under nocodazole. PAN2 knockdown in human cells produces multipolar spindles, demonstrating evolutionary conservation. Cytoplasmic deadenylation by PAN2-PAN3 and CCR4-NOT regulates mRNA stability and translation, but the contribution of individual deadenylases to mitotic progression is poorly defined. Here we show that PAN2-PAN3 catalytic activity is selectively required for mitotic robustness under microtubule stress. In budding yeast, the catalytically inactive Pan2-D1020A mutant fails to complement pan2{Delta} nocodazole sensitivity, leading to G2/M arrest, spindle defects, and increased cell death. pan2{Delta} exhibits synthetic genetic interactions with tubulin and tubulin-folding genes, and RNA-seq reveals dysregulation of cell-cycle transcripts including the cyclins CLB1 and CLN3. The phenotype is stress-conditional: pan2{Delta} cells proliferate normally without drug. In mammalian cells, PAN2 depletion sensitizes HEK293A cells to nocodazole and colchicine and produces prolonged metaphase with multipolar spindles. Together, these findings define a conserved, stress-conditional role for PAN2-PAN3 deadenylase activity in safeguarding mitotic fidelity when microtubules are compromised. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/591760v3_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19e6662org.highwire.dtl.DTLVardef@53ba18org.highwire.dtl.DTLVardef@de9c99org.highwire.dtl.DTLVardef@fb0f9d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO See accompanying graphical abstract file. C_FIG

cell biology↗

bioRxiv2024

Structural Models for a Series of Allosteric Inhibitors of IGF1R Kinase

The allosteric inhibition of Insulin-like Growth Factor Receptor 1 Kinase (IGF1RK) is a potential strategy to overcome selectivity barriers in targeting receptor tyrosine kinases. We constructed structural models of a series of 12 indole-butyl-amine derivatives which have been reported as allosteric inhibitors of IGF1RK. We further studied dynamics and interactions of each inhibitor in the allosteric pocket via all-atom explicit-solvent molecular dynamics (MD) simulations. We discovered that a bulky carbonyl substitution at the R1 indole ring is structurally unfavorable for inhibitor binding in the IGF1RK allosteric pocket. Moreover, we found that the most potent derivative (termed C11) acquires a distinct conformation, forming an allosteric pocket channel with better shape complementarity and interactions with the receptor. In addition to a hydrogen bonding interaction with V1063, the cyano derivative C11 forms a stable hydrogen bond with M1156, which is responsible for its unique binding conformation in the allosteric pocket. Our findings show that the position of chemical substituents at the R1 indole ring with different pharmacophore features influences molecular interactions and binding conformations of the indole-butyl-amine derivatives, hence dramatically affecting their potencies. Our results provide a structural framework for the design of allosteric inhibitors with improved affinities and specificities against IGF1RK.

biophysics↗

bioRxiv2023

Computational Modeling of Hyperpolarizing Astrocytic Influence on Cortical Up-Down State Transitions

The Up-Down dynamics seen in cortical structures during non-rapid-eye-movement (NREM) sleep, anesthetized states, and quiet wakefulness is the spontaneous alternation between phases of heightened firing activity (referred to as the Up state) and periods of neuronal inactivity (termed the Down state) within neural networks. By leveraging bistable dynamics imposed by a depolarising astrocyte population, in the current paper, we introduced a hyperpolarising astrocyte population to an existing model of Up-Down dynamics to account for biological relevance. We created a computational rate model that includes populations of depolarizing and hyperpolarizing astrocytes and neurons. To optimize model parameters, we used the Elementary Effects (EE) test. It was followed by linear stability analysis to locate bistable regimes in the parameter hyperspace. The addition of hyperpolarizing gliotransmission perturbed model dynamics, indicating its sensitivity to qualitatively differing architectures. We then identified a bistable regime within the dynamics spectrum. According to the EE test, the strength of cell population coupling is a low-sensitivity parameter, possibly due to neuroplastic changes. We also found that the threshold of excitatory cell populations and the strength of adaptation are high-sensitivity parameters, whereas the threshold of inhibitory cell populations is low-sensitivity. Our model enables the possibility of testing biologically relevant theories of hyperpolarizing gliotransmission, where data remains scant.

neuroscience↗

bioRxiv2022

Structural insight into antibody evasion of SARS-CoV-2 omicron variant

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to mutate and evolve with the emergence of omicron (B.1.1.529) as the new variant of concern. The rapid spread of this variant regionally and globally could be an allusion to increased infectivity, transmissibility, and antibody resistance. The omicron variant has a large set of mutations in its spike protein, specifically in the receptor binding domain (RBD), reflecting their significance in ACE2 interaction and antibody recognition. We have carried out the present study to understand how these mutations structurally impact the binding of the antibodies to their target epitope. We have computationally evaluated the binding of different classes of RBD targeted antibodies, namely, CB6 (etesevimab), REGN10933 (casirivimab), S309 (sotrovimab), and S2X259 to the omicron mutation-induced RBD. Molecular dynamics simulations and binding free energy calculations unveil the binding affinity and stability of the antibody-RBD complexes. All the four antibodies show reduced binding affinity towards the omicron RBD. The therapeutic antibody CB6 aka etesevimab was substantially affected due to numerous omicron mutations occurring in its target epitope. This study provides a structural insight into the reduced efficacy of RBD targeting antibodies against the SARS-CoV-2 omicron variant.

bioinformatics↗

bioRxiv2021

Autophagy-related protein Atg11 preserves asymmetric inheritance of spindle microtubule-organizing centers

Emerging studies hint at the roles of autophagy-related proteins in various cellular processes in a eukaryotic cell. To understand if autophagy-related proteins influence genome stability, we examined a cohort of 35 autophagy mutants in Saccharomyces cerevisiae. We observed cells lacking Atg11 show poor mitotic stability of minichromosomes. Atg11 molecules dynamically localize to the spindle pole bodies (SPBs). Loss of Atg11 leads to a delayed cell cycle progression. Such cells accumulate at metaphase at an elevated temperature that is relieved when the spindle assembly checkpoint is inactivated. Indeed, atg11{Delta} cells have stabilized securin levels, that prevent anaphase onset, confirming chromosome biorientation defects associated with the mutant. Atg11 functions in the Kar9-dependent spindle positioning pathway and maintains Kar9 asymmetry by facilitating proper dynamic instability of astral microtubules (aMTs). Taken together, this study uncovers a non-canonical role of Atg11 in facilitating MT dynamics crucial for chromosome segregation.

genetics↗

bioRxiv2020

Evidence for the plant recruitment of beneficial microbes to suppress soil-borne pathogen

O_LIEmerging experimental framework suggests that plants under biotic stress may actively seek help from soil microbes, but empirical evidence underlying such a cry for help strategy is limited. C_LIO_LIWe used integrated microbial community profiling, pathogen and plant transcriptive gene quantification and culture-based methods to systematically investigate a three-way interaction between the wheat plant, wheat-associated microbiomes and Fusarium pseudograminearum (Fp). C_LIO_LIA clear enrichment of a dominant bacterium, Stenotrophomonas rhizophila (SR80), was observed in both the rhizosphere and root endosphere of Fp-infected wheat. SR80 reached 3.7x107 cells g-1 in the rhizosphere and accounted for up to 11.4% of the microbes in the root endosphere. Its abundance had a positive linear correlation with the pathogen load at base stems and expression of multiple defense genes in top leaves. Upon re-introduction in soils, SR80 enhanced plant growth, both the below- and above-ground, and induced strong disease resistance by priming plant defense in the aboveground plant parts, but only when the pathogen was present C_LIO_LITogether, the bacterium SR80 seems to have acted as an early warning system for plant defense. This work provides novel evidence for the potential protection of plants against pathogens by an enriched beneficial microbe via modulation of the plant immune system. C_LI

microbiology↗

bioRxiv2017

Comparison of one-stage and two-stage genome-wide association studies

Linear mixed models are widely used in humans, animals, and plants to conduct genome-wide association studies (GWAS). A characteristic of experimental designs for plants is that experimental units are typically multiple-plant plots of families or lines that are replicated across environments. This structure can present computational challenges to conducting a genome scan on raw (plot-level) data. Two-stage methods have been proposed to reduce the complexity and increase the computational speed of whole-genome scans. The first stage of the analysis fits raw data to a model including environment and line effects, but no individual marker effects. The second stage involves the whole genome scan of marker tests using summary values for each line as the dependent variable. Missing data and unbalanced experimental designs can result in biased estimates of marker association effects from two-stage analyses. In this study, we developed a weighted two-stage analysis to reduce bias and improve power of GWAS while maintaining the computational efficiency of two-stage analyses. Simulation based on real marker data of a diverse panel of maize inbred lines was used to compare power and false discovery rate of the new weighted two-stage method to single-stage and other two-stage analyses and to compare different two-stage models. In the case of severely unbalanced data, only the weighted two-stage GWAS has power and false discovery rate similar to the one-stage analysis. The weighted GWAS method has been implemented in the open-source software TASSEL.

genetics↗