Biology subjects

Srivastava, S.

Publications and source records attributed to Srivastava, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

A mechanistic understanding of the effect of Staphylococcus aureus VraS histidine kinase single point mutation on antibiotic resistance

Bacterial genomic mutations in Staphylococcus aureus (S. aureus) have been detected in isolated resistant clinical strains, yet their mechanistic effect on the development of antimicrobial resistance remains unclear. The resistance-associated regulatory systems acquire adaptive mutations under stress conditions that may lead to a gain of function effect and contribute to the resistance phenotype. Here, we investigate the effect of a single-point mutation (T331I) in VraS histidine kinase, part of the VraSR two-component system in S. aureus. VraSR senses and responds to environmental stress signals by upregulating gene expression for cell wall synthesis. A combination of enzyme kinetics, microbiological, and transcriptomic analysis revealed the mechanistic effect of the mutation on VraS and S. aureus. Michaelis Mentens kinetics show that the VraS mutation caused an increase in the autophosphorylation rate of VraS and enhanced its catalytic efficiency. The introduction of the mutation through recombineering coupled with CRISPR-Cas9 counterselection to the Newman strain wild-type (WT) genome doubled the minimum inhibitory concentration of three cell wall-targeting antibiotics. The mutation caused an enhanced S. aureus growth rate at sub-lethal doses of the antibiotics, confirming the causative effect of mutation on bacterial persistence. Transcriptomic analysis showed a genome-wide alteration in gene expression levels and protein-protein interaction network of the mutant compared to the WT strain after exposure to vancomycin. The results suggest that vraS mutation causes several mechanistic changes at the protein and cellular levels that favor bacterial survival under antibiotic stress and cause the mutation-harboring strains to become the dominant population during infection. ImportanceRising antimicrobial resistance (AMR) is a global health problem. Mutations in the two- component system have been linked to drug- resistance in Staphylococcus aureus, yet the exact mechanism through which these mutations work is understudied. We investigated the T331I mutation in the vraS gene linked to sensing and responding to cell wall stress. The mutation caused changes at the protein level by increasing the catalytic efficiency of VraS kinase activity. Introducing the mutation to the genome of an S. aureus strain resulted in changes in the phenotypic antibiotic susceptibility, growth kinetics, and genome-wide transcriptomic alterations. By a combination of enzyme kinetics, microbiological, and transcriptomic approaches, we highlight how small genetic changes can significantly impact bacterial physiology and survival under antibiotic stress. Understanding the mechanistic basis of antibiotic resistance is crucial to guide the development of novel therapeutic agents to combat AMR.

microbiology↗

bioRxiv2024

Cell type transcriptional identities are maintained in cultured ex vivo human brain tissue

It is becoming more broadly accepted that human-based models are needed to better understand the complexities of the human nervous system and its diseases. The recently developed human brain organotypic culture model is one highly promising model that requires the involvement of neurosurgeons and neurosurgical patients. Studies have investigated the electrophysiological properties of neurons in such ex vivo human tissues, but the maintenance of other cell types within explanted brain remains largely unknown. Here, using single-nucleus RNA sequencing, we systematically evaluate the transcriptional identities of the various cell types found in six patient samples after fourteen days in culture (83,501 nuclei from day 0 samples and 45,738 nuclei from day 14 samples). We used two pediatric temporal lobectomy samples, an adult frontal cortex sample, two IDH wild-type glioblastoma samples, and one medulloblastoma sample. We found remarkably high correlations of day 14 transcriptional identities to day 0 tissue, especially in tumor cells (r = 0.90 to 0.93), though microglia (r = 0.86), oligodendrocytes (r = 0.80), pericytes (r = 0.77), endothelial cells (r = 0.78), and fibroblasts (r = 0.76) showed strong preservation of their transcriptional profiles as well. Astrocytes and excitatory neurons showed more moderate preservation (r = 0.66 and 0.47, respectively). Because the main difficulty with organotypic brain cultures is the acquisition of human tissue, which is readily available to neurosurgeons, this model is easily accessible to neurosurgeon-scientists and neurosurgeons affiliated with research laboratories. Broad uptake of this more representative model should prompt advances in our understanding of many uniquely human diseases, lead to more reliable clinical trial performance, and ultimately yield better therapies for our patients.

neuroscience↗

bioRxiv2024

Computational prediction of cyclotides from Viola odorata as potential inhibitors against the neuraminidase of Streptococcus pneumoniae

Cyclotides are naturally occurring peptides characterized by a cyclic cystine knot, which provides them with exceptional structural stability. In addition to their stability, cyclotides exhibit diverse therapeutic activities including antimicrobial, antiviral and antitumor activities, making them promising candidates in drug discovery. However, computational studies aimed at identifying cyclotide-based inhibitors for infectious diseases remain limited. To this end, we conducted a virtual screening of cyclotides from an Indian medicinal plant Viola odorata to identify potential inhibitors against a bacterial pathogen causing respiratory infections. We compiled a library of 93 cyclotides by retrieving their structures from public domain or predicting them using the AlphaFold server. We then docked these cyclotides against the neuraminidase protein of Streptococcus pneumoniae and analyzed the interacting residues and binding energies to identify top five potential inhibitors namely, kalata S, kalata B1, cycloviolacin O15, vodo L12, and cycloviolacin O36. Thereafter, we performed molecular dynamics simulations of the protein-cyclotide complexes, and observed that the cyclotides remained stable within the complex. Altogether, this study is the first computational effort to identify potential cyclotide inhibitors against bacterial pathogen causing respiratory diseases, which can be further pursued for experimental validation to develop novel therapeutic agents for respiratory infections.

pharmacology and toxicology↗

bioRxiv2024

Benzene metabolites increase vascular permeability by activating heat shock proteins and Rho GTPases

Benzene is a ubiquitous environmental and occupational pollutant abundant in household products, petrochemicals, and cigarette smoke. It is also a well-known carcinogen and hematopoietic toxin. Population-based studies indicate an increased risk of heart failure in subjects exposed to inhaled benzene, which coincides with the infiltration of immune cells into the myocardium. However, the mechanisms of benzene-induced cardiovascular disease remain unknown. Our data suggests that benzene metabolites trans,trans-muconaldehyde (MA), and hydroquinone (HQ) propagate endothelial activation and apoptosis analyzed by endothelial-specific microparticles in C57BL/6J mice plasma. Subcutaneous injections of MA and HQ increased vascular permeability by 1.54 fold and 1.27 fold correspondingly. In addition, the exposure of primary cardiac microvascular endothelial cells to MA increased vascular permeability detected by transendothelial monolayer resistance and by fluorescently labeled dextrans diffusion. The bulk RNA sequencing of endothelial cells exposed to MA for 2, 6, and 24 hours showed MA-dependent upregulation of heat shock-related pathways at 2 and 6 hours, dysregulation of GTPases at 6 hours, and altered cytoskeleton organization at 24 hours of exposure. We found that the HSP70 protein induced by MA in endothelial cells is colocalized with F-actin foci. HSP70 inhibitor 17AAG and HSP90 inhibitor JG98 attenuated MA-induced endothelial permeability, while HSP activator TRC enhanced endothelial leakage. Moreover, MA induced Rac1 GTPase activity, while Rho GTPase inhibitor Y-27632 attenuated MA-induced endothelial permeability. We showed that benzene metabolites compromised the endothelial barrier by altering HSP- and GTPase-related signaling pathways.

pharmacology and toxicology↗

bioRxiv2024

Common AAV gene therapy vectors show indiscriminate transduction of living human brain cell types

The development of cell-type-specific gene therapy vectors for treating neurological diseases holds great promise, but has relied on animal models with limited translational utility. We have adapted an ex vivo organotypic model to evaluate adeno-associated virus (AAV) transduction properties in living slices of human brain tissue. Using fluorescent reporter expression and single-nucleus RNA sequencing, we found that common AAV vectors show broad transduction of normal cell types, with protein expression most apparent in astrocytes; this work introduces a pipeline for identifying and optimizing AAV gene therapy vectors in human brain samples.

bioengineering↗

bioRxiv2024

Peripheral CGRP engages brain-wide electrical network activity of migraine

BackgroundMigraine is a disorder of severe, recurrent headaches and debilitating sensory, cognitive and affective symptoms, often triggered by stress. Early life stress in childhood has been shown to increase the likelihood of migraine in adulthood in humans. Calcitonin-gene relate peptide (CGRP) has been shown to reliably and acutely induce migraine or migraine-like behavior in both humans and rodent models. Here we investigate the impact of early life stress and CGRP on migraine-related neural circuitry, as well as the impact of early life stress on CGRP-mediated migraine-like activity in order to better understand the mechanisms by which early life stress predisposes neural circuitry to migraine brain activity. MethodsWe implemented an early life stress paradigm in the outbred strain of mice, CD1. We evaluated the impact of peripheral CGRP on migraine-like behavior and employed multi-site in vivo neurophysiology in freely behaving mice. A changepoint analysis was used to dissect differences in individual CGRP-induced responses. ResultsWe found that early life stress exacerbated migraine-related behavioral and network physiology. CGRP alone caused disruptions in neural oscillatory activity across a network of brain regions including the anterior cingulate cortex (ACC), amygdala (AMY), thalamus (Po, VPM, and MDthal), and parabrachial nucleus (PBN). We found that power across the network was lowered within 10 minutes of peripheral CGRP exposure, which was sustained for [~]40-50 min. Coherence was mostly disrupted in amygdalar brain region pairings, and took on a shorter timecourse, with partial rescue of these responses by migraine abortive, sumatriptan. We found that early life stress exacerbated most of these responses, especially AMY-thalamic coherence pairings, although early life stress in the absence of CGRP demonstrated no impact on the network overall. We further identified individual mice with brain-network activity hypersusceptible to migraine. ConclusionsOur findings demonstrate that early life stress confers vulnerability to migraine, simultaneously impacting behavior and brain network activity responses to peripheral CGRP.

neuroscience↗

bioRxiv2024

DrugProtAI: A guide to the future research of investigational target proteins

Drug design and development are central to clinical research, yet ninety percent of drugs fail to reach the clinic, often due to inappropriate selection of drug targets. Conventional methods for target identification lack precision and sensitivity. While various computational tools have been developed to predict the druggability of proteins, they often focus on limited subsets of the human proteome or rely solely on amino acid properties. To address the challenge of class imbalance between proteins with and without approved drugs, we propose a novel Partitioning Method. We evaluated the druggability potential of 20,273 reviewed human proteins, of which 2,636 have approved drugs. Our comprehensive analysis of 183 features, encompassing biophysical and sequence-derived properties, achieved a median AUC of 0.86 in target predictions. We utilize SHAP (Shapley Additive Explanations) scores to identify key predictors and interpret their contribution to druggability. We have reviewed and evaluated 688 investigational proteins from DrugBank (https://go.drugbank.com/) using our tool, DrugProtAI (https://drugprotai.pythonanywhere.com/). Our tool offers druggability predictions and access to 2M+ publications on drug targets and their effects, aiding in the selection of target proteins for drug development. We believe that insights into key predictors will significantly advance drug development and propel the field forward.

bioinformatics↗

bioRxiv2024

Characterizing the effect of impeller design in plant cell fermentations using CFD modeling

Cultivation of plant cell cultures in conventional bioreactors designed for microbial cells often results in the decrease of biomass productivity as compared to that in shake flasks, presumably due to the imbalance between the mass transfer requirements and compromise with cell viability. Hit and trial methods are usually adapted to achieve high biomass productivity in the bioreactor. In this study, a rational approach has been developed to choose a suitable impeller for Viola odorata cell suspension culture using computational fluid dynamics (CFD). A two-phase CFD model was employed to characterize the non-Newtonian fluid dynamics in a stirred tank reactor using different impeller designs, a setric, Rushton and marine impeller. The simulations were performed adopting Euler-Euler approach for the two-phase flow and dispersed{kappa} - {varepsilon} turbulence model. The numerical model was validated with good agreement with experimental determination of volumetric mass transfer coefficient. The impact of impeller design was then investigated on critical process parameters like mixing, oxygen mass transfer and shear. The developed CFD model demonstrated that setric impeller is a suitable choice for V. odorata cell cultivation offering low-shear environment at equivalent velocity magnitudes at reactor bottom with higher cell-lift capabilities which is preferable in high cell-density plant cell cultivations.

bioengineering↗

bioRxiv2024

Model-based fed-batch cultivation of Viola odorata plant cells exhibiting antimalarial and anticancer activity

Viola odorata is used in the indigenous medicine to treat respiratory tract disorders and is limited in availability. Bioprocess principles can be applied to develop sustainable methods to produce high-quality V. odorata biomass. To this effect, a modified stirred tank reactor and a balloon-type bubble column reactor were used to improve biomass production. Nutrient feeding strategies were developed using first principle-based mathematical modelling to achieve higher cell density in the reactor. Experimental validation of the fed-batch model-predicted strategy resulted in a two-fold enhancement in biomass production (32.2 g DW L-1) at the bioreactor level. Also, bioreactor-cultivated biomass extracts were tested for in vitro hemolytic, cytotoxic, anti-inflammatory, and in vivo antiplasmodial activities. This is the first report on fed-batch cultivation in bioreactors and the antiplasmodial activity of V. odorata. Overall, the bioactive potential of the in vitro-generated biomass extracts is found to be similar to that in the natural plant biomass extracts. HighlightsO_LICultivation of V. odorata cell suspension culture using a modified stirred tank and balloon-type bubble column bioreactors. C_LIO_LIBatch kinetic models were developed and extrapolated into a fed-batch model. C_LIO_LIEnhanced biomass production (32.2 g DW L-1) in bioreactors using a nutrient-feeding strategy. C_LIO_LIExtracts showed anti-inflammatory effects and up to 80 % inhibition of parasite growth, with no hemolytic activity. C_LIO_LIConfirmed antiplasmodial activity in vivo, effective alongside artesunate. C_LIO_LIIn vitro-generated biomass extracts showed comparable bioactive potential to that of natural plants. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/618783v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@171c39aorg.highwire.dtl.DTLVardef@1e0eda5org.highwire.dtl.DTLVardef@121eddorg.highwire.dtl.DTLVardef@1a86215_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

PANOMIQ: A Unified Approach to Whole-Genome, Exome, and Microbiome Data Analysis

The integration of whole-genome sequencing (WGS), whole-exome sequencing (WES), and microbiome analysis has become essential for advancing our understanding of complex biological systems. However, the fragmented nature of current analytical tools often complicates the process, leading to inefficiencies and potential data loss. To address this challenge, we present PANOMIQ, a comprehensive software solution that unifies the analysis of WGS, WES, and microbiome data into a single, streamlined pipeline. PANOMIQ is designed to facilitate the entire analysis process from raw data to interpretable results. It is the fastest algorithm that can achieve results much more quickly compared to traditional pipeline approaches of WGS and WES analysis. It incorporates advanced algorithms for high-accuracy variant calling in both WGS and WES, along with robust tools for characterizing microbial communities. The softwares modular architecture allows for seamless integration of these diverse data types, enabling researchers to uncover complex interactions between host genomics and microbiomes. In this study, we demonstrate the capabilities of PANOMIQ by applying it to a series of datasets encompassing a wide range of applications, including disease association studies and environmental microbiome profiling. Our results highlight PANOMIQs ability to deliver comprehensive insights, significantly reducing the time and computational resources required for multi-omic analysis. By providing a unified platform for WGS, WES, and microbiome analysis, PANOMIQ offers a powerful tool for researchers aiming to explore the full spectrum of genomic and microbial diversity. This software not only simplifies the analytical workflow but also enhances the depth of biological interpretation, paving the way for more integrated and holistic studies in genomics and microbiology.

bioinformatics↗

bioRxiv2024

Targeting Liver Epsins Ameliorates Dyslipidemia in Atherosclerosis

BackgroundThe low-density lipoprotein receptor (LDLR) in the liver plays a crucial role in clearing low-density lipoprotein cholesterol (LDL-C) from the bloodstream. This process takes place mainly in the liver. Under atherogenic conditions, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), secreted by the liver, binds to LDLR on hepatocytes, preventing its recycling and enhancing its lysosomal degradation. This process reduces LDL-C clearance, promoting hypercholesterolemia. Epsins, a family of ubiquitin-binding endocytic adaptors, are key regulators of atherogenesis in lesional cells, including endothelial cells and macrophages. Given epsins canonical role in regulating endocytosis of cell surface receptors, we aimed to determine whether and how liver epsins contribute to PCSK9-mediated LDLR endocytosis and degradation, thereby impairing LDL-C clearance and accelerating atherosclerosis. MethodsLiver-specific epsin knockout (Liver-DKO) atherosclerotic models were generated in ApoE-/- and PCSK9-AAV8-induced atheroprone mice fed on a Western diet. We utilized single-cell RNA sequencing, along with molecular, cellular, and biochemical analyses, to investigate the physiological role of liver epsins in PCSK9-mediated LDLR degradation. Additionally, we explored the therapeutic potential of nanoparticle-encapsulated siRNAs targeting epsins 1 and 2 in ApoE-/- mice with established atherosclerosis. ResultsWestern diet (WD)-induced atherosclerosis was significantly attenuated in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, as well as in PCSK9-AAV8-induced Liver-DKO mice compared with PCSK9-AAV8-induced wild-type (WT) mice accompanied by reductions in blood cholesterol and triglyceride levels. Mechanistically, single-cell RNA sequencing of hepatocytes and aortas isolated from atherosclerotic ApoE-/- and ApoE-/-/Liver-DKO mice revealed epsin-deficient Ldlrhi hepatocytes with diminished lipogenic potential. Notably, pathway analysis of hepatocytes showed increased LDL particle clearance and enhanced LDLR-cholesterol interactions under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, correlating with decreased plasma LDL-C levels. Furthermore, pathway analysis of the aortas showed attenuated inflammation and endothelial activation, coupled with reduced lipid uptake, and enhanced cholesterol efflux under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls. Moreover, the absence of liver epsins led to an upregulation of LDLR protein expression in hepatocytes. We further demonstrated that epsins bind LDLR via the ubiquitin-interacting motif (UIM), enabling PCSK9-mediated LDLR degradation. Depleting epsins abolished this degradation, thereby preventing atheroma progression. Lastly, targeting liver epsins with nanoparticle-encapsulated epsins siRNAs effectively ameliorates dyslipidemia and inhibits atherosclerosis progression. These results are consistent with findings showing an increased epsin1 and epsin2 expression in atherosclerotic cardiovascular disease patients. ConclusionsLiver epsins drive atherogenesis by promoting PCSK9-mediated LDLR degradation, thereby elevating circulating LDL-C levels and heightening lesional inflammation. As such, targeting epsins in the liver represents a promising therapeutic strategy to mitigate atherosclerosis by preserving LDLR and enhancing LDL-C clearance in the liver.

cell biology↗

bioRxiv2024

Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

Veins have emerged as the origin of all other endothelial cell subtypes needed to expand vascular networks during developmental and pathological neoangiogenesis. Here, we uncover the significant role of the angioneurin Fibronectin Leucine Rich Transmembrane protein (FLRT) 2 in central nervous system (CNS) vascular development in the mouse. Early postnatal FLRT2 deletion reveals specific defects in retinal veins, impacting endothelial cell proliferation, sprouting and polarity that result in reduced tip cells at the vascular front. FLRT2 interacts with VE-cadherin and together with the endocytic adaptor protein Numb contribute to the modulation of adherens junction morphology in both retina and cerebral cortex in vivo. Utilizing expansion microscopy, we visualize the altered dynamic distribution of VE-cadherin in tissue of FLRT2 endothelial mutants. Additionally, FLRT2 in cortical vessels regulates the crosstalk between adherens and tight junctions, influencing blood-brain barrier development. Our findings position FLRT2 as a vein-specific crucial regulator of CNS vascular development.

neuroscience↗

bioRxiv2024

Conclusions about Neural Network to Brain Alignment are Profoundly Impacted by the Similarity Measure

Deep neural networks are widely used to model biological perception and behavior, making the similarities and differences between artificial and biological systems consequential. If a principle (e.g. self-supervised learning) produces a model resembling biology, this is taken as evidence the same principle shapes the biological system. But what it means for an artificial system to be similar to a biological one is complex. A popular approach compares representations of identical stimuli using a similarity measure like Representational Similarity Analysis. Yet scientific questions rarely specify which measure is appropriate, raising a key question: do conclusions depend on this choice? Focusing on vision, we show that measure choice influences both hierarchical correspondence between systems and the ranking of which artificial systems are most biological. Reanalyzing prior studies, we find the choice hugely consequential: models best under one measure are often worst under another, and prior conclusions can flip or dissolve. Different metrics capture fundamentally different aspects of similarity, warranting healthy skepticism toward such comparisons.

neuroscience↗

bioRxiv2024

Bioenergetic stress potentiates antimicrobial resistance and persistence

Antimicrobial resistance (AMR) is a global health crisis and there is an urgent need to better understand AMR mechanisms. Antibiotic treatment alters several aspects of bacterial physiology, including increased ATP utilization, carbon metabolism, and reactive oxygen species (ROS) formation. However, how the "bioenergetic stress" induced by increased ATP utilization affects treatment outcomes is unknown. Here we utilized a synthetic biology approach to study the direct effects of bioenergetic stress on antibiotic efficacy. We engineered a genetic system that constitutively hydrolyzes ATP or NADH in Escherichia coli. We found that bioenergetic stress potentiates AMR evolution via enhanced ROS production, mutagenic break repair, and transcription-coupled repair. We also find that bioenergetic stress potentiates antimicrobial persistence via potentiated stringent response activation. We propose a unifying model that antibiotic-induced antimicrobial resistance and persistence is caused by antibiotic-induced. This has important implications for preventing or curbing the spread of AMR infections.

microbiology↗

bioRxiv2024

Spatially Resolved Tumor Ecosystems and Cell States in Gastric Adenocarcinoma Progression and Evolution

Gastric cancer (GC) is a major cause of global cancer mortality with high heterogeneity levels. To explore geospatial interactions in tumor ecosystems, we integrated 1,563 spatial transcriptomic regions-of-interest (ROIs) with 152,423 single-cell expression profiles across 130 GC samples from 70 patients. We observed pervasive expression-based intratumor heterogeneity, recapitulating tumor progression through spatially localized and functionally ordered subgroups with specific immune microenvironments and immune checkpoint profiles. Evolutionary phylogenetic analysis revealed two different evolutionary trajectories (branched evolution and diaspora evolution) associated with distinct molecular subtypes, clinical prognoses, stromal neighborhoods including VWF+ ACKR1+ endothelial cells, and genetic drivers such as SOX9. Spatial analysis of tumor-stromal interfaces across multiple GCs highlighted new ecosystem states not attributable to mere tumor/stroma admixture, landmarked by increased GREM1 expression. Our results provide insights into how the cellular ecosystems of individual GCs are sculpted by tumor intrinsic and extrinsic selective pressures, culminating in individualized patient-specific cancer cartographies.

cancer biology↗

bioRxiv2024

Radiation resistance in cervical cancer: An insight on mitochondrial associated membranes and its related proteins.

Radiation therapy proves ineffective against radiation-resistant cancers, exacerbating the disease. The Mitochondrial Associated Membrane (MAM) forms through the tethering of the endoplasmic reticulum to the mitochondrial membrane. Under stress conditions such as radiation, mitochondrial function becomes impaired, prompting the recruitment of endoplasmic reticulum proteins for cell survival. Therefore, understanding the role of ER and mitochondria interactions is crucial for the development of inhibitors or drugs to sensitize cancer cells to radiation. In this study, we focused on ultrastructural alterations in radiation-resistant cells. The main structural changes observed included mitochondrial elongation, mitophagy, the presence of lipid droplets, and shortened ER segments. While MAM formation (the interaction between the ER and mitochondria) was observed in SiHa cells not exposed to radiation, it was disrupted in radiation-resistant cells. We analysed genes associated with mitochondrial elongation, mitophagy, lipid droplet formation, short ER fragments, and ER stress using a dataset from cervical cancer patients in The Cancer Genome Atlas (TCGA). Our analyses suggest that BNIP3L is implicated in therapy failure and tumour recurrence. In future, inhibiting BNIP3L could potentially sensitize resistant cells to radiation therapy.

cancer biology↗

bioRxiv2024

Investigating REV-ERBβ Binding Pocket Dynamics with Implications for Rational Design of Small Molecule Modulators

ABSTRACTREV-ERB{beta} is a nuclear receptor (NR) with heme as an endogenous ligand that regulates its transcriptional activity. With key role in cellular functions such as glucose metabolism, immune response, and dysregulation in pathologies such as Type-2 diabetes mellitus and obesity, small molecule agonists and antagonists targeting REV-ERBs have been discovered. However, due to lack of crystal structures in complex with these compounds, the structural and dynamical basis of these activities still remains elusive and hinders rational design of molecules targeting REV-ERB. Using molecular dynamics simulations and docking studies, we have characterized the dynamics of REV-ERB{beta} ligand-binding domain (LBD) in different conformational states. The presence of heme in the binding pocket within LBD was found to stabilize its dynamics as well as nuclear co-repressor (NCoR) peptide binding. We further show that the binding of antagonist destabilizes the NCoR peptide binding to LBD mediated by loss of interactions with residues at the NCoR-REV-ERB{beta} interface. These findings could be utilized to design molecular scaffolds with better activity and selectivity against REV-ERB{beta}.

bioinformatics↗

bioRxiv2024

Quantitative differences in anaerobic growth and metabolism of three different E. coli strains using 13C-Metabolic Flux Analysis

Escherichia coli are model organisms for biotechnological research owing to their abilities to grow on defined media and consume a variety of carbon sources, as well as a vast knowledge base and tools available for their manipulation. Several E. coli strains and their derivatives are available. However, the quantitative metabolic differences between the strains could be better characterized. In this study, we investigated the differences in the intracellular flux distribution between three commonly used strains of E. coli (K-12 MG1655, C, and W) using 13C-Metabolic flux analysis (13C-MFA), the gold standard in fluxomics. The labeling data was analyzed using FluxPyt, our previously developed Python-based tool. Threefold differences in the growth rates of these strains were observed when growing anaerobically on glucose in M9 medium, with E. coli W being the fastest ( = 0.62 {+/-} 0.01 h-1) and E. coli C the slowest ( = 0.22 {+/-} 0.01 h-1). The strains growth rates correlate with glucose uptake and flux through the oxidative pentose phosphate pathway. Interestingly, succinate was a major product in E. coli C only. 13C-MFA revealed that under these conditions, Entner-Doudoroff (ED) pathway was active in E. coli C and E. coli W but not in E. coli K-12 MG1655. In E. coli K-12 MG1655 and E. coli C, the Tricarboxylic Acid (TCA) cycle ran incompletely. Also, the glyoxylate shunt was inactive in all three strains. Growth, xylose intake rates, and product profiles on xylose mirrored those of glucose. Thus, this study has highlighted significant differences in the growth and metabolism of the E. coli strains on glucose and xylose. This will form a basis for identifying the best strain for a particular application.

systems biology↗