Biology subjects

Roychowdhury, A.

Publications and source records attributed to Roychowdhury, A..

11 recordsLinked to original sources

bioRxiv2026

A highly stringent high-throughput screening assay for identifying transcription-modulating agents of HIV-1 latency

HIV-1 latency remains a central obstacle to curing infection, and current latency-modulating agents (LMAs) suffer from poor specificity and inconsistent efficacy. To enable discovery of small molecules (SMs) that directly target the viral master transcriptional regulatory circuit (MTRC), we developed a highly stringent, dual-reporter high-throughput screening (HTS) assay based on a natural HIV-1 subtype C long terminal repeat (LTR) variant, LRhR-HC, which exhibits markedly reduced transcriptional noise and a high activation threshold. We engineered Jurkat cells to stably harbour two independent reporter cassettes driven by the tough-to-activate LRhR-HC-LTR and the canonical LR-HHC-LTR, enabling simultaneous detection of latency-promoting and latency-reversing activities through both fluorescent and secreted enzymatic reporters. This dual-reporter line responded robustly and predictably to conventional latency-reversal agents (LRAs) and latency-promoting agents (LPAs), validating assay responsiveness. Z'-factor measurements demonstrated excellent assay performance, with values ranging from approximately 0.7 across different formats, confirming a strong dynamic range and reproducibility. Screening of the 1,520-compound Prestwick chemical library (PCL), which includes FDA-approved drugs, identified 27 candidate LPAs, including known agents such as Spironolactone and Aminacrine, thereby validating the assay specificity while revealing several novel inhibitory molecules. A mechanistically focused panel of 10 additional compounds yielded two putative LPAs and one LRA, with secondary analyses confirming their latency-modulating activities and cytotoxicity profiles. Collectively, this HIV-1C-derived dual-reporter platform provides a stringent and flexible HTS system for identifying LMAs with potential applications in both block-and-lock and shock-and-kill cure strategies.

molecular biology↗

bioRxiv2024

Complete Genome Sequence, Metabolic Profiling and Functional Studies reveal Ligilactobacillus salivarius LS-ARS2 is a Promising Biofilm-forming Probiotic with Significant Antioxidant, Antibacterial, and Antibiofilm Potential

BACKGROUNDProbiotics restore microbial balance and prevent gut-inflammation. Therefore, finding out novel probiotic strains is a demand. As gut-microbe, benefits of Ligilactobacillus salivarius (LS) are established. However, strain-specific detailed studies are limited. Here, we illustrate probiotic attributes of novel LS-ARS2 for its potential application as food-supplement and/or therapeutic to improve gut-health. METHODSWhole genome sequencing (WGS) and phylogenetic analysis confirm the strain as LS. To establish probiotic properties, acid-bile tolerance, auto-aggregation, cell-surface-hydrophobicity, biofilm-formation and adhesion-assays are performed. To ensure safety attributes, antibiotic-susceptibility, haemolytic, DNase, trypan-blue and MTT assays are done. ABTS, DPPH, superoxide, hydroxyl free radical scavenging assays are used to determine anti-oxidant potential. Antibacterial assays including co-culture assay with pathogen and pathogenic biofilm-inhibition assays are performed to explore antibacterial efficacy. To characterize metabolic-profile of LS-ARS2-derived cell-free-supernatant (CFS), HRMS analysis are carried out. Consequently, WGS-analyses predict potential molecular associations related to functional outcomes. RESULTSWe find LS-ARS2 as remarkable fast-growing strain that shows acid and bile tolerance (>60% survival rate) indicating promising gut-sustainability. High auto-aggregation capacity (>80%), robust cell-surface hydrophobicity (>85%), and adhesion efficacy to Caco-2 cells illustrate significant potential of LS-ARS2 for gut colonization. Fascinatingly, LS-ARS2 is able to form biofilm within 24 h (p<0.0001), rare among LS strains, indicating the potential of the strain for efficient stay in the gut. The strain ensures safety attributes. LS-ARS2-WGS analysis recognizes probiotic-specific determinants, predicts genomic stability, identifies orthologous-clusters for diverse functions, and predicts metabolites and bacteriocins. HRMS-studies with LS-ARS2-CFS further validate the presence of diverse beneficial metabolites with antimicrobial and immunomodulatory potential. LS-ARS2 shows significant antioxidant properties in ABTS (>60%), DPPH (>10U/mL), superoxide (>70%), and hydroxyl free radical scavenging assays (>70%). Further, LS-ARS2 shows antimicrobial activities against Gram-positive Methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative multidrug-resistant clinical strains enterotoxigenic Escherichia coli, Vibrio cholerae, and Shigella flexneri. Anti-Salmonella effect of LS-ARS2 is prominent (p<0.0001). Most interestingly, LS-ARS2-CFS inhibits MRSA-biofilm (p<0.0001), again rare among LS strains. CONCLUSIONS LS-ARS2 is a novel fast-growing, biofilm-forming probiotic with significant antioxidant, antibacterial, and anti-biofilm potentials, suggesting the promising applications of LS-ARS2 for combating pathogenic biofilms and improving gut-health. However, further in vivo studies would facilitate their potential applications.

microbiology↗

bioRxiv2024

Subtractive proteomics unravel the potency of D-Alanine-D-Alanine Ligase as the drug target for Burkholderia pseudomallei

Melioidosis, also known as Whitmores disease, is caused by the deadly pathogen Burkholderia pseudomallei and remains a significant global health concern, particularly in South Asia. The disease is contracted through exposure to contaminated soil, water, air, and food. Infected individuals often present with abscesses in internal organs such as the lungs, spleen, and liver, and in soft tissues, with severe cases leading to septic shock and acute pneumonia. The rising incidence and mortality rates, coupled with B. pseudomalleis ability to form biofilms and develop resistance to antibiotics like cephalosporins, make treatment increasingly challenging. This highlights the urgent need for novel therapeutic approaches. D-Alanine-D-Alanine ligase (Ddl), a crucial enzyme involved in the final stage of bacterial cell wall synthesis, which protects the pathogen from the hostile cellular environment of the host. While many bacteria have two isoforms of this enzyme, B. pseudomallei possesses only the DdlB isoform, presenting a significant vulnerability. Our study represents the first successful attempt to target DdlB through a combination of molecular docking and molecular dynamics simulations. These investigations provide strong evidence that Conivaptan acts as an effective inhibitor of DdlB, offering a novel therapeutic approach for combating melioidosis.

bioinformatics↗

bioRxiv2024

Segregation, Finite Time Elastic Singularities and Coarsening in Renewable Active Matter

Material renewability in active living systems, such as in cells and tissues, can drive the large-scale patterning of forces, with distinctive phenotypic consequences. This is especially significant in the cell cytoskeleton, where multiple species of myosin bound to actin, apply differential contractile stresses and undergo differential turnover, giving rise to patterned force channeling. Here we study the dynamical patterning of stresses that emerge in a hydrodynamic description of a renewable active actomyosin elastomer comprising two myosin species. Our analytical framework also holds for an actomyosin elastomer with a single myosin species. We find that a uniform active contractile elastomer spontaneously segregates into spinodal stress patterns, followed by a finite-time collapse into tension carrying singular structures that display self-similar scaling and caustics. Our numerical analysis carried out in 1D, shows that these singular structures move and merge, and gradually result in a slow coarsening dynamics. We discuss the implications of our findings to the emergence of stress fibers and the spatial patterning of actomyosin. Our study suggests, that with state-dependent turnover of crosslinkers and myosin, the in vivo cytoskeleton can navigate through the space of material parameters to achieve a variety of functional phenotypes.

biophysics↗

bioRxiv2024

Designing a novel Scaffold-Based Multi-Epitope Vaccine to Combat Melioidosis Caused by Burkholderia pseudomallei: An In-silico and Immunoinformatics approach

Burkholderia pseudomallei, the gram-negative bacteria causing melioidosis, is becoming a serious threat to healthcare settings. In recent years, B. pseudomallei has been identified as an emerging and significant etiological agent responsible for localized pyogenic infections primarily observed in India and South Asia. At present, no vaccine against melioidosis is available in the treatment system. This study has undertaken an in-silico reverse vaccinology approach to design a novel multi-epitope vaccine for treating B. pseudomallei-mediated infections. B-cell and T-cell epitopes have been predicted and stitched to develop a multi-epitope vaccine. The predicted vaccine is found to be non-toxic, non-allergic, and immunogenic in nature. Immune simulation results indicate that the designed vaccine can generate an immune response resembling a real-life scenario. The 610 amino-acid long vaccine construct has been codon-optimized and could be cloned in the E. coli K12 system. These findings from this immunoinformatics study offer a foundation for developing a tailored, safe, and potent vaccine targeting B. pseudomallei.

bioinformatics↗

bioRxiv2024

PfHT1 controls glucose uptake in malaria parasite: molecular dynamics study in plasma membrane like environment reveals ligand induced conformational changes trigger closed conformation and alters binding pocket geometry

Plasmodium falciparum hexose transporter 1 protein or PfHT1 is the major glucose transporter of the parasite and indispensable for its survival at the blood stage of infection. PfHT1 transports the hexose sugar to meet the energy demand of the parasite. Studying the mechanism of transport and designing structure specific inhibitors against PfHT1 is an intelligent strategy to kill malaria parasite by starving it out because at the blood stage the sole carbon source of Plasmodium falciparum is glucose. In this study the conformational dynamics of PfHT1 has been studied in detail in the apo and holo (inhibitor bound) form using Molecular Dynamics Simulation. Which reveals that PfHT1 undergoes ligand induced closed state if compared with the apo form. The geometry of ligand binding pocket also shifts from the apo form in the presence of inhibitors. A de novo drug designing approach based on the skeleton of leads obtained from screening nearly 4500 compounds, has produced inhibitors of PfHT1 with higher specificity and affinity. The drug screening data as well as the conformational dynamics study was validated using Molecular Dynamics Simulation platform where a near physiological atmosphere was created for PfHT1 by constructing a lipid phase (phospholipid bilayer) sandwiched between aqueous phases (mimics extracellular and cytosolic polar environment).

bioinformatics↗

bioRxiv2024

Designing a Novel 3D Scaffold for Multiepitope Vaccine Development: Engineering Ag85a Protein for Enhanced Stability and Antigenicity

Designing multi epitope vaccine (MEV) by reverse vaccinology has become immensely important in the area of vaccine research due to the emergence of new pathogens as well as rise of multi drug resistant old evils like tuberculosis. Administering a vaccine may have the best possibility to save mankind from these unforeseeable events. The strategy of designing a MEV in-silico lies in a few basic steps, including procuring the amino acid sequence of the B cell and T cell epitopes from literature search, bioinformatics approach to construct a potent immunogen capable of eliciting both humoral and cell mediated response. But the challenge lies in the construction of a stable protein with a compact tertiary structure. Merely joining the epitopes one after another, may not be sufficient to achieve this. In this study, a methodology has been detailed to tackle this great challenge using a simple approach of protein engineering. A scaffold based MEV has been designed for the very first time against Mtb by converting a vaccine candidate protein, Ag85A into a scaffold by truncating its non-immunogenic regions so the gaps could be filled by the highly immunogenic epitopes. The stability of the MEV was estimated by molecular dynamics simulation.

biochemistry↗

bioRxiv2023

De novo drug designing coupled with brute force screening and structure guided lead optimization gives highly specific inhibitor of METTL3: a potential cure for Acute Myeloid Leukaemia

Expression of METTL3, a SAM dependent methyltransferase, which deposits m6A on mRNA is linked to poor prognosis in Acute Myeloid Leukaemia and other type of cancers. Down regulation of this epitranscriptomic regulator has been found to inhibit cancer progression. Silencing the methyltransferase activity of METTL3 is a lucrative strategy to design anticancer drugs. In this study 3600 commercially available molecules were screened against METTL3 using brute force screening approach. However, none of these compounds take advantage of the unique Y-shaped binding cavity of the protein, raising the need for de novo drug designing strategies. As such, 125 branched, Y-shaped molecules were designed by "stitching" together the chemical fragments of the best inhibitors that interact strongly with the METTL3 binding pocket. This results in molecules that have the three-dimensional structure and functional groups which enable it to fit in the METTL3 cavity like fingers in a glove, having unprecedented selectivity and binding affinities. The designed compounds were further refined based on Lipinskis rule, docking score and synthetic accessibility. The molecules faring well in these criteria were simulated for 100ns to check the stability of the protein inhibitor complex followed by binding free energy calculation.

bioinformatics↗

bioRxiv2022

Identification of potential natural compound inhibitors and drug like molecules against human METTL3 by docking and molecular dynamics simulation

Nucleotide level chemical modification in transcriptome is critical in regulating different cellular processes, including cancer. The most investigated epitranscriptomic modification is methylation at the N6-position of adenosine (m6A). This dynamic modification process is carried out by: writer, reader and eraser proteins. Writers are methyltransferases, METTL3 is the major writer that works in association with METTL14, an accessory protein. Extensive study revealed that cancer progression for acute myeloid leukaemia, gastric cancer, colorectal cancer, hepatocellular carcinoma, and lung cancer is directly contributed by irregular expression of METTL3. Targeting METTL3, has opened a new window in the development of new inhibitors/drugs. In this study, 80 commercially available compounds were found from an unbiased screening by molecular docking, showing better score when compared with the existing substrate/substrate-analogue and the inhibitor bound crystal structures in terms of docking score and binding energy calculation. Among this pool of compounds, the best seven small molecules, AMF, RAD, JNJ, MEH, ECP, MHN, SGI, have been selected and further validated by different computational tools like binding energy calculation, molecular dynamics simulation etc. The novel hits found in this study can function as lead compounds which can be developed into inhibitors as well as drugs, specific against METTL3.

bioinformatics↗

bioRxiv2020

In vitro and in silico analyses of the angiotensin-I converting enzyme inhibitory activity of peptides identified from Bellamya bengalensis protein hydrolysates

The study focuses on identification of ACE-inhibitory peptides from the proteolytic digests of muscle protein of Bellamya bengalensis and its underlying mechanism. 120 min Alcalase-hydrolysates were ultrafiltered to isolate the small peptide fraction (<3kDa) and in vitro ACE-inhibitory activity was analyzed. The IC50 value of the 120 min hydrolysate ultafiltered fraction was found to be 86.74 {+/-} 0.575 {micro}g/mL, while the IC50 of Lisinopril is 0.31 {+/-} 0.07 {micro}g/mL. This fraction was assessed in MALDI-ToF mass-spectrometer and five peptides were sequenced via de novo sequencing. The ACE-inhibitory potential of the peptides have a positive correlation with the hydrophobicity of the amino acids. Synthetic analogue of the peptide (IC50 value 8.52 {+/-} 0.779 {micro}g/mL) was used to understand the thermodynamics of the inhibition by checking the binding affinity of the peptide to ACE by Isothermal titration calorimetry compared with lisinopril, and further substantiated by in silico site specific molecular docking study.

biochemistry↗

bioRxiv2019

LITE-1 Mediates X-ray Avoidance Response in C. elegans

We report the finding that C. elegans display X-ray avoidance behavior at high but well tolerated doses, and that this behavior appears to require LITE-1, a gustatory receptor that has been implicated in UV avoidance behavior. We recorded acute behavioral responses of wildtype worms to increasing intensities of X-ray stimulation and found a positive stimulation-response relationship. Mutant strains of worms with dysfunctional photoreceptor proteins LITE-1 and GUR-3 were assayed, and the X-ray avoidance response was found to be nearly absent in LITE-1 mutants but not GUR-3 mutants, suggesting a prominent role for LITE-1 in the detection of X-rays. These findings may be important for developing optogenetics tools to stimulate cells in deep tissue using X-rays, for understanding the mechanism of LITE-1 signaling, and for understanding how organisms may respond to radiation.

neuroscience↗