Biology subjects

Rakshit, S.

Publications and source records attributed to Rakshit, S..

13 recordsLinked to original sources

bioRxiv2026

A multifunctional polyketide synthase in nematodes produces divergent families of signaling molecules that control different developmental arrests

To improve their chances of reproductive success, nematodes not only must arrest their development in response to adverse growth conditions but also must quickly recover if conditions improve. A polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid assembly line that is expressed in the canal-associated neurons (CANs) of Caenorhabditis elegans promotes recovery from starvation-induced larval arrest. Here, we show that in the predatory nematode Pristionchus pacificus this assembly line produces a suite of secondary metabolites, including a family of hybrid polyketide-nonribosomal peptides known as the nemamides, the related nemaketides, and a family of ascarylose-modified polyketides named ascarenes. Depending on the starter unit that is loaded onto the PKS, the assembly line can produce dramatically different downstream products. Whereas the nemamides promote recovery from starvation-induced larval arrest, the ascarenes inhibit development of the dauer larval stage and promote recovery. This dichotomy suggests that the PKS-NRPS megasynthetase serves as a signaling hub in the CANs, controlling multiple developmental events. The PKS-NRPS assembly line is highly conserved across many nematode species, and identification of these chemical signals will help to elucidate the signaling pathways that control development in the worm and lead to novel anthelmintics.

biochemistry↗

bioRxiv2025

Chromosome-Scale, Telomere-to-Telomere Assembly of Winged Bean (Psophocarpus tetragonolobus (L.) DC.) Genome Reveals Lipid Biosynthetic Hotspots and Gene Family Dynamics

Winged bean (Psophocarpus tetragonolobus) is a nutritionally rich but cytologically and genomically underexplored legume with potential for climate-resilient agriculture. We report a telomere-to-telomere, chromosome-scale genome assembly ([~]697.69 Mb; N50 = 85.98 Mb; [~]98.25% of the cytometric estimate) of the cultivar AKWB1 using a combination of PacBio HiFi, Illumina, BioNano, and Hi-C technologies. Cytogenetics revealed a diploid karyotype (2n = 18) with exclusively metacentric chromosomes, establishing the first chromosomal ideotype map for the species. The high-quality assembly anchors 98.28% of bases to nine pseudochromosomes and includes 53,745 annotated protein-coding genes, with 97.6% BUSCO completeness. Repeat elements comprise [~]60% of the genome, dominated by LINEs, LTRs, and DNA transposons. KEGG and GO annotations reveal diverse biosynthetic capabilities across metabolic and stress-response pathways. Comparative genomics with nine legumes demonstrates conserved chromosomal synteny with Glycine max, absence of recent whole-genome duplication, and a contraction-dominated gene family history. Lipid metabolism analysis identified 750+ genes across 12 KEGG pathways, including fatty acid biosynthesis, elongation, and triacylglycerol assembly. Chromosomes 2, 3, and 5 showed enrichment of lipid biosynthetic loci, suggesting evolutionary hotspots. This reference genome positions P. tetragonolobus as a valuable model for studying genome evolution and engineering underutilized legumes for oil yield, protein enrichment, and climate adaptability.

genomics↗

bioRxiv2024

Structure-Function Relationship of the Ryanodine Receptor Cluster Network in Sinoatrial Node Cells

The rate of spontaneous action potentials (APs) generated by sinoatrial node cells (SANC) is regulated by local Ca2+ release (LCR) from the sarcoplasmic reticulum via Ca2+ release channels (ryanodine receptors, RyRs). LCR events propagate and self-organize within the network of RyR clusters (Ca release units, CRUs) via Ca-induced-Ca-release (CICR) that depends on CRU sizes and locations: while larger CRUs generate stronger release signals, the networks topology governs signal diffusion and propagation. This study used super-resolution structured illumination microscopy to image the 3D network of CRUs in rabbit SANC. The peripheral CRUs formed a spatial mesh, reflecting the cell surface geometry. Two distinct subpopulations of CRUs were identified within each cell, with size distributions conforming to a two component Gamma mixture model. Furthermore, neighboring CRUs exhibited repulsive behavior. Functional properties of the CRU network were further examined in a novel numerical SANC model developed using our experimental data. Model simulations revealed that heterogeneities in both CRU sizes and locations facilitate CICR and increase AP firing rate in a cooperative manner. However, these heterogeneities reduce the effect of {beta}-adrenergic stimulation in terms of its relative change in AP firing rate. The presence of heterogeneities in both sizes and locations allows SANC to reach higher absolute AP firing rates during {beta}-adrenergic stimulation. Thus, the CICR facilitation by heterogeneities in CRU sizes and locations regulates and optimizes cardiac pacemaker cell operation under various physiological conditions. Dysfunction of this optimization could be a key factor in heart rate reserve decline in aging and disease.

biophysics↗

bioRxiv2024

De-novo design of actively spinning and gyrating spherical micro-vesicles

Engineering spherical self-propelled swimmers that exhibit rotation and directed translation has posed a significant experimental challenge in biomedicine design. Often a secondary external field or asymmetric geometry is employed to generate rotation, complicating the design process. In this work, we utilize spherical Giant Unilamellar Vesicles (GUVs) as chassis and enzymes undergoing cyclic, non-reciprocal conformational changes as power units to establish design principles to synthesize autonomous spherical micro({micro})-rotors. Leveraging transient interactions, we induce spontaneous symmetry-breaking in enzyme distribution on GUVs, enabling diverse movements from pure spinning to spiral 3D trajectories. With this design, we now open new avenues for advancing self-propelled systems with biocompatible materials, unlocking innovations in biomedical applications.

biophysics↗

bioRxiv2023

A host AAA-ATPase exhibits bacteriolytic activity for clearance of microbial infection

An array of host cytosol guarding factors impede bacterial proliferation and preserve cellular sterility. Amongst them, proteasomal degradation of ubiquitinated pathogens has emerged as a critical mechanism for ensuring cytosolic sanctity. We wondered how proteasomes, with their small size and inability to extract membrane-bound proteins, can eradicate pathogens. Here, we unveil a unique strategy, wherein VCP/p97, a host AAA-ATPase, eliminates pathogens by exerting mechanical force that physically unfolds and pulls out ubiquitinated proteins from bacterial membrane. Combining a single-molecule approach along with molecular dynamic simulation and in-vitro reconstitution, we demonstrate that protein extraction by p97 causes extensive membrane lysis and release of cytosolic contents from phylogenetically diverse microbes. Additionally, in an in-vivo mouse sepsis model, this segregase-dependent bactericidal effect of p97 abrogated microbial proliferation in host tissues. Overall, we discovered a distinct innate antimicrobial function of p97, that protects the host against lethal bacterial infections. One Sentence SummaryA host AAA-ATPase exhibits bacteriolytic activity.

microbiology↗

bioRxiv2023

Metagenomic analysis of Pigs' faecal microbiome and its functional response associated with dietary fibre

Pig husbandry is the most valued and economically sustainable husbandry in the livestock farming system. The performance and productivity of the pig are chiefly dependent on the nutritional factor steered by gut beneficial bacteria. The swine gut microbiome has a direct relationship with feed efficiency. As a consequence, identifying microbial taxonomy and functional capacity is critical for proper nutrient digestion. In the present investigation, eighteen grower pigs aged 3 months and weighing 30{+/-}0.55 kg were allocated into three different groups using a randomized block layout and supplemented with QPM maize fodder of 0, 5, and 10% to the basal diet by substituting (wt/wt on DM) the maize grains, and named as T0, T1, and T2 to probe the increase in gut beneficial bacteria found in the faecal contents. For comparison with T0, T1, and T2, a random faecal sample designated R was collected from six grower pigs of the same age group and bodyweight fed a maize-soya bean-based diet. The experiment is being carried out to investigate the effect of various maize fodder levels on the metagenomic profiles of pig gut microbiota using the 16S rRNA gene. All of the experimental diets were iso-nitrogenous, with protein content ranging from 18.37 to 18.63 per cent. The data generated by 16S rRNA amplicon analysis was 68, 56, 61, and 39 Mb in R, T0, T1, and T2 samples, respectively. From taxonomic distribution, bacterial phyla namely Firmicutes, Bacteroidetes, Proteobacteria, and Spirochetes are found in descending order of relative abundance in the R, T0, and T1 groups, respectively, while Spirochaetes, Proteobacteria, Fibrobacteres, Bacteroidetes, and Firmicutesare found in descending order of relative abundance in the T2 group. The relative profusion of Fibrobacter succinogenes is 0.89% and 16.84% in the T1 and T2 groups. According to the findings, a higher level of maize fodder in the diet of grower pigs promotes the growth of fibre-degrading bacteria in the gut microbiota, particularly Fibrobacter succinogenes. Moreover, feeding green maize has decreased the population of methanobacteria in the gut of pigs, which in turn has limited the production of methane.

genomics↗

bioRxiv2022

Differences in anti-viral immune responses in individuals of Indian and European origin: relevance for the COVID-19 pandemic

During the COVID-19 pandemic, large differences in susceptibility and mortality due to SARS-CoV-2 infection have been reported between populations in Europe and South Asia. While both host and environmental factors (including BCG vaccination) have been proposed to explain this, the potential biological substrate of these differences is unknown. We purified peripheral blood mononuclear cells from individuals living in India and the Netherlands at baseline and 10-12 weeks after BCG vaccination. We compared chromatin accessibility between the two populations at baseline, as well as gene transcription profiles and cytokine production capacities upon viral stimulation with influenza and SARS-CoV-2. The chromatin accessibility of genes important for adaptive immunity was higher in Indians compared to Europeans, while the latter had more accessible chromatin regions in genes of the innate immune system. At the transcriptional level, we observed that Indian volunteers displayed a more tolerant immune response to viral stimulation, in contrast to a more exaggerated response in Europeans. BCG vaccination strengthened the tolerance program in Indians, but not in Europeans. These differences may partly explain the different impact of COVID-19 on the two populations.

immunology↗

bioRxiv2022

Redefining the structure of tip-links in hair-cells

Tip-link, the gating-spring in hearing, modulates the mechanoelectrotransduction process and protects the hearing sensory machineries from overexcited auditory inputs. Tip-links are formed by two exceptionally long cadherins, cadherin-23 and protocadherin-15. It is proposed that both the cadherins first independently form cis-dimers through lateral interactions and finally, engage in trans-interactions to form the heterotetrameric assembly in tip-links. In fact, the molecular details of the protocadherin cis-dimers are well-understood. However, little structural and molecular details are known for independent cis-dimer of cadherin-23. Using photo-induced cross-linking of unmodified proteins in solution and on lipid-membranes, we observed no trace of cadherin-23 cis-dimers, rather we captured trans-homodimers of cadherin-23. We thus, redefine the tip-link structure where protocadherin-15 remains as cis-dimers, however, two individual cadherin-23 binds to protocadherin-15 cis-dimer for tip linking.

biochemistry↗

bioRxiv2022

Cis-clustering of cadherin-23 controls the kinetics of cell-cell adhesion

Cis and trans-interactions among cadherins secure multicellularity. While the molecular structure of trans-interactions of cadherins is well understood, identifying the molecular cues that spread the cis-interactions two-dimensionally is still ongoing. Here, we report that transient, weak, multivalent, and spatially distributed hydrophobic interactions, requisite for liquid-liquid phase separations of biomolecules in solution, alone can drive the lateral-clustering of cadherin-23 on a membrane. No specific cis-dimer interactions are required for the lateral clustering. In cellulo, the cis-clustering accelerates the cell-cell adhesion and, thus, contributes in cell-adhesion kinetics along with strengthening the junction. Although the physiological connection of cis-clustering with rapid adhesion is yet to be explored, M2-macrophages that predominantly express cadherin-23 undergo fast attachments to circulatory tumor cells during metastasis.

cell biology↗

bioRxiv2021

Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis

A fundamental challenge in HIV eradication is to understand how the virus establishes latency, maintains stable cellular reservoirs, and promotes rebound upon interruption of antiretroviral treatment (ART). Here, we discovered an unexpected role of the ubiquitous gasotransmitter hydrogen sulfide (H2S) in HIV latency and reactivation. We show that reactivation of HIV-1 is associated with down-regulation of the key H2S producing enzyme cystathionine-{gamma}-lyase (CTH) and reduction in endogenous H2S. Genetic silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, and remodels the transcriptome of latent cells to trigger HIV reactivation. Chemical complementation of CTH activity using a slow-releasing H2S donor, GYY4137, suppressed HIV reactivation and diminished virus replication. Mechanistically, GYY4137 blocked HIV reactivation by inducing the Keap1-Nrf2 pathway, inhibiting NF-{kappa}B, and recruiting the epigenetic silencer, YY1, to the HIV promoter. In latently infected CD4+ T cells from ART-suppressed human subjects, GYY4137 in combination with ART prevented viral rebound and improved mitochondrial bioenergetics. Moreover, prolonged exposure to GYY4137 exhibited no adverse influence on proviral content or CD4+ T cell subsets, indicating that diminished viral rebound is due to a loss of transcription rather than a selective loss of infected cells. In summary, this work provides mechanistic insight into H2S-mediated suppression of viral rebound and suggests the inclusion of an H2S donor in the current ART regimen to achieve a functional HIV-1 cure.

microbiology↗

bioRxiv2019

Variable Mutations at the p53-R273 Oncogeic Hotspot Position Leads to Altered Properties

Mutations in p53 protein, especially in the DNA binding domain is one of the major hallmarks of cancer. The R273 position is a DNA contact position and has several oncogenic variants. Surprisingly, cancer patients carrying different mutant-variants of R273 in p53 have different survival rates indicating that the DNA contact inhibition may not be the sole reason for reduced survival with R273 variants. Here, we probed the structural properties of three major oncogenic variants of the R273: ([R273L], [R273H], and [R273C])p53. Using a series of biophysical, biochemical and theoretical simulation studies, we observe that these oncogenic variants of the p53 not only suffer a loss in DNA binding, but also show distinct structural stabilty, aggregation and toxicity profiles. [R273C]p53 shows maximum amyloidogenicity while [R273L]p53 shows maximum aggregation. Further probe in the aggregation mechanism show that [R273C]p53 aggregation is disulphide mediated whereas hydrophobic interactions dominate self-assembly in [R273L]p53. MD simulation studies clearly show that -helical intermediates are observed in [R273C]p53 whereas {beta}-sheets are observed for [R273L]p53. Our study indicates that each of the R273 variant has its own distinct property of stability and self-assembly, the molecular basis of which, may lead to different types of cancer pathogenesis in vivo. These studies will aid the design of therapeutic strategies for cancer using residue specific or process specific protein aggregation as target.\n\nStatement of significanceThe present work stems from an interesting observation that genetic mutations that results in switching of one amino acid to different variants at the same codon show different cancer cell progression. We are trying to understand the molecular reason behind the different gain-of-function opted by these variants. With the help of biophysical and biochemical experiments, and computational studies we have observed that the different thermal stability, unique mechanism of unfolding and self-assembly might be one of the crucial parameters for their different oncogenic effect. These studies thus call for the need of developing therapeutic strategies that consider the resultant mutant-variant as a target rather than mutation position. This is an important lead towards the understanding of cancer.

biophysics↗

bioRxiv2019

Broken force dispersal network in tip-links by the mutations induces hearing-loss

Tip-link as force-sensor in the hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in inner-ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an AFM. AFM allowed us to mimic the high and wide range of force ramps (103 - 106 pN.s-1) as experienced in the inner ear. We performed in silico network analyses to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.\n\nSignificance StatementForce-sensors in inner ear are the key components in the hearing. Mutations in force-sensors often lead to congenital hearing loss. Loss of hearing has become a threat to humanity, with over 5% of world population suffering from deafness and 40% of which is congenital, primarily due to mutations in the sensory machinery in inner-ear. A better understanding of the molecular mechanism of the underlined hearing loss due to mutations is, therefore, necessary for better therapeutics to deaf. Here with a zoomed region of the force-sensors, we pointed out the differences in the force-propagation properties of the mutant and wild-type force-sensors. Our observation on restoring of functions of mutants in Ca2+-rich buffer indicates methods of developing low-cost therapeutic strategies against deafness.

biophysics↗

bioRxiv2017

Molecular mechanism of cell-cell adhesion mediated by cadherin-23

Adherin-junctions are traditionally described by the homophilic-interactions of classical cadherin-proteins at the extracellular region. However, the role of long-chain non-classical cadherins like cadherin-23(Cdh23) is not explored as yet even though it is implicated in tissue-morphogenesis, cancer, and force-sensing in neuronal tissues. Here, we identified a novel antiparallel-binding interface of Cdh23 homodimer in solution by combining biophysical and computational methods, in-vitro cell-binding, and mutational modifications. The dimer consists of two electrostatic-based interfaces extended up to two terminal domains, atypical to classical-cadherins known so far, and forms the strongest interactions in cadherin-family as measured using single-molecule force-spectroscopy. We further identified single point-mutation, E78K, that completely disrupts this binding. Interestingly, the mutation, S77L, found in skin cancers falls within the binding interface of the antiparallel-dimer. Overall, we provide the molecular architecture of Cdh23 at the cell-cell junctions which are likely to have far-reaching applications in the fields of mechanobiology and cancer.

biophysics↗