Biology subjects

Acharya, R.

Publications and source records attributed to Acharya, R..

11 recordsLinked to original sources

bioRxiv2026

A Robust and Scalable Workflow for the Production of Circular Single-Stranded DNA for Genome Engineering Applications

Circular single-stranded DNA (cssDNA) is a versatile biomolecule with applications spanning genome editing, DNA nanotechnology, synthetic biology, molecular diagnostics, and aptamer development. Compared with linear single-stranded DNA, cssDNA offers enhanced structural stability, resistance to in-cellulo degradation by exonucleases and enables the generation of long, sequence-defined DNA molecules that are difficult to obtain through conventional chemical synthesis methods. Despite its growing utility, widespread adoption of cssDNA has been limited by the lack of accessible, scalable, and cost-effective production methods, with many existing workflows relying on specialised reagents, extensive optimisation, or commercially synthesised DNA. Here, we present a streamlined, end-to-end protocol for the laboratory-scale production of high-purity cssDNA using an M13 phagemid-based system and standard molecular biology laboratory infrastructure. The workflow encompasses bacterial culture, phage amplification, nuclease treatment, phage precipitation, anion-exchange purification, and quality control, with practical optimisations to improve yield, reproducibility, and scalability. Using this approach, yields range from 120-195 {micro}g of purified cssDNA from 300 mL of culture supernatant. The protocol provides detailed guidance on critical process parameters, troubleshooting, and quality assessment, enabling reliable production of cssDNA suitable for a wide range of downstream molecular biology and genome engineering applications.

molecular biology↗

bioRxiv2025

Defying Salinity, Drought, and pH Extremes: A Multifunctional Rhizobacterium, Burkholderia gladioli ST3M-39a, Matches Fertilizer Efficacy in Wheat via Phosphate Solubilization

Global phosphorus scarcity and the environmental impacts of chemical fertilizers necessitate sustainable microbial alternatives for agriculture. We characterized Burkholderia gladioli ST3M-39a, a maize rhizosphere isolate, as a multifunctional plant growth-promoting rhizobacterium with exceptional climate resilience. The strain achieved rapid phosphate solubilization (177.96 {+/-} 5.26 {micro}g/mL within 24 h; molybdenum-antimony assay), zinc solubilization, and ammonia production, EPS production, and produced stress-alleviating enzymes (cellulase and protease). Crucially, it maintained robust growth and phosphate-mobilizing capacity under extreme abiotic stresses: pH 4.5-8.5, 7.5% NaCl salinity, and drought-mimicking low water activity (aw 0.950, 32% sorbitol). In wheat trials, ST3M-39a inoculation significantly increased the growth parameters (p < 0.05 vs. those of the uninoculated controls), resulting in 85-92% of the biomass stimulation observed with diammonium phosphate (DAP) fertilizer. This multifunctional stress tolerance, coupled with its near-fertilizer efficacy, positioned ST3M-39a as a transformative bioinoculant for degraded soils. Field validation of its agricultural deployment and ecological impact is now pivotal.

microbiology↗

bioRxiv2025

Multi Omics Driven Polypharmacology for Osteoarthritis Joint Regeneration

Osteoarthritis (OA) is a common degenerative condition of major joints with no effective cure. Yograj Guggulu (YG) is a well-established polyherbal formulation in Indian traditional medicine for the management of joint disorders. However, its mode of action remains unclear. We integrated multi omics network analysis with a rat model to investigate the effects of oral administration of YG on osteoarthritic joints. YG administration significantly alleviated pain and improved joint function. Imaging and histological analyses reveal that YG protects against structural degeneration in the joint with OA. Proteomic and metabolomic profiling, along with network analysis, uncovered that multiple components of YG alter protein expression in key pathways related to inflammation and cartilage degradation, thus validating the observed clinical effects. The efficacy of YG seems to be the result of the synergistic action of its diverse constituents. Our findings support the use of YG as a multitarget therapy for OA and encourage efforts for the potential repurposing of its active components.

systems biology↗

bioRxiv2024

Insights into the conformational dynamics of the cytoplasmic domain of metal-sensing sensor histidine kinase ZraS

ZraS is a metal sensor integral to ZraPSR, a two-component signaling system found in enterobacters. It belongs to a family of bifunctional sensor histidine kinases (SHKs) and is speculated to sense zinc-induced stress on the bacterial envelope. Information on the structure-function relationship of sensor kinases is elusive due to the lack of full-length structures, intrinsically dynamic behavior, and difficulty trapping them in active state conformations. While the kinase domains of a few sensor histidine kinases are well-characterized, they exhibit significant functional diversity attributed to their modular multi-domain arrangement in the cytoplasmic region, combined with other signal transducing elements such as simple helices, HAMP and PAS domains. We report the crystal structure of the entire cytoplasmic region of Escherichia coli ZraS (EcZraS-CD) resolved at resolution 2.49 [A], comprising of a unique helical linker and the kinase domain. In the asymmetric unit, four molecules of ZraS assemble as homodimers trapped as two ligand-bound occluded conformers. Our analysis using these conformers show that modulation of the dimer bundle through segmental helical bending, sliding, and rotation leads to reorganization of the dimerization interface during kinase activation. Further, our analysis reveals the significance of aromatic amino acid interactions and loop residues at the dimer base in regulating the directionality of rotation during autophosphorylation. We also performed an invitro coupled assay to determine ATPase activity. Overall, our findings provide structure-based mechanistic insights into the process of autophosphorylation in trans-acting sensor histidine kinases.

biochemistry↗

bioRxiv2024

MALT1 protease inhibition restrains glioblastoma progression by reversing tumor-associated macrophage-dependent immunosuppression

MALT1 protease is an intracellular signaling molecule that promotes tumor progression via cancer cell-intrinsic and cancer cell-extrinsic mechanisms. MALT1 has been mostly studied in lymphocytes, and little is known about its role in tumor-associated macrophages. Here, we show that MALT1 plays a key role in glioblastoma (GBM)-associated macrophages. Mechanistically, GBM tumor cells induce a MALT1-NF-{kappa}B signaling axis within macrophages, leading to macrophage migration and polarization toward an immunosuppressive phenotype. Inactivation of MALT1 protease promotes transcriptional reprogramming that reduces migration and restores a macrophage "M1-like" phenotype. Preclinical in vivo analysis shows that MALT1 inhibitor treatment results in increased immuno-reactivity of GBM-associated macrophages and reduced GBM tumor growth. Further, the addition of MALT1 inhibitor to temozolomide reduces immunosuppression in the tumor microenvironment, which may enhance the efficacy of this standard-of-care chemotherapeutic. Together, our findings suggest that MALT1 protease inhibition represents a promising macrophage-targeted immunotherapeutic strategy for the treatment of GBM. Graphical abstract. O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/614808v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@18c969eorg.highwire.dtl.DTLVardef@f48f0dorg.highwire.dtl.DTLVardef@1dbbbb1org.highwire.dtl.DTLVardef@ff8237_HPS_FORMAT_FIGEXP M_FIG The effects of tumor cell-induced CARD9-BCL10-MALT1 (CBM) activation (left) and MALT1 protease inhibition (right) on GBM associated macrophages in the tumor microenvironment. Cartoon of cellular components of a GBM tumor with an immunosuppressive TME characterized by "M2-like macrophages" (left) and conversion to a more immune-reactive tumor microenvironment characterized by "M1-like macrophages and increased effector T-cells (right) as a result of MALT1 protease inhibition. C_FIG

cancer biology↗

bioRxiv2023

A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of SignificanceWe developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

cancer biology↗

bioRxiv2023

Structural analyses of 'substrate-pH of activity' pairing observed in Polysaccharide lyases

Anionic polysaccharides found in nature are functionally and structurally diverse, and so are the polysaccharide lyases (PLs) which catalyse their degradation. Atomic superposition of various PL folds according to their cleavable substrate structure confirm the occurrence of structural convergence at PL active sites. This suggests that various PL folds have emerged to cleave a particular class of anionic polysaccharide during the course of evolution. While structural and mechanistic similarity of PLs active site have been highlighted in earlier studies, a detailed understanding regarding functional properties of this catalytic convergence remains an open question, especially the role of extrinsic factors such as pH in the context of substrate binding and catalysis. Our earlier structural and functional work on pH directed multi-substrate specificity of Smlt1473 inspired us to regroup PLs according to substrate type to analyse the pH dependence of their catalytic activity. Interestingly, we find that particular groups of substrates are cleaved in a particular pH range (acidic/neutral/basic) irrespective of PLs fold, boosting the idea of functional convergence as well. Based on this observation, we set out to define structurally and computationally the key constituents of an active site among PL families. This study delineates the structural determinants of conserved substrate-pH activity pairing within and between PL families.

biochemistry↗

bioRxiv2021

The ultrafast snap of a finger is mediated by skin friction

The snap of a finger has been used as a form of communication and music for millennia across human cultures. However, a systematic analysis of the dynamics of this rapid motion has not yet been performed. Using high-speed imaging and force sensors, we analyze the dynamics of the finger snap. Our analysis reveals the central role of skin friction in mediating the snap dynamics by acting as a latch to control the resulting high velocities. We evaluate the role of this frictional latch experimentally, by covering the thumb and middle finger with different materials to produce different friction coefficients and varying compressibility. In doing so, we reveal that the compressible, frictional latch of the finger pads likely operate in a regime optimally tuned for both friction and compression. We also develop a soft, compressible friction-based latch-mediated spring actuated (LaMSA) model to further elucidate the key role of friction and how it interacts with a compressible latch. Our mathematical model reveals that friction plays a dual role in the finger snap, both aiding in force loading and energy storage while hindering energy release. Our work reveals how friction between surfaces can be harnessed as a tunable latch system and provide design insight towards the frictional complexity in many robotics and ultra-fast energy-release structures.

biophysics↗

bioRxiv2021

Virtual meetings promise to eliminate the geographical and administrative barriers and increase accessibility, diversity, and inclusivity

The COVID-19 pandemic brought a new set of unprecedented challenges not only for healthcare, education, and everyday jobs but also in terms of academic conferences. In this study, we investigate the effect of the broad adoption of virtual platforms for academic conferences as a response to COVID-19 restrictions. We show that virtual platforms enable higher participation from underrepresented minority groups, increased inclusion, and broader geographic distribution. We also discuss emerging challenges associated with the virtual conference format resulting in a decreased engagement of social activities, limited possibilities of cross-fertilization between participants, and reduced peer-to-peer interactions. Lastly, we conclude that a novel comprehensive approach needs to be adopted by the conference organizers to ensure increased accessibility, diversity, and inclusivity of post-pandemic conferences. Our findings provide evidence favoring a hybrid format for future conferences, marrying the strength of both in-person and virtual platforms.

scientific communication and education↗

bioRxiv2020

A Tunable, Simplified Model for Biological Latch Mediated Spring Actuated Systems

We develop a model of latch-mediated spring actuated (LaMSA) systems relevant to comparative biomechanics and bioinspired design. The model contains five components: two motors (muscles), a spring, a latch, and a load mass. One motor loads the spring to store elastic energy and the second motor subsequently removes the latch, which releases the spring and causes movement of the load mass. We develop open-source software to accompany the model, which provides an extensible framework for simulating LaMSA systems. Output from the simulation includes information from the loading and release phases of motion, which can be used to calculate kinematic performance metrics that are important for biomechanical function. In parallel, we simulate a comparable, directly actuated system that uses the same motor and mass combinations as the LaMSA simulations. By rapidly iterating through biologically relevant input parameters to the model, simulated kinematic performance differences between LaMSA and directly actuated systems can be used to explore the evolutionary dynamics of biological LaMSA systems and uncover design principles for bioinspired LaMSA systems. As proof of principle of this concept, we compare a LaMSA simulation to a directly actuated simulation that includes a either Hill-type force-velocity trade-off or muscle activation dynamics, or both. For the biologically-relevant range of parameters explored, we find that the muscle force-velocity trade-off and muscle activation have similar effects on directly actuated performance. Including both of these dynamic muscle properties increases the accelerated mass range where a LaMSA system outperforms a directly actuated one.

biophysics↗

bioRxiv2018

A Computational Framework To Assess Genome-Wide Distribution Of Polymorphic Human Endogenous Retrovirus-K In Human Populations

Human Endogenous Retrovirus type K (HERV-K) is the only HERV known to be insertionally polymorphic. It is possible that HERV-Ks contribute to human disease because people differ in both number and genomic location of these retroviruses. Indeed viral transcripts, proteins, and antibody against HERV-K are detected in cancers, auto-immune, and neurodegenerative diseases. However, attempts to link a polymorphic HERV-K with any disease have been frustrated in part because population frequency of HERV-K provirus at each site is lacking and it is challenging to identify closely related elements such as HERV-K from short read sequence data. We present an integrated and computationally robust approach that uses whole genome short read data to determine the occupation status at all sites reported to contain a HERV-K provirus. Our method estimates the proportion of fixed length genomic sequence (k-mers) from whole genome sequence data matching a reference set of k-mers unique to each HERV-K loci and applies mixture model-based clustering to account for low depth sequence data. Our analysis of 1000 Genomes Project Data (KGP) reveals numerous differences among the five KGP super-populations in the frequency of individual and co-occurring HERV-K proviruses; we provide a visualization tool to easily depict the prevalence of any combination of HERV-K among KGP populations. Further, the genome burden of polymorphic HERV-K is variable in humans, with East Asian (EAS) individuals having the fewest integration sites. Our study identifies population-specific sequence variation for several HERV-K proviruses. We expect these resources will advance research on HERV-K contributions to human diseases.\n\nAuthor summaryHuman Endogenous Retrovirus type K (HERV-K) is the youngest of retrovirus families in the human genome and is the only group that is polymorphic; a HERV-K can be present in one individual but absent from others. HERV-Ks could contribute to disease risk but establishing a link of a polymorphic HERV-K to a specific disease has been difficult. We develop an easy to use method that reveals the considerable variation existing among global populations in the frequency of individual and co-occurring polymorphic HERV-K, and in the total number of HERV-K that any individual has in their genome. Our study provides a global reference set of HERV-K genomic diversity and tools needed to determine the genomic landscape of HERV-K in any patient population.

genomics↗