Biology subjects

Sarkar, B.

Publications and source records attributed to Sarkar, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Avidity-controlled biotherapeutic delivery systems for the treatment of ischemia-reperfusion acute kidney injury

Regulatory T cells (Tregs) are key mediators of immune resolution and tissue repair following acute kidney injury (AKI), yet strategies to selectively enrich Tregs within the renal microenvironment remain limited. Interleukin-2 (IL-2) is essential for Treg survival and function, but systemic IL-2 delivery is constrained by a short half-life, narrow therapeutic window, and risk of off-target activation of cytotoxic T cells. Effective Treg-based immunomodulation may therefore require both localized cytokine signaling and targeted recruitment of precursor immune populations. Here, we present a sequential, biomaterial-based immunomodulatory strategy that integrates immune cell recruitment with sustained cytokine delivery. An injectable granular hydrogel physically encapsulating the chemokine CCL21 was used to initially recruit CD4+ T cells to the injured kidney. Subsequently, supramolecular guest-host interactions were leveraged to deliver IL-2 modified with the hydrophobic "guest" moiety adamantane, enabling prolonged local cytokine release over 28 days. In healthy mice, we observed early CD4+ T cell recruitment, followed by a pronounced increase in Treg frequency, consistent with immune reprogramming driven by our sequential delivery system. In a murine model of bilateral ischemia-reperfusion (BiAKI), this injectable biomaterial induced a local increase in the proportion of FOXP3-expressing Tregs and a concomitant decrease in the levels of AKI markers. This two-step immunomodulatory approach minimizes acute immune activation while promoting long-term enrichment of Tregs within the renal microenvironment. Together, these findings demonstrate that granular hydrogels with supramolecular host capability can orchestrate immune recruitment and differentiation in a spatiotemporally controlled manner, providing a versatile immunomodulatory platform for applicability in kidney disease.

bioengineering↗

bioRxiv2026

A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common form of kidney cancer. It is also one of deadliest cancers, with a 5-year survival rate of less than 20% in advanced cancer patients with distant metastasis. Current treatments rely on targeted therapies, such as sunitinib, which are limited in eHicacy. Recently, antibody drug conjugates (ADCs) have emerged as a promising treatment modality by delivering cytotoxic payloads specifically to cancer cells. Here, we describe the engineering of a novel ADC (LT-025), where the antibody targets the kidney injury molecule (KIM) -1 receptor over-expressed on RCC cells to deliver a toxic maytansinoid (DM1) payload. Unlike prior attempts to engineer a KIM-1 targeted ADC that were limited by a heterogenous product, here we used a microbial transglutaminase (MTGase)-based conjugation strategy, which achieved a site-specific conjugation of the drug-linker to the antibody, resulting in a homogenous ADC with a drug-to-antibody ratio (DAR) of 2. The ADC exhibited prolonged stability, excellent antigen-binding capability, and KIM-1 expression-dependent cellular internalization and cytotoxicity in RCC, including in sunitinib-resistant RCC. Excitingly, LT-025 was well tolerated in vivo, and combining LT-025 and sunitinib exhibited a synergistic antitumor eHicacy in a RCC mouse model. Combining an ADC with a targeted therapeutic could emerge as a paradigm shift in the management of advanced RCC.

cancer biology↗

bioRxiv2025

Investigating the prognostic value of KCNN4 gene expression in human pancreatic adenocarcinoma by bioinformatic analysis

Finding accurate biomarkers for early detection and prognosis is critical because pancreatic adenocarcinoma (PAAD) is a deadly cancer with a poor prognosis. Though its function in PAAD is still unknown, the gene KCNN4, which codes for a calcium-activated potassium channel, has been linked to a number of malignancies. In this work, the cancer studies of the CPTAC, TCGA, and GTEx projects were targeted using a thorough database mining technique. Several analytical methods were used to examine full-scale molecular, expression, and prognostic profiles of the KCNN4 gene in pancreatic adenocarcinoma tissues. It was discovered that the majority of the cancerous tissue had different levels of mRNA and protein expression for this gene. The results of comparative immunohistochemistry also revealed that normal pancreatic tissues had reduced expression of the KCNN4 protein. An analysis overview of copy-number alterations and KCNN4 mutations in PAAD samples was also conducted. Furthermore, there was a correlation found between the expression of KCNN4 and both overall and disease-free survival in pancreatic adenocarcinoma. In pancreatic adenocarcinoma tissue, co-expressed genes of KCNN4 were found to be involved in cell maintenance-related functions, according to gene coexpression, additional ontology, and pathway analysis. When it comes to cancer diagnosis and treatment, the experimental findings of this study should aid in integrating KCNN4 into clinical applications.

bioinformatics↗

bioRxiv2025

Computational Evaluation of Phytochemicals as Potential Anti-HIV Drugs Targeting CCR5 and CXCR4 Receptors

HIV is a major worldwide health concern; hence new therapeutic approaches are needed to fight viral resistance and enhance treatment results. HIV entrance into host cells depends on the CCR5 and CXCR4 receptors, which makes them potential targets for antiviral medication development. The objective of this study is to computationally evaluate 53 phytochemicals that target CCR5 and CXCR4 as potential anti-HIV medications. Effective anti-HIV medications were projected to be phytochemicals that may inhibit these receptors and so interfere with the HIV life cycle. AutoDock Vina was used to perform the molecular docking investigation from which six phytochemicals capable of inhibiting CCR5 and CXCR4 were identified based on the lowest docking score. i.e., Withaferin A, Oleanolic Acid, Ursolic Acid, Theaflavine, Camptothecin, and Hypericin. The SWISSADME server was utilized to decide their druglikeness properties, the ADMETlab server to predict different pharmacokinetic and pharmacodynamic properties, the PASS-Way2Drug server to evaluate their activity spectra, and the RS-WebPredictor server to figure out the metabolism in the body. They adhered to Lipinskis rule of five and had promising ADME/toxicity study result along with favorable molecular dynamics simulation. Overall, the above-mentioned six phytochemicals might have the potential to be used as alternative HIV therapeutics.

bioinformatics↗

bioRxiv2025

A Chlorinated Diketopiperazine Antibiotic Targets Mycobacterium Tuberculosis DNA Gyrase

We describe a novel macrocyclic peptide, speirobactin, produced by Photorhabdus that selectively kills Mycobacterium tuberculosis. A non-ribosomal peptide synthase (NRPS) containing two linear modules codes for the synthesis of speirobactin. The biosynthetic operon contains a pentapeptide-repeat protein as a resistance gene. Genomic analysis of speirobactin-resistant mutants of M. tuberculosis led to identification of DNA gyrase as the molecular target. The mutations were recreated by allelic replacement and show that DNA gyrase is the only target. Transcriptome analysis of M. tuberculosis treated with antibiotics shows that speirobactin clusters close to fluoroquinolones, supporting its action against the DNA gyrase.

cell biology↗

bioRxiv2024

Identification of novel thiazole derivatives as flaviviral protease inhibitors against DENV and JEV

Flaviviruses are the causative agents of viral hemorrhagic fever (VHF) globally and have demonstrated the capacity to result in fatal outcomes if not managed effectively. Among different types of flaviviruses, dengue (DENV) and Japanese encephalitis (JEV) are the most common in tropical and subtropical countries. Given the scarcity of effective vaccines against flaviviral infections, small molecule agents that target crucial viral proteins emerge as the sole feasible option. This study outlines the synthesis of novel thiazole compounds as flavivirus NS2B-NS3 protease inhibitor and characterization of their antiviral activity against DENV and JEV. We synthesized a heterocyclic template derived from a substrate-based retrotripeptide dengue protease inhibitor, leading to forty-eight thiazole derivatives. Two compounds demonstrated significant inhibition of dengue virus protease activity in vitro. Comprehensive characterization of these two compounds was conducted through biochemical assay which revealed an uncompetitive mode of inhibition. Subsequent cell-based assays using Dengue and Japanese encephalitis viruses as representatives Flaviviruses imparted the potential of these compounds to block viral RNA synthesis, and viral replication. Finally time-course experiments unveiled that the two compounds impeded the accumulation of viral genomic RNA primarily at later stages of infection, aligning with their capacity to hinder NS2B-NS3 protease activity, polyprotein processing and viral genomic RNA replication. Together our work presents the development and validation of flavivirus protease inhibitors with therapeutic potential against Dengue and Japanese encephalitis virus.

microbiology↗

bioRxiv2024

Notch and LIM-homeodomain protein Arrowhead regulate each other in a feedback mechanism to play a role in wing and neuronal development in Drosophila

Notch pathway is an evolutionarily conserved signaling system that operates to influence an astonishing array of cell fate decisions in different developmental contexts. To identify novel effectors of Notch signaling, we analyzed the whole transcriptome of Drosophila wing and eye imaginal discs in which an activated form of Notch was overexpressed. A LIM homeodomain protein Arrowhead (Awh) was identified as a novel candidate which plays a crucial role in Notch mediated developmental events. Awh alleles show strong genetic interaction with Notch pathway components. Awh loss-of-function upregulates Notch targets Cut and Wingless. Awh gain-of-function downregulates Notch targets by reducing the expression of ligand, Delta. Consequently, the expression of Wingless effector molecule Armadillo and its downstream targets, Senseless and Vestigial, also gets downregulated. Awh overexpression leads to ectopicexpression of engrailed, a segment polarity gene in the anterior region of wing disc, leading to patterning defects. Additionally, Notch gain-of-function mediated neuronal defects get significantly rescued with Awh overexpression. Activated Notch inhibits Awh activity, suggesting a regulatory loop between Awh and Notch. Additionally, the defects caused by Awh gain-of-function were remarkably rescued by Chip, a LIM interaction domain containing transcriptional co-factor. The present study highlights the novel feedback regulation between Awh and Notch.

developmental biology↗

bioRxiv2024

A Mycobacterium tuberculosis secreted virulence factor disrupts host snRNP biogenesis

We earlier reported that Mycobacterium tuberculosis (Mtb) alters host RNA splicing to help its intracellular growth. Here, we report an unprecedented mechanism whereby a secreted virulence factor from Mtb interferes with the biogenesis of key spliceosomal components, causing an altered RNA splicing pattern. A high-throughput yeast-2-hybrid screen identified several Mtb-secreted proteins that can interact with the host RNA splicing factors (SFs). Through custom-designed in-cell assays, we show that one of those proteins, Rv1435c, targets specific exon-skipping events to alter RNA splicing. We show that Rv14345c or host splicing regulator 1 (hsr1) facilitates direct interaction between Mtb phagosomes and U5 snRNA and SNRPF, key components of the snRNPs. Genetic deletion of hsr1 reverses the specific exon-skipping events caused by WT Mtb infection. The{Delta} hsr1 strain shows compromised growth during ex vivo infection in the macrophages and in vivo infection in the mice. Tissue sections from the WT Mtb or{Delta} hsr1-infected mice showed significant hsr1-dependent SNRPF staining, a phenomenon also noted in the human intestinal tuberculosis (ITB) biopsies. We infer that hsr1 is a virulence factor which alters RNA splicing by interacting with U5snRNA and SNRPF. The splicing regulators from the host and pathogen are novel targets for anti-tuberculosis therapy.

molecular biology↗

bioRxiv2024

PPARγ mediated enhanced lipid biogenesis fuels Mycobacterium tuberculosis growth in hepatocytes.

Mycobacterium tuberculosis (Mtb) infection of the lungs, besides producing prolonged cough with mucus, also causes progressive fatigue and cachexia with debilitating loss of muscle mass. While anti-tuberculosis (TB) drug therapy is directed toward eliminating bacilli, the treatment regimen ignores the systemic pathogenic derailments that probably dictate TB-associated mortality and morbidity. Presently, it is not understood whether Mtb spreads to metabolic organs and brings about these impairments. Here we show that Mtb creates a replication-conducive milieu of lipid droplets in hepatocytes by upregulating transcription factor PPAR{gamma} and scavenging lipids from the host cells. In hepatocytes, Mtb shields itself against the common anti-TB drugs by inducing drug-metabolizing enzymes. Infection of the hepatocytes in the in vivo aerosol mice model can be consistently observed post-week 4 along with enhanced expression of PPAR{gamma} and drug-metabolizing enzymes. Moreover, histopathological analysis indeed shows the presence of Mtb in hepatocytes along with granuloma-like structures in human biopsied liver sections. Hepatotropism of Mtb during the chronic infectious cycle results in immuno-metabolic dysregulation that could magnify local and systemic pathogenicity, altering clinical presentations.

microbiology↗

bioRxiv2023

A novel vitamin C analog acts as a potent bio-enhancer to augment the activities of anti-tuberculosis drugs against Mycobacterium tuberculosis

Mycobacterium tuberculosis is a deadly pathogen that claims millions of lives every year. Current research focuses on finding new anti-tuberculosis drugs that are safe and effective, with lesser side effects and toxicity. One important approach is to identify bio-enhancers that can improve the effectiveness of anti-tuberculosis drugs, resulting in reduced doses and shortened treatment times. We investigated the use of vitamin C-derived isotetrones as bio- enhancer agents. In this context, our results revealed that the lead compound C11 inhibits growth, improves MIC/MBC, and enhances the killing of M. tuberculosis H37Rv strain when used in combination with first-line and injectable anti-TB drugs in a dose-dependent manner. The combination of C11 and rifampicin also reduced the generation of spontaneous mutants against rifampicin and reached a mutation prevention concentration (MPC) with moderate rifampicin concentrations. The identified compounds were proven to be effective against the MDR strain of M. tuberculosis and non-cytotoxic in HepG2 cells. We also found that C11 induced the generation of reactive oxygen species (ROS) inside macrophages and within bacteria, resulting in better efficacy.

microbiology↗

bioRxiv2023

Neutrophil-derived IL-17 limits protective host responses and promotes tuberculosis pathogenesis

The protective correlates of Mycobacterium tuberculosis (Mtb) infection-elicited host immune responses are incompletely understood. Here, we report pro-pathogenic crosstalk involving Ly6G+granulocytes (Ly6G+Gra), IL-17 and COX2. We show that in the lungs of Mtb-infected wildtype mice, either BCG-vaccinated or not, most intracellular bacilli are Ly6G+Gra-resident four weeks post-infection onwards. In the genetically susceptible IFN{psi} -/- mice, excessive Ly6G+Gra infiltration correlates with severe bacteraemia. Neutralizing IL-17 (anti-IL17mAb) and COX2 inhibition by celecoxib reverse Ly6G+Gra infiltration, associated pathology and death in IFN{psi} -/- mice. Surprisingly, Ly6G+Gra also serves as the major source of IL-17 in the lungs of Mtb-infected WT or IFN{psi} -/- mice. The IL-17-COX2-Ly6G+Gra interplay also operates in WT mice. Inhibiting ROR{psi}t, the key transcription factor for IL-17 production or COX2, reduces the bacterial burden in Ly6G+Gra, leading to reduced bacterial burden and pathology in the lungs of WT mice. In the Mtb-infected WT mice, COX2 inhibition abrogates IL-17 levels in the lung homogenates and significantly enhances BCGs protective efficacy, mainly by targeting the Ly6G+Gra-resident Mtb pool, a phenotype also observed when IL-17 is blocked by ROR{psi}t inhibitor. Furthermore, in pulmonary TB patients, high neutrophil count and IL-17 correlated with adverse treatment outcomes. Together, our results suggest that IL-17 and PGE2 are the negative correlates of protection, and we propose targeting the pro-pathogenic IL-17-COX2-Ly6G+Gra axis for TB prevention and therapy.

immunology↗

bioRxiv2023

A hepatocyte-specific transcriptional program driven by Rela and Stat3 exacerbates experimental colitis in mice by modulating bile synthesis

Hepatic factors secreted by the liver promote homeostasis and are pivotal for maintaining the liver-gut axis. Bile acid metabolism is one such example wherein, bile acid synthesis occurs in the liver and its biotransformation happens in the intestine. Dysfunctional interactions between the liver and the intestine stimulate varied pathological outcomes through its bidirectional portal communication. Indeed, aberrant bile acid metabolism has been reported in inflammatory bowel disease (IBD). However, the molecular mechanisms underlying these crosstalks that perpetuate intestinal permeability and inflammation remain obscure. Here, we identify a novel hepatic gene program regulated by Rela and Stat3 that accentuates the inflammation in an acute experimental colitis model. Hepatocyte-specific ablation of Rela and Stat3 reduces the levels of primary bile acids in both the liver and the gut and shows a restricted colitogenic phenotype. On supplementation of chenodeoxycholic acid (CDCA), knock-out mice exhibit enhanced colitis-induced alterations. This study provides persuasive evidence for the development of multi-organ strategies for treating IBD and identifies a hepatocyte-specific rela-stat3 network as a promising therapeutic target.

immunology↗

bioRxiv2023

Apolipoprotein-E transforms intracellular Amyloid-β oligomers to a more toxic state

It is poorly understood why ApoE variants are major genetic risk factors in Alzheimers disease (AD), which is associated with the aggregation of amyloid beta (A{beta}). Here we directly image specific changes in small A{beta} oligomers in rat brain cells that correlate with the cellular ApoE content. An inhibitor of A{beta}-ApoE interaction suppresses this change and concomitantly reduces A{beta} toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and the stoichiometry of the oligomers. hiPSC-derived neural stem cells from Alzheimers patients also show similar changes. Interaction with ApoE therefore changes the oligomeric state, membrane affinity, and toxicity of A{beta} oligomers, and can be directly read out in live cells. Our findings suggest a rapid and quantitative assay for AD drug discovery. One-sentence summaryApoE causes specific toxicogenic modifications of A{beta} oligomers, and these changes can be directly imaged in live cells.

biochemistry↗

bioRxiv2023

Second messenger c-di-AMP regulates multiple antibiotic sensitivity pathways in Mycobacterium smegmatis by discrete mechanisms

One of the debilitating causes of high mortality in the case of tuberculosis and other bacterial infections is the resistance development against standard drugs. There are limited studies so far to describe how a bacterial second messenger molecule can directly participate in distinctive antibiotic tolerance characteristics of a cell in a mechanism-dependent manner. Here we show that intracellular cyclic di-AMP (c-di-AMP) concentration can modulate drug sensitivity of Mycobacterium smegmatis by directly interacting with either a protein effector or with the 5-UTR regions in mRNA of the genes and thus causing transcriptional downregulation of important genes in the pathways. We studied four antibiotics with different mechanisms of action: rifampicin, ciprofloxacin, erythromycin, and tobramycin and subsequently found that the level of drug sensitivity of the bacteria is directly proportional to the c-di-AMP concentration inside the cell. Further, we unraveled the underlying molecular mechanisms to delineate the specific genes and pathways regulated by c-di-AMP and hence result in differential drug sensitivity in M. smegmatis. Thus, our findings of c-di-AMP messenger controlling drug resistance phenotypes of mycobacteria against four different classes of antibiotics is a unique observation that will contribute to scientific advancement in the field. One Sentence SummaryHere we describe how second messenger c-di-AMP modulates the antibiotic sensitivity and resistance profile of M. smegmatis by diverse mechanisms.

microbiology↗

bioRxiv2023

E3 ubiquitin ligase Deltex facilitates the expansion of Wingless gradient and antagonizes Wingless signaling through a conserved mechanism of transcriptional effector Armadillo/β-catenin degradation

The Wnt/Wg pathway controls myriads of biological phenomena throughout the development and adult life of all organisms across the phyla. Thus, an aberrant Wnt signaling is associated with a wide range of pathologies in humans. Tight regulation of Wnt/Wg signaling is required to maintain proper cellular homeostasis. Here we report a novel role of E3 ubiquitin ligase Deltex in Wg signaling regulation. Drosophila dx genetically interacts with wg and its pathway components. Further, Dx LOF results in a reduced spreading of Wg while its over-expression expands the diffusion gradient of the morphogen. We attribute this change in Wg gradient to the endocytosis of Wg through Dx which directly affects the short and long-range Wg targets. We also demonstrate the role of Dx in regulating Wg effector Armadillo where Dx down-regulates Arm through proteasomal degradation. We also showed the conservation of Dx function in the mammalian system where DTX1 is shown to bind with {beta}-catenin and facilitates its proteolytic degradation, spotlighting a novel step that potentially modulates Wnt/Wg signaling cascade.

developmental biology↗

bioRxiv2022

Single Photon smFRET. III. Application to Pulsed Illumination

Forster resonance energy transfer (FRET) using pulsed illumination has been pivotal in leveraging lifetime information in FRET analysis. However, there remain major challenges in quantitative single photon, single molecule FRET (smFRET) data analysis under pulsed illumination including: 1) simultaneously deducing kinetics and number of system states; 2) providing uncertainties over estimates, particularly uncertainty over the number of system states; 3) taking into account detector noise sources such as crosstalk, and the instrument response function contributing to uncertainty; in addition to 4) other experimental noise sources such as background. Here, we implement the Bayesian nonparametric framework described in the first companion manuscript that addresses all aforementioned issues in smFRET data analysis specialized for the case of pulsed illumination. Furthermore, we apply our method to both synthetic as well as experimental data acquired using Holliday junctions. Why It MattersIn the first companion manuscript of this series, we developed new methods to analyze noisy smFRET data. These methods eliminate the requirement of a priori specifying the dimensionality of the physical model describing a molecular complexs kinetics. Here, we apply these methods to experimentally obtained datasets with samples illuminated by laser pulses at regular time intervals. In particular, we study conformational dynamics of Holliday junctions.

biophysics↗

bioRxiv2022

Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway

Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.

cell biology↗

bioRxiv2022

Exploring different virulent proteins of human respiratory syncytial virus for designing a novel epitope-based polyvalent vaccine: Immunoinformatics and molecular dynamics approaches

Human Respiratory Syncytial Virus (RSV) is one of the most prominent causes of lower respiratory tract infections (LRTI), contributory to infecting people from all age groups - a majority of which comprises infants and children. The implicated severe RSV infections lead to numerous deaths of multitudes of the overall population, predominantly the children, every year. Consequently, despite several distinctive efforts to develop a vaccine against the RSV as a potential countermeasure, there is no approved or licensed vaccine available yet, to control the RSV infection effectively. Therefore, through the utilization of immunoinformatics tools, a computational approach was taken in this study, to design and construct a multi-epitope polyvalent vaccine against the RSV-A and RSV-B strains of the virus. Potential predictions of the T-cell and B-cell epitopes were followed by extensive tests of antigenicity, allergenicity, toxicity, conservancy, homology to human proteome, transmembrane topology, and cytokine-inducing ability. The most promising epitopes (i.e. 13 CTL epitopes, 9 HTL epitopes, and 10 LBL epitopes) exhibiting full conservancy were then selected for designing the peptide fusion with appropriate linkers, having hBD-3 as the adjuvant. The peptide vaccine was modeled, refined, and validated to further improve the structural attributes. Following this, molecular docking analysis with specific TLRs was carried out which revealed excellent interactions and global binding energies. Additionally, molecular dynamics (MD) simulation was conducted which ensured the stability of the interactions between vaccine and TLR. Furthermore, mechanistic approaches to imitate and predict the potential immune response generated by the administration of vaccines were determined through immune simulations. Owing to an overall evaluation, in silico cloning was carried out in efforts to generate recombinant pETite plasmid vectors for subsequent mass production of the vaccine peptide, incorporated within E.coli. However, more in vitro and in vivo experiments can further validate its efficacy against RSV infections.

immunology↗