Biology subjects

Bhandari, S.

Publications and source records attributed to Bhandari, S..

15 recordsLinked to original sources

bioRxiv2026

Genetic interaction approaches reveal emerging roles of innexins in development : Insights from a novel pannier–innexin-2 interaction during Drosophila embryogenesis

Effective communication between cells is essential for the typical development and behaviour of an organism. In this context, gap junctions represent the most universally preserved components at cellular membranes of multicellular organisms, facilitating metabolic and electrical connections between cells. Disruptions in these junctions have been linked to various developmental abnormalities and pathological conditions in humans. The invertebrate gap junction proteins, referred to as innexins, exhibit conserved cellular and molecular mechanisms of functioning with their vertebrate counterparts, known as connexins. Consequently, they provide valuable means for studying and understanding the functions of gap junctions in development. In the Drosophila embryo, innexin-2 is expressed in the amnioserosa and ectoderm, where it is required for epithelial morphogenesis. Genetic depletion of innexin-2 results in cuticular defects and embryonic lethality. Pannier, a GATA family transcription factor, is a key regulator of dorsal tissue development in Drosophila and is expressed in the amnioserosa, dorsal ectoderm and the dorsal vessel during embryogenesis. Pannier mutants exhibit defects in dorsal closure, cuticle formation, and cardiac specification. Although substantial evidence from vertebrate systems indicate that connexin expression is regulated by transcription factors such as GATA4, Nkx2.5, Tbx2, Tbx3, and Tbx5, whether a similar regulatory relationship exists between these transcription factors and gap junction proteins in Drosophila remains unknown. In this study, we investigate how innexin mediated intercellular communication impacts pannier dependent morphogenetic processes during Drosophila embryogenesis.

Developmental Biology↗

bioRxiv2026

Escherichia coli and Mammalian Cells Follow Divergent Rules for Lipid-Driven Cytosolic Accumulation

Lipidation is a widely used strategy to promote membrane permeation, yet whether the factors governing lipid-driven accumulation are shared across mammalian and Gram-negative envelopes remains unresolved. Here, we apply the Chloroalkane Azide-based Membrane Penetration (CHAMP) assay to a systematically designed library of lipid conjugates in both HeLa and E. coli cells. CHAMP, developed by our group, pairs a minimally disruptive azide tag with a cytosolically anchored HaloTag to quantify cytosolic accumulation directly. The two systems show divergent trends: most lipid modifications reduce E. coli cytosolic accumulation, whereas larger, more hydrophobic conjugates, including medium-chain, cyclized, and heteroatom-containing lipids, are preferentially internalized by mammalian cells. By systematically editing the compound scaffold and, separately, perturbing individual layers of the bacterial envelope, we resolve how charge, scaffold composition, and specific envelope barriers shape these patterns. Together, these results indicate that lipidation is a context-dependent permeation strategy rather than a general one. By showing that hydrophobic modifications frequently hinder cytosolic entry into E. coli, this work offers a permeability-based hypothesis for the scarcity of lipidated antibacterials with cytosolic targets, and delineates where lipophilicity-driven optimization is likely to fail in the diderm envelope.

pharmacology and toxicology↗

bioRxiv2026

Molecular Determinants Governing the Antitubercular Activity of Griselimycin

The emergence of drug-resistant strains of Mycobacterium tuberculosis makes anti-tubercular drug development a critical priority. Griselimycin is a cyclic peptide that targets the essential DNA sliding clamp of M. tuberculosis. While griselimycin is a promising antimycobacterial compound, its poorly understood structure-activity relationship has stalled derivatization. To investigate the contribution of each amino acid towards its activity, we assessed the antibiotic activity of an alanine scan library in M. tuberculosis. Incorporation of an N-terminal fluorophore enabled visualization of griselimycin accumulation inside of mycobacteria-infected macrophage cells. Derivatives containing altered cyclization chemistries, unnatural amino acids, expanded proline side chains, and alkyne handles were assessed, with a final design combining the most potent edits showing increased antibiotic activity. These findings present the comprehensive structure-activity investigation of griselimycin and can be used to rationally design future analogues.

pharmacology and toxicology↗

bioRxiv2026

Mapping the Modification Landscape of MHC-I Epitopes: A Framework for Immunogenic Peptidomimetic Antigen Design

Peptide-based cancer vaccines offer a promising strategy to target tumor-specific neoantigens. This approach is increasingly critical as post-translationally modified peptides, driven by altered tumor metabolism, emerge as a unique class of neoantigens. Because these chemically distinct epitopes cannot be genetically encoded by mRNA or viral platforms, synthetic peptide vaccines are poised to be the primary route to target these types of neoantigens. Yet, their clinical translation is restricted by poor metabolic stability, limited intracellular permeability, and structural requirements for MHC-I binding and T cell receptor (TCR) recognition. Although peptidomimetic modifications have been widely explored to improve pharmacokinetics, their impact on antigen presentation and immune recognition remains poorly understood. Here, we undertook a comprehensive evaluation of peptidomimetics geared at MHC-I neoantigens and generated a diverse library of systematically modified peptides that incorporate backbone N-methylation, peptoid substitution, and stereochemical inversion. Integrated assays revealed a highly position-dependent tolerance to peptidomimetic modifications, while subsequent combinatorial designs demonstrated non-additive effects on the balance between immunogenicity and pharmacokinetics. Collectively, these findings establish design principles and provide a framework for balancing immune recognition with enhanced stability and permeability in peptidomimetic antigen design.

immunology↗

bioRxiv2025

Split-luciferin Assay for Real-time Measurement of Cytosolic Drug Accumulation in Live Mycobacteria

Tuberculosis causes over one million deaths annually and remains the leading cause of death from a single infectious agent. The emergence of multidrug-resistant Mycobacterium tuberculosis strains highlights the urgent need for new antibiotics, a pursuit hindered by the bacteriums complex cell envelope. As most anti-tuberculosis agents act on intracellular targets, assessing cytosolic drug accumulation is critical. Conventional approaches generally quantify whole-cell association without resolving subcellular localization. Moreover, no current method permits real-time monitoring of drug accumulation in live mycobacterial cells. Here, we present a split-luciferin-based assay to quantify molecular accumulation in mycobacteria. Using this approach, we quantified the cytosolic accumulation of diverse small-molecule antibiotics and polyarginine peptides conjugated via a disulfide-linked D-cysteine tag. We also show the localization of a polyarginine peptide inside of mycobacteria in infected macrophage cells, demonstrating that these peptides can cross multiple accumulation barriers. Our findings establish the first assay for real-time quantification of cytosolic molecular accumulation in live mycobacteria, addressing a longstanding methodological gap and enabling mechanistic insights into intracellular drug uptake.

microbiology↗

bioRxiv2025

Honey Bee Queen Susceptibility to Viral Infection Varies Across Developmental Stages in Queen Rearing Operations

Viruses are a large class of honey bee pathogens that negatively affect colony health, yet their prevalence and transmission dynamics in commercial queen production operations remain poorly understood. To address this gap, we conducted a series of controlled queen monitoring experiments and surveys to understand the prevalence and viral loads of seven viruses across developmental stages of queens, drones, royal jelly, and workers from associated colonies. All viruses except SBV were detected, with BQCV, DWV-B, and LSVs showing consistently high prevalence. Eggs were frequently infected with LSVs, DWV-B, and CBPV, suggesting vertical virus transmission, and highlighting the importance of selecting healthy breeder queens. BQCV, on the other hand, dominated in queen larvae, pupae, and adult stages. Mated queens, particularly those maintained in bank colonies, exhibited higher prevalence and viral loads than virgin queens, with DWV-B and BQCV being most abundant. Worker bees from bank colonies also showed slightly higher viral loads compared to other colonies, indicating potential risks associated with queen banking. Drone samples revealed high BQCV and DWV-B prevalence, indicative of their potential role in venereal transmission. The results from hierarchical clustering and correlation analyses provided evidence that viral profiles of queens did not necessarily match those of their resident colonies, highlighting complex viral transmission dynamics. Collectively, these findings provide novel insights into virus transmission dynamics during queen production and emphasize the need to improve queen health. Author summaryHoney bee queen failure is a frequent challenge in beekeeping operations, and viral infections are increasingly recognized as an important contributor. Since no chemical therapeutics are available for managing viral diseases in honey bees, understanding routes of viral transmission is critical for developing strategies to minimize infections. However, the diversity of viruses infecting queens, their prevalence, and their main transmission routes have not been systematically investigated within the queen production industry. Therefore, in this study we investigated viral prevalence, loads, and transmission dynamics in commercial queen production operations, using a diverse array of samples, including queens at different developmental stages, worker bees from associated colonies, drones, and Varroa mites. Our analysis consistently indicates that vertical transmission from infected breeder queens, natural mating, and bank colonies with high Varroa infestations are key points where queens are most vulnerable and become infected with viruses. These findings offer novel insights into virus transmission dynamics in queens, emphasizing the importance of selecting healthy breeder queens and controlling Varroa infestations in drone source and bank colonies.

pathology↗

bioRxiv2025

Spatial Expression Pattern and Cellular Organisation of Gap Junctions in Third Instar Wing Imaginal Discs of Drosophila melanogaster

The Drosophila wing imaginal disc serves as a powerful model to study intercellular communication during development. In our study, we report and discuss the expression pattern and cellular distribution of innexin-1, innexin-2 and innexin-3 in the cells of the wing imaginal discs. Our immunohistochemical data show that all three innexins are broadly expressed across the membranes of both the disc proper and peripodial epithelial cells of the wing disc. The stainings further reveal that, within the disc proper epithelium, junctional proteins are arranged in a clear apico-basal hierarchy: cadherins at the apical surface, followed by septate junction proteins, with innexins localised sub-apically beneath these components. All three innexins are enriched within this sub-apical domain, and are additionally detected at mid-and baso-lateral sites in varying levels. Notably, innexin-2 exhibits partial colocalization with coracle, a septate junction-associated protein, suggesting a functional association. In the peripodial epithelium, innexins are detected in distinct punctate patterns across cell membranes, implying heterogeneity in their molecular characteristics. To validate these expression patterns, we carried out tissue-specific RNAi-mediated knockdowns using the pannier-Gal4 driver targeting the notum, a structurally and functionally important but underexplored region in innexin research. Knockdown of innexin-2 and innexin-3 led to complete loss of their expression within this region. Notably, silencing of innexin-2 also affected the expression of septate junction associated proteins and innexin-3 knockdown was accompanied by a significant reduction in disc size and altered morphology. These findings depict and confirm the presence of innexins in the notum region and also indicate that individual innexins may have distinct or shared functional roles within the same tissue domain of expression. Their localization to specific membrane domains is likely to underlie their differential modes of action. Although previous studies have demonstrated the functional involvement of gap junctions in various aspects of normal wing development in Drosophila, a description of the arrangement of innexins on the third instar wing discs is required for better understanding of their roles. Our study addresses this gap by providing a comprehensive analysis of the cellular localisation and organisation of gap junctions, specifically innexin-1,-2 and-3, within the third instar wing discs, thereby supporting and extending existing knowledge.

cell biology↗

bioRxiv2025

RICTOR regulates an interspecies crosstalk that influences longevity through a novel methionine cycle-mitophagy axis

Adaptive modulation of physiological traits in response to environmental variability, particularly dietary fluctuations, is essential for organismal fitness. Such adaptability is governed by complex gene-diet interactions, yet the molecular circuits integrating microbe-derived metabolites with host metabolic and stress response pathways remain less explored. Here, we identify the conserved mechanistic target of rapamycin complex 2 (mTORC2) component, RICTOR, as a critical regulator of dietary plasticity in Caenorhabditis elegans, specifically in response to bacterially derived vitamin B12 (B12). Loss of rict-1, the C. elegans ortholog of RICTOR, confers enhanced osmotic stress tolerance and longevity on B12-rich bacterial diets. These phenotypic adaptations require two B12-dependent enzymes: methionine synthase (METR-1), functioning in the folate-methionine cycle (Met-C), and methylmalonyl-CoA mutase (MMCM-1), a mitochondrial enzyme essential for propionate catabolism. The latter catalyzes the formation of succinyl-CoA, subsequently converted to succinate via the tricarboxylic acid (TCA) cycle. Elevated succinate levels were found to induce mitochondrial fragmentation, thereby activating mitophagy, an autophagic process indispensable for the increased stress resilience and longevity observed in the rict-1 mutants. Crucially, this Met-C-mitophagy axis is modulated by microbial inputs, with B12 and methionine acting as proximal dietary signals. Our findings delineate a mechanistic framework through which RICTOR restrains host sensitivity to microbial-derived metabolites, thus maintaining mitochondrial homeostasis and regulating lifespan. This work reveals a pivotal role for RICTOR in insulating host physiology from environmental nutrient-driven perturbations by modulating organellar quality control pathways.

cell biology↗

bioRxiv2025

Profiling Cytosolic Drug Delivery in Mammalian Cells: A Generalizable Assay for Intracellular Accumulation

The ability of biologically active molecules to access intracellular targets remains a critical barrier in drug development. While assays for measuring cellular uptake exist, they often fail to distinguish between membrane-associated or endosomal trapped compounds and those that successfully reach the cytosol. Here, we present the Chloroalkane HaloTag Azide-based Membrane Penetration (CHAMP) Assay, a novel high-throughput method that employs a minimally disruptive azide tag to report the cytosolic accumulation of diverse molecules in mammalian cells. The CHAMP assay utilizes HaloTag-expressing cells and strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry to quantify the presence of azide-tagged test compounds in the cytosol. We demonstrate the versatility of this approach by evaluating the accumulation profiles of small molecules, peptides, and proteins, revealing how structural variations and stereochemical differences influence cytosolic penetration. Our findings with cell-penetrating peptides confirm established structure-activity relationships, with longer polyarginine sequences showing enhanced accumulation. Additionally, we observed that C-terminal amidation and D-amino acid substitutions significantly impact cellular penetration. When applied to supercharged proteins and antibiotics, CHAMP successfully discriminates between compounds with varying accumulation capabilities. This method provides a robust platform for screening cytosolic accumulation while minimizing the confounding effects of large tags on molecular permeability, potentially accelerating the development of therapeutics targeting intracellular pathways.

biochemistry↗

bioRxiv2025

A Temporal and Spatial Atlas of Adaptive Immune Responses in the Lymph Node Following Viral Infection

The spatial organization of adaptive immune cells within lymph nodes is critical for understanding immune responses during infection and disease. Here, we introduce AIR-SPACE, an integrative approach that combines high-resolution spatial transcriptomics with paired, high-fidelity long-read sequencing of T and B cell receptors. This method enables the simultaneous analysis of cellular transcriptomes and adaptive immune receptor (AIR) repertoires within their native spatial context. We applied AIR-SPACE to mouse popliteal lymph nodes at five distinct time points after Vaccinia virus footpad infection and constructed a comprehensive map of the developing adaptive immune response. Our analysis revealed heterogeneous activation niches, characterized by Interferon-gamma (IFN-{gamma}) production, during the early stages of infection. At later stages, we delineated sub-anatomical structures within the germinal center (GC) and observed evidence that antibody-producing plasma cells differentiate and exit the GC through the dark zone. Furthermore, by combining clonotype data with spatial lineage tracing, we demonstrate that B cell clones are shared among multiple GCs within the same lymph node, reinforcing the concept of a dynamic, interconnected network of GCs. Overall, our study demonstrates how AIR-SPACE can be used to gain insight into the spatial dynamics of infection responses within lymphoid organs.

immunology↗

bioRxiv2024

Loop-mediated Isothermal Amplification (LAMP) assay for reliable detection of Xanthomonas axonopodis pv. vasculorum

Xanthomonas axonopodis pv. vasculorum (Xav), the causative agent of sugarcane gumming disease, represents a significant threat to global sugarcane production due to its systemic and destructive nature. Despite the economic implications, a field-deployable, Xav-specific diagnostic tool has not been developed. This resulted in a loop-mediated isothermal amplification (LAMP) assay targeting the pelL gene, unique to Xav strains, as a rapid and precise diagnostic assay. The selection of the pelL gene was informed by comprehensive in silico analyses of Xav genomes and related Xanthomonas species and other close relatives. Validation against the NCBI GenBank database and internally sequenced genomes confirmed the genes exclusivity to Xav. Subsequent primers for both endpoint PCR and LAMP assays were designed using the pelL gene region. The LAMP assay underwent extensive testing against inclusivity and exclusivity panels. Use of exclusivity panel, comprising 81 strains from related species, other bacterial genera, and host genomes, demonstrated the assays specificity with no false positives. The assay exhibited a detection limit of 1 pg, and its effectiveness was unimpeded by crude host lysate (sugarcane). Further validation through multi-device and multi-operator testing underscored the assays 100% reproducibility and robustness. Application to infected plant samples resulted in the detection of all infected specimens without any false positives or negatives. This novel LAMP assay is accurate and reliable tool for Xav detection, with promising applications in routine diagnostics, biosecurity measures, microbial forensics, and epidemiological research.

plant biology↗

bioRxiv2023

Poor biosafety and biosecurity practices and haphazard antibiotics usage in poultry farms in Nepal hindering antimicrobial stewardship

Poultry industry in Nepal has experienced remarkable growth in the last decade, but farm biosafety and biosecurity measures are often overlooked by farmers due to lack of knowledge or to save cost. As a result, farms often suffer from sporadic and regular outbreaks of many zoonotic diseases such as highly pathogenic avian influenza (HPAI), impacting production and creating public health challenges. Poor farm management practices, including overuse of antibiotics for prophylaxis and therapeutics, can complicate the spread of poultry diseases by creating and enhancing antimicrobial resistance (AMR) that is threating to both, poultry, and human health. We assessed biosafety, biosecurity risks and AMR stewardship in sixteen poultry farms located in four districts (Ramechhap, Nuwakot, Sindhupalchowk, and Kavre) surrounding densely populated Kathmandu valley. Risk assessment and AMR stewardship evaluation questionnaire were administered to formulate biosafety and biosecurity compliance matrix (BBCM). Risk assessment checklist assessed facility operations, personnel and standard operating procedures, water supply, cleaning and maintenance, rodent/pest control and farm record keeping. Oral and cloacal samples from the poultry were collected, pooled, and screened for eight poultry pathogens using Polymerase Chain Reaction (PCR) tests. Based on BBCM, we identified one of the farms in Sindhupalchowk (Farm 4) having the most (BBCM score= 67%) and a farm in Kavre (Farm 3) to having the least (BBCM score= 12%) biosafety and biosecurity compliance. Although most of the farms (61.6%) followed general poultry farming practices, only half had clean and well-maintained farms. Personal safety standard procedure compliance (BBCM score = 42.4%) and rodent control (BBCM score = 3.1%) were the biggest gaps. At least one of either bacterial or viral pathogen was detected in all farms. Mycoplasma gallisepticum was the most common disease detected in all but one farm, followed by Mycoplasma synoviae. Although more than half of the farmers considered AMR a threat, over 26% of them used antibiotics as a preventive measure and 81% did not consider withdrawal period for antibiotics prior to processing of their meat products. Additionally, antibiotics classified as Watch and Restrict by the WHO were frequently used by the farmers to treat bacterial infections in their farms. Lack of awareness and inadequate enforcement of regulations have exacerbated the risk of disease transmission in farms and compromised antimicrobial stewardship.

microbiology↗

bioRxiv2022

Population History and Genome Wide Association Studies of Birth Weight in a Native High Altitude Ladakhi Population

Pathological low birth weight due to fetal growth restriction (FGR)is an important predictor of adverse obstetric and neonatal outcomes. It is more common amongst native lowlanders when gestating in the hypoxic environment of high altitude, whilst populations who have resided at high altitude for many generations are relatively protected. Genetic study of pregnant populations at high altitude permits exploration of the role of hypoxic in FGR pathogenesis, and perhaps of FGR pathogenesis more broadly. We studied the umbilical cord blood DNA of 316 neonates born to pregnant women managed at the Sonam Norboo Memorial Hospital, Ladakh (altitude 3540m) between February 2017-January 2019. Principal component, admixture and genome wide association studies (GWAS) were applied to dense single nucleotide polymorphism (SNP) genetic data, to explore ancestry and genetic predictors of low birth weight. Our findings support Tibetan ancestry in the Ladakhi population, with subsequent admixture with neighboring Indo-Aryan populations. Fetal growth protection was evident in Ladakhi neonates. Although no variants achieved genome wide significance, we observed nominal association of seven variants across genes (ZBTB38, ZFP36L2, HMGA2, CDKAL1, PLCG1) previously associated with birthweight.

genetics↗

bioRxiv2020

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next generation sequencing

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance and for determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for detection of SARS-CoV-2, with an additional advantage of enabling genetic epidemiology of SARS-CoV-2.

genomics↗

bioRxiv2019

Identification of peptide coatings that enhance diffusive transport of nanoparticles through the tumor microenvironment

In solid tumors, increasing drug penetration promotes their regression and improves the therapeutic index of compounds. However, the heterogeneous extracellular matrix (ECM) acts a steric and interaction barrier that hinders effective transport of therapeutics, including nanomedicines. Specifically, the interactions between the ECM and surface physicochemical properties of nanomedicines (e.g. charge, hydrophobicity) impedes their diffusion and penetration. To address the challenges using existing surface chemistries, we used peptide-presenting phage libraries as a high-throughput approach to screen and identify peptides as coatings with desired physicochemical properties that improve diffusive transport through the tumor microenvironment. Through iterative screening against the ECM and identification by next-generation DNA sequencing and analysis, we selected individual clones and measured their transport by diffusion assays. Here, we identified a net-neutral charge, hydrophilic peptide P4 that facilitates significantly higher diffusive transport of phage than negative control through in vitro tumor ECM. Through alanine mutagenesis, we confirmed that the hydrophilicity, charge, and their spatial ordering impact diffusive transport. P4 phage clone exhibited almost 200-fold improved uptake in ex vivo pancreatic tumor xenografts compared to the negative control. Nanoparticles coated with P4 exhibited [~]40-fold improvement in diffusivity in pancreatic tumor tissues, and P4-coated particles demonstrated less hindered diffusivity through the ECM compared to particles functionalized with gold standard poly(ethylene) glycol or iRGD peptide ligand. By leveraging the power of molecular diversity using phage display, we can greatly expand the chemical space of surface chemistries that can improve the transport of nanomedicines through the complex tumor microenvironment to ultimately improve their efficacy.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC=\"FIGDIR/small/659524v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (23K):\norg.highwire.dtl.DTLVardef@18bc04forg.highwire.dtl.DTLVardef@1feb597org.highwire.dtl.DTLVardef@14403e5org.highwire.dtl.DTLVardef@5bbf6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗