Biology subjects

Mukherjee, S.

Publications and source records attributed to Mukherjee, S..

At least 19 recordsLinked to original sources

bioRxiv2026

A composited genome approach to analyse cistrome diversity in barley

Motivation: Functional regulatory variants are important determinants of heritable diversity, yet their systematic identification requires both genome-wide maps of transcription factor (TF) occupancy and suitable genome sequences for mapping. While chromosome-scale assemblies remain costly for large association panels, short-read sequencing data are widely available. We investigated whether draft genomes reconstructed from short reads are sufficient for comparative MOA-seq analyses of TF occupancy. Results: We generated genome-wide TF footprint maps for the barley cultivars Bowman, Morex, and Golden Promise, identifying approximately 240,000 loci per genotype. Roughly half of all footprints occurred in intergenic regions. Footprints conserved between genotypes were depleted for sequence variation and located closer to genes. Meanwhile, genotype-specific footprints were enriched for SNP-containing and gene-distal regions, highlighting the extensive diversity of regulatory landscapes among barley cultivars. We further compared two short-read-based draft genome assembly strategies: i) a SNP-replacement and ii) a Practical Haplotype Graph (PHG)-based composite genome approach, to enable MOA-seq analyses without chromosome-scale genome assemblies. Compared to the SNP-replacement strategy, the composite genome displayed 2.3-fold higher local sequence concordance for SNPs/INDELs relative to the published reference. Both draft genomes accurately reproduced MOA-seq profiles, recovering approximately 80 % of reference peak bases with per-base coverage correlations of 0.79-0.90 relative to the assembled reference. While the composite genome performed best across the genome overall, the simpler SNP-replacement strategy more accurately recovered TF occupancy within regulatory peaks due to fewer structural alignment artefacts. Availability: Pipelines consist of existing open-source tools, Bash, GAWK and Java, and are available at [zenodo; tbd].

genomics↗

bioRxiv2026

Transient Splicing-Associated Isoform Remodeling Defines Early MAPKi Resistance Adaptation in Melanoma

A long-standing question in targeted therapy resistance is how tumor cells rapidly enter drug-tolerant states before stable resistance emerges. In BRAF-mutant melanoma, early adaptation to MAPK inhibitor (MAPKi) therapy is accompanied by profound phenotypic plasticity, yet the molecular mechanisms that enable this transition remain poorly understood. Here, we show that MAPKi adaptation is characterized by a transient transcript isoform remodeling that is largely invisible to conventional gene-expression analysis. We integrated and analyzed multiple different datasets, including bulk and single-cell RNA-seq datasets from melanoma cell lines, murine models, patient-derived xenografts and patient tumors, spanning no-treatment, on-treatment/minimal residual disease, and acquired-resistant states. We find that differential transcript usage peaks during the adaptive on-treatment phase and then is markedly attenuated when resistance was established. These dynamics were conserved across experimental systems and supported by independent exon-usage analyses. Notably, most genes undergoing differential transcript usage showed no corresponding change in total gene expression, and genes with isoform-level changes more strongly highlighted MAPKi resistance-associated pathways, including PI3K/AKT/mTOR and receptor tyrosine kinase signaling. Further, classifiers trained on cell-line transcript-usage features identified the adaptive state more accurately than gene-expression-based models in independent PDX and patient datasets. Interestingly, MAPKi-associated isoform changes recapitulated melanoblast-associated developmental splicing programs and were linked to a few key splicing regulators. Together, these findings identify an underappreciated regulatory layer connecting drug tolerance, developmental plasticity, and acquired resistance. Beyond the current context, our results suggest that comprehensively tracking isoform and splicing alterations may potentially advance our understanding of the emergence of treatment resistance in additional indications and treatment regimes.

cancer biology↗

bioRxiv2026

Genomic foundation models do not yet boost common variant discovery beyond standard functional annotations

Genomic foundation models (GFMs) use large-scale AI to model genetic sequences and their influence on molecular phenotypes. While GFMs have shown promise in rare variant analysis, their utility for common variant discovery remains untested. Using the surrogate functional false discovery rate framework for functionally informed GWAS discovery, we compared standard functional annotations (baselineLD) with annotations derived from three leading GFMs: the sequence-to-function models AlphaGenome and Borzoi, and the genomic language model GPN-Star. Across 27 UK Biobank traits, GPN-Star decreased locus discovery relative to a standard GWAS, whereas AlphaGenome and Borzoi increased discovery but by less than baselineLD (medians 1.8%, 3.8%, and 5.1%). When we combined the GFM and baselineLD annotations, there was no increase over baselineLD alone (median changes -0.1% to 0.2%), suggesting that AlphaGenome and Borzoi mostly capture information already in baselineLD. Thus, GFMs may require larger corpora or trait-specific feature engineering to improve discovery beyond standard functional annotations.

genomics↗

bioRxiv2026

Gut bacterium of Leishmania donovani-non-transmitting sand fly induces IL-23~Th17 axis to protect hosts from drug-resistant visceral leishmaniasis

Background: Visceral leishmaniasis (VL) is caused by Leishmania donovani and differentiates under the influence of biotope-specific gut bacteria in the Leishmania donovani-transmitting sand fly vector, Phlebotomus argentipes, which coexists with the Leishmania donovani-non-transmitting Sergentomyia species in the Indian subcontinent. Hypothesis: Sergentomyia are primarily herpetophilic. Interestingly, we identified a specific population of anthropophilic Sergentomyia babu babu, a proven naturally non-competent vector for transmitting Leishmania donovani, with high abundance and human-bite incidences in VL-non-endemic regions, compared with VL-endemic regions of India. Thus, the possibility of egesting gut bacteria to humans from VL-non-transmitting Sergentomyia and inducing downstream immune cascades remains undeniable. Innovation: We characterized gut bacteria from anthropophilic Sergentomyia babu babu in both VL-endemic and VL-non-endemic regions of India and found that a heat-inactivated formulation of Heyndrickxia sporothermodurans [NCBI Sequence Read Archive (SRA): PRJNA1403236], isolated from anthropophilic Sergentomyia babu babu from a VL-non-endemic region, prevented and cured L. donovani infection by inducing IL-23~Th17. More importantly, it also prevented the initial parasite establishment by inducing phagocytosis, ROS, and TNF- in circulatory Ly6G-CD11b+Ly6Chi monocytes. To the best of our knowledge, this is the first report confirming that the gut bacteria of VL-non-transmitting sand flies are critical and need to be investigated in depth for developing therapeutics and/or prophylactics against visceral leishmaniasis (VL), and suggests that possible bite-transfer of gut bacteria from disease-non-transmitting vectors to hosts may restrict disease pathogenicity. Further clinical trials are needed to warrant (Indian patent: 202511041013, April 28, 2025).

immunology↗

bioRxiv2026

Serotonin2A receptors in the medial prefrontal cortex do not drive DOI-induced head-twitch responses in rodents

Background The head-twitch response (HTR) is a serotonin2A receptor (5-HT2AR) dependent behavioral correlate of serotonergic psychedelic action in rodent models. Although cortical 5-HT2ARs have been implicated in HTRs, the contribution of specific cortical circuits to this behavior remains poorly defined, with a few reports implicating the medial prefrontal cortex (mPFC) in the generation of HTRs. Results The serotonergic psychedelic, 2,5-dimethoxy-4-iodoamphetamine (DOI), induced head-twitch responses (HTRs) in male and female rats and mice, with sex differences noted only in mice. DOI also increased ear-scratch responses (ESRs), and reduced digging and grooming behaviors only in mice. While the DOI-evoked HTRs and ESRs were absent in 5-HT2AR knock-out (5-HT2AR KO) mice, reduced digging and grooming occurred in both wild-type and 5-HT2AR KO mice. We assessed the role of 5-HT2ARs in the mPFC in regulating DOI-induced HTRs using pharmacological and genetic approaches. Targeted DOI microinfusion into the mPFC did not induce HTRs in rats, in contrast to the robust increase in HTRs noted following systemic DOI administration. Local delivery of the selective 5-HT2AR antagonist, MDL100907, into the mPFC also had no effect on the systemic DOI-evoked increase in HTRs in rats. Experiments capitalizing on genetic strategies to perturb 5-HT2AR expression in the mPFC, indicated that viral restoration of 5-HT2AR expression in mPFC CaMKII-positive excitatory neurons was not sufficient to reinstate DOI-evoked HTRs or ESRs in 5-HT2AR KO mice. Further, conditional 5-HT2AR deletion in CaMKII-positive excitatory neurons in the mPFC did not alter systemic DOI-induced HTRs and ESRs in mice. Conclusions These findings indicate that the serotonergic psychedelic DOI induces species- and sex-specific behavioral effects in rodent models, with a robust 5-HT2AR dependence noted for DOI-evoked HTRs and ESRs. Further, we show that 5-HT2ARs in the mPFC do not appear to contribute to the enhanced HTRs and ESRs elicited by DOI.

neuroscience↗

bioRxiv2026

mCranberry: A rationally engineered monomeric red fluorescent protein with 85% quantum yield

Fluorescent proteins (FPs) are the workhorse of modern-day cellular imaging and biosensing. However, rationally predicting and engineering the photophysical properties of FPs remain challenging, as these are governed by complex three-dimensional microenvironments and post-translational modifications. Here, we present a novel surface-mutation-guided engineering strategy combined with iterative rounds of rational mutagenesis to improve key photophysical properties of red fluorescent proteins (RFPs). This approach generated two bright, monomeric RFP variants from the mFruits lineage, mCranberry and mCranberry2, with quantum yields of 85% and 80%, respectively. Strategic incorporation of charged amino acid residues on the protein surface enhanced fluorescence quantum yield without compromising photostability. Comprehensive photophysical characterization and live-cell microscopy demonstrate enhanced performance of these variants and establish their utility as robust probes for fluorescence imaging. However, the exact mechanism of such improvements is subject to further investigation. Collectively, this work introduces a strategy for optimizing fluorescence properties and provides a framework that may be broadly applicable to other pigment-protein systems such as genetically encoded metal-ion indicators and photosynthetic light-harvesting complexes.

biophysics↗

bioRxiv2026

A Nanomechanical Fingerprint of Centromeric Methylation Co-Localization in TNBC Chromosomal Instability

Centromeric heterochromatin is maintained by a self-reinforcing loop between DNA methylation (5mC) and histone H3 lysine 9 trimethylation (H3K9me3), yet whether this coupling is preserved at the level of individual chromosomes in cancer has been inaccessible to conventional epigenomic assays, which average signals across cell populations and cannot resolve single-chromosome heterogeneity. Here, we use antibody-functionalized atomic force spectroscopy to simultaneously map 5mC and H3K9me3 at the centromeres of individual metaphase chromosomes from non-tumorigenic, luminal, and triple-negative breast cancer (TNBC) cell models. Co-localized 5mC/H3K9me3 sites are mechanically stiffer than sites carrying either mark alone across the panel, revealing a cooperative biophysical signature of epigenetic crosstalk. Luminal chromosomes acquire the highest mark density, co-localization, and stiffness, whereas TNBC chromosomes show reduced density and diminished, though not absent, co-localization and stiffness, converging toward the low baseline of non-tumorigenic epithelium. TGF-{beta}1-induced epithelial-mesenchymal transition reproduces this attenuated phenotype in luminal cells, linking it directly to a process central to TNBC aggressiveness. These biophysical parameters classify cancer versus non-tumorigenic chromosomes with near-perfect, cross-validated accuracy, and further resolve the two TNBC subtypes using colocalization-derived features alone (macro-F1 = 0.933). These results establish the mechanical consequence of epigenetic mark co-localization as a single-chromosome-resolvable signature of centromeric organization, attenuated in aggressive breast cancer, and demonstrate a force-spectroscopy platform for mechano-epigenetic profiling of cancer chromosomes.

biophysics↗

bioRxiv2026

Repeated exposure to viral and bacterial danger signals in Parkin deficient mice induces inflammation but fails to trigger parkinsonism

Experimental models of Parkinson's disease (PD) traditionally involve direct perturbations of the functioning of dopamine (DA) neurons. However, PD is increasingly recognized as multifactorial in origin and involving both cell-autonomous vulnerability factors and non-cell-autonomous triggers including inflammatory signals deriving from bacterial or viral infections. Notably, genes associated with early-onset familial PD as PRKN (Parkin) and PINK1, are ubiquitously expressed and have been implicated in immune regulation, further supporting a role for inflammation in disease etiology. In the present study, we tested the hypothesis that alternate Polyinosinic:polycytidylic acid (Poly(I:C)), mimicking viral infection, and lipopolysaccharides (LPS), mimicking bacterial infection, may induce sufficient brain inflammation to impair the DA system in Parkin-deficient mice and recapitulate some of the systemic features of PD. We developed a protocol with alternating Poly(I:C) and LPS, inducing acute peripheral inflammation in both genotypes, with consistent body weight loss and an increase of fecal lipocalin-2. Modest levels of neuroinflammation were present six months after the treatment, with higher levels of the microglial marker Iba1 in the dorsal striatum and reduced levels of the astrocyte marker GFAP in the mesencephalon, potentially indicating reduced neuronal support in the longer term. Finally, we find that this alternating Poly(I:C)/LPS treatment does not induce dopaminergic denervation or motor dysfunctions, even in Parkin KO mice. Taken together, our findings suggest that repeated short-term exposure to pathogen-derived signals is insufficient to induce DA system impairment in young adult Parkin deficient mice. Instead, induction of PD-like pathology may require a convergence of factors, including a prolonged or recurrent inflammatory burden, aging, genetic susceptibility, and potentially additional environmental or cellular stressors, that together create a pathogenic "perfect storm". Thus, in its current form, the paradigm developed in the present study should not be regarded as a fully representative model of PD. Rather, it provides proof of concept that temporally alternating inflammatory stimuli can shape non-motor symptom-like phenotypes and underscores the need for future animal model research to incorporate interacting risk factors, disease duration, and age-dependent vulnerability.

neuroscience↗

bioRxiv2026

Different forms of autophagy restrict neurite outgrowth in disparate compartments in a single neuron

Autophagy is a degradative pathway that is critical in neurons to maintain their homeostasis and to direct nervous system development. Neurons are large, highly polarized cells with distinct compartments that perform discrete functions. How an individual neuron can differentially mobilize autophagy in the axon, dendrite, and cell body is unknown. Here we interrogate the role of autophagy in the neurodevelopment of a single neuron in Caenorhabditis elegans to identify how the spatial compartmentalization of neuronal autophagy ultimately restricts neurite outgrowth in multiple neuronal compartments. However, while canonical autophagy restricts dendrite outgrowth, noncanonical forms of autophagy appear to restrain neurite outgrowth in the axon and soma. Through mutant analysis of the autophagy pathway, we identify that WIPI2-independent autophagy modulates ectopic neurite formation in the soma and that ATG9-independent autophagy regulates axon arborization. Further, we find that unrelated lipid scramblases can compensate for the loss of ATG9 in axon arborization. Our data indicate that neurons marshal both canonical and non-canonical autophagy to spatially control development of separate compartments.

neuroscience↗

bioRxiv2026

Colloidal DNA nanoaggregates applied towards file-partitioning for information storage and dynamic data obfuscation

The molecular programmability of nucleic acids has facilitated the development of architected DNA/RNA nanostructures and their applications in novel materials and technologies. We report how different types of DNA and RNA nanoaggregates, bundling digital information encoded into oligo libraries, can be formed by manipulating the ionic strength of the solution. As DNA or RNA suspensions are immersed in solutions of increasing salt concentrations, we observe the onset of aggregation. Further increase in ionic strength leads to the formation of stable, reproducible, and well-defined aggregates. We show that these nanoaggregates are kinetically trapped at room temperature, stably partition DNA libraries that encode image files, and support file-specific random access by bundling DNA libraries with unique address oligos. The nanoaggregate files can be disrupted and reformed into scrambled bundles using simple external fluid shear or temperature annealing, rapidly obfuscating the data. We term these nanoaggregates nucleic acid PACKeTs: Partitioned Aggregates of Colloidal DNA/RNA through Kinetic Trapping. Overall, the results demonstrate how gaining fundamental insights into ionic colloidal aggregation enables new forms of manipulation of DNA and RNA libraries. This understanding could lead to novel functionalities including kinetically trapped data partitioning, random access, and data encryption or obfuscation.

bioengineering↗

bioRxiv2026

An extended N-terminus restrains the plant cell death-inducing ability of the catalytically competent ribonuclease domain in a pea powdery mildew RALPH effector

RALPH (RNase-like proteins associated with haustoria) effectors, which are preferentially expressed in haustoria and structurally resemble fungal T1/F1 RNases, constitute one of the largest effector families in powdery mildew (PM) fungi, yet their functions in dicot-adapted PM species remain poorly understood. Unlike cereal PM RALPHs, which lack the catalytic residues required for RNase activity, some dicot PM RALPHs retain these residues. Here, we performed a comprehensive structural and expression-based characterization of the pea PM Erysiphe pisi RALPH (EpRALPH) repertoire and functionally characterized EpRALPH11, a RALPH effector with partial conservation of the catalytic residues of T1/F1 fungal RNases. Comparative analyses identified multi-RNase-domain RALPHs as a conserved feature of the Erysiphe lineage, while expression profiling showed that many EpRALPHs are preferentially expressed in haustoria during early host colonization. AlphaFold 3-based structural analyses revealed a conserved T1/F1 RNase-like fold despite substantial sequence and surface charge divergence, indicating functional diversification among EpRALPHs. EpRALPH11 enhanced susceptibility to E. pisi in Medicago truncatula, localized to the nucleolus, and induced nucleolar fragmentation when heterologously expressed in Nicotiana benthamiana leaves. Its RNase domain exhibited T1 RNase activity in vitro, supporting the retention of a catalytically competent RNase domain and, together with its nucleolar localization, suggesting that EpRALPH11 targets plant rRNA and disrupts nucleolar functions. The RNase domain induced cell death in N. benthamiana, whereas the full-length protein and catalytic mutants did not. Cell death induction required exclusive nucleolar localization of the RNase domain, and an extended N-terminal intrinsically disordered region suppressed this activity in the full-length protein. Together, our findings reveal a previously unrecognized mechanis regulating RNase activity in a dicot PM RALPH effector and provide new insights into the functional diversification of RALPHs and their adaptation to obligate biotrophy.

plant biology↗

bioRxiv2026

KDM6A Loss Confers an Invasive Phenotype in Osteosarcoma by Activating Cytoplasmic YAP-Dependent β-catenin Stabilisation

Osteosarcoma (OS) is the most common primary malignant bone tumour and is characterised by aggressive growth, early metastasis, and a very stagnant clinical outcome. Although epigenetic dysregulation has been implicated in OS progression, the mechanisms linking epigenetic alterations to metastatic signalling remain unclear. Here, we identified the lysine-protein demethylase 6A (KDM6A/UTX) as a critical suppressor of OS metastasis and uncovered a novel regulatory axis involving the Hippo/YAP and Wnt/{beta}-catenin signalling. Initial bioinformatics analyses revealed frequent KDM6A alterations and significantly reduced expression in OS patient datasets, which correlated with metastatic disease and poor prognosis. Functional inhibition of KDM6A by pharmacological inhibitors and siRNA induced a hyper-invasive phenotype, marked by elevated mesenchymal markers, enhanced cytoskeletal remodelling, increased transendothelial adhesion and decreased chemotherapeutic drug sensitivity. Importantly, restoration of KDM6A expression effectively counteracted these effects. Mechanistically, KDM6A loss activated Wnt/{beta}-catenin signalling, resulting in nuclear translocation of {beta}-catenin and transcriptional activation of genes associated with stemness and invasion. Therefore, inhibition of {beta}-catenin reversed the invasive phenotype. Further analysis revealed that KDM6A regulated Hippo signalling through epigenetic control of the negative regulator of Yes-Associated Protein (YAP)-LATS1. KDM6A inhibition led to enrichment of H3K27me3, a repressive mark, at the LATS1 promoter. Accumulated YAP was predominantly localised in the cytoplasm, where it interacted with GSK3{beta} and contributed to the stabilisation of {beta}-catenin by preventing its proteasomal degradation. Collectively, our findings identify a novel KDM6A-LATS1-YAP-{beta}-catenin signalling axis that drives metastatic progression in OS.

cancer biology↗

bioRxiv2026

Tumor-specific Kinase Motif Enrichment Analysis Identifies Personalized Therapeutic Cancer Targets

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are an uncommon and poorly understood malignancy with low mutational burden, lacking well-defined oncogenic drivers. GEP-NET mortality frequently results from extensive hepatic metastases. Accordingly, we interrogated phosphoproteomic data from GEP-NET liver metastases and patient-matched uninvolved liver to identify tumor-specific signaling and targetable tumor vulnerabilities using Kinase Motif Enrichment Analysis (KMEA), a new tool leveraging the recent Kinase Library compendium of the substrate motif specificity for nearly the entire human kinome. KMEA identified patient tumor-specific upregulation of mTOR or casein kinase 2 (CK2) activity that would be undiscoverable by standard personalized genomic and transcriptomic approaches. Striking concordance was observed between KMEA predictions for specific tumors, and their sensitivity to inhibitors of mTOR or CK2 using patient tumor-derived organoids. These findings reveal potential clinically-actionable protein kinases hyperactivated in GEP-NETs, and more broadly indicate a general method for personalized cancer treatment using phosphoproteomics and KMEA-derived kinase activity signatures.

cancer biology↗

bioRxiv2026

Kaptin controls neuronal circuit assembly by limiting actin-driven axon collateral branching

Axon collateral branching is a fundamental determinant of neuronal connectivity, enabling individual neurons to innervate multiple targets and establish functional neural circuits. Although branch initiation requires local actin remodelling within the axon shaft, the mechanisms that restrict actin assembly to prevent excessive branching remain poorly understood. Here, we identify Kaptin (Kptn) as a conserved negative regulator of axon collateral formation that limits the maturation of axonal actin patches. Using primary neuronal cultures, quantitative live-cell imaging, and zebrafish genetics, we show that loss of Kptn enhances the conversion of actin patches into filopodial protrusions, leading to excessive collateral branching. Kptn-deficient zebrafish exhibit increased motor axon arborisation, elevated neuromuscular junction density and impaired motor behaviour. Mechanistically, Kptn antagonises the actin elongation factor Formin-2 (Fmn2) to regulate actin filament barbed-end dynamics, thereby controlling the threshold for productive branch initiation. Importantly, these branching defects occur independently of Kptns established role in mTORC1 signalling, revealing a distinct physiological function during neuronal development. Our findings identify Kptn as a key molecular brake that constrains axon collateral branching and establish negative regulation of actin patch maturation as a fundamental mechanism controlling neuronal circuit assembly. This work provides a mechanistic framework for understanding how KPTN mutations associated with intellectual disability and epilepsy disrupt neuronal connectivity.

neuroscience↗

bioRxiv2026

DCX enables branching of subpellicular microtubules in Plasmodium falciparum gametocytes and is required for mosquito colonisation

Plasmodium falciparum, the causative agent of malaria, relies on specialised tubulin-based cytoskeletal structures to support its parasitic lifestyle. These include the conoid required for parasite motility and host-cell invasion, as well as subpellicular microtubules (SPMTs) that support parasite shape and rigidity. Here, we investigate the function of the doublecortin-domain protein DCX, a microtubule-binding protein previously associated with the Plasmodium conoid. We first show that, in P. falciparum, DCX is not expressed in the merozoite stage and is not required for the invasion of human erythrocytes. By contrast, DCX is expressed in ookinetes, the motile stage responsible for infecting the mosquito vector, where it associates with conoid tubulin fibres, consistent with a role in stabilising the conoid architecture. Unexpectedly, we find that DCX is required for P. falciparum transmission to the mosquito independently of conoid function. We further link this requirement to the distinctive organisation of SPMTs in P. falciparum gametocytes, which display an unusual branching architecture comprising multiple microtubules of 15 to 18 protofilaments. Deletion of DCX leads to a reduction in SPMT branching and is associated with higher protofilament numbers, revealing a previously unrecognised role for DCX in shaping the ultrastructure of SPMTs in P. falciparum gametocytes. Altogether, our findings uncover the repurposing of DCX across distinct microtubule systems in transmission stages and identify DCX as a key factor mediating microtubule branching and stabilisation in SPMTs required for efficient mosquito transmission.

microbiology↗

bioRxiv2026

G-LATO: Inference of Spatial Latent Ordering via Deep Gaussian Processes

Spatial transcriptomics enables the study of cells within their native tissue context, yet identifying gradients of cellular development remains challenging. We introduce a deep Gaussian process model to address this gap. Our method recovers spatially smooth gradients explaining observed gene expression. We illustrate our method on healthy liver and glioblastoma data in reconstructing known spatial organisation and uncovering new pathological gradients, thus providing robust inference for spatial biology.

bioinformatics↗

bioRxiv2026

A C-terminal Processing Protease Implicated in Flagellin Turnover and Developmental Progression in a Bacterial Predator

Carboxy-terminal processing proteases (CTPs) are widely conserved bacterial proteases implicated in protein maturation, quality control, and stress responses, yet many family members functions remain unclear. Here, we characterise Bd0967, a previously unstudied CTP from the predatory bacterium Bdellovibrio bacteriovorus, and its role in flagellar function during predatory development. Crystal structures reveal a self-compartmentalised protease in which a PDZ domain forms a lid over a large internal cavity accessed through a proteolytic tunnel. Co-purifying peptides occupied two distinct substrate-binding sites, suggesting a coordinated recognition mechanism. Affinity pulldowns coupled with mass spectrometry identified several Bdellovibrio flagellins as candidate substrates, which were subsequently validated by biochemical assays and shown to be selectively degraded through recognition of a conserved C-terminal motif. A Bd0967-mCherry translational fusion localised to periplasmic foci during the intracellular predatory growth, consistent with flagellar resorption and flagellin turnover following prey invasion. Deletion of bd0967 caused developmental defects, including aberrant Bdellovibrio cell morphology and reduced predation efficiency. Together, these findings establish Bd0967 as a specialised CTP that couples flagellin degradation and developmental progression in a predatory bacterium.

biochemistry↗

bioRxiv2026

Shape, Shrink, Spheroid: A DIY High-throughput Spheroid Generation Device

3D spheroids, which closely replicate three-dimensional cell-cell and cell-extracellular matrix interactions, offer superior predictive capabilities compared to conventional 2D monolayer cultures, positioning them as forward-looking platforms in drug testing, cancer biology, and regenerative medicine. However, high-throughput generation of uniform sized spheroids is still a technological challenge. In one hand, the use of conventional ultra-low attachment (ULA) multiwell plates for this purpose is labour intensive and complex. On the other hand, the use of microfabricated facilities demands cutting-edge infrastructure such as clean room, photolithography, and microfluidic setup which are often unavailable for the resource constrained laboratories. In this study, we addressed these problems by developing a low-cost Do-It-Yourself (DIY), polydimethylsiloxane (PDMS) and agarose-based spheroid generation device, capable of producing and maintaining hundreds of spheroids with minimal user intervention. We have demonstrated two variants based on their size, termed here as S1 and S2 devices which fit into 6-well and 12-well plates, and can generate 600 and 1200 uniform-sized spheroids respectively. We validated our device with various cell lines including primary and cancerous cell lines. We further demonstrated the drug testing capabilities of the device by estimating the IC50 value of the anticancer drug Temozolomide on U87-MG. The value was comparable with the same obtained from the spheroids generated using conventional ULA plates. Additional attachment of a perfusion system made the device suitable for long-term spheroid culture without much user intervention. Furthermore, the devices can also be used for the production of spheroids with gradually changing diameters in a controlled manner, resembling a size gradient. This feature is useful for checking the effect of drugs on different-sized spheroids and for co-culturing spheroids with varying cell densities, mimicking the disease architecture. We have co-cultured two types of the placental trophoblast cells, i.e., extravillous trophoblast (HTR-8) and syncytiotrophoblast (BeWo) with varying densities. In summary, this paper demonstrates a unique DIY method for a high-throughput uniform-sized spheroid generation at a fraction of cost which can be deployed to resource-constrained labs.

bioengineering↗