Biology subjects

Roy, P.

Publications and source records attributed to Roy, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Biogeography and cryptic diversity of the ancient centipede genus Digitipes (Scolopendromorpha) in South and Southeast Asia

Understanding the relative roles of vicariance and dispersal in shaping diversity and distribution patterns is central to historical biogeography. In this study, we investigate the historical biogeography of the ancient centipede genus Digitipes Attems, 1930 from South and Southeast Asia. First, we determined the phylogenetic position of the genus Digitipes within the Order Scolopendromorpha (n=414) by assembling primary and published sequences (n=30) for two mtDNA markers (COI, 16S) and one nuclear marker (28S) using Maximum Likelihood and Bayesian inference. We further used single-locus and multi-locus coalescent-based species delimitation methods to identify putative species within the genus Digitipes. We then used three fossil calibrations to estimate divergence times in a Bayesian framework, and used the resulting time-calibrated phylogeny for biogeographic analysis in a likelihood framework (BioGeoBEARS). The genus Digitipes was monophyletic with strong support, with SEA lineages nested within the Indian clade and sister to the D. barnabasi species complex from the Western Ghats. D. pruthii, the Eastern Ghats species, was nested with the Western Ghats species clade. A single-locus mPTP-based species-delimitation method suggested the presence of 24 putative species, far exceeding the number of morphologically described species, indicating an underestimation of species diversity. Divergence time estimates suggest that Digitipes began diversifying around 126 mya (100-159 mya), affirming its Gondwanan origin. Time-stratified ancestral area reconstruction suggested that early vicariance, followed by jump dispersal and range expansion, shaped the distribution of the genus Digitipes in South and Southeast Asia. There was one dispersal event from India to Southeast Asia, following a transient land connection between them, around 50 mya, supporting the Out-of-India hypothesis. Additionally, three jump dispersal events and five range expansions explained diversification within peninsular India. Particularly, D. pruthii originated from a jump dispersal event from the Central Western Ghats to the Eastern Ghats around 37 mya. Our results highlight the importance of an integrative taxonomic framework to delineate hidden diversity and to obtain robust species hypotheses for testing biogeographic hypotheses.

evolutionary biology↗

bioRxiv2026

The Nematicide Tioxazafen Disrupts Proteasome Function via Cytochrome P450 Bioactivation

Tioxazafen is an effective nematicide whose commercialization was halted because handlers reportedly developed rashes after working with seeds coated with a tioxazafen-laced cocktail. Here, we show that tioxazafen is bioactivated into toxic products by nematode and human cytochrome P450s. Through systematic analyses, we found that bioactivated tioxazafen disrupts proteasome function, leading to the accumulation of the NRF1 transcription factor ortholog SKN-1A in the nematode C. elegans, and a bounce-back transcriptional up-regulation of proteasome components. Genetic upregulation of the C. elegans proteasome supresses tioxazafens lethality, indicating that proteotoxicity is a key contributor to death. A survey of human P450s revealed that skin-expressed CYP1A1 toxifies tioxazafen and may account for the reaction to tioxazafen-coated seeds. We also found that rabbit CYP1A1 fails to bioactivate tioxazafen, which may explain the pre-market failure to detect robust adverse skin reactions. Our work highlights vulnerabilities in pre-market toxicological assays and a provides potential solution to prospectively identifying P450 toxication events. One-Sentence SummaryTioxazafen is bioactivated by cytochrome P450s into a proteasome disruptor.

pharmacology and toxicology↗

bioRxiv2026

Profilin-1 Deficiency Activates STING to Drive T Cell-Mediated Anti-Tumor Immunity in Breast Cancer

Dysregulation of actin-binding protein Profilin1 (Pfn1) in tumor cells has prominent impacts on the tumor-intrinsic aspects of tumor progression. However, whether and how modulation of Pfn1 expression in tumor cells influences immune surveillance in cancer is not known. We utilized an inducible CRISPR/Cas9 knockout (KO) model to first demonstrate that triggering Pfn1 depletion in breast cancer cells leads to features of genomic instability (polyploidy, micronuclei, and DNA damage) and intrinsic defects in both homologous-recombination- and non-homologous end-joining-mediated double-stranded DNA repair. Pfn1-deficient breast cancer cells exhibit nuclear envelope abnormality and the accumulation of cytosolic DNA. This leads to activation of the nucleic acid-sensing cGAS-STING pathway and the type-I interferon (IFN) response including STING-mediated upregulation of pro-inflammatory chemokines. In an immunocompetent mouse model of breast cancer, triggering Pfn1 loss selectively in tumor cells promotes an immunogenic tumor microenvironment marked by a striking increase in intratumoral presence of CD8 T cells, leading to a robust tumor regression. Pfn1 knockout-induced tumor regression requires an intact immune system and can also be reversed by CD8+ T cell depletion. Based on these findings, we conclude that Pfn1 loss in tumor cells leverages a type I IFN response to drive a T-cell-mediated anti-tumor response in breast cancer. These findings for the first time reveal promising therapeutic opportunities in targeting Pfn1-driven pathways to enhance immunotherapeutic outcomes in breast cancer. Significance StatementExpression of actin-binding protein Profilin-1 is frequently altered in cancer; yet how these changes impact the immune response against tumors is unclear. Here we show that triggering Profilin-1 depletion in breast cancer cells promotes features of genomic instability, defects in DNA repair, and cytosolic release of DNA. This activates the cGAS-STING pathway, triggering a type I interferon response and immune-cell-attracting signals that drive a CD8+ T cell-mediated anti-tumor immune response and tumor regression in vivo. Therefore, Profilin-1 could be a novel actionable target for achieving immunological benefit in breast cancer. On a broader level, our studies establish a conceptual framework of how dysregulation of actin cytoskeletal proteins can harness nuclear damage-sensing signaling to augment anti-tumor immune response in cancer.

cancer biology↗

bioRxiv2026

Mechanochemical Decoupling of ATP Hydrolysis and RNA Translocation in SARS-CoV-2 nsp13 by the L405D Mutation

SARS-CoV-2 nonstructural protein 13 (nsp13) is a highly conserved helicase that couples ATP hydrolysis to RNA translocation through long-range allosteric communication between its ATPase and RNA-binding domains. In prior work, we identified L405 as a key regulator of interdomain motions and proposed that the L405D mutation would disrupt this coupling by perturbing conformational translocations required for translocation [J. Phys. Chem. B 2024 v128 492-503]. Subsequent experiments confirmed that L405D attenuates helicase activity while largely preserving ATPase activity, implicating a breakdown in ATP-to-RNA coupling [J. Biol. Chem. 2026 v302 111198]. Here, we provide a data-driven explanation for this decoupling by combining Gaussian accelerated molecular dynamics (GaMD) simulations with Shape-GMM clustering and linear discriminant analysis. Whereas wild-type nsp13 exhibits both conformational selection and induction, L405D collapses the conformational landscape to operate predominantly through selection, eliminating ATP-induced structural transitions required for efficient catalytic cycling. This loss of induction traps the ATP-binding pocket in a mid-open conformation, impairing product release and reducing ATP turnover, while simultaneously disrupting coordinated motif-RNA interactions required for inchworm translocation. These findings establish that mutation-induced reshaping of conformational ensembles can modulate access to reaction-competent states, providing a general framework for understanding how targeted mutations disrupt catalytic function through allosteric ensemble remodeling in motor proteins.

biophysics↗

bioRxiv2026

A Phosphorylation Switch Modulates Configurational Codes in the Oncofetal IGF2BP RNA Binding Paralogs

The insulin like growth factor 2 mRNA binding proteins (IGF2BP1-3) are oncofetal RNA regulators that control translation, stability, and localization of several transcripts, yet display paralog specific functions despite high structural similarity. Each paralog contains six RNA binding domains (two RRMs and four KH domains) linked by intrinsically disordered segments. mTORC2 phosphorylates IGF2BP1 and IGF2BP3 at a single conserved serine within the disordered linker between the RRM2 and KH1 domains, a modification required for proper regulation of mRNA translational fate. Pairing site specific phosphoserine incorporation with structural and biophysical interrogations, we show that this phosphorylation acts as a configurational switch that reorganizes long range arrangements of RNA binding domains and linkers without altering the secondary structure, and with only modest effects on RNA binding affinity. Critically, pSer driven rearrangements occur both in the RNA free state and upon RNA engagement, and the resulting architectures differ markedly between IGF2BP1 and IGF2BP3 despite >70% sequence identity. These paralog specific, phosphorylation dependent configurational landscapes likely underlie differences in mRNA recognition modes and functional outcomes. Our work identifies a post translational mechanism that tunes IGF2BP paralog dynamics across free and RNA bound states to program target mRNA selection, processing, and translational fate.

biochemistry↗

bioRxiv2026

Wnt stimulation and inhibition in the development and phenotype of patient-derived gallbladder organoids

Gallbladder cancer (GBC) is a highly lethal malignancy with limited experimental models to study disease biology or evaluate therapeutic responses. Although canonical Wnt activation is commonly used for patient-derived organoid (PDO) development and expansion, gallbladder PDOs has also been generated under Wnt-inhibitory conditions. No comparative assessment has determined how Wnt pathway modulation influences gallbladder PDO development, phenotype or drug response. This study systematically compared the impact of canonical Wnt activation (WNTAct medium containing CHIR99021) versus inhibition (WNTInh medium containing DKK1) on the establishment, propagation, molecular features and therapeutic responses of PDOs generated from malignant or non-malignant gallbladder tissues derived from the same patient. Both media supported successful PDO generation with comparable efficiency, preserving biliary epithelial functions and marker expression. Transcriptomic profiling confirmed selective enrichment of canonical Wnt target genes in PDOs generated in WNTAct cultures. WNTAct conditions enabled markedly superior long-term propagation, whereas WNTInh cultures more consistently retained the dysplastic features in malignant samples. Gemcitabine response assays demonstrated significantly greater drug sensitivity in PDOs grown in WNTAct medium, a phenotype reversible upon media switching but requiring extended adaptation, indicating a dynamic and context-dependent influence of Wnt signaling on chemotherapeutic vulnerability. Collectively, the findings reveal a trade-off between long-term propagation and histological fidelity in gallbladder PDOs and show that Wnt signaling modulates gemcitabine sensitivity in a reversible manner. This comparative framework provides practical guidance for selecting culture conditions for gallbladder PDO based disease modelling and precision oncology applications.

cell biology↗

bioRxiv2026

Divergent venoms among two closely related co-distributed centipede species, Scolopendra morsitans and S. hardwickei in tropical Asia

Venom is an important functional trait that helps predatory animals capture prey. Centipede predatory venoms are complex cocktails of multiple proteins, such as neurotoxins (scoloptoxins), cytotoxins, {beta}-pore-forming toxins, and enzymes. We examined venom phenotypes in two closely related and co-occurring centipede species, Scolopendra morsitans (n=28) and S. hardwickei (n=11), in peninsular India to determine whether their venoms are similar or dissimilar. An integrated proteo-transcriptomic approach was used to characterise the venom phenotypes of the two species across multiple individuals in peninsular India. We used species occurrence records and species distribution models to assess the distributional overlap among these species within the peninsular Indian region. The species showed significant overlap in their current and projected geographical ranges, corresponding with their co-occurrence. We characterised the venom profiles of both species and found that the venoms were cocktails of enzymes, {beta}-pore-forming toxins, and neurotoxins comprising 110 and 84 proteins in S. morsitans and S. hardwickei, respectively. However, the venom composition of both species differed significantly in toxin abundance and species-specific protein repertoires. This indicates trait divergence in venom phenotypes, suggesting that distinct venom compositions may facilitate coexistence among ecologically similar predatory centipedes. The observed variation in venom phenotypes among co-distributed species opens up important avenues for future research into their ecological roles and functional significance. In this study, we provided a detailed account of venom composition across multiple individuals from the species geographic range and highlighted the importance of investigating the role of venom as a trait that could influence species interactions and shape communities in these diverse tropical forests.

ecology↗

bioRxiv2026

A Cationic Amphiphilic Drug (CAD) Defense System in the Nematode Caenorhabditis elegans

Cationic Amphiphilic Drugs (CADs) severely disrupt lysosomal function, which leads to a cellular pathology characterized by excess phospholipids called phospholipidosis. Through a forward genetic screen and mining of published datasets, we discovered that CADs induce the expression of the CYP-35B family of cytochrome P450s and the PGP-13 p-glycoprotein pump via the nuclear receptors NHR-70 and NHR-107 in the nematode C. elegans. A pgp-13 fluorescent reporter revealed hundreds of human drugs that upregulate the CAD defense system in vivo. Chemoinformatic analyses indicate that the pgp-13 reporter may be useful in identifying CADs that have pathogenic potential in humans. Mutant analyses coupled to metabolomics and structural modeling show that the CYP-35Bs are necessary and sufficient for CAD metabolism, and that CYP-35B2 D311 is key in mediating electrostatic interactions with the positively charged CADs. We also show that CAD metabolites are effluxed via PGP-13 acting partially redundantly with PGP-14 and that an intact defense system is necessary to resist CAD-induced pathology. Finally, we demonstrate that bacteria that likely cohabitate with C. elegans in nature trigger the CAD defense system, providing a plausible explanation for why a pathway that protects against anthropogenic small molecules exists in nematodes.

pharmacology and toxicology↗

bioRxiv2025

Molecular insights into Profilin1-dependent regulation of cellular phosphatidylinositol-(4,5)-bisphosphate

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

cell biology↗

bioRxiv2025

Visualization of Bluetongue virus RNA segment networks in infected cells: multipartite genomic RNA assortment is independent of viral proteins NS2 and VP6

Bluetongue virus (BTV), with a genome of ten double-stranded RNA segments (S1-S10), is an emerging animal pathogen causing major economic losses in livestock worldwide. BTV replication involves RNA-RNA and RNA-protein interactions, with RNA-binding proteins, VP6 and NS2 playing key roles in genome assembly and RNA packaging. To explore the dynamics of RNA segment interactions and the roles of VP6 and NS2 in RNA complex formation, we used RNA fluorescence in-situ Hybridisation Chain Reaction (HCR), along with site-specific mutagenesis and reverse genetics. We found that RNA segments interact sequentially, from the smallest (S10) to the largest (S1), forming a single complex that includes the entire genome. This process is independent of VP6 or NS2, although NS2 enhances the assembly of larger segments. Additionally, we show that VP6 binds to +ssRNAs before their incorporation into viral assembly factory (inclusion bodies/VIBs). These findings reveal that RNA-RNA interactions, rather than primary replicase proteins, govern the sorting and recruitment of genome segments. Our data offer new insights into BTV RNA packaging, showing that genome segments destined for packaging and dsRNA synthesis are segregated through complex formation, distinct from +ssRNAs used in protein synthesis, including those encoding the replicase complex.

microbiology↗

bioRxiv2025

Development of a Synthetic Hydrogel to Foster Microvascularization of an Endometriosis Microphysiological System

The ascent of novel alternative methods (NAMs) in drug development spotlights the dual needs for improved biological fidelity to in vivo along with reproducibility, especially in regulatory applications. The need for pre-clinical models of patient-derived endometriosis lesions motivates development of a vascularizable completely synthetic extracellular matrix (v-CS-ECM) that supports morphogenesis of perfusable microvasculature in a microfluidic device, in the context of relevant lesion cells. This paper describes v-CS-ECM, a peptide-modified polyethylene glycol-based hydrogel crosslinked with a cell-degradable peptide that achieves these dual goals. Vessels form by morphogenesis after the liquid v-CS-ECM precursor, containing endothelial cells and fibroblasts, is injected into the tissue compartment to encapsulate cells. Vessel formation is influenced by ECM biochemical and biophysical properties, source of vascular cells, and microphysiological system (MPS) operating conditions. The v-CS-ECM also supports co-culture of endometrial epithelial organoids (EEOs) and fibroblasts, and formation of microvascularized endometriosis lesion-like structures when all cell types are co-encapsulated in a microfluidic device with constant flow. Hence, v-CS-ECM overcomes limitations of reproducibility and biological function inherent in the fibrin-based ECM typically employed for microvascular morphogenesis, as well as Matrigel for organoid culture, thus offering promise for NAMs evaluating endometriosis drugs in the preclinical setting.

bioengineering↗

bioRxiv2025

Phytoremediation of 25+ Years Weathered Petroleum Contamination with Symbiotic Arbuscular Mycorrhizal Fungi and Bacillus subtilis ATCC 21332

Petroleum hydrocarbon (PHC) contamination affects approximately 60% of Canadian contaminated sites, with the boreal ecozone facing acute exposure from industrial activities. This study evaluated the long-term effectiveness of plant-microbe enhanced phytoremediation of 25+ years weathered PHC-contaminated field soil over a 24-month period. Five native or naturalized plant species (three tufted grasses, a forb, and a tree) were tested across two contaminated soils and a control to compare remediation efficacy and plant health: 25,700 mg/kg total petroleum hydrocarbons (TPH) and a 1:1 contaminated and background site soil mixture (12,600 mg/kg TPH). Soil treatments included a Plants-Only Control, Arbuscular Mycorrhizal Fungi (AMF), plant-growth-promoting-rhizobacterium - Bacillus subtilis, and AMF+B. subtilis. Plant inoculation significantly improved remediation compared to natural attenuation (No Plants Control) in both PHC-contaminated soils. AMF-based treatments significantly remediated both soils: AMF in the 25,700 mg/kg TPH soil (74% mean TPH remediation) and AMF+B. subtilis in the 12,600 mg/kg TPH soil (96% remediation). In the 12,600 mg/kg TPH soil, all treatments successfully reduced PHC levels to below federal and provincial thresholds ([≥]95% removal) within 17 months. Picea mariana (black spruce) and Andropogon gerardii (big bluestem) above- and below-ground biomasses were not significantly impaired by the 25,700 and 12,600 mg/kg TPH soils, indicating their suitability for site remediation. B. subtilis significantly improved above-ground biomass and plant height and is therefore recommended for co-inoculation with AMF. These findings demonstrate that AMF-enhanced phytoremediation represents a highly effective, sustainable approach for long-term PHC remediation in Canadian boreal ecosystems, offering significant advantages over natural attenuation processes alone. HighlightsO_LIAMF-based treatments achieved highest PHC removal in 25+ year weathered boreal soils C_LIO_LIPrairie grasses remediated 12,600 mg/kg TPH soil by >95% in AMF+B. subtilis treatment C_LIO_LIEnhanced Picea mariana (black spruce) biomass and moisture in 25,700 mg/kg TPH soil C_LIO_LIBacillus subtilis significantly enhanced plant above-ground biomass and height C_LIO_LIAMF helped maximize PHC remediation, while B. subtilis promoted plant growth C_LI

ecology↗

bioRxiv2025

Petroleum Hydrocarbon Concentration as the Primary Driver of Soil Bacterial Community Dynamics During Mycorrhizal Fungi and Rhizobacteria Assisted Phytoremediation

Petroleum hydrocarbon (PHC) contamination poses widespread environmental risks in Canada, where it is known to contribute to 60% of existing contaminated sites. It requires effective remediation strategies in the boreal ecozone due to the cold climate resulting in slow site weathering processes and soil nutrient cycling. This study utilized PHC-contaminated field soil (25,700 mg/kg total petroleum hydrocarbons - TPH) and background soil (<120 mg/kg TPH) collected from a Canadian boreal site where PHCs had weathered in place for 25+ years. Additionally, a 1:1 diluted soil was prepared (12,600 mg/kg TPH), and with the field contaminated soil, used to investigate the effects on rhizobacterial community composition. In two-year greenhouse experiments (2023 - 2025), the soils were planted with native or na turalized plant species and inoculated with i) arbuscular mycorrhizal fungi (AMF), ii) the biosurfactant-producing and plant-growth-promoting rhizobacterium (PGPR) - Bacillus subtilis ATCC 21332, or iii) both AMF and B. subtilis 21332. PHC contamination was determined to exert a dominant influence on rhizobacterial community composition. Alpha- and beta-diversity analyses revealed that neither plant species nor microbial inoculants significantly altered bacterial diversity or community structure beyond the overriding effect of PHC concentration. Proteobacteria, Actinobacteriota, Acidobacteriota, and Chloroflexi dominated across all soils, with hydrocarbon-degrading genera such as KCM-B 112 and Sphingomonas significantly enriched in the 25,700 mg/kg TPH soil. Functional gene profiling identified widespread aerobic (e.g., alkB, CYP153, assA, nahAc, pheA, xylM, xylE, todC1, bphA1, and pahE) hydrocarbon-degradation pathways across phyla, suggesting extensive horizontal gene transfer and functional redundancy across treatments and plant species. Exogenously introduced B. subtilis ATCC 21132 established only in the background soil, suggesting competitive dominance of indigenous PHC-degraders. These findings underscore the magnitude of PHC concentration as the primary driver of rhizobacterial dynamics and indicate that augmenting native microbial capacity through soil carbon enhancement, rather than bioaugmentation, may significantly impact soil bacterial community composition during rhizodegradation of boreal soils.

molecular biology↗

bioRxiv2025

A Phosphoinositide Interacting Protein Coordinates Stress Precursor Activities

Samd14 is crucial for cell signaling and survival in mouse models of acute anemia. Samd14 has an N-terminal actin capping protein (CP) and a C-terminal sterile alpha motif (SAM) to coordinate stem cell factor/Kit and erythropoietin receptor signaling pathways during terminal differentiation of red blood cell precursors. Here we present new findings that Samd14 expression is needed to maintain balanced autophagy in red blood cell precursors following acute anemia. Autophagy gene signatures and protein levels are markedly altered within the context of acute anemia when red blood cell precursors accelerate the process of erythropoiesis. Samd14 interacted via its SAM domain with phosphatidylinositol 3-phosphate (PI3P) which is an integral component of endosomal and autophagic membranes. We tested PI3Ps role in red blood cell differentiation using a small molecule inhibitor of the Class III PI 3-kinase VPS34, which is the sole kinase responsible for PI3P genesis. SAR405 treatment blocked red blood cell formation. In the absence of Samd14, higher doses of VPS34 inhibition were required to block erythroid differentiation. Given the critical roles of autophagy in normal differentiation, Samd14s stress-dependent activation and roles in autophagy suggest that this mechanism is needed to maintain progenitor levels and balance the production of healthy, mature red blood cells. Key Points- Autophagy genes/proteins are deregulated in red blood cell progenitors after hemolysis-induced acute anemia. - The anemia-activated Samd14 protein interacts with the PI3P lipid moiety which is integral to endosomal and autophagic membrane functions. - VPS34 inhibition blocks terminal erythroid maturation in a Samd14-dependent manner.

molecular biology↗

bioRxiv2025

Adaptive trade-offs between niche-driven defence system selection and horizontal gene transfer suggests clinical success in Acinetobacter spp.

2.IntroductionBacteria face relentless selective pressure from both antibiotics and bacteriophages, driving the evolution of diverse defence systems and shaping horizontal gene transfer (HGT) dynamics. Gap StatementThe relationship between bacterial defence system repertoires and horizontal gene transfer in clinically relevant Acinetobacter species remains poorly understood, limiting our ability to predict resistance evolution and design targeted phage therapies. AimTo characterize defence arsenal across Acinetobacter species and investigate their associations with different HGT markers MethodologyWe performed a comparative genomic analysis of 132 genomes from 18 Acinetobacter species, focusing on the interplay between defence architectures and HGT markers. ResultsOur result reveals pronounced differences in defence system repertoires between lineages: while most Acinetobacter spp. harbour a multilayered arsenal, the clinically dominant A. baumannii international clone 2 (IC2) exhibits a depleted but specialised defence landscape, notably enriched in the phosphorothioation-based SspBCDE system and nearly devoid of restriction-modification systems. Strikingly, many defence systems display mutual exclusivity, and defence system is tightly linked to the presence of mobile elements, antibiotics, and heavy metal resistance. Plasmid-borne defence systems, especially BREX, are prevalent, highlighting the role of mobile elements in distributing both immunity and resistance traits. These patterns suggest that successful clinical lineages finely balance immune defences and genetic plasticity, facilitating rapid adaptation under antimicrobial selection and phage threats. ConclusionThese findings provide insights into evolutionary trade-offs underpinning multidrug resistance and have implications for designing targeted phage therapies against recalcitrant Acinetobacter infections. 3. Data summaryAll bioinformatics analysis workflows, custom scripts, and R code are publicly available at https://github.com/vikos77/Acinetobacter-defence-systems. The repository includes bash scripts for genome downloading and tool execution (scripts 1-10), R scripts for statistical analysis and visualization (scripts 1-9), and conda environment files for reproducible software installation. Complete analysis protocols are documented in the repository README and individual script headers. All software versions and parameters are specified in the code documentation. 4. Impact StatementThis study reveals how defence system composition in Acinetobacter species is shaped by ecological niche and linked to horizontal gene transfer, antibiotic resistance, and mobile genetic elements. Notably, the clinically dominant A. baumannii IC2 lineage exhibits a streamlined defence repertoire favouring genetic plasticity, facilitating rapid adaptation under antibiotic and phage pressure. These findings enhance our understanding of resistance evolution and support the rational design of phage-based therapies targeting multidrug-resistant pathogens.

bioinformatics↗

bioRxiv2025

Small molecule intervention of actin-binding protein profilin1 reduces tumor angiogenesis in renal cell carcinoma

Angiogenesis plays a key role in the development and progression of renal cell carcinoma (RCC). Actin-binding protein profilin-1 (Pfn1) is overexpressed in clear cell RCC predominantly in tumor-associated vascular endothelial cells (ECs). We previously demonstrated that that EC-selective (over)expression of Pfn1 accelerates RCC progression, and conversely, genetic loss of EC-Pfn1 dramatically inhibits tumor angiogenesis impeding tumor initiation and/or progression in RCC, suggesting that Pfn1 could be an actionable therapeutic target in RCC. In this study, we demonstrate that 4,4-((4-bromophenyl)methylene)bis(3,5-dimethyl-1H-pyrazole), a small molecule that we had previously identified as an inhibitor of Pfn1-actin interaction, directly binds to Pfn1 and attenuates tumor angiogenesis when directly administered into subcutaneous RCC tumors. Next, we undertook a chemical optimization approach to design and synthesize 4,4-((4-(trifluoromethyl)phenyl)methylene)bis(3,5-dimethyl-1H-pyrazole), a structural analog of our originally identified inhibitor, that exhibits improved anti-angiogenic efficacy in vitro and in vivo. Finally, we demonstrate that Pfn1 inhibitor is amenable to lipid microbubble encapsulation and release in the tumor microenvironment (TME) by ultrasound-mediated disruption of circulating microbubbles to achieve anti-angiogenic and anti-tumor benefit. In summary, our findings suggest that tumor-localized release of Pfn1 inhibitor could be a potential therapeutic strategy in RCC.

cell biology↗

bioRxiv2025

Comparative clinical transcriptome of pir genes in severe Plasmodium vivax malaria

vir genes, a multigene family in Plasmodium vivax that are a part of a larger superfamily of genes called the pir (Plasmodium interspersed repeat) genes, have been reported earlier to be potentially involved in cyto-adherence and evasion of splenic clearance. Plasmodium vivax, historically characterised as a "benign" malaria parasite, has been associated with clinical outcomes including hepatic dysfunction, renal failure, and cerebral malaria in India and several global regions. It constitutes an economic burden and presents a public health challenge alongside other Plasmodium species. Here, we present a part of global transcriptomic studies using custom-designed microarrays that compare the transcriptome of the parasite responsible for severe Plasmodium vivax manifestations, specifically hepatic dysfunction and cerebral malaria from India, with an emphasis on the pir genes, some of which are reported to play a role in cyto-adherence. The RNA of the parasite isolated from 23 patients (uncomplicated group = 6, hepatic dysfunction group = 12, and cerebral malaria group = 5) was subjected to microarray hybridisation, and the data obtained showed a wide range of pir subfamilies to have been differentially expressed. We report the upregulation of 24 pir genes in the cerebral malaria group (n = 5) and 28 pir genes in the hepatic dysfunction group (n = 12), which belong to different subfamilies in at least 50% of the severe malaria patients group. Out of the upregulated pir genes in the cerebral malaria group, members of vir subfamily E (n=8 genes) and the pvpir subfamily H (n=6 genes) are expressed in a higher proportion compared to the hepatic dysfunction group, where members of vir subfamily E (n=9) and C (n=6) are expressed in a major proportion. Author SummaryPlasmodium vivax, considered to cause benign infections, has been reported over the past couple of decades as manifesting with severe symptoms like cerebral malaria, hepatic dysfunction, and others. This paper reports the analysis of a selected pir gene repertoire from microarray hybridisation performed with custom-designed 15K microarrays. Differences between pir genes, which are specifically upregulated in either hepatic dysfunction or cerebral malaria, the two manifestations considered in addition to uncomplicated vivax malaria samples, are highlighted. The latter provided the control values for comparison with the disease groups. The data presented in this study show differentially upregulated pir genes, some of which are also common to both manifestations. Literature has reported one of the VIR proteins, encoded by vir14 C gene, to be involved in adhesion to ICAM-1. The future scope of this work includes validation with a larger sample size and characterising the products of the pir gene, as depicted with reference to their role in cytoadherence or other mechanisms that could lead to severe disease. It would be possible to devise strategies based on this data that could lead to the use of some of these molecules as potential biomarkers or for therapeutic intervention.

genomics↗

bioRxiv2025

Allosteric Regulation of RNA Affinity by Motif V-VI Coupling in West Nile Virus NS3 Helicase

The rise of flaviviral diseases, including West Nile virus (WNV), presents a growing threat to global public health and underscores the urgent need for new therapeutic strategies. The non-structural protein 3 helicase (NS3h) of the Orthoflavivirus genus, including WNV, is essential for viral replication and a promising antiviral target. Previously [Roy et al., Nucleic Acids Research, 52, 13, 2024, 7447-7464], we showed that the motif VI loop (VIL) in WNV NS3h functions as a nucleotide valve, regulating ADP affinity during hydrolysis. In this study, we uncover an ATP-dependent coupling between nucleotide affinity at motif VIL and RNA affinity at motifs IVa and V, suggesting a coordinated mechanism of ssRNA translocation. Using microsecond-scale all-atom molecular dynamics simulations of hydrolysis-cycle intermediates, we find that key VIL residues (R461, R464) correlate strongly with RNA phosphate affinity of motif V. Structural analyses reveal an ATP-sensitive interaction between E413 (motif V) and R461 (motif VIL) that modulates the conformation of the motif V 310-helix, thereby influencing RNA binding. This dynamic interaction is lost in catalytically deficient VIL mutants, which have been experimentally shown to impair hydrolysis and attenuate viral replication. These findings provide mechanistic insights into NS3h function and identify new opportunities for structure-based antiviral design.

biophysics↗