Biology subjects

Kumar, S.

Publications and source records attributed to Kumar, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

In vivo CAR-T cell therapy eliminates manufacturing complexities associated with ex vivo autologous approaches, but safety concerns have limited adoption. We developed viroVbot, a next-generation in vivo CAR-T platform, by combining computational immunogenicity prediction (CIMMEXTM) with envelope engineering. Screening 22,562 glycoprotein sequences, we identified 641 vesiculovirus homologs, from which we selected Piry virus glycoprotein (PIRYV) as the optimal candidate. PIRYV exhibited lower MHC-epitope density, reduced human seroprevalence, with decreased T cell activation compared to VSV-G. To enhance targeting specificity, we engineered receptor-binding-deficient PIRYV (ePIRYVRBD) displaying CD3/CD7 nanobodies for T cell-selective transduction. To maximize safety, we engineered CAR-TRAP producer cells to eliminate unwanted B cell transduction and incorporated machine learning-optimized T cell-specific promoters that restrict CAR activation exclusively to lymphocytes. Additional modifications suppressed hepatocyte expression and prevented phagocytic uptake. In humanized xenograft models, viroVbot3 generated potent BCMA/CD19 specific CAR-T responses against multiple myeloma and Claudin18.2-targeting gastric cancer, demonstrating sequential redosing with alternative envelopes. Critically, viroVbot3 exhibited minimal off-target organ biodistribution with CAR expression restricted to T lymphocytes. These findings establish viroVbot as a low-immunogenic platform for scalable in vivo CAR-T manufacturing with capability for sequential redosing across hematologic and solid tumors.

bioengineering↗

bioRxiv2026

Tiny Subsamples and Upsampling Tame Big Data Evolutionary Analysis in Phylogenomics

Long runtimes, high memory demands, and reliance on high-performance computing impede phylogenomic analyses. We review a scalable phylogenomic subsampling with upsampling (PSU) framework, in which small subsamples of sites from a concatenated alignment are expanded by upsampling before inference, and the resulting analyses are then aggregated to obtain evolutionary estimates. PSU harnesses the fact that the computational cost of maximum likelihood analysis is strongly influenced by the number of distinct site patterns in the concatenated alignment, whereas statistical power depends primarily on the amount of evolutionary information represented by the total number of sites and substitutions. By reducing the former while restoring the latter through upsampling, PSU can approximate many full-data analyses at substantially lower computational cost. Analysis of simulated and empirical datasets shows that PSU can accurately estimate bootstrap support values, select the optimal substitution model, test evolutionary hypotheses, and infer branch lengths, divergence times, and associated uncertainty measures, while reducing runtime and memory requirements by orders of magnitude. PSU also provides distributions of inferred clade support across independent subsamples, enabling detection of conflicting phylogenetic signals that may remain hidden in conventional bootstrap analysis. Automated tuning of subsample size, the number of subsamples, and the number of upsampling replicates make PSU practical across diverse datasets. We suggest that PSU is a general strategy for scalable phylogenomic inference using a broad range of statistical methods. By enabling analyses of genome-scale alignments on commodity hardware, PSU broadens research access and reduces environmental and infrastructural costs of big-data phylogenomics.

evolutionary biology↗

bioRxiv2026

Menin regulates oncogenic cell identity transcriptional networks in multiple myeloma

Multiple myeloma (MM) is a heterogenous cancer that remains mostly incurable. A unifying feature of MM cells is a highly interconnected network of transcription factors and co-factors that coordinate the activity of super-enhancers to enforce myeloma cell identity. Targeting this network offers a promising avenue for therapeutic intervention across genetically diverse myeloma sub-types. Using integrated molecular and functional genomic approaches we identified Menin as a key driver of oncogenic gene expression in MM cells. Menin and its co-factor KMT2A bind at super-enhancers and maintain expression of essential myeloma genes, including IRF4. We demonstrate that Menin inhibitors, which have recently been approved for treatment of acute myeloid leukaemia, are highly active in myeloma cell lines and in vivo models. Combining Menin inhibitors with other super-enhancer targeting therapies such EP300/CREBBP inhibitors or immunomodulatory drugs (IMiDs) overcomes epigenetic plasticity in cells resistant to single agent treatment.

Cancer Biology↗

bioRxiv2026

STITCH links cellular morphology and gene expression in spatial transcriptomics

In situ spatial (ISS) sequencing can uncover co-variation between cellular morphology and gene expression in vivo. However, a principled and interpretable mathematical representation of morphology has not yet been applied in this context. In particular, current deep learning-based representations of cell images confound a cell's shape with its size. We present an interpretable representation of cellular boundary contours, based on tangent principal component analysis (TPCA) in a Kendall shape manifold, that captures size-independent contour shape features. This approach successfully recovers shape-perturbing genes in an RNAi screen than a previous metric geometry-based approach. We build on TPCA to develop KSTITCH (Kendall Shape-TranscriptomIc Correlation and Harmonization), an approach to reveal covariation between cell morphology with gene expression in ISS datasets. In a Xenium dataset, KSTITCH recovers known morphology-transcriptomic relationships in keratinocytes, macrophages and endothelial cells. Across samples in a melanoma CosMx dataset, KSTITCH reproducibly associates elongated and triangular fibroblasts with proximity to malignant cells and myofibroblast-like transcriptional program. Finally, KSTITCH independently recovers a known link between mesenchymal-like malignant cell states and increased cell area in two melanoma cohorts. KSTITCH can thus yield interpretable morphology-transcriptome relationships across cell types, patients, and spatial transcriptomics platforms. KSTITCH is available at https://github.com/vishakagopalan/kstitch .

bioinformatics↗

bioRxiv2026

Amylo-Pipe: an integrated web server for mechanistic and kinetic prediction of protein and peptide aggregation

Protein aggregation is central to amyloid-related disorders and remains a major developability challenge for protein therapeutics. Over the past two decades, significant advances have been made to predict aggregation-prone regions (APRs) and estimate aggregation propensity in proteins and peptides. In contrast, the prediction of aggregation kinetics has received relatively less attention due to the limited availability and heterogeneity of experimental data. Consequently, aggregation propensities from APR prediction algorithms were widely accepted as a means to predict relative changes in the aggregation kinetics of proteins and mutants. Previous studies have demonstrated, using large-scale datasets, that aggregation propensity shows a weak or inconsistent correlation with aggregation kinetics. In the present study, we have integrated complementary state-of-the-art mechanistic and kinetic prediction tools for protein aggregation into a unified, user-friendly web framework entitled "Amylo-Pipe". Amylo-Pipe also implements practical features that are especially useful for protein engineering, such as gatekeeper-residue mutational scanning to support the design of aggregation-resistant variants. By consolidating multiple prediction tasks in a single interface, Amylo-Pipe enables a more comprehensive assessment of aggregation behavior than APR-only workflows. The web server is freely accessible at: https://web.iitm.ac.in/bioinfo2/amylopipe/.

bioinformatics↗

bioRxiv2026

Integrative genomics reveals shared and stress-specific adaptive pathways underlying acidic soil-associated metal toxicity in rice

Soil acidity-associated toxicities of aluminum (Al), cadmium (Cd), and manganese (Mn) severely constrain rice productivity in upland ecosystems. To investigate the genomic basis of adaptation to acidic soil-related metal stress, we conducted an integrated meta-QTL (M-QTL) and functional genomics analysis in rice. Meta-analysis of 681 QTLs and MTAs from 53 QTL mapping and GWAS studies identified 79 robust M-QTLs, including ten overlapping regions associated with Al-, Cd-, and Mn-responsive traits. A multi-criteria prioritization framework identified 98 candidate genes supported by positional overlap, transcriptomic recurrence, and functional annotation, enriched for ion transport, detoxification, and redox regulation pathways. M-QTL10.9 emerged as a major hotspot enriched for glutathione-S-transferase genes, whereas M-QTL9.5 contained the highest density of prioritized candidates linked to Al and Cd responses. Comparative physiological & biochemical analyses of the contrasting rice genotypes Sahasarang and IR64 revealed genotype-dependent differences in antioxidant responses, metal partitioning, metabolic regulation, and cell wall remodeling under individual and combined metal stresses. Expression profiling of prioritized candidate genes, including OsACO family genes, OsZIP10, and OsGSTU10, further revealed genotype-dependent transcriptional divergence under combined stress. The identification of overlapping M-QTLs across Al, Cd, and Mn datasets suggests both shared and stress-specific adaptive responses to acidic soil-associated metal stress in rice.

genomics↗

bioRxiv2026

Niche-specific contribution of the branched-chain amino acid biosynthesis protein IlvD in Streptococcus pneumoniae infection

Systemic pneumococcal infections are a major cause of morbidity and mortality. Pneumococci isolated from bacteraemia lack clear genetic signatures distinguishing them from isolates recovered from other disease sites. The bloodstream is an evolutionary dead end, providing no means of onward transmission, and acute bacteraemic events offer limited opportunity for within-host evolution. Nonetheless, we reasoned that it might be possible to identify genetic determinants of virulence in blood through genomic comparison of matched lung and blood isolates from individual infections. Using samples from a mouse invasive pneumonia model, we observed frequent occurrence of low population frequency mutations in ilvD - encoding a branched-chain amino acid (BCAA) biosynthesis protein - in bloodstream isolates. These mutations were absent from the lung-resident bacterial population. The single-nucleotide polymorphisms in ilvD clustered in a short stretch of nucleotides that are highly conserved across bacteria, suggesting they might disrupt protein function. Deletion of ilvD in pneumococcal strain D39 conferred competitive advantages in both in vitro environments mimicking the bloodstream and in mouse and Drosophila systemic infection models. Improved survival of ilvD mutants within phagocytic cells was dependent on iron availability. Conversely, disruption of ilvD reduced fitness in the nasopharynx, suggesting BCAA biosynthesis is required for upper airway colonisation but is a liability in the context of systemic infection. Nasopharyngeal colonisation defects could be rescued by administering intranasal BCAA to infected mice, suggesting loss of capacity for de novo BCAA synthesis in ilvD mutants accounted for reduced upper airway fitness. In support of a critical role for BCAA biosynthesis in the primary commensal lifestyle of pneumococcus, genomic analysis of clinical isolates demonstrated that ilvD is under purifying selection. Collectively, these findings highlight the context-specific role of IlvD and BCAA biosynthesis in infection, with a requirement for protein function in the BCAA-restricted environment of nasopharynx, but fitness benefits deriving from loss of IlvD and its associated iron-sulfur cluster, in the BCAA-rich conditions of blood.

microbiology↗

bioRxiv2026

A Multi-Epitope Vaccine Design for Human Pasteurellosis using Outer Membrane β-barrel Proteins of Pasteurella multocida

Pasteurella multocida is a facultative anaerobic, Gram-negative coccobacillus that causes pasteurellosis in companion animals, livestock, and poultry and poses a significant zoonotic risk to humans through bite wounds, scratches, licking, and transfer of bodily fluids. Although vaccines are available for livestock and poultry, no vaccine is currently licensed for human use. In this study, we systematically identified and characterized 29 outer membrane {beta}-barrel (OMBB) proteins in P. multocida Past9 proteome and classified them into functional categories, including TonB-dependent receptors, porins, autotransporters, adhesins, and efflux pumps. B-cell, cytotoxic T-lymphocyte (CTL), and helper T-lymphocyte (HTL) epitopes were predicted from the identified proteins and screened based on antigenicity, non-allergenicity, and non-toxicity. Epitopes conserved across eight human-infecting P. multocida strains and located within the extracellular loop (ECL) region were incorporated into a multi-epitope vaccine (MEV) construct. The designed MEV was predicted to be antigenic, non-allergenic, and soluble. Its tertiary structural model was iteratively refined and validated. Molecular docking with human toll-like receptors 4/2 (TLR4/TLR2) predicted stable interactions, further supported by 100 ns molecular dynamics simulations. Immune simulation of the MEV construct predicted a strong simulated immune response. Furthermore, codon optimization and in silico cloning supported the feasibility of recombinant MEV expression in E. coli. The construct was benchmarked against OmpH, a well-known antigenic protein and exhibited broadly comparable predicted immune responses and receptor-binding energetics. This study proposes a designed MEV candidate against human pasteurellosis and highlights OMBB proteins as potential immunogenic targets for vaccine development.

bioinformatics↗

bioRxiv2026

Multiple versus pairwise sequence alignments for protein phylogenetics using foundation models

Phylogenetic inference is a common task in molecular and evolutionary biology and has conventionally required a multiple sequence alignment (MSA), a statistical model of amino acid substitutions, and an optimality principle. Recently, global models of amino acid substitutions have been inferred from millions of MSAs using transformer-based deep learning, resulting in protein foundation models (pFMs), also known as protein language models (PLMs). Training pFMs on MSAs hypothetically enables them to encode residue dependencies and the phylogenetic structure of the MSA collection. In contrast, pFMs trained on individual sequences lack access to such phylogenetic structure. Here, we assess the phylogeny inference gains offered by the use of MSA for training pFMs by comparing the relative accuracies of phylogenies inferred using two types of pFMs: one trained on a large collection of MSAs (msat-pFM, [1]) and the other trained using a collection of single sequences (esm-pFM). For msat-pFM analysis, we inferred neighbor-joining trees using pairwise distances estimated directly from the sequence attention matrices. For esm-pFM [2], pairwise distances were obtained using the correlation of attentions of homologous residues, where pairwise sequence alignments (PSA) were used to establish residue homologies. Surprisingly, MSA phylogenies inferred using the msat-pFM were less accurate than esm-pFMs. This pattern was seen across datasets spanning both small and large numbers of species and proteins. Also, PSA phylogenies obtained using residue attentions from early ESM-PFM layers were much more accurate. These results suggest that the multiple sequence alignment step, which is obligatory to establish residue homologies across multiple sequences, may not add information when using evolutionary distances based on attentions in pFMs.

bioinformatics↗

bioRxiv2026

Design of a Multi-epitope Vaccine Against Human Glanders Targeting Outer Membrane β-barrel Proteins of Burkholderia mallei

Burkholderia mallei, a facultative intracellular Gram-negative pathogen, is the causative agent of glanders that primarily affects solipeds and is sporadically transmitted to humans. Current interventions mainly rely on antibiotics; however, increasing antimicrobial resistance and the lack of a licensed vaccine further complicate disease management. Surface exposed outer membrane {beta}-barrel (OMBB) proteins serve as excellent targets for vaccine development. In the present study, a consensus-based computational framework was employed on the B. mallei turkey2 proteome that identified 59 OMBB proteins - including porins, TonB receptors, autotransporters, and efflux components. These OMBB proteins were leveraged to predict B- and T-cell epitopes which were manually curated, and mapped onto the corresponding protein models to identify surface-exposed epitopes with direct accessibility to the host immune cells. These epitopes were linked together to construct a multi-epitope vaccine (MEV) that was predicted to be antigenic, and soluble upon overexpression. The tertiary structure of the MEV was generated which was used for molecular docking with TLR4 and TLR2. Molecular dynamics simulation and flexibility analysis confirmed the structural stability of the MEV-TLR4/TLR2 complexes. In-silico immune simulation showed the capability of MEV to induce a strong immune response. Codon optimization and in-silico cloning were performed to evaluate its efficient expression in the E. coli host. The findings suggest that surface exposed OMBB proteins can serve as promising antigenic candidates for designing an MEV construct.

bioinformatics↗

bioRxiv2026

Emergent Collective Bats Oscillation Dynamics on Tree Branches

The flying fox bats roost in large colonies, suspended upside-down with minimal grip efforts from tree branches that are exposed to environmental disturbances. In this study, we investigate the oscillation dynamics of bats hanging from tree branches under natural conditions with wind. Bats modulate their grips to control the oscillation during wind disturbances and actively transform their postures. Using field observations, we analyze the angular deformation, speed, and phase of individual and collective bats swaying motions in response to environmental perturbations. We observed the mechanical coupling-based synchronization of collective bat oscillations on a tree branch. To rationalize this new phenomenon of bats synchronization behavior, we perform a table-top experiment of a physical model using active oscillators and passive systems. This work could inform the design of bio-inspired suspension systems and contribute to our understanding of animal balance and collective behavior in unsteady and complex environments.

biophysics↗

bioRxiv2026

Lung surfactants as a component of lipid nanoparticles for pulmonary mRNA delivery

Pulmonary delivery of lipid nanoparticles (LNPs) remains an area of significant interest, given the broad range of genetic disorders that could be addressed through localized administration of therapeutic nucleic acids to the lung. In this study, we investigated how incorporation of the clinically used lung surfactant cocktail Poractant alfa affects the in vitro and in vivo transfection performance of mRNA-loaded LNPs. The resulting lung surfactant-enhanced LNPs (Surf-LNPs) exhibited substantial improvements in particle assembly, yielding an order of magnitude higher particle concentration at equivalent input conditions compared to conventional (Onpattro-like) LNP formulations. In vitro, Surf-LNPs demonstrated several-fold increases in mRNA transfection efficiency and protein expression while maintaining excellent cytocompatibility. These enhancements are attributed to an elevated apparent pKa and the surface-active properties of surfactant protein B (SP-B), which promote more rapid and efficient endosomal escape relative to conventional LNPs. In vivo evaluation following intranasal administration further revealed enhanced mCherry expression in the lungs of mice treated with Surf-LNPs compared to conventional LNPs. Ultimately, these findings establish lung surfactant incorporation as a simple yet powerful formulation strategy to improve pulmonary gene delivery using LNPs, with the potential to significantly advance the translation of inhaled nucleic acid therapeutics.

bioengineering↗

bioRxiv2026

Transforming off-the-shelf personal glucose meter into a sustainable and decentralized label-free nucleic acid and NAAT detection platform

In todays world, point-of-care nucleic acid detection still remains extensively constrained and limited by the heavy dependence on centralized urban instrumentation facilities and complex assay workflows. Here, we elucidate a glucometer-based analytical platform that enables label-free detection of nucleic acids and the nucleic acid amplification products through a simple redox-mediated mechanism. The approach leverages the potassium ferricyanide (K3[Fe(CN)6])/ potassium ferrocyanide (K4[Fe(CN)6]), redox system, which is intrinsic to commercial glucometers, complementing with interactions between methylene blue (MB) and nucleic acids. These interactions transduce concentration differences in nucleic acids into quantifiable electrochemical signal readouts. Distinct varied signal outputs are observed between single-stranded and double-stranded DNA, enabling the direct detection as well as integration with nucleic acid amplification tests (NAATs), including polymerase chain reaction, rolling circle amplification, and loop-mediated isothermal amplification. Optimization of reaction parameters and conditions leads to enhancement of the overall signal discrimination and sensitivity across various assay formats. This innovation repurposes widely available off-the-shelf glucometers as a low-cost, portable nucleic acid detectors, thus eliminating the need for any specialized instrumentation. Our results enumerate and establish a generalized and scalable strategy for nucleic acid sensing. The platform thus supports sustainable and environmentally responsible point-of-care testing, thereby enabling improved accessibility and public health monitoring at resource-limited and remote settings.

biochemistry↗

bioRxiv2026

Experience-Dependent Gain Modulation Drives Thermosensory Responses in Behavior

Sensory neurons must extract behaviorally relevant features from dynamic environments while maintaining sensitivity across wide stimulus ranges. To understand how sensory encoding adapts to experience during behavior, we combine long-duration calcium imaging in freely moving C. elegans with a temperature-trajectory playback paradigm to determine how the thermosensory neuron AFD extracts behaviorally relevant sensory features during navigation. We observe that AFD functions as a leaky integrator of recently experienced temperature changes, accumulating thermal inputs over a rolling window of tens of seconds, resulting in calcium levels that represent recent temperature dynamics during runs. Importantly, we determine that AFD selectively amplifies responses to temperature changes near its learned preferred temperature. This experience-dependent gain control aligns encoding with the navigational goal, providing a mechanism for representing temperature preference within a derivative-based sensory system. A minimal mathematical model incorporating derivative detection, leaky integration, and temperature-dependent gain captures the calcium dynamics over a range of stimuli, and a simulation based on the mathematical model predicts goal-oriented locomotor strategies across stimulus regimes. Together, these findings show how gain control allows a derivative-based sensory code to represent an absolute goal and guide locomotory strategies during navigation. HighlightsO_LIAFD encodes recent temperature changes over behaviorally relevant timescales. C_LIO_LIAFD amplifies temperature-change responses within a learned temperature-centered gain window. C_LIO_LIGain control provides a mechanism for representing distance to temperature preference within the derivative-based sensory system. C_LIO_LIA mathematical model and simulation of derivative sensing, leaky integration, and gain control predicts AFD calcium responses and goal-oriented locomotory strategies across stimulus regimes. C_LI

neuroscience↗

bioRxiv2026

Immunological imprinting shapes the cross-reactive antibody responses to the KP.2 and LP.8.1 vaccine doses

The emergence of the SARS-CoV-2 Omicron BA.2.86 subvariant, a lineage derived from the BA.2 strain, led to the 2024-2025 COVID-19 vaccine update to include KP.2 or related JN.1-lineage spike antigens. We evaluated the magnitude, breadth, and durability of humoral immune responses following a single KP.2 vaccine dose in a longitudinal cohort of 21 individuals up to six months. KP.2 vaccination increased spike-specific binding and neutralizing antibodies against the ancestral WA1 strain, alongside the BA.5, XBB.1.5, and KP.2 variants. Power law modeling estimated half-lives for WA1- and KP.2-specific IgG responses at 770 and 248 days, respectively. Additionally, the KP.2 dose increased IgG1 and IgG4 subclasses more than IgG2 and IgG3 responses to both spike proteins. Serum depletion experiments using WA1 or KP.2 proteins demonstrated most vaccine-elicited antibodies were cross-reactive. Consequently, KP.2 vaccine-induced antibodies retained broad neutralizing activity against recently circulating Omicron subvariants (BA.2.86, KP.3.1.1, XEC, LP.8.1, LF.7, XFG.3.12, PQ.1, BA.3.2.1, and RE.2). Using a live virus neutralization assay, XFG.3.12 showed the greatest reduction in neutralizing titers relative to KP.2 (4.2-fold). In a small subset, an LP.8.1 vaccine dose increased neutralizing activity against the matched variant while maintaining WA1 and KP.2 cross-reactivity, but only modestly increased antibodies to divergent variants BA.3.2.1 and RE.2. Ultimately, these data indicate the KP.2 mRNA vaccine generates durable, cross-reactive responses against current Omicron subvariants. However, ongoing spike evolution impacts neutralization of emerging lineages, highlighting the need for continued viral monitoring and timely vaccine updates. IMPORTANCESARS-CoV-2 continues to evolve, raising ongoing concerns about how well updated vaccines protect against emerging variants. This study evaluates antibody responses after KP.2 spike mRNA vaccine dose and shows that a single dose induces durable and broadly cross-reactive immunity against both earlier strains and recently circulating Omicron subvariants. Despite this breadth, reduced neutralizing activity against certain emerging variants indicates that ongoing antigenic changes can impact vaccine induced antibody effectiveness. These findings provide insight into how current vaccines perform over time and highlight the need to track viral evolution and update vaccine antigens to maintain broad protection against severe disease, hospitalization, and death.

immunology↗

bioRxiv2026

Mutations in MreB suppress β-lactam sensitivity upon c-di-AMP accumulation in Listeria monocytogenes

Cyclic di-AMP (c-di-AMP) is an essential second messenger in Listeria monocytogenes, but its accumulation is detrimental as it disrupts cell wall homeostasis and attenuates virulence. The mechanisms underlying this toxicity remain poorly understood. To understand the molecular basis of this toxicity, we performed a forward genetic screen to identify suppressor mutations that restore {beta}-lactam resistance in a {Delta}pdeA {Delta}pgpH ({Delta}PDE) mutant, which accumulates high c-di-AMP and is susceptible to cell wall-targeting {beta}-lactam antibiotics. We found that the majority of suppressor mutants carried mutations in the mreB gene, which encodes the bacterial actin-like cytoskeletal protein, MreB, that directs lateral peptidoglycan synthesis during cell elongation. These mutations restored {beta}-lactam resistance and ex vivo virulence while still retaining high intracellular c-di-AMP levels. Microscopy analyses indicate that these suppressor mutations reduce MreB activity, as evidenced by cell widening, and that they phenocopy sublethal treatment with the MreB inhibitor A22. Consistently, A22 treatment also rescued {beta}-lactam sensitivity in the {Delta}PDE mutant, supporting a functional link between MreB activity and c-di-AMP toxicity. Mechanistically, c-di-AMP accumulation impaired cell division/septation and reduced peptidoglycan synthesis under cell wall stress, whereas MreB mutations restored both transglycosylation and transpeptidation activities and promoted cell division. These effects were independent of potassium homeostasis, suggesting a distinct pathway linking c-di-AMP to cell wall regulation in L. monocytogenes. Together, our findings demonstrate that dysregulated MreB activity contributes to cell wall defects at elevated c-di-AMP levels and highlight the importance of coordinating cytoskeletal dynamics with cell division to maintain cell envelope integrity.

microbiology↗

bioRxiv2026

Deciphering the Molecular Structure of the Type III Secretion System in Chlamydia trachomatis for Structure-Based Therapeutic Targeting

Chlamydia trachomatis is an obligate intracellular pathogen that causes sexually transmitted infections and trachoma. Its persistent forms show reduced antibiotic susceptibility, creating a need for new antivirulence strategies. The C. trachomatis injectisome or type III Secretion System (T3SS) injects effector proteins into host cells, yet the molecular structure of the complete apparatus is unknown. Here, we identify and validate all 13 T3SS constituent proteins using TXSSScan and reverse-BLAST analysis, and model and assemble them into the complete T3SS apparatus using an integrated computational pipeline. Template-based modeling revealed a conserved architecture despite low sequence identity (18-46%), and the assemblies were validated stereochemically and energetically against homolog controls. Targeting the oligomerization interface of the CdsN ATPase through structure-based virtual screening of the e-Drug3D and IMPPAT libraries, followed by ADMET filtering, including predicted membrane permeability for intracellular targeting, identified three repurposable FDA-approved candidates: M Roflumilast, Elacestrant, and Tecovirimat. MM-GBSA calculations and molecular dynamics simulations confirmed stable complexes with two adjacent CdsN monomers. Elacestrant showed the most favorable binding free energy. These results provide the first molecular-level map of the C. trachomatis T3SS and a rational basis for antivirulence drug repurposing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/723290v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@6893c8org.highwire.dtl.DTLVardef@1be5eddorg.highwire.dtl.DTLVardef@1462b25org.highwire.dtl.DTLVardef@1baadab_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

Integrin-independent Tie2 activation using de novo designed proteins

The Angiopoietin-Tie2 pathway is a key regulator of vascular stability, but therapeutic exploitation has been limited by the poor developability of Angiopoietin-1 (Ang1), and there are key unresolved mechanistic questions. Ang1 binds both Tie2 and 5{beta}1 integrin, and the role of the 5{beta}1 interaction in signaling has been unclear. We used RFdiffusion to design a stable, high-affinity Tie2 minibinder that has no affinity for 5{beta}1. The binder is a selective antagonist in monomeric form, and a potent agonist when assembled into an octavalent architecture (H8mb) which drives Tie2 clustering. H8mb signals as potently as Ang1, indicating that integrin engagement is not required for Tie2 activation. However, the duration of signaling is reduced, and internalization of H8mb-Tie2 complexes is more rapid, suggesting that integrin may function as a co-receptor for Ang1 that prolongs signaling by extending the lifetime of the receptor ligand complex on the cell surface. In a mouse model of acute respiratory distress syndrome (ARDS), H8mb markedly improved survival. These results demonstrate that de novo designed receptor binders can enable dissection of co-receptor control of signaling dynamics, and the more stable and manufacturable H8mb provides routes to more developable therapeutic candidates.

biochemistry↗