Biology subjects

Srivastava, A.

Publications and source records attributed to Srivastava, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Inverse Protocol Prediction from Spheroid Microscopy Imaging via Morphology-Aware Structured Learning

We introduce Inverse Protocol Prediction (IPP), which is a task of inferring experimental culture conditions directly from a single bright-field spheroid image. We formulate IPP as a structured multi-label prediction problem and propose a protocol-aware learning framework that integrates morphology extraction, multimodal representation learning, and dependency-aware inference. Morphometric descriptors derived from automated spheroid segmentation are fused with deep visual embeddings via a morphometry vision fusion module. To capture biological and procedural dependencies, we develop a Hierarchical Multi-Task Transformer that conditions predictions across protocol attributes. The framework is trained with domainadversarial supervision and morphology-preserving augmentation to improve robustness to acquisition variability. Hybrid convolution attention encoders achieve the best performance, reaching 95.7% multi-attribute accuracy. We further evaluate cross dataset transfer and temporal morphology forecasting. Results demonstrate that structured, dependency-aware modeling enables reliable reconstruction of experimental protocols from imaging alone, supporting reproducibility auditing and protocol validation in 3D cell culture systems.

bioinformatics↗

bioRxiv2026

A Tissue Culture Free Genome Editing Strategy in Plants Using Broad-Host-Range Viral Vectors Derived from Geminiviruses

The use of viral vectors offers a promising alternative to traditional transformation methods for creating gene-edited plants. In this study, we developed a novel plant genome editing system by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB). Cas9, Cas12f, and Cas12j nucleases were efficiently expressed along with corresponding guide RNAs under viral promoters. By leveraging tRNA spacers in place of external promoters and terminators, we significantly reduced the overall cargo size, streamlining vector design. Additionally, we compared the traditional AtU6-driven gRNA delivery with a novel spacer:gRNA:spacer format in Cas9-expressing lines and observed comparable editing efficiencies. The broad host range of WDIV and AYLCB, combined with this tissue culture-free genome editing platform, opens up possibilities for editing across a wide range of plant species.

plant biology↗

bioRxiv2026

Serum free expansion and transduction of human Vγ9δ2 T cells for adoptive immunotherapy

Background and AimGamma delta T cells are multivalent immune cells with innate and adaptive features that sense a broad spectrum of tumor-associated stress patterns to lyse them without prior potentiation. They are therefore a good option for developing an off-the-shelf therapeutic immune cell product with antitumor functions. Therefore, we aimed to develop an optimized cGMP compatible protocol for expanding V gamma 9-V delta 2 T cells in a serum-free media for adoptive immunotherapy applications. Methods and ResultsPeripheral blood mononuclear cells (PBMC) from healthy donors were activated with Zoledronic Acid (ZOL) and IL-2 for 14 days. Cell proliferation, fold expansion, and phenotype were monitored. Among the regular donors, the baseline levels (day 0) of CD3+Vgamma9+delta2+ T cells were (05.10+/-0.74%). There was a robust expansion of V gamma 9-V delta 2 T cells in the serum-free media (110+/-29.89-fold). During processing, an abrupt reduction of {beta} T cells was observed as early as day +07. After two weeks, 87.82+/-5.11% (n=8) of T cells were CD3+Vgamma9+ in Optimizer with 97.15+/-0.7% of the CD3+Vgamma9+ T cells positive for delta2. The CD3+Vgamma9+ NKG2D+ increased during expansion, reaching 93.76+/-1.55% expression on day +14. V gamma 9-V delta 2 T cells expanded in the serum-free (Optimizer) media had relatively reduced variance and reduced over all yield and fold expansion but comparable percentage of Vgamma9+ and {beta} TCR+ T cells on day 14. A flow cytometry-based tumor-toxicity assay gauged the antitumor functions against K562 cell lines. Pretreatment of K562 cells with ZOL differentially enhanced (2.48+/-0.76 fold) the cytotoxic capacity of V gamma 9-V delta 2 T cells in a donor-dependent manner. The conditions for lentiviral transduction and transgene expressions of V gamma 9-V delta 2 T cells were improvised as gauged by GFP expression driven by CMV promoter (39.09+/- 8.94%) without compromising the viability. ConclusionWe have optimized a cGMP compatible protocol for expansion of human V gamma 9-V delta 2 T cells in a serum-free media with high purity and viability in as early as 07 days. Pretreatment of target tumor cells with ZOL enhanced the cytotoxicity, revealing the impact of V gamma 9-V delta 2 T cell intrinsic factors in tumor lysis. This protocol, may be scaled up for clinical translation for adoptive immunotherapy.

bioengineering↗

bioRxiv2026

Autophagy Cholesterol Axis Remodeling Supports Malignant Progression and Chemoresistance in Glioma

Glioma progression and resistance to temozolomide (TMZ) remain major clinical challenges. Here, we investigated whether dysregulated autophagy and cholesterol metabolism are coordinately remodeled during glioma progression and TMZ resistance. Tissue microarray analysis of astrocytoma and glioblastoma specimens revealed progressive autophagosome accumulation, reflected by increased LC3{beta} puncta, coupled with impaired autophagic flux compared with adjacent normal brain tissue. These alterations intensified with tumor grade and were associated with upregulation of farnesyl diphosphate synthase (FDPS), linking malignant progression to cholesterol pathway remodeling. TMZ-resistant (R) glioblastoma cells exhibited epithelial-to-mesenchymal transition, mitotic quiescence, and mitochondrial remodeling consistent with a therapy-tolerant phenotype. Bioenergetic profiling demonstrated reduced respiratory reserve, diminished ATP-linked respiration, and elevated proton leak, indicating constrained metabolic flexibility. In parallel, impaired autophagy flux was associated with suppression of de novo cholesterol synthesis and transcriptional downregulation of SREBP-2 and LDL-R. Comprehensive lipidomic profiling revealed marked cholesterol metabolic reprogramming in R cells, characterized by accumulation of specific cholesteryl esters, including CE 22:5, CE 22:6, CE 22:4, and CE 20:4, despite reduced cholesterol biosynthesis. Pharmacologic inhibition of the mevalonate pathway with simvastatin significantly altered cholesteryl ester profiles but failed to restore autophagy flux or sensitize R cells to TMZ-induced apoptosis, even under combined TMZ-simvastatin treatment. Lay AbstractAs gliomas progress from astrocytoma to glioblastoma, autophagy becomes dysregulated and cholesterol metabolism is rewired. This coordinated remodeling supports tumor survival, metabolic plasticity, and resistance to temozolomide therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/697885v2_ufig1.gif" ALT="Figure 1000"> View larger version (79K): org.highwire.dtl.DTLVardef@1183dd2org.highwire.dtl.DTLVardef@82e20dorg.highwire.dtl.DTLVardef@c6c8dforg.highwire.dtl.DTLVardef@adb427_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsAutophagy flux blockade intensifies during progression from astrocytoma to glioblastoma Dysregulated autophagy is coupled to altered cholesterol metabolism in malignant gliomas TMZ-resistant glioblastoma cells undergo epithelial-to-mesenchymal transition and mitotic quiescence Resistant cells exhibit constrained bioenergetic capacity and mitochondrial remodeling Impaired autophagy suppresses de novo cholesterol synthesis and lipid recycling Lipidomics reveals accumulation of long-chain cholesteryl esters in TMZ-resistant cells Statin-based cholesterol inhibition fails to resensitize glioblastoma cells to temozolomide

cancer biology↗

bioRxiv2025

Charge reversal at the Lhcb2 N-terminus impairs phosphorylation and PSI-LHCII complex formation

State transitions balance excitation-energy distribution between Photosystem I and Photosystem II in higher plants. Stn7-mediated phosphorylation of the N-terminus of the light-harvesting complex II protein Lhcb2 plays a central role in photosynthetic state transitions. However, it remains unclear how the intrinsic charge of this region, independent of its phosphorylation status, influences state transitions and thylakoid membrane organization. Here, we introduced specific charge-altering mutations in the Lhcb2 N-terminus of Arabidopsis thaliana in the lhcb2 knock-out background and analyzed their effects on LHCII phosphorylation, state transition dynamics, PSI-LHCII complex formation, and thylakoid ultrastructure. Substitution of a conserved positively charged arginine with a negatively charged glutamate (R2E) markedly reduced Lhcb1 and Lhcb2 phosphorylation and state transition efficiency, and abolished PSI-LHCII complex formation. In contrast, introducing a negative charge at a downstream position (Q9E) had no detectable effects. Electron microscopy revealed no significant changes in thylakoid organization in either mutant compared to WT Lhcb2 plants. Despite strongly reduced Lhcb1 and Lhcb2 phosphorylation in the R2E mutant, residual state transitions persisted, potentially mediated by Stn7-dependent phosphorylation of other target proteins. Together, these results provide insight into the role of N-terminal LHCII electrostatics in state transitions and thylakoid membrane organization in plants.

plant biology↗

bioRxiv2025

SWARM: A Single-Molecule Workflow for High-Precision Profiling of RNA Modifications

Nanopore direct RNA sequencing promises to decode the epitranscriptome by detecting multiple modifications on individual RNA molecules, but its potential for biological discovery is hampered by high false-positive rates. We present SWARM, an AI-based framework designed to overcome this fundamental limitation. Its key innovation is a crosstalk-aware training strategy that incorporates non-target modifications and orthogonally validated cellular signals, enabling high-precision detection of m6A, pseudouridine ({Psi}), and m5C at single-nucleotide and single-molecule resolution. Using rigorous in vitro and cellular RNA benchmarks, SWARM outperforms existing tools and maintains strong agreement with orthogonal methods. Applying SWARM across mammalian tissues reveals thousands of novel modification sites with confirmed motifs and localisation patterns. Our high-resolution multi-tissue modification map revealed no evidence of widespread m6A-{Psi} interplay in predominant writer contexts, challenging models of a coordinated epitranscriptomic code. We further discovered a previously unrecognised splicing-shaped mode of {Psi} deposition, whereby TRUB1-mediated pseudouridylation preferentially occurs after exon-exon ligation, consistent with local RNA structure stabilisation. SWARM provides a robust, universally applicable tool for epitranscriptome discovery.

bioinformatics↗

bioRxiv2025

EBV INFECTION OUTCOMES DETERMINED BY MONOCYTE AND TREG-DRIVEN IMMUNE DYNAMICS IN AN EX VIVO PBMC MODEL

Epstein-Barr virus (EBV) infects >95% of the adult population with diverse outcomes ranging from benign latency to cancers and autoimmune diseases. Immunological control of EBV infection is known to be an important determinant of EBV infection outcomes. However, species-specific viral tropism and limited infection models have impeded mechanistic insights into early host-immune control of EBV infection. Here, we use ex vivo infection of peripheral blood mononuclear cells (PBMCs), rather than routinely used B cell enriched culture systems, to study immune and viral dynamics during primary EBV infection. We combined bulk RNA sequencing, EBV transcript enrichment, and flow cytometry to characterize cellular responses across Days 1, 7-8, and 14 post-infection. Early infection triggered a monocyte-specific antiviral response marked by changes in the expression of genes associated with lipid metabolism (LIPA, lysosomal acid lipase) and chemotaxis (CCR1 and CCR2). Inhibitors of LIPA increased EBV titers during primary infection, indicating that LIPA is part of an early monocyte-driven antiviral response. At later timepoints post-infection, donor-dependent variability in lymphoblastoid cell line (LCL) outgrowth was associated with divergent immune states. Donors that failed to generate LCLs demonstrated increased frequencies of CD8+ T cells and reduced numbers of regulatory T cells (CD4CD25FOXP3). EBV transcriptomics revealed that LCL-failed donors exhibited elevated early lytic gene expression but did not establish a type III latency program. Our findings suggest that individual variations in immune cell composition and gene expression may account for differences in the immune response to EBV. These findings define temporal immune and viral signatures that predict transformation outcome and highlight intact PBMCs as a tractable model to study EBV pathogenesis in a genetically diverse, human-specific context. AUTHOR SUMMARYIndividual variation in response to Epstein-Barr virus (EBV) infection can lead to diverse pathogenic outcomes, ranging from cancers to autoimmune disease. To study this variation, we analyzed immune cell response and viral dynamics during the ex vivo primary EBV infection of peripheral blood mononuclear cells (PBMCs) from donors that either fail or succeed to generate lymphoblastoid cell lines (LCLs). Flow cytometry and RNA-seq revealed a rapid monocyte-specific antiviral response among all donors marked by genes associated with lipid metabolism (LIPA) and chemotaxis (CCR1 and CCR2). LIPA inhibition increased EBV titers during primary infection, demonstrating a functional antiviral role. At later timepoints, donor-specific differences in CD8+ T cells and Treg subsets, along with EBV gene expression, were correlated with successful LCL outgrowth. Treatment with the Treg-depleting antibody RG6292 suppressed viral transformation in donors that otherwise supported LCL outgrowth, confirming a functional role for Tregs in shaping early EBV infection outcomes. Viral transcript enrichment-seq revealed an upregulation of early lytic and failure to sustain latent gene expression correlating with failure to generate LCL. These findings highlight intact PBMCs as a tractable model to study EBV viral-host interaction in a genetically diverse, human-specific context, and that Tregs play a key determining role in viral transformation.

immunology↗

bioRxiv2025

Calcitonin-Driven Reactivation of the Hippo Tumor-Suppressor Cascade Attenuates YAP/TAZ Oncogenic Signaling in Glioblastoma

Activation of WT calcitonin receptor (WT CTR) by Calcitonin (CT) acts as growth inhibitory axis in GBM, while the patient-derived loss-of-function CTR mutants promote tumor growth and lower patient survival. The precise mechanisms that orchestrate CTR activation, modulate its downstream signaling dynamics, and give rise to structural anomalies in mutant variants remain incompletely defined and warrant comprehensive investigation. Here, we found that salmon CT treatment suppressed the growth of patient-derived glioma stem cells (GSCs) by activating the Hippo pathway through the CTR/cAMP/PKA/LATS1 signaling cascade, while simultaneously inhibiting the oncogenic YAP/TAZ transcription factors. However, GSCs expressing a phosphorylation-resistant YAP mutant were refractory to growth inhibition by CT in vitro. GSCs, unlike differentiated glioma cells (DGCs), expressed high levels of CTR thus making them suitable for CT-based therapy. Furthermore, the intranasal delivery of salmon CT inhibited glioma growth initiated by GSCs in an intracranial orthotopic mouse model, resulting in increased survival in the mice. In addition, unlike the WT, the CTR mutants failed to activate the Hippo pathway. A large-scale, microsecond-long all-atom molecular dynamics simulation study of mutant CTR systems revealed remarkable changes in CTR interactions with the CT and G-GTPase domains. Packing and conformational dynamics data from simulation studies of the WT and mutant CTR systems could explain the perturbed cAMP/PKA signaling that may compromise downstream signaling. Together, we demonstrate that the CT/CTR axis inhibits glioma by activating the Hippo pathway, provide evidence for the structural defects in the loss-of-function CTR mutants, and propose a CT-based therapeutic strategy for GBM. Statement of significanceCT/CTR axis targets YAP/TAZ through Hippo pathway activation. Patient-derived GSCs express higher CTR levels, and intranasally delivered CT suppresses GSC-initiated tumors. All-atom molecular dynamics simulation uncovers structural perturbations in the CTR mutants.

cancer biology↗

bioRxiv2025

Blind Prediction of Complex Water and Ion Ensembles Around RNA in CASP16

Biomolecules rely on water and ions for stable folding, but these interactions are often transient, dynamic, or disordered and thus hidden from experiments and evaluation challenges that represent biomolecules as single, ordered structures. Here, we compare blindly predicted ensembles of water and ion structure to the cryo-EM densities observed around the Tetrahymena ribozyme at 2.2-2.3 [A] resolution, collected through target R1260 in the CASP16 competition. 26 groups participated in this solvation cryo-ensemble prediction challenge, submitting over 350 million atoms in total, offering the first opportunity to compare blind predictions of dynamic solvent shell ensembles to cryo-EM density. Predicted atomic ensembles were converted to density through local alignment and these densities were compared to the cryo-EM densities using Pearson correlation, Spearman correlation, mutual information, and precision-recall curves. These predictions show that an ensemble representation is able to capture information of transient or dynamic water and ions better than traditional atomic models, but there remains a large accuracy gap to the performance ceiling set by experimental uncertainty. Overall, molecular dynamics approaches best matched the cryo-EM density, with blind predictions from bussilab_plain_md, SoutheRNA, bussilab_replex, coogs2, and coogs3 outperforming the baseline molecular dynamics prediction. This study indicates that simulations of water and ions can be quantitatively evaluated with cryo-EM maps. We propose that further community-wide blind challenges can drive and evaluate progress in modeling water, ions and other previously hidden components of biomolecular systems.

biophysics↗

bioRxiv2025

Statistical thermodynamics based design principles into the temperature induced fold switching of a metamorphic protein

Fold-switching metamorphic protein sequences defy the classical "one sequence - one fold" paradigm. The ability of metamorphic proteins to reversibly switch between distinct folds make them attractive de novo protein engineering candidates since they can function as environment-sensitive molecular switches. However, the underlying thermodynamic design principles that drive their fold-switching behaviour is poorly understood. Gaining insights into the molecular driving forces leading to fold switching behavior is crucial for the rational design of new metamorphic proteins based molecular switches, sensors and stimulus responsive nanomaterials. In this study, we perform a detailed thermodynamic analysis of a designed fold-switching protein [PNAS 2023, 120 (4), e2215418120] 1 that transitions between a 3 fold and /{beta} fold upon changes in temperature. We use an efficient advanced sampling molecular simulation based free energy calculation approach called Confine-Desolvate-Convert-Solvate-Release (CDCSR), which subjects the protein through the complete range of thermodynamic cycle and deconvolutes the enthalpic and entropic driving forces at each stage of the cycle. We find that while 3 fold is stabilized at low temperatures by enthalpic contributions from favorable water-water and protein-water interactions, the transition to the /{beta} fold at high temperatures is driven by the gain of entropy from the release of ordered water molecules surrounding the 3 fold. Our study elucidates the molecular driving forces governing temperature-induced fold-switching behavior and provides a rigorous statistical thermodynamic framework that can help in the design and engineering of future synthetic and functional metamorphic proteins. SignificanceRecent success in the de novo protein design for molecular function is powered by the transformative AI methods and interpreted using the classical wisdom from the Physics-based modelling approaches. Stimuli-sensitive fold switching proteins that can take multiples shapes based on environmental factors are the next frontier in de novo protein engineering. In this work, we unravel the thermodynamic driving forces behind a recently designed temperature-sensitive artificial protein and provide a Physics-based framework to understand the design principles leading to fold switching behavior. Our work compliments the ongoing AI methods that are being explored to unravel the hidden evolutionary embeddings inherent in fold-switching proteins and also highlights the importance of thinking in terms of entropy-based design principles for natural system.

biophysics↗

bioRxiv2025

Palmitoylation of PSD95 flips the bilayer juxtaposed domain and drives cluster formation on membrane

Postsynaptic density protein 95 (PSD95) is a highly abundant scaffolding protein at excitatory synapses, where it organizes the molecular architecture of the postsynaptic density (PSD) by anchoring neurotransmitter receptors, ion channels, adhesion molecules, and signaling enzymes. Emerging evidence indicates that PSD95 forms transient "nanodomains" essential for the fidelity of synaptic transmission and plasticity, and disruptions in its clustering are linked to neuropsychiatric disorders. Palmitoylation, a reversible post-translational modification involving the covalent attachment of palmitate to Cysteine residues, has been implicated in the localization and clustering of the PSD95 at the plasma membrane, but its structural and mechanistic role remains unclear. Here, we use a multiscale molecular dynamics approach that combines all-atom and coarse-grained simulations to investigate how palmitoylation regulates PSD95 conformational dynamics, membrane association, and self-assembly. Our results show that palmitoylation promotes an open conformation in solution and stabilizes an extended assembly-primed state at the membrane. In the unmodified state, the PDZ1 domain remains distal while PDZ2 preferentially engages with the membrane; palmitoylation reverses this arrangement, anchoring PDZ1 to the membrane and enhancing clustering propensity. Membrane lipid composition further modulates this behavior, with negatively charged lipids influencing favorable domains orientation through electrostatic interactions. Finally, coarse-grained simulations reveal that palmitoylated PSD95 forms stable dimers on the membrane surface, whereas unmodified molecules fail to associate persistently. Together, these findings provide a mechanistic model for how palmitoylation facilitates the early stages of PSD95 clustering, offering nanoscale insights into synaptic organization that complement experimental observations. Significance StatementPSD95 is a key scaffolding protein that organizes synaptic components essential for neuronal communication. Its ability to cluster at the membrane is critical for postsynaptic architecture, yet the molecular determinants of this process remain unclear. Using multiscale molecular dynamics simulations we reveal how palmitoylation, a reversible lipid modification, acts as a molecular switch that anchors PSD95 to the membrane and reorients its domains to favor self-association. We further demonstrate that membrane lipid composition modulates these effects through electrostatic interactions. Our work offers a mechanistic model for the initial steps of PSD95 clustering, highlighting how post-translational modifications and membrane environment together regulate synaptic protein organization. These insights bridge molecular structure with synaptic function and are not easily accessible through current experimental methods. Graphical Abstract Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/679381v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1411deaorg.highwire.dtl.DTLVardef@132cddorg.highwire.dtl.DTLVardef@e69aa6org.highwire.dtl.DTLVardef@1b34753_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Deciphering Spatially Resolved Pathway Heterogeneity in Ovarian Cancer Post-Neoadjuvant Chemotherapy

High-grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of ovarian cancer, characterized by high recurrence rates and limited treatment options following chemotherapy resistance. Its significant heterogeneity poses major challenges for effective therapy and clinical outcomes. In this study, we present a systems-level analysis of spatial transcriptomics data to characterize tumor heterogeneity in post-neoadjuvant chemotherapy HGSOC patients. By integrating gene expression profiles with spatial localization and histological context, we quantified hallmark pathway activities across tissue regions. The computed pathway scores were then used for clustering to investigate intra-tumoral heterogeneity. We also constructed gene co-expression network within tumor-enriched regions. Finally, we examined the association of these co-expressed modules with treatment response. Clustering based on pathway activity scores revealed spatially distinct regions enriched for different hallmark pathways, uncovering functionally diverse cellular subpopulations within the tumor microenvironment. Tumor cell-enriched clusters show difference in pathways related to proliferation, metabolism, immune signaling and stress response, while fibroblast-rich regions exhibit upregulation of epithelial-mesenchymal transition (EMT). Unsupervised co-expression analysis further revealed gene modules associated with both biological processes and clinical phenotypes. Poor responders exhibit higher expression of gene modules involved in stress response, ribosomal function, oxidative phosphorylation, and cell-cycle regulation. In contrast, good responders show elevated activity in modules enriched for immune activation, extracellular matrix (ECM) remodeling, and inflammatory signaling. Our findings provide insights into spatially resolved functional states, tumor heterogeneity, and molecular features associated with treatment response, offering a foundation for precision oncology approaches in ovarian cancer.

systems biology↗

bioRxiv2025

Lipid Dynamics and Organization Around Voltage-Gated Sodium Channels: A Coarse-Grained Perspective

Lipid-ion channel interactions play a critical role in channel function and membrane structural organization. Despite this importance, the mechanisms behind the rearrangement of lipids around ion channels are still unclear. To investigate this, we conducted coarse grained (CG) molecular dynamics simulations of voltage gated sodium ion channels (NavAB) embedded in a ternary lipid bilayer composed of 1,2-dilinoleoyl-sn- glycero-3-phosphocholine (DIPC), 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC), and cholesterol (CHOL) at varying CHOL concentrations (6.62%, 17.62% and 30.00%). By analyzing lipid organization and membrane structure, we examined how membrane composition and channel state (activated and resting) influence lipid redistribution near the channel interface. Our key finding is a pronounced preference for DIPC for the channel vicinity, observed consistently for all CHOL concentrations and channel states. Our simulations reveal that hydrophobic mismatch dictates lipid sorting near NavABs. The hydrphobic thickness of the channel favors flexible DIPC lipids, which are packed efficiently around it, while excluding thicker DPPC lipids. This exclusion drives DPPC and cholesterol to form ordered domains farther from the channel interface. Mixing entropy analysis supports local lipid de-mixing near the channel, aligning with the emergence of phase-separated domains. Notably, the hydrophobic thickness of NavAB remained stable and in close agreement with the experimental values, indicating that lipid-specific properties drive reorganization near the channel. Overall, our findings demonstrate that hydrophobic mismatch is a key driver of lipid reorganization and domain formation around ion channels, regardless of CHOL concentration or channel conformational state.

biophysics↗

bioRxiv2025

Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

Cancer dissemination by perineural invasion (PNI) is associated with poor outcomes in epithelial malignancies, yet the cellular interactions critical to this process are not fully defined. Single-cell transcriptomic analysis of cutaneous squamous cell carcinoma (cSCC) with PNI highlighted KITLG-KIT signaling activation between keratinocytes and mast cells. Additionally, we observed increased expression of HRH1 on tumor cells, which encodes the H1 histamine receptor. We confirmed these changes in other cancers, including PNI-positive hepatobiliary and oral mucosal tumors. Histamine enhanced tumor invasion into nerve in organoids that recapitulate key features of PNI, an effect rescued by H1-antihistamines. Mechanistically, broad upregulation of matrix metalloproteinases (MMPs) through P38 activation underscored histamine-mediated PNI by enabling collagen degradation in the nerve sheath. Examination of multiple cancer types with PNI showed co-expression of HRH1 and MMP2 on tumor cells as well as type IV collagen loss in involved nerves. Retrospective analysis of >15,000 patient health records in two independent databases demonstrated that H1-antihistamine use improved overall survival and immunotherapy response in cancers that spread by PNI. Together, these data reveal a critical role for mast cells in PNI and present a new therapeutic strategy for targeting this process in epithelial cancers.

cancer biology↗

bioRxiv2025

Comparing PAS domain coupled intrinsic dynamics in bHLH PAS domain transcription factor complexes

The basic Helix-Loop-Helix-Per-Arnt-Sim (bHLH-PAS) transcription factors (TFs) are regulators of several critical cellular functions such as circadian rhythm, hypoxia response and neuronal development. These proteins contain tandemly repeated PAS domains that mediate heterodimer formation. While PAS domains adopt a conserved fold, recent studies suggest that their interaction interfaces differ distinctly in different TF complexes. However, the implications of these differences on the intrinsic dynamics of PAS domains remain unclear. In this study, we performed a comparative analysis of PAS domain dynamics across multiple bHLH-PAS TF complexes using all-atom Elastic Network Models (ENMs) and molecular dynamics (MD) simulations. We decomposed the intrinsic dynamics of PAS domains into self-coupled (internal domain dynamics) and directly coupled (interaction partner-influenced dynamics) motions using a projection-based approach. Our results show that self-coupled motions are more conserved across PAS domains than structure or sequence alone, while directly coupled motions capture the context-specific influence of partner proteins. Furthermore, hierarchical clustering of the overall covariance-based similarity scores revealed distinct grouping of CLOCK:BMAL1-type and HIF:ARNT-type complexes, which were not captured by sequence or structural comparisons. Root mean square fluctuation profiles derived from both MD and ENM approaches showed strong correspondence, validating the utility of ENMs in capturing biologically relevant dynamics, even in cases where the structural complexes were modelled using AlphaFold3. PAS-B domains were generally found to be less flexible than PAS-A domains for all the complexes analysed. Regions with high directly coupled flexibility were generally localized regions with high interface propensity in class I PAS-B domains, suggesting a higher level of coupled dynamics between PAS-B domains. Our results highlight how PAS domain intrinsic dynamics are shaped by both their internal architecture and complex-specific interactions, offering new insights into the functional diversification of bHLH-PAS transcription factors. Statement of SignificancePAS domains are ubiquitous across all domains of life with diverse functions attributed to them. As part of bHLH-PAS transcription factors (TFs), they enable dimerization of Class I and Class II TFs. In this work, we investigated the effect of dimerization of PAS domains on their flexibility by using a method that allows us to isolate the intrinsic dynamics internal to a target domain and the intrinsic dynamics linked to the crosstalk between domains, from the all-atom elastic network model-based normal modes of the whole TF complex. Our findings reveal a context specific conservation of intrinsic dynamics based on the type of heterodimer complex. We also find a strong agreement between more-detailed MD simulations and the coarse-grained method used.

biophysics↗

bioRxiv2025

Division of labor in cargo and membrane recognition by SNX1-SNX5: Insights from multiscale modelling

Sorting Nexins (SNXs) are a large group of diverse cellular trafficking proteins that play essential roles in membrane remodeling and cargo sorting between organelles. SNX proteins comprise a banana-shaped BAR domain that acts as a curvature-inducing scaffold and a phosphoinositide lipids sensitive Phox-Homology Domain (PXD) that interacts with the membrane to ensure specific and efficient organelle binding. In concert with the larger retromer machinery, these proteins traffic and recycle cargo between the endosomal membrane, trans-Golgi network, and plasma membrane. Interestingly, the SNX1-SNX5 heterodimeric construct forms a part of the newly discovered pathway where cargo sorting and membrane remodeling can take place in a retromer-independent fashion. In this work, we use molecular dynamics and continuum mechanics simulations to understand the features of SNX1-SNX5 heterodimer, especially the molecular determinants at PXDs, which impart organelle membrane specificity and retromer-independent cargo recognition ability to these proteins. Our all-atom molecular dynamics simulations with isolated PXDs and full-length SNX1-SNX5 on bilayers show that SNX1-PXD has robust membrane-binding features that are largely insensitive to single or double mutation of the basic residues on its surface. Comparing the simulation-based binding poses against the recently solved cryo-EM structures of tubular membrane-bound SNX1 homodimer and SNX1-SNX5 heterodimer also provided interesting insights into the association profile of isolated PXDs when they have the freedom to explore different membrane-binding poses. Our protein-protein simulations of SNX5-PXD with the tail region of the CI-MPR transmembrane cargo protein using metadynamics simulations reveal aromatic residue-rich{pi} - {pi} interactions between the two proteins, and a favorable and kinetically accessible binding free energy profile for SNX5. To model the emergent behavior of cargo sequestration and endosomal tube formation by SNX1-SNX5 heterodimer, we also performed Dynamically Triangulated Surface (DTS) based mesoscopic simulations by developing an augmented Helfrich-like continuum-mechanics Hamiltonian to incorporate transmembrane proteins in DTS models. SIGNIFICANCESorting Nexins (SNXs) are a conserved family of endosomal coat proteins that drive cargo-rich tubule formation for receptor transport and recycling within the cell. The cargo-recognition and sorting in these pathways are mediated by the associated retromer complex. SNX1-SNX5 heterodimer is unique to the SNXs family as it has been shown to sequester cargo for trafficking in a retromer-independent manner. The canonical membrane adaptor Phox-homology domain of SNX5 has evolved to carry out this special function. Using molecular dynamics and advanced sampling metadynamics calculations as well as Helfrich-like Hamiltonian-based mesoscopic modelling, we explore the molecular and thermodynamic driving forces that makes the SNX1-SNX5 heterodimeric constructs multifunctional in character.

biophysics↗

bioRxiv2025

Gastrointestinal colonization as a source of Staphylococcus aureus in atopic dermatitis

Atopic dermatitis (AD) is a prevalent inflammatory skin disease with complex pathogenesis. Both skin and gut microbiota influence AD, with Staphylococcus aureus, in particular, exacerbating the disease. However, the relationship between S. aureus colonization in the gut and skin, and whether it affects AD, remains unclear. Using a combination of culture-based methods, microbiome analysis, and genome sequencing of S. aureus from multiple body sites of children with and without AD, we found that the gut represents a major reservoir of genetically diverse S. aureus that is transmitted to the skin, including mutants associated with worse disease. We validated this association between S. aureus gastrointestinal colonization and AD in an independent human cohort and demonstrated its direct effect on disease in an infantile AD mouse model, wherein S. aureus gastrointestinal colonization worsened skin inflammation. Overall, this study identifies a previously unrecognized S. aureus reservoir, with implications for microbiota-targeting therapies in AD.

microbiology↗

bioRxiv2025

Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response

The heat shock response (HSR) is the major defense mechanism against proteotoxic stress in the cytosol and nucleus of eukaryotic cells. Initiation and attenuation of the response are mediated by stress-dependent regulation of heat shock transcription factors (HSFs). Saccharomyces cerevisiae encodes a single HSF (Hsf1), facilitating the analysis of HSR regulation. Hsf1 is repressed by Hsp70 chaperones under non-stress conditions, and becomes activated under proteotoxic stress, directly linking protein damage and its repair to the HSR. J-domain proteins (JDPs) are essential for targeting of Hsp70s to their substrates, yet the specific JDP(s) regulating Hsf1 and connecting protein damage to HSR activation remain unclear. Here we show that the yeast nuclear JDP Apj1 primarily controls the attenuation phase of the HSR by promoting Hsf1s displacement from heat shock elements in target DNA. In apj1{Delta} cells, HSR attenuation is significantly impaired. Additionally, yeast cells lacking both Apj1 and the major JDP Ydj1 exhibit increased HSR activation even in non-stress conditions, indicating their distinct regulatory roles. Apj1s role in both nuclear protein quality control and Hsf1 regulation underscores its role in directly linking nuclear proteostasis to HSR regulation. Together these findings establish the nucleus as key stress-sensing signaling hub.

molecular biology↗