Biology subjects

Sharma, D.

Publications and source records attributed to Sharma, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Transmural Single-Nucleus Transcriptomics Nominates Candidate Disease Programs in Human Necrotizing Enterocolitis

Background & Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease of premature infants marked by inflammation, ischemia, mucosal injury, and, in advanced cases, necrosis extending into deeper layers of the intestinal wall. Because NEC affects specialized, interacting cells in a developing intestine, we used single-nucleus transcriptomics to build a transmural, cell-type-resolved map and nominate hypothesis-generating disease programs distinct from maturation. Methods: Full-thickness intestinal specimens from fetal/prenatal controls, acute NEC cases, and infant controls were analyzed by single-nucleus RNA sequencing (snRNA-seq). Fetal and infant samples served as developmental references. Analyses included cell annotation, NEC-versus-infant differential expression, gene ontology (GO), cross-lineage recurrence, cell death signatures, and developmental maturity. Results: We profiled 53,735 nuclei by snRNA-seq and resolved 34 epithelial, stromal, vascular, immune, neuronal, glial, and smooth muscle populations. We used a morphology-based nuclei scoring system for sample selection that predicted high-quality transcript recovery. NEC nuclei were represented across this cellular landscape, with recurrent gene-expression changes suggesting shared inflammatory recruitment, antimicrobial/redox defense, stress adaptation, complement/vascular activation, reduced homeostatic signaling, and cell death-associated regulation. Developmental maturity analysis identified fetal-like expression levels in subsets of NEC-regulated genes. Proliferative crypt populations retained DNA repair/cell-cycle programs with inflammatory, hypoxic, and ER-stress features, while absorptive enterocytes showed reduced digestive, transport, detoxification, and terminal villus-function programs. Secretory-lineage changes implicated goblet mucus-barrier responses with reduced glycosylation, trafficking, and hydration; enteroendocrine changes in epithelial defense, metabolic stress, and neural signaling; and Paneth cell retention of antimicrobial effector transcripts despite altered ER quality control, autophagy, and granule delivery in NEC. Non-epithelial programs implicated stromal Wnt/BMP niche loss, impaired lymphatic drainage/scavenging, macrophage cytokine activation with reduced clearance machinery, neuroglial circuit defects, and altered smooth muscle contraction-relaxation balance. Conclusions: This single nuclear intestinal cell atlas nominates multicellular programs that may govern NEC initiation, progression toward tissue destruction, and resolution toward functional recovery.

pathology↗

bioRxiv2026

To mix or to de-mix?: Cation-π suppresses heterogeneity in local structure and dynamics of heterotypic Condensates

Heterotypic biomolecular condensates can exhibit pronounced spatial and dynamic heterogeneity because chemically distinct components compete between self- and cross-association. Here, we use coarse-grained simulations to investigate how cation-{pi} interactions modulate this competition in condensates formed by peptides with systematic variation of hydrophobicity-to-charge ratios in binary peptide pairs. By independently tuning sequence heterogeneity, peptide composition, and cation-{pi} interaction strength, we establish how an additional short-range interaction reshapes condensate stability, internal organization, and molecular dynamics. In the absence of cation-{pi} interactions, highly heterogeneous peptide pairs form distinct core-shell architectures, with charge- and hydrophobicity-rich sequences preferentially occupying different regions of the condensate. Introducing cation-{pi} interactions progressively enhances phase stability and promotes molecular mixing, with stronger interactions substantially increasing the critical temperature and condensate density. Energetic analysis reveals that cation-{pi} interactions partially compensate for the loss of electrostatic and hydrophobic stabilization, providing an additional and tuneable source of cohesive interactions. This enhanced cross-association also suppresses spatial and dynamic heterogeneity, yielding more homogeneous peptide distributions, slower translational diffusion, and more uniform chain reconfiguration within the dense phase. Collectively, our results demonstrate that cation-{pi} interactions provide a molecular mechanism for coupling condensate stability to spatial organization and internal dynamics, enabling sequence heterogeneity and composition to be translated into distinct condensate states.

biophysics↗

bioRxiv2026

Structural basis of interaction between Pol1 and DnaN from Mycobacterium tuberculosis

DNA polymerases generally interact with the processivity clamp (DnaN/{beta}-clamp) during replication via clamp-binding motifs (CBMs). Since a CBM was not detected in DNA Polymerase I (PolI) from E. coli, it is generally believed that PolI performs its functions in genome replication and repair, independently of the {beta}-clamp. We observe that, while PolI and {beta}-clamp proteins from E. coli do not interact, their homologs from Mycobacterium tuberculosis bind strongly, and the binding site localises to the Klenow fragment of PolI. The cryo-EM structure (4.9 [A]) of the Mtb-PolI-Klenow: Mtb-{beta}-clamp complex, coupled with continuous heterogeneity analysis of the cryo-EM data and binding experiments, showed that the primary CBM in PolI may be defined by the stretch 457QLSLLDD463. The crystal structure (2.9 [A]) of the {beta}-clamp with the CBM peptide revealed specific interactions that stabilise the peptide within the {beta}-clamp's canonical binding site. Phylogenetic analyses across different bacteria showed that the identified CBM is present only in PolI from members of the order Mycobacteriales. Overall, the interaction between PolI with the {beta}-clamp, mediated by a CBM unique to the Mycobacteriales order, highlights the mechanistic distinctiveness of genome replication in Mtb and phylogenetically related organisms, and has important implications for drug discovery.

biochemistry↗

bioRxiv2026

Engineering Heterotypic Biomolecular Condensates with Synthetic Peptides for Controlled Spatial Organization and Liquid-like Nature

Sequence heterogeneity is a defining feature of cellular biomolecular condensates, yet how competing interaction motifs encode their thermodynamic stability, internal organization, and dynamics remains poorly understood. Here, we systematically tune the hydrophobicity mismatch between intrinsically disordered peptide pairs to establish sequence hydrophobicity as a programmable determinant of heterotypic condensate behaviour. We show that heterotypic condensates are thermodynamically more stable than homotypic ones having same average hydrophobicity through the cooperative interplay of short-range hydrophobic and long-range electrostatic interactions. Increasing hydrophobicity mismatch drives a composition-dependent transition from homogeneous condensates to core-shell architectures accompanied by pronounced spatial and dynamical heterogeneity, whereas reducing sequence disparity restores homogeneous organization and nearly uniform dynamics. Our results establish a direct molecular link between sequence chemistry, phase stability, condensate architecture, and transport dynamics, providing predictive design principles for engineering synthetic biomolecular condensates with programmable organization and material properties. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/743201v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@12e7fborg.highwire.dtl.DTLVardef@13c31a0org.highwire.dtl.DTLVardef@de3453org.highwire.dtl.DTLVardef@3d4600_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

An Open-Source End-to-End Pipeline for Large-Scale EEG-Based Brain Age Modelling

Aging affects individuals at varying biological rates, prompting the development of the Brain Age Index (BAI) to quantify neurobiological health relative to chronological age and disease risk. While structural MRI has dominated brain age prediction, its high cost, immobility, and low temporal resolution restrict its clinical scalability and responsiveness to transient neurophysiological changes. Electroencephalography (EEG) offers a highly scalable, portable, and temporally precise alternative capable of capturing dynamic brain states. However, the transition of EEG-based models to clinical biomarkers is impeded by methodological limitations, including small or biased datasets, inconsistent preprocessing pipelines, and a distinct lack of interpretable machine learning approaches. To address these persistent challenges, this paper presents a comprehensive, open-source, end-to-end pipeline for large-scale EEG-based brain age modeling. Developed using the Temple University Hospital EEG Corpus (TUEG) the largest publicly available resting-state EEG dataset. The pipeline encompasses rigorous data engineering, reproducible preprocessing, and robust feature extraction. Following quality control and subject-level dataset partitioning to definitively prevent data leakage, exactly 41,181 recordings were successfully retained. Two independent feature sets were extracted: the Catch22 time-series characteristics and a comprehensive set of spectral, aperiodic, and non-linear dynamics from the CCS toolbox. The methodology evaluates seven regression models, optimized via Optuna for hyperparameter tuning, and integrates SHAP (SHapley Additive exPlanations) for transparent feature importance analysis. By making this infrastructure publicly available, this work lowers the barrier to entry for large-cohort studies, fostering reproducible development and clinical validation of dynamic brain age biomarkers.

neuroscience↗

bioRxiv2026

Snow leopard-mediated apparent competition between wild ungulates and domestic livestock in the Trans-Himalaya

Across High Asia, snow leopards Panthera uncia share habitats with pastoralists and consume both wild and domestic herbivores. Increasing wild prey is vital for predator conservation, but its effect on livestock depredation is unclear: it could decrease depredation through apparent facilitation or increase it through apparent competition. We investigated this association using a 13-year time series dataset from Upper Spiti Landscape in the Indian Trans-Himalaya. Long-term data on elusive carnivores are rare, making this time series especially valuable for testing predator-mediated interactions. We found higher wild ungulate density was accompanied by significantly greater depredation of free-ranging livestock, and this association remained positive after bootstrapping to account for uncertainty in density estimates. These results suggest snow leopard-mediated apparent competition between wild and domestic herbivores. We recommend conservation efforts that increase wild prey must also integrate effective livestock protection and support strategies to enhance coexistence between predators, prey and pastoralists.

ecology↗

bioRxiv2026

Seizure-related gene 6 (SEZ6) encodes a Cancer stem cell-specific proangiogenic molecule: A novel glioma therapeutic target

Cancer stem-like cells (CSCs) not only initiate tumors but also orchestrate angiogenesis through multiple mechanisms, thereby sustaining vascularization and promoting tumor growth. In glioblastoma (GBM; WHO grade IV glioma), the most aggressive adult brain tumor, glioma stem-like cells (GSCs; glioma CSCs) are the main drivers of tumor progression, drug resistance, and recurrence. An interrogated multi-omic secretome analysis identified Wnt-{beta}-catenin signaling-regulated, membrane-localized Seizure Related 6 Homolog (SEZ6) as a patient-derived GSC-specific proangiogenic molecule. Silencing of SEZ6 inhibited the ability of the GSC secretome to induce angiogenic network formation by brain- and lung-derived endothelial cells, but not GSC growth as neurospheres in vitro. SEZ6 silencing also suppressed patient-derived GSC-initiated glioma tumors, resulting in reduced tumor vasculature in an orthotopic mouse model. We also found that SEZ6 induces TGF{beta}-dependent IL-8 expression in endothelial cells to promote angiogenesis. Coimmunoprecipitation and molecular dynamics simulation experiments determined that the Sushi 3 domain of SEZ6 mediates the interaction with TGF{beta} RII to activate the TGF{beta} pathway. Pharmacological inhibition of BACE1 with an in-house-developed small molecule, or blockade of the SEZ6-TGF{beta} RII interaction using a rationally designed peptide, markedly attenuated SEZ6-driven TGF{beta} signaling, and suppressed angiogenesis. Thus, our findings identify SEZ6 as a novel CSC-specific therapeutic target for GBM.

cancer biology↗

bioRxiv2026

PAD4-mediated histone citrullination contributes to enhanced NETosis in type 2 diabetes but not in type 1 diabetes

Neutrophil extracellular traps (NETs) are web-like structures released by activated neutrophils through a process known as NETosis, which help immobilize, trap, and eliminate invading pathogens. While NETs play a critical role in host defense, excessive or dysregulated NET formation can contribute to chronic inflammatory diseases such as diabetes and its complications. Elevated levels of NETs and increased expression of its mediator, protein-arginine deiminase type 4 (PAD4), have been reported in diabetes. However, the underlying molecular mechanisms governing the enhanced NETosis in type 1 (T1D) and type 2 diabetes (T2D) remain unclear. Using mouse models and human patient samples, we show that neutrophils undergo enhanced NETosis in T1D and T2D through distinct pathways. We found enhanced NETosis in T1D occurs in a PAD4-independent manner, driven by robust cytosolic ROS production by NADPH oxidase (NOX). This leads to myeloperoxidase activation and chromatin decondensation. We further confirm the PAD4 independent mechanism in neutrophils from STZ-induced PAD4-/- mice. In contrast, in T2D, neutrophils undergo NOX-independent NETosis, which relies on calcium-mediated mitochondrial ROS production, PAD4 activation, and hyper-histone citrullination. Taken together, our findings reveal previously uncharacterized distinct mechanisms of enhanced NETosis in T1D and T2D.

Molecular Biology↗

bioRxiv2026

TifBERT: a self-supervised foundation model for normalization-robust bulk RNA-seq representation learning

Bulk RNA sequencing remains central to translational genomics, yet foundation-model development has largely focused on single-cell data. Existing transformer approaches for bulk RNA-seq often rely on expression discretization, numerical reconstruction, external gene embeddings, or restricted gene sets, limiting robustness across normalization schemes and cohorts. Here, we introduce TifBERT, a self-supervised framework for full-transcriptome bulk RNA-seq representation learning. TifBERT converts each unordered expression profile into a sample-specific gene sequence using term frequency-inverse document frequency (TF-IDF) ordering, prioritizing genes that are both highly expressed within a sample and selectively expressed across the cohort. It is then pretrained using masked gene modeling, predicting gene identities from transcriptomic context rather than reconstructing expression values. Pretrained on harmonized TCGA Pan-Cancer data spanning five RNA-seq normalization schemes, TifBERT learns contextual representations across approximately 10,000 genes without expression binning, landmark-gene restriction, or external biological embeddings. Across 33 TCGA cancer types, TifBERT achieved 90.83% accuracy, 0.996 macro AUC-ROC, and 0.903 MCC. It also captured pathway-level biology, achieving mean sample-wise and pathway-wise Pearson correlations of 0.754 and 0.762 across 1,387 PARADIGM pathway activities. Independent evaluation on GTEx healthy tissues showed preservation of tissue-level transcriptomic structure without retraining. In comparison with existing models, TifBERT achieves competitive subtype discrimination with substantially greater stability and produces markedly richer embedding geometry (effective rank 95.6 versus 6.3), without requiring expression discretization or in-distribution pretraining exposure. Together, TifBERT provides a scalable, normalization-independent foundation model for reusable bulk transcriptomic representation learning.

bioinformatics↗

bioRxiv2026

LINE-1 Retrotransposon Activation and 5-Gene Dysregulation as a Common Pathological Signature in Post-Traumatic and Status Epilepticus Models of Epilepsy.

The transition from an initial brain insult to chronic epilepsy remains a critical challenge in clinical neuroscience, often involving complex molecular shifts that are difficult to capture through single-gene analysis. This study investigated the existence of a convergent pathway of genomic instability across two distinct etiological models: iron-induced (FeCl3) post-traumatic epilepsy (PTE) and lithium-pilocarpine-induced (LiCl-Pilo) status epilepticus (SE). By integrating Electrophysiology (EEG), {gamma}-oscillation spectral analysis, multi-unit activity (MUA) recordings with behavioural assessments Morris Water Maze (MWM) and Open Field Test (OFT) we validated and established the chronic hyperexcitability and associated cognitive- emotional co-morbidities of these two models. Which further served as a functional backdrop for investigating a targeted 8-gene panel consisting of GABRA2, KCNN2, KCNAB1, GRIK1, BCL11A, BRD7, STX1B, and PNPO. RT-PCR analysis demonstrated a systemic dysregulation of 5 genes out of these 8 genes, mirroring the simultaneous collapse of inhibitory signalling, membrane stability, and neurotransmitter metabolism seen in human phenotypes. While the clinical roles of these genes are well documented, our study demonstrates that these rat models provide a high-fidelity platform for complex multigene studies and gene-level therapeutic exploration. Furthermore, in-silico docking identified Fisetin as a high-affinity ligand for the dysregulated proteins, offering a promising multi-target strategy for stabilizing the epileptic genome.

neuroscience↗

bioRxiv2026

Combinatorial base editing couples disease correction with lineage amplification in hematopoietic stem and progenitor cells

First-generation genome editing therapies have largely focused on correcting or compensating for pathogenic variants. However, as these approaches enter the clinic, emerging biological constraints limit maximal therapeutic impact. Because globin genes are activated late during erythroid differentiation, genome-corrected hematopoietic stem and progenitor cells (HSPCs) gain little selective advantage in the bone marrow. Here, we establish a strategy that links therapeutic genome edits to an erythroid fitness-enhancing allele to amplify the output of clinically relevant cells. We develop a multiplex base editing strategy that couples fetal hemoglobin (HbF) reactivation with erythroid lineage expansion. Introduction of a naturally occurring erythropoietin receptor truncation (tEPOR) associated with benign erythrocytosis increased erythroid cell production without impairing viability or differentiation. Combinatorial editing of tEPOR together with the BCL11A erythroid enhancer and HBG1/2 promoters in healthy donor, sickle cell disease, and {beta}-thalassemia HSPCs synergistically increased erythroid proliferation and HbF expression beyond single base-edited or Casgevy-treated controls. Multiplex base-edited HSPCs retained long-term lineage repopulation and engraftment capacity in vivo, establishing a modular strategy that pairs disease correction with lineage amplification to improve therapeutic potency.

cell biology↗

bioRxiv2026

Small Molecule Regulation of CLOCK:BMAL1 DNA Binding Activity

CLOCK:BMAL1 is a bHLH-PAS transcription factor complex that utilizes its bHLH (basic helix-loop-helix) domains to bind E-box motifs in DNA and tandem PAS (PER-ARNT-SIM) domains to heterodimerize and interact with regulatory proteins to generate circadian rhythms. PAS domains are evolutionarily conserved modules that frequently bind small molecule ligands within buried cavities to perform sensory and signal transduction functions. CLOCK and BMAL1 PAS domains have cavities that could be leveraged to regulate the transcription factor, and consequently, the circadian clock. Using NMR spectroscopy, we identified small molecules that bind within a cavity inside the PAS-A domain of CLOCK and its paralog NPAS2, which sits at an important flexible junction in the structured core of the heterodimer. We identified a gatekeeping mutant in the core of CLOCK PAS-A that significantly decreased ligand binding affinity. High-pressure NMR studies showed that ligand binding or the gatekeeping mutant significantly stabilized the domain. Finally, we showed that ligands induced dose-dependent displacement of CLOCK:BMAL1 from DNA in vitro. Together, these data demonstrate that small molecules can regulate DNA binding by the circadian transcription factor CLOCK:BMAL1 through occupancy of a PAS domain cavity.

biochemistry↗

bioRxiv2026

Aberrant oxidative metabolism selects for TET2-deficient hematopoietic stem and progenitor cells

The mechanism(s) driving selective expansion of mutant hematopoietic stem and progenitor cells (HSPC) in clonal hematopoiesis (CH) are incompletely understood. Here, we address the role of metabolism in selection for HSPC with loss of function mutations in TET2. Loss of Tet2 in murine HSPC triggers overexpression of glycolysis and oxidative phosphorylation genes and increased oxidative metabolism via an enlarged mitochondrial network. However, Tet2-deficient HSPC maintain a normal redox state. Strikingly, compound loss of the rate-limiting pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) triggers increased reactive oxygen species and impairs the fitness of Tet2-deficient HSPC. We find that aberrant oxidative metabolism is also a feature of HSPC in human CH and clonal cytopenia of unknown significance (CCUS). Overall, our data point to aberrant metabolism as a critical and conserved driver of selection in TET2-deficient CH and identify the PPP as a crucial compensatory pathway needed to maintain their selective advantage. Statement of SignificanceThis study identifies oxidative metabolism as a critical driver of selection for TET2-deficient HSPC in clonal hematopoiesis (CH). It also demonstrates that cellular redox state is a vulnerability that impairs their fitness. These insights establish targetable metabolic pathway(s) that could be exploited in the setting of TET2 mutant CH.

cancer biology↗

bioRxiv2026

In silico transcriptomic analysis reveals shared molecular signatures and immune-associated pathways between Hashimotos thyroiditis and type 2 diabetes with exploratory drug repurposing

The management of Hashimotos thyroiditis (HT), one of the most prevalent autoimmune disorders worldwide, becomes more complex when it coexists with type 2 diabetes (T2D) compared with the management of either disease alone. This complexity may arise from overlapping genetic, metabolic, and immune dysregulation, as well as potential therapeutic conflicts. Although HT and T2D are known to co-occur and share immune and metabolic features, the molecular characteristics underlying these overlaps have not been systematically explored. This study aimed to identify shared gene expression signatures and associated biological pathways between HT and T2D using an in silico, hypothesis-generating approach, and to explore candidate compounds that may be relevant to both conditions. Independent transcriptomic datasets (GSE138198 for control/HT and GSE29231 for control/T2D) were analyzed, leading to the identification of 59 genes that were differentially expressed in both HT and T2D compared with control samples. Protein-protein interaction (PPI) network analysis prioritized five shared key genes (sKGs): CDC42, CD74, FOS, RAC2, and YWHAB. Functional enrichment analysis of these sKGs revealed overlapping biological processes, molecular functions, cellular components, and immune-related signaling pathways, as well as shared regulatory networks involving transcription factors (FOXC1 and HNF4A) and microRNAs (hsa-miR-221-3p and hsa-miR-29a-3p). Immune infiltration analysis demonstrated broadly similar patterns of immune dysregulation in both diseases, providing additional biological context for the observed shared molecular signatures. Finally, an exploratory in silico drug repurposing pipeline incorporating molecular docking, ADMET profiling, drug-likeness assessment, and molecular dynamics simulations prioritized three candidate compounds: gliquidone, oleanolic acid, and glipizide for further investigation. Overall, this study provides a hypothesis-generating framework highlighting shared molecular features between HT and T2D, which may inform future experimental validation and clinical research. Author SummaryIn this work, we wanted to better understand why Hashimotos thyroiditis, an autoimmune condition that affects the thyroid gland, is often seen in people who also have type 2 diabetes. Treating patients who live with both conditions can be difficult, and we were interested in finding out whether they share common biological causes. To do this, we examined genetic data from individuals with each disease and looked for patterns that appeared in both groups. We discovered several genes that seem to act in similar ways in the two conditions, particularly genes linked to immune system activity and associated pathways. This finding suggests that shared molecular signatures and immune-associated pathways may play a role in the development of both diseases. We also explored how these shared genetic features influence larger biological processes and immune responses. The similarities we found support the idea that the two diseases may be connected through related biological pathways. In addition, we used computer-based screening methods to identify existing drugs that might influence these shared pathways. While these results need further testing, we hope our findings help open new directions for research and eventually contribute to better care for patients affected by both conditions.

bioinformatics↗

bioRxiv2026

Cellular mechanisms of acquired drug resistance against regorafenib in colon cancer cells.

Colorectal cancer is the third most common cancer across the world. Acquired resistance to therapeutics is one of the major challenges in cancer cure. With the development of resistance to organometallic drugs there was switch for the usage of inhibitors of kinases which play a crucial role in cellular activities. Regorafenib is a multi-kinase inhibitor used as an oral anti-cancer drug for treating advanced colorectal cancer (CRC). With increasing reports of acquired resistance to regorafenib in long-term use it is inevitable to understand the mechanisms underlying in the development of resistance. To understand the molecular mechanism of acquired drug resistance towards regorafenib we have developed a regorafenib resistant HCT116 cell line (Reg-R-HCT116) and an integrated quantitative proteomic and phospho-proteomics approach is used to elucidate the molecular signaling mechanisms that help in drug tolerance. Proteome and Phosphoproteome analysis revealed an extensive remodelling of signaling pathways associated with metabolism, protein synthesis and stress adaptations. This also revealed a large set of phosphorylated proteins as well as proteins that might be associated with aberrant activation or differential alteration of PI3K-AKT-mTOR, EIF2, HIF-1, Apoptosis inhibition, Glucose metabolism, amino acid metabolism and DNA-repair-associated signaling. Differential phosphorylation of downstream molecules of the mentioned pathways NDRG1, ACINUS and RICTOR and enhanced cell survival further confirmed their role in drug tolerance. Targeted inhibition of both the mTOR complexes using Torin1 resensitized the cells to regorafenib. Combination treatment of regorafenib with torin1 showed a synergistic cytotoxicity and attenuated the expression of key survival proteins. These findings provide mechanistic insight into acquired resistance to regorafenib in colorectal cancer and identified mTOR/eIF2 signaling as one of the critical drivers of resistance phenotype. The results suggest combinatorial targeting of these pathways could be an effective strategy to overcome regorafenib resistance and improve clinical outcomes in CRC.

cancer biology↗

bioRxiv2026

Dual Effect of RNA-like Polyelectrolytes on Stability and Dynamics of Biomolecular Condensates: A Tale of Competitive Short and Long-range Interactions

Heterotypic biomolecular condensates underpin the spatiotemporal organization of cellular components and enable precise biological regulation. These condensates frequently comprise proteins and RNA at variable stoichiometries, with RNA acting as a key modulator through its ability to engage in both long-range electrostatic and short-range specific interactions. How such RNA-like components regulate condensate stability and dynamics across distinct interaction regimes, however, remains unclear. Here, we examine the role of RNA-like polyelectrolytes in tuning the stability and material properties of heterotypic condensates formed by designed short peptide sequences spanning a continuum from long-range electrostatics-to short-range hydrophobic interactions. By systematically strengthening short-range hydrophobic interactions while varying polyelectrolyte composition, we uncover a dual, regime-dependent role of RNA-like species. In electrostatics-dominated condensates, excessive polyelectrolyte concentration rapidly destabilizes droplets due to enhanced long-range repulsive interactions while the droplets retain stability until intermediate polyelectrolyte concentration fairly well. In contrast, in strongly hydrophobic condensates, polyelectrolytes function as multivalent sticker hubs, stabilizing condensates through favourable peptide- polyelectrolyte interactions. Polyelectrolyte enrichment within condensates is nonlinear with respect to mixing fraction and saturates at largest cluster mole fractions of [~]0.2-0.25. Condensate dynamics reflect this interplay: polyelectrolyte diffusivity is 20-40% higher in electrostatics dominated systems than in hydrophobic ones, while in extremely hydrophobic condensates, polyelectrolytes diffuse [~]50% more slowly than peptides, indicative of scaffold-like behaviour. Together, these results reveal tuneable and opposing roles of RNA-like polyelectrolytes in shaping condensate stability, dynamics, and morphology across diverse interaction regimes.

biophysics↗

bioRxiv2025

Ewing Sarcoma Breakpoint Region 1 (EWSR1) protein binds to the non-structural protein-2 and the non-coding regions of the Chikungunya virus genome to inhibit the virus replication

Chikungunya virus (CHIKV) is an alphavirus with a single-stranded RNA genome. Conserved sequence elements within the 5'- and 3'-non-coding regions (NCRs) of the CHIKV genome interact with host proteins to regulate viral RNA replication. Electrophoretic mobility shift assays (EMSA) and mass spectrometry identified several host RNA-binding proteins that associate with these conserved NCR elements, among which EWSR1 uniquely bound the 3'-ends of both the plus-sense genomic RNA and the minus-sense replication intermediate RNA. EMSA and biolayer interferometry demonstrated that EWSR1 binds strongly to two RNA elements: a 48-nucleotide region in the plus-strand 3'-NCR (3NCR48) and a 50-nucleotide region at the 3'-end of the minus-strand replication intermediate (5NCR-RC50), which is complementary to the genomes 5'-NCR and its adjacent sequence. RNA immunoprecipitation from CHIKV-infected cells confirmed that EWSR1 binds CHIKV RNA in vivo. Functional analyses revealed that CHIKV infection downregulates EWSR1 expression and causes its cytoplasmic accumulation. Knockdown or knockout of EWSR1 expression increased CHIKV genomic RNA levels and viral titers, whereas its overexpression suppressed both. EWSR1 reduced the synthesis of both plus- and minus-sense CHIKV RNAs in infected cells. Additionally, EWSR1 was found to bind CHIKV nsP2 and inhibit its helicase activity, which is critical for viral genome replication. The antiviral activity of EWSR1 extended to Ross River virus but not to Sindbis virus, consistent with conservation of the 48-nucleotide sequence in their respective 3'-NCRs. Together, these findings identify EWSR1 as a novel host restriction factor that limits CHIKV replication through specific interactions with viral RNA and the nsP2 protein. IMPORTANCEChikungunya virus (CHIKV) poses a significant medical challenge, necessitating the development of novel antiviral strategies. This study demonstrates that the host protein EWSR1 plays a key role in controlling CHIKV replication. By binding to specific regions of viral RNA and interacting with nsP2, a critical viral enzyme, EWSR1 inhibits viral RNA synthesis and reduces viral titers. In the absence of EWSR1, CHIKV replicates more efficiently, underscoring its antiviral function. These findings establish EWSR1 as a novel host restriction factor that limits CHIKV replication. Understanding this interaction enhances our knowledge of host-virus dynamics and may guide the design of new antiviral approaches against CHIKV and related alphaviruses.

microbiology↗

bioRxiv2025

The Dynein-Dynactin complex influences amnioserosa cell morphodynamics and adhesion during Drosophila dorsal closure

The contraction of the amnioserosa, which provides the major force for Drosophila dorsal closure, is powered by apical constriction. While much work has demonstrated the requirement for the spatiotemporal regulation of intracellular forces generated by the actomyosin cytoskeleton in patterning apical constriction, the role of the microtubule cytoskeleton remains poorly understood. We previously uncovered the necessity of apical microtubule meshwork reorganisation in patterning apical constriction dynamics, and demonstrated its reliance on EB1-dependent microtubule growth and Patronin-dependent non-centrosomal nucleation. Here we discover the requirement for Dynein-Dynactin complex functions in patterning cell morphodynamics in the amnioserosa. We show that perturbing this complex in multiple ways, including the use of a mutant that likely disrupts microtubule anchorage rather than transport, alters apical constriction morphodynamics and modulates adherens junction remodelling during amnioserosa cell delamination. The defects we observe are additionally sensitive to ECadherin levels and lead to compromised amnioserosa integrity and adverse dorsal closure outcomes. We suggest that the Dynein-Dynactin complex influences cell morphodynamics in the amnioserosa by enabling the regulation and spatiotemporal coordination of cell adhesion, the mechanistic basis for which remains to be discovered.

cell biology↗