Biology subjects

Das, R.

Publications and source records attributed to Das, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Megalin-mediated deterministic pseudotransduction in proximal tubules fundamentally challenges current understanding of AAV kidney transduction

Genetic kidney diseases are promising targets for gene therapy. Emerging AAV capsids that reportedly enable effective gene delivery to proximal tubules (PTs) following intravenous injection have raised hopes for treating diseases affecting renal tubules. Despite these promising developments, the scientific literature contains contradictory and non-reproducible findings, which prompted us to conduct a detailed and rigorous investigation of the mechanisms of AAV vector-mediated PT transduction. Here we provide compelling evidence that previously reported effective PT transduction following intravenous AAV-GFP or AAV-mCherry administration largely reflects megalin-mediated deterministic pseudotransduction rather than stochastic vector-mediated gene delivery. We demonstrate that this process is mediated by megalin-dependent uptake of circulating reporter proteins released from transduced organs including the liver and filtered through the glomeruli. These observations highlight the ongoing challenge of achieving true proximal tubule transduction via systemic AAV delivery and caution against relying on small reporters like GFP or mCherry, which can produce misleading signals.

bioengineering↗

bioRxiv2026

Topoisomerase IIβ Kinase: A protein processed from HIV-1 envelope by cysteine proteases

Topoisomerase II{beta} (TopoII{beta}) is activated by TopoII{beta} kinase (TopoII{beta}K) during HIV-1 infection. This study demonstrates that TopoII{beta}K is produced within 15-30 minutes post-infection and is conserved across the major HIV-1 subtypes (A, B, C, and D), although its formation kinetics vary among subtypes. Immunological mapping using specific anti-gp120 antibodies suggests that TopoII{beta}K is derived from the N-terminal region of gp120. Notably, TopoII{beta}K co-localizes with endolysosomes shortly after viral entry. Inhibition of cysteine proteases using E64d and Z-FY-CHO abrogates TopoII{beta}K production in both HIV-1-infected and envelope-transfected cells. These findings suggest that TopoII{beta}K is a conserved polypeptide product of gp120 processing within endolysosomes during the early stages of HIV-1 infection, highlighting a potentially conserved mechanism that regulates early viral infection across major HIV-1 subtypes.

molecular biology↗

bioRxiv2026

Neurogenic colonic motility patterns and gut transit disrupted in a mouse model of Rett Syndrome

Gastrointestinal dysfunction is a prominent feature of neurodevelopmental disorders and contributes significantly to visceral discomfort, but the specific etiology is unclear. In particular, it is unknown which cell types within central, autonomic or enteric nervous systems, gut muscle, epithelium or immune system are the principal drivers of gut dysfunction. We report that the Mecp2 mouse model of Rett syndrome, a neurodevelopmental disorder with profound gut dysmotility, exhibits impaired whole gut transit times and constipation in vivo as well as striking alterations to propagating neurogenic contractions in ex vivo colons. Selective elimination of Mecp2 in neurons, but not glia or smooth muscle, recapitulates these dysmotility phenotypes. Bulk RNA sequencing of colonic external muscle, containing the myenteric compartment of the enteric nervous system, showed significant expression changes in specific neurochemical signaling pathways. Pharmacological modulation of one, the M1 acetylcholine receptor, modified the aberrant neurogenic contractile phenotype in mutant mice and reproduced it in wild-type mice. These results identify disruptions to neuronal muscarinic signaling in the enteric nervous system in a mouse model of Rett syndrome and provide an experimental strategy to identify translationally relevant cellular and molecular pathways underlying gastrointestinal dysmotility in neurodevelopmental disorders.

physiology↗

bioRxiv2026

FAST-MaP: Chemical Mapping of RNA Structures Using Primer-less Sequencing

RNA structure governs the function of non-coding RNAs and influences mRNA stability and translation, making testing of models of experimental RNA structure relevant to fields spanning structural biology, virology, and molecular therapeutics. Here we present FAST-MaP (Fast and Accessible Sequencing Technology for Mutational Profiling), a protocol that enables per-nucleotide RNA structure characterization using standard molecular biology equipment and requiring no sequencing infrastructure or bioinformatics expertise. RNA is chemically modified with orthogonal probes (2A3 and DMS), reverse-transcribed, and PCR-amplified to produce dsDNA amplicons that are submitted directly to a commercial primer-less sequencing service. Returned FASTQ files are processed through a freely accessible web server to generate normalized reactivity profiles within minutes. The complete protocol, from DNA template to structural data, can be completed in approximately one week. We illustrate the workflow on a 659-nucleotide RNA, demonstrating how to test structure preservation across buffers, and how to test specific secondary and tertiary structure predictions of the RNA from computational modeling or cryo-electron microscopy. The protocol requires only standard molecular-biology skills and does not require sequencing or bioinformatics expertise.

biochemistry↗

bioRxiv2026

Shear effects in active models of normal and cancer cells

Mechanical properties of biological tissues, driven by passive and active forces, play a vital role in several processes ranging from development to cancer metastasis. However, the dynamical responses of cells in tissues, subject to mechanical deformations such as shear and the associated rheological properties, are not well characterized. Here, we use three-dimensional agent-based models for normal and cancer tissues to investigate their responses to simple shear as a function of cell stiffness and stochastic active forces. In the normal epithelium, with uniform strength of active force, the yield stress as a function of shear rate follows the Herschel-Bulkley form over a range of cell volume fraction. Strikingly, the shear rate dependence and the elasticity-dependent changes in the yield stress fall on master curves upon suitable scaling. To model cancer-like behavior, a certain fraction (Np) of cells was chosen to have enhanced activity and decreased stiffness. As Np increases, the extent of collective cell movement decreases, transitioning from affine (collective) to non-affine (individualistic) movement, a finding that is in accord with imaging experiments. Simulations of a model of a stiff solid tumor, with radius Rs embedded in normal tissue, show that as Rs increases, the yield stress increases. Interestingly, the cells migrate collectively as Rs increases. A Gaussian Mixture Model (GMM) and a mean field theory quantitatively account for the simulation as well as experimental results on cancerous, non-cancerous, and a mixture of these two types. The combined theoretical and experimental study establishes that heterogeneity in stiffness and activity determines non-affine movements in normal and cancer tissues.

biophysics↗

bioRxiv2026

Loss of PTPRB function remodels VEGFR1 activation in tumors overexpressing the receptor tyrosine kinase

The VEGF Receptor-1 (VEGFR1) is a deceptive receptor tyrosine kinase (RTK). In early embryonic development, VEGFR1 negatively regulates angiogenesis by acting like a decoy receptor. Ligand binding transiently phosphorylates the receptor and induces a weak activation, even at high receptor density. Yet, in multiple cancers, overexpression of VEGFR1 plays a central role in tumor vascularization and growth. Unlike many pro-oncogenic RTKs, extensive patient data analysis revealed no somatic mutation in VEGFR1 that may spontaneously activate the tyrosine kinase. The mechanism by which VEGFR1 is activated in cancers has remained an open question for more than two decades. Here, we evaluated the multi-omics profiles of VEGFR1 and its regulators in a pan-cancer database. We observed an inverse correlation between VEGFR1 and PTPRB phosphatase expression in KIRC patients and disease outcome. We observed that patients overexpressing VEGFR1 and deficient in PTPRB expression have a lower likelihood of survival. Using super-resolution single-cell imaging, we discovered that inhibiting PTPRB spontaneously activates VEGFR1 by inducing ligand-independent dimerization, possibly by shifting the equilibrium toward the active state. PTPRB inhibition induces sustained, ligand-dependent phosphorylation of VEGFR1, which may promote tumor vascularization. We conclude that a subtle phosphatase imbalance is fundamental in determining VEGFR1s role in pathological angiogenesis in tumors.

biochemistry↗

bioRxiv2026

Cryo-EM of a nucleotide-polymerizing ribozyme enables its predictive improvement

Ribozymes capable of self-replication from nucleotides would have been central to the hypothesized RNA World. The leading laboratory models for such molecules were converted from a class I ligase by in vitro evolution but then developed without 3D structures. Here, scaffolded cryo-EM of the substrate-free tC19Z RNA polymerase ribozyme at 3.1 [A] resolution shows how this conversion was achieved. An accessory domain evolved from random sequence grips the ancestral ligase through a loop-loop contact, a seam of magnesium ions, and a six-base stack, and rebuilds the ligases substrate binding site from different residues of its own. A previously unrecognized pairing, present before substrate binds, sequesters the 5' end that must otherwise pair with the template. Compensatory mutations to the ribozyme and template, designed to break this ectopic pairing, increase the extension rate. These results suggest that accelerating RNA structure determination may speed progress toward nucleotide-based self-replication.

biochemistry↗

bioRxiv2026

Type V Collagen Controls Decidual Extracellular Matrix Organization and Angiogenesis During Embryo Implantation

Extracellular matrix (ECM) remodeling and angiogenesis are essential processes underlying endometrial decidualization during embryo implantation. Fibrillar collagens, the major structural components of the ECM, form the architectural framework of the decidua and undergo dynamic reorganization during this process. However, the functional role of type V collagen--a key regulator of collagen fibrillogenesis-- remains undefined. Here, we demonstrate that Col5a1 is highly expressed in decidual stromal and endothelial cells of the mouse uterus. Conditional deletion of Col5a1 leads to progressive uterine hemorrhage beginning at gestation day 8, culminating in complete embryo resorption and pregnancy loss by day 12. Col5a1-deficient decidua exhibits severe structural distortion, marked disorganization of fibrillar collagen, shallow and misdirected embryo invasion, and profound disruption of decidual angiogenesis and vascular network formation. Transcriptomic profiling further reveals distinct gene expression signatures and signaling pathways regulated by COL5A1 in the decidua. Collectively, these findings identify type V collagen as a critical ECM regulator required for maintaining decidual integrity, supporting angiogenesis, and ensuring successful pregnancy.

physiology↗

bioRxiv2026

Advancing Plant Health in Sub-Saharan Africa: Understanding National Agricultural Research Institutions capacities for Strategic Investment Priorities to Boost Genetic Gains Against Crop Biotic Stress

Plant health research within National Agricultural Research and Extension Systems (NARES) represents a critical yet neglected pillar of agricultural resilience in sub-Saharan Africa (SSA), where crop productivity remains substantially below global averages due to persistent biotic and abiotic stresses. Although plant health units have a strategic mandate to support crop protection, disease surveillance, and varietal improvement, the institutional capacity of these units across NARES in SSA is not defined. Here, we assessed 36 plant health units across 26 SSA countries to determine research capacity, identify operational constraints, and examine their contributions to crop improvement and food security. Our findings reveal the existence of a strong human capital operating under structurally constrained systems. More than 65% of personnel possess postgraduate qualifications, indicating the presence of a highly trained scientific workforce with considerable potential to drive plant health innovation. However, this expertise is undermined by severe infrastructural and institutional limitations that restrict research delivery. Access to essential facilities remains low, whereby only 53% of respondents had access to plant pathology laboratories, 43% had molecular biology platforms, and 37% had glass/screenhouses, while only 30% of respondents maintained phenotyping infrastructure such as sick plots or endemic/hot spot disease sites. These deficiencies undermine plant health research in areas like biotic stress resistance screening, pathogen diagnostics, and diversity. It was noted that plant health units primarily target staple food crops central to regional food systems, including cereals (maize, rice, sorghum, and millets), legumes (groundnut and cowpea), and root and tuber crops (cassava and potato). All these crops are affected by diverse biotic stresses that include fungi, bacteria, viruses, insect pests, and parasitic weeds. Yet research effectiveness to address these challenges is further constrained by unclear or fragmented protocols, poorly equipped laboratories, weak data capture, and management systems. Critical gaps were consistently identified in pathogen isolation and characterization, disease phenotyping, surveillance and mapping, experimental design, and data management. Workforce composition raises additional concerns regarding long-term system sustainability. Women account for only 18% of plant health personnel, while researchers younger than 35 years represent just 10% of the workforce, reflecting weak generational succession and gender inequity. These demographic imbalances constitute an existential threat to institutional continuity and innovation capacity. In summary, closing these gaps will require coordinated investment in infrastructure, technical training, institutional strengthening, and digital modernization. Equally important is the development of inclusive workforce strategies that improve gender representation and strengthen succession pathways. Unlocking the potential of NARES plant health networks is essential for accelerating resilient crop development, strengthening agricultural productivity, and advancing food security across SSA.

pathology↗

bioRxiv2026

Electrostatically Assembled Layer-by-Layer Plasmonic Implant for Combination Therapy against Multidrug-Resistant Chronic Wound Biofilms

Although persistent infection from chronic wound lowers the efficacy of single therapy; But combination therapy with prolonged drug release have shown promising effect, such as elimination of heavy bacterial film and multi/single drug resistance with minimal side effects. One such breakthrough is multilayered scaffold that are chemically and physiologically stable. To achieve this objective, we demonstrated construction of layer-by-layer (LBL) assembly aligned by alternate deposition of (PEI/PSS and PAA/PSS) based on electrostatic force. These films exhibited loading of Ibu and gen which can be altered depending on different parameter such dipping time, pH and number of layers. Briefly, gen was encapsulated onto PPGNRs, as GNRs have good stability and their antibacterial properties can be enhanced by tunning surface upon adding chemical drugs. Such functionalized GNRs showed excellent photo thermal property which assisted controlled release of gen via deconstruction of thin layers, whereas burst release of ibu. Furthermore, complete disruption of bacterial colonies when combined with near infrared irradiation (NIR). The formed LBL endowed the great healing capability, controlled antibiotic release solve the problems of bacterial resistance due to synergistic effect. Taken together, the antibacterial, cytocompatibility, and stimuli responsive characteristics of this robust multilayer assembly can be promising multifunctional drug delivery system in different medical aliments.

microbiology↗

bioRxiv2026

De novo design of RNA pseudoknots with deep learning

RNA design has been hindered by the limited accuracy of 3D structure prediction. Here, we show that intricate RNA structures can be generated with current deep learning tools through accurate de novo design of pseudoknot secondary structures. In an Eterna competition involving 57 pseudoknots, generative AI methods matched experienced human designers in solving most blind challenges, evaluated by single-nucleotide-resolution chemical mapping, compensatory mutagenesis, and cryogenic electron microscopy. AI-generated molecules with accurate secondary structures formed well-ordered 3D folds stabilized by noncanonical tertiary interactions not modeled during design. Success was guided by an RNet foundation model trained on prior chemical mapping data, suggesting that some difficult RNA design tasks may be tractable without first solving RNA 3D structure prediction.

biophysics↗

bioRxiv2026

Virus-host interactions and viral population dynamics across atmospheric cloud events

BackgroundCloud water harbors diverse microbial communities despite its extreme oligotrophic conditions. However, the ecological and evolutionary dynamics of viruses in these transient atmospheric habitats remain poorly understood. Clouds have traditionally been regarded primarily as passive carriers of microorganisms rather than as active ecological environments supporting microbial interactions. In this study, cloud water was sampled at Mount Verde, Cape Verde Islands (744 m a.s.l.). We performed metagenomic analyses of iron-flocculated cloud water alongside genome analyses of a bacterial isolate and metagenome-assembled genomes using established bioinformatic approaches. Viral diversity, virus-host interactions, metabolic functions, genetic adaptations, and viral population dynamics across cloud events were investigated. In addition, UV-B resistance experiments were conducted for a novel cloud-water isolate. ResultsWe isolated 24 cloud water bacteria, including four novel species lineages, and recovered 62 high-quality metagenome-assembled genomes, including 10 novel species lineages. We identified 458 viral operational taxonomic units and 237 virus-host linkages across diverse prokaryotic hosts, revealing active viral predation across diverse bacterial taxa. In addition, CRISPR spacer matches from isolates of novel bacterial lineages such as Deinococcus nubigenus MPC36 were found. Viruses carried genes involved in host adaptation to environmental stressors, including cold-shock response, UV radiation resistance, and osmotic stress. In addition, viral populations exhibited SNP-level microdiversity and shifts in single-nucleotide variant composition across temporally proximate cloud events, indicating rapid population turnover. Experimental characterization of the cloud isolate Curtobacterium nubigenum MPC39 further revealed pronounced resistance to UV-B radiation and the presence of an inducible prophage, Curtobacterium phage vB_CnuS_Cirrus1 assigned to the new viral family Nebulaviridae, which could be validated in transmission electron microscopy. Reconstructed genomes from cloud-associated bacteria encoded carbon monoxide dehydrogenase genes and UV resistance genes, suggesting trace gas metabolism and enhanced UV protection as survival strategies in oligotrophic cloud droplets. In silico replication rates estimated using iRep were consistent with active bacterial replication at the time of sampling. ConclusionsTogether, these findings demonstrate that clouds are not merely passive carriers of microorganisms, but dynamic atmospheric ecosystems in which virus-host interactions shape microbial diversity and contribute to microbial turnover, atmospheric dispersal, and cloud-water biogeochemistry.

microbiology↗

bioRxiv2026

Dysregulation of anti-Ro60 B cell autoreactivity in systemic lupus erythematosus

To understand the dysregulation of autoreactive B cells in SLE, we tracked Ro60-specific cells in seropositive (SP) and seronegative (SN) patients and healthy donors (HD), using flow cytometry and monoclonal antibodies. Consistent with permissive central tolerance, Ro60+ naive B cells were present in all groups with increased anergy in HD. HD and SN SLE also had greatly decreased or absent Ro60+ memory and ASC, which were greatly increased in active SP SLE, thereby indicating defective distal tolerance in the latter group. Notably, Ro60 autoreactivity was strictly purged from naive-derived extra-follicular B cells in HD and SN SLE, but expanded in SP SLE, suggesting the importance of autoreactivity censoring in this pathway. SLE clustering of the distribution of Ro60+ B cells identified disease heterogeneity in tolerance enforcement in SLE. Finally, we demonstrate a much higher degree of polyreactivity against other lupus antigens in SLE Ro60+ naive cells, which is greatly attenuated in memory cells. Our work represents the first systematic study of antigen-specific autoreactive B cells and ASC in SLE. It enhances our understanding of human B cell tolerance and defines new approaches to measuring autoimmune activity in the course of SLE, including the assessment of immune resetting after B cell depletion therapies.

immunology↗

bioRxiv2026

SRSA-VAE: Self-Attention-Based Feature Learning for Single-Cell Multimodal Clustering

Clustering plays a critical role in the analysis of single-cell omics data for identifying cellular heterogeneity and uncovering biological mechanisms. However, the high dimensionality, sparsity, and multimodal nature of single-cell datasets such as single-cell RNA sequencing (scRNA-seq) and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) pose significant challenges for effective feature learning and representation learning. Traditional dimensionality reduction methods often rely on linear transformations and fail to capture complex nonlinear relationships between gene and protein expression profiles. In this work, we propose SRSA-VAE, a scalable variational autoencoder framework that integrates a residual self-attention encoder for context-aware feature learning and multimodal representation learning. The proposed model dynamically contextualizes gene and protein representations through a self-attention mechanism, enabling the encoder to capture inter-cell relationships and emphasize biologically informative signals. A scalable residual connection further stabilizes training and preserves essential input information during latent representation learning. We evaluate SRSA-VAE on five large-scale publicly available single-cell datasets, including both scRNA-seq and CITE-seq data, and compare its performance with established deep generative models. Experimental results demonstrate that SRSA-VAE consistently outperforms existing methods in Adjusted Rand Index (ARI) across benchmark datasets, with particularly strong gains on complex immune cell populations. Ablation studies further confirm the importance of the self-attention mechanism and residual connection in enhancing model stability and clustering accuracy. The proposed model offers a generalizable, robust, and scalable solution for single-cell clustering tasks. Code Repositoryhttps://github.com/rangan2510/srsa-vae

bioinformatics↗

bioRxiv2026

An autologous cell-based therapeutic vaccine expressing IL6/1 fusokine drives robust anti-tumor response against ovarian cancer.

BackgroundCytokines are immunomodulatory proteins that play central roles in regulating immune responses and represent attractive targets for cancer therapy. However, as single agents, cytokines have shown limited clinical benefit due to systemic toxicities and a short in vivo half-life. Our group has focused on engineering fusion cytokines (fusokines) that couple two cytokines into a single biologic to reprogram immune cell responses by enforcing non-canonical receptor engagement and signaling. A chimeric IL-6/IL-1{beta} fusokine was engineered to test the hypothesis that enforced co-engagement of IL-6 and IL-1{beta} signaling pathways would confer a gain-of-function phenotype in T cells and promote robust anti-tumor immunity. Here, we describe the immunomodulatory properties of IL6/1 fusokine and a method to deliver this fusokine to produce inhibition of ovarian tumor growth in a pre-clinical mouse model. MethodsLentiviral vectors encoding murine or human IL6/1 were designed using Vector Builder and expressed in either HEK293, CHO or ID8-F3 (p53-/-) cells depending on the downstream experiment to be conducted. IL6/1 expression was validated by ELISA and flow cytometry. Effects of human IL6/1 (hIL6/1) on T cell function (proliferation, memory phenotype, activation induced apoptosis) were monitored by flow cytometry. For in vivo studies, ID8-F3 murine ovarian cancer cells expressing mouse IL6/1 (mIL6/1) were administered intraperitoneally (I.P.) as a cell-based therapy to C57BL/6 female mice bearing established ID8-F3 luciferase tumors. Tumor progression was monitored by bioluminescence (BLI) imaging, and overall survival was evaluated. ResultshIL6/1 significantly enhanced T cell survival and selectively promoted activation and expansion of CD45RO memory T cells. mIL6/1 expressing ID8-F3 cells (ID8IL6/1) demonstrated stable transduction and sustained cytokine secretion. In vivo, ID8IL6/1 cell therapy significantly reduced tumor growth and improved overall survival compared to control groups, with 2 of 8 mice achieving complete tumor clearance. ConclusionThese findings indicate that IL6/1 fusokine enhances T cell survival and proliferation while promoting memory responses. Engineered cancer cells (ID8-F3) expressing mIL6/1 fusokine induced a strong anti-tumor response when delivered as a therapeutic vaccine in ovarian cancer mouse model. What is already known on this topicO_LIFusokines are a class of bifunctional proteins designed to achieve synergistic immune modulation. Previous studies in our lab have shown fusokine exhibit gain-of-function immunomodulating activity. Individually, IL-6 and IL-1{beta} are recognized for their roles in promoting T-cell proliferation and effector function. However, the potential for a fused IL-6/1 fusokine to reprogram the immune system and elicit a superior anti-tumor response in vivo in ovarian cancer model is not yet studied. C_LI What this study addsO_LIThis study develops a novel fusion cytokine (fusokine), combining IL-6 and IL-1{beta}, and demonstrate robust activation of T cells. In a preclinical ovarian cancer model, engineered cancer cells expressing IL6/1 used as a therapeutic vaccine showed significant tumor reduction and improved overall survival. C_LI How this study might affect research, practice or policyO_LIThis study demonstrates that in comparison to individual cytokines, fusokines have greater potential to activate T cell function and when delivered as a cell therapy, achieve clear therapeutic efficacy in an ovarian cancer model. Further translational and clinical studies may enable the development of novel and more effective fusokine cell therapy approaches for patients with ovarian cancer. C_LI

immunology↗

bioRxiv2026

Gαi2 coordinates neuronal microtubule and neurofilament networks to regulate axon initiation in vivo

Neurons possess a highly polarised morphology, established through axon formation. However, the mechanisms regulating stable axon initiation during embryonic development remain poorly understood. Here, using fixed-tissue super-resolution and high-resolution live-tissue imaging in developing chick spinal cord, we demonstrate that the multifunctional G-protein Gi2 coordinates the interconnected neurofilament and microtubule networks to achieve stable axon outgrowth. Before axon initiation, microtubule network orientation shifts towards the site of the future axon where coiled Gi2-associated neurofilaments accumulate asymmetrically before unfurling into the initiating axon behind microtubules. Crucially, Gi2 is associated with neurofilaments and microtubules at points of contact. Gi2 depletion reduces engagement between these cytoskeletal networks, and leads to impaired passage of neurofilaments into initiating axons and disrupted axon outgrowth, supporting a role for Gi2 in regulating microtubule-driven incorporation of neurofilaments in nascent axons. These findings advance mechanistic understanding of polarity establishment and offer an enriched view of cytoskeletal regulation during axon formation.

developmental biology↗

bioRxiv2026

Ciliary cAMP regulates Shh signal interpretation to drive polarisation of differentiating neurons

Cellular differentiation is characterised by transitions in cell-state through reinterpretation of extracellular signals. However, the mechanisms facilitating context-dependent signal interpretation remain poorly understood. Differentiating neurons remodel a molecularly distinct primary cilium as they switch from canonical to non-canonical Shh signalling. Here, using long-term live-tissue imaging, we demonstrate that the opposing Shh signalling modulators Smo and GPR161 simultaneously accumulate in the remodelled primary cilium. The correct balance of Smo and GPR161 leads to elevated ciliary cAMP levels, which suppresses Gli transcription factor activation and regulates actin dynamics to drive neuron polarisation. Notably, disrupting this balance through Smo hyperactivation or GPR161 depletion results in reduced ciliary cAMP, inappropriate activation of canonical Shh signalling, dysregulated actin dynamics and initiation of multiple unstable axon-like projections. Thus, this study identifies shifts in ciliary cAMP levels as a key regulator of cellular signal interpretation, and links primary cilium-mediated signal transduction to precise control of cytoskeletal organisation.

developmental biology↗

bioRxiv2026

Investigating the activity of Varicella Zoster Virus (VZV) SUMO-targeted Ubiquitin Ligase ORF61

Varicella Zoster Virus (VZV) is a dsDNA virus that infects dermal cells and causes characteristic cutaneous lesions. The virus undergoes neurotropism and later causes secondary cycles of infection. In the host nucleus, Promyelocytic Leukaemia Nuclear Bodies (PML-NBs) spontaneously form around the VZV genome to repress viral gene expression. VZV encodes for a ubiquitin E3 ligase ORF61 to disperse PML-NBs and alleviate repression. ORF61 functions as a ubiquitin E3 ligase with a conserved RING domain at the N-terminal end. It carries three SUMO-interacting motifs (SIMs) that mediate interactions with SUMOylated proteins within PML bodies. The mechanism by which ORF61 disperses PML-NBs is poorly understood. To understand how ORF61 interacts with SUMOylated proteins, we investigated its interaction with SUMO and studied its SUMO-Targeted Ubiquitin Ligase (STUbL) activity. Our studies reveal that ORF61 co-opts the E2D family for ubiquitination activity. A specific network of interactions between the E2 enzyme, ORF61, and Ub facilitates polyubiquitination. ORF61 can synthesize branched polyubiquitin chains of K11, K48, and K63 linkages. The C-terminal SIM in ORF61 is a high-affinity binder of SUMO chains. Utilizing the SIM, ORF61 targets specific lysines on SUMO chains for ubiquitination. These studies provide crucial insights into the functional mechanism of viral STUbL ORF61.

biochemistry↗