Biology subjects

Vasudevan, A.

Publications and source records attributed to Vasudevan, A..

17 recordsLinked to original sources

bioRxiv2026

Lipid nanoparticle induced model endosomal membrane disruption

Endosomal escape remains the central bottleneck limiting lipid nanoparticle (LNP)-mediated mRNA delivery. In this study, we use giant unilamellar vesicles (GUVs) mimicking the endosomal membrane to directly visualize the interaction and membrane disruptions caused by two model LNPs constituted by two different ionizable lipids: LP01 and BiP-20 (a Branched ionizable Phospholipid) that are both benchmark ionizable lipids for in vivo gene editing in liver. Despite identical TNS-measured apparent pKa (6.6) for both lipids in our conditions, LP01 LNP showed maximal membrane engagement at pH 5.8, while BiP20 LNP required pH 5.1, demonstrating that bulk pKa alone cannot predict the pH at which functional membrane interaction occurs. Using confocal microscopy, we show that the LNP engagement with the endosomal membrane proceeds through first electrostatic attraction, clustering, membrane disruption by forming highly curved morphologies, and finally leading to the rupture of the whole endosomal mimicking membrane. Notably, the mRNA cargo is released only upon complete lysis of the membrane. This indicates that the membrane destabilization and cargo release are mechanistically distinct steps. These findings establish a cell-free platform for dissecting individual sub-steps of endosomal escape.

biophysics↗

bioRxiv2026

Leaf and cluster spectral signatures reveal trait-dependent prediction performance for grapevine cluster architecture and juice quality

Grapevine cluster architecture is a key selection target in breeding programs because it influences disease susceptibility, yield stability and juice quality. High-throughput phenotyping offers a rapid and non-destructive approach to capture biochemical and structural variation in these traits, yet the influence of plant organ reflectance and data partitioning strategies on trait prediction remains poorly understood. In this study, we evaluated how hyperspectral reflectance from different grapevine organs contributes to the prediction of cluster architecture and juice quality traits in two clonal populations of Riesling and Pinot. Using partial least squares regression (PLSR), we assessed the prediction accuracy of eight cluster architecture and six juice quality traits under two data partitioning strategies. Models based on cluster reflectance outperformed those using dry leaf reflectance for most traits, except for pH. Partitioning the dataset by cluster type increased trait variance and improved predictions for number of berries (R{superscript 2} = 0.53), berry diameter (R{superscript 2} = 0.79), and total acidity (R{superscript 2} = 0.48). Visible, red-edge and NIR spectra were most informative regions to predict the traits studied. Together, our results highlight the importance of organ-specific data and appropriate calibration strategies to improve phenomic models for the development of scalable proxies for grapevine improvement. HighlightSpectral phenomics reveals that prediction accuracy in grapevine depends on organ spectral signatures and traits, with cluster reflectance outperforming leaves, informing new phenotyping strategies for breeding improvement.

plant biology↗

bioRxiv2025

OCRL regulates lysosomal function and endolysosomal homeostasis in Drosophila nephrocytes

The OCRL gene encodes a lipid phosphatase that dephosphorylates phosphatidylinositol 4,5 bisphosphate [(PI(4,5)P2]. Mutations in OCRL lead to a rare human genetic disorder, Lowe syndrome (LS) that affects the eye, kidney and brain. OCRL is widely expressed in cells and is localized to multiple organelles, including the plasma membrane, endosomes, Golgi and lysosomes. Although multiple defects in the endo-lysosomal system have been reported in OCRL depleted cells, the primary site of action of OCRL is unclear. Here we present a Drosophila nephrocyte model of LS; depletion of Drosophila OCRL (dOCRL) manifests with defects in endocytic uptake, altered endosomal compartments as well as expanded but dysfunctional lysosomes. Reconstitution of dOCRL depleted nephrocytes with a lysosome targeted version of the enzyme rescues not only the lysosomal defects but surprisingly also defects in endosomal structure and function. These findings suggest that the primary defect in LS cells is likely to be altered lysosome structure and function. Therefore, regulation of PI(4,5)P2 homeostasis at the lysosome membrane by OCRL is critical to homeostasis of the endosomal system.

cell biology↗

bioRxiv2025

On-target toxicity limits the efficacy of CDK11 inhibition against cancers with 1p36 deletions

The cyclin-dependent kinase CDK11 is an understudied kinase that has been the subject of conflicting reports regarding its function in cancer. Here, we combine genetic and pharmacological approaches to demonstrate that CDK11 is a critical regulator of cancer cell survival that is required for RNA splicing and the expression of homologous recombination genes. Inhibition of CDK11 disrupts genome stability, promotes the retention of intronic sequences in mature mRNAs, and induces synthetic lethality with PARP inhibitors. Through integrative analysis of functional genomics datasets, we identify heterozygous deletions of chromosome 1p36 - which encompasses CDK11 and its activating cyclin CCNL2 - as a recurrent and predictive biomarker of sensitivity to CDK11 inhibition. To assess the therapeutic potential of CDK11, we develop MEL-495R, a selective and orally bioavailable CDK11 inhibitor. Additionally, we establish a genetically-engineered mouse model that allows us to differentiate between the on-target and off-target effects of CDK11 inhibitors in vivo. Using this platform, we demonstrate that MEL-495R induces widespread on-target toxicity, revealing a narrow therapeutic index. Together, these findings define CDK11 as a core cancer dependency, uncover a chromosomal deletion that sensitizes tumors to CDK11 inhibition, and provide a generalizable strategy for deconvolving drug efficacy and toxicity in vivo for novel oncology targets.

cancer biology↗

bioRxiv2025

Proteome-Wide Discovery of Degradable Proteins Using Bifunctional Molecules

Targeted protein degradation (TPD) is an emergent therapeutic strategy with the potential to circumvent challenges associated with targets unamenable to conventional pharmacological inhibition. Among TPD approaches, Proteolysis Targeting Chimeras (PROTACs) have shown marked advancement with numerous candidates in clinical development. Despite their potential, most PROTACs utilize advanced small molecule inhibitors, inherently limiting the scope of this approach. More generally, the fraction of the proteome tractable to PROTAC-type strategies is unknown. Here, we describe a chemical proteomic strategy for the agnostic discovery of degradable human proteins in cells using a new class of bifunctional degrader molecules called "AgnoTACs". Proteome-wide screening of 72 AgnoTACs in human cells uncovered downregulation events spanning >50 functionally and structurally diverse proteins, most of which lack chemical probes. Our findings highlight the potential of function-biased chemical libraries coupled with proteomic profiling to discover degrader starting points as well as furnish a blueprint for expanding our understanding of the chemically degradable proteome.

biochemistry↗

bioRxiv2025

N2B27 media formulations influence gastruloid development

Gastruloids are 3D aggregates of pluripotent stem cells grown in suspension culture that mimic many aspects of gastrulation and early axial elongation. The N2B27 basal medium in which mouse gastruloids are cultured can either be home-made (HM-N2B27) with materials of known origin, or commercially sourced (NDiff227), where the exact formulation is unknown. In this study we examined whether these formulations resulted in significant differences in gastruloid development. Our results reveal that while both media enable the standard gastruloid elongation, HM-N2B27 results in gastruloids that start the elongation process earlier, have higher number of cells and an increased anterior domain. RNAseq analysis showed significant differences in cell fate specification, with HM-N2B27 gastruloids exhibiting higher expression of spinal cord-related genes, while NDiff227 favours mesodermal differentiation. Furthermore, differential gene enrichment analysis suggests that changes in key signalling pathways underline the differences between HM-N2B27 and NDiff227 gastruloids. These findings highlight the importance of basal media composition for gastruloid development, underscoring the need for careful media selection during in vitro engineering of stem cell-based embryo models. Summary statementIn this work we explored the cellular and molecular sensitiveness of the mouse gastruloid model system to variations in N2B27 media formulations.

developmental biology↗

bioRxiv2024

An in-cell approach to evaluate E3 ligases for use in targeted protein degradation.

One of the major challenges in evaluating the suitability of potential [~]700 E3 ligases for target protein degradation (TPD) is the lack of binders specific to each E3 ligase. Here we apply genetic code expansion (GCE) to encode a tetrazine-containing non-canonical amino acid (Tet-ncAA) site-specifically into the E3 ligase, which can be conjugated with strained trans-cyclooctene (sTCO) tethered to a neo-substrate protein binder by click chemistry within living cells. The resulting E3 ligase minimally modified and functionalized in an E3-ligand free (ELF) manner, can be evaluated for TPD of the neo-substrate. We demonstrate that CRBN encoded with clickable Tet-ncAA, either in the known immunomodulatory drug (IMiD)-binding pocket or across surface, can be covalently tethered to sTCO-linker-JQ1 and recruit BRD2/4 for CRBN mediated degradation, indicating the high plasticity of CRBN for TPD. The degradation efficiency is dependent on location of the Tet-ncAA encoding on CRBN as well as the length of the linker, showing the capability of this approach to map the surface of E3 ligase for identifying optimal TPD pockets. This ELF-degrader approach has the advantages of not only maintaining the native state of E3 ligase, but also allowing the interrogation of E3 ligases and target protein partners under intracellular conditions and can be applied to any known E3 ligase.

cell biology↗

bioRxiv2024

Harnessing the FBXW7 somatic mutant R465C for targeted protein degradation

Targeted protein degradation (TPD) is a pharmacological strategy that eliminates specific proteins from cells by harnessing cellular proteolytic degradation machinery. In proteasome-dependent TPD, expanding the repertoire of E3 ligases compatible with this approach could enhance the applicability of this strategy across various biological contexts. In this study, we discovered that a somatic mutant of FBXW7, R465C, can be exploited by heterobifunctional compounds for targeted protein degradation. This work demonstrates the potential of utilizing mutant E3 ligases that occur exclusively in diseased cells for TPD applications.

biochemistry↗

bioRxiv2024

Physicochemical Principles Driving Small Molecule Binding to RNA

The possibility of using RNA-targeting small molecules to treat diseases is gaining traction as the next frontier of drug discovery and development. The chemical characteristics of small molecules that bind to RNA are still relatively poorly understood, particularly in comparison to protein-targeting small molecules. To fill this gap, we have generated an unprecedented amount of RNA-small molecule binding data, and used it to derive physicochemical rules of thumb that could be used to define areas of chemical space enriched for RNA binders - the Small molecules Targeting RNA (STaR) rules of thumb. These rules have been applied to publicly available RNA-small molecule datasets and found to be largely generalizable. Furthermore, a number of patented RNA-targeting compounds and FDA-approved compounds also pass these rules, as well as key RNA binding approved drug case studies including Risdiplam. We anticipate this work will significantly accelerate the exploration of the RNA-targeted chemical space, towards unlocking RNAs potential as a small molecule drug target. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=130 HEIGHT=200 SRC="FIGDIR/small/578268v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@15ced07org.highwire.dtl.DTLVardef@1cd891eorg.highwire.dtl.DTLVardef@e51599org.highwire.dtl.DTLVardef@1ecfa86_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

A genetic and physiological model of renal dysfunction in Lowe syndrome

Lowe syndrome (LS) is an X-linked recessive genetic disorder characterized by renal dysfunction, neurodevelopmental defects, and cataract. The affected gene, OCRL encodes for a polyphosphoinositide 5-phosphatase. OCRL is localized to multiple sub-cellular locations in the endolysosomal system and defects in these organelles have been described in human cells depleted of OCRL. However, the relationship of the endolysosomal defects in OCRL depleted cells to the altered physiology of kidney cells of LS patients has not been completely determined. Here we model the kidney phenotypes of LS using a Drosophila nephrocyte model. Using this model system, we demonstrate that OCRL plays a cell-autonomous role in nephrocyte function. Deletion of the only OCRL ortholog in Drosophila (dOCRL) leads to cell-autonomous defects in larval nephrocyte structure and function. Null mutants of dOCRL (dOCRLKO) show defects in the endolysosomal system of larval nephrocytes that are associated with physiological defects in nephrocyte function. These defects could be rescued by reconstitution with a human OCRL transgene but not with a phosphatase dead version or a human LS patient derived mutation. Overall, this work provides a model system to understand the mechanisms by which the sub-cellular changes from loss of OCRL leads to defects in kidney function in human patients.

cell biology↗

bioRxiv2023

Preferential transport of synaptic vesicles across neuronal branches is regulated by the levels of the anterograde motor UNC-104/KIF1A

Asymmetric transport of cargo across axonal branches is a field of active research. Mechanisms contributing to preferential cargo transport along specific branches in vivo in wild type neurons are poorly understood. We find that anterograde synaptic vesicles preferentially enter the synaptic branch or pause at the branch point in C. elegans PLM neurons. The anterograde motor UNC-104/KIF1A regulates this vesicle behaviour at the branch point. Reduced levels of functional UNC-104 cause vesicles to predominantly pause at the branch point and lose their preference for turning into the synaptic branch. SAM- 4/Myrlysin, which aids in recruitment/activation of UNC-104 on synaptic vesicles, regulates vesicle behaviour at the branch point similar to UNC-104. Increasing the levels of UNC-104 increases the preference of vesicles to go straight towards the asynaptic end. This suggests that the neuron optimises UNC-104 levels on the cargo surface to maximise the fraction of vesicles entering the branch and minimise the fraction going to the asynaptic end.

cell biology↗

bioRxiv2021

A human stem cell resource to decipher the biochemical and cellular basis of neurodevelopmental defects in Lowe Syndrome

Human brain development is a complex process where multiple cellular and developmental events are co-ordinated to generate normal structure and function. Alteration in any of these events can impact brain development, manifesting clinically as neurodevelopmental disorders. Human genetic disorders of lipid metabolism often present with features of altered brain function. Lowe syndrome (LS), is a X-linked recessive disease with features of altered brain function. LS results from mutations in OCRL1 that encodes a phosphoinositide 5-phosphatase enzyme. However, the cellular mechanisms by which loss of OCRL1 leads to brain defects remain unknown. Human brain development involves several cellular and developmental features not conserved in other species and understanding such mechanisms remains a challenge. Rodent models of LS have been generated, but failed to recapitulate features of the human disease. Here we describe the generation of human stem cell lines from LS patients. Further, we present biochemical characterization of lipid metabolism in patient cell lines and demonstrate their use as a "disease-in-a-dish" model for understanding the mechanism by which loss of OCRL1 leads to altered cellular and physiological brain development.

cell biology↗

bioRxiv2021

Genomic sequencing of Lowe syndrome trios reveal a mechanism for the heterogeneity of neurodevelopmental phenotypes

Lowe syndrome is an X-linked recessive monogenic disorder resulting from mutations in the OCRL gene that encodes a phosphatidylinositol 4,5 bisphosphate 5-phosphatase. The disease affects three organs-the kidney, brain and eye and clinically manifests as proximal renal tubule dysfunction, neurodevelopmental delay and congenital cataract. Although Lowe syndrome is a monogenic disorder, there is considerable heterogeneity in clinical presentation; some individuals show primarily renal symptoms with minimal neurodevelopmental impact whereas others show neurodevelopmental defect with minimal renal symptoms. However, the molecular and cellular mechanisms underlying this clinical heterogeneity remain unknown. Here we analyze a Lowe syndrome family in whom affected members show clinical heterogeneity with respect to the neurodevelopmental phenotype despite carrying an identical mutation in the OCRL gene. Genome sequencing and variant analysis in this family identified a large number of damaging variants in each patient. Using novel analytical pipelines and segregation analysis we prioritize variants uniquely present in the patient with the severe neurodevelopmental phenotype compared to those with milder clinical features. The identity of genes carrying such variants underscore the role of additional gene products enriched in the brain or highly expressed during brain development that may be determinants of the neurodevelopmental phenotype in Lowe syndrome. We also identify a heterozygous variant in CEP290, previously implicated in ciliopathies that underscores the potential role of OCRL in regulating ciliary function that may impact brain development. More generally, our findings demonstrate analytic approaches to identify high-confidence genetic variants that could underpin the phenotypic heterogeneity observed in monogenic disorders.

genomics↗

bioRxiv2021

Combustible and electronic cigarette exposures increase ACE2 activity and SARS-CoV-2 Spike binding

The outbreak of coronavirus disease 2019 (COVID-19) has extensively impacted global health. The causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a transmembrane metallo-carboxypeptidase that is expressed in both membrane-anchored (mACE2) and soluble (sACE2) forms in the lung. Tobacco use has been speculated as a vulnerability factor for contracting SARS-CoV-2 infection and subsequent disease severity, whilst electronic cigarettes (e-cigarettes) have been shown to induce harmful proteomic and immune changes in the lungs of vapers. We therefore tested the hypothesis that combustible tobacco (e.g. cigarettes) and non-combustible e-cigarettes could affect ACE2 activity and subsequent SARS-CoV-2 infection. We observed that sACE2 activity was significantly higher in bronchoalveolar lavage fluid from both smokers and vapers compared to age-matched non-smokers. Exposure to cigarette smoke increased ACE2 levels, mACE2 activity, and sACE2 in primary bronchial epithelial cultures. Finally, treatment with either cigarette smoke condensate or JUUL e-liquid increased infections with a spike-coated SARS-CoV-2 pseudovirus. Overall, these observations suggest that tobacco product use elevates ACE2 activity and increases the potential for SARS-CoV-2 infection through enhanced spike protein binding.

physiology↗

bioRxiv2021

Cargo crowding, stationary clusters and dynamical reservoirs in axonal transport

Molecular motors drive the directed transport of presynaptic vesicles along the narrow axons of nerve cells. Stationary clusters of such vesicles are a prominent feature of axonal transport, but little is known about their physiological and functional relevance. Here, we develop a simulation model describing key features of axonal cargo transport with a view to addressing this question, benchmarking the model against our experiments in the touch neurons of C. elegans. Our simulations provide for multiple microtubule tracks and varied cargo motion states while also incorporating cargo-cargo interactions. Our model also incorporates obstacles to vesicle transport in the form of microtubule ends, stalled vesicles, and stationary mitochondria. We devise computational methodologies to simulate both axonal bleaching and axotomy, showing that our results reproduce the properties of both moving as well as stationary cargo in vivo. Increasing vesicle numbers leads to larger and more long-lived stationary clusters of vesicular cargo. Vesicle clusters are dynamically stable, explaining why they are ubiquitously seen. Modulating the rates of cargo motion-state switching allows cluster lifetimes and flux to be tuned both in simulations and experiments. We demonstrate, both in simulations and in an experimental system, that suppressing reversals leads to larger stationary vesicle clusters being formed while also reducing flux. Our simulation results support the view that the physiological significance of clusters is located in their role as dynamic reservoirs of cargo vesicles, capable of being released or sequestered on demand.

biophysics↗

bioRxiv2020

NAD+ prevents septic shock by non-canonical inflammasome blockade and IL-10

Septic shock is characterized by an excessive inflammatory response depicted in a cytokine storm that results from invasive bacterial, fungi, protozoa, and viral infections. Non-canonical inflammasome activation is crucial in the development of septic shock promoting pyroptosis and pro-inflammatory cytokine production via caspase-11 and Gasdermin-D (GSDMD). Here, we show that NAD+ treatment protected mice towards bacterial and LPS induced endotoxic shock by blocking the non-canonical inflammasome specifically. NAD+ administration impeded systemic IL-1{beta} and IL-18 production and GSDMD-mediated pyroptosis of macrophages via the IFN-{beta}/STAT-1 signaling machinery. More importantly, NAD+ administration not only improved casp-11 KO (knockout) survival but rendered WT mice completely resistant to septic shock via the IL-10 signaling pathway that was independent from the non-canonical inflammasome. Here, we delineated a two-sided effect of NAD+ blocking septic shock through a specific inhibition of the non-canonical inflammasome and promoting immune homeostasis via IL-10, underscoring its unique therapeutic potential.

immunology↗

bioRxiv2019

Single chromosome gains can function as metastasis suppressors and metastasis promoters

Most human tumors display chromosome-scale copy number alterations, and high levels of aneuploidy are frequently associated with advanced disease and poor patient prognosis. To examine the relationship between aneuploidy and cancer progression, we generated and analyzed a series of congenic human cell lines that harbor single extra chromosomes. We find that different aneuploidies can have distinct effects on invasive behavior: across 13 different cell lines, 12 trisomies suppressed invasiveness or were largely neutral, while a single trisomy increased metastatic behavior by triggering a partial epithelial-mesenchymal transition. In contrast, chromosomal instability, which can lead to the development of aneuploidy, uniformly suppressed cellular invasion. By analyzing genomic copy number and survival data from 10,133 cancer patients, we demonstrate that specific aneuploidies are associated with distinct clinical outcomes, and the acquisition of certain aneuploidies is in fact linked with a favorable prognosis. Thus, aneuploidy is not a uniform driver of malignancy, and different chromosome copy number changes can uniquely influence tumor progression. At the same time, the gain of a single chromosome is capable of inducing a profound cell state transition, underscoring how genomic plasticity can engender phenotypic plasticity and lead to the acquisition of enhanced metastatic properties.

cancer biology↗