Biology subjects

Mohan, K.

Publications and source records attributed to Mohan, K..

14 recordsLinked to original sources

bioRxiv2026

A Multi-Kinase Inhibitor Screen Identifies mTOR Signaling as a Vulnerability in Advanced Prostate Cancer.

The receptor tyrosine kinase AXL has context-dependent roles in prostate cancer (PCa). Although AXL signaling has been implicated in PCa progression, it can also promote disseminated tumor cell dormancy. Paradoxically, AXL expression has been reported to be lost in metastatic PCa. This raises two critical questions: Is AXL loss associated with aggressive disease, and does it create therapeutically exploitable vulnerabilities? In this study, we comprehensively analyzed AXL expression across pan-cancer and PCa cohorts and found that AXL downregulation was significantly associated with treatment resistance, aggressive PCa phenotypes, and shorter overall survival. To investigate the functional consequences of AXL loss, we used CRISPR-Cas9 based gene editing to generate isogenic AXLKO cells and characterized their proliferative and migratory properties. To identify therapeutically exploitable vulnerabilities associated with AXL loss, we screened these cells against a library of 1,814 kinase inhibitors using the isogenic cell lines. We identified Torin-2 as a candidate therapeutic agent and validated its activity using dose-response assays, which demonstrated an ~ 18-fold lower IC50 in AXLKO cells than in AXLWT cells. In parallel, PRISM/DepMap analysis provided orthogonal evidence of drug dependencies associated with low AXL expression and revealed a selective vulnerability of AXLKO cells to PI3K/AKT/mTOR pathway inhibition. Collectively, our findings link AXL downregulation to aggressive PCa phenotypes and identify mTOR inhibition as a potential therapeutic strategy for AXL-deficient PCa.

cancer biology↗

bioRxiv2026

Retrieval of binding sites across the AlphaFold human proteome using protein language model representations

Protein language models (PLMs) provide powerful representations of protein sequence, but their utility for proteome-scale binding-site retrieval remains unclear. Here, we present PocketScope, a training-free framework that represents cavity-lining residues using frozen ESM-C 600M embeddings and retrieves related binding sites through exhaustive late-interaction MaxSim, without pooling or approximate nearest-neighbor search. PocketScope identified 153,805 cavities across 37,682 proteins in the AlphaFold human proteome and recovered documented drug off-targets across a curated set of pharmacological pairs. On the ProSPECCTs benchmark, PocketScope ranks 1st of 23 methods by mean rank across the ten collections. PocketScope provides a practical framework for proteome-scale off-target prediction. PocketScope is open source and also freely available as a web server at https://www.bhargavaresearch.org/pocketscope.

bioinformatics↗

bioRxiv2026

SOX2 can associate with chromatin directly by binding to DNA or indirectly via association with other chromatin-bound proteins

It is widely assumed that SOX2 regulates gene expression and facilitates the opening of chromatin by binding directly at SOX motifs. To test this assumption, we created a SOX2 DNA binding mutant to determine whether other regions of SOX2 contribute to gene target specificity. When exogenously expressed in cells, this SOX2 mutant [SOX2(G76P)], like elevated unmodified SOX2, dramatically alters the transcriptome, but it does so by regulating vastly different gene sets and gene networks than SOX2. Consistent with their differential effects on the transcriptome, ChIP-seq analysis demonstrates that SOX2 and SOX2(G76P) associate primarily with different genomic loci, and motif analysis indicates that SOX2 binds primarily at SOX motifs, whereas SOX2(G76P) associates with chromatin at non-SOX motifs, including AP-1 motifs. Additionally, ATAC-seq analysis indicates that SOX2 substantially increases chromatin accessibility, but SOX2(G76P) does not. The findings presented lead to the conclusion that SOX2(G76P) associates with chromatin indirectly by a "piggyback" mechanism through its association with other chromatin-associated proteins, including AP-1 complexes. Remarkably, we also show that SOX2 and SOX2(G76P) each associate with a subset of the same gene loci that contain several different DNA motifs, including AP-1 motifs, but no high confidence SOX motifs. Overall, our findings provide new perspectives on SOX2 and lead to two important conclusions: 1) selection of gene targets by SOX2 is not solely determined by its DNA binding domain, and 2) SOX2 not only associates with chromatin directly by binding to SOX motifs but can also associate with a subset of gene loci indirectly through its association with other chromatin-associated proteins.

molecular biology↗

bioRxiv2026

NDST1 as a substrate-reduction target in Mucopolysaccharidosis type IIIC: virtual screening, microsecond molecular dynamics, and peptide design

Mucopolysaccharidosis IIIC (Sanfilippo syndrome type C) is a rare lysosomal storage disorder caused by loss-of-function mutations in HGSNAT, which encodes an enzyme involved in heparan sulfate (HS) degradation, leading to impaired HS catabolism, lysosomal accumulation, and progressive neurodegeneration. Because enzyme replacement therapies have limited penetration across the blood-brain barrier, substrate-reduction therapy represents an alternative therapeutic strategy. Here, N-deacetylase/N-sulfotransferase 1 (NDST1), a key enzyme responsible for HS biosynthesis, was investigated as a potential substrate-reduction target. A structure-based computational pipeline was used to identify and evaluate inhibitors targeting the NDST1 sulfotransferase domain. Approximately 4.1 million drug-like compounds and FDA-approved drugs were screened by molecular docking, followed by pharmacokinetic filtering, molecular dynamics simulations, and MM/PBSA binding free energy calculations. In parallel, peptide binders targeting the same site were generated using diffusion-based protein design and evaluated using molecular dynamics and MM/GBSA analysis. Four chemically distinct small-molecule scaffolds and three peptide candidates were identified as stable binders to the NDST1 active site. The lead small-molecule candidate exhibited a predicted binding free energy of -13.36 {+/-} 5.87 kcal mol-1. These provide a focused set of candidates for further investigation and support the feasibility of targeting NDST1 as a substrate-reduction strategy for MPS IIIC.

biophysics↗

bioRxiv2026

An Integrated Preclinical Platform for Lethal Neuroendocrine Prostate Cancer from Rapid Autopsy Bone and Liver Metastases.

Treatment-emergent neuroendocrine prostate cancer (NEPC) is an aggressive, therapy-resistant disease arising in up to 20% of castration resistant prostate cancers, yet robust biologically relevant preclinical models remain scarce. Here, we describe a technical blueprint for establishing an integrated platform of patient-derived models from visceral and bone metastases collected through a prostate cancer rapid autopsy program (PC RAP). We report the establishment and characterization of patient-derived xenograft (PDX) models from liver metastasis tissue, liver and bone metastasis-derived organoid lines (PDOs), and corresponding patient-derived organoid xenograft (PDOX) models. In addition, we established, to our knowledge, the first mesenchymal stem cell (MSC) cultures derived from neuroendocrine prostate cancer (NEPC) bone metastases. The PDOs preserved intratumoral heterogeneity, displaying both CRPC-NE and CRPC-adenocarcinoma features. These organoids retained neuroendocrine identity across multiple passages, with transcriptomic profiles concordant with the original patient tissue and matched PDX models generated at our institution and at the National Cancer Institute (NCI Patient-Derived Models Repository). To model the bone metastatic microenvironment, we generated novel organoid-based New Approach Methodologies (NAMs) by co-culturing PDOs with iPSC-derived bone marrow organoids, establishing a physiologically relevant vascularized organotypic model of PC bone metastasis. To extend our studies in vivo, we established preclinical models using the liver and bone metastasis-derived organoid models. The PDOX models were tumorigenic and developed spontaneous lymph node metastases, providing clinically relevant models for investigating lethal NEPC biology. Together, these complementary patient-derived models provide a robust and versatile platform for investigating NEPC biology, metastatic progression, and evaluating new therapeutic strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/740121v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1c641f1org.highwire.dtl.DTLVardef@113696aorg.highwire.dtl.DTLVardef@16dcc72org.highwire.dtl.DTLVardef@1898d9f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINovel preclinical models of visceral and bone metastases established from a prostate cancer rapid autopsy program. C_LIO_LIThis study is the first to establish mesenchymal stem cell cultures from NEPC bone metastases. C_LIO_LIPDOs preserve heterogeneity, showing both CRPC-NE and CRPC-Adeno features, with transcriptomic profiles concordant with originator tissue and PDX models. C_LIO_LIPC RAP-derived organoids are tumorigenic in vivo and generate spontaneous lymph node metastases. C_LI

cancer biology↗

bioRxiv2024

Visual image familiarity learning at multiple timescales in the primate inferotemporal cortex

Humans and other primates can rapidly detect familiar objects and distinguish them from never-before-seen novel objects. We have an astonishing capacity to remember the details of visual scenes even after a single, fleeting experience. This ability is thought to rely in part on experience-dependent changes in the primate inferotemporal cortex (IT). Single neurons in IT encode visual familiarity by discriminating between novel and familiar stimuli, with stronger neural activity on average for novel images. However, key open questions are to understand how neural encoding in IT changes as images progress from novel to highly familiar, and what learning rules and computations can account for learning-dependent changes in IT activity. Here, we investigate the timescales over which novel stimuli become familiar by recording in IT as initially novel images become increasingly familiar both within and across days. We identified salient and persistent memory-related signals in IT that spanned multiple timescales of minutes, hours, and days. Average neural activity progressively decreased with familiarity as firing rates were strongest for novel, weaker for intermediately familiar, and weakest for highly familiar images. Neural signatures of familiarity learning were slow to develop as response reductions to initially-novel images emerged gradually over multiple days (or hundreds of views) of visual experience. In addition to slow changes that emerged across sessions, neural responses to novel images showed rapid decreases with familiarity within single sessions. To gain insight into the mechanisms underlying changes of visual responses with familiarity, we use computational modeling to investigate which plasticity rules are consistent with these changes. Learning rules inferred from the neural data revealed a strong diversity with many neurons following a negative plasticity rule as they exhibited synaptic depression over the course of learning across multiple days. A recurrent network model with two plasticity time constants - a slow time constant for long timescales and a fast time constant for short timescales - captured key dynamic features accompanying the transition from novel to familiar, including a gradual decrease in firing rates over multiple sessions, and a rapid decrease in firing rates within single sessions. Our findings suggest that distinct and complementary plasticity rules operating at different timescales may underlie the inferotemporal code for visual familiarity.

neuroscience↗

bioRxiv2023

Age-associated changes in endothelial transcriptome and chromatin landscape correlate with elevated risk of hemorrhage

Intracerebral hemorrhage (ICH), a devastating form of stroke, is a leading global cause of human death and disability. The major risk factors for ICH include increasing age, hypertension and cerebral amyloid angiopathy. Despite high mortality and morbidity associated with ICH, the mechanisms leading to blood-brain barrier (BBB) dysfunction with age and development of hemorrhagic stroke is poorly understood. In the vasculature of the central nervous system, endothelial cells (ECs) constitute the core component of the BBB and provide a physical barrier due to tight junctions, adherens junctions, and basement membrane layers. In this study, we show in brains of mice that incidents of intracerebral bleeding increase with advancing age. After isolation of an enriched population of cerebral ECs, we studied gene expression in ECs isolated from murine brains of increasing ages of 2, 6, 12, 18, and 24 months. The study reveals agedependent dysregulation of 1388 genes in the ECs, including many involved in the maintenance of BBB and vascular integrity. Since epigenetic mechanisms regulate gene expression, we also investigated age-dependent changes at the levels of CpG methylation and accessible chromatin in cerebral ECs. Our study reveals correlations between age-dependent changes in chromatin structure and gene expression. We find significant age-dependent downregulation of the apelin receptor (Aplnr) gene along with an age-dependent reduction in chromatin accessibility of the promoter of this gene. Aplnr is known to play a crucial role in positive regulation of vasodilation and is implicated in vascular health. Interestingly, we also observe an age-dependent reduction in the protein expression levels of the apelin receptor in the brain, potentially implicating the apelin receptor to be critical for the increased risk of intracerebral hemorrhage with ageing.

molecular biology↗

bioRxiv2022

Near-chromosomal de novo assembly of Bengal tiger genome reveals genetic hallmarks of apex-predation

The tiger, a poster child for conservation, remains an endangered apex predator. Continued survival and recovery will require a comprehensive understanding of their genetic diversity and the use of such information for population management. A high-quality tiger genome assembly will be an important tool for conservation genetics, especially for the Indian tiger, the most abundant subspecies in the wild. Here, we present high-quality near-chromosomal genome assemblies of a female and a male wild Indian tiger (Panthera tigris tigris). Our assemblies had a scaffold N50 of >140{square}Mb, with 19{square}scaffolds, corresponding to the 19 numbered chromosomes, containing 95% of the genome. Our assemblies also enabled detection of longer stretches of runs of homozygosity compared to previous assemblies which will improve estimates of genomic inbreeding. Comprehensive genome annotation identified 26,068 protein-coding genes, including several gene families involved in key morphological features such as the teeth, claws, vision, olfaction, taste and body stripes. We also identified 301 microRNAs, 365 small nucleolar RNAs, 632 tRNAs and other noncoding RNA elements, several of which are predicted to regulate key biological pathways that likely contribute to tigers apex predatory traits. We identify signatures of positive selection in the tiger genome that are consistent with the Panthera lineage. Our high-quality genome will enable use of non-invasive samples for comprehensive assessment of genetic diversity, thus supporting effective conservation and management of wild tiger populations.

genomics↗

bioRxiv2021

Cheating the cheater: Suppressing false positive enrichment during biosensor-guided biocatalyst engineering

Transcription factor (TF)-based biosensors are very desirable reagents for high-throughput enzyme and strain engineering campaigns. Despite their potential, they are often difficult to deploy effectively as the small molecules being detected can leak out of high-producer cells, into low-producer cells, and activate the biosensor therein. This crosstalk leads to the overrepresentation of false positive/cheater cells in the enriched population. While the host cell can be engineered to minimize crosstalk (e.g., by deleting responsible transporters), this is not easily applicable to all molecules of interest, particularly those that can diffuse passively. One such biosensor recently reported for trans-cinnamic acid (tCA) suffers from crosstalk when used for phenylalanine ammonia-lyase (PAL) enzyme engineering by directed evolution. We report that desensitizing the biosensor (i.e., increasing the limit of detection, LOD) suppresses cheater population enrichment. Further we show that, if we couple the biosensor-based screen with an orthogonal pre-screen that eliminates a large fraction of true negatives, we can successfully reduce the cheater population during the fluorescence-activated cell sorting (FACS). Using the approach developed here, we were successfully able to isolate PAL variants with ~70% high kcat after a single sort. These mutants have tremendous potential in Phenylketonuria (PKU) treatment and flavonoid production.

synthetic biology↗

bioRxiv2021

In-depth sequence-function characterization reveals multiple paths to enhance phenylalanine ammonia-lyase (PAL) activity.

Phenylalanine ammonia-lyases (PALs) deaminate L-phenylalanine to trans-cinnamic acid and ammonium and have idespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from Anabaena variabilis (Trichormus variabilis) has garnered significant attention as the active ingredient in Pegvaliase(R), the only FDA-approved drug treating classical phenylketonuria (PKU). Although an extensive body of literature exists on structure, substrate-specificity, and catalytic mechanism, protein-wide sequence determinants of function remain unknown, which limits the ability to rationally engineer these enzymes. Previously, we developed a high-throughput screen (HTS) for PAL, and here, we leverage it to create a detailed sequence-function landscape of PAL by performing deep mutational scanning (DMS). Our method revealed 79 hotspots that affected a positive change in enzyme fitness, many of which have not been reported previously. Using fitness values and structure-function analysis, we picked a subset of residues for comprehensive single- and multi-site saturation mutagenesis to improve the catalytic activity of PAL and identified combinations of mutations that led to improvement in reaction kinetics in cell-free and cellular contexts. To understand the mechanistic role of the most beneficial mutations, we performed QM/MM and MD and observed that different mutants confer improved catalytic activity via different mechanisms, including stabilizing first transition and intermediate states and improving substrate diffusion into the active site, and decreased product inhibition. Thus, this work provides a comprehensive sequence-function relationship for PAL, identifies positions that improve PAL activity when mutated and assesses their mechanisms of action.

bioengineering↗

bioRxiv2020

Distributed Visual Category Processing Across Medial Superior Temporal and Lateral Intraparietal Cortices

Categorization is an essential cognitive and perceptual process for recognition and decision making. The posterior parietal cortex (PPC), particularly the lateral intraparietal (LIP) area has been suggested to transform visual feature encoding into cognitive or abstract category representations. By contrast, areas closer to sensory input, such as the middle temporal (MT) area, encode stimulus features but not more abstract categorical information during categorization tasks. Here, we compare the contributions of PPC subregions in category computation by recording neuronal activity in the medial superior temporal (MST) and LIP areas during a categorization task. MST is a core motion processing area interconnected with MT, and often considered an intermediate processing stage between MT and LIP. Here we show that MST shows robust decision-correlated category encoding and working memory encoding similar to LIP, suggesting that MST plays a substantial role in cognitive computation, extending beyond its widely recognized role in visual motion processing.

neuroscience↗

bioRxiv2020

Interaction between neuronal encoding and population dynamics during categorization task switching in parietal cortex

Primates excel at categorization, a cognitive process for assigning stimuli into behaviorally relevant groups. Categories are encoded in multiple brain areas and tasks, yet it remains unclear how neural encoding and dynamics support cognitive tasks with different demands. We recorded from parietal cortex during flexible switching between categorization tasks with distinct cognitive and motor demands, and also studied recurrent neural networks (RNNs) trained on the same tasks. In the one-interval categorization task (OIC), monkeys rapidly reported their decisions with a saccade. In the delayed match-to-category (DMC) task, monkeys decided whether sequentially presented stimuli were categorical matches. Neuronal category encoding generalized across tasks, but categorical encoding was more binary-like in the DMC task and more graded in the OIC task. Furthermore, analysis of the trained RNNs supports the hypothesis that binary-like encoding in the DMC task arises through compression of graded feature encoding by population attractor dynamics underlying short-term working memory.

neuroscience↗

bioRxiv2020

Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities

There is broad interest in engineering phenylalanine ammonia-lyase (PAL) for its biocatalytic applications in industry (fine-chemicals and natural product synthesis) and medicine (phenylketoruria/PKU and cancer treatment). While site-specific mutagenesis has been employed to improve PAL stability or substrate specificity, a more comprehensive mutational landscape has yet to be explored for this class of enzymes. Here, we report development of a directed evolution technique to engineer PAL enzymes. Central to this approach is a high-throughput enrichment that couples E. coli growth to PAL activity. Using the clinically-relevant PAL from Anabaena variabilis, which is used on the formulation of pegvaliase for PKU therapy, we identified mutations at residues previously unknown as relevant for function that increase turnover frequency almost twofold after only a single round of engineering. This work demonstrates the power our technique for ammonia-lyase enzyme engineering.

bioengineering↗

bioRxiv2016

Distinct cAMP signaling modalities mediate behavioral flexibility and consolidation of social status in Drosophila aggression.

Social rituals, like male-male aggression in Drosophila, are often stereotyped and the component behavioral patterns modular. The likelihood of transition from one behavioral pattern to another is malleable by experience and confers flexibility to the behavioral repertoire. Experience-dependent modification of innate aggressive behavior in flies alters fighting strategies during fights and establishes dominant-subordinate relationships. Dominance hierarchies resulting from agonistic encounters are consolidated to longer lasting social status-dependent behavioral modifications resulting in a robust loser effect.\n\nWe show that cyclic adenosine monophosphate (cAMP) dynamics regulated by the calcium/calmodulin-dependent adenylyl cyclase, Rut and the cAMP phosphodiesterase, Dnc but not the Amn gene product, in specific neuronal groups of the mushroom body and central complex, mediate behavioral plasticity necessary to establish dominant- subordinate relationships. rut and dnc mutant flies are unable to alter fighting strategies and establish dominance relationships during agonistic interactions. This real-time flexibility during a fight is independent of changes in aggression levels. Longer-term consolidation of social status in the form of a loser effect, however, requires additional Amn-dependent inputs to cAMP signaling and involves a circuit-level association between the /{beta} and {gamma} neurons of the mushroom body.\n\nOur findings implicate cAMP signaling in mediating plasticity of behavioral patterns in aggressive behavior and in the generation of a temporally stable memory trace that manifests as a loser effect.\n\nSUMMARY STATEMENTPhasic recruitment of different cAMP signaling modalities in specific neuronal groups lead to the formation of temporally distinct components of learning and memory in fly aggression.

neuroscience↗