Biology subjects

Walsh, M.

Publications and source records attributed to Walsh, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Massively scaled variant effect mapping across the MAPK pathway reveals the importance of molecular and cellular context

Variation alters inherent protein properties like thermodynamic stability and catalytic activity, and these intrinsic effects are shaped by the complex cellular environment. Signaling cascades typify this complexity, with tens or hundreds of proteins acting in concert through regulated states and interactions to transduce signals. Here, we measure the effect of 186,676 variants across 17 proteins in the RAS-MAPK pathway on protein abundance and pathway activity in human cells under diverse conditions, for a total of 503,099 variant effect measurements. We discovered 14,412 dominant negative variants across 12 proteins, nearly all previously unknown, that disrupt specific domains or interaction interfaces while leaving other functions intact and that are depleted from the human population. Measuring the abundance of 106,642 variants across 10 proteins under HSP90 inhibition showed that HSP90 shapes the effect of 67% of missense variants across the pathway, buffering destabilizing variants in both strong and weak clients. Our variant effect data, collected in the context of an intact proteome, enabled us to assess the functional importance of 395 experimentally and computationally modeled MAPK protein interactions. Using additional mutational scans, we resolved how the promiscuous switch II helix binding surface of KRAS accommodates overlapping interactions with LZTR1 and SOS1, revealing distinct allosteric networks that support each interaction. Thus, we establish a framework for measuring variant effects at unprecedented scale in human cells, illuminating fundamental MAPK pathway signaling mechanisms and demonstrating that cellular context profoundly shapes the effect of most variants.

genomics↗

bioRxiv2026

DNMT1-Mediated Epigenetic Reprogramming Drives KMT2A Amplifications and Rearrangements

MLL/KMT2A amplifications and rearrangements are prevalent in infant, adult, and therapy-induced leukemia; however, the molecular contributors controlling these alterations remain elusive. Here, we reveal that rapid CTCF degradation generates local genome structure alterations at KMT2A and copy gains and rearrangements. We then established a conserved, coordinated interplay between DNA and histone methylation pathways that control CTCF occupancy, and in turn, KMT2A locus stability. For example, DNMT1 overexpression promotes increased H3K9 methylation, reduced CTCF occupancy, and causes KMT2A alterations. However, DNMT1 inhibition suppresses these events and KMT2A alterations caused by topoisomerase II inhibition. Locus-specific epigenome targeting demonstrated that DNA or H3K9 methylation promotes KMT2A copy gains and rearrangement, whereas targeted TET activity suppresses these events upon methylation perturbation or doxorubicin treatment. These findings identify a conserved, coordinated DNA-histone methylation axis governing KMT2A amplifications and rearrangement susceptibility, revealing biomarkers and therapeutic targets to predict and intercept these events in cancer.

cancer biology↗

bioRxiv2026

Over-the-counter fiber supplementation associates with metabolic and microbial shifts in rectal mucosa

Fiber supplements are the first line of treatment for all patients with benign anorectal disease. In addition, fiber has been found to support gut microbiota and reduce inflammation; however, their integrated effect on the colonic mucosal environment is incompletely defined. We evaluated how mixed-fiber formulations may collectively shape the rectal mucosal environment through coordinated effects on microbiota composition, short-chain fatty acid (SCFA) handling, and host transcription. We evaluated multi-compartment responses to a 28-day mixed-fiber intervention in 37 participants with benign anorectal disease (~15 g/day; 7.5g psyllium husk, 8g wheat dextrin) and assessed the microbiota (16S rRNA), the metabolome (GC-MS and LC-MS), and transcriptome (subset n=10) before (PRE) and after (POST) supplementation. Changes in KEGG pathways were tested with gene set enrichment analysis (GSEA). Associations were tested by Spearman correlation, and microbiota-based prediction of SCFA responses by random-forest regression. Microbiota diversity was stable after the fiber intervention, however, 10 mucosal ASVs were differentially abundant including Parabacteroides and Lachnospiraceae NK4A136 group while SCFAs, propionate and isobutyrate, both decreased in rectal tissue and butyrate decreased in serum. To understand these shifts in the metabolome, we proceeded with the transcriptome where we identified 130 differentially expressed genes and enrichment of metabolic and SCFA-related pathways. Random forest regression captured a modest signal for serum butyrate. Taken together, short-term mixed-fiber supplementation produced selective rectal mucosal taxonomic shifts and enriched mucosal metabolic and SCFA-metabolism program despite reduced SCFA pools, suggesting that even brief over-the-counter fiber supplementation may reshape the mucosal metabolic environment.

microbiology↗

bioRxiv2026

Organoid-T cell co-cultures functionally stratify tumor-reactive T cells and their responses to immune checkpoint inhibitors

Tumor-reactive T cells (TRTs) are critical for anti-tumor immunity but are incompletely captured by current assays, which fail to reproduce tumor-specific antigen diversity. Here, we show that multiplex functional profiling of patient-derived tumor organoid-T cell co-cultures (PDOTs) enables robust identification of TRTs across CD8, CD4, and double-negative (DN) T cell populations. Single activation markers underestimated TRT responses, whereas integrated analysis revealed broader functional repertoire. MHCI blockade abrogated CD8 and DN TRT responses while preserving CD4 reactivity, supporting antigen-dependent recognition across T cell lineages. Tumor PDO expressed MHCI and MHCII, and PDOTs enabled generation and detection of TRTs from peripheral blood. PD1 blockade induced heterogeneous responses, enhancing CD8 and DN activity and unexpectedly augmenting CD4 reactivity. PDOTs further identified additional inhibitory pathways whose therapeutic targeting in combination with PD1 blockade increased TRT responses. These findings establish PDOTs as a platform to identify TRTs and functionally stratify patient-specific tumor-T cell responses to checkpoint immunotherapy.

immunology↗

bioRxiv2026

A genetically encoded local learning rule enables physical learning in engineered bacteria

Training physical neural networks directly in matter remains difficult because most platforms do not store and update weights within the same physical substrate. Here we show that engineered Escherichia coli can implement a genetically encoded local learning rule acting on persistent biological memory. We introduce memregulons: bacterial memory elements in which coupled plasmids store an analogue weight as the relative abundance of P1 within the P1+P2 plasmid pool. Environmental inputs activate local promoters, and a shared sublethal kanamycin signal converts promoter activity into differential growth that lowers the stored P1 fraction. In single strains, flow-cytometry trajectories across distinct promoters support the predicted dependence of weight change on learning-channel activity and standing population variance. At the single-cell level, repeated negative learning reshapes the stored weight distribution as it approaches the lower boundary. In mixed populations and co-cultures, one shared negative learning signal selectively rewrites active memregulons, enabling externally routed supervised tic-tac-toe lessons. We then generalise the architecture across orthogonal chemical inputs and combinatorial promoters, and use experimentally measured updates in hybrid analyses of winner-take-all and nonlinear-classifier tasks. These results establish physical learning in living matter through local negative updates of genetically encoded analogue weights.

synthetic biology↗

bioRxiv2026

LINE-1 retrotransposition is a recurrent cause of MET exon 14 skipping in cancer

MET exon 14 skipping is a pathogenic event that results in decreased ubiquitin-mediated degradation of the MET receptor, sustained oncogenic signaling, and conferred sensitivity to MET tyrosine kinase inhibitors. While exon 14 skipping is most commonly caused by somatically acquired base substitutions and small indels near the exon 14 splice sites, here we report nine cases in which long interspersed element-1 (LINE-1, L1)-mediated insertions within or adjacent to MET exon 14, including one case of a LINE-1-mediated pseudogene insertion, appear to cause exon 14 skipping. These describe the first recurrent and clinically actionable mutations caused by LINE-1 retrotransposition in cancer.

cancer biology↗

bioRxiv2025

Four new Duchenne muscular dystrophy mouse models with clinically relevant exon deletions in the human DMD gene

Mutation specific therapeutic approaches, like exon skipping or gene-editing, hold promise for the treatment of Duchenne muscular dystrophy (DMD). Translatability of preclinical studies investigating these approaches could greatly be improved through the use of humanized mouse models, as these allow preclinical testing of human specific sequences. We developed four novel humanized DMD mouse models with either a deletion of exon 44, 45, 51 or 53 in the human DMD gene, in a mouse dystrophin negative background (mdx mouse; exon 23 nonsense mutation). Our optimized prescreening pipeline allowed us to do so very efficiently with the CRISPR-Cas9 technology. We confirmed either complete lack of dystrophin, or expression of trace levels, which led to development of muscle pathology consisting of muscle fiber de-, and regeneration, inflammation and fibrosis in young adult mice. Intramuscular treatment with vivo-morpholinos targeting a flanking exon induced exon skipping in the DMD strains, which restored the disrupted open reading frame and subsequently dystrophin expression. This validates these models as valuable tools for preclinical studies investigating human sequence specific therapeutic approaches for DMD. Summary statementHumanized Duchenne muscular dystrophy mouse models were created with deletions of exon 44, 45, 51 or 53 in the human DMD gene. These dystrophic models allow preclinical testing of human-specific dystrophin restoring approaches.

neuroscience↗

bioRxiv2025

The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

In type 1 diabetes (T1D), the innate and adaptive immune systems attack and eventually destroy the insulin-secreting pancreatic {beta} cells. During this process, {beta} cells activate inflammatory signaling pathways that augment the dysfunction and destruction imposed by cellular autoimmunity. The 12-lipoxygenase (12-LOX) pathway produces the pro-inflammatory eicosanoid 12-HETE, which induces oxidative and endoplasmic reticulum stress and results in diminished insulin secretion and apoptosis. The G protein-coupled receptor GPR31 has been identified as a putative receptor for 12-HETE. In this study, we generated conventional GPR31 knockout (KO) mice on the C57BL/6J background. To interrogate the role of GPR31 in {beta} cells, we treated islets from wildtype and Gpr31b KO mice with pro-inflammatory cytokines and subjected the islets to RNA sequencing. Differentially expressed pathways in Gpr31b KO islets included those pertaining to inflammation and oxidative stress, consistent with functional studies that demonstrated reduced cytokine-induced oxidative stress in Gpr31b KO islets compared to wildtype controls. Bone marrow-derived macrophages from Gpr31b KO mice showed reduced macrophage migration and decreased inflammatory IFN- and IFN-{gamma} signaling by RNA sequencing. To mimic islet and macrophage inflammation as seen in T1D, wildtype and Gpr31b KO mice were treated with the diabetogenic toxin streptozotocin. Compared to wildtype, Gpr31b KO mice had improved glucose tolerance and preserved {beta}-cell mass. siRNA knockdown of Gpr31b in non-obese diabetic (NOD) mice reduced insulitis, macrophage infiltration, and oxidative stress. Collectively, these findings are consistent with previously published data using 12/15-LOX KO mice and suggest that GPR31 mediates the pro-inflammatory responses of 12-HETE in both {beta} cells and macrophages.

molecular biology↗

bioRxiv2024

Spatial analysis of HPV associated cervical intraepithelial neoplastic tissues demonstrate distinct immune signatures associated with cervical cancer progression

Cervical cancer remains the fourth most common cancer affecting women worldwide, and incidences of other HPV-related cancers continue to rise. For the development of effective prevention strategies in high-risk patients, we aimed to better understand the roles of inflammatory pathways and the tumour microenvironment as the main driver of progression to malignancy in HPV-infected tissues. We analysed the spatial organisation of seven samples of HPV+ high-grade squamous intraepithelial lesion (HSIL) and cervical intraepithelial neoplasia 3 (CIN3), comparing tumour heterogeneity and immune microenvironments between pre-malignant (neoplastic) and adjacent cervical tissues. We observed evidence of immune suppression within the neoplastic regions across all samples and identified distinct immune clusters for each dysplastic lesion. Previous single-cell data analyses in an HPV16 E7 oncoprotein-driven transgenic mouse model suggested a potential role for IL34-CSF1R signalling in immune modulation, where low IL34 expression was associated with Langerhans cell dysfunction, and, in cervical cancer, with poor patient outcome. Here, we observed that IL34-CSF1R co-expression was absent within HPV-associated neoplastic tissues but present in adjacent normal tissue regions. Additionally, we identified enrichment of an M2 gene signature in neoplastic tissue, while adjacent tissue was enriched with a pro-inflammatory M1 gene signature. Our findings provide bio-pathological insights into the spatial cellular and molecular mechanisms underlying HPV-associated cervical cancer immune regulation and suggest a strategy to modulate the immune system in HPV-positive neoplastic cervical and other tissues.

genomics↗

bioRxiv2024

DNA polymerase theta-mediated DNA repair is a functional dependency and therapeutic vulnerability in DNMT3A deficient leukemia cells

Myeloid malignancies carrying somatic DNMT3A mutations (DNMT3Amut) may be refractory to standard therapy. DNMT3Amut leukemia cells accumulate toxic DNA double strand breaks (DSBs) and stalled replication forks, rendering them dependent on DNA damage response (DDR). We report here that DNA polymerase theta (Pol{theta}), a key element in DSB repair by end-joining (TMEJ) and in fork restarting, is essential for survival and proliferation of DNMT3Amut leukemia cells. Pol{theta} is overexpressed in DNMT3Amut leukemia cells due to abrogation of PARP1 PARylation-dependent UBE2O E3 ligase-mediated ubiquitination and proteasomal degradation of Pol{theta}. In addition, PARP1-mediated recruitment of the SMARCAD1-MSH2/MSH3 repressive complex to DSBs was diminished in DNMT3Amut leukemia cells which facilitated loading of Pol{theta} on DNA damage and promoting TMEJ and replication fork restart. Pol{theta} inhibitors enhanced the anti-leukemic effects of standard drugs such as FLT3 kinase inhibitor quizartinib, cytarabine +/- doxorubicin, and etoposide in vitro and in mice with DNMT3Amut leukemia. Altogether, Pol{theta} is an attractive target in DNMT3Amut hematological malignancies.

cancer biology↗

bioRxiv2024

Crystallographic fragment screen of Enterovirus D68 3C protease and iterative design of lead-like compounds using structure-guided expansions

The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics. One cause for concern is the currently emerging pathogen Enterovirus D68 (EV-D68) which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but, in severe cases, acute flaccid myelitis. The 3C protease of EV-D68 (3Cpro) is a potential target for the development of antiviral drugs due to its essential role in the viral life cycle and high sequence conservation amongst family members. In this study, we describe the identification of fragments which bind to3Cpro using crystallographic screening and the expansion of these into more lead-like compounds. The hits revealed interesting directions for hit-to-lead progression, specifically the importance of the pocket occupied by the conserved glutamine sidechain of the substrates and the interactions formed. Additionally, two pockets could be joined by not following the backbone of the native substrates, thus circumventing the screening issues arising from the flexibility of the catalytic triad. These observations of the novel binding modes of the chemical matter found by this screen can help shape future drug design campaigns against 3C proteases.

biochemistry↗

bioRxiv2024

High-throughput crystallographic fragment screening of Zika virus NS3 Helicase

The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barre syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. Here we applied high-throughput crystallographic fragment screening on ZIKV NS3Hel, which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.

biophysics↗

bioRxiv2024

Crystallographic Fragment Screen of Coxsackievirus A16 2A Protease identifies new opportunities for the development of broad-spectrum anti-enterovirals

Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16. Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity.

biochemistry↗

bioRxiv2024

A scoping review of evidence of naturally occurring Japanese encephalitis infection in vertebrate animals other than humans, ardeid birds and pigs

Japanese encephalitis virus (JEV) is the leading cause of human encephalitis in Asia. JEV is a vector-borne disease, mainly transmitted by Culex mosquitoes, with Ardeidae birds as maintenance hosts and pigs as amplifying hosts. Other vertebrate animal hosts have been suggested to play a role in the epidemiology of JEV. This scoping review followed PRISMA guidelines to identify species in which evidence of naturally occurring JEV infection was detected in vertebrates other than ardeid birds, pigs and people. Following systematic searches, 4372 records were screened, and data were extracted from 62 eligible studies. Direct evidence (virus, viral antigen or viral RNA) of JEV infection was identified in a variety of mammals and birds (not always identified to the species level), including bats, passerine birds (family Turdidae), livestock (cattle [Bos taurus] and a goat [Capra hircus]), carnivores (two meerkats [Suricata suricatta]), and one horse (Equus caballus). Bat families included Pteropodidae, Vespertilionidae, Rhinolophidae, Miniopteridae, Hipposideridae. Indirect evidence suggestive of JEV infection (antibodies) was detected in several mammalian and avian orders, as well as reported in two reptile species. However, a major limitation of the evidence of JEV infection identified in this review was diagnostic test accuracy, particularly for serological testing. Studies generally did not report diagnostic sensitivity or specificity. Although we hypothesise that bats and passerine birds could play an underappreciated role in JEV epidemiology, development of diagnostic tests to differentiate JEV from other orthoflaviviruses will be critical for effective surveillance in these, as well as the companion and livestock species that could be used to evaluate JEV control measures in currently endemic regions.

ecology↗

bioRxiv2024

Comparison of compartmental analytical BOLD fMRI models against Monte Carlo simulations performed over cortical micro-angiograms

BOLD fMRI arises from a physiological and physical cascade of events taking place at the level of the cortical microvasculature which constitutes a medium with complex geometry. Several analytical models of the BOLD contrast have been developed but these have not been compared directly against detailed bottom-up modeling methods. Using a 3D modeling method based on experimentally measured images of mice microvasculature and Monte Carlo simulations, we quantified the accuracy of two analytical models to predict the amplitude of the BOLD response from 1.5T to 7T, for different TE and for both gradient echo and spin echo acquisition protocols. We also showed that accounting for the tridimensional structure of the microvasculature results in more accurate prediction of the BOLD amplitude, even if the values for SO2 were averaged across individual vascular compartments. A secondary finding is that modeling the venous compartment as two individual compartments results in more accurate prediction of the BOLD amplitude compared to standard homogenous venous modeling, arising from the bimodal distribution of venous SO2 across the microvasculature in our data.

biophysics↗

bioRxiv2023

Mapping the landscape pool of ardeid species to landscape structure associated with Japanese encephalitis virus in Australia

Japanese encephalitis virus (JEV), a zoonotic, mosquito-borne virus, has broad circulation across the Central Indo-Pacific biogeographical region (CIPBR), which recently expanded dramatically within this region across southeastern Australia over the summer of 2021-2022. Preliminary investigation of the landscape epidemiology of the outbreaks of JEV in Australian piggeries found associations with particular landscape structure as well as ardeid species richness. The ways in which waterbird species from diverse taxonomic pools with substantial functional variation might couple with JEV-associated landscape structure was not explored, and therefore, key questions regarding the landscape epidemiology and infection ecology of JEV remain unanswered. Moreover, given the established presence of JEV within the CIBPR, the extent to which waterbird species pools in JEV-associated landscapes in Australia reflect broader regional patterns in functional biogeography presents a further knowledge gap particularly with respect to potential virus dispersal via maintenance hosts. The current study investigated waterbird species presence, ecological traits, and functional diversity distribution at landscape scale, and how these aligned with confirmed JEV detections in eastern Australia and the wider CIPBR. The results showed that waterbird habitat associated with JEV detection in Australia in 2022 and more widely across the CIPBR over the last 20 years reflects a range of species representing 8 families in 4 orders (ardeids, anatids, rallids, phalacrocoracids, threskiornithids, gruids, and pelecanids). Increasing waterbird functional diversity (trait-based mean pairwise dissimilarity) was associated with landscapes delineating JEV occurrence, while only one individual trait, high hand-wing index, was consistently associated with species presence in these JEV-associated landscapes in both Australia and the broader CIPBR. This suggests that dispersal capacity among the waterbird species pools that dominate JEV-associated landscapes might be important. By taking an agnostic approach to JEV maintenance host status, this study indicates a relatively large, CIPBR-wide pool of waterbird families associated with JEV landscapes, challenging the narrow view that JEV maintenance is limited to ardeid birds. In addition, these findings highlight the potential for leveraging functional biogeography in high-risk landscapes across broad geographic extent to guide landscape-specific selection of species for JEV surveillance.

ecology↗

bioRxiv2023

Structural Analysis of Genomic and Proteomic Signatures Reveal Dynamic Expression of Intrinsically Disordered Regions in Breast Cancer and Tissue

Structural features of proteins capture underlying information about protein evolution and function, which enhances the analysis of proteomic and transcriptomic data. Here we develop Structural Analysis of Gene and protein Expression Signatures (SAGES), a method that describes expression data using features calculated from sequence-based prediction methods and 3D structural models. We used SAGES, along with machine learning, to characterize tissues from healthy individuals and those with breast cancer. We analyzed gene expression data from 23 breast cancer patients and genetic mutation data from the COSMIC database as well as 17 breast tumor protein expression profiles. We identified prominent expression of intrinsically disordered regions in breast cancer proteins as well as relationships between drug perturbation signatures and breast cancer disease signatures. Our results suggest that SAGES is generally applicable to describe diverse biological phenomena including disease states and drug effects.

pharmacology and toxicology↗

bioRxiv2022

Engineered gene circuits with reinforcement learning allow bacteria to master gameplaying

Gene circuits enable cells to make decisions by controlling the expression of genes in reaction to specific environmental factors1. These circuits can be designed to encode logical operations2-7, but implementation of more complex algorithms has proved more challenging. Directed evolution optimizes gene circuits8 without the need for design knowledge9, but adjusting multiple genes and conditions10 in genotype searches poses challenges11. Here we show a multicellular sensor system, AdaptoCells, in Escherichia coli, that can evolve complex behavior through an accelerated adaptation to chemical environments. AdaptoCells recognize chemical patterns and act as a decision-making system. Using an iterative improvement method, we demonstrate that the AdaptoCells can evolve to achieve mastery in the game of tic-tac-toe, demonstrating an unprecedented level of complexity for engineered living cells. We provide an effective and straightforward way to encode complexity in gene circuits, allowing for fast adaptation in response to dynamic environments and leading to optimal decisions.

synthetic biology↗