Biology subjects

Marsh, A.

Publications and source records attributed to Marsh, A..

7 recordsLinked to original sources

bioRxiv2026

Data-Efficient Exploration of Enzyme Function Using Family-Specific Machine Learning

Enzymes are essential biocatalysts across diverse industries, driving demand for high-performing variants. Foundation models are attractive for guiding enzyme discovery, but often lack the resolution to model subtle variations driving function within homologous families. Navigating these rugged functional landscapes to identify elite variants remains challenging and experimentally costly, even when guided by such models. Here we show that coupling dense, family-specific experimental screening with targeted, sequence-based deep learning provides a data-efficient discovery strategy. We experimentally screened 1,513 natural homologues from an esterase superfamily (>7,500 assays) and used this functional landscape to train task-specific models that predict activity, thermostability, and substrate specificity from sequence alone. Prospective experimental validation of previously untested sequences demonstrated that these task-specific models significantly outperformed generalist pre-trained and physics-based models in enriching for target traits. Residue-level attribution further indicated that the models captured sequence patterns consistent with underlying structural features. Finally, retrospective simulations showed that iterative retraining compresses the search space, discovering 60% of top-tier hits using nearly half the samples required by pre-trained baseline models. Together, these results highlight that machine learning can provide mechanistic insight, and that integrating targeted data acquisition with iterative machine learning provides a more data-efficient discovery strategy than relying on generic model scale.

bioengineering↗

bioRxiv2025

Comprehensively Testing the Function of Missense Variation in the STK11 Tumour Suppressor

The tumor suppressor gene STK11 encoding Serine/Threonine Kinase 11 (STK11) is associated with Peutz-Jeghers Syndrome (PJS), a heritable gastrointestinal disease that increases lifetime cancer risk, and with somatic variation that contributes to [~]30% of lung and 20% of cervical cancers. Although identifying pathogenic variants is clinically actionable, over 94% of STK11 missense variants that have been observed clinically lack a definitive classification. We therefore measured the impact of STK11 variants at scale in a mammalian cell-based assay, scoring 6,026 (73% of all possible) amino acid substitutions across the full-length gene. Functional scores--which were consistent with biochemical properties, smaller-scale assays, and pathogenicity annotations--identified a subset of PJS patients with germline STK11 variants diagnosed later in life, as well as somatic STK11 variants found in cancer patients that had comparable overall survival estimates to wild-type STK11. Our scores provided new evidence for 350 annotated VUS STK11 missense variants and [~]80% of missense variants that have not yet been reported clinically, but we might expect to observe in the future. Thus, our effect map provides a proactive resource for gaining sequence-structure-function insights and evidence for actionable interpretation of clinical missense variants.

cancer biology↗

bioRxiv2025

Saturation mapping of MUTYH variant effects using DNA repair reporters

Variants of uncertain significance (VUS) limit the actionability of genetic testing. A prominent example is MUTYH, a base excision repair factor associated with polyposis and colorectal cancer, which has a pathogenic variant carrier rate approaching 1 in 50 individuals in some populations. To systematically interrogate variant function in MUTYH, we coupled deep mutational scanning with a DNA repair reporter containing its lesion substrate, 8OG:A. Our variant-to-function map covers >97% of all possible MUTYH point variants (n=10,941) and achieves 100% accuracy classifying the pathogenicity of known clinical variants (n=247). Leveraging a large clinical registry, we observe significant associations with colorectal polyps and cancer, with more severely impaired missense variants conferring greater risk. We recapitulate known functional differences between pathogenic founder alleles, and highlight sites of complete missense intolerance, including residues that intercalate DNA and coordinate essential Zn2+ or Fe-S clusters. This map provides a resource to resolve the 1,032 existing missense VUS and 90 variants with conflicting interpretations in MUTYH, and demonstrates a scalable strategy to interrogate other clinically relevant DNA repair factors.

genomics↗

bioRxiv2024

Rodent Gut Bacteria Coexisting with an Insect Gut Virus in Parasitic Cysts: Metagenomic Evidence of Microbial Translocation and Co-adaptation in Spatially-Confined Niches

In medicine, parasitic cysts or cysticerci (fluid-filled cysts, larval stage of tapeworms) are believed to be sterile (no bacteria), and therein, the treatment of cysticerci infestations of deep extra-intestinal tissues (e.g., brain) relies almost exclusively on the use of antiparasitic medications, and rarely antibiotics. To date, however, it is unclear why common post-treatment complications include abscessation. This study quantified the microbial composition of parasitic cyst contents in a higher-order rodent host, using multi-kingdom shotgun metagenomics, to improve our understanding of gut microbial translocation and adaptation strategies in wild environments. Analysis was conducted on DNA from two hepatic parasitic cysts (Hydatigera (Taeenia) taeniaeformis) in an adult vole mouse (Microtus arvalis), and from feces, liver, and peritoneal fluid of three other vole family members living in a vegetable garden in Ohio, USA. Bacterial metagenomics revealed the presence of gut commensal/opportunistic species, including Parabacteroides distasonis, Klebsiella variicola, Enterococcus faecium, and Lactobacillus acidophilus, inhabiting the cysts. Parabacteroides distasonis and other species were also present outside the cyst in the peritoneal fluid. Remarkably, viral metagenomics revealed various murine viral species, but unexpectedly, it detected an insect-origin virus from the army moth (Pseudaletia/Mythimna unipuncta) known as Mythimna unipuncta granulovirus A (MyunGV-A) in both cysts, and in one fecal and one peritoneal sample from two different voles, indicating survival of the insect virus and adaption in voles. Metagenomics also revealed a significantly lower probability of fungal detection in the cysts compared to other samples (peritoneal fluid, p<0.05; and feces p<0.05), with single taxon detection in each cyst for Malassezia and Pseudophaeomoniella oleicola. The samples with a higher probability of fungi were the peritoneal fluid. In conclusion, commensal/pathobiont bacterial species can inhabit parasitic tapeworm cysts, which needs to be considered during therapeutic decisions of cysticerci or other chronic disease scenarios where immune privileged and spatially restricted ecosystems with limited nutrients and minimal presence of immune cells could facilitate microbial adaptation, such as within gut wall cavitating micropathologies in Crohns disease.

microbiology↗

bioRxiv2023

Vps18 contributes to phagosome membrane integrity in Mycobacterium tuberculosis-infected macrophages.

Mycobacterium tuberculosis (Mtb) has evolved to be exquisitely adapted to survive within host macrophages. The capacity to damage the phagosomal membrane has emerged as central to Mtb virulence. While Mtb factors driving membrane damage have been described, host factors that repair that damage to contain the pathogen remain largely unknown. We used a genome-wide CRISPR screen to identify novel host factors required to repair Mtb-damaged phagosomal membranes. Vacuolar protein sorting-associated protein 18 (Vps18), a member of the HOPS and CORVET trafficking complexes, was among the top hits. Vps18 colocalized with Mtb in macrophages beginning shortly after infection, and Vps18-knockout macrophages demonstrated increased damage of Mtb-containing phagosomes without impaired autophagy. Mtb grew more robustly in Vps18-knockout cells, and the first-line anti-tuberculosis antibiotic pyrazinamide was less effective. Our results identify Vps18 as required for phagosomal membrane integrity in Mtb-infected cells and suggest that modulating phagosome integrity may hold promise for improving the efficacy of antibiotic treatment for TB.

microbiology↗

bioRxiv2021

A New Bioink For Improved 3D Bioprinting Of Bone-Like Constructs

Bone tissue loss can occur due to disease, trauma or following surgery, in each case treatment involving the use of bone grafts or biomaterials is usually required. Recent development of three-dimensional (3D) bioprinting (3DBP) has enabled the printing of customized bone substitutes. Bioinks used for bone 3DBP employ various particulate phases such as ceramic and bioactive glass particles embedded in the bioink creating a composite. When composite bioinks are used for 3DBP based on extrusion, particles are heterogeneously distributed causing damage to cells due to stresses created during flow in the matrix of the composite. Therefore, the objective of this study was to develop cell-friendly osteopromotive bioink mitigating the risk of cell damage due to the flow of particles. Towards this end, we have linked organic and inorganic components, gelatin methacryloyl (GelMA) and Ag-doped bioactive glass (Ag-BaG), to produce a hybrid material, GelMA-Ag-BaG (GAB). The distribution of the elements present in the Ag-BaG in the resulting hybrid GAB structure was examined. Rheological properties of the resulting hydrogel and its printability, as well as the degree of swelling and degradation over time, were also evaluated. GAB was compared to GelMA alone and GelMA-Ag-BaG nanocomposites. Results showed the superiority of the hybrid GAB bioink in terms of homogenous distribution of the elements in the structure, rheological properties, printability, and degradation profiles. Accordingly, this new bioink represents a major advance for bone 3DBP.

bioengineering↗

bioRxiv2017

Cytosine methylation within marine sediment microbial communities: potential epigenetic adaptation to the environment

Marine sediments harbor a vast amount of Earths microbial biomass, yet little is understood regarding how cells subsist in this low-energy, presumably slow-growth environment. Cells in marine sediments may require additional methods for genetic regulation, such as epigenetic modification via DNA methylation. We investigated this potential phenomenon within a shallow estuary sediment core spanning 100 years of age across its depth. Here we provide evidence of dynamic community m5-cytosine methylation within estuarine sediment metagenomes using a methylation-sensitive Illumina assay. The methylation states of individual CpG sites were reconstructed and quantified across three depths within the sediment core. A total of 6254 CpG sites were aligned for direct comparison of methylation states between samples, with 4235 sites mapped to taxa and genes. Our results demonstrate the presence of differential methylation within environmental CpG sites across an age/depth gradient of sediment. We show that epigenetic modification can be detected within complex environmental communities. The change in methylation state of environmentally relevant genes across depths may indicate a dynamic role of DNA methylation in biogeochemical processes.

microbiology↗