Biology subjects

Butcher, J.

Publications and source records attributed to Butcher, J..

14 recordsLinked to original sources

bioRxiv2026

Reliable and Efficient Agentic Protein Design with Toolset Harnessing

Computational protein design workflows require the orchestration of multiple tools, data formats, and analysis steps that are often fragmented. Here, we introduce Chaperone, a lightweight and customizable framework that enables seamless integration of RFdiffusion3 (RFD3) with downstream sequence design, structure prediction, and evaluation tools, including ProteinMPNN, AlphaFold3 (AF3), and more than 50 other protein design tools. By combining a programmable agent-friendly interface with parallelized execution on high-performance computing systems, Chaperone as a harness enables rapid iteration and scalable exploration of complex protein design workflows. Across an evaluation of nine biomolecular design tasks, we find using Chaperone reduces total token usage relative to baseline agents by 86% and GPU costs by 64%, while increasing the overall computational design pass rate. We demonstrate Chaperones utility for large-scale design-space exploration through a two-week end-to-end small-molecule binder design campaign, yielding low nanomolar binders to cortisol zero shot. We further demonstrate its versatility across distinct design challenges, including the design of a symmetric 12-subunit self-assembly nanoparticle and a linker that mediates interactions between C2- and D3-symmetric protein complexes to form crystalline assemblies. Together, these results demonstrate how agent harnesses built around standardized tool interfaces can substantially improve the efficiency and success of complex, long-horizon agentic design workflows in biology. We make Chaperone freely available to the community on GitHub: https://github.com/RosettaCommons/chaperone.

biochemistry↗

bioRxiv2026

Finite afterload during asymmetrically cycled preload promotes fetal ventricular growth, maturation, and contractile function while suppressing fibrotic remodeling

Myocardial development requires precise modulation of its growth and maturation by the mechanical loads within cardiac cycle, including preload and afterload. However, the mechanisms by which these natural cardiac loads interact to simultaneously govern growth and maturation of the developing myocardium in both cellular and mesoscale levels remain poorly understood. Here, we developed a naturally engineered fetal ventricular tissue (NFVT) platform that enables the application of afterload under dynamic preload using cyclic stretching with an asymmetrical duty cycle (asymmetrically cycled preload) to better replicate the natural cardiac loading in chick NFVT. Our results showed that low afterload (LA) enhanced NFVT contractile function with sustained tissue growth and improved cardiomyocyte maturation while suppressing fibrosis phenotypes. These effects were associated with reduced YAP1 and NOTCH activation in cardiomyocytes and enhanced tissue architecture. In contrast, high afterload (HA) induced contractile impairment with fibrotic remodeling through activation of cardiomyocyte PIEZO1/YAP1 signaling and sustained fibroblast entanglement. TeaserLow afterload under asymmetrically cycled preload promotes NFVTs contractile function with sustained tissue growth and cardiomyocyte maturation while suppressing fibrosis phenotypes through regulation of cellular mechanotransduction, including minimal PIEZO1 expression and inhibited YAP1 and NOTCH activation in cardiomyocytes, and improvement of collective cellular organization.

Cell Biology↗

bioRxiv2026

Directional bone matrix mineralization in the CAM assay is governed by vascular integration and matrix remodeling

Bone matrix mineralization plays an important role in maintaining bone health but is a highly dynamic process for which few physiologically relevant model systems exist. The chorioallantoic membrane (CAM) assay is one such assay and has been used to study biomineralization; however, the mechanisms controlling mineral deposition in the CAM assay remain poorly understood. Here, we implanted decellularized, organic bone matrices onto the CAM and investigated their mineralization using a combination of micro-computed tomography, Raman spectroscopy, scanning electron microscopy, and histochemistry. These studies revealed increased mineralization and microvessel density on the shell-facing interface of the implanted matrices, while their embryo-facing counterpart lacked mineralization but was densely infiltrated by cells. Seeding organic bone matrices with mesenchymal stromal cells (MSCs) prior to CAM-implantation prevented mineralization by altering matrix micro- and nanostructure and limiting vessel integration. Collectively, our results suggest that mineral precursors from the eggshell and vasculature combine to mineralize organic bone matrix in the CAM assay, while proteolytic remodeling by invaded or implanted stromal cells inhibits that process. Our findings provide critical new insights into the interplay between acellular and cellular drivers of bone matrix mineralization, and will inform future studies of biomineralization using the CAM assay.

bioengineering↗

bioRxiv2026

Kinemomics: spatiotemporal morphodynamic mapping of ventricular kinematic subpopulations in organotypic fetal heart slices

Recent advancements in omics technologies have deepened our understanding of complex biological systems and the interplay among gene expression, mechanical cues, and cellular metabolism. However, capturing the spatiotemporal dynamics of live cellular behavior within intact tissue contexts remains a major challenge. Here, we introduce Kinemomics, a framework for spatiotemporal mapping of morphogenetic kinematic states in living tissue. Using a novel fetal cardiac organotypic slice culture system that preserves both beating and morphogenesis, Kinemomics integrates live fluorescent labeling, timelapse imaging, and endpoint immunofluorescence to generate morphodynamic profiles across full-thickness developing myocardium. We validate Kinemomics under controlled Yap-1 signaling perturbations and demonstrate its ability not only to intrinsically decode region- and treatment-specific phenotypes, but also to resolve their spatiotemporal evolution during development and malformation. Kinemomics further reveals localized emergent developmental and malforming fronts that drive system-level behavior, establishing a generalizable platform for investigating computational morphodynamics and cross-scale interactions in complex spatiotemporal systems.

developmental biology↗

bioRxiv2026

Generative design of sequence specific DNA binding proteins

De novo protein design has advanced rapidly in recent years, yet the programmable recognition of specific DNA sequences remains a longstanding challenge. Here we describe a deep learning based approach for designing sequence selective DNA binding proteins. Our method combines structure generation using RFdiffusion3 with explicit screening against off-target interactions using AlphaFold3. We test this approach by generating 96 designs for each of 15 diverse DNA targets and identify specific binders for 7 targets, representing a ~100-fold improvement in success rates over previous approaches. We further characterize the binding landscape using variant competition assays and randomized library screening, revealing robust sequence discrimination across diverse targets. Together, these results represent a significant step forward in de novo sequence specific DNA binder design.

synthetic biology↗

bioRxiv2025

Spatiotemporal Atlas of Heart Development Reveals Blood-Flow-Dependent Cellular, Structural, Metabolic, and Spatial Remodeling

Embryonic heart development depends on coordinated interactions between cellular programs, molecular signaling, and biomechanical forces, yet how mechanical cues shape cellular and molecular pathways remains incompletely understood. We perturbed cardiac blood flow by partial left or right atrial ligation (LAL/RAL) in chick embryos, generating chamber-specific hemodynamic gain- or loss-of-function states. Using single-cell and unbiased, high-resolution spatial transcriptomics, we generated a spatiotemporal atlas of flow-dependent tissue development, enabling systematic investigations of bidirectional interactions between blood-flow mechanics and tissue development. Spatially resolved analyses recapitulated key features of normal morphogenesis including regional maturation and the cellular neighborhood. We further revealed flow-specific remodeling across molecular, cellular, and architectural levels. Altered flow induced LOX-expressing cardiomyocyte and endocardial states, disrupted ventricular layer organization, and delayed maturation, alongside transient metabolic and ion-transport adaptations. Together, these findings define how redistributed blood flow reshapes developing cardiac tissues and provide a framework for studying flow-dependent remodeling in morphogenesis and malformation.

developmental biology↗

bioRxiv2025

Longitudinal Live Imaging Derived 4D Hemodynamics and Dynamic Tissue Mechanics Across Outflow Tract Morphogenesis

Growth and remodeling of the cardiac outflow tract (OFT) is poorly understood but associated with serious congenital heart defects (CHD). While only a minority of CHDs have identifiable genetic causes, the functional roles of mechanical forces in OFT remodeling are far less characterized. A key barrier has been the lack of longitudinal investigations examining the interplay between dynamic blood flow and wall motion across clinically relevant stages. Here, we developed a live high-frequency ultrasound derived 4D moving-domain computational fluid dynamics (CFD) simulation approach, enabling longitudinal quantification of OFT hemodynamics and tissue mechanics in the same embryos across Hamburger-Hamilton (HH) stage 21 to HH27. We found that rising wall shear stress (WSS) strongly correlates with tissue extension in the distal OFT, whereas the proximal OFT experiences increasing expansive strains and higher hydrostatic stress with heartbeats. Additionally, we identified a double-helical flow pattern in the OFT lumen, possibly reflecting an evolutionary legacy for directing oxidized and non-oxidized blood flow before great vessel septation. Together, our results advanced insights in how hemodynamic forces and tissue stress contribute to OFT remodeling and septation.

developmental biology↗

bioRxiv2025

Accelerating Biomolecular Modeling with AtomWorks andRF3

Deep learning methods trained on protein structure databases have revolutionized biomolecular structure prediction, but developing and training new models remains a considerable challenge. To facilitate the development of new models, we present AtomWorks: a broadly applicable data framework for developing state-of-the-art biomolecular foundation models spanning diverse tasks, including structure prediction, generative protein design, and fixed backbone sequence design. We use AtomWorks to train RosettaFold-3 (RF3), a structure prediction network capable of predicting arbitrary biomolecular complexes with an improved treatment of chirality that narrows the performance gap between closed-source AlphaFold3 (AF3) and existing open-source implementations. We expect that AtomWorks will accelerate the next generation of open-source biomolecular machine learning models and that RF3 will be broadly useful as a structure prediction tool. To this end, we release the AtomWorks framework (https://github.com/RosettaCommons/atomworks), together with curated training data, code and model weights for RF3 (https://github.com/RosettaCommons/modelforge) under a permissive BSD license.

biochemistry↗

bioRxiv2025

Valve endothelial monolayer fissuring via RhoA activity induces 3D calcific aortic valve lesion emergence as revealed by a longitudinal live-imaging platform

Calcific aortic valve disease (CAVD) is a degenerative disease with wide prevalence in the aging population and a low survival rate after onset of symptoms, yet there are no effective pharmacological treatments. Many patients present to the clinic with symptoms at end-stages of CAVD, when the disease may be irreversible. The ability to identify and live-trace calcific lesion emergence in-vivo would allow for the identification of disease biomarkers and discovery of therapeutic targets at earlier, more treatable stages. In this work, we establish a new multimodal in-vitro CAVD platform consisting of lineage traced-VEC and VIC cells in a 3D model combined with live optical coherence and fluorescence microscopy to unravel VEC, VIC, and matrix transitional events during calcific lesion formation. We discover that fissuring of the endothelial monolayer combined with the formation of dense aggregates in adjacent regions is a key biomarker of the onset of lesion formation. This coincides with the formation of dense VIC and ECM conglomerates under endothelial aggregates, an additional biomarker of pathogenesis. Further, we discover that fibrotic tissue compaction is correlated with but not necessary for lesion formation. Additionally, we identify RhoA activation in disease-treated samples. We demonstrate that RhoA inhibition through ROCK, but not Rac1 inhibition, prevents delamination of the endothelial monolayer, fibrotic remodeling, and emergence of calcific lesions. Together, this work establishes a new longitudinal live-imaging platform that identifies emergent cell and matrix biological signatures of CAVD onset and enables the evaluation of therapeutic interventions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/660489v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@dc378org.highwire.dtl.DTLVardef@19c37d5org.highwire.dtl.DTLVardef@11581c6org.highwire.dtl.DTLVardef@fb0a6a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Systematic Metaproteomics Mapping Reveals Functional and Ecological Landscapes of Human Gut Microbiota Responses to Therapeutic Drugs

Therapeutic compounds exert impacts on gut microbiota; however, how they affect the community functional ecology, especially as reflected at the protein level, remains largely unexplored. In this study, we systematically map metaproteomic responses of ex vivo human gut microbiota to 312 compounds, generating 4.6 million microbial protein responses, available as an interactive resource (https://shiny.imetalab.ca/MPR_Viz/). Protein-level analyses identify significant metaproteomic shifts induced by 47 compounds, with neuropharmaceuticals as the sole drug class significantly enriched among these hits. Further analyses on the community level reveal a tri-stability pattern in microbial composition and the emergence of three distinct functional states, based on a functional beta-diversity metric. Notably, neuropharmaceuticals cause particularly strong effects on the microbiomes, lowering the proteome-level functional redundancy and raising the level of antimicrobial resistance proteins, ultimately pushing the microbiome into an alternative functional state. Preliminary validation suggests that enhancing functional redundancy may contribute to maintaining microbiota resilience against neuropharmaceutical-induced antimicrobial resistance. Overall, this work establishes a comprehensive view of how drugs influence gut microbiome function and ecology at the protein level, proposes a landscape-based framework for interpreting community resilience, and highlights the need to consider protein-level and ecological responses in the evaluation of therapeutic interventions.

microbiology↗

bioRxiv2023

Dry surface biofilm formation by Candida auris facilitates persistence and tolerance to sodium hypochlorite

Candida auris is an enigmatic fungal pathogen, recently elevated to the critical priority group of pathogens by the World Health Organization. Of key concern is its ability to cause outbreaks within intensive and chronic care units, facilitated through its environmental persistence. We investigated the susceptibility of phenotypically distinct C. auris isolates to sodium hypochlorite (NaOCl) disinfection, and the subsequent role of biofilms in surviving disinfection using a dry-surface biofilm (DSB) model and transcriptomic profiling. Planktonic cells were tested for susceptibility to NaOCl in suspension, with biofilm formation using the DSB model consisting of consecutive 48 hr cycles with/without media across a 12-day period, assessed using viable counts, biomass assays, and microscopy. Disinfection efficacy was assessed using clinically relevant protocols of 500-1000ppm for 1-5min. RNA-sequencing was performed on untreated DSBs in comparison to planktonic cells. Isolates were found to be sensitive to NaOCl planktonically at concentrations [≤]62.5 ppm, and grew robust biofilms using the DSB protocol. Biofilms developed tolerance to all NaOCl treatment parameters, with only 2-4 log10-reductions in viable cells observed at highest concentrations. Transcriptomics identified ABC transporters and iron acquisition pathways as strongly upregulated in DSBs relative to planktonic cells. Our novel findings have optimised a DSB protocol in which C. auris biofilms can mediate tolerance to adverse conditions such as NaOCl disinfection, suggesting a lifestyle through which this problematic yeast can environmentally persist and transmit. Mechanistically it has been shown for the first time that upregulation of small-molecule and iron transport pathways are potential facilitators of environmental survival. IMPORTANCECandida auris is a pathogenic yeast that has been responsible for outbreaks in healthcare facilities across the globe, predominantly affecting vulnerable patients. This organism displays a concerning ability to persist within the healthcare environment that is likely facilitated by attaching onto surfaces and developing protective microbial communities knows as biofilms. These communities allow cells to survive and tolerate disinfection with bleach. In this study, we show that C. auris forms robust biofilms on surfaces which promote survival up to 12 days, even with prolonged drying periods. We also demonstrate that development of these biofilms over time significantly reduces the efficacy of hypochlorite disinfection. By investigating the molecular mechanisms of biofilms, we have shown that these biofilms express efflux pumps, which may actively remove hypochlorite molecules from cells, allowing them to tolerate disinfection, and that uptake of iron from the external environment is also important for survival of these communities.

microbiology↗

bioRxiv2021

Revealing Protein-Level Functional Redundancy in the Human Gut Microbiome using Ultra-deep Metaproteomics

Functional redundancy is a key property of ecosystems and represents the fact that phylogenetically unrelated taxa can play similar functional roles within an ecosystem. The redundancy of potential functions of human microbiome has been recently quantified using metagenomics data. Yet, the redundancy of functions which are actually expressed within the human microbiome remains largely unexplored. Here, we quantify the protein-level functional redundancy in the human gut microbiome using metaproteomics and network approaches. In particular, our ultra-deep metaproteomics approach revealed high protein-level functional redundancy and high nestedness in proteomic content networks - bipartite graphs that connect taxa with their expressed functions. We further examined multiple metaproteomics datasets and showed that various environmental factors, including individuality, biogeography, xenobiotics, and disease, significantly altered the protein-level functional redundancy. Finally, by projecting the bipartite proteomic content networks into unipartite weighted genus networks, functional hub genera across individual microbiomes were discovered, suggesting that there may be a universal principle of functional organization in microbiome assembly. HighlightsO_LIUltra-deep metaproteomics reveals high protein-level functional redundancy in the human gut microbiome C_LIO_LIWithin-sample proteomic content networks display universal topology C_LIO_LIVarious environmental factors influence the redundancy of expressed functions C_LIO_LIFunctional hub genera are present across different datasets C_LI

systems biology↗

bioRxiv2021

Examining the effects of an anti-Salmonella bacteriophage preparation, BAFASAL, on ex vivo human gut microbiome composition and function using a multi-omics approach

Salmonella infections (salmonellosis) pose serious health risks to humans, usually via contamination in our food chain. This foodborne pathogen causes major food losses and human illnesses that result in significant economic impacts. Pathogens such as Salmonella have traditionally been kept at bay through the use of antibiotics, but antibiotic overuse within the food industry has led to the development of numerous multidrug-resistant bacterial strains. Thus, governments are now restricting antibiotic use, forcing the industry to search for alternatives to secure safe food chains. Bacteriophages, viruses that infect and kill bacteria, are currently being investigated and used as replacement treatments and prophylactics due to their specificity and efficacy. They are generally regarded as safe alternatives to antibiotics as they are natural components of the ecosystem. One example is BAFASEL, a commercial bacteriophage mixture that specifically targets Salmonella and is currently approved for use in poultry farming. However, when specifically used in the industry they can also make their way into humans through our food chain or exposure as is the case for antibiotics. In particular, agricultural workers could be repeatedly exposed to bacteriophages supplemented in animal feeds. To the best of our knowledge, no studies have investigated the effects of such exposure to bacteriophages on the human gut microbiome. In this study, we used a novel in vitro assay called RapidAIM to investigate BAFASALs potential impact on five individual human gut microbiomes. Multi-omics analyses, including 16S rRNA gene sequencing and metaproteomic, revealed that ex vivo human gut microbiota composition and function were unaffected by BAFASAL treatment providing an additional measure for its safety. Due to the critical role of the gut microbiome in human health and the known role of bacteriophages in regulation of microbiome composition and function, we suggest assaying the impact of bacteriophage-cocktails on the human gut microbiome as a part of their safety assessment. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2020

Experimental-based Computational Modeling Distinguishes Early Cardiac Outflow Tract Compensation Mechanisms

Mechanical forces are essential for proper growth and remodeling of the primitive pharyngeal arch arteries (PAAs) into the great vessels of the heart. Despite general acknowledgement of a link between abnormal hemodynamics and cardiac malformations, the direct correlation between hemodynamics and pharyngeal arch artery morphogenesis remains poorly understood. The elusiveness behind understanding hemodynamic-malformation links is largely due to the difficulty of performing isolated hemodynamic perturbations and quantifying key hemodynamic indices in-vivo. To overcome this issue, minimally invasive occlusion experiments were combined with three-dimensional anatomical models of development and in-silico testing of experimental phenomenon. This combined experimental-computational approach led to a mechanistic understanding of physiological compensation mechanisms in abnormal cardiac morphogenesis. Using our experimental-based framework, we detail morphological and hemodynamic changes twenty-four hours post vessel occlusion. To gain mechanistic insights into the dynamic vessel adaptation process, we perform in-silico occlusions which allow for quantification of instantaneous changes in mechanical loading. We follow the propagation of small defects in a single embryo Hamburger Hamilton (HH) Stage 18 embryo to a more serious defect in an HH29 embryo. Results demonstrate that abnormal PAA hemodynamics can precipitate abnormal cardiac function given the correct timing and location of injury. Following vessel occlusion, morphology changes along the arches are no longer a simple flow-mediated response but rather work to maintain a range of wall shear stress values. Occlusion of the presumptive aortic arch overrides natural growth mechanisms and prevents it from becoming the dominant arch of the aorta. Author SummaryThe developing great vessels transport flow from the heart to the rest of the body. Proper spatial temporal morphogenesis of the primitive paired vessels into the definitive outflow tract of the heart is critical for normal cardiac function. Malpatterning of the great vessels is highly prevalent in congenital heart defects and occurs in conjunction with other intracardiac malformations, such as impaired ventricle and valve development. In this work, we combine experimental-based computational modeling with theoretical adaptation principles. Our combined experimental-computational framework allows for the delineation of immediate and longer-term vascular remodeling as well as the physical mechanisms behind such changes. We show that a small flow obstruction originating within the developing vessels can propagate into structurally serious malformations with impaired functionality.

developmental biology↗