Biology subjects

Hall, M.

Publications and source records attributed to Hall, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Liposomal DAGLβ Inhibitor Shows Substantial Antinociceptive Potency Mediated by AMPK in a Neuropathic Pain Mouse Models

Diacylglycerol lipase-beta (DAGL{beta}), a biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), is highly expressed in macrophages and serves as a potential contributor to inflammatory and hypernociceptive responses. Moreover, the DAGL{beta} inhibitor KT109 produces antinociceptive effects in various rodent pain models, and its liposome formulation dramatically increases its antinociceptive potency in a lipopolysaccharide mouse model of inflammatory pain compared to free KT109. Here, we further optimized the liposomal KT109 formulation through adjustment of lipid composition and improved processing, achieving 100-fold higher drug encapsulation efficiency than the previous formulation, uniform size distribution, and excellent storage stability. Further, the second generation liposomal KT109 showed remarkable potency (ED50 [95% CL] = 0.226 [0.113-0.454] mg/kg) in a mouse model of paclitaxel-induced neuropathic pain, compared to free KT109 (ED50 [95% CL] = 31 [21-46] mg/kg). Liposomal KT109 (0.4 mg/kg) fully reversed paclitaxel-induced mechanical hypersensitivity for up to 24 hours, and its antinociceptive effects were sustained following a week of daily injections, with no detectable tolerance. In addition, the adenosine monophosphate-activated protein kinase (AMPK) inhibitor, dorsomorphin, prevented the antinociceptive effects of KT109, which is consistent with previous work demonstrating AMPK activation mediates the antinociceptive phenotype of DAGL{beta} knockout mice.

pharmacology and toxicology↗

bioRxiv2026

Probing the sequence variability tolerance in a de novo α-helical barrel biocatalyst

De novo-designed enzymes have recently achieved high catalytic activity and stereoselectivity while demonstrating exceptional thermostability in entirely novel protein scaffolds. Among these, -helical barrel protein scaffolds are attractive structures for biocatalysis due to their structural simplicity, high thermostability, and rationalizable sequence patterning. However, enabling major structural reengineering of these scaffolds while maintaining the structure, stability and catalytic activity while also improving soluble protein production remain major challenges and pose the fundamental question how engineerable a de novo backbone-sequence pair is. Here, we combine deep learning based and classic computational protein design to modify and optimize de novo -helical barrel biocatalysts. Using the previously reported six-helical barrel 6H5L as a model scaffold, AlphaFold2-guided RosettaRemodel enabled the design of a truncated variant, whose crystal structure closely matches the design model. Additional sequence-redesign using ProteinMPNN generated a variant with a tenfold increase of soluble protein yield in Escherichia coli. Biochemical, biophysical, and structural analyses showed that both variants retained the overall barrel architecture, high thermal stability, and catalytic activity for both purified protein and whole-cell systems. Detailed kinetic analysis on the variants showed both variation in kcat and Km, reflecting changes in catalytic turnover and substrate binding. Together, these approaches provide new insights and possibilities for the further engineering of functional de novo -helical barrels, their ability to withstand dramatically large sequence changes and their broader application in biocatalysis and biotechnology.

biochemistry↗

bioRxiv2026

Hidden diversity and host specificity of Cladocopium symbionts revealed by phylotranscriptomics in giant sea anemones.

Giant sea anemones form an intricate, metabolically linked symbiosis with anemonefish (genus Amphiprion) and dinoflagellates (family Symbiodiniaceae). While the association between fish and anemone is well documented, the identity and distribution of resident Symbiodiniaceae remains poorly characterized. Here, we used RNA-sequencing of anemone tentacles and post-assembly filtering to recover Symbiodiniaceae sequences from Japanese anemones. Using transcriptome-wide phylogenetic approaches, we identified distinct Symbiodiniaceae strains in seven sea anemone species and four cryptic lineages of Entacmaea quadricolor. Phylogenies of 1300 and 214 nuclear genes resolved relationships among these symbionts with greater sensitivity than traditional molecular markers such as the Internal Transcribed Spacer 2 (ITS2) or partial 28S ribosomal DNA. Marker-based assessments indicate these Symbiodiniaceae as novel and specific to sea anemone hosts. Our transcriptome-wide phylogenies reveal previously undescribed Cladocopium (Clade C) strains associated with giant sea anemones, with symbionts clustering according to host species and lineage. Cophylogenetic analysis revealed significant phylogenetic congruence between anemone and Symbiodiniaceae, indicating a nonrandom association between host and symbiont evolutionary histories. Two sympatric cryptic lineages of E. quadricolor previously identified through host transcriptomics harbor distinct, specific Symbiodiniaceae assemblages. Analysis of genes under positive selection indicates functional divergence between these Symbiodiniaceae; three-dimensional reconstructions of symbiont cells using serial block-face scanning electron microscopy show consistent differences in cellular organization between two strains, further supporting their distinct functional identity. Together, our results uncover a hidden diversity of host-specific Symbiodiniaceae and suggest that cryptic diversification in giant sea anemones is mirrored by diversification of their algal symbionts within this tripartite symbiosis.

evolutionary biology↗

bioRxiv2026

Deinococcus radiodurans HU: a versatile architect of nucleoid structure and plasticity

Nucleoid-associated proteins are known to compact and organise bacterial genomes, but how their local DNA binding shapes higher-order chromatin architecture remains unclear. Using in vitro and in situ approaches, we investigated how Deinococcus radiodurans HU (DrHU) orchestrates nucleoid organisation. DrHU engages DNA through two synergistic binding modes: high-affinity {beta}-hairpin-mediated stabilisation of DNA loops and low-affinity, multivalent interaction via its N-terminal tail, driving parallel, uniformly spaced DNA alignment. In vitro, DrHU organises supercoiled plasmid DNA into regular 2D lattices of double-spirals, while in situ, UV-C light induces a nucleoid transition from a loose DNA mesh to a blue liquid crystalline phase characterised by DNA swirls. The striking structural similarity and shared inter-filament spacing of these two arrangements suggests that DrHU plays a pivotal role in genome organisation through its versatile binding properties that enable DNA loop stabilisation, protection and DNA bridging, linking molecular interactions to 3D genome ordering and stress-induced remodelling.

microbiology↗

bioRxiv2026

Phenotypic screening for small molecules that lower PrP in cultured cells

PrP lowering is a validated therapeutic hypothesis in prion disease. To identify small molecules that reduce PrP levels, we performed phenotypic screening in cultured cells. To prioritize PrP specificity in our primary screen, we generated mouse N2a cells stably expressing GFP and used high content imaging analysis to select compounds that lowered PrP without affecting GFP signal or cell viability. Screening a curated library of 3,492 compounds with annotated mechanisms of action identified two small molecules, EYH (PubChem CID: 71678945) and LCZ (PubChem CID: 24970350), that selectively and dose-dependently lowered PrP. Proteomics on whole cell lysates identified PrP as the #1 or #2 most potently downregulated out of 8,722 proteins detected. Both compounds minimally affected Prnp mRNA, reduced expression of exogenously transfected PrP, and remained potent in non-dividing primary cells, consistent with a post-translational mechanism. Co-treatment with the proteasome inhibitor MG132 yielded accumulation of unglycosylated PrP, demonstrating proteasome clearance of PrP. However, both compounds showed limited or no activity in human cell lines, and failed to reduce PrP in vivo after 14 days of treatment. These findings highlight the challenges associated with mechanism-agnostic phenotypic screening for PrP-lowering compounds and support prioritizing compounds with known mechanisms of action.

neuroscience↗

bioRxiv2026

Identification and Masking of Artefactual and Misleading Within-Host Variants in Deep-Sequencing SARS-CoV-2 Data

Deep sequencing data are increasingly used to study within-host viral diversity and to inform evolutionary inference. For SARS-CoV-2, analyses based on intra-host single-nucleotide variants (iSNVs) have been widely applied to quantify within-host diversity and infer transmission dynamics. However, these applications critically depend on the reliable identification of low-frequency variants, which remain vulnerable to systematic and technical artefacts. In this study, we show that recurrent artefactual iSNVs are common in large-scale SARS-CoV-2 sequencing data and can persist even under conservative minor allele frequency (MAF) thresholds. Using data from the UKs Office for National Statistics COVID-19 Infection Survey, we demonstrate that such artefacts are predominantly sequencing centre-rather than protocol-specific. Each centre exhibits a modest, distinct set of recurrent artefactual variants showing little overlap with sites routinely masked at the consensus level. To address this, we developed a systematic, dataset-aware framework that uses recurrence within sequencing datasets to identify small, noise-adapted sets of artefactual iSNVs to mask. Applying this framework reduces spurious sharing of low-frequency variants between samples and qualitatively alters downstream inferences, including estimates of within-host diversity and transmission bottleneck sizes. Together, these findings highlight the importance of explicit, dataset-aware artefact control for robust inference from within-host variation, particularly as genomic studies increasingly seek to exploit sub-consensus diversity in rapidly evolving pathogens such as SARS-CoV-2.

genomics↗

bioRxiv2026

Exposure to a low dose mixture of endocrine disrupting chemicals alters the brain transcriptome and animal behavior

Exposures to pervasive chemical toxicants such as endocrine disrupting chemicals (EDCs) are associated with adverse neurological and neurodevelopmental deficits. Although EDCs are widespread as sparse mixtures in the environment, most research has focused on single chemicals at high concentrations. Here, we studied the effects of ldEDC: a low-dose mixture of widely prevalent toxicants at doses representative of normal human exposure levels. Primary cultured mouse neurons treated with ldEDC exhibited altered gene expression compared to vehicle controls in genes critical for neuron activity, indicating low doses EDCs can affect neuronal function directly. We next tested persistent exposure through the maternal diet to define perinatal effects on offspring. Exposed offspring exhibited differences in development, tactile sensitivity, and sex-specific changes in motor behavior. Cortical single-nuclei sequencing identified broad transcriptomic changes, particularly in distinct cortical layer subpopulations, excitatory neurons, and astrocytes. Cell-cell signaling between neurons and non-neuronal populations were altered in exposed mice, specifically in pathways associated with cellular adhesion. Transcriptomic differences were also sex-specific. Together, these in vitro and in vivo findings reveal molecular and phenotypic consequences of EDC exposure at a mixture of doses well below commonly studied levels and highlights common functional pathways of susceptibility.

neuroscience↗

bioRxiv2026

Bright calcium-modulated bioluminescent indicators for activity imaging and red photon-assisted synaptic transmission

Bioluminescent calcium sensors have unique uses in neuroscience and neuroengineering, enabling noninvasive imaging of neuronal activity and contactless activation of opsin-expressing neurons. However, the speed, range, and robustness of non-invasive imaging and rewiring or neuronal activity are limited by the radiance and dynamic range of existing bioluminescent calcium sensors. Here, we report the stepwise engineering of improved cyan-excitable red fluorescent proteins (mCyRFP4 and dCyRFP4), an improved red bioluminescent protein based on NanoLuc and CyRFP4 (Antares3), and an improved bioluminescent calcium sensor based on Antares3 (CaMBI3). Antares3 is 3-fold brighter than its predecessor, while CaMBI3 responds with an overall dynamic range of 24-fold, enabling high-sensitivity detection of calcium dynamics in muscle and neuronal tissues in vivo. Finally, a CaMBI3 variant and a red-shifted opsin ChRmine enabled red photon-assisted synaptic transmission in C. elegans. CaMBI3 thus facilitates genetically targeted non-invasive imaging and rewiring of neural activity in living animals.

neuroscience↗

bioRxiv2025

Bark-associated diazotroph communities are a cryptic source of nitrogen in forests

Nitrogen is an essential nutrient limiting forest productivity. Plants cannot access atmospheric N2 directly and rely on diazotrophic bacteria to fix nitrogen into bioavailable forms such as ammonium. Within forests, biological nitrogen fixation (BNF) occurs primarily in soils and root nodules. However it is unclear whether the extensive microbial communities recently discovered in tree bark can also fix nitrogen. Here we combine field measurements, metagenomic profiling, and biogeochemical assays to show that bark-dwelling diazotroph communities are abundant and active across diverse tree species. Bark from eight Australian tree species showed exceptionally high C:N ratios and depleted nitrogen stable isotope signatures ({delta}15N) consistent with locally fixed nitrogen, suggesting strong selection for diazotrophs. Consistently, bark microbial communities harbour phylogenetically and physiologically diverse diazotrophs, averaging [~]1012 cells m-2 and in higher relative abundance than underlying soils. Canonical and alternative nitrogenases were detected across eight bacterial phyla, primarily bark-adapted Alphaproteobacteria, Acidobacteriota, and Verrucomicrobiota, with strong signatures of purifying selection. Stable isotope labelling experiments demonstrated that bark-dwelling diazotrophs fix nitrogen at rates varying with tree species and habitat. In line with the presence of methane-oxidising diazotrophs, BNF was strongly stimulated by methane addition and suppressed by methanotroph inhibitors. Initial upscaling suggests bark-associated nitrogen fixation contributes up to 3.8 Tg N yr-1 globally ([~]6% of natural terrestrial BNF), with further studies required to constrain this budget and its contribution to tree nitrogen demands. Altogether, this discovery of substantial above-ground nitrogen inputs revises our understanding of forest nutrient cycles and redefines the functional scope of the caulosphere.

microbiology↗

bioRxiv2025

Ampyrone (4-Aminoantipyrine) is a Direct Agonist of Human Tyrosinase and Potential Therapeutic for Oculocutaneous Albinism and Disorders of Hypopigmentation.

Significant loss of pigmentation can increase visual disability, skin cancer risk, and psychosocial stress. Tyrosinase (TYR) catalyzes the first and rate-limiting step of melanin synthesis. Inhibitors of TYR are well established and are currently used in clinical settings; however, there is a dearth of direct activators of TYR. Here, using a unique human TYR construct, high-throughput screening, and computational analysis techniques, we identified ampyrone as a TYR activator. Ampyrone increased the in vitro catalytic activity of the intramelanosomal domain of human TYR (hTYR) and its hypomorphic variant, P406L, a cause of oculocutaneous albinism type 1B (OCA1B). Moreover, ampyrone induced melanin synthesis in both wild-type and OCA1B human melanocytes, as well as 3-dimension (3D) human skin cultures. Our results reveal ampyrone as a lead compound for first-in-class TYR activators, potentially accelerating the discovery of novel therapies for patients with genetic and acquired diseases of hypopigmentation.

pharmacology and toxicology↗

bioRxiv2025

Streptococcus pyogenes nuclease A interferes with host complement functions

Bacterial pathogens deploy diverse virulence factors to subvert host immunity, yet the molecular details of these interactions often remain unresolved. Here, we investigate the structure and host interactome of the Streptococcus pyogenes nuclease A (SpnA). We characterized the structure and dynamics of SpnA using hydrogen-deuterium exchange mass spectrometry and single-particle electron cryo microscopy, yielding the first structural insights to this protein. This allowed us to identify an additional oligonucleotide-binding domain whose flexible structure may play an important function in the nucleolytic activity of SpnA. Affinity-pulldown mass spectrometry identified the complement system membrane attack complex C5b67 components as key interactors in human plasma. Cross-linking mass spectrometry combined with integrative modeling identified the direct binding interfaces between SpnA and C5b67. These interfaces are highly conserved among genetically diverse S. pyogenes strains. The interaction between SpnA and C5b67 is suggested to prevent the assembly of a functional membrane attack complex. Taken together, our findings uncover a novel function of SpnA in complement inhibition and identifies new potential targets to prevent and treat S. pyogenes infections.

microbiology↗

bioRxiv2025

Metabolically flexible microorganisms rapidly establish glacial foreland ecosystems

An overriding question in ecology is how new ecosystems form. This question can be tested by studying colonisation of environments with little to no pre-existing life. Here, we investigated the functional basis of microbial colonisation in the forelands of a maritime Antarctic and an alpine Swiss retreating glacier, by integrating quantitative ecology, genome-resolved metagenomics, and biogeochemical measurements. Habitat generalists and opportunists rapidly colonize both forelands and persist across soil depth and decadal chronosequences that serve as proxies for temporal community dynamics. These microbes are metabolically flexible chemotrophic aerobes that overcome oligotrophic conditions by using both organic and inorganic compounds, including atmospheric trace gases and sulfur substrates, for energy and carbon acquisition. They co-exist with metabolically flexible early-colonising opportunists and metabolically restricted later-colonising specialists, including photosynthetic Cyanobacteria, ammonia-oxidising archaea, and obligate predatory and symbiotic bacteria, that exhibit narrower habitat distributions. Analysis of 589 species-level metagenome-assembled genomes reveals early colonisation both by generalists and opportunists is strongly associated with metabolic flexibility. Field- and laboratory-based biogeochemical measurements reveal the activity of metabolically flexible microbes rapidly commenced in the forelands. Altogether, these findings suggest primary succession in glacial foreland soils is driven by self-sufficient metabolically flexible bacteria that mediate chemosynthetic primary production and likely provide a more hospitable soil environment for subsequent colonisation.

microbiology↗

bioRxiv2024

A novel combination of CDK4/6 and PI3K inhibitors exhibits highly synergistic activity and translational potential in Ewing sarcoma.

Ewing sarcoma is a highly aggressive solid malignancy affecting children and young adults. Ewing sarcoma is driven primarily by EWSR1::FLI1, a fusion oncoprotein that has been notoriously difficult to target with traditional pharmacologic agents. There are numerous examples of preclinical promising combinations of small molecules that are never tested in pediatric clinical trials because agents fail to reach the market due to limited efficacy for common adult cancers. Moreover, the effectiveness of single-agent therapies for cancer treatment is often limited. To address these limitations, we selected 28 compounds that were largely FDA approved or in late stages of clinical development and known to regulate important pathways in Ewing sarcoma. We performed a drug screen in Ewing sarcoma cell lines with 180 combinations of tyrosine kinase inhibitors, cell cycle inhibitors, and conventional chemotherapy. The results of the screen revealed that a PI3K inhibitor, copanlisib, combined with a CDK4/6 inhibitor, ribociclib, exhibited strong synergistic anti-Ewing sarcoma activity. Using proteomic methods such as a reverse-phase protein array and western immunoblotting, we demonstrated that this combination induced a downregulation of the PI3K/AKT pathway as well as proteins involved in cell cycle regulation. We further confirmed these in vitro data using bulk RNA-sequencing. To evaluate the phenotypic effect of the PI3K/CDK4/6 inhibition in Ewing sarcoma lines, we performed apoptosis and cell cycle analyses using flow cytometry and demonstrated that ribociclib primarily induced a G0/G1 arrest with minimal effect on Ewing cell viability but significantly enhanced the apoptotic effect of copanlisib treatment. In a xenograft model of Ewing sarcoma, we demonstrated that the combination therapy significantly prolonged survival compared to treatment with either vehicle or single-agent therapy alone. Our findings identify a new candidate therapy combination for Ewing sarcoma using FDA-approved drugs and provide a resource of additional potential synergistic combinations for future validation. Statement of translational relevanceTreatment of patients with newly diagnosed Ewing sarcoma involves intensive multi- agent chemotherapy, radiation, and surgery, often leading to long-term toxicities. Children with relapsed or metastatic disease experience poor outcomes with five-year overall survival rates of only 15 to 30 percent. Therefore, new therapeutic approaches are urgently needed. Our drug screen revealed several promising combinations with synergistic anti-Ewing sarcoma activity including the PI3K inhibitor copanlisib with the CDK4/6 inhibitor ribociclib. This combination also significantly improved survival in a mouse xenograft model of Ewing sarcoma compared to treatment with vehicle or either drug alone. Pre-clinical validation of a combination therapy composed of two FDA- approved drugs nominates this combination for early phase trials for patients with with relapsed or refractory Ewing sarcoma, a group of patients greatly in need of new therapeutic opportunities. The data generated by our drug screen may also be used as a resource to identify other promising combinations for future validation as Ewing sarcoma therapies.

cancer biology↗

bioRxiv2024

Lineage-aware evolutionary analysis of hepatitis C virus within-host dynamics

Analysis of viral genetic data has previously revealed distinct within-host population structures in both untreated and interferon-treated chronic hepatitis C virus (HCV) infections. While multiple subpopulations persisted during the infection, each subpopulation was observed only intermittently. However, it was unknown whether similar patterns were also present after Direct Acting Antiviral (DAA) treatment, where viral populations were often assumed to go through narrow bottlenecks. Here we tested for the maintenance of population structure after DAA treatment failure. We analysed whole-genome next-generation sequencing data generated from a randomised study using DAAs (the BOSON study). We focused on samples collected from patients (N=84) who did not achieve sustained virological response (i.e. treatment failure) and had sequenced virus from multiple timepoints. For each individual, we tracked concordance in nucleotide variant frequencies through time. Using a sliding window approach, we applied sequenced-based and tree-based clustering algorithms across the entire HCV genome. Finally, we reconstructed viral haplotypes and estimated lineage specific within-host divergence rates from the haplotype phylogenies. Distinct viral subpopulations were maintained among a high proportion of individuals post DAA treatment failure. Using maximum likelihood modelling and model comparison, we found an overdispersion of viral evolutionary rates among individuals, and significant differences in evolutionary rates between lineages within individuals. These results suggest the virus is compartmentalised within individuals, with the varying evolutionary rates due to different viral replication rates or different selection pressures. We propose lineage awareness in future analyses of HCV evolution and infections to avoid conflating patterns from distinct lineages, and to recognise the likely existence of unsampled subpopulations.

evolutionary biology↗

bioRxiv2024

Computational design of a thermostable de novo biocatalyst for whole cell biotransformations

Over the last decades, transformative catalytic strategies have emerged, with biocatalysis cur-rently exerting a substantial influence on the pharmaceutical and fine chemical sectors. Fast pro-gress in the design of efficient enzymatic processes, however, suffers from the lack of readily available, stable, and customizable protein scaffolds that can be adapted to different catalytic functions. Here, we detail the design and experimental characterization of computationally de-signed de novo proteins with a non-natural fold for biocatalytic applications. The initial design and several variants form a helical barrel structure comprised of six antiparallel straight helices con-nected by five loops, creating an open central channel with two accessible cavities. To demon-strate the versatility of this scaffold, we designed variants with catalytic sites positioned at differ-ent locations along the central channel. All designs show high thermal stability and excellent agreement between experimental and calculated scattering profiles from small-angle X-ray scat-tering, while a crystal structure of a surface-redesigned variant confirms the close match be-tween the designed and experimental structures. Importantly, repositioning and engineering the catalytic sites enables substantial modulation of catalytic activity, with the best variant showing an approximately 11-fold increase in catalytic efficiency compared with the original design. Finally, the designs can be used for whole-cell biotransformations and tolerate up to 20% organic sol-vent. These results establish a stable de novo protein scaffold with tunable functional sites, offer-ing a versatile platform for biocatalysis, biosensing, and biosynthetic systems.

biochemistry↗

bioRxiv2024

Extracellular Vesicles heterogeneity through the lens of multiomics

Extracellular vesicles (EVs) are heterogenous in size, biogenesis, cargo and function. Beside small EVs, aggressive tumor cells release a population of particularly large EVs, namely large oncosomes (LO). This study provides the first resource of label-free quantitative proteomics of LO and small EVs obtained from distinct cancer cell types (prostate, breast, and glioma). This dataset was integrated with a SWATH Proteomic assay on LO, rigorously isolated from the plasma of patients with metastatic prostate cancer (PC). Proteins enriched in LO, which were identified also at the RNA level, and found in plasma LO significantly correlated with PC progression. Single EV RNA-Seq of the PC cell-derived LO confirmed some of the main findings from the bulk RNA-Seq, providing first evidence that single cell technologies can be successfully applied to EVs. This multiomics resource of cancer EVs can be leveraged for developing a multi-analyte approach for liquid biopsy.

cancer biology↗

bioRxiv2024

HCM-associated mutations in MYH6/7 drive pathologic expression of TGF-β1 in cardiomyocytes within weeks of developmental specification

Hypertrophic cardiomyopathy (HCM) is characterized by myocyte hypertrophy, sarcomere disarray, and myocardial fibrosis, leading to significant morbidity and mortality. As the most common inherited cardiomyopathy, HCM largely results from mutations in sarcomeric protein genes. Current treatments for HCM primarily focus on alleviating late-stage symptoms, with a critical gap in the detailed understanding of early-stage deficiencies that drive disease progression. We recently showed, in monolayers of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) with MYH7 R723C and MYH6 R725C mutations, altered expression of several extracellular matrix (ECM)-related genes with associated defects in cardiomyocyte-ECM adhesion. To better evaluate the cardiomyocyte-ECM interface and pathological ECM dynamics in early-stage HCM, here we adopted a 3D engineered heart tissue (EHT) model containing both cardiomyocytes and fibroblasts, the primary contributor to ECM remodeling. Mutant EHTs showed aberrant cardiomyocyte distribution, augmented calcium handling, and force generation compared to controls. Altered proteoglycan deposition and increased phosphorylated focal adhesion kinase (pFAK) further indicated changes in ECM composition and connectivity. Elevated transforming growth factor beta-1 (TGF-{beta}1) secretion and a higher proportion of activated fibroblasts were identified in mutant EHTs, along with sustained TGF-{beta}1 transcription specifically in mutant cardiomyocytes. Remarkably, blocking TGF-{beta}1 receptor signaling reduced fibroblast activation and contraction force to control levels. This study underscores the early interplay of mutant hiPSC-CMs with fibroblasts, wherein mutant cardiomyocytes initiate fibroblast activation via TGF-{beta}1 overexpression, independent of the immune system. These findings provide a promising foundation for developing and implementing novel strategies to treat HCM well before the manifestation of clinically detectable fibrosis and cardiac dysfunction.

bioengineering↗

bioRxiv2024

Computational design of highly active de novo enzymes

Enzymes are broadly used as biocatalysts in industry and medicine due to their coverage of vast areas of chemical space, their exquisite selectivity and efficiency as well as the mild reaction conditions at which they operate. Custom designed enzymes can produce tailor-made biocatalysts with potential applications extending beyond natural reactions. However, current design methods require testing of high numbers of designs and mostly produce de novo enzymes with low catalytic activities. As a result, they require costly experimental optimization and high-throughput screening to be industrially viable. Here we present rotamer inverted fragment finder-diffusion (Riff-Diff), a hybrid machine learning and atomistic modelling strategy for scaffolding catalytic arrays in de novo proteins. We highlight the general applicability of Riff-Diff by designing enzymes for two mechanistically distinct chemical transformations, the retro-aldol reaction and the Morita-Baylis-Hillman reaction. We show that in both cases it is possible to generate catalysts exhibiting activities rivalling those optimized by in-vitro evolution, along with exquisite stereoselectivity. High resolution structures of six of the designs revealed an angstrom level of active site design precision. The design strategy can, in principle, be applied to any catalytically competent amino acid constellation. These findings enable the practical applicability of de novo protein catalysts in synthesis and shed light on fundamental principles of protein design and enzyme catalysis.

biochemistry↗