Biology subjects

Holmes, D.

Publications and source records attributed to Holmes, D..

8 recordsLinked to original sources

bioRxiv2026

A scalable approach to resolving variants of uncertain significance

Over 90% of missense variants across [~]4,000 disease-associated genes are variants of uncertain significance (VUS). Experimental variant effect measurements provide critical evidence about pathogenicity and inform disease biology, but most variants lack data and clinical translation has been limited. The Impact of Genomic Variation on Function Consortium generated experimental data for 62,215 variants across ten genes using multiplexed assays and 1,407 variants across 163 genes using arrayed assays, curated 193,139 additional community-generated variant effect measurements across 30 additional genes, and developed automated calibration methods for translating experimental data and variant effect predictions into clinical evidence. To reduce current VUS, we developed a scalable workflow using only experimental and predictive evidence, enabling reclassification of 75% of the 16,115 VUS in these genes as pathogenic or benign with <1% error. To minimize future VUS, we analyzed >90,000 unobserved variants; 62% had enough evidence to be "preclassified" as pathogenic or benign. We validated our data, evidence and classifications using All of Us and created interactive resources to enable clinical use of the calibrated data. Thus, for 40 genes, representing 1% of the clinical genome, we resolve most existing VUS and future variants, illustrating how systematic use of scalable evidence can empower genomic medicine.

genomics↗

bioRxiv2025

Machine learning-based development of Gadolinium binding peptides

Gadolinium-based contrast agents (GBCAs) are indispensable tools in magnetic resonance imaging (MRI), yet their clinical use is limited by non-specific tissue accumulation, low molecular specificity, and safety concerns. Protein and peptide scaffolds provide a promising alternative because they can bind metal ions with high selectivity and enable precise molecular targeting. However, identifying short peptide motifs with optimal gadolinium (Gd3) coordination and high relaxivity remains a major challenge. Here, we used a machine-learning-driven peptide evolution platform, the Protein Optimization Engineering Tool (POET), to design and optimize short Gd-binding motifs that enhance longitudinal relaxivity (r). Two algorithmic strategies were tested; motif-based and regular-expression-based representations, both were trained on an initial set of 74 twelve-amino-acid peptides derived from natural EF-hand scaffolds. Through two rounds of directed evolution and experimental screening, we identified peptides with up to a 24% increase in r ratio compared with the best natural EF-hand, and a 55% improvement in absolute r after removal of unbound Gd. Further analysis revealed that peptides exhibiting higher relaxivity generally possessed a more negative net charge and lower isoelectric point than the buffer pH, indicating stronger electrostatic stabilization of Gd3. Sequence enrichment analysis showed that acidic and small polar residues, particularly aspartic acid, glycine, and threonine, were selectively favored during evolution, while bulky hydrophobic and basic residues were depleted. These compositional trends align with improved solubility and enhanced metal coordination. Together, these results demonstrate a generalizable framework that integrates computational evolution with biophysical screening to discover new biologically derived Gd-binding motifs. This approach provides a scalable route to engineer responsive, tunable, and biocompatible MRI contrast tags for precision imaging and molecular diagnostics.

biophysics↗

bioRxiv2025

Rapid High-Resolution Analysis of Polysaccharide-Lignin Interactions via Proton-Detected Solid-State NMR with Application to Eucalyptus and Spruce

The plant secondary cell wall, a complex matrix composed of cellulose, hemicellulose, and lignin, is crucial for the mechanical strength and water-proofing properties of plant tissues, and serves as a primary source of biomass for biorenewable energy and biomaterials. Structural analysis of these polymers and their interactions within the secondary cell wall has been heavily relying on 13C-based solid-state NMR techniques. In this study, we explore the application of 1H-detected solid-state NMR techniques for rapid, high-resolution structural characterization of polysaccharides and lignin, demonstrated on the stems of hardwood eucalyptus and softwood spruce. We employed several strategies, including the use of synthesized 2D spectra to resolve central 1H resonances and the combined application of 3D hCCH and hCHH experiments for complete resonance assignment and unambiguous identification of lignin-carbohydrate interactions. Our findings emphasize the central role of acetylated three-fold xylan conformers, rather than two-fold, in stabilizing the carbohydrate-lignin interface, with glucuronic acid sidechains in eucalyptus glucuronoxylan colocalizing with lignin, revised cellulose-lignin interactions involving uncoated microfibril surfaces, and pectin-lignin interactions indicative of early-stage lignification. We also carefully evaluated the interference from tannins in softwood species. These results present a novel approach for rapid structural analysis of lignocellulosic biomaterials without the need for solubilization or extraction.

biophysics↗

bioRxiv2024

CYP76BK1 orthologs catalyze furan and lactone ring formation in clerodane diterpenoids across the mint family

The Lamiaceae (mint family) is the largest known source of furanoclerodanes, a subset of clerodane diterpenoids with broad bioactivities including insect antifeedant properties. The Ajugoideae subfamily, in particular, accumulates significant numbers of structurally related furanoclerodanes. The biosynthetic capacity for formation of these diterpenoids is retained across most Lamiaceae subfamilies, including the early-diverging Callicarpoideae which forms a sister clade to the rest of Lamiaceae. VacCYP76BK1, a cytochrome P450 monooxygenase from Vitex agnus-castus, was previously found to catalyze the formation of the proposed precursor to furan and lactone-containing labdane diterpenoids. Through transcriptome-guided pathway exploration, we identified orthologs of VacCYP76BK1 in Ajuga reptans and Callicarpa americana. Functional characterization demonstrated that both could catalyze the oxidative cyclization of clerodane backbones to yield a furan ring. Subsequent investigation revealed a total of ten CYP76BK1 orthologs across six Lamiaceae subfamilies. Through analysis of available chromosome-scale genomes, we identified four CYP76BK1 members as syntelogs within a conserved syntenic block across divergent subfamilies. This suggests an evolutionary lineage that predates the speciation of the Lamiaceae. Functional characterization of the CYP76BK1 orthologs affirmed conservation of function, as all catalyzed furan ring formation. Additionally, some orthologs yielded two novel lactone ring moieties. The presence of the CYP76BK1 orthologs across Lamiaceae subfamilies closely overlaps with the distribution of reported furanoclerodanes. Together, the activities and distribution of the CYP76BK1 orthologs identified here support their central role in furanoclerodane biosynthesis within the Lamiaceae family. Our findings lay the groundwork for biotechnological applications to harness the economic potential of this promising class of compounds. Significance StatementThe discovery and functional characterization of CYP76BK1 orthologs across diverse Lamiaceae subfamilies revealed novel chemistry and their central role in furanoclerodane biosynthesis, providing insights into the metabolic landscape and dynamic evolution of this plant family over approximately 50 million years. These findings pave the way for targeted biosynthetic engineering efforts and the sustainable production of furanoclerodane compounds, offering promising prospects for agricultural and pharmaceutical applications.

plant biology↗

bioRxiv2022

Different Routes for Direct Interspecies Electron Transfer with Diverse Electron-Accepting Partners

Direct interspecies electron transfer (DIET) may be most important in methanogenic environments, but studies to date that have examined the mechanisms for electron transfer from electron-donating partners have primarily focused on co-cultures in which fumarate was the terminal electron acceptor. To better understand DIET with methanogens, the transcriptome of Geobacter metallireducens during DIET-based growth with Geobacter sulfurreducens reducing fumarate was compared with the transcriptome of G. metallireducens grown in co-culture with diverse Methanosarcina species. The transcriptome of G. metallireducens co-cultured with G. sulfurreducens was significantly different from those with Methanosarcina. Furthermore, the transcriptome of G. metallireducens grown with Methanosarcina barkeri, which lacks outer-surface cytochromes, was different from the transcriptome of G. metallireducens co-cultured with Methanosarcina acetivorans or Methanosarcina subterranea, which have an outer-surface c-type cytochrome that serves as an electrical connect for DIET. Differences in G. metallireducens expression patterns for c-type cytochrome genes were particularly notable. The impact of deleting G. metallireducens c-type cytochrome genes depended upon the electron-accepting partner. Multiple cytochromes that G. metallireducens required for DIET with G. sulfurreducens and for Fe(III) oxide reduction were not required for DIET with the Methanosarcina species. Porin-cytochrome complexes and e-pili were required for optimal DIET with all partners. Shewanella oneidensis, another electroactive microbe with abundant outer-surface c-type cytochromes, did not grow via DIET. The results demonstrate that the presence of outer-surface cytochromes does not necessarily confer the capacity for DIET, and also emphasize the substantial impact of the physiology of the electron-accepting partner on the physiology of the electron-donating DIET partner.

microbiology↗

bioRxiv2021

Mechanisms for Electron Uptake by Methanosarcina acetivorans During Direct Interspecies Electron Transfer

Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in co-culture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in M. acetivorans grown in co-culture versus pure culture growth on acetate revealed that transcripts for the outer-surface, multi-heme, c-type cytochrome MmcA were higher during DIET-based growth. Deletion of mmcA inhibited DIET. The high aromatic amino acid content of M. acetivorans archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based co-culture as well as the archaella-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based co-cultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen. ImportanceThe conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters. Potential electron donors for carbon dioxide reduction are H2 or electrons derived from direct interspecies electron transfer (DIET) between bacteria and methanogens. Elucidating the relative importance of these electron donors has been difficult due to a lack of information on the electrical connects on the outer surface of methanogens and how they process the electrons received from DIET. Transcriptomic patterns and gene deletion phenotypes reported here provide insight into how a group of Methanosarcina that play an important role in methane production in soils and sediments participate in DIET.

microbiology↗

bioRxiv2020

Development of a small molecule that corrects misfolding and increases secretion of Z α1-antitrypsin

Severe 1-antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant 1-antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high throughput screen to identify small molecules that bind to, and stabilise Z 1-antitrypsin. The lead compound blocks Z 1-antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z 1-antitrypsin three-fold in mammalian cells including an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerization pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z 1-antitrypsin into the plasma by 7-fold. There was no observable clearance of hepatic inclusions with respect to controls. This study provides proof-of-principle that mutation ameliorating small molecules are a viable approach to treat protein conformational diseases.

pharmacology and toxicology↗

bioRxiv2019

A Membrane-Bound Cytochrome Enables Methanosarcina acetivorans to Conserve Energy to Support Growth from Extracellular Electron Transfer

Conservation of energy to support growth solely from extracellular electron transfer was demonstrated for the first time in a methanogen. Methanosarcina acetivorans grew with methanol as the sole electron donor and the extracellular electron acceptor anthraquione-2,6-disulfonate (AQDS) as the sole electron acceptor when methane production was inhibited with bromoethanesulfonate. Transcriptomics revealed that transcripts for the gene for the transmembrane, multi-heme, c-type cytochrome MmcA were 4-fold higher in AQDS-respiring cells versus methanogenic cells. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction whereas strains in which other cytochrome genes were deleted grew as well as the wild-type strain. The MmcA-deficient strain grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and components of the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenezyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in Archaea.\n\nImportanceExtracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and can enhance the speed and stability of anaerobic digestion, an important bioenergy strategy. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea has been suspected for some time, but the studies with Methanosarcina acetivorans reported here provide the first genetic evidence supporting this hypothesis. The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens, but possibly also for anaerobic methane oxidation.

microbiology↗