Biology subjects

Lee, M. A.

Publications and source records attributed to Lee, M. A..

10 recordsLinked to original sources

bioRxiv2025

metaboprep v2: Broadening the application of the metaboprep beyond metabolomics

High-throughput multiplex assays for metabolomics and proteomics offer opportunities for biomarker discovery and disease stratification in epidemiological research. The complexity of these datasets requires robust, standardized and transparent preprocessing workflows to ensure reproducibility and comparability across studies. We present an updated and enhanced version of the metaboprep R package. Originally designed for metabolomics data, it has now been extended to support proteomics datasets from platforms such as Olink(R) and SomaScan(R). This release introduces a user-friendly, modular, object-oriented architecture using Rs S7 system, enabling improved input format flexibility, streamlined report generation and increased compatibility with other third-party tools. The updated pipeline is structured in three parts: data import, filtering and summary, and output generation. This structure provides a reproducible yet customizable framework for pre-analysis data preparation with utility across multiple omics platforms and particular value in supporting multi-cohort epidemiological research. Availability and implementationThe metaboprep package is implemented in R and freely available at: https://github.com/MRCIEU/metaboprep.

bioinformatics↗

bioRxiv2025

PSMtags improve peptide sequencing and throughput in sensitive proteomics

Mass spectrometry-based proteomics enables comprehensive characterization of protein abundance, function, and interactions. Label-free approaches are simple to implement but challenging to scale to thousands of samples per day. Multiplexed techniques, such as plexDIA, can address these limitations but remain restricted by the lack of mass tags optimized for data-independent acquisition (DIA) workflows. Here, we present a systematic approach screening a library of 576 compounds that identifies several small molecules that, when conjugated to peptides, improve their detection and sequence identification by mass spectrometry. The lead molecule, PSMtag, substantially increases the detection of fragment b-ions, which increases the confidence of sequence identification and enhances de novo sequencing. PSMtags allow 9-plexDIA, using only stable isotopes of carbon, oxygen and nitrogen. As a result, it allows simultaneously increasing proteome coverage and sample throughput for plexDIA workflows without compromising quantitative accuracy. We demonstrate 240 samples-per-day with 9-plexDIA, while acquiring 28,359 protein data points in the same time label-free methods acquire 4,340. Our approach constitutes an expandable framework for designing mass tags to overcome existing limitations in multiplexed proteomics and provides plexDIA reagents capable of analyzing over 1,000 samples per day when using 10 minute runs. By facilitating higher throughput and improved identification, this innovation holds significant potential for accelerating proteomic studies across diverse biological and clinical applications.

bioengineering↗

bioRxiv2025

Pathobiont-triggered induction of epithelial IDO1 drives regional susceptibility to Inflammatory Bowel Disease

The structure and function of the mammalian gut vary by region, yet why inflammatory diseases manifest in specific regions and not others remains unclear. We use a TNF-overexpressing Crohns disease (CD) model (Tnf{Delta}ARE/+), which typically presents in the terminal ileum (TI), to investigate how environmental factors interact with the hosts immune susceptibility to drive region-specific disease. We identified Chlamydia muridarum, an intracellular bacterium and murine counterpart to the human sexually transmitted C. trachomatis, as necessary and sufficient to trigger disease manifestation in the ascending colon (AC), another common site of human CD. Disease manifestation in the AC depends on indoleamine 2,3-dioxygenase (IDO1) activity induced by hypersensitive surface secretory cells in genetically susceptible hosts. Single-cell and microbial analyses of human specimens also implicates this pathobiont-epithelial IDO1 pathway in patients with a history of CD in the AC. Our findings demonstrate that genetic and microbial factors can independently drive region-specific disease and provide a unique model to study CD specific to the AC.

cell biology↗

bioRxiv2024

Small Molecule Modulators of Beta-arrestins

{beta}-arrestins are multifunctional regulators of G protein-coupled receptor (GPCR) signaling, orchestrating diverse downstream signaling events and physiological responses across the vast GPCR superfamily. While GPCR pharmacology has advanced to target orthosteric and allosteric sites, as well as G proteins and GRKs, comparable chemical tools to study {beta}-arrestins remain lacking. Here, we report the discovery of small-molecule inhibitors that selectively target {beta}-arrestins and delineate their mechanism of action through integrated pharmacological, biochemical, biophysical, and structural analyses. These inhibitors disrupt {beta}-arrestin-engagement with agonist-activated GPCRs, impairing desensitization, internalization, and {beta}-arrestin-dependent functions while sparing G protein-receptor coupling. Cryo-EM, MD simulations, and structure-guided mutagenesis reveal that one modulator, Cmpd-5, engages a cryptic pocket formed by the middle, C-, and lariat loops of {beta}-arrestin1--a critical receptor-binding interface--stabilizing a distinct conformation incompatible with GPCR engagement. Together, these findings provide a mechanistic framework for {beta}-arrestin modulation, introducing transducer-targeted strategies to fine-tune GPCR signaling and guide the development of pathway-specific therapeutics.

biochemistry↗

bioRxiv2022

The structure of Vibrio cholerae FeoC reveals conservation of the helix-turn-helix motif but not the cluster-binding domain

Most pathogenic bacteria require ferrous iron (Fe2+) in order to sustain infection within hosts. The ferrous iron transport (Feo) system is the most highly-conserved prokaryotic transporter of Fe2+, but its mechanism remains to be fully characterized. Most Feo systems are composed of two proteins: FeoA, a soluble SH3-like accessory protein and FeoB, a membrane protein that translocates Fe2+ across a lipid bilayer. Some bacterial feo operons encode FeoC, a third soluble, winged-helix protein that remains enigmatic in function. We previously demonstrated that select FeoC proteins bind O2-sensitive [4Fe-4S] clusters via Cys residues, leading to the proposal that some FeoCs could sense O2 to regulate Fe2+ transport. However, not all FeoCs conserve these Cys residues, and FeoC from the causative agent of cholera (Vibrio cholerae) notably lacks any Cys residues, precluding cluster binding. In this work, we determined the NMR structure of VcFeoC, which is monomeric and conserves the helix-turn-helix domain seen in other FeoCs. In contrast, however, the structure of VcFeoC reveals a truncated winged {beta}-sheet in which the cluster-binding domain is notably absent. To test the interactions of VcFeoC with VcFeoB, we used NMR to demonstrate that these proteins interact in a 1:1 stoichiometry with a Kd of ca. 25 M. Finally, using homology modeling, we predicted the structure of VcNFeoB and used docking to identify an interaction site with VcFeoC, which is confirmed by NMR spectroscopy. These findings provide the first atomic-level structure of VcFeoC and contribute to a better understanding of its role vis-a-vis FeoB.

biochemistry↗

bioRxiv2022

CyclinD2-mediated regulation of neurogenic output from the retinal ciliary margin is perturbed in albinism

In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. We focus here on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ has the competence to generate predominantly ipsilaterally-projecting RGCs, but this competence is altered in the albino visual system due to CyclinD2 downregulation and disturbed temporal control of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit a diminished ipsilateral retinogeniculate projection and compromised depth perception. Pharmacological stimulation of calcium channels in albino mice, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs, and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit. HighlightsO_LIThe mouse ventral CMZ produces predominantly ipsilateral RGCs. C_LIO_LIIn the albino visual system, CyclinD2 downregulation leads to delayed G1/S transition toward mitotic exit of CMZ progenitors. C_LIO_LIPerturbations in the temporal control of cell cycle by CyclinD2 lead to reduced Zic2+ RGCs and consequently, a diminished ipsilateral retinogeniculate projection and compromised depth perception. C_LIO_LICalcium channel modulation during embryogenesis normalizes the levels of CyclinD2 and restores binocular vision in albino mice. C_LI

developmental biology↗

bioRxiv2021

metaboprep: an R package for pre-analysis data description and processing.

MotivationMetabolomics is an increasingly common part of health research and there is need for pre-analytical data processing. Researchers typically need to characterize the data and to exclude errors within the context of the intended analysis. While some pre-processing steps are common, there is currently a lack of standardization and reporting transparency for these procedures. ResultsHere we introduce metaboprep, a standardized data processing workflow to extract and characterize high quality metabolomics data sets. The package extracts data from pre-formed worksheets, provides summary statistics and enables the user to select samples and metabolites for their analysis based on a set of quality metrics. A report summarizing quality metrics and the influence of available batch variables on the data is generated for the purpose of open disclosure. Where possible, we provide users flexibility in defining their own selection thresholds. Availability and implementationmetaboprep is an open-source R package available at https://github.com/MRCIEU/metaboprep Contactd.a.hughes@bristol.ac.uk or laura.corbin@bristol.ac.uk

bioinformatics↗

bioRxiv2020

Common Marmoset Gut Microbiome Profiles in Health and Intestinal Disease

Chronic gastrointestinal (GI) diseases are the most common diseases in captive marmosets. The gut microbiome of healthy (n=91), inflammatory bowel disease (IBD) (n=59), and duodenal ulcer/stricture (n=23) captive marmosets was characterized. Healthy marmosets exhibited a "humanized," Bacteroidetes-dominant microbiome. Despite standardized conditions, cohorts subdivided into Prevotella- and Bacteroides-dominant groups based on marmoset source. IBD was highest in a Prevotella-dominant cohort while strictures were highest in a Bacteroides-dominant cohort. Stricture-associated dysbiosis was characterized by Anaerobiospirillum loss and Clostridium perfringens increases. Stricture tissue presented upregulation of lipid metabolism genes and increased abundance of C. perfringens, a causative agent of GI diseases and intestinal strictures in humans. IBD was associated with a lower Bacteroides:P. copri ratio within each source. Consistent with Prevotella-linked diseases, pro-inflammatory genes were upregulated. This report highlights the humanization of the captive marmoset microbiome and its potential as a "humanized" animal model of C. perfringens-induced enteritis/strictures and P. copri-associated IBD.

microbiology↗

bioRxiv2020

WNT inhibition creates a BRCA-like state in Wnt-addicted cancer

Wnt signaling maintains diverse adult stem cell compartments and is implicated in chemotherapy resistance in cancer. PORCN inhibitors that block Wnt secretion have proven effective in Wnt-addicted preclinical cancer models and are in clinical trials. In a survey for potential combination therapies, we found that Wnt inhibition synergizes with the PARP inhibitor olaparib in Wnt-addicted cancers. Mechanistically, we find that multiple genes in the homologous recombination and Fanconi anemia repair pathways, including BRCA1, FANCD2, and RAD51 are dependent on Wnt/{beta}-catenin signaling in Wnt-high cancers, and treatment with a PORCN inhibitor creates a BRCA-like state. This coherent regulation of DNA repair genes occurs via a Wnt/{beta}-catenin/MYBL2 axis. Importantly, this pathway also functions in intestinal crypts, where high expression of BRCA and Fanconi anemia genes is seen in intestinal stem cells, with further upregulation in Wnt high APCmin mutant polyps. Our findings suggest a general paradigm that Wnt/{beta}-catenin signaling enhances DNA repair in stem cells and cancers to maintain genomic integrity. Conversely, interventions that block Wnt signaling may sensitize cancers to radiation and other DNA damaging agents.

cancer biology↗

bioRxiv2019

Large-scale death of retinal astrocytes duringnormal development mediated by microglia

Naturally-occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the central nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type - the astrocyte - has rarely been studied. It is presently unclear whether astrocytes are subject to significant amounts of developmental death, or how it occurs. Here we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia kill and engulf astrocytes to mediate their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally-occurring cell death, and demonstrate its importance for formation and integrity of the retinal gliovascular network.

neuroscience↗