Biology subjects

Mitchell, C. M.

Publications and source records attributed to Mitchell, C. M..

5 recordsLinked to original sources

bioRxiv2026

Principles of in situ protein sequencing: expansion microscopy-adapted Edman degradation and amino acid recognition

The ability to map protein identity, with resolution sufficient to infer interactions, would support analysis of how proteins work together, or malfunction, in biological processes and diseases. Although several emerging technologies aim towards single-molecule protein sequencing, they require proteins to be removed from the nanoscale spatial context of cells and tissues. Expansion microscopy (ExM) has facilitated a diversity of chemical analyses by isotropically separating molecules throughout a specimen after permeation via a charged hydrogel, followed by gel swelling. Here, we adapt key protein sequencing steps - Edman degradation and amino acid recognition - to the ExM gel context. Using testbed peptides in ExM gels, we show that N-terminal amino acids can be recognized over multiple cycles of in-gel Edman degradation. This principle-oriented study demonstrates sequencing chemistry on defined synthetic constructs, rather than endogenous proteins in biological samples. These results establish principles of in situ protein sequencing and provide a framework for future in situ protein sequencing developments, including the development of higher specificity and affinity amino acid binders.

biochemistry↗

bioRxiv2025

Synergistic therapeutic effects of endolysin BNT331 and a Lactobacillus crispatus consortium in a human vagina chip model of bacterial vaginosis

Bacterial vaginosis (BV) is a vaginal infection caused by an imbalance in the vaginal microbiome characterized by a decrease in healthy bacteria dominated by Lactobacillus crispatus along with a concomitant increase in dysbiotic bacteria, such as Gardnerella. Current treatments commonly fail to fully eradicate BV, and the lack of more effective therapies is due in part to the absence of relevant human models. Here, we used a human vagina-on-a-chip (Vagina Chip) microfluidic culture device that has been previously shown to faithfully recapitulate the human vaginal microenvironment as well as the inflammatory and injurious effects of G. vaginalis to test the therapeutic efficacy of a consortium of L. crispatus alone or in combination with endolysin BNT331, which specifically targets and lyses Gardnerella. These studies revealed that when L. crispatus was added alone, it suppressed inflammation even though it failed to engraft or displace the G. vaginalis bacteria. In contrast, BNT331 effectively killed Gardnerella in dysbiotic Vagina Chip. Importantly, the combined administration of both treatments resulted in restoration of a healthier vaginal microenvironment, as indicated by higher engraftment of L. crispatus on-chip, inhibition of G. vaginalis, and a reduction in inflammation. Similar effects of this combined treatment were observed when administered to Vagina Chips infected with vaginal swab samples from BV patients. These data suggest that combination of a live biotherapeutic product composed of a L. crispatus consortium with a potent antimicrobial agent that targets G. vaginalis, such as BNT331, may offer an effective therapeutic strategy for patients with BV. One Sentence SummaryEndolysin BNT331 combined with Lactobacillus crispatus reduces bacterial load and suppresses inflammation in a human vagina chip model of bacterial vaginosis.

pathology↗

bioRxiv2025

Maternal exercise during lactation reprograms obesity-related changes in mammary metabolism to optimize milk fatty acids and offspring energy expenditure

Maternal obesity alters breast milk composition in ways that may predispose infants to excess adiposity. While maternal exercise during lactation has been associated with favorable shifts in milk metabolites in humans, the mechanisms by which exercise remodels the mammary gland and milk lipid profile to influence offspring metabolism remain unclear. We developed a mouse model incorporating daily moderate treadmill exercise during lactation, indirect calorimetry, stable isotope tracer respirometry, and mammary epithelial cell (MEC) proteomics in lean (LN) and diet-induced obese (OB) dams. Maternal obesity broadly remodeled the MEC proteome, reducing enzymes of de novo fatty acid synthesis and altering lipid transport and oxidative pathways. These molecular adaptations corresponded to higher milk triglyceride content and shifts in fatty acid composition, including an elevated omega-6 to omega-3 fatty acid ratio. The exercise (EX) intervention during lactation reset MEC protein networks, enhancing translational and vesicle transport pathways while reducing fatty acid desaturation, relative to the sedentary (SED) group. In OB dams, exercise increased milk medium-chain fatty acid (MCFA) levels and partially corrected the n6/n3 FA ratio. Offspring nursed by OB-EX dams exhibited higher whole-body energy expenditure, increased fatty acid oxidation, and improved metabolic flexibility compared to litters consuming OB-SED milk. Together, maternal exercise during lactation remodels mammary metabolism and milk fatty acid composition in obese dams, enhancing neonatal lipid oxidation and energy expenditure. These findings highlight lactation as a modifiable window, wherein maternal activity influences milk composition and infant metabolic health. New and noteworthyMaternal obesity alters milk fatty acid composition, with consequences for infant metabolism. Exercise during lactation in obese dams remodeled the mammary epithelial cell proteome, increasing medium-chain fatty acids in milk and enhancing lipid oxidation and energy expenditure in offspring.

physiology↗

bioRxiv2025

StrainFacts accurately quantifies both endogenous and live biotherapeutic product strain abundances in simulated and clinical vaginal microbiota samples

Live biotherapeutic products (LBPs) deliver microbial strains to modulate the host microbiota in order to promote health or treat and prevent disease. Since endogenous strains are already present, accurately evaluating LBP efficacy and mechanism of action requires distinguishing administered from endogenous strains. Although computational tools exist for inferring strains from short-read metagenomic data, few have been rigorously tested in the context of LBP treatment. Here, we assess the ability of StrainFacts, a computational tool for inferring strains from short-read metagenomic data, to estimate strain abundances and genotypes of endogenous and administered strains. We performed a simulation study of a single-strain LBP trial, modeling serial samples across a range of administered strain abundance, co-occurring endogenous strains, and sequencing depths. We found that StrainFacts accurately estimated both LBP and endogenous strain abundances and genotypes within simulated samples. We further validated methods using human vaginal microbiota samples spiked with CTV-05, the Lactobacillus crispatus strain contained in the LBP LACTIN-V, which has been shown to reduce recurrent bacterial vaginosis. Our findings demonstrate that StrainFacts can robustly assess LBP and endogenous strain colonization, abundance, and dynamics in simulated and experimental microbiota samples, supporting its utility as an analysis tool for vaginal LBP therapeutic trial data.

bioinformatics↗

bioRxiv2021

Cysteine dependence in Lactobacillus iners constitutes a novel therapeutic target to modify the vaginal microbiota

Vaginal microbiota composition affects several important reproductive health outcomes. Lactobacillus crispatus-dominant bacterial communities have favorable associations whereas anaerobe-dominant communities deficient of lactobacilli are linked to poor outcomes, including bacterial vaginosis (BV). Lactobacillus iners, the most abundant vaginal species worldwide, has adverse associations compared to L. crispatus, but standard metronidazole treatment for BV promotes L. iners-dominance, likely contributing to post-treatment relapse. L. iners is under-studied because it fails to grow in standard Lactobacillus media in vitro. Here we trace this in vitro phenotype to a species-specific cysteine requirement associated with limitations in cysteine-related transport mechanisms and show that vaginal cysteine concentrations correlate with Lactobacillus abundance in vivo. We demonstrate that cystine uptake inhibitors selectively impede L. iners growth and that combining an inhibitor with metronidazole thus promotes L. crispatus dominance of defined BV-like communities. These findings identify a novel target for therapeutic vaginal microbiota modulation to improve reproductive health.

microbiology↗