Biology subjects

Wright, A. T.

Publications and source records attributed to Wright, A. T..

13 recordsLinked to original sources

bioRxiv2026

Perturbation-based propagation of bacterial composition uncertainty improves robustness and reproducibility of microbiome differential abundance analysis.

Differential abundance (DA) analysis remains the analytical bottleneck of microbiome science; despite dozens of available methods, results remain poorly reproducible across tools, datasets, and study designs. Three intertwined challenges drive this failure: compositional data structure, stochastic count uncertainty, and complex experimental design; yet no existing method addresses all three simultaneously. Here, we introduce PARALLAX (Perturbation-based Assessment of Robustness Across Latent Logical Axes), a unified DA framework that resolves this gap by propagating compositional uncertainty through per-replicate linear mixed modeling across a structured scale perturbation grid, then summarizes inference through two stability-based metrics: Ppost (effect consistency across Monte Carlo replicates) and Rscale (robustness to scale perturbation). PARALLAX retains uncertainty through the complete inferential pipeline while natively supporting repeated measures and multifactorial designs. Under repeated subsampling, PARALLAX identified fully stable DA taxa in 14.4% of cases, versus 1.7% and 0% for comparators. We applied PARALLAX to two independent longitudinal shotgun metagenomic datasets from mechanistically distinct mouse models: Gulf War toxicant exposure and repetitive head trauma. In both, PARALLAX resolved robust, sex-specific, and time-dependent genus-level shifts while accommodating repeated measures and multifactorial design, tasks that challenged both comparator frameworks. PARALLAX is released as an open-source, AI-compatible R workflow, lowering the barrier to rigorous microbiome DA analysis across complex study designs.

bioinformatics↗

bioRxiv2026

Chronic stress remodels gut bacterial ecology and polysaccharide metabolism with neuroendocrine consequences despite unchanged behavior

The gut-microbiome-brain axis (GMBA) links the gut and brain through metabolic, immunological, and neuroendocrine pathways, and can become dysregulated during psychological stress (PS). While previous studies primarily infer microbial function and use physical stressors that incompletely model human PS, here, functional multi-omics was applied to a chronic social-isolation mouse model. Although isolation caused no measurable behavioral changes, brain proteomics revealed stress-associated molecular responses. Taxonomic and short-chain fatty acid analyses suggested limited microbiome changes; however, metaproteomics uncovered sex-specific restructuring of bacterial ecology and carbohydrate-active enzymes. In females, isolation increased Petralouisia muris proteins involved in bacterial microcompartment metabolism and {beta}-glucuronidases across Lachnospiraceae taxa. Enriched {beta}-glucuronidase abundance coincided with reduced circulating glucuronides and altered brain estrogen-receptor activity, suggesting a microbial deconjugation-host estrogen-signaling interaction. These findings demonstrate that functional GMBA alterations may precede or occur independently of overt behavioral phenotypes and highlight the value of direct functional profiling in PS.

microbiology↗

bioRxiv2026

Commensal Bacteria in the Gut Microbiome Encode Previously Unrecognized Aromatic Hydrocarbon Catabolic Machinery

Introduction. Polycyclic aromatic hydrocarbon (PAH) toxicology has been primarily defined by host metabolic bioactivation to reactive intermediates. Yet ingested PAHs and biliary-delivered metabolites also enter the intestinal lumen, where they encounter the gut microbiota, an additional but understudied metabolic compartment that may alter toxicant fate. Although PAH interactions are well characterized in environmental microbes, resulting in defined enzymatic pathways for their catabolism, whether gut commensal organisms express related machinery in vivo remains poorly understood. To address this gap, we used an oral murine-exposure model of naphthalene and integrated compartment-resolved 16S rRNA gene profiling, global metaproteomics, and chemoproteomics to resolve the PAH-processing repertoire of the gut microbiota and its spatial distribution. Results. Naphthalene, a common environmental toxicant and representative PAH, elicited a compartment-dependent functional response that was most pronounced in the large intestine. Distally, exposure was associated with 2,386 differential proteins and accounted for 48.4% of metaproteomic variation, compared with 7.9% of 16S rRNA gene compositional variation. The small intestine showed a reduced proteomic response, with 994 differentially abundant proteins. Hydrocarbon catabolic markers showed a 4.8-fold distal expansion (36 to 172 proteins), including the naphthalene-catabolic nahE, proteins associated with phenylacetate, {beta}-ketoadipate, catechol/TOL, and anaerobic CoA-linked redox machinery. The small-intestinal repertoire decreased from 19 to 14 proteins, though aromatic-catabolic markers doubled, indicating a selective expansion. Critically, these homologs concentrated in Lachnospiraceae, a gut family that is prevalent in humans, primarily known for anaerobic fermentation, with no priorly described PAH-processing capacity. An uncultured Acetatifactor showed the largest hydrocarbon hits (14 to 82 protein increase), with additional contributions from Kineothrix, Eisenbergiella, and Petralouisia. Chemoproteomics, using a naphthalene-based probe, revealed enrichment of an orthogonal proteome of proteins interacting with naphthalene, capturing 171 small-intestinal and 67 large-intestinal proteins that were undetected in the global metaproteomes. Conclusions. Gut commensals express homologs of aromatic-hydrocarbon catabolic enzymes characterized in environmental microorganisms, and naphthalene amplifies this repertoire most strongly in the large intestine. The decoupling of taxonomic structure, expressed-protein representation, and probe engagement identifies a functional dimension of PAH exposure that composition-based profiling does not resolve and positions microbiome variation as a candidate modifier of PAH metabolic fate.

microbiology↗

bioRxiv2025

Modeling MEK-inhibitor Associated Retinopathy in vitro using human induced-1 pluripotent stem cell-derived retinal pigment epithelial cells

Pharmacologic inhibitors of MEK are important anti-cancer drugs but can result in MEK inhibitor-Associated Retinopathy (MEKAR) in which vision is lost due to serous retinal detachments that form via an unknown mechanism. We hypothesized that the cause of this side effect is drug-induced dysfunction of retinal pigment epithelial (RPE) cells. To test this hypothesis, we used human induced pluripotent stem cell-derived RPE cells. We treated mature, hiPSC-derived RPE cells with selumetinib and measured impacts on RPE-specific function, structure, and gene expression. Selumetinib increases the ability of hiPSC-derived RPE to internalize bovine rod outer segments (1.9 vs 3.0, p=0.0024). It also decreases expression of aquaporin 1 during the first 10 days of treatment (2.7 vs 1.1, p=0.0015). It has no effect on the ability of hiPSC-derived RPE to maintain membrane integrity. Selumetinib alters gene expression of hiPSC-derived RPE, with significant changes in genes involved in transport of ions and small molecules regulating cell volume and lysosomal acidification. Selumetinib may lead to subretinal fluid accumulation by both increasing secretions into this space and decreasing outflow.

molecular biology↗

bioRxiv2025

Production of clinical grade patient iPSC-derived 3D retinal organoids containing transplantable photoreceptor cells

Neurodegenerative conditions that affect the retina are currently the leading cause of incurable blindness in the developed world. Although gene and drug therapies are being developed to slow disease progression in some cases, restorative cell replacement approaches are needed for patients with significant vision impairment due to retinal degeneration. While a variety of different cell types have been evaluated in the context of retinal cell replacement, induced pluripotent stem cells (iPSCs), which can be generated and delivered as an autologous therapeutic, are in many ways the most attractive donor cell source currently available. Like embryonic stem cells, iPSCs must be differentiated into the target therapeutic cell type prior to transplantation. For instance, for patients with retinitis pigmentosa who have primary photoreceptor cell disease, photoreceptor cell derivation and enrichment are required prior to transplantation. Although other effective retinal differentiation protocols exist, they are often not fully compatible with clinical manufacturing. In this study, we report development of a xeno-free 3D retinal differentiation protocol based on the most robust adherent/non-adherent 3D differentiation strategies published to date. In addition, we demonstrate that while iPSC reprogramming efficiency is enhanced under reduced oxygen tension (i.e., 5%), efficient embryoid body and subsequent retinal organoid production require standard oxygen levels (i.e., 21%). Finally, we show that photoreceptor precursor cells obtained from 3D retinal organoids derived using the developed protocol under current good manufacturing practices (cGMP) survive in the subretinal space of dystrophic Pde6b-null rats for 1-month post-transplantation and form new synaptic connections with host bipolar neurons.

neuroscience↗

bioRxiv2025

Comprehensive identification of β-lactam antibiotic polypharmacology in Mycobacterium tuberculosis

Infections with Mycobacterium tuberculosis (Mtb) cause tuberculosis (TB), which requires at least six months of treatment with multiple antibiotics. There is emergent interest in using {beta}-lactam antibiotics to improve treatment outcomes for patients. These drugs target cell wall biosynthesis, but a comprehensive list of enzymes inhibited by {beta}-lactams in Mtb is lacking. In the current study, we sought to identify and characterize Mtb enzymes inhibited by {beta}-lactam antibiotics using physiological conditions representative of both acute and chronic TB disease. We used new activity-based probes based on the {beta}-lactam antibiotic meropenem due to its approval by the World Health Organization for TB treatment. Activity-based probes label enzymes based on both substrate specificity and catalytic mechanism, enabling precise identification of drug targets. We identified previously undiscovered targets of meropenem in addition to known cell wall biosynthetic enzymes. We validated {beta}-lactam binding and hydrolysis for six newly identified targets: Rv1723, Rv2257c, Rv0309, DapE (Rv1202), MurI (Rv1338), and LipD (Rv1923). Our results demonstrate that there are at least 30 enzymes in Mtb vulnerable to inhibition by meropenem. This is many more {beta}-lactam targets than historically described, suggesting that efficacy in Mtb is a direct result of polypharmacology.

biochemistry↗

bioRxiv2025

Discovery of non-opioid receptor protein targets of fentanyl across tissues from animal models and humans using photoaffinity probes

Synthetic opioids such as fentanyl and related analogs have been widely used for pain management. However, their negative side effects, including respiratory depression and high potential for addiction, underscore the need for a deeper understanding of fentanyls interactions with proteins throughout the human body. Fentanyl analogs bind and activate opioid receptors in the central and peripheral nervous systems, triggering numerous downstream signaling pathways. Increasingly, fentanyl has been shown to interact with non-opioid receptors, and elucidation of these non-canonical fentanyl-protein interactions may provide insights into the mechanisms contributing to fentanyls adverse effects and illuminate novel countermeasure strategies. To identify proteins in mammalian tissues that may interact with fentanyl, we designed and synthesized three affinity-based probes (AfBPs) that include the fentanyl core and feature a diazirine photoaffinity group and alkyne handle for click chemistry at different positions. Molecular docking simulations predicted that these AfBPs bind the mu opioid receptor similarly to fentanyl. Affinity-based protein profiling using the FA-T1 probe in vitro in tissues from six animal species identified histamine N-methyltransferase (HNMT), endophilin-B1 (SH3GLB1), fructosamine-3-kinase (FN3K), cutA divalent cation tolerance analog (CUTA), and monoamine oxidase B (MAOB) among the top proteins that bind fentanyl in multiple species and tissue types. Molecular docking of fentanyl and remifentanil with these protein structures identified putative binding sites. The interaction of fentanyl with specific proteins was empirically assessed through protein structural analyses. These findings highlight potential fentanyl-protein interactions that may contribute to the acute and long-term impacts of fentanyl exposures.

pharmacology and toxicology↗

bioRxiv2024

Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids

Autologous photoreceptor cell replacement therapy shows great promise for treating patients with multiple forms of inherited retinal degenerative blindness. Specifically, in disorders such as retinitis pigmentosa and Stargardts disease, selective death of photoreceptor cells results in irreversible blindness. Induced pluripotent stem cell (iPSC) derived retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of photoreceptor cells required for these therapies. However, in addition to photoreceptor cells, retinal organoids also contain many other retinal cell types. Therefore, approaches for isolating fate committed photoreceptors from dissociated retinal organoids are desirable to produce photoreceptor cell replacement therapies. In this work, we present a partial dissociation strategy, which leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. Given that this technique can be performed with only standard plasticware and cGMP compliant reagents, it is an ideal candidate for use in the preparation of clinical cell therapies.

bioengineering↗

bioRxiv2024

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

plant biology↗

bioRxiv2023

Glutathione transferase photoaffinity labeling demonstrates GST activation by safeners and NPR1-independent activation by BTH

Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. Here, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study global GST activation in various plant species. The small molecule probe contains glutathione, a photoreactive group, and a minitag for coupling to reporter tags via click chemistry. Under UV irradiation, this probe quickly and robustly labels GSTs in crude protein extracts. Enrichment and MS analysis of labeled proteins from Arabidopsis identified ten GSTs from the Phi(F) and Tau(U) classes. Photoaffinity labeling of GSTs demonstrated GST activation in wheat seedlings upon treatment with safeners, and in Arabidopsis leaves upon infection with avirulent bacteria. Photoaffinity labeling and proteomics identified six Phi- and Tau-class GSTs that are induced upon treatment with salicylic acid (SA) analog benzothiadiazole (BTH) and these were tested for enhancing immunity in disease assays. Our data confirm that BTH-induced GST activation is independent of NPR1, the master regulator of SA signaling.

plant biology↗

bioRxiv2019

Selection, succession and stabilization of soil microbial consortia

Soil microorganisms play fundamental roles in cycling of soil carbon, nitrogen and other nutrients, yet we have a poor understanding of how soil microbiomes are shaped by their nutritional and physical environment. Here we investigated the successional dynamics of a soil microbiome during 21-weeks of enrichment on chitin and its monomer, N-acetylglucosamine. We examined succession of the soil communities in a physically heterogeneous soil matrix as well as a highly mixed liquid medium. The guiding hypothesis was that the initial species richness would influence the tendency for the selected consortia to stabilize and maintain relatively constant community structure over time. We also hypothesized that long term, substrate-driven growth would result in consortia with reduced species richness as compared to the parent microbiome and that this process would be deterministic with relatively little variation between replicates. We found that the initial species richness does influence the long-term community stability in both liquid media and soil and that lower initial richness results in a more rapid convergence to stability. Despite use of the same soil inoculum and access to the same major substrate, the resulting community composition differed greatly in soil compared to liquid medium. Hence, distinct selective pressures in soils relative to homogenous liquid media exist and can control community succession dynamics. This difference is likely related to the fact that soil microbiomes are more likely to thrive, with fewer compositional changes, in a soil matrix compared to liquid environments. IMPORTANCEThe soil microbiome carries out important ecosystem functions, but interactions between soil microbial communities have been difficult to study due to the high microbial diversity and complexity of the soil habitat. Here we successfully obtained stable consortia with reduced complexity that contained species found in the original source soil. These consortia and the methods used to obtain them can be a valuable resource for exploration of specific mechanisms underlying soil microbial community ecology. The results of this study also provide new experimental context to better inform how soil microbial communities are shaped by new environments and how a combination of initial taxonomic structure and physical environment influences stability.

microbiology↗

bioRxiv2018

Lactobacillus acidophilus disrupts collaborative multispecies bile acid metabolism

Bile acids are metabolic links between hosts and their gut microbiomes, yet little is known about the roles they play in microbe-to-microbe interactions. Here we present a study designed to investigate the effect that a common probiotic, Lactobacillus acidophilus, has on microbial interactions that lead to formation of secondary bile acids. A model microbial consortium was built from three human gut isolates, Clostridium scindens, Collinsella aerofaciens, and Blautia obeum, and cultured under different bile acid and probiotic treatments. A multi-omics platform that included mass spectrometry-based metabolomics and activity-based proteomic probes was used to produce two major results. The first, was that an uncommon secondary bile acid - ursocholate - was produced by a multi-species chemical synthesis pathway. This result highlights a new microbe-to-microbe interaction mediated by bile acids. The second finding was that the probiotic strain, L. acidophilus, quenched the observed interactions and effectively halted consortial synthesis of ursocholate. Little is known about the role that ursocholate plays in human health and development. However, we did discover that a decrease in ursocholate abundance corresponded with successful weight loss in patients after gastric bypass surgery versus those who did not lose weight after surgery. Hence, this study uncovered basic knowledge that may aid future designs of custom probiotic therapies to combat obesity.

microbiology↗

bioRxiv2018

Proximity-dependent proteomics of the Chlamydia trachomatis inclusion membrane reveals functional interactions with endoplasmic reticulum exit sites

Chlamydia trachomatis is the most common bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia-host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX system of proximity-dependent biotinylation. APEX is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX fused to known inclusion membrane proteins, allowing biotinylation and pull-down of inclusion-associated proteins. Using quantitative mass spectrometry against APEX labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological and genetic disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. APEX is therefore a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth.

microbiology↗