Deciphering molecular mechanisms stabilizing the reovirus-binding complex.

Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics. Primary attachment of reovirus to host cells is mainly mediated by the trimeric viral protein, σ1, which engages cell-surface glycans, followed by high-affinity binding to junctional adhesion molecule-A (JAM-A). This multistep process is thought to be accompanied by major conformational changes in σ1, but direct evidence is lacking. By combining biophysical, molecular, and simulation approaches, we define how viral capsid protein mechanics influence virus-binding capacity and infectivity. Single-virus force spectroscopy experiments corroborated by in silico simulations show that GM2 increases the affinity of σ1 for JAM-A by providing a more stable contact interface. We demonstrate that conformational changes in σ1 that lead to an extended rigid conformation also significantly increase avidity for JAM-A. Although its associated lower flexibility impairs multivalent cell attachment, our findings suggest that diminished σ1 flexibility enhances infectivity, indicating that fine-tuning of σ1 conformational changes is required to successfully initiate infection. Understanding properties underlying the nanomechanics of viral attachment proteins offers perspectives in the development of antiviral drugs and improved oncolytic vectors.

Single-Molecule Analysis of SARS-CoV-2 Binding to C-Type Lectin Receptors.

Despite intense scrutiny throughout the pandemic, development of efficacious drugs against SARS-CoV-2 spread remains hindered. Understanding the underlying mechanisms of viral infection is fundamental for developing novel treatments. While angiotensin converting enzyme 2 (ACE2) is accepted as the key entry receptor of the virus, other infection mechanisms exist. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and its counterpart DC-SIGN-related (DC-SIGNR, also known as L-SIGN) have been recognized as possessing functional roles in COVID-19 disease and binding to SARS-CoV-2 has been demonstrated previously with ensemble and qualitative techniques. Here we examine the thermodynamic and kinetic parameters of the ligand-receptor interaction between these C-type lectins and the SARS-CoV-2 S1 protein using force-distance curve-based AFM and biolayer interferometry. We evidence that the S1 receptor binding domain is likely involved in this bond formation. Further, we employed deglycosidases and examined a nonglycosylated S1 variant to confirm the significance of glycosylation in this interaction. We demonstrate that the high affinity interactions observed occur through a mechanism distinct from that of ACE2.

Modulating Macrophage Clearance of Nanoparticles: Comparison of Small-Molecule and Biologic Drugs as Pharmacokinetic Modifiers of Soft Nanomaterials.

Nanomedicines show benefits in overcoming the limitations of conventional drug delivery systems by reducing side effects, toxicity, and exhibiting enhanced pharmacokinetic (PK) profiles to improve the therapeutic window of small-molecule drugs. However, upon administration, many nanoparticles (NPs) prompt induction of host innate immune responses, which in combination with other clearance pathways such as renal and hepatic, eliminate up to 99% of the administered dose. Here, we explore a drug predosing strategy to transiently suppress the mononuclear phagocyte system (MPS), subsequently improving the PK profile and biological behaviors exhibited by a model NP system [hyperbranched polymers (HBPs)] in an immunocompetent mouse model. In vitro assays allowed the identification of five drug candidates that attenuated cellular association. Predosing of lead compounds chloroquine (CQ) and zoledronic acid (ZA) further showed increased HBP retention within the circulatory system of mice, as shown by both fluorescence imaging and positron emission tomography-computed tomography. Flow cytometric evaluation of spleen and liver tissue cells following intravenous administration further demonstrated that CQ and ZA significantly reduced HBP association with myeloid cells by 23 and 16%, respectively. The results of this study support the use of CQ to pharmacologically suppress the MPS to improve NP PKs.

Associations between conflicting nutrition information, nutrition confusion and backlash among consumers in the UK.

OBJECTIVE: To examine the effects of exposure to conflicting nutritional information (CNI) through different forms of media on nutrition-related confusion and backlash among consumers in the UK. DESIGN: Cross-sectional survey administered via Qualtrics among 18-75-year-old participants in the UK. The sample was stratified by age and gender with quotas defined according to the 2011 UK census distribution. SETTING: Qualtrics' Online panel of respondents in the UK. PARTICIPANTS: 676 participants comprising nearly an equal number of females (n 341) and males (n 335) and a majority (58·6 %) from households whose income was <£30 000. RESULTS: Our findings showed that nearly 40 % of respondents were exposed to some or a lot of CNI. We found that while exposure to CNI from TV and online news increased nutrition confusion, CNI from health professionals increased backlash. Exposure to CNI from social media and health websites was associated with reduced backlash. We also found that nutrition confusion and backlash were negatively associated with exercise behaviour and fruit and vegetable consumption, respectively. CONCLUSIONS: Our study supports the theoretical pathways that explain the influence of CNI exposure on nutrition-related cognitive and behavioural outcomes. Additionally, different types of online information sources are associated with these outcomes to varying degrees. In the context of obesity and diabetes rates in the UK, our findings call for (a) further experimental research into the effects of CNI on consumers' diet-related cognitions and behaviours and (b) multi-stakeholder, interdisciplinary approaches to address this problem.

Structural Insights into Endobiotic Reactivation by Human Gut Microbiome-Encoded Sulfatases.

Phase II drug metabolism inactivates xenobiotics and endobiotics through the addition of either a glucuronic acid or sulfate moiety prior to excretion, often via the gastrointestinal tract. While the human gut microbial ß-glucuronidase enzymes that reactivate glucuronide conjugates in the intestines are becoming well characterized and even controlled by targeted inhibitors, the sulfatases encoded by the human gut microbiome have not been comprehensively examined. Gut microbial sulfatases are poised to reactivate xenobiotics and endobiotics, which are then capable of undergoing enterohepatic recirculation or exerting local effects on the gut epithelium. Here, using protein structure-guided methods, we identify 728 distinct microbiome-encoded sulfatase proteins from the 4.8 million unique proteins present in the Human Microbiome Project Stool Sample database and 1766 gut microbial sulfatases from the 9.9 million sequences in the Integrated Gene Catalogue. We purify a representative set of these sulfatases, elucidate crystal structures, and pinpoint unique structural motifs essential to endobiotic sulfate processing. Gut microbial sulfatases differentially process sulfated forms of the neurotransmitters serotonin and dopamine, and the hormones melatonin, estrone, dehydroepiandrosterone, and thyroxine in a manner dependent both on variabilities in active site architecture and on markedly distinct oligomeric states. Taken together, these data provide initial insights into the structural and functional diversity of gut microbial sulfatases, providing a path toward defining the roles these enzymes play in health and disease.

Effects of Surface Charge of Hyperbranched Polymers on Cytotoxicity, Dynamic Cellular Uptake and Localization, Hemotoxicity, and Pharmacokinetics in Mice.

Nanoscaled polymeric materials are increasingly being investigated as pharmaceutical products, drug/gene delivery vectors, or health-monitoring devices. Surface charge is one of the dominant parameters that regulates nanomaterial behavior in vivo. In this paper, we demonstrated how control over chemical synthesis allowed manipulation of nanoparticle surface charge, which in turn greatly influenced the in vivo behavior. Three methacrylate/methacrylamide-based monomers were used to synthesize well-defined hyperbranched polymers (HBP) by reversible addition-fragmentation chain transfer (RAFT) polymerization. Each HBP had a hydrodynamic diameter of approximately 5 nm as determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Incorporation of a fluorescent moiety within the polymeric nanoparticles allowed determination of how charge affected the in vivo pharmacokinetic behavior of the nanomaterials and the biological response to them. A direct correlation between surface charge, cellular uptake, and cytotoxicity was observed, with cationic HBPs exhibiting higher cellular uptake and cytotoxicity than their neutral and anionic counterparts. Evaluation of the distribution of the differently charged HBPs within macrophages showed that all HBPs accumulated in the cytoplasm, but cationic HBPs also trafficked to, and accumulated within, the nucleus. Although cationic HBPs caused slight hemolysis, this was generally below accepted levels for in vivo safety. Analysis of pharmacokinetic behavior showed that cationic and anionic HBPs had short blood half-lives of 1.82 ± 0.51 and 2.34 ± 0.93 h respectively, compared with 5.99 ± 2.30 h for neutral HBPs. This was attributed to the fact that positively charged surfaces are more readily covered with opsonin proteins and thus more visible to phagocytic cells. This was supported by in vitro flow cytometric and qualitative live cell imaging studies, which showed that cationic HBPs tended to be taken up by macrophages more effectively and rapidly than neutral and anionic particles.

Using Peptide Aptamer Targeted Polymers as a Model Nanomedicine for Investigating Drug Distribution in Cancer Nanotheranostics.

Theranostics is a strategy that combines multiple functions such as targeting, stimulus-responsive drug release, and diagnostic imaging into a single platform, often with the aim of developing personalized medicine.1,2 Based on this concept, several well-established hyperbranched polymeric theranostic nanoparticles were synthesized and characterized as model nanomedicines to investigate how their properties affect the distribution of loaded drugs at both the cell and whole animal levels. An 8-mer peptide aptamer was covalently bound to the periphery of the nanoparticles to achieve both targeting and potential chemosensitization functionality against heat shock protein 70 (Hsp70). Doxorubicin was also bound to the polymeric carrier as a model chemotherapeutic drug through a degradable hydrazone bond, enabling pH-controlled release under the mildly acid conditions that are found in the intracellular compartments of tumor cells. In order to track the nanoparticles, cyanine-5 (Cy5) was incorporated into the polymer as an optical imaging agent. In vitro cellular uptake was assessed for the hyperbranched polymer containing both doxorubicin (DOX) and Hsp70 targeted peptide aptamer in live MDA-MB-468 cells, and was found to be greater than that of either the untargeted, DOX-loaded polymer or polymer alone due to the specific affinity of the peptide aptamer for the breast cancer cells. This was also validated in vivo with the targeted polymers showing much higher accumulation within the tumor 48 h postinjection than the untargeted analogue. More detailed assessment of the nanomedicine distribution was achieved by directly following the polymeric carrier and the doxorubicin at both the in vitro cellular level via compartmental analysis of confocal images of live cells and in whole tumors ex vivo using confocal imaging to visualize the distribution of the drug in tumor tissue as a function of distance from blood vessels. Our results indicate that this polymeric carrier shows promise as a cancer theranostic, demonstrating active targeting to tumor cells with the capability for simultaneous drug release.

Single-Molecule Investigation of the Binding Interface Stability of SARS-CoV-2 Variants with ACE2.

The SARS-CoV-2 pandemic spurred numerous research endeavors to comprehend the virus and mitigate its global severity. Understanding the binding interface between the virus and human receptors is pivotal to these efforts and paramount to curbing infection and transmission. Here we employ atomic force microscopy and steered molecular dynamics simulation to explore SARS-CoV-2 receptor binding domain (RBD) variants and angiotensin-converting enzyme 2 (ACE2), examining the impact of mutations at key residues upon binding affinity. Our results show that the Omicron and Delta variants possess strengthened binding affinity in comparison to the Mu variant. Further, using sera from individuals either vaccinated or with acquired immunity following Delta strain infection, we assess the impact of immunity upon variant RBD/ACE2 complex formation. Single-molecule force spectroscopy analysis suggests that vaccination before infection may provide stronger protection across variants. These results underscore the need to monitor antigenic changes in order to continue developing innovative and effective SARS-CoV-2 abrogation strategies.

Gut microbial ß-glucuronidases influence endobiotic homeostasis and are modulated by diverse therapeutics.

Hormones and neurotransmitters are essential to homeostasis, and their disruptions are connected to diseases ranging from cancer to anxiety. The differential reactivation of endobiotic glucuronides by gut microbial ß-glucuronidase (GUS) enzymes may influence interindividual differences in the onset and treatment of disease. Using multi-omic, in vitro, and in vivo approaches, we show that germ-free mice have reduced levels of active endobiotics and that distinct gut microbial Loop 1 and FMN GUS enzymes drive hormone and neurotransmitter reactivation. We demonstrate that a range of FDA-approved drugs prevent this reactivation by intercepting the catalytic cycle of the enzymes in a conserved fashion. Finally, we find that inhibiting GUS in conventional mice reduces free serotonin and increases its inactive glucuronide in the serum and intestines. Our results illuminate the indispensability of gut microbial enzymes in sustaining endobiotic homeostasis and indicate that therapeutic disruptions of this metabolism promote interindividual response variabilities.

Diverse but desolate landscape of gut microbial azoreductases: A rationale for idiopathic IBD drug response.

Prodrugs reliant on microbial activation are widely used but exhibit a range of efficacies that remain poorly understood. The anti-inflammatory compound 5-aminosalicylic acid (5-ASA), which is packaged in a variety of azo-linked prodrugs provided to most Ulcerative Colitis (UC) patients, shows confounding inter-individual variabilities in response. Such prodrugs must be activated by azo-bond reduction to form 5-ASA, a process that has been attributed to both enzymatic and non-enzymatic catalysis. Gut microbial azoreductases (AzoRs) are the first catalysts shown to activate azo-linked drugs and to metabolize toxic azo-chemicals. Here, we chart the scope of the structural and functional diversity of AzoRs in health and in patients with the inflammatory bowel diseases (IBDs) UC and Crohn's Disease (CD). Using structural metagenomics, we define the landscape of gut microbial AzoRs in 413 healthy donor and 1059 IBD patient fecal samples. Firmicutes encode a significantly higher number of unique AzoRs compared to other phyla. However, structural and biochemical analyses of distinct AzoRs from the human microbiome reveal significant differences between prevalent orthologs in the processing of toxic azo-dyes, and their generally poor activation of IBD prodrugs. Furthermore, while individuals with IBD show higher abundances of AzoR-encoding gut microbial taxa than healthy controls, the overall abundance of AzoR-encoding microbes is markedly low in both disease and health. Together, these results establish that gut microbial AzoRs are functionally diverse but sparse in both health and disease, factors that may contribute to non-optimal processing of azo-linked prodrugs and idiopathic IBD drug responses.

Enhanced drug delivery to cancer cells through a pH-sensitive polycarbonate platform.

Polymer-drug conjugates are widely investigated to enhance the selectivity of therapeutic drugs to cancer cells, as well as increase circulation lifetime and solubility of poorly soluble drugs. In order to direct these structures selectively to cancer cells, targeting agents are often conjugated to the nanoparticle surface as a strategy to limit drug accumulation in non-cancerous cells and therefore reduce systemic toxicity. Here, we report a simple procedure to generate biodegradable polycarbonate graft copolymer nanoparticles that allows for highly efficient conjugation and intracellular release of S-(+)-camptothecin, a topoisomerase I inhibitor widely used in cancer therapy. The drug-polymer conjugate showed strong efficacy in inhibiting cell proliferation across a range of cancer cell lines over non-cancerous phenotypes, as a consequence of the increased intracellular accumulation and subsequent drug release specifically in cancer cells. The enhanced drug delivery towards cancer cells in vitro demonstrates the potential of this platform for selective treatments without the addition of targeting ligands.

Mechanism-based inhibition of gut microbial tryptophanases reduces serum indoxyl sulfate.

Indoxyl sulfate is a microbially derived uremic toxin that accumulates in late-stage chronic kidney disease and contributes to both renal and cardiovascular toxicity. Indoxyl sulfate is generated by the metabolism of indole, a compound created solely by gut microbial tryptophanases. Here, we characterize the landscape of tryptophanase enzymes in the human gut microbiome and find remarkable structural and functional similarities across diverse taxa. We leverage this homology through a medicinal chemistry campaign to create a potent pan-inhibitor, (3S) ALG-05, and validate its action as a transition-state analog. (3S) ALG-05 successfully reduces indole production in microbial culture and displays minimal toxicity against microbial and mammalian cells. Mice treated with (3S) ALG-05 show reduced cecal indole and serum indoxyl sulfate levels with minimal changes in other tryptophan-metabolizing pathways. These studies present a non-bactericidal pan-inhibitor of gut microbial tryptophanases with potential promise for reducing indoxyl sulfate in chronic kidney disease.

Microbial ß-glucuronidases drive human periodontal disease etiology.

Periodontitis is a chronic inflammatory disease associated with persistent oral microbial dysbiosis. The human ß-glucuronidase (GUS) degrades constituents of the periodontium and is used as a biomarker for periodontitis severity. However, the human microbiome also encodes GUS enzymes, and the role of these factors in periodontal disease is poorly understood. Here, we define the 53 unique GUSs in the human oral microbiome and examine diverse GUS orthologs from periodontitis-associated pathogens. Oral bacterial GUS enzymes are more efficient polysaccharide degraders and processers of biomarker substrates than the human enzyme, particularly at pHs associated with disease progression. Using a microbial GUS-selective inhibitor, we show that GUS activity is reduced in clinical samples obtained from individuals with untreated periodontitis and that the degree of inhibition correlates with disease severity. Together, these results establish oral GUS activity as a biomarker that captures both host and microbial contributions to periodontitis, facilitating more efficient clinical monitoring and treatment paradigms for this common inflammatory disease.

Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus.

Mammalian orthoreovirus (reovirus) infects most mammals and is associated with celiac disease in humans. In mice, reovirus infects the intestine and disseminates systemically to cause serotype-specific patterns of disease in the brain. To identify receptors conferring reovirus serotype-dependent neuropathogenesis, we conducted a genome-wide CRISPRa screen and identified paired immunoglobulin-like receptor B (PirB) as a receptor candidate. Ectopic expression of PirB allowed reovirus binding and infection. PirB extracelluar D3D4 region is required for reovirus attachment and infectivity. Reovirus binds to PirB with nM affinity as determined by single molecule force spectroscopy. Efficient reovirus endocytosis requires PirB signaling motifs. In inoculated mice, PirB is required for maximal replication in the brain and full neuropathogenicity of neurotropic serotype 3 (T3) reovirus. In primary cortical neurons, PirB expression contributes to T3 reovirus infectivity. Thus, PirB is an entry receptor for reovirus and contributes to T3 reovirus replication and pathogenesis in the murine brain.

Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut.

Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile.

Grade 1 Internal Carotid Artery Blunt Cerebrovascular Injury Persistence Risks Stroke With Current Management: An EAST Multicenter Study.

BACKGROUND: Higher blunt cerebrovascular injury (BCVI) grade and lack of medical therapy are associated with stroke. Knowledge of stroke risk factors specific to individual grades may help tailor BCVI therapy to specific injury characteristics. METHODS: A post-hoc analysis of a 16 center, prospective, observational trial (2018-2020) was performed including grade 1 internal carotid artery (ICA) BCVI. Repeat imaging was considered the second imaging occurrence only. RESULTS: From 145 grade 1 ICA BCVI included, 8 (5.5%) suffered a stroke. Grade 1 ICA BCVI with stroke were more commonly treated with mixed anticoagulation and antiplatelet therapy (75.0% vs 9.6%, P <.001) and less commonly antiplatelet therapy (25.0% vs 82.5%, P = .001) compared to injuries without stroke. Of the 8 grade 1 ICA BCVI with stroke, 4 (50.0%) had stroke after medical therapy was started. In comparing injuries with resolution at repeat imaging to those without, stroke occurred in 7 (15.9%) injuries without resolution and 0 (0%) injuries with resolution (P = .005). At repeat imaging in grade 1 ICA BCVI with stroke, grade of injury was grade 1 in 2 injuries, grade 2 in 3 injuries, grade 3 in 1 injury, and grade 5 in one injury. DISCUSSION: While the stroke rate for grade 1 ICA BCVI is low overall, injury persistence appears to heighten stroke risk. Some strokes occurred despite initiation of medical therapy. Repeat imaging is needed in grade 1 ICA BCVI to evaluate for injury progression or resolution.

A structural metagenomics pipeline for examining the gut microbiome.

Metagenomic sequencing data provide a rich resource from which to expand our understanding of differential protein functions involved in human health. Here, we outline a pipeline that combines microbial whole genome sequencing with protein structure data to yield a structural metagenomics-informed atlas of microbial enzyme families of interest. Visualizing metagenomics data through a structural lens facilitates downstream studies including targeted inhibition and probe-based proteomics to define at the molecular level how different enzyme orthologs impact in vivo function. Application of this pipeline to gut microbial enzymes like glucuronidases, TMA lyases, and bile salt hydrolases is expected to pinpoint their involvement in health and disease and may aid in the development of therapeutics that target specific enzymes within the microbiome.

Multi-omic analysis of host-microbial interactions central to the gut-brain axis.

The gut microbiota impact numerous aspects of human physiology, including the central nervous system (CNS). Emerging work is now focusing on the microbial factors underlying the bi-directional communication network linking host and microbial systems within the gastrointestinal tract to the CNS, the "gut-brain axis". Neurotransmitters are key coordinators of this network, and their dysregulation has been linked to numerous neurological disease states. As the bioavailability of neurotransmitters is modified by gut microbes, it is critical to unravel the influence of the microbiota on neurotransmitters in the context of the gut-brain axis. Here we review foundational studies that defined molecular relationships between the microbiota, neurotransmitters, and the gut-brain axis. We examine links between the gut microbiome, behavior, and neurological diseases, as well as microbial influences on neurotransmitter bioavailability and physiology. Finally, we review multi-omics technologies uniquely applicable to this area, including high-throughput genetics, modern metabolomics, structure-guided metagenomics, targeted proteomics, and chemogenetics. Interdisciplinary studies will continue to drive the discovery of molecular mechanisms linking the gut microbiota to clinical manifestations of neurobiology.

Atomic force microscopy applied to interrogate nanoscale cellular chemistry and supramolecular bond dynamics for biomedical applications.

Understanding biological interactions at a molecular level grants valuable information relevant to improving medical treatments and outcomes. Among the suite of technologies available, Atomic Force Microscopy (AFM) is unique in its ability to quantitatively probe forces and receptor-ligand interactions in real-time. The ability to assess the formation of supramolecular bonds and intermediates in real-time on surfaces and living cells generates important information relevant to understanding biological phenomena. Combining AFM with fluorescence-based techniques allows for an unprecedented level of insight not only concerning the formation and rupture of bonds, but understanding medically relevant interactions at a molecular level. As the ability of AFM to probe cells and more complex models improves, being able to assess binding kinetics, chemical topographies, and garner spectroscopic information will likely become key to developing further improvements in fields such as cancer, nanomaterials, and virology. The rapid response to the COVID-19 crisis, producing information regarding not just receptor affinities, but also strain-dependent efficacy of neutralizing nanobodies, demonstrates just how viable and integral to the pre-clinical development of information AFM techniques are in this era of medicine.

Metagenomics combined with activity-based proteomics point to gut bacterial enzymes that reactivate mycophenolate.

Mycophenolate mofetil (MMF) is an important immunosuppressant prodrug prescribed to prevent organ transplant rejection and to treat autoimmune diseases. MMF usage, however, is limited by severe gastrointestinal toxicity that is observed in approximately 45% of MMF recipients. The active form of the drug, mycophenolic acid (MPA), undergoes extensive enterohepatic recirculation by bacterial ß-glucuronidase (GUS) enzymes, which reactivate MPA from mycophenolate glucuronide (MPAG) within the gastrointestinal tract. GUS enzymes demonstrate distinct substrate preferences based on their structural features, and gut microbial GUS enzymes that reactivate MPA have not been identified. Here, we compare the fecal microbiomes of transplant recipients receiving MMF to healthy individuals using shotgun metagenomic sequencing. We find that neither microbial composition nor the presence of specific structural classes of GUS genes are sufficient to explain the differences in MPA reactivation measured between fecal samples from the two cohorts. We next employed a GUS-specific activity-based chemical probe and targeted metaproteomics to identify and quantify the GUS proteins present in the human fecal samples. The identification of specific GUS enzymes was improved by using the metagenomics data collected from the fecal samples. We found that the presence of GUS enzymes that bind the flavin mononucleotide (FMN) is significantly correlated with efficient MPA reactivation. Furthermore, structural analysis identified motifs unique to these FMN-binding GUS enzymes that provide molecular support for their ability to process this drug glucuronide. These results indicate that FMN-binding GUS enzymes may be responsible for reactivation of MPA and could be a driving force behind MPA-induced GI toxicity.