Deciphering molecular mechanisms underlying the inhibition of ß-glucuronidase by xanthones from Centaurium spicatum.

Herein, we scrutinized the inhibitory potential of five xanthones and a flavonoid, sourced from Centaurium spicatum, against ß-glucuronidase activity. The results showed that gentisin and azaleatin emerged as the most potent inhibitors, with significantly lower IC50 values of 0.96 ± 0.10 and 0.57 ± 0.04 µM, respectively. The evaluation of enzyme kinetics unveiled that the isolated xanthones manifested inhibition of ß-glucuronidase through a mixed inhibition mode, whereas azaleatin exhibited a noncompetitive inhibition mechanism. The findings from molecular docking analysis unveiled that the compounds under investigation, particularly azaleatin, displayed comparatively diminished binding affinities towards ß-glucuronidase. Furthermore, the tested drugs were shown to occupy a common binding site as the employed reference drug. Our comprehensive Molecular Dynamics (MD) simulations analysis revealed consistent trajectories for the investigated drugs, wherein azaleatin and gentisin demonstrated notable stabilization of energy levels. Analysis of various MD parameters revealed that drugs with the lowest IC50 values maintained relatively stable interactions with ß-glucuronidase. These drugs were shown to exert notable alterations in their conformation or flexibility upon complexation with the target enzyme. Conversely, the flexibility and accessibility of ß-glucuronidase was reduced upon drug binding, particularly with azaleatin and gentisin, underscoring the stability of the drug-enzyme complexes. Analysis of Coul-SR and LJ-SR interaction energies unveiled consistent and stable interactions between certain isolated drugs and ß-glucuronidase. Azaleatin notably displayed the lowest average Coul-SR interaction energy, suggesting strong electrostatic interactions with the enzyme's active site and significant conformational variability during simulation. Remarkably, LJ-SR interaction energies across different xanthones complexes were more negative than their Coul-SR counterparts, emphasizing the predominant role of van der Waals interactions, encompassing attractive dispersion and repulsive forces, in stabilizing the drug-enzyme complexes rather than electrostatic interactions.

Gut microbiota biotransformation of drug glucuronides leading to gastrointestinal toxicity: Therapeutic potential of bacterial ß-glucuronidase inhibition in mycophenolate-induced enteropathy.

AIMS: Drug-induced enteropathy is often associated with the therapeutic use of certain glucuronidated drugs. One such drug is mycophenolic acid (MPA), a well-established immunosuppressant of which gastrointestinal adverse effects are a major concern. The role of bacterial ß-glucuronidase (ß-G) from the gut microbiota in MPA-induced enteropathy has recently been discovered. Bacterial ß-G hydrolyzes MPAG, the glucuronide metabolite of MPA excreted in the bile, leading to the digestive accumulation of MPA that would favor in turn these adverse events. We therefore hypothesized that taming bacterial ß-G activity might reduce MPA digestive exposure and prevent its toxicity. MAIN METHODS: By using a multiscale approach, we evaluated the effect of increasing concentrations of MPA on intestinal epithelial cells (Caco-2 cell line) viability, proliferation, and migration. Then, we investigated the inhibitory properties of amoxapine, a previously described bacterial ß-G inhibitor, by using molecular dynamics simulations, and evaluated its efficiency in blocking MPAG hydrolysis in an Escherichia coli-based ß-G activity assay. The pharmacological effect of amoxapine was evaluated in a mouse model. KEY FINDINGS: We observed that MPA impairs intestinal epithelial cell homeostasis. Amoxapine efficiently blocks the hydrolysis of MPAG to MPA and significantly reduces digestive exposure to MPA in mice. As a result, administration of amoxapine in MPA-treated mice significantly attenuated gastrointestinal lesions. SIGNIFICANCE: Collectively, these results suggest that the digestive accumulation of MPA is involved in the pathophysiology of MPA-gastrointestinal adverse effects. This study provides a proof-of-concept of the therapeutic potential of bacterial ß-G inhibitors in glucuronidated drug-induced enteropathy.

Microbial ß-Glucuronidase Hydrogel Beads Activate Chemotherapeutic Prodrug.

Bacteria-assisted chemotherapeutics have been highlighted as an alternative or supplementary approach to treating cancer. However, dynamic cancer-microbe studies at the in vitro level have remained a challenge to show the impact and effectiveness of microbial therapeutics due to the lack of relevant coculture models. Here, we demonstrate a hydrogel-based compartmentalized system for prodrug activation of a natural ingredient of licorice root, glycyrrhizin, by microbial ß-glucuronidase (GUS). Hydrogel containment with Lactococcus lactis provides a favorable niche to encode GUS enzymes with excellent permeability and can serve as an independent ecosystem in the transformation of pro-apoptotic materials. Based on the confinement system of GUS expressing microbes, we quantitatively evaluated chemotherapeutic effects enhanced by microbial GUS enzyme in two dynamic coculture models in vitro (i.e., 2D monolayered cancer cells and 3D tumor spheroids). Our findings support the processes of prodrug conversion mediated by bacterial GUS enzyme which can enhance the therapeutic efficacy of a chemotherapy drug under dynamic coculture conditions. We expect our in vitro coculture platforms can be used for the evaluation of pharmacological properties and biological activity of xenobiotics as well as the potential impact of microbes on cancer therapeutics.

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.

Supplementation with a Probiotic Formula Having ß-Glucuronidase Activity Modulates Serum Estrogen Levels in Healthy Peri- and Postmenopausal Women.

Declines in estrogen levels occur in women transitioning to menopause. Estrogen hormones play important roles in multiple systems of the body, and estrogen loss is associated with a variety of symptoms that can decrease quality of life. The gut microbiota is involved in regulating endogenous estrogen levels. A portion of estrogen glucuronides can be reactivated in the gut by the microbial enzyme ß-glucuronidase, and the resulting free estrogens can return to the bloodstream. Here, we carried out in vitro screening of ß-glucuronidase activities for 84 strains belonging to 16 different species of lactic acid bacteria and bifidobacteria and found that one and three strains of Levilactobacillus brevis and Lacticasebacillus rhamnosus, respectively, can deconjugate estrogens. Among these strains, L. brevis KABP052 had the highest ß-glucuronidase activity. Moreover, in an exploratory, randomized, double-blind, placebo-controlled trial, we demonstrated that serum estrogen levels in healthy peri- and postmenopausal women given a probiotic formula containing KABP052 were maintained over time, whereas levels significantly decreased in the group given a placebo. Significantly higher levels of estradiol (31.62 ± 7.97 pg/mL vs. 25.12 ± 8.17 pg/mL) and estrone (21.38 ± 8.57 pg/mL vs. 13.18 ± 8.77 pg/mL) were observed in the probiotic versus placebo group after 12 weeks of intervention. This clinical study demonstrated for the first time the estrogen modulation capacity of a probiotic formula containing a bacterial strain having ß-glucuronidase activity in women during the menopausal transition and formed the basis for future investigations using probiotics in the menopausal population.

Disease characteristics, effectiveness, and safety of vestronidase alfa for the treatment of patients with mucopolysaccharidosis VII in a novel, longitudinal, multicenter disease monitoring program.

BACKGROUND: Mucopolysaccharidosis VII (MPS VII) is an ultra-rare, autosomal recessive, debilitating, progressive lysosomal storage disease caused by reduced activity of ß-glucuronidase (GUS) enzyme. Vestronidase alfa (recombinant human GUS) intravenous enzyme replacement therapy is an approved treatment for patients with MPS VII. METHODS: This disease monitoring program (DMP) is an ongoing, multicenter observational study collecting standardized real-world data from patients with MPS VII (N ≈ 50 planned) treated with vestronidase alfa or any other management approach. Data are monitored and recorded in compliance with Good Clinical Practice guidelines and planned interim analyses of captured data are performed annually. Here we summarize the safety and efficacy outcomes as of 17 November 2022. RESULTS: As of the data cutoff date, 35 patients were enrolled: 28 in the Treated Group and seven in the Untreated Group. Mean (SD) age at MPS VII diagnosis was 4.5 (4.0) years (range, 0.0 to 12.4 years), and mean (SD) age at DMP enrollment was 13.9 (11.1) years (range, 1.5 to 50.2 years). Ten patients (29%) had a history of nonimmune hydrops fetalis. In the 23 patients who initiated treatment prior to DMP enrollment, substantial changes in mean excretion from initial baseline to DMP enrollment were observed for the three urinary glycosaminoglycans (uGAGs): dermatan sulfate (DS), -84%; chondroitin sulfate (CS), -55%; heparan sulfate (HS), -42%. Also in this group, mean reduction from initial baseline to months 6, 12, and 24 were maintained for uGAG DS (-84%, -87%, -89%, respectively), CS (-70%, -71%, -76%, respectively), and HS (+ 3%, -32%, and - 41%, respectively). All adverse events (AEs) were consistent with the known vestronidase alfa safety profile. No patients discontinued vestronidase alfa. One patient died. CONCLUSIONS: To date, the DMP has collected invaluable MPS VII disease characteristic data. The benefit-risk profile of vestronidase alfa remains unchanged and favorable for its use in the treatment of pediatric and adult patients with MPS VII. Reductions in DS and CS uGAG demonstrate effectiveness of vestronidase alfa to Month 24. Enrollment is ongoing.

Secondary metabolites from Penicillium sp. HS-11, a fungal endophyte of Huperzia serrata.

Three new sorbicillinoids sorbicatechols E-G (1-3), along with seven known compounds 4-10, were obtained from the ethanol extract of Penicillium sp. HS-11, a fungal endophyte of the medicinal plant Huperzia serrata. The structures of 1-3 were established by detailed interpretation of the spectroscopic data and their absolute configurations were established by comparative analyses of the ECD spectra. Sorbicatechol G (3) represented the first hybrid sorbicillinoid bearing a tetralone skeleton. In the in-vitro bioassay, trichodimerol (5) exhibited moderate inhibitory activity against the Escherichia coli ß-glucuronidase (EcGUS) with an IC50 value of 92.0 ± 9.4 µM.

Development of an LC-MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates.

PURPOSE: Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline). METHODS: The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation. RESULTS: We found that the recombinant ß-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3 min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%. CONCLUSIONS: We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.

Enhancing SN38 prodrug delivery using a self-immolative linker and endogenous albumin transport.

Enhancing the delivery and release efficiency of hydroxyl agents, constrained by high pKa values and issues of release rate or unstable linkage, is a critical challenge. To address this, a self-immolative linker, composed of a modifiable p-hydroxybenzyl ether and a fast cyclization adapter (N-(ortho-hydroxyphenyl)-N-methylcarbamate) was strategically designed, for the synthesis of prodrugs. The innovative linker not only provides a side chain modification but also facilitates the rapid release of the active payloads, thereby enabling precise drug delivery. Particularly, five prodrug model compounds (J1, J2, J3, J5 and J6) were synthesized to evaluate the release rates by using ß-glucuronic acid as trigger and five hydroxyl compounds as model payloads. Significantly, all prodrug model compounds could efficiently release the hydroxyl payloads under the action of ß-glucuronidase, validating the robustness of the linker. And then, to assess the drug delivery and release efficiency using endogenous albumin as a transport vehicle, J1148, a SN38 prodrug modified with maleimide side chain was synthesized. Results demonstrated that J1148 covalently bound to plasma albumin through in situ Michael addition, effectively targeting the tumor microenvironment. Activated by ß-glucuronidase, J1148 underwent a classical 1, 6-elimination, followed by rapid cyclization of the adapter, thereby releasing SN38. Impressively, J1148 showed excellent therapeutic efficacy against human colonic cancer xenograft model, leading to a significant reduction or even disappearance of tumors (3/6 of mice cured). These findings underscore the potential of the designed linker in the delivery system of hydroxyl agents, positioning it at the forefront of advancements in drug delivery technology.

Tumoral acidosis promotes adipose tissue depletion by fostering adipocyte lipolysis.

OBJECTIVE: Tumour progression drives profound alterations in host metabolism, such as adipose tissue depletion, an early event of cancer cachexia. As fatty acid consumption by cancer cells increases upon acidosis of the tumour microenvironment, we reasoned that fatty acids derived from distant adipose lipolysis may sustain tumour fatty acid craving, leading to the adipose tissue loss observed in cancer cachexia. METHODS: To evaluate the pro-lipolytic capacities of acid-exposed cancer cells, primary mouse adipocytes from subcutaneous and visceral adipose tissue were exposed to pH-matched conditioned medium from human and murine acid-exposed cancer cells (pH 6.5), compared to naive cancer cells (pH 7.4). To further address the role of tumoral acidosis on adipose tissue loss, a pH-low insertion peptide was injected into tumour-bearing mice, and tumoral acidosis was neutralised with a sodium bicarbonate buffer. Prolipolytic mediators were identified by transcriptomic approaches and validated on murine and human adipocytes. RESULTS: Here, we reveal that acid-exposed cancer cells promote lipolysis from subcutaneous and visceral adipocytes and that dampening acidosis in vivo inhibits adipose tissue depletion. We further found a set of well-known prolipolytic factors enhanced upon acidosis adaptation and unravelled a role for ß-glucuronidase (GUSB) as a promising new actor in adipocyte lipolysis. CONCLUSIONS: Tumoral acidosis promotes the mobilization of fatty acids derived from adipocytes via the release of soluble factors by cancer cells. Our work paves the way for therapeutic approaches aimed at tackling cachexia by targeting the tumour acidic compartment.

Glucuronic acid confers colonization advantage to enteric pathogens.

Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces ß-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial ß-glucuronidase inhibitor (GUSi) decreased C. rodentium's colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for ß-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn't activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial ß-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.

A first-in-class ß-glucuronidase responsive conjugate for selective dual targeted and photodynamic therapy of bladder cancer.

In this report, we present a novel prodrug strategy that can significantly improve the efficiency and selectivity of combined therapy for bladder cancer. Our approach involved the synthesis of a conjugate based on a chlorin-e6 photosensitizer and a derivative of the tyrosine kinase inhibitor cabozantinib, linked by a ß-glucuronidase-responsive linker. Upon activation by ß-glucuronidase, which is overproduced in various tumors and localized in lysosomes, this conjugate released both therapeutic modules within targeted cells. This activation was accompanied by the recovery of its fluorescence and the generation of reactive oxygen species. Investigation of photodynamic and dark toxicity in vitro revealed that the novel conjugate had an excellent safety profile and was able to inhibit tumor cells proliferation at submicromolar concentrations. Additionally, combined therapy effects were also observed in 3D models of tumor growth, demonstrating synergistic suppression through the activation of both photodynamic and targeted therapy.

Draft genome sequences of two ß-glucuronidase positive strains of Salmonella enterica subspecies salamae isolated from reptile feces in KwaZulu-Natal, South Africa.

Two Salmonella enterica isolates obtained from reptile feces displayed ß-glucuronidase activity. Nearly complete genome sequences were obtained after shotgun sequencing and de novo genome assembly. By comparison to reference genomes, both isolates were identified as Salmonella enterica subspecies salamae with the sequence type identified as 1208 and the serotype as 42:r:-.

ß-Glucuronidase-triggered reaction for fluorometric and colorimetric dual-mode assay based on the in situ formation of silicon nanoparticles.

BACKGROUND: ß-Glucuronidase (GUS) is considered as a promising biomarker for primary cancer. Thus, the reliable detection of GUS has great practical significance in the discovery and diagnosis of cancer. Compared with traditional organic probes, silicon nanoparticles (Si NPs) have emerged as robust optical nanomaterials due to their facile preparation, superior photobleaching resistance and excellent biocompatibility. However, most nanomaterials-based methods only output a single signal which is easily influenced by external factors in complex systems. Hence, developing nanomaterial-based multi-signal optical assays for highly sensitive GUS determination is still urgently desired. RESULTS: In this study, we developed a simple and efficient one-step method for the in situ preparation of yellow color and yellow-green fluorescent Si NPs. This was achieved by combining 3-[2-(2-aminoethylamino) ethylamino] propyl-trimethoxysilane with p-aminophenol (AP) in an aqueous solution. The obtained Si NPs showed yellow-green fluorescence at 535 nm when excited at 380 nm, while also exhibiting an absorption peak at a wavelength of 490 nm. Taking inspiration from the easy synthesis step regulated by AP, which is generated through the hydrolysis of 4-aminophenyl ß-D-glucuronide catalyzed by GUS, we constructed a direct fluorometric and colorimetric dual-mode method to measure GUS activity. The developed fluorometric and colorimetric sensing platform showed high sensitivity and accuracy with detection limits for GUS determination as low as 0.0093 and 0.081 U/L, respectively. SIGNIFICANCE: This study provides a facile dual-mode fluorometric and colorimetric approach for determination of GUS activity based on novel Si NPs for the first time. This designed sensing approach was successfully employed for the quantification of GUS in human serum samples and screening of GUS inhibitors, indicating the feasibility and potential applications in clinical cancer diagnosis and anti-cancer drug discovery.

Deconjugation potentials of natural estrogen conjugates in sewage and wastewater treatment plant: New insights from model prediction and on-site investigations.

Natural estrogen conjugates play important roles in municipal wastewater treatment plant (WWTP), but their deconjugation potentials are poorly understood. This work is the first to investigate the relationships between the enzyme activities of arylsulfatase/ß-glucuronidase and deconjugation potentials of natural estrogen conjugates. This work led to three important findings. First, the enzyme activity of ß-glucuronidase in sewage is far higher than that of arylsulfatase, while their corresponding activities in activated sludge were similar. Second, a model based on ß-glucuronidase could successfully predict the deconjugation potentials of natural estrogen glucuronide conjugates in sewage. Third, the enzyme activity of arylsulfatase in sewage was too low to lead to evident deconjugation of sulfate conjugates, which means that the deconjugation rate of estrogen sulfates can be regarded as zero. By comparing their theoretical removal based on enzyme activity and on-site investigation, it is reasonable to conclude that reverse deconjugation of estrogen conjugates (i.e., conjugation of natural estrogens to form conjugated estrogens) likely exist in WWTP, which explains well why natural estrogen conjugates cannot be effectively removed in WWTP. Meanwhile, this work provides new insights how to improve the removal performance of WWTP on natural estrogen conjugates. SYNOPSIS: This work is the first to show how arylsulfatase/ß-glucuronidase could affect deconjugation of natural estrogen conjugates and possible way to enhance their removal in wastewater treatment plant.

The Relationship Between Gut Microbiome Estrobolome and Breast Cancer: A Systematic Review of Current Evidences.

Breast cancer is the most frequent kind of cancer and the second leading cause of mortality worldwide, behind heart disease. Next-generation sequencing technologies enables for unprecedented enumeration of human resident gut microorganisms, conferring novel insights into the role of the microbiota in health and individuals with breast cancer. A growing body of research on microbial dysbiosis seems to indicate an elevated risk of health complications including cancer. Although several dysbiosis indices have been proposed, their underlying methodology, as well as the cohorts and conditions of breast cancer patients are significantly different. To date, these indices have not yet been thoroughly reviewed especially when it comes to researching the estrogen-gut microbiota axis. Instead of providing a thorough rating of the most effective diversity measurements, the current work aims to be used to assess the relevance of each study's findings across the demographic data, different subtypes, and stages of breast cancer, and tie them to the estrobolome, which controls the amount of oestrogen that circulates through humans. This review will cover 11 studies which will go into a detailed discussion for the microbiome results of the mentioned studies, leaving to the user the final choice of the most suited indices as well as highlight the observed bacteria found to be related to the estrobolome in hopes of giving the reader a better understanding for the biological cross-talk between gut microbiome and breast cancer progression. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01135-z.

Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing.

BACKGROUND: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. RESULTS: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-ß-xylanase and ß-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA ß-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L- 1 after 48 h under oxygen limited condition in bioreactor fermentations. CONCLUSION: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Astraoleanosides E-P, oleanane-type triterpenoid saponins from the aerial parts of Astragalus membranaceus Bunge and their ß-glucuronidase inhibitory activity.

Historically, Astragalus membranaceus Bunge has been used as a beneficial medicinal plant, particularly in the Asian traditional medical systems, for the treatment of various human diseases such as stomach ulcers, diarrhea, and respiratory issues associated with phlegm. In this study, a phytochemical characterization of the aerial parts of A. membranaceusled to the isolation of 29 oleanane-type triterpenoid saponins, including 11 new compounds named astraoleanosides E-P (6-9, 13, 14, 18-22), as well as 18 known ones. The structures of these compounds were elucidated using nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry. Among them, astraoleanoside H (9) and cloversaponin III (15) demonstrated the most potent ß-glucuronidase inhibitory activities, with IC50 values of 21.20 ± 0.75 and 9.05 ± 0.47 µM, respectively, compared to the positive control d-saccharic acid 1,4-lactone (IC50 = 20.62 ± 1.61 µM). Enzyme kinetics studies were then conducted to investigate the type of inhibition exhibited by these active compounds. In addition, the binding mechanism, key interactions, binding stability, and dynamic behavior of protein-ligand complexes were investigated through in silico approaches, such as molecular docking and molecular dynamics simulations. These findings highlight the promising potential of triterpenoid saponins from A. membranaceus as lead compounds for ß-glucuronidase inhibitors, offering new possibilities for the development of therapeutic agents targeting various diseases where ß-glucuronidase plays a crucial role.

Targeted inhibition of gut bacterial ß-glucuronidases by octyl gallate alleviates mycophenolate mofetil-induced gastrointestinal toxicity.

Mycophenolate mofetil (MMF) is a viable therapeutic option against various immune disorders as a chemotherapeutic agent. Nevertheless, its application has been undermined by the gastrotoxic metabolites (mycophenolic acid glucuronide, MPAG) produced by microbiome-associated ß-glucuronidase (ßGUS). Therefore, controlling microbiota-produced ßGUS underlines the potential strategy to improve MMF efficacy by overcoming the dosage limitation. In this study, the octyl gallate (OG) was identified with promising inhibitory activity on hydrolysis of PNPG in our high throughput screening based on a chemical collection of approximately 2000 natural products. Furthermore, OG was also found to inhibit a broad spectrum of BGUSs, including mini-Loop1, Loop 2, mini-Loop 2, and mini-Loop1,2. The further in vivo experiments demonstrated that administration of 20 mg/kg OG resulted in predominant reduction in the activity of BGUSs while displayed no impact on the overall fecal microbiome in mice. Furthermore, in the MMF-induced colitis model, the administration of OG at a dosage of 20 mg/kg effectively mitigated the gastrointestinal toxicity, and systematically reverted the colitis phenotypes. These findings indicate that the OG holds promising clinical potential for the prevention of MMF-induced gastrointestinal toxicity by inhibition of BGUSs and could be developed as a combinatorial therapy with MFF for better clinical outcomes.

ß-D-glucuronidase activity triggered monitoring of fecal contamination using microbial and chemical source tracking markers at drinking water intakes.

Intense rainfall and snowmelt events may affect the safety of drinking water, as large quantities of fecal material can be discharged from storm or sewage overflows or washed from the catchment into drinking water sources. This study used ß-d-glucuronidase activity (GLUC) with microbial source tracking (MST) markers: human, bovine, porcine mitochondrial DNA markers (mtDNA) and human-associated Bacteroidales HF183 and chemical source tracking (CST) markers including caffeine, carbamazepine, theophylline and acetaminophen, pathogens (Giardia, Cryptosporidium, adenovirus, rotavirus and enterovirus), water quality indicators (Escherichia coli, turbidity) and hydrometeorological data (flowrate, precipitation) to assess the vulnerability of 3 drinking water intakes (DWIs) and identify sources of fecal contamination. Water samples were collected under baseline, snow and rain events conditions in urban and agricultural catchments (Québec, Canada). Dynamics of E. coli, HF183 and WWMPs were similar during contamination events, and concentrations generally varied over 1 order of magnitude during each event. Elevated human-associated marker levels during events demonstrated that urban DWIs were impacted by recent contamination from an upstream municipal water resource recovery facility (WRRF). In the agricultural catchment, mixed fecal pollution was observed with the occurrences and increases of enteric viruses, human bovine and porcine mtDNA during peak contaminating events. Bovine mtDNA qPCR concentrations were indicative of runoff of cattle-derived fecal pollutants to the DWI from diffuse sources following rain events. This study demonstrated that the suitability of a given MST or CST indicator depend on river and catchment characteristics. The sampling strategy using continuous online GLUC activity coupled with MST and CST markers analysis was a more reliable source indicator than turbidity to identify peak events at drinking water intakes.