Background Probiotics could decrease irinotecan-induced diarrhea due to the reduction of intestinal beta-d-glucuronidase activity. This study included a combined analysis of two clinical trials aimed ...to determine the effectiveness of the probiotics in the prophylaxis of irinotecan-induced diarrhea in metastatic colorectal cancer (CRC) patients. Methods This combined analysis included 46 patients with CRC enrolled in the Probio-SK-003 (NCT01410955) and 233 patients from Probio-SK-005 (NCT02819960) starting a new line of irinotecan-based therapy with identical eligibility criteria. Patients were randomized in a ratio 1:1 to probiotic formulas vs. placebo administered for 12 and 6 weeks, respectively. Due to the different durations of study treatments, only the first 6 weeks of therapy were used for analysis. Results In total, 279 patients were randomized, including 142 patients in the placebo and 137 participants in the probiotic arm. Administration of probiotics did not significantly reduce the incidence of grade 3/4 diarrhea compared to placebo (placebo 12.7% vs. probiotics 6.6%, p = 0.11). Neither the overall incidence of diarrhea (placebo 48.6% vs. probiotics 41.6%, p = 0.28) nor the incidence of enterocolitis (placebo 4.2% vs. probiotics 0.7%, p = 0.12) was different in the placebo vs. probiotic arm. However, subgroup analysis revealed that patients with a colostomy who received a placebo had a significantly higher incidence of any diarrhea (placebo 51.2% vs. probiotics 25.7%, p = 0.028) and grade 3/4 diarrhea (placebo 14.6% vs. probiotics 0.0%, p = 0.03) compared to the probiotic arm. Conclusions This combined analysis suggests that probiotics could be beneficial in the prevention of irinotecan-induced diarrhea in colorectal cancer patients with colostomy.
Gut microbial β-glucuronidase (GUS) enzymes have been suggested to be involved in the estrobolome, the collection of microbial reactions involving estrogens. Furthermore, bacterial GUS enzymes within ...the gastrointestinal tract have been postulated to be a contributing factor in hormone-driven cancers. However, to date, there has been no experimental evidence to support these hypotheses. Here we provide the first in vitro analysis of the ability of 35 human gut microbial GUS enzymes to reactivate two distinct estrogen glucuronides, estrone-3-glucuronide and estradiol-17-glucuronide, to estrone and estradiol, respectively. We show that certain members within the Loop 1, mini-Loop 1, and FMN-binding classes of gut microbial GUS enzymes can reactivate estrogens from their inactive glucuronides. We provide molecular details of key interactions that facilitate these catalytic processes and present the structures of two novel human gut microbial GUS enzymes related to the estrobolome. Further, we demonstrate that estrogen reactivation by Loop 1 bacterial GUS enzymes can be inhibited both in purified enzymes and in fecal preparations of mixed murine fecal microbiota. Finally, however, despite these in vitro and ex vivo data, we show that a Loop 1 GUS-specific inhibitor is not capable of reducing the development of tumors in the PyMT mouse model of breast cancer. These findings validate that gut microbial GUS enzymes participate in the estrobolome but also suggest that the estrobolome is a multidimensional set of processes on-going within the mammalian gastrointestinal tract that likely involves many enzymes, including several distinct types of GUS proteins.
The thiadiazole-based benzothioate (3a-f) and benzenesulfonothioate (5a-f) hybrid derivatives were designed and synthesized in a single step reaction. All the synthesized compounds structures were ...confirmed through spectroscopic techniques including 1H NMR, 13C NMR and HRMS (ESI). Then, the synthesized compounds were subjected to in vitro thymidine phosphorylase and beta-glucuronidase inhibition profile. All the newly synthesized moieties (3a-f) & (5a-f) were found potent and showed moderately to good inhibition profile. Among benzothioate analogues (3a-f), the analogues 3d and 3c were identified to be active inhibitors of thymidine phosphorylase and beta-glucuronidase enzymes having IC50 values of 2.11 ± 0.31 μM & 3.28 ± 0.20 μM (against thymidine phosphorylase) and 2.50 ± 0.20 μM & 3.71 ± 0.10 μM (against beta-glucuronidase) respectively. However, among benzenesulfonothioate (5a-f) analogues, analogues 5a and 5d were found to be significantly active, even more than standard drugs with IC50 values of 2.23 ± 0.13 μM & 3.10 ± 0.17 μM (against thymidine phosphorylase) and 3.12 ± 0.20 μM & 5.60 ± 0.34 μM respectively. The results were compared to standard drugs such as D-saccharic acid with IC50 values of 10.10 ± 0.10 µM (for benzothioate series) & 8.50 ± 0.10 µM (for benzenesulfonothioate series) and 7-Deazaxanthine having IC50 values of 12.20 ± 0.10 µM (for benzothioate series) & 9.20 ± 0.30 µM (benzenesulfonothioate series) respectively. From structure-activity (SAR) analysis, it was confirmed that any variation found in inhibitory activities of both thymidine phosphorylase beta-glucuronidase enzymes was due to different substitution pattern of substituent(s) at variable position of aryl ring. Moreover, a good protein-ligand interaction (PLI) subjected to molecular docking study against shown by the most active analogues against corresponding target with key binding interactions with least binding energies. These findings reveal that thiadiazole-containing benzothioate (3a-f) and benzenesulfonothioate (5a-f) act as thymidine phosphorylase beta-glucuronidase inhibitors to develop novel therapeutics agents for the purpose of drug discovery.
Popularity of hyaluronan (HA) in the cosmetics and pharmaceutical industries, led to the investigation and development of new HA-based materials, with enzymes playing a key role. ...Beta-D-glucuronidases catalyze the hydrolysis of a beta-D-glucuronic acid residue from the non-reducing end of various substrates. However, lack of specificity towards HA for most beta-D-glucuronidases, in addition to the high cost and low purity of those active on HA, have prevented their widespread application. In this study, we investigated a recombinant beta-glucuronidase from Bacteroides fragilis (rBfGUS). We demonstrated the rBfGUS's activity on native, modified, and derivatized HA oligosaccharides (oHAs). Using chromogenic beta-glucuronidase substrate and oHAs, we characterized the enzyme's optimal conditions and kinetic parameters. Additionally, we evaluated rBfGUS's activity towards oHAs of various sizes and types. To increase reusability and ensure the preparation of enzyme-free oHA products, rBfGUS was immobilized on two types of magnetic macroporous bead cellulose particles. Both immobilized forms of rBfGUS demonstrated suitable operational and storage stabilities, and their activity parameters were comparable to the free form. Our findings suggest that native and derivatized oHAs can be prepared using this bacterial beta-glucuronidase, and a novel biocatalyst with enhanced operational parameters has been developed with a potential for industrial use.
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Background
The incidence of irinotecan-induced diarrhea varies between 60-90%, by which the incidence of severe diarrhea is 20-40%. The objective of this phase III trial was to determine the ...effectiveness of the probiotic mixture containing
Bifidobacterium
, BB-12
®
and
Lactobacillus rhamnosus
, LGG
®
in the prophylaxis of irinotecan-induced diarrhea in metastatic colorectal cancer patients due to a reduction in the activity of intestinal beta-D-glucuronidase.
Methods
From March 2016 to May 2022, a total of 242 patients with colorectal cancer starting a new line of irinotecan-based therapy were registered to the study in 11 cancer centers in Slovakia. Patients were randomized in a ratio 1:1 to probiotic formula vs. placebo that was administered for 6 weeks. Each capsule of Probio-Tec
®
BG-Vcap-6.5 contained 2.7x10
9
colony-forming units (CFU) of 2 lyophilized probiotic strains
Bifidobacterium
, BB-12
®
(50%) and
Lactobacillus rhamnosus
GG, LGG
®
(50%).
Results
Administration of probiotics compared to placebo was not associated with a significant reduction of grade 3/4 diarrhea (placebo arm 11.8% vs. probiotic arm 7.9%, p=0.38). Neither the overall incidence of diarrhea (46.2% vs. 41.2%, p=0.51) nor the incidence of enterocolitis (3.4% vs. 0.9%, p=0.37) was different in the placebo vs. probiotic arm. Subgroup analysis revealed that patients with colostomy had higher incidence of any diarrhea and grade 3/4 diarrhea in the placebo arm compared to the probiotic arm (48.5% vs. 22.2%, p=0.06 and 15.2% vs. 0%, p=0.06, respectively). Moreover, patients on probiotic arm had significantly better diarrhea-free survival (HR = 0.41, 95%CI 0.18 – 0.95, p=0.05) and needed less loperamide (p=0.01) compared to patients on placebo arm. We did not observe any infection caused by probiotic strains used in this study.
Conclusion
This study failed to achieve its primary endpoint, and results suggest a lack of benefit of administered probiotic formula for the prevention of irinotecan-induced diarrhea. However, subgroup analysis suggests a possible benefit in patients with colostomy.
Ethylene-response factors (ERFs) play an important role in regulating gene expression in plant responses to biotic and abiotic stresses. In this study, a new ERF transcription factor, GmERF7, was ...isolated from soybean. Sequence analysis showed that GmERF7 contained an AP2/ERF domain with 58 amino acids, two putative nuclear localization signal (NLS) domains, an acidic amino acid-rich transcriptional activation domain and a conserved N-terminal motif MCGGAI(I/L). The expression of GmERF7 was induced by drought, salt, methyl jasmonate (MeJA), ethylene (ETH) and abscisic acid (ABA) treatments. However, the expression of GmERF7 decreased under cold treatment. GmERF7 localized to the nucleus when transiently expressed in onion epidermal cells. Furthermore, GmERF7 protein bound to the GCC-box element in vitro and activated the expression of the β-glucuronidase (GUS) reporter gene in tobacco leaves. Activities of GmERF7 promoter (GmERF7P) upregulated in tobacco leaves with 10h drought, salt and ETH treatments. However, activities of GmERF7P decreased with 10h cold and ABA treatments. Overexpression of GmERF7 in tobacco plants led to higher levels of chlorophyll and soluble carbohydrates and a lower level of malondialdehyde compared with wild-type tobacco plants under salt stress conditions, which indicated that GmERF7 enhanced salt tolerance in transgenic plants.
► GmERF7 acts as a transcription activator in the nucleus. ► GmERF7 is involved in multiple signal transduction pathways. ► GmERF7 binds to the GCC-box to activate the expression of stress-related genes. ► Overexpression of GmERF7 enhanced salt tolerance in tobacco plants.
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 β-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.
Background
Black‐shouldered kites (BSK, Elanus caeruleus), Brahminy kites (BrK, Haliastur indus), and black kites (BK, Milvus migrans govinda) are medium‐sized hawks found in Thailand, and little is ...known about the hematology of these three kite species.
Objective
This study reports basic hematologic values and describes the light microscopic, cytochemical, and ultrastructural characteristics of blood cells in these kites.
Methods
Blood samples were collected from 113 healthy kites (50 BSKs, 53 BrKs, and 10 BKs) from January 2012 to December 2017. Complete blood cell counts, cytochemical staining (Sudan black B, peroxidase PO, periodic acid‐Schiff, α‐naphthyl acetate esterase, and β‐glucuronidase), and transmission electron microscopy were performed using standard methods.
Results
Hematology, morphometry, and cytochemical staining patterns of blood cells were tabulated. BSK erythrocytes were smaller than BrK and BK erythrocytes. Heterophils, the largest granulocytes, were the most prevalent leukocytes in all kites. Cytochemical reactions in blood cells from these three kite species were the same, except that heterophils from BrKs were the only cells positive for PO. The ultrastructure of heterophil and eosinophil granules from the BSKs were similar in their homogeneous electron densities but differed in shape. The eosinophil granules from BrKs and BKs revealed heterogeneous electron densities with central pallor in some granules. Basophils had different granular electron densities, and some granules were electron‐lucent.
Conclusion
The 23 baseline hematologic values and morphologic, cytochemical, and ultrastructural characteristics of all blood cell types in this study provide reference data for future kite healthcare.
The serine-threonine kinase CK2, which targets over 300 cellular proteins, is overexpressed in all cancers, presumably reflecting its ability to promote proliferation, spread, and survival through a ...wide range of complementary mechanisms. Via an activating phosphorylation of Cdc373, a co-chaperone which partners with Hsp90, CK2 prolongs the half-life of protein kinases that promote proliferation and survival in many cancers, including Akt, Src, EGFR, Raf-1, and several cyclin-dependent kinases. CK2 works in other ways to boost the activity of signaling pathways that promote cancer aggressiveness and chemoresistance, including those driven by Akt, NF-kappaB, hypoxia-inducible factor-1, beta-catenin, TGF-beta, STAT3, hedgehog, Notch1, and the androgen receptor; it promotes the epidermal-mesenchymal transition and aids efficiency of DNA repair. Several potent and relatively specific inhibitors of CK2 are now being evaluated as potential cancer drugs; CX-4945 has shown impressive activity in cell culture studies and xenograft models, and is now entering clinical trials. Moreover, it has long been recognized that the natural flavone apigenin can inhibit CK2, with a Ki near 1 µM; more recent work indicates that a range of flavones and flavonols, characterized by a planar structure and hydroxylations at the 7 and 4′ positions – including apigenin, luteolin kaempferol, fisetin, quercetin, and myricetin - can inhibit CK2 with Ki s in the sub-micromolar range. This finding is particularly intriguing in light of the numerous studies demonstrating that each of these agents can inhibit the growth of cancer cells lines in vitro and of human xenografts in nude mice. These studies attribute the cancer-retardant efficacy of flavones/flavonols to impacts on a bewildering array of cellular targets, including those whose activities are boosted by CK2; it is reasonable to suspect that, at least in physiologically achievable concentrations, these agents may be achieving these effects primarily via CK2 inhibition. Inefficient absorption and rapid conjugation limit the bioefficacy of orally administered flavonoids; however, the increased extracellular beta-glucuronidase of many tumors may give tumors privileged access to glucuronidated flavonoids, and nanopartical technology can improve the bioavailability of these agents. Enzymatically modified isoquercitrin has particular promise as a delivery vehicle for quercetin. Hence, it may be worthwhile to explore the clinical potential of flavones/flavonols as CK2 inhibitors for cancer therapy.
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