Glycoside-metabolizing oxidoreductase D3dgpA from human gut bacterium.

The Gfo/Idh/MocA family enzyme DgpA was known to catalyze the regiospecific oxidation of puerarin to 3"-oxo-puerarin in the presence of 3-oxo-glucose. Here, we discovered that D3dgpA, dgpA cloned from the human gut bacterium Dorea sp. MRG-IFC3, catalyzed the regiospecific oxidation of various C-/O-glycosides, including puerarin, in the presence of methyl ß-D-3-oxo-glucopyranoside. While C-glycosides were converted to 3"- and 2"-oxo-products by D3dgpA, O-glycosides resulted in the formation of aglycones and hexose enediolone from the 3"-oxo-products. From DFT calculations, it was found that isomerization of 3"-oxo-puerarin to 2"-oxo-puerarin required a small activation energy of 9.86 kcal/mol, and the O-glycosidic bond cleavage of 3"-oxo-products was also thermodynamically favored with a small activation energy of 3.49 kcal/mol. In addition, the reaction mechanism of D3dgpA was discussed in comparison to those of Gfo/Idh/MocA and GMC family enzymes. The robust reactivity of D3dgpA was proposed as a new general route for derivatization of glycosides.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-ß-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum ß-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of ß-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-ß-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and ß-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

Potentiation of Antibiotic Activity of Aztreonam against Metallo-ß-Lactamase-Producing Multidrug-Resistant Pseudomonas aeruginosa by 3-O-Substituted Difluoroquercetin Derivatives.

The combination of aztreonam (ATM) and ceftazidime-avibactam (CAZ-AVI; CZA) has shown therapeutic potential against serine-ß-lactamase (SBL)- and metallo-ß-lactamase (MBL)-producing Enterobacterales. However, the ability of CZA to restore the antibiotic activity of ATM is severely limited in MBL-producing multidrug-resistant (MDR) Pseudomonas aeruginosa strains because of the myriad of intrinsic and acquired resistance mechanisms associated with this pathogen. We reasoned that the simultaneous inhibition of multiple targets associated with multidrug resistance mechanisms may potentiate the antibiotic activity of ATM against MBL-producing P. aeruginosa. During a search for the multitarget inhibitors through a molecular docking study, we discovered that di-F-Q, the previously reported efflux pump inhibitor of MDR P. aeruginosa, binds to the active sites of the efflux pump (MexB), as well as various ß-lactamases, and these sites are open to the 3-O-position of di-F-Q. The 3-O-substituted di-F-Q derivatives were thus synthesized and showed hereto unknown multitarget MDR inhibitory activity against various ATM-hydrolyzing ß-lactamases (AmpC, KPC, and New Delhi metallo-ß-lactamase (NDM)) and the efflux pump of P. aeruginosa, presumably by forming additional hydrophobic contacts with the targets. The multitarget MDR inhibitor 27 effectively potentiated the antimicrobial activity of ATM and reduced the MIC of ATM more than four-fold in 19 out of 21 MBL-producing P. aeruginosa clinical strains, including the NDM-producing strains which were highly resistant to various combinations of ATM with ß-lactamase inhibitors and/or efflux pump inhibitors. Our findings suggest that the simultaneous inhibition of multiple MDR targets might provide new avenues for the discovery of safe and efficient MDR reversal agents which can be used in combination with ATM against MBL-producing MDR P. aeruginosa.

Production of Four Flavonoid C-Glucosides in Escherichia coli.

Flavonoid C-glucosides, which are found in several plant families, are characterized by several biological properties, including antioxidant, anticancer, anti-inflammatory, neuroprotective, hepatoprotective, cardioprotective, antibacterial, antihyperalgesic, antiviral, and antinociceptive activities. The biosynthetic pathway of flavonoid C-glucosides in plants has been elucidated. In the present study, a pathway was introduced to Escherichia coli to synthesize four flavonoid C-glucosides, namely, isovitexin, vitexin, kaempferol 6-C-glucoside, and kaempferol 8-C-glucoside. A five- or six-step metabolic pathway for synthesizing flavonoid aglycones from tyrosine was constructed and two regioselective flavonoid C-glycosyltransferases from Wasabia japonica (WjGT1) and Trollius chinensis (TcCGT) were used. Additionally, the best shikimate gene module construct was selected to maximize the titer of each C-glucoside flavonoid. Isovitexin (30.2 mg/L), vitexin (93.9 mg/L), kaempferol 6-C-glucoside (14.4 mg/L), and kaempferol 8-C-glucoside (38.6 mg/L) were synthesized using these approaches. The flavonoid C-glucosides synthesized in this study provide a basis for investigating and unraveling their novel biological properties.

Protection of Skin Fibroblasts from Infrared-A-Induced Photo-Damage Using Ginsenoside Rg3(S)-Incorporated Soybean Lecithin Liposomes.

Protection of skin cells from chronic infrared-A (IRA) irradiation is crucial for anti-photoaging of the skin. In this study, we investigated the protective activity of Rg3(S) and Rg3(S)-incorporated anionic soybean lecithin liposomes (Rg3/Lipo) with a size of approximately 150 nm against IRA-induced photodamage in human fibroblasts. The formulated Rg3/Lipo showed increased solubility in aqueous solution up to a concentration of 200 µg/ml, compared to free Rg3(S). In addition, Rg3/Lipo exhibited superior colloidal stability in aqueous solutions and biocompatibility for normal human dermal fibroblasts (NHDFs). After repeated IRA irradiation on NHDFs, elevated levels of cellular and mitochondrial reactive oxygen species (ROS) were greatly reduced by Rg3(S) and Rg3/Lipo. In addition, cells treated with Rg3/Lipo exhibited noticeably reduced apoptotic signals following IRA irradiation compared to untreated cells. Thus, considering aqueous solubility and cellular responses, Rg3/Lipo could serve as a promising infrared protector for healthy aging of skin cells.

Phenolic C-glycoside synthesis using microbial systems.

Plants produce different types of phenolic compounds. The majority of these compounds are glycosylated. Phenolic O-glycosides are also common. Recently, C-glycosylation of phenolic compounds has received attention because of the biological importance of phenolic C-glycosides. To date, three classes of C-glycosyltransferases (CGTs) have been characterized based on the type of sugar acceptor: flavonoid CGT, coumarin CGT, and xanthone CGT. Phylogenetic analysis of glycosyltransferases has revealed that CGTs form a distinct class that is clearly different from that of O-glycosyltransferases. The characterized CGTs have been introduced into microbial systems to synthesize phenolic C-glycosides. Here, we review recent progress in the development of CGTs and their application in the synthesis of phenolic C-glycosides using microbial systems.

Exosome-mediated delivery of transforming growth factor-ß receptor 1 kinase inhibitors and toll-like receptor 7/8 agonists for combination therapy of tumors.

In this study, combination therapy with the transforming growth factor-ß receptor I (TGFßRI) kinase inhibitor SD-208 and a toll-like receptor (TLR)-7/8 agonist resiquimod (R848) was examined along with serum-derived exosomes (EXOs) as versatile carriers. SD-208-encapsulated EXOs (SD-208/EXOs) and R848-encapsulated EXOs (R848/EXOs) were successfully prepared with a size of 87 ± 8 nm and 51 ± 4 nm, respectively, which were stable in aqueous solution at pH 7.4. SD-208/EXOs and R848/EXOs reduced the migration of cancer cells (B16F10 and PC-3) and triggered the release of proinflammatory cytokines from stimulated macrophages and dendritic cells, respectively. The fluorescent dye-labeled EXOs showed significantly improved penetration through the PC-3/fibroblast co-culture spheroids and enhanced accumulation in the B16F10 mouse tumor model compared with the free fluorescent dye. In addition, the combination therapy of R848/EXOs (R848 dose of 0.36 mg/kg) and SD-208/EXOs (SD-208 dose of 0.75 mg/kg) reduced tumor growth and improved survival rate at low doses in the B16F10 tumor xenograft model. Taken together, the combination therapy using the TGFßRI kinase inhibitor and TLR 7/8 agonist with EXOs may serve as a promising strategy to treat melanoma and prostate cancer. STATEMENT OF SIGNIFICANCE: Owing to the prevalence of several non-responding cancers that resist treatment, it is necessary to identify a novel combined treatment strategy with biomaterials to maximize therapeutic efficacy and minimize the undesirable side effects. In this study, we aimed to examine the use of the TGFßRI kinase inhibitor SD-208 and the TLR7/8 agonist resiquimod (R848) encapsulated within serum-derived EXOs for their synergistic antitumor effects. We first demonstrated that combined treatment with SD-208 and R848 can be a convincing strategy to circumvent tumor growth in vivo using serum-derived exosomes as promising carriers. Therefore, we believe this manuscript would be of great interest to the biomaterial communities especially who are studying immunotherapy.

Synthesis of chlorogenic acid and p-coumaroyl shikimate by expressing shikimate gene modules in Escherichia coli.

AIM: Chlorogenic acid and p-coumaroyl shikimate are hydroxycinnamic acid derivatives. These compounds are nutraceutical supplements due to their biological activities including prevention of cardiovascular disease and cancers. These two compounds were synthesized in Escherichia coli through two-culture system using two mutants, which are biochemically interdependent. The aim of this work was to improve the titres of their production in a single E. coli mutant in which all necessary genes were introduced. This was done by testing various shikimate gene combinations to determine the optimal gene combination for the synthesis of chlorogenic acid and p-coumaroyl shikimate. METHODS AND RESULTS: A series of gene modules harbouring shikimate pathway genes were constructs. Six gene module constructs for chlorogenic acid synthesis and eight constructs for p-coumaric acid synthesis were tested in order to find the best one. Chlorogenic acid synthesis showed highest with the gene module construct containing ydiB, aroB, aroGf , ppsA and tktA. Using the E. coli strain, 109.7 mg L-1 chlorogenic acid was synthesized. The best gene module construct for the p-coumaroyl shikimate synthesis contained aroD and aroGf . In addition, we used two E. coli deletion mutant strains (ΔaroK and ΔaroL) to increase the final titre. The E. coli ΔaroK mutant harbouring this gene module construct synthesized 713.4 mg L-1 of p-coumaroyl shikimate. CONCLUSION: The chlorogenic acid synthesis using the current system was approximately 35.4% higher of the titre than titres obtained with an alternative method that depends on co-cultivation of two mutants. At the same time, production of p-coumaroyl shikimate increased 5.8 times. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study's findings indicate that our selection of the shikimate gene module contributed to increases in the levels of the substrates and could be applied to synthesize other compounds whose synthesis requires intermediates of the shikimate pathway.

Role of ginseng in the neurovascular unit of neuroinflammatory diseases focused on the blood-brain barrier.

Ginseng has long been considered as an herbal medicine. Recent data suggest that ginseng has anti-inflammatory properties and can improve learning- and memory-related function in the central nervous system (CNS) following the development of CNS neuroinflammatory diseases such as Alzheimer's disease, cerebral ischemia, and other neurological disorders. In this review, we discuss the role of ginseng in the neurovascular unit, which is composed of endothelial cells surrounded by astrocytes, pericytes, microglia, neural stem cells, oligodendrocytes, and neurons, especially their blood-brain barrier maintenance, anti-inflammatory effects and regenerative functions. In addition, cell-cell communication enhanced by ginseng may be attributed to regeneration via induction of neurogenesis and angiogenesis in CNS diseases. Thus, ginseng may have therapeutic potential to exert cognitive improvement in neuroinflammatory diseases such as stroke, traumatic brain injury, multiple sclerosis, Parkinson's disease, and Alzheimer's disease.

Synthesis of 4-Hydroxybenzoic Acid Derivatives in Escherichia coli.

Hydroxybenzoic acids (HBAs) such as 4-hydroxybenzoic acid (4-HBA) and 3,4-dihydroxybenzoic acid (DHB; protocatechuic acid) and its ester with methanol (methylparaben [MP]) are known to have various functional biological properties, including antibacterial, anticancer, antidiabetic, antiaging, antiviral, and anti-inflammatory activities. Since these compounds are widely used in cosmetic, food, and pharmaceutical industries, the use of renewable feedstocks for the production of HBAs is an area of growing interest. In this study, we used Escherichia coli to synthesize these three hydroxybenzoic acid derivatives (4-HBA, DHB, and MP). We overexpressed ubiC in E. coli to synthesize 4-HBA from chorismate, a substrate that is produced by the shikimate pathway in E. coli. For the synthesis of DHB, an additional gene (pobA) was introduced, while hbad and EHT1 were co-expressed to synthesize MP. To supply more chorismate, we introduced the shikimate gene module construct and selected the best construct for increased yields. Using this approach, 723.5 mg/L 4-HBA, 942.0 mg/L DHB, and 347.7 mg/L MP were synthesized. Our study showed that the shikimate gene module constructs can be applicable to increase the yields of HBA derivatives in HBA-tolerant microorganisms.