New insights into metabolomics profile generation in fermented tea: the relevance of bacteria and metabolites in Fuzhuan brick tea.

BACKGROUND: The contribution of bacteria to fermented tea is not clear and the associated research is relatively limited. To reveal the role of microorganisms in fermented tea processing, the microbial community and metabolites of Fuzhuan brick tea (FBT), a Chinese traditional fermented tea, were revealed via high-throughput sequencing and liquid chromatography-mass spectrometry (LC-MS). RESULTS: In FBT, bacterial communities had a higher abundance and diversity, Lactococcus and Bacillus were the main bacteria, and Eurotium was the predominant fungus. The predictive metabolic function indicated the pathways of cellular growth, environmental information, genetics and material metabolism of bacterial communities were abundant, whereas the fungal community predictive metabolic function was almost saprotroph. Using LC-MS, 1143 and 536 metabolites were defined in positive and negative ion mode, respectively. There were essential correlations between bacterial populations and metabolites, such that Bacillus was correlated significantly with 44 metabolites (P < 0.05) and Enterococcus was significantly associated with 15 metabolites (P < 0.05). Some of the main active components were significantly correlated with the bacteria, such as Enterococcus, Lactococcus and Carnobacterium. CONCLUSION: Not only Eurotium, but also the bacteria were involved in the changes of metabolomics profile in fermented FBT. The present study assists in providing new insights into metabolomics profile generation in fermented tea. The present research lays a foundation for controlling the FBT fermentation by artificial inoculation to improve quality. © 2021 Society of Chemical Industry.

MrgX2 is a promiscuous receptor for basic peptides causing mast cell pseudo-allergic and anaphylactoid reactions.

Activation of MrgX2, an orphan G protein-coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine-like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by Mrgprb2. MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug-like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug-like molecules activated MrgX2 (pEC50 ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl-Arg9-amide) and TAT (49-60), a fragment of HIV-1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo-allergic reactions known as red man syndrome, is an agonist of MrgX2.

A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic ß-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.

Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

Sickle cell anemia (SCA) is a genetic disorder of the ß-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.

Intermittent treatment with parathyroid hormone (PTH) as well as a non-peptide small molecule agonist of the PTH1 receptor inhibits adipocyte differentiation in human bone marrow stromal cells.

Whereas continuous PTH infusion increases bone resorption and bone loss, intermittent PTH treatment stimulates bone formation, in part, via reactivation of quiescent bone surfaces and reducing osteoblast apoptosis. We investigated the possibility that intermittent and continuous PTH treatment also differentially regulates osteogenic and adipocytic lineage commitment of bone marrow stromal progenitor/mesenchymal stem cells (MSC). The MSC were cultured under mildly adipogenic conditions in medium supplemented with dexamethasone, insulin, isobutyl-methylxanthine and troglitazone (DIIT), and treated with 50 nM human PTH(1-34) for either 1 h/day or continuously (PTH replenished every 48 h). After 6 days, cells treated with PTH for 1 h/day retained their normal fibroblastic appearance whereas those treated continuously adopted a polygonal, irregular morphology. After 12-18 days numerous lipid vacuole and oil red O-positive adipocytes had developed in cultures treated with DIIT alone, or with DIIT and continuous PTH. In contrast, adipocyte number was reduced and alkaline phosphatase staining increased in the cultures treated with DIIT and 1 h/day PTH, indicating suppression of adipogenesis and possible promotion of early osteoblastic differentiation. Furthermore, intermittent but not continuous PTH treatment suppressed markers of differentiated adipocytes such as mRNA expression of lipoprotein lipase and PPARgamma as well as glycerol 3-phosphate dehydrogenase activity. All of these effects of intermittent PTH were also produced by a 1 h/day treatment with AH3960 (30 microM), a small molecule, non-peptide agonist of the PTH1 receptor. AH3960, like PTH, activates both the cAMP and calcium signaling pathways. Treatment with the adenylyl cyclase activator forskolin for 1 h/day, mimicked the anti-adipogenic effect of intermittent PTH, whereas pretreatment with the protein kinase-A inhibitor H89 prior to intermittent PTH resulted in almost complete conversion to adipocytes. In contrast, the MAP kinase inhibitor PD 98059 failed to prevent the anti-adipocytic effect of intermittent PTH, suggesting that the inhibitory effect of PTH on adipocyte differentiation is predominantly cAMP-dependent. These results demonstrate a differential effect of PTH1 receptor agonists on the adipocytic commitment and differentiation of adult human bone marrow mesenchymal stem cells. This response may represent an additional mechanism that contributes to the overall bone anabolic action of intermittent PTH.