Customized media based on miniaturized screening improve growth rate and cell yield of methane-oxidizing bacteria of the genus Methylomonas

The growth of twelve methanotrophic strains within the genus Methylomonas, including the type strains of Methylomonas methanica and Methylomonas koyamae, was evaluated with 40 different variations of standard diluted nitrate mineral salts medium in 96-well microtiter plates. Unique profiles of growth preference were observed for each strain, showing a strong strain dependency for optimal growth conditions, especially with regards to the preferred concentration and nature of the nitrogen source. Based on the miniaturized screening results, a customized medium was designed for each strain, allowing the improvement of the growth of several strains in a batch setup, either by a reduction of the lag phase or by faster biomass accumulation. As such, the maintenance of fastidious strains could be facilitated while the growth of fast-growing Methylomonas strains could be further improved. Methylomonas sp. R-45378 displayed a 50 % increase in cell dry weight when grown in its customized medium and showed the lowest observed nitrogen and oxygen requirement of all tested strains. We demonstrate that the presented miniaturized approach for medium optimization is a simple tool allowing the quick generation of strain-specific growth preference data that can be applied downstream of an isolation campaign. This approach can also be applied as a first step in the search for strains with biotechnological potential, to facilitate cultivation of fastidious strains or to steer future isolation campaigns.     

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.     

The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae.

We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair.     

Isotopically sensitive branching in the formation of cyclic monoterpenes: proof that (-)-alpha-pinene and (-)-beta-pinene are synthesized by the same monoterpene cyclase via deprotonation of a common intermediate

To determine whether the bicyclic monoterpene olefins (-)-alpha-pinene and (-)-beta-pinene arise biosynthetically from the same monoterpene cyclase by alternate deprotonations of a common carbocationic intermediate, the product distributions arising from the acyclic precursor [10-2H3,1-3H]geranyl pyrophosphate were compared with those resulting from incubation of [1-3H]geranyl pyrophosphate with (-)-pinene cyclase from Salvia officinalis. Alteration in proportions of the olefinic products generated by the partially purified pinene cyclase resulted from the suppression of the formation of (-)-beta-pinene (C10 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of (-)-alpha-pinene (C4 deprotonation). (-)-Pinene cyclase as well as (+)-pinene cyclase also exhibited a decrease in the proportion of the acyclic olefin myrcene generated from the deuteriated substrate, accompanied by a corresponding increase in the commitment to cyclized products. The observation of isotopically sensitive branching, in conjunction with quantitation of the magnitude of the secondary deuterium isotope effect on the overall rate of product formation by the (+)- and (-)-pinene cyclases as well as two other monoterpene cyclases from the same tissue, supports the biosynthetic origin of (-)-alpha-pinene and (-)-beta-pinene by alternative deprotonations of a common enzymatic intermediate. A biogenetic scheme consistent with these results is presented, and alternate proposals for the origin of the pinenes are addressed.     

Impact of vaginal microbiome communities on HIV antiretroviral-based pre-exposure prophylaxis (PrEP) drug metabolism

Despite the efficacy of antiretroviral-based pre-exposure prophylactics (PrEP) in men who have sex with men, studies in women have produced widely varying outcomes. Recent evidence demonstrates that vaginal microbial communities are associated with increased HIV acquisition risk and may impact PrEP efficacy. Here, we investigate the mechanisms underlying how vaginal bacteria alter PrEP drug levels and impact HIV infection rates ex vivo. Using cervicovaginal lavages (CVLs) from women with or without bacterial vaginosis (BV), we identified microbial metabolism of PrEP drugs in BV samples through LC-MS/MS analysis of soluble drug levels and metabolite formation in dual T-cell cultures. CVL samples were assessed for microbiome analysis using sequencing of bacterial 16S rRNA genes. We also observed non-Lactobacillus bacteria that are associated with BV may potentially impact PrEP efficacy through increased HIV infection rates in co-cultures containing Lactobacillus or BV bacteria, PrEP drugs, CEM-GFP cells, and HIV-1_LAI virus. Finally, we used these data to develop a novel predictive mathematical simulation modeling system to predict these drug interactions for future trials. These studies demonstrate how dysbiotic vaginal microbiota may impact PrEP drugs and provides evidence linking vaginal bacteria to PrEP efficacy in women.     

Simultaneous Near-Infrared Radiant Heating and UV Radiation for Inactivating Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Powdered Red Pepper (Capsicum annuum L.)

This study was conducted to investigate the efficacy of the simultaneous application of near-infrared (NIR) heating and UV irradiation for reducing populations of food-borne pathogens, including Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 in red pepper powder and to clarify the mechanisms of the lethal effect of the NIR-UV combined treatment. Also, the effect of the combination treatment on quality was determined by measuring changes in color and pungency constituents. Simultaneous NIR-UV combined treatment for 5 min achieved 3.34- and 2.78-log CFU reductions in S. Typhimurium and E. coli O157:H7, respectively, which involved 1.86- and 1.31-log CFU reductions, respectively, which were attributed to the synergistic effect. Through qualitative and quantitative analyses, damage to the cell envelope was identified as the main factor contributing to the synergistic lethal effect of NIR-UV combined treatment. Color values and capsaicin and dihydrocapsaicin content of NIR-UV simultaneously treated red pepper powder were not significantly (P > 0.05) different from those of untreated samples. These results suggest that simultaneous application of NIR and UV treatment can be effectively used to control food-borne pathogens in powdered red pepper without affecting quality.     

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.