Potential Application of Bacteriophages in Enrichment Culture for Improved Prenatal Streptococcus agalactiae Screening.

Vertical transmission of Streptococcus agalactiae can cause neonatal infections. A culture test in the late stage of pregnancy is used to screen for the presence of maternal S. agalactiae for intrapartum antibiotic prophylaxis. For the test, a vaginal⁻rectal sample is recommended to be enriched, followed by bacterial identification. In some cases, Enterococcus faecalis overgrows in the enrichment culture. Consequently, the identification test yields false-negative results. Bacteriophages (phages) can be used as antimicrobial materials. Here, we explored the feasibility of using phages to minimize false-negative results in an experimental setting. Phage mixture was prepared using three phages that specifically infect E. faecalis: phiEF24C, phiEF17H, and phiM1EF22. The mixture inhibited the growth of 86.7% (26/30) of vaginal E. faecalis strains. The simple coculture of E. faecalis and S. agalactiae was used as an experimental enrichment model. Phage mixture treatment led to suppression of E. faecalis growth and facilitation of S. agalactiae growth. In addition, testing several sets of S. agalactiae and E. faecalis strains, the treatment with phage mixture in the enrichment improved S. agalactiae detection on chromogenic agar. Our results suggest that the phage mixture can be usefully employed in the S. agalactiae culture test to increase test accuracy.

Treatment of oral leukoplakia with a low-dose of beta-carotene and vitamin C supplements: a randomized controlled trial.

Management of oral leukoplakia-a potentially malignant disorder-is currently not evidence-based. Of the few randomized trials that have been reported, most have negative data. Therefore, a multi-centre, randomized, double-blind controlled trial (RCT) was undertaken to evaluate the use of low-dose beta-carotene combined with vitamin C supplements for the treatment and to prevent malignant transformation of oral leukoplakia. 46 Japanese participants with oral leukoplakia were allocated randomly either to an experimental arm (10 mg day(-1) of beta-carotene and 500 mg day(-1) of vitamin C) or placebo arm (50 mg day(-1) of vitamin C). Current or ex-smokers within 3 months of cessation were excluded. The supplements were continued over a period of 1 year. The primary endpoint was clinical remission at 1-year and the likelihood of malignant transformation during a 5-year follow-up period as a secondary endpoint. The overall clinical response rate in the experimental arm was 17.4% (4/23) and 4.3% (1/23) in the placebo arm (p = 0.346). During the median 60-month follow-up period, two subjects in the experimental arm and three in the control arm developed oral cancer. Under the intention-to-treat principle, relative risk by supplementing with beta-carotene and vitamin C was 0.77 (95%CI: 0.28-1.89) (p = 0.580) by the Cox proportional hazards model. No unfavorable side-effects were noted. Beta-carotene (10 mg day(-1) ) and vitamin C were neither effective for clinical remission, nor for protection against the development of cancer. Data from this RCT does not support the hypothesis that chemoprevention with this treatment is effective for oral leukoplakia.

Procyanidin C1 from Cinnamomi Cortex inhibits TGF-ß-induced epithelial-to-mesenchymal transition in the A549 lung cancer cell line.

Cancer metastasis is one of the most critical events in cancer patients, and the median overall survival of stage IIIb or IV patients with metastatic lung cancer in the TNM classification is only 8 or 5 months, respectively. We previously demonstrated that Juzentaihoto, a Japanese traditional medicine, can inhibit cancer metastasis through the activation of macrophages and T cells in mouse cancer metastatic models; however, the mechanism(s) through which Juzentaihoto directly affects tumor cells during the metastasis process and which herbal components from Juzentaihoto inhibit the metastatic potential have not been elucidated. In this study, we focused on the epithelial-to-mesenchymal transition (EMT), which plays an important role in the formation of cancer metastasis. We newly determined that only the Cinnamomi Cortex (CC) extract, one of 10 herbal components of Juzentaihoto, inhibits TGF-ß-induced EMT. Moreover, the contents of catechin trimer in CC extracts were significantly correlated with the efficacy of inhibiting TGF-ß-induced EMT. Finally, the structure of the catechin trimer from CC extract was chemically identified as procyanidin C1 and the compound showed inhibitory activity against TGF-ß-induced EMT. This illustrates that procyanidin C1 is the main active compound in the CC extract responsible for EMT inhibition and that procyanidin C1 could be useful as a lead compound to develop inhibitors of cancer metastasis and other diseases related to EMT.

Mn tolerance in rice is mediated by MTP8.1, a member of the cation diffusion facilitator family.

Manganese (Mn) is an essential micronutrient for plants, but is toxic when present in excess. The rice plant (Oryza sativa L.) accumulates high concentrations of Mn in the aerial parts; however, the molecular basis for Mn tolerance is poorly understood. In the present study, genes encoding Mn tolerance were screened for by expressing cDNAs of genes from rice shoots in Saccharomyces cerevisiae. A gene encoding a cation diffusion facilitator (CDF) family member, OsMTP8.1, was isolated, and its expression was found to enhance Mn accumulation and tolerance in S. cerevisiae. In plants, OsMTP8.1 and its transcript were mainly detected in shoots. High or low supply of Mn moderately induced an increase or decrease in the accumulation of OsMTP8.1, respectively. OsMTP8.1 was detected in all cells of leaf blades through immunohistochemistry. OsMTP8.1 fused to green fluorescent protein was localized to the tonoplast. Disruption of OsMTP8.1 resulted in decreased chlorophyll levels, growth inhibition in the presence of high concentrations of Mn, and decreased accumulation of Mn in shoots and roots. However, there was no difference in the accumulation of other metals, including Zn, Cu, Fe, Mg, Ca, and K. These results suggest that OsMTP8.1 is an Mn-specific transporter that sequesters Mn into vacuoles in rice and is required for Mn tolerance in shoots.

Improved adsorption of an Enterococcus faecalis bacteriophage ΦEF24C with a spontaneous point mutation.

Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component.

A phagocytotic inducer from herbal constituent, pentagalloylglucose enhances lipoplex-mediated gene transfection in dendritic cells.

Antigen-presenting cells are key vehicles for delivering antigens in tumor immunotherapy, and the most potent of them are dendritic cells (DCs). Recent studies have demonstrated the usefulness of DCs genetically modified by lipofection in tumor immune therapy, although sufficient gene transduction into DCs is quite difficult. Here, we show that Paeoniae radix, herbal medicine, and the constituent, 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG), have an attractive function to enhance phagocytosis in murine dendritic cell lines, DC2.4 cells. In particular, PGG in combination with lipofectin (LPF) enhanced phagocytic activity. Furthermore, PGG enhanced lipofection efficacy in DC2.4 cells, but not in colorectal carcinoma cell lines, Colon26. In other words, PGG synergistically enhanced the effect of lipofectin-dependent phagocytosis on phagocytic cells. Hence, according to our data, PGG could be an effective aid in lipofection using dendritic cells. Furthermore, these findings provide an expectation that constituents from herbal plant enhance lipofection efficacy.

Characterization of a NifS-like chloroplast protein from Arabidopsis. Implications for its role in sulfur and selenium metabolism.

NifS-like proteins catalyze the formation of elemental sulfur (S) and alanine from cysteine (Cys) or of elemental selenium (Se) and alanine from seleno-Cys. Cys desulfurase activity is required to produce the S of iron (Fe)-S clusters, whereas seleno-Cys lyase activity is needed for the incorporation of Se in selenoproteins. In plants, the chloroplast is the location of (seleno) Cys formation and a location of Fe-S cluster formation. The goal of these studies was to identify and characterize chloroplast NifS-like proteins. Using seleno-Cys as a substrate, it was found that 25% to 30% of the NifS activity in green tissue in Arabidopsis is present in chloroplasts. A cDNA encoding a putative chloroplast NifS-like protein, AtCpNifS, was cloned, and its chloroplast localization was confirmed using immunoblot analysis and in vitro import. AtCpNIFS is expressed in all major tissue types. The protein was expressed in Escherichia coli and purified. The enzyme contains a pyridoxal 5' phosphate cofactor and is a dimer. It is a type II NifS-like protein, more similar to bacterial seleno-Cys lyases than to Cys desulfurases. The enzyme is active on both seleno-Cys and Cys but has a much higher activity toward the Se substrate. The possible role of AtCpNifS in plastidic Fe-S cluster formation or in Se metabolism is discussed.