Green Tea-Derived Catechins Suppress the Acid Productions of Streptococcus mutans and Enhance the Efficiency of Fluoride.

Green tea-derived catechins, which can be divided into galloylated (epicatechin gallate: ECG, epigallocatechin gallate: EGCG) and non-galloylated (catechin: C, epicatechin: EC, epigallocatechin: EGC) catechins, are considered to be the main contributors to the caries control potential of green tea. In this study, we intended to compare the antimicrobial effects of these representative green tea-derived catechins and their combined effects with fluoride on the acid production and aggregation of Streptococcus mutans. The effects of different catechins on the growth, aggregation and acid production of S. mutans, and the combined effect of catechins and potassium fluoride (2 mm at pH 7.0, 0.3 mm at pH 5.5) on S. mutans acid production were measured by anaerobic culture, turbidity changes due to aggregation, and pH-stat methods. Molecular docking simulations were also performed to investigate the interactions between catechins and membrane-embedded enzyme II complex (EIIC), a component of the phosphoenolpyruvate-dependent phosphotransferase system (sugar uptake-related enzyme). ECG or EGCG at 1 mg/mL significantly inhibited the growth of S. mutans, induced bacterial aggregation, and decreased glucose-induced acid production (p < 0.05). All catechins were able to bind to EIIC in silico, in the following order of affinity: EGCG, ECG, EGC, EC, and C. Furthermore, they enhanced the inhibitory effects of fluoride at pH 5.5 and significantly inhibited S. mutans acid production by 47.5-86.6% (p < 0.05). These results suggest that both galloylated and non-galloylated catechins exhibit antimicrobial activity, although the former type demonstrates stronger activity, and that the caries control effects of green tea may be due to the combined effects of multiple components, such as catechins and fluoride. The detailed mechanisms underlying these phenomena and the in vivo effect need to be explored further.

Development and evaluation of a chewing gum containing antimicrobial peptide GH12 for caries prevention.

The purpose of this study was to develop a chewing gum containing a novel antimicrobial peptide GH12 and evaluate its biocompatibility, antimicrobial activity, and caries-preventive effects in vivo and in vitro. GH12 chewing gum was developed using a conventional method and its extracts were prepared in artificial saliva. GH12 concentration in the extracts was determined by high-performance liquid chromatography; extracts were used for growth curve assay, time-kill assay, crystal violet staining assay, scanning electron microscopy, and Cell Counting Kit-8 assay. A rat caries model was established, and molars were treated topically with extracts for 5 weeks. Weight gain monitoring, hematoxylin-eosin staining, micro-computed tomography, and Keyes scoring were conducted. Significant inhibition of Streptococcus mutans growth and biofilm formation was observed. Extracts displayed low cytotoxicity against human gingival epithelial cells. No significant differences in weight gain or signs of harm to the mucosal tissues in any of the rats were observed. Keyes scores of caries lesions in the GH12 chewing gum group were lower than those of the negative control group. It was concluded that GH12 chewing gum showed good biocompatibility, antimicrobial activity, and caries-preventive effects, exhibiting great potential to prevent dental caries as an adjuvant to regular oral hygiene.

Green Tea-Derived Epigallocatechin Gallate Inhibits Acid Production and Promotes the Aggregation of Streptococcus mutans and Non-Mutans Streptococci.

It has been suggested that green tea-derived epigallocatechin gallate (EGCG), which has antimicrobial properties, might help prevent dental caries. However, the detailed properties of EGCG remain unclear. In this study, the antimicrobial properties of EGCG were evaluated by examining its bactericidal activity, its inhibitory effects against bacterial growth, acid production, acidic end-product formation, and sugar uptake (phosphoenolpyruvate-dependent phosphotransferase system, PEP-PTS activity), and its effects on bacterial aggregation, using monocultured planktonic cells of Streptococcus mutans and non-mutans streptococci. Coincubating S. mutans with EGCG (1 mg/mL) for 4 h had no bactericidal effects, while it decreased the growth and acid production of S. mutans by inhibiting the activity of the PEP-PTS. EGCG (2 mg/mL) caused rapid bacterial cell aggregation and had reduced the optical density of S. mutans cell suspension by 86.7% at pH 7.0 and 90.7% at pH 5.5 after 2 h. EGCG also reduced the acid production of non-mutans streptococci, including S. sanguinis, S. gordonii, and S. salivarius, and promoted the aggregation of these non-mutans streptococci. Furthermore, these antimicrobial effects of short-term EGCG treatment persisted in the presence of saliva. These results suggest that EGCG might have short-term antibacterial effects on caries-associated streptococci in the oral cavity.

New approach to evaluating the effects of a drug on protein complexes with quantitative proteomics, using the SILAC method and bioinformatic approach.

Protein-protein interactions (PPIs) lead the formation of protein complexes that perform biochemical reactions that maintain the living state of the living cell. Although therapeutic drugs should influence the formation of protein complexes in addition to PPI network, the methodology analyzing such influences remain to be developed. Here, we demonstrate that a new approach combining HPLC (high performance liquid chromatography) for separating protein complexes, and the SILAC (stable isotope labeling using amino acids in cell culture) method for relative protein quantification, enable us to identify the protein complexes influenced by a drug. We applied this approach to the analysis of thalidomide action on HepG2 cells, assessed the identified proteins by clustering data analyses, and assigned 135 novel protein complexes affected by the drug. We propose that this approach is applicable to elucidating the mechanisms of actions of other therapeutic drugs on the PPI network, and the formation of protein complexes.

Post-Translational Regulation of the Dicing Activities of Arabidopsis DICER-LIKE 3 and 4 by Inorganic Phosphate and the Redox State.

In Arabidopsis thaliana, small interfering RNAs (siRNAs) generated by two Dicer isoforms, DCL3 and DCL4, function in distinct epigenetic processes, i.e. RNA-directed DNA methylation and post-transcriptional gene silencing, respectively. Plants often respond to their environment by producing a distinct set of small RNAs; however, the mechanism for controlling the production of different siRNAs from the same dsRNA substrate remains unclear. We established a simple biochemical method to visualize the dsRNA-cleaving activities of DCL3 and DCL4 in cell-free extracts prepared from Arabidopsis seedlings. Here, we demonstrate that different nutrient statuses of a host plant affect the post-translational regulation of the dicing activity of DCL3 and DCL4. Phosphate deficiency inhibited DCL3, and the activity of DCL3 was directly activated by inorganic phosphate. Sulfur deficiency inhibited DCL4 but not DCL3, and the activity of DCL4 was recovered by supplementation of the cell-free extracts with reductants containing a thiol group. Immunopurified DCL4 was activated by recombinant Arabidopsis thioredoxin-h1 with dithiothreitol. Therefore, DCL4 is subject to redox regulation. These results demonstrate that post-translational regulation of DCL activities fine-tunes the balance between branches of the gene silencing pathway according to the growth environment.

Formation of bioactive N-doped TiO2 on Ti with visible light-induced antibacterial activity using NaOH, hot water, and subsequent ammonia atmospheric heat treatment.

Titanium (Ti) treated with NaOH and hot water, and heated in an ammmonia (NH3) gas atmosphere for 1 or 3h exhibited in vitro apatite formation within 7days when soaked in simulated body fluid (SBF). Moreover, the treated Ti decomposed methylene blue and showed excellent bactericidal activity against Escherichia coli under visible light irradiation. The surface treatment resulted in the formation of a fine network of N-doped anatase-type titania (TiO2-xNx) on the Ti surface, which was responsible for both the apatite formation in SBF and the visible light-induced antibacterial activity. These preliminary results highlight the efficacy of our simple method for producing novel bioactive Ti with visible light-induced antibacterial activity, which could be applied to orthopaedic and dental implants without the risk of infection.

Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.

A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.