TRMT10A dysfunction perturbs codon translation of initiator methionine and glutamine and impairs brain functions in mice.

In higher eukaryotes, tRNA methyltransferase 10A (TRMT10A) is responsible for N1-methylguanosine modification at position nine of various cytoplasmic tRNAs. Pathogenic mutations in TRMT10A cause intellectual disability, microcephaly, diabetes, and short stature in humans, and generate cytotoxic tRNA fragments in cultured cells; however, it is not clear how TRMT10A supports codon translation or brain functions. Here, we generated Trmt10a null mice and showed that tRNAGln(CUG) and initiator methionine tRNA levels were universally decreased in various tissues; the same was true in a human cell line lacking TRMT10A. Ribosome profiling of mouse brain revealed that dysfunction of TRMT10A causes ribosome slowdown at the Gln(CAG) codon and increases translation of Atf4 due to higher frequency of leaky scanning of its upstream open reading frames. Broadly speaking, translation of a subset of mRNAs, especially those for neuronal structures, is perturbed in the mutant brain. Despite not showing discernable defects in the pancreas, liver, or kidney, Trmt10a null mice showed lower body weight and smaller hippocampal postsynaptic densities, which is associated with defective synaptic plasticity and memory. Taken together, our study provides mechanistic insight into the roles of TRMT10A in the brain, and exemplifies the importance of universal tRNA modification during translation of specific codons.

Candida dubliniensis in Japanese Oral Microbiota: A Cross-Sectional Study of Six Geographic Regions in Japan.

INTRODUCTION: Candida dubliniensis was reclassified from the C. albicans genotype D, and reports show its frequent detection in HIV-positive individuals and easy acquisition of antifungal drug resistance. However, the oral carriage rate in healthy people and contribution to candidiasis in Japan is unclear. METHODS: We conducted a cross-sectional survey of the C. dubliniensis carriage rate, performed genotyping and tested antifungal drug susceptibility and protease productivity. Specimens from 2432 Japanese subjects in six regions (1902 healthy individuals, 423 with candidiasis individuals, 107 HIV-positive individuals) were cultured using CHROMagarTMCandida, and the species was confirmed via 25S rDNA amplification and ITS sequences analyzed for genotyping. RESULTS: The C. dubliniensis carriage rate in healthy Japanese was low in the central mainland (0-15%) but high in the most northerly and southerly areas (30-40%). The distribution of these frequencies did not differ depending on age or disease (HIV-infection, candidiasis). Genotype I, previously identified in other countries, was most frequent in Japan, but novel genotypes were also observed. Six antifungal drugs showed higher susceptibility against C. albicans, but protease productivity was low. CONCLUSIONS: Oral C. dubliniensis has low pathogenicity with distribution properties attributed to geography and not dependent on age or disease status.

Association Between Dental Caries Prevalence and Stress Levels in Japanese Children.

Introduction Early life stress (ELS) caused by abuse and bullying has increased dramatically, however, effective means for accurate detection have not been found. Some decades ago, an association between stress and dental caries was suggested. However, even now, stress is not recognized widely as a potential risk factor for caries. Therefore, the purpose of this study was to verify the possible effects of stress by comparing them to the effects of saliva factors and the microorganisms that pose a general caries risk. Methods We conducted cross-sectional observation research on 30 children with mental problems, diagnosed as 'stressed,' and 30 age-matched unstressed children in the same elementary school. An oral examination (dental caries diagnosis) and an oral environment survey (saliva test) were carried out in 2007. Further, the concentration and activity of salivary stress proteins were measured. All variables were statistically analyzed using the Mann-Whitney U test, correlation, and multivariate analysis. Results The dental caries experience ratio was significantly higher in the stress group, and only the concentration of CgA, a salivary stress protein, showed a significant difference. Unexpectedly, we did not detect any differences in the rates and counts of cariogenic bacteria or salivary buffering activity. Binomial logistic regression analysis only showed significance in the presence or absence of ELS. Conclusion Stress factors may have a stronger influence on caries development in the stress group than in the general caries risk. Therefore, long-term stress, causing changes in the children's bodies, might hint at important factors leading to the development of dental caries.

Proteome analysis of high affinity mouse saliva proteins to hydroxyapatite.

Caries sensitivity varies between the two strains of inbred mice, BALB/cA has high sensitivity and C3H/HeN has low sensitivity. One potential reason seems to be a difference in pellicle-forming saliva protein composition. Here, we performed a proteomic analysis in order to identify differences of hydroxyapatite (HAP) adsorbed saliva proteins between these two mouse strains. HAP column chromatography revealed twice the quantity of high-affinity saliva proteins in C3H/HeN compared to BALB/cA. One- and two-dimensional electrophoresis showed 2 bands/spots with deviating migration. They were identified as murine carbonic anhydrase VI (CAVI) by peptide mass fingerprinting and confirmed with western blotting using a specific polyclonal antibody. Total RNA from the salivary glands of both mouse strains, PCR amplification of cDNA with a CAVI specific primer, and sequence analysis revealed one different base in codon 96, resulting in one different amino acid. Glyco-chains of CAVI deviate in one N-glycan, confirmed by mass analysis. CAVI activity was estimated from distinct circular dichroism spectra of the molecules and found higher in C3H/HeN mice. In summary, the CAVI composition of BALB/cA and C3H/HeN differs in one amino acid and a glyco-chain modification. Further, saliva from caries resistant C3H/HeN mice displayed higher CAVI activity and also overall hydroxyapatite adsorption, suggesting a relationship with caries susceptibility.

Limosilactobacillus (Lactobacillus) fermentum ALAL020, a Probiotic Candidate Bacterium, Produces a Cyclic Dipeptide That Suppresses the Periodontal Pathogens Porphyromonas gingivalis and Prevotella intermedia.

Periodontal disease develops as a result of oral microbiota in dysbiosis, followed by the growth of periodontal pathogens such as Porphyromonas gingivalis and Prevotella intermedia. In case of acute symptoms, antibacterial agents and disinfectants are administered, however the appearance of drug-resistant bacteria and allergies cause problems. In recent years, studies on the effects of probiotics have been conducted as an alternative therapy for periodontitis. However, the basic mechanism of the inhibitory effect of probiotic bacteria on periodontal disease has not been clearly elucidated. To clarify the antibacterial mechanism of probiotics against periodontal pathogens, we used Limosilactobacillus (Lactobacillus) fermentum ALAL020, which showed the strongest antibacterial activity against P. gingivalis and P. intermedia among 50 screened lactic acid bacteria strains. The antibacterial substances produced were identified and structurally analyzed. After neutralizing the MRS liquid culture supernatant of ALAL020 strain, the molecular weight (m/z) of the main antibacterial substance separated by gel filtration column chromatography and reverse phase HPLC was 226.131. This low molecular weight compound was analyzed by LC-MS and disclosed the composition formula C11H18O3N2, however the molecular structure remained unknown. Then, structural analysis by NMR revealed C11H18O3N2 as the cyclic dipeptide, "hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dion cyclo (Hyp-Leu) ". Based on the results of this analysis, cyclo (Hyp-Leu) was chemically synthesized and the antibacterial activity against P. gingivalis and P. intermedia was measured. The minimum inhibitory concentration (MIC) was 2.5 g/L and the minimum bactericidal concentration (MBC) was shown to be less than 5 g/L. In addition, an in vitro epithelial tissue irritation test at 10 g/L showed no tissue toxicity. So far there are no reports of this peptide being produced by probiotic bacteria. Furthermore, antibacterial activity of this cyclic dipeptide against periodontal disease bacteria has not been confirmed. The results of this study might lead to a comprehensive understanding of the antibacterial mechanism against periodontal disease bacteria in future, and are considered applicable for the prevention of periodontal disease.

A field survey on elution of lead and nickel from taps used in homes and analysis of product test results.

The elution of lead, and nickel from water supply devices into water is a potential health concern. This study was performed to examine the actual concentrations of nickel and lead in the water from taps in homes and offices, focusing on the differences between first flush and fully flushed water. The water quality management target value and water quality standard in Japan specify nickel and lead concentrations in drinking water <20 and <10 µg/L, respectively. Nickel concentration in the first flush water (100 mL) from 110 household taps revealed 22 cases (20%) > 20 µg/L, while the fully flushed water satisfied the standard after running 5000 mL of water. The nickel concentration decreased gradually in sequential sampling of each 100 mL from the taps. Lead concentration in the first flush water exceeded the standard in 32 cases (29%), while the fully flushed water was below the target value. The concentration in the first flush water tended to decrease with time since the tap installation, and this was significant after 10 years for nickel but not significant for lead. It is important to flush retained water out of the tap after several hours without use. No significant correlation was found with the volume of the test faucet in the market, but bronze-based products showed higher nickel concentrations than brass and plastic products.

Plaque-removal effect of ultrafine bubble water: Oral application in patients undergoing orthodontic treatment.

During orthodontic treatment, plaque tends to form around fixed orthodontic appliances, which increases the risk of dental caries. It has been reported that ultarafine bubble with a diameter <1 µm water (UFBW) effectively removes organic matter. In addition, UFBW is harmless and stable for at least one month with refrigeration. The aim of this study was to examine the plaque-removal effect of ultrafine bubble water (UFBW) to establish a new method to prevent dental caries in patients during orthodontic treatment procedures. The in vitro study examined different concentrations of UFBW and compared the cleaning effect to that of existing mouthwashes. High-concentration UFBW (HUFBW) was most effective in cleaning. In the subsequent clinical study, HUFBW showed a significantly higher plaque-removal effect compared to distilled water (p<0.01). Thus, supplementary use of HUFBW could decrease the incidence of dental caries during orthodontic treatment.

A photometric pH assay for microplate bacterial cultures.

A photometric pH assay for sugar-fermenting bacterial culture on a 96-well plate was developed. This assay can save time and effort in repeat handlings. Its use could decrease the risk of bacterial contamination in measurement devices and leakage into the environment. The assay's pH estimation range was pH 4.2-7.6.

[Efficacy of Slightly Acidic Electrolyzed Water against Contamination of Water Line of Dental Units].

OBJECTIVES: The purpose of this study was to evaluate the efficacy of slightly acidic electrolyzed water (SAEW) against the contamination of the water line of dental units and the effects of SAEW on the water line. MATERIALS AND METHODS: The experimental material was a prototype dental unit equipped with a SAEW generator. SAEW is directly supplied to each device or part of this unit system. Experimental SAEW samples were collected from a high-speed handpiece (HS-1), an ultrasonic scaler, and a cup filler of the prototype dental unit. Control samples were taken before and after the prescribed flushing from another high-speed handpiece (HS-2) that is directly supplied with tap water in the same dental unit. The samples were analyzed for free chlorine and heterotrophic bacteria for 7 years to assess the efficacy and effects of SAEW. The substances eluted in SAEW were examined to investigate the effect of SAEW on the water line. A questionnaire survey was conducted on patients on whom dental uints supplied with SAEW were used. RESULTS: SAEW always showed a higher free chlorine concentration than tap water during the observation period of 7 years. In HS-2 supplied with tap water, the free chlorine concentration increased significantly owing to the prescribed flushing. SAEW always showed a significantly smaller number of heterotrophic bacteria than tap water. No abnormal levels values of water line components eluted into SAEW were observed. There were few negative comments from patients on whom dental units supplied with SAEW were used. CONCLUSIONS: SAEW continuously used for 7 years was effective for contamination control in the water line of dental units.

Analysis of plaque microbiota and salivary proteins adhering to dental materials.

OBJECTIVES: Plaque causes oral diseases and aspiration-pneumonia in the elderly. It is not known whether pellicle-like attached salivary proteins and microbiota on dental materials are identical to those on teeth. The purpose of this study was to determine the properties of salivary proteins and microbiota that attach to dental materials. METHODS: Eight subjects wore removable oral splints with pieces of pure-titanium, cobalt-chromium alloy, silver-palladium-copper-gold-alloy, denture-base-resin, and hydroxyapatite for 24 h. The bacteria that adhered to each material were analyzed using 16S rRNA sequencing simultaneously. Each material sample was then immersed in pooled saliva, and the attached proteins were collected. Salivary proteins were analyzed using MALDI-TOF/MS, and high molecular weight proteins were identified using peptide mass fingerprinting. RESULTS: Among the dental materials, the α- and ß-diversity of adherent flora were similar. The bacterial species that adhered easily to materials were Streptococcus sp. oral taxon 058, Neisseria mucosa, Gemella haemolysans, and Rothia dentocariosa. Regardless of material, the peaks or spots of attached salivary proteins had similar patterns, containing functioning proteins such as anchoring receptors for early colonizers. CONCLUSIONS: There were no significant differences in microbiota and protein adherence in hydroxyapatite compared to the dental materials. Therefore, similar microbiota was determined to have formed on the similar pellicle-like proteins. In our study, the characteristics of plaque adhesion on both hydroxyapatite and dental materials were clarified. Based on this study, the creation of new methods of inhibiting plaque adhesion to prevent aspiration-pneumonia and oral infections can be undertaken.

Analysis of oral microbiota in Japanese oral cancer patients using 16S rRNA sequencing.

OBJECTIVES: It is important to determine the cause of increasing oral cancer occurrence and mortality rates in Japan, because the mortality rate has recently decreased in other developed countries. The impact of microbiota in carcinogenesis, especially in the digestive tract has been reported. This study aimed to clarify the relationship between oral cancer and oral microbiota in Japanese patients. METHODS: DNA was extracted from salivary samples of 60 oral cancer patients and 80 non-cancer individuals as controls. We performed metagenomic analysis using 16S rRNA amplicon sequencing. Statistical analysis in this study was performed using R (version 3.5.0). RESULTS: Oral cancer patients showed higher α-diversity compared to the control group, and the ß-diversity between the two groups differed significantly. Further, there was a significant difference in the abundance ratio of bacterial genera between the two groups. Peptostreptococcus, Fusobacterium, Alloprevotella, and Capnocytophaga were more abundant in the cancer group compared to the control, whereas Rothia and Haemophilus were less abundant (p < 0.01). A negative correlation in the microbiota composition was confirmed between the operational taxonomic units (OTU) of genus Rothia and T-stage progression using the TNM classification method. We performed logistic regression analysis to investigate the impact factor for the oral cancer group, and the result showed that Chao 1 index and sex are statistically significant variables. CONCLUSIONS: In this study, we observed an increased bacterial diversity in oral cancer patients and found distribution changes for some bacteria.

A Proposal of Remedies for Oral Diseases Caused by Candida: A Mini Review.

An opportunistic pathogen, Candida is not only related to oral problems such as oral candidiasis and denture stomatitis, but also to systemic diseases such as aspiration pneumonia and fungemia. The carriage rate of Candida species in the oral cavity of individuals wearing dentures and with removable orthodontic appliances, has increased. Moreover, it is one of the causal pathogens in refractory infected root canals because of its resistance to antifungal drugs in root canal therapy and poses a great challenge during the treatment of patients. This problem has led to the search for alternative strategies for the treatment and management of C. albicans infections. In this mini review, recent preventive strategies against Candida infection in the oral mucosa with natural product-derived antifungal molecules were discussed. Inhibitory strategies by introducing competitive naturally-derived antifungal peptide molecules with Candida adhesion molecules were specifically introduced. In addition, novel sterilization methods for Candida-infected root canals and tooth structures in the oral cavity were considered, with focused attention on the activities of reactive oxygen species. The possibility of application of these novel strategies in clinical treatments and daily life was also proposed.

Rapid screening method for detecting highly pathogenic Streptococcus intermedius strains carrying a mutation in the lacR gene.

Streptococcus intermedius is a member of the normal human commensal flora and secretes a human-specific cytolysin intermedilysin (ILY) as a major virulence factor. Expression of ily is repressed by LacR and loss-of-function mutations of LacR are observed in many ILY high-producing strains isolated from deep-seated abscesses, suggesting that high ILY production is necessary for increased virulence. However, because ILY exhibits no ß-hemolysis on animal blood agar plates, differentiating ILY high- and low-producing strains using conventional laboratory methods is not possible. Interestingly, S. intermedius also produces glycosidases, including MsgA and NanA, which exhibit N-acetyl-ß-d-glucosaminidase and neuraminidase activities, respectively. Moreover, MsgA expression, but not NanA, is negatively regulated by LacR. Here we measured the activities of MsgA, NanA and ILY in strains isolated from clinical specimens and dental plaque to determine the correlation between these glycosidase activities and ILY hemolytic activity. Hemolytic activity showed a strong positive correlation with MsgA and a weak negative correlation with NanA activities. Therefore, we calculated the ratio of MsgA and NanA activity (M/N ratio). This value showed a stronger positive correlation (r = 0.81) with ILY hemolytic activity and many strains with high M/N ratios (>2) were ILY-high producers with loss-of-function mutations in LacR.

The Candida species that are important for the development of atrophic glossitis in xerostomia patients.

BACKGROUND: The purpose of this study was to clarify the species of Candida that are important for the development of atrophic glossitis in xerostomia patients. METHODS: A total of 231 patients with subjective dry mouth were enrolled in the present study. Logistic regression analysis was performed to clarify the contribution of each Candida species and other variables to the development of atrophic glossitis. The dependent variable was the absence/presence of atrophic glossitis. The Candida colony-forming units (CFU) of C. albicans, C. glabrata, C. tropicalis, and C. krusei, as well as age, gender, resting (RSFR) and stimulated (SSFR) whole salivary flow rate, and denture-wearing status, were treated as explanatory variables. RESULTS: Logistic regression analysis showed that two factors were closely associated with the presence of atrophic glossitis: an increase in C. albicans CFU and a decrease in the SSFR. CONCLUSIONS: C. albicans, but not non-albicans Candida, was associated with atrophic glossitis in xerostomia patients who had no systemic predisposing factors, indicating that C. albicans remains a treatment target for Candida-related atrophic glossitis.

Plasma-treated water eliminates Streptococcus mutans in infected dentin model.

Non-mechanical procedures for removing caries-infected dentin are warranted in dentistry. We previously demonstrated the marked sterilization effect for direct irradiation of low-temperature plasma using dentin model infected with Streptococcus mutans. However it requires 180 s of intraoral plasma irradiation to eliminate bacteria. We alternatively investigated whether plasma-treated water (PTW), i.e., pure water exposed to plasma in an atmosphere, has a same bactericidal activity with the plasma irradiation. In the infected dentin model, the viable S. mutans counts recovered by bur at depth of 0.8-2.4 mm from the cavity floor were 104-106 CFU/round bur. After PTW application for only 10 s, the count was significantly decreased to below the detection limit (2.5 CFU/round bur) or 3.0±5.0 CFU/round bur. Since the bactericidal activity of PTW is rapidly deactivated at body temperature (37°C), PTW is likely to be biocompatible and holds significant potential for non-mechanical procedures for removing caries-infected dentin.

Therapeutic Application of Synbiotics, a Fusion of Probiotics and Prebiotics, and Biogenics as a New Concept for Oral Candida Infections: A Mini Review.

Candida is a major human fungal pathogen causing infectious conditions predominantly in the elderly and immunocompromised hosts. Although Candida resides as a member of the oral indigenous microbiota in symbiosis, some circumstances may cause microbial imbalance leading to dysbiosis and resultant oral candidiasis. Therefore, oral microbial symbiosis that suppresses the overgrowth of Candida is important for a healthy oral ecosystem. In this regard, probiotics, prebiotics, and synbiotics can be considered a potential therapeutic and preventive strategy against oral candidiasis. Prebiotics have a direct effect on microbial growth as they stimulate the growth of beneficial bacteria and suppress the growth of pathogens. Probiotics render a local protective effect against pathogens and a systemic indirect effect on immunological amelioration. Synbiotics are fusion products of prebiotics and probiotics. This mini review discusses the potential use and associated limitations of probiotics, prebiotics, and synbiotics for the prevention and treatment of oral candidiasis. We will also introduce biogenics, a recent concept derived from the work on probiotics. Biogenics advocates the use of beneficial bioactive substances produced by probiotic bacteria, whose activities are independent from the viability of probiotic bacteria in human bodies.

Factors associated with the presence of atrophic tongue in patients with dry mouth.

PURPOSE: This study aimed to identify factors associated with atrophic tongue in patients with dry mouth. METHODS: Discriminant analysis was performed in 1265 patients with dry mouth to identify factors that might influence the risk of developing atrophic tongue. The dependent variable was the presence of atrophic tongue, while patient age, resting saliva flow rate, stimulated saliva flow rate and Candida colony-forming units (CFU) were used as the independent variables. RESULTS: The standardised linear discriminant coefficients showed that Candida CFU, stimulated saliva flow rate and age were significantly associated with the presence of atrophic tongue. The following linear discriminant function was obtained: z = 0.024 × age - 0.63 × (resting saliva flow rate) - 0.81 × (stimulated saliva flow rate) + 0.002 × Candida CFU - 0.611. CONCLUSION: High Candida CFU, low stimulated saliva flow rate and advanced age were identified as closely associated factors for the risk of development of atrophic tongue.

An analysis of the biofilms adhered to framework alloys using in vitro denture plaque models.

The purpose of this study was to perform a quantitative and qualitative evaluation of the biofilms formed on framework alloys in vitro. The biofilms formed by unfiltered fresh human saliva or Streptococcus mutans and/or Candida albicans on commercially pure titanium and gold-copper-platinum demonstrated higher amounts than other alloy and resin samples. In contrast, the silverpalladium-copper-gold showed a significantly decreased level of biofilm formation. Although the adhesion level of Streptococcus mutans on cobalt-chromium was high, that of Candida albicans was less extensive. A T-RFLP analysis and qualitative PCR of the microbes in the biofilms were performed. In a cluster analysis of all T-RFLP profiles, the cobalt-chromium pattern was integrated into one cluster. On qualitative PCR, the existence of microorganisms related to caries, preriodontitis and aspiration pneumonia was observed. Our results showed that the biofilm formation on each framework alloy was different in terms of both the quantity and quality.

Identification and characterization of a novel secreted glycosidase with multiple glycosidase activities in Streptococcus intermedius.

Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit ß-d-galactosidase and N-acetyl-ß-d-hexosaminidase activities. We, therefore, named this protein "multisubstrate glycosidase A" (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated ß-d-galactosidase, ß-d-fucosidase, N-acetyl-ß-d-glucosaminidase, and N-acetyl-ß-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-ß-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked ß-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had ß-d-galactosidase and ß-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-ß-d-glucosaminidase and N-acetyl-ß-d-galactosaminidase activities. The ß-d-galactosidase and ß-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-ß-d-glucosaminidase and N-acetyl-ß-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.

Inhibitory effect of coated mannan against the adhesion of Candida biofilms to denture base resin.

The adherence of Candida on dentures is related to diseases such as denture stomatitis and aspiration pneumonia. Mannan is a major component of the Candida cell surface, and contributes to the cell adherence. A previous report indicated that the adherence of C. albicans to culture dishes was inhibited by the coating them with mannan. The purpose of this study was to examine the adhesion inhibitory effect of mannan coating on acrylic denture surfaces against C. albicans and C. glabrata. The amount of Candida attached on the acrylic surfaces coated with mannan was calibrated by culture methods. Mannan showed significant inhibitory effects on Candida adhesion in both the yeast and hyphal form in a concentration-dependent manner, and the durability of the inhibitory effect continued for three days. These results suggest that mannan coating on the denture base acrylic can prevent Candida adhesion on the denture.