Oranart Matangkasombut

Detection of LINE-1s hypomethylation in oral rinses of oral squamous cell carcinoma patients.

This study aimed to (i) investigate long interspersed nuclear element-1 (LINE-1) methylation levels of oral squamous cell carcinomas (OSCCs), the major type of oral malignancies; and (ii) investigate whether the hypomethylation of LINE-1s can be detected in oral rinses of OSCC patients. The combined bisulfite restriction analysis polymerase chain reaction (PCR) of LINE-1s (COBRALINE-1) was used. We found that tissues from OSCC specimens had lower methylation levels of LINE-1s than cells collected from the oral rinses of normal volunteers. Interestingly, cells collected from oral rinses of OSCC patients also revealed hypomethylated LINE-1s at the same level as OSCC tissues. There was no difference in the level of hypomethylation due to stages, locations, histological grades, and history of betel chewing, smoking and/or alcohol consumption. In conclusion, OSCCs possessed global hypomethylation and this alteration could be detected from oral rinses of OSCC patients by a simple PCR technique, COBRALINE-1. Therefore, COBRALINE-1 of oral rinses may be applied for non-invasive detection of oral malignancies.

LINE-1 methylation status of endogenous DNA double-strand breaks

DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.

Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans Induces DNA Damage, S/G2 Cell Cycle Arrest, and Caspase- Independent Death in a Saccharomyces cerevisiae Model

ABSTRACT Cytolethal distending toxin (CDT) is a bacterial toxin that induces G2/M cell cycle arrest, cell distension, and/or apoptosis in mammalian cells. It is produced by several Gram-negative species and may contribute to their pathogenicity. The catalytic subunit CdtB has homology with DNase I and may act as a genotoxin. However, the mechanism by which CdtB leads to cell death is not yet clearly understood. Here, we used Saccharomyces cerevisiae as a model to study the molecular pathways involved in the function of CdtB from Aggregatibacter actinomycetemcomitans, a cause of aggressive periodontitis. We show that A.actinomycetemcomitans CdtB (AaCdtB) expression induces S/G2 arrest and death in a DNase-catalytic residue and nuclear localization-dependent manner in haploid yeasts. Yeast strains defective in homologous recombination (HR) repair, but not other DNA repair pathways, are hypersensitive to AaCdtB, suggesting that HR is required for survival upon CdtB expression. In addition, yeast does not harbor the substrate for the other activity proposed for CdtB function, which is phosphatidylinositol-3,4,5-triphosphate phosphatase. Thus, these results suggest that direct DNA-damaging activity alone is sufficient for CdtB toxicity. To investigate how CdtB induces cell death, we examined the effect of CdtB in yeast strains with mutations in apoptotic regulators. Our results suggest that yeast death occurs independently of the yeast metacaspase gene YCA1 and the apoptosis-inducing factor AIF1 but is partially dependent on histone H2B serine 10 phosphorylation. Therefore, we report here the evidence that AaCdtB causes DNA damage that leads to nonapoptotic death in yeast and the first mutation that confers resistance to CdtB.

Oral Candida carriage and immune status in Thai human immunodeficiency virus-infected individuals.

Oral candidiasis is a common opportunistic infection among human immunodeficiency virus (HIV)-infected individuals, with growing concerns about the emergence of non-albicans species with resistance to antifungal agents. This cross-sectional study determined the prevalence of oral Candida species in Thai HIV-infected adults and factors affecting their colonization. Candida species were identified from oral rinse samples of 60 HIV-infected participants of the MTCT-Plus initiative and 49 healthy controls by culture-based and molecular assays. The prevalence of oral Candida carriage was similar in HIV-infected patients (56.6 %) and in controls (55.1 %, P = 0.87). Candida albicans was the most predominant species in both groups (94.1 % of Candida carriers in HIV, 88.9 % in control). Interestingly, Candida dubliniensis was the second most common species in controls (29.6 %) and the third in HIV-infected patients (11.8 %, P = 0.08). Multivariate analysis showed that, amongst HIV-infected individuals, CD4 count <200 cells mm(-3) was associated with increased prevalence of oral carriage of both C. albicans (P = 0.03) and non-albicans species (P = 0.03). Moreover, patients with tuberculosis infection had a higher prevalence of the non-albicans species than those without (P = 0.03). Intriguingly, contraceptive use was also associated positively with non-albicans and multi-species carriage (P = 0.04 for both). However, use of antiretroviral drugs protected the patients from Candida carriage (P = 0.03), especially from C. albicans (P = 0.02). In conclusion, while HIV-infected individuals had a similar prevalence of oral Candida carriage to that of the control group, host immune status, tuberculosis infection, and contraceptive use may influence oral colonization of Candida, especially of the non-albicans species.

Replication-Independent Endogenous DNA Double-Strand Breaks in Saccharomyces cerevisiae Model

Without exposure to any DNA-damaging agents, non-dividing eukaryotic cells carry endogenous DNA double-strand breaks (EDSBs), or Replication-Independent (RIND)-EDSBs. In human cells, RIND-EDSBs are enriched in the methylated heterochromatic areas of the genome and are repaired by an ATM-dependent non-homologous end-joining pathway (NHEJ). Here, we showed that Saccharomyces cerevisiae similarly possess RIND-EDSBs. Various levels of EDSBs were detected during different phases of the cell cycle, including G0. Using a collection of mutant yeast strains, we investigated various DNA metabolic and DNA repair pathways that might be involved in the maintenance of RIND-EDSB levels. We found that the RIND-EDSB levels increased significantly in yeast strains lacking proteins involved in NHEJ DNA repair and in suppression of heterochromatin formation. RIND-EDSB levels were also upregulated when genes encoding histone deacetylase, endonucleases, topoisomerase, and DNA repair regulators were deleted. In contrast, RIND-EDSB levels were downregulated in the mutants that lack chromatin-condensing proteins, such as the high-mobility group box proteins, and Sir2. Likewise, RIND-EDSB levels were also decreased in human cells lacking HMGB1. Therefore, we conclude that the genomic levels of RIND-EDSBs are evolutionally conserved, dynamically regulated, and may be influenced by genome topology, chromatin structure, and the efficiency of DNA repair systems.

Prevalence of oral Candida carriage in Thai adolescents

AIM Oral candidiasis is among the most common AIDS-associated opportunistic infections. Adolescents remain at the highest risk of HIV infection and could suffer from oral candidiasis. However, information on oral Candida carriage in this population is limited. This study aims to evaluate the prevalence of oral Candida in Thai adolescents. METHODS Oral rinse samples from 80 healthy Thais (age: 15-17 years) were collected and analyzed for the prevalence of Candida species using culture-based and polymerase chain reaction assays. RESULTS Twenty six adolescents (32.5%) carried Candida in the oral cavity. Candida albicans was detected in 28.75% (23/80). Non-albicans Candida species were detected in 6.25% (5/80). The majority (92.3%, 24/26) of adolescents with Candida carried a single species. Two carried two species: one with Candida glabrata and Candida albicans, and the other with Candida parapsilosis and Candida albicans. Three adolescents harbored only non-albicans species, with one carrying Candida tropicalis and two carrying Candida parapsilosis. Candida dubliniensis was not detected in this population. Most adolescents carried Candida at a low level (<500 c.f.u./mL). CONCLUSIONS Oral Candida was present in approximately one-third of adolescents. Candida albicans was the most prevalent (88.5%), and non-albicans species were present in 19.2% of those with oral Candida.

In-office bleaching gel with 35% hydrogen peroxide enhanced biofilm formation of early colonizing streptococci on human enamel

OBJECTIVES To compare the effects of 25% and 35% hydrogen peroxide in-office bleaching systems on surface roughness and streptococcal biofilm formation on human enamel. METHODS Enamel specimens (3mm×3mm×2mm, n=162) from human permanent teeth were randomly divided into 3 treatment groups (n=54 each): (1) control, (2) bleached with 25% hydrogen peroxide (Zoom2™), and (3) bleached with 35% hydrogen peroxide (Beyond™). The enamel surface roughness was measured by a profilometer before and after treatments. Subsequently, the treated enamel specimens were randomly placed into 3 subgroups (n=18 each) and incubated with: (1) trypticase soy broth control, (2) Streptococcus mutans culture and (3) Streptococcus sanguinis culture for 24h. Biofilm formation was quantified by crystal violet staining. The biofilm structure on three specimens from each group was visualized by scanning electron microscopy. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests with Bonferroni corrections. Significance level was set at p<0.05. RESULTS Both bleaching systems significantly reduced enamel surface roughness comparing to the control group (p<0.001), but there was no difference between the two treatment groups. Remarkably, S. sanguinis biofilm formation was significantly higher on enamel specimens bleached with 35% hydrogen peroxide than other treatments (p<0.001), but was lower on those bleached with 25% hydrogen peroxide (p<0.001). In contrast, no difference in S. mutans biofilm formation was observed among the three treatment groups. CONCLUSION Both 25% and 35% hydrogen peroxide caused similar degrees of reduction in enamel surface roughness. Nevertheless, bleaching with 35% hydrogen peroxide appeared to markedly promote S. sanguinis biofilm formation. CLINICAL SIGNIFICANCE The increase of early colonizer biofilm raised concerns over adverse effects of in-office bleaching on plaque formation. This should be further investigated in vivo and efficient plaque control should be emphasized after bleaching with high concentrations of hydrogen peroxide.

Effectiveness of a motionless ultrasonic toothbrush in reducing plaque and gingival inflammation in patients with fixed orthodontic appliances

OBJECTIVE To compare the effectiveness of a motionless ultrasonic toothbrush to a manual toothbrush in reducing dental plaque, gingival inflammation, and mutans streptococci in patients with fixed orthodontic appliances. MATERIALS AND METHODS Twenty-five orthodontic patients were recruited to this crossover study. The patients were randomized into two groups starting with manual or motionless ultrasonic toothbrushes for 30 days. After a 30-day washout period, the patients switched to the other toothbrush type for 30 days. Plaque and gingival indices were evaluated by two calibrated-blinded examiners before and after each 30-day period of brushing. Salivary samples were also collected for quantification of mutans streptococci. RESULTS On the bracket side, the motionless ultrasonic toothbrush showed a significantly higher mean plaque index bracket score after 30-day usage than baseline (P = .049), while the manual toothbrush group showed no difference between the before and after brushing periods (P = .10). The changes in plaque index bracket score were significantly more favorable in the manual toothbrush group than in the ultrasonic toothbrush group (P = .04). In contrast, no difference was observed on the nonbracket side. There was no significant difference in the changes of gingival index or the numbers of mutans streptococci between the two groups. CONCLUSION Manual toothbrushing performed better than brushing with the motionless ultrasonic toothbrush in plaque removal on the bracket side in orthodontic patients. However, no difference was observed in terms of gingival status and the numbers of mutans streptococci.

Separation and detection of mutans streptococci by using magnetic nanoparticles stabilized with a cell wall binding domain-conjugated polymer

A number of salivary mutans streptococci (MS: Streptococcus mutans and Streptococcus sobrinus) are used in dental caries risk assessment. In this study, a simple, yet effective assay was developed for MS detection. Magnetic nanoparticles (MNPs) were first grafted with poly(acrylic acid) that bears active carboxyl groups available for conjugation with the cell wall binding domain (CWBD) of automutanolysin which specifically binds to MS. The binding efficiency of CWBD-conjugated MNPs to MS was tested with pure cultures of streptococcal standard strains. After mixing CWBD-conjugated MNPs with culture, bacteria-bound particles were separated from unbound cells using a magnet and filtered through a cellulose acetate membrane (pore-size 0.8 μm). The color intensity of particles remaining on the membrane represents the number of bound bacteria. The CWBD-conjugated MNPs showed higher efficiency in binding to S. mutans and S. sobrinus than to non-mutans streptococci (S. sanguinis and S. salivarius) with capture efficiencies of 77 and 69% for MS and 38 and 15% for non-MS. Moreover, this method can quantify the number of MS in the range of 102 to 107 colony-forming units (CFU) mL−1, which covers the range of MS levels used in caries risk assessment. The calculated limit of detection of the assay was 16 and 72 CFU mL−1 for S. mutans and S. sobrinus, respectively. Furthermore, the CWBD-conjugated MNPs could be used to efficiently quantify the number of MS in human saliva samples containing highly complex mixtures of bacterial species. These results suggest that the assay could be applicable as a simple tool for MS determination in not only clinical settings but also community fields without clinical expert requirement.

CdtC-induced processing of membrane-bound CdtA is a crucial step in Aggregatibacter actinomycetemcomitans CDT holotoxin formation

ABSTRACT Aggregatibacter actinomycetemcomitans is an oral pathogen causing periodontal disease and bacterial endocarditis. It produces cytolethal distending toxin (CDT) that could damage mammalian cells and tissues. CDT is a tripartite protein toxin composed of CdtA, CdtB, and CdtC. We have previously indicated that CdtA is a lipoprotein and that the proteolytic processing of CdtA is important for biogenesis and secretion of CDT holotoxin. Here, we established an in vitro processing assay of CdtA and investigated the interactions of CdtA with other Cdt subunits. This assay demonstrated that incubation of membrane-bound CdtA (MCdtA), CdtB, and CdtC immediately generated a processed form of CdtA (CdtA′), which is recovered from the soluble fraction. In contrast, incubation of soluble membrane-unbound CdtA with CdtB and CdtC did not yield any CdtA′. Furthermore, incubation of CdtC with MCdtA was enough to induce rapid processing of MCdtA, whereas CdtB alone was unable to induce the processing. Coimmunoprecipitation demonstrated that CdtA′ and CdtC formed a complex. Furthermore, subsequent addition of CdtB to this reaction mixture resulted in complete CDT holotoxin complex. The cytolethal distending activity assay demonstrated that CDT complex containing CdtA′ showed far stronger cytotoxicity than that containing CdtA. Collectively, our data suggest that CDT holotoxin formation in vivo is a sequential event: interaction of MCdtA and CdtC induces proteolytic processing of MCdtA, and the released CdtA′ forms a complex with CdtC. Subsequent binding of CdtB to the CdtA′/CdtC complex results in CDT holotoxin formation.