Genome-Wide Identification of Host Genes Required for Toxicity of Bacterial Cytolethal Distending Toxin in a Yeast Model

BackgroundAggregatibacter actinomycetemcomitans, a periodontal pathogen, secretes a cytolethal distending toxin (AaCDT) that causes host cell cycle arrest and cell death. Although CDT could be an important virulence factor, it is unclear how it enters the nucleus to exert its cytotoxicity.ObjectiveTo investigate the mechanisms of AaCDT by genome-wide screening for host mutations that confer resistance to the catalytic subunit, AaCdtB, in a Saccharomyces cerevisiae model.MethodsWe transformed the yeast haploid deletion library, a collection of yeast mutants with single gene deletions of virtually all non-essential ORFs in the genome, with plasmids carrying galactose-inducible AaCdtB. Yeast mutants that showed resistance to AaCdtB were selected and rescreened by a spotting assay. AaCdtB expression was confirmed by western blot analysis; any mutants that showed no or weak expression of AaCdtB were omitted from the analysis. The lists of genes whose mutations confer resistance to AaCdtB were analyzed for Gene Ontology (GO) term enrichments. Localization of AaCdtB-EGFP was examined using fluorescent microscopy. Nuclear localization relative to EGFP control was calculated and compared to wild-type.ResultsOut of approximately 5,000 deletion mutants, we isolated 243 mutants that are resistant to AaCdtB. GO analyses indicated that genes associated with organic anion transport are significantly enriched (16 genes). Furthermore, several genes associated with the nucleus and endoplasmic reticulum (ER) were identified. Localization studies of AaCdtB, in mutants with the deletion of genes associated with the GO term organic anion transport, showed lower nuclear localization than wild-type. The results suggest that these genes may be required for AaCdtB translocation into the nucleus and its cytotoxicity.ConclusionThe genome-wide screen in the yeast deletion library allowed us to identify a large number of host genes required for AaCdtB cytotoxicity. Further investigation could lead to more insights into the mechanisms of CdtB intoxication

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

Hyposalivation, oral health, and Candida colonization in independent dentate elders.

Hyposalivation is an important problem in elders and could interfere with several oral functions and microbial ecology. While the number of independent elders who retain more natural teeth increases worldwide, few studies examined hyposalivation in this population. Thus, this study aims to examine relationships between hyposalivation, oral health conditions and oral Candida colonization in independent dentate elders and evaluate factors associated with salivary flow and Candida carriage. We conducted a cross-sectional study in fifty-three dentate elders (≥65 years old with at least 4 pairs of posterior occlusal contacts) with no, or well-controlled, systemic conditions. Participants were interviewed for medical history, subjective dry mouth symptoms, oral hygiene practices and denture information. Unstimulated and stimulated salivary flow rates, objective dry mouth signs, gingival, tongue-coating, and root-caries indices were recorded. Stimulated saliva was cultured on Sabouraud-dextrose agar for Candida counts. Candida species were identified using chromogenic Candida agar and polymerase chain reaction. Statistical significance level was set at p<0.05. The results showed that hyposalivation was associated with higher gingival and tongue-coating indices (p = 0.003 and 0.015, respectively), but not root-caries index. Hyposalivation was also associated with higher prevalence of oral Candida colonization (p = 0.010; adjusted OR = 4.36, 95% confidence interval = 1.29-14.72). These two indices and Candida load were negatively correlated with unstimulated and stimulated salivary flow rates. Interestingly, non-albicans Candida species were more prevalent in denture wearers (p = 0.017). Hence, hyposalivation is a risk factor for poorer oral health and oral Candida colonization in independent dentate elders. Because of its potential adverse effects on oral and systemic health, hyposalivation should be carefully monitored in elders

Effects of blue-light LED toothbrush on reducing dental plaque and gingival inflammation in orthodontic patients with fixed appliances: a crossover randomized controlled trial

Abstract Background Patients with fixed orthodontic appliances have higher plaque accumulation and gingival inflammation. Our aim was to compare the effectiveness of a light emitting diode (LED) toothbrush with a manual toothbrush in reducing dental plaque and gingival inflammation in orthodontic patients with fixed appliances, and to investigate the effect of the LED toothbrush on Streptococcus mutans (S. mutans) biofilm in vitro. Methods Twenty-four orthodontic patients were recruited and randomly assigned into 2 groups: (1) started with manual and (2) started with LED toothbrushes. After a 28-day usage and 28-day wash-out period, the patients switched to the other intervention. The plaque and gingival indices were determined at baseline and 28 days after each intervention. The patients’ compliance and satisfaction scores were collected using questionnaires. For the in vitro experiments, S. mutans biofilm was divided into 5 groups (n = 6) with 15-, 30-, 60-, or 120-sec LED exposure, and without LED exposure as a control group. Results There was no significant difference in the gingival index between the manual and LED toothbrush groups. The manual toothbrush was significantly more effective in reducing the plaque index in the proximal area on the bracket side (P = 0.031). However, no significant difference was found between the two groups in other areas around the brackets or on the non-bracket side. After LED exposure in vitro, the percentages of bacterial viability after LED exposure for 15–120 s were significantly lower compared with the control (P = 0.006). Conclusion Clinically, the LED toothbrush was not more effective in reducing dental plaque or gingival inflammation than the manual toothbrush in orthodontic patients with fixed appliances. However, the blue light from the LED toothbrush significantly reduced the number of S. mutans in biofilm when it was exposed to the light for at least 15 s in vitro. Clinical Trial Registration Thai Clinical Trials Registry (TCTR20210510004). Registered 10/05/2021

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

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

EDSBs were detected in different DNA preparations including HMW DNA (cell→gel), liquid DNA (cell→liquid), and liquid DNA extracted from in-gel HMW DNA (cell→gel→liquid).

<p>(A) The levels of EDSBs from different DNA preparation methods. (B) Subtracted DSBs levels between liquid DNA and other methods. When comparing cell→gel→liquid with cell→liquid, adding in gel preparation step did not increase the number of DSBs significantly. The average levels of EDSBs from 9 independent experiments are shown as histograms with error bars representing SEM.</p

Streaming Spectral Processing with Consumer-level Graphics Processing Units

This paper describes the implementation of a streaming spectral processing system for realtime audio in a consumer-level onboard GPU (Graphics Processing Unit) attached to an off-the-shelf laptop computer. It explores the implementation of four processes: standard phase vocoder analysis and synthesis, additive synthesis and the sliding phase vocoder. These were developed under the CUDA development environment as plugins for the Csound 6 audio programming language. Following a detailed exposition of the GPU code, results of performance tests are discussed for each algorithm. They demonstrate that such a system is capable of realtime audio, even under the restrictions imposed by a limited GPU capability

Yeast strains used in this study.

<p>Yeast strains used in this study.</p