Effects of tongue cleaning on bacterial flora in tongue coating and dental plaque: a crossover study

BACKGROUND: The effects of tongue cleaning on reconstruction of bacterial flora in dental plaque and tongue coating itself are obscure. We assessed changes in the amounts of total bacteria as well as Fusobacterium nucleatum in tongue coating and dental plaque specimens obtained with and without tongue cleaning. METHODS: We conducted a randomized examiner-blind crossover study using 30 volunteers (average 23.7 ± 3.2 years old) without periodontitis. After dividing randomly into 2 groups, 1 group was instructed to clean the tongue, while the other did not. On days 1 (baseline), 3, and 10, tongue coating and dental plaque samples were collected after recording tongue coating score (Winkel tongue coating index: WTCI). After a washout period of 3 weeks, the same examinations were performed with the subjects allocated to the alternate group. Genomic DNA was purified from the samples and applied to SYBR® Green-based real-time PCR to quantify the amounts of total bacteria and F. nucleatum. RESULTS: After 3 days, the WTCI score recovered to baseline, though the amount of total bacteria in tongue coating was significantly lower as compared to the baseline. In plaque samples, the bacterial amounts on day 3 and 10 were significantly lower than the baseline with and without tongue cleaning. Principal component analysis showed that variations of bacterial amounts in the tongue coating and dental plaque samples were independent from each other. Furthermore, we found a strong association between amounts of total bacteria and F. nucleatum in specimens both. CONCLUSIONS: Tongue cleaning reduced the amount of bacteria in tongue coating. However, the cleaning had no obvious contribution to inhibit dental plaque formation. Furthermore, recovery of the total bacterial amount induced an increase in F. nucleatum in both tongue coating and dental plaque. Thus, it is recommended that tongue cleaning and tooth brushing should both be performed for promoting oral health

Glaucomatous Visual Field Defect Severity and the Prevalence of Motor Vehicle Collisions in Japanese: A Hospital/Clinic-Based Cross-Sectional Study

Purpose. This study examined the association between the severity of visual field defects and the prevalence of motor vehicle collisions (MVCs) in subjects with primary open-angle glaucoma (POAG). Methods. This is a cross-sectional study. Japanese patients who have had driver’s licence between 40 and 85 years of age were screened for eligibility. Participants answered a questionnaire about MVCs experienced during the previous 5 years. Subjects with POAG were classified as having mild, moderate, or severe visual field defect. We evaluated associations between the severity of POAG and the prevalence of MVCs by logistic regression models. Results. The prevalence of MVCs was significantly associated with the severity of POAG categorized by worse eye MD (control: 30/187 = 16.0%; mild POAG: 17/92 = 18.5%; moderate POAG: 14/60 = 23.3%; severe POAG: 14/47 = 29.8%; P=0.025, Cochran-Armitage trend test). Compared to the control group, the adjusted OR for MVC prevalence in subjects with mild, moderate, or severe POAG in the worse eye was 1.07 (95% CI: 0.55 to 2.10), 1.44 (95% CI: 0.68 to 3.08), and 2.28 (95% CI: 1.07 to 4.88). Conclusions. There is a significant association between the severity of glaucoma in the worse eye MD and the prevalence of MVCs

Accumulation of Squalene in a Microalga <i>Chlamydomonas reinhardtii</i> by Genetic Modification of Squalene Synthase and Squalene Epoxidase Genes

<div><p>Several microalgae accumulate high levels of squalene, and as such provide a potentially valuable source of this useful compound. However, the molecular mechanism of squalene biosynthesis in microalgae is still largely unknown. We obtained the sequences of two enzymes involved in squalene synthesis and metabolism, squalene synthase (CrSQS) and squalene epoxidase (CrSQE), from the model green alga <i>Chlamydomonas reinhardtii</i>. CrSQS was functionally characterized by expression in <i>Escherichia coli</i> and CrSQE by complementation of a budding yeast <i>erg1</i> mutant. Transient expression of CrSQS and CrSQE fused with fluorescent proteins in onion epidermal tissue suggested that both proteins were co-localized in the endoplasmic reticulum. <i>CrSQS</i>-overexpression increased the rate of conversion of ¹⁴C-labeled farnesylpyrophosphate into squalene but did not lead to over-accumulation of squalene. Addition of terbinafine caused the accumulation of squalene and suppression of cell survival. On the other hand, in <i>CrSQE</i>-knockdown lines, the expression level of <i>CrSQE</i> was reduced by 59–76% of that in wild-type cells, and significant levels of squalene (0.9–1.1 μg mg^–1 cell dry weight) accumulated without any growth inhibition. In co-transformation lines with <i>CrSQS</i>-overexpression and <i>CrSQE</i>-knockdown, the level of squalene was not increased significantly compared with that in solitary <i>CrSQE</i>-knockdown lines. These results indicated that partial knockdown of <i>CrSQE</i> is an effective strategy to increase squalene production in <i>C</i>. <i>reinhardtii</i> cells.</p></div

Knockdown of <i>CrSQE</i> gene in parental line (PL) UVM4.

<p>PL UVM4 and five knockdown lines (SQEKD-1 to SQEKD-5) were analyzed. A) qRT-PCR analysis of the <i>CrSQE</i> gene expression of each gene was normalized to that of the <i>CRY1</i> gene. Asterisks above the bars indicate significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01). B) Squalene content was measured in each line. DW; cell dry weight. ND; not detected. C) Contents of ergosterol (grey bars), putative 7-dehydroporiferasterol (open bars) and squalene (hatched bars) were measured in each line. D) Doubling time in each line was calculated by measuring absorbance at 730 nm. Data in all experiments indicate mean value ± SD from three biological replicates.</p

Functional characterization of CrSQS in <i>E</i>. <i>coli</i>.

<p>A) SDS-polyacrylamide gel electrophoresis of crude extracts from cells harboring the empty vector pET-21b (lane 1), cells expressing <i>CrSQS</i> (lane 2) and purified CrSQS using a TALON Metal affinity chromatography. B) Normal-phase thin-layer chromatogram of the reaction products derived from (1–¹⁴C) farnesyl diphosphate (FPP). Crude extracts from cells harboring the empty vector (lanes 1–3), from cells expressing CrSQS (lanes 4–6) and purified CrSQS protein (lanes 7–9) were assayed for SQS activity using (1–¹⁴C) FPP and Mg²⁺ with NADPH (lanes 1–2, 4–5, and 7–8 are experimental replicates) or without NADPH (lanes 3, 6, 9). Authentic (1–¹⁴C) FPP was loaded in lane 10 as a negative control. The positions of origin, solvent front and authentic squalene are indicated on the left. Open triangles on the right indicate position of signals from putative dehydrosqualene and 12-hydroxysqualene in lane 9.</p

Effects of terbinafine (TBF) on squalene and sterol biosynthesis and cell viability.

<p>A) Contents of ergosterol (grey bars), putative 7-dehydroporiferasterol (open bars) and squalene (hatched bars) in the cells treated with different concentrations of TBF. DW; cell dry weight. B) Images of cultures treated with TBF. C) Chlorophyll content in cells treated with TBF. Data in all experiments are mean value ± SD from three biological replicates.</p