Effect of students' determination of testing time on their test performance.

This article investigates whether giving students control over preparing for and the moment of taking a test affects their test results in comparison with when the school is in control of the amount of training followed by a predetermined test moment. The students participated in training for manual dexterity. After the training, the students performed a test. The results of the test were stored in a database. Students from the group with freedom to select the moment of the test performed much better than those in the other group who did not have the freedom to select the moment for the test, with significantly fewer students requiring three attempts to pass the test. The fact that students when given the responsibility to develop manual skills performed better than when guided by the policy of the school is hopeful in the sense that students can learn in an early stage of their study to take responsibility for learning.

Identification and differential gene expression of adhesin-like wall proteins in Candida glabrata biofilms.

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.

Effects of different kinds of fluorides on enolase and ATPase activity of a fluoride-sensitive and fluoride-resistant Streptococcus mutans strain.

Enolase and ATPase are sensitive to fluoride. It is unclear whether this sensitivity differs for F-sensitive and F-resistant cells or for different types of fluoride. Permeabilized cells of the fluoride-sensitive strain Streptococcus mutans C180-2 and its fluoride-resistant mutant strain C180-2 FR were preincubated at pH 7 or 4 with NaF, the amine fluorides Olaflur and Dectaflur and amine chloride controls. After preincubations, enolase and ATPase activities of the cells were assessed. Enolase activity was more inhibited after preincubation at pH 7 with NaF than with Olaflur. Amine chloride stimulated, although not with statistical significance, the enolase activity of both strains. After preincubation at pH 4 the enolases were strongly inactivated, but the fluoride-resistant strain's enolase to a lesser extent. The results suggested that amine acts to protect enolase activity against the detrimental low pH effect. Gene sequencing showed that the enolase genes of the fluoride-resistant and fluoride-sensitive strain were identical. ATPase activity was not reduced after NaF preincubation at either pH 7 or pH 4. The amine fluorides and their chloride controls in the preincubation mixture reduced the ATPase activity significantly at both pH values. In conclusion, our results showed that preincubation with amine fluoride did not inhibit enolase activity more effectively than NaF. The amine part of the molecule may protect enolase activity against preincubations at low pH. ATPase activity was not inhibited by NaF preincubation but was significantly inhibited after preincubation with amine fluorides and amine chlorides.

Effect of Psidium cattleianum leaf extract on Streptococcus mutans viability, protein expression and acid production.

Plants naturally produce secondary metabolites that can be used as antimicrobials. The aim of this study was to assess the effects of Psidium cattleianum leaf extract on Streptococcus mutans. The extract (100%) was obtained by decoction of 100 g of leaves in 600 ml of deionized water. To assess killing, S. mutans biofilms were treated with water (negative control) or various extract dilutions [100, 50, 25% (v/v) in water] for 5 or 60 min. To evaluate the effect on protein expression, biofilms were exposed to water or 1.6% (v/v) extract for 120 min, proteins were extracted and submitted to 2-dimensional difference gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry. The effect of 1.6% (v/v) extract on acid production was determined by pH measurements and compared to a water control. Viability was similar after 5 min of treatment with the 100% extract or 60 min with the 50% extract (about 0.03% survival). There were no differences in viability between the biofilms exposed to the 25 or 50% extract after 60 min of treatment (about 0.02% survival). Treatment with the 1.6% extract significantly changed protein expression. The abundance of 24 spots was decreased compared to water (p < 0.05). The extract significantly inhibited acid production (p < 0.05). It is concluded that P. cattleianum leaf extract kills S. mutans grown in biofilms when applied at high concentrations. At low concentrations it inhibits S. mutans acid production and reduces the expression of proteins involved in general metabolism, glycolysis and lactic acid production.

[Microbial genetics. New possibilities for the prevention and treatment of (oral) infections]. / Microbiële genetica nieuwe mogelijkheden voor preventie en behandeling van (orale) infecties.

New insights in the microbial genetics of pathogenic oral micro-organisms and the development of a new array of molecular genetic techniques together have led to alternative strategies in the development of antimicrobial agents. In this article the importance of insights in microbial molecular biology for the prevention and treatment of (oral) infectious diseases is illustrated. Following the introduction of relevant terminology, the role of microbial genetics in developing of target-based anti-microbial drugs for prevention and treatment of (oral) infections is discussed. Subsequently, the impact of microbial genetics on vaccine development and several, mainly still experimental, prevention strategies are discussed.

The VicRK system of Streptococcus mutans responds to oxidative stress.

In Streptococcus mutans, virulence and cariogenicity may be modulated via the two-component regulatory system VicRK. Environmental signals, sensed by VicK, inducing this modulation are still unclear, however, and were investigated in the present study. We found that VicRK displays homology with protein-domains that, in other bacteria, are involved in redox-sensing. After constructing a VicRK-promoter GFP-reporter strain, we showed increased fluorescence intensity under oxidative stress. Potential interference of alternative signals and experimental conditions on GFP expression was excluded by the use of negative and positive control strains. Finally, we constructed a clean vicK knockout mutant, which proved to be more sensitive to H(2)O(2) than the wild-type. In conclusion, this study showed that the VicRK system responds to and protects against oxidative stress. As a result, a link between oxidative/redox stress and the cariogenic nature of S. mutans can be hypothesized.

Zinc and cadmium accumulation and tolerance in populations of Sedum alfredii.

To investigate the variation of Zn and Cd accumulation and tolerance of Sedum alfredii (a newly reported Zn/Cd hyperaccumulator), field surveys and hydroponic experiments were conducted among three populations of this species: two originating from old Pb/Zn mines in Zhejiang (ZJ) and Hunan (HN) Provinces and one from a "clean" site in Guangdong (GD) Province, China. Under field conditions, up to 12,524 and 12,253 mg kg(-1) Zn, and 1400 and 97 mg kg(-1) Cd in shoots of ZJ and HN plants were recorded respectively. Under hydroponic conditions, ZJ and HN plants accumulated significantly higher Zn and Cd in their leaves and stems, and possessed significantly higher Zn and Cd tolerance than GD plants. Among the two contaminated populations, ZJ plants showed higher Cd tolerance and accumulation (in leaves) than HN plants. The present results indicate that significant differences in Zn and Cd accumulation and tolerance exist in populations of S. alfredii.

[Effect of three surface modification techniques of pure titanium on bacteria adhesion and ultrasonic cleaning efficacy].

OBJECTIVE: To evaluate the bacteria adhesion behavior and ultrasonic cleaning efficacy on pure titanium modified with 3 different techiques. METHODS: Pure titanium disks with mechanically polished surfaces(MP), titania nanotube surfaces(TNT)and sandblast-large grit and acid-etched surfaces(SLA)were used as substrates. The surface characteristics of the 3 types of specimens were detected. The disks of all groups were co-cultured with Porphyromonas gingivalis(Pg)and microcosm for 1 day and 5 days respectively. The cell viabilities of bacteria attached to the 3 types of surfaces were tested. The remaining bacteria on different surfaces after ultrasonic treatment were observed through live/dead bacteria staining. RESULTS: MP and SLA surfaces demonstrated a micro-scale structure, while TNT surfaces showed a nano-scale structure. The surface roughness of SLA specimen was the highest([1.62 ± 0.13]µm), and that of MP([0.81 ± 0.10]µm)and TNT specimen([0.792 ± 0.080]µm)were relatively lower and showed no statistical difference(P >0.05). At 1 and 5 d, the cell viability and the biomass of Pg attached to MP surfaces were as low as 1 829±210 and 13 811±3 110 and A570 value were 0.80±0.35 and 1.56±0.30 respectively. At 1 d, the cell viability of microcosm adhered on MP and TNT surfaces were lower(63 943±6 990 and 69 860±5 555)than that on the SLA surface, and the biomass of microcosm adhered on MP surfaces demonstrated the lowest value(A570 value 5.84±0.60). At 5 d, both the cell viability and biomass of micorcosm adhered on the three surfaces were of no statistical difference(P<0.05). The remaining bacteria on TNT surfaces were the least in the three groups and distributed sporadically after ultrasonic treatment. The remaining bacteria on all surfaces increased with culture time. CONCLUSIONS: Both surface topography and roughness affect early bacteria adhesion. However, this effect can be weakened as the biofilm getting mature. The surface topography can significantly affect the mechanical cleaning efficacy of the biofilm. TNT surface reveals a lower adhesion of microcosm and a higher efficacy of ultrasonic cleaning compared to MP and SLA surfaces.

Biofilm layers affect the treatment outcomes of NaF and Nano-hydroxyapatite.

During caries formation, dental biofilms function not only as acid producers but also as reservoirs and diffusion barriers for active caries-preventive components. The aim of this study was to investigate the influence of biofilms as a stagnant layer on the efficacy of NaF and nano-hydroxyapatite (nHA). Biofilms of Streptococcus mutans C180-2 were formed on the surfaces of artificially demineralized enamel in an active attachment biofilm model. After 2 days of biofilm formation, the model was subjected to a pH-cycling schedule, together with a control group without biofilms. Specimens were treated for 5 min twice daily with water, a 10% nHA slurry, or 18.4 mM NaF. At the end of the pH-cycling period, the biofilms were removed for the determination of the viable counts, the lactic acid production, and the calcium content. The mineral changes in the demineralized enamel blocks were analyzed by transversal microradiography. No differences in the biofilm viable counts and lactic acid production were found in the different treatment groups. The mean calcium content of the biofilms in the nHA group was 60.7 ± 15.3 mmol/g wet weight, which was approximately 8-fold higher than in the other 2 groups. The application of NaF resulted in net remineralization, but in the presence of a biofilm, net demineralization was observed. In contrast, nHA treatment reduced further demineralization compared with the water treatment, but the presence of a biofilm enhanced this effect. In conclusion, the presence of biofilms clearly influenced the treatment outcomes of anticaries products. Biofilms could either enhance or impede their efficacy. This result implies that biofilms should be included in the in vitro tests for the preclinical screening of caries-protective agents.

Survival of glass ionomer restorations placed in primary molars using atraumatic restorative treatment (ART) and conventional cavity preparations: 2-year results.

OBJECTIVE: To compare the survival of glass ionomer cement (GIC) restorations placed in a dental clinic setting using both the atraumatic restorative treatment (ART) approach with hand instruments, and conventional cavity preparation with rotary instruments. METHOD AND MATERIALS: Two encapsulated high-strength conventional GICs (Fuji IX GP, Ketac-Molar Aplicap) were placed in 82 Class I and 53 Class II preparations and one encapsulated non-gamma 2 amalgam alloy (GK-amalgam) was placed in 32 Class I preparations, in the primary molars of 60 Chinese children with a mean age of 7.40 +/- 1.24 (SD) years. Thus, 9 treatment groups were formed. RESULTS: After two years, there were no significant survival differences found among 7 of the 9 treatment groups (p = 0.99). However, two groups comprising Fuji IX GP and Ketac-Molar Aplicap placed in Class II cavities prepared using the ART approach showed significantly lower restoration survivals (p < 0.001). Only 3 of the 72 initially sealed fissures adjacent to the restorations appeared to retain any GIC material. CONCLUSIONS: In a clinic setting, both the ART hand instrument and conventional rotary instrument methods were equally suitable for high Class I restoration survival, but not for Class II restoration survival where the conventional cavity preparation method was preferable.

Diverse outcomes of Photodynamic Antimicrobial Chemotherapy on five Enterococcus faecalis strains.

OBJECTIVES: In the present study, the effectiveness of Photodynamic Antimicrobial Chemotherapy (PACT) was evaluated on planktonic cells and biofilms of five Enterococcus faecalis clinical isolates. METHODS: Planktonic cells and biofilms of E. faecalis E2, E3, ER3/2s, OS16 and AA-OR34 were grown in SDMY medium plus 0.4% glucose. Approximately 5.0×10(7)CFU planktonic cells and 24h biofilms were subjected to PACT using the combination of Light Emitting Diodes (LEDs, Biotable(®)) and Photogem(®). The metabolic activity of bacterial cells was evaluated by a resazurin assay. Biomass values of the biofilms were determined by a crystal violet assay. RESULTS: Compared to the water-treated control group, gradual increases of light energy led to greater reduction of metabolic activity of planktonic cells and biofilms of E. faecalis when the combination of LEDs and Photogem(®) was applied. Photogem(®) alone significantly reduced the metabolic activity of planktonic cells, whereas LEDs or Photogem(®) alone did not result in biofilm viability changes. PACT yielded similar antimicrobial outcomes on planktonic cells of all tested E. faecalis strains, whereas biofilms of E. faecalis E3, ER3/2s and OS16 were more resistant to PACT than biofilms of E. faecalis E2 and AA-OR34. CONCLUSIONS: The efficacy of PACT on E. faecalis biofilms was strain dependent. PACT demonstrated its potential as an adjuvant antimicrobial treatment by killing of E. faecalis planktonic and biofilm cells.

Effects of Streptococcus mutans on dendritic cell activation and function.

Despite existing preventive and therapeutic measures, caries remains a ubiquitous infectious disease. Vaccine studies suggest that an adaptive immune response, culminating in effective antibody production, may reduce an individual's susceptibility to caries. However, the efficacy of the immune response elicited by mutans streptococci in the oral cavity remains controversial. A greater understanding of the early stages of the adaptive immune response to cariogenic bacteria may potentially assist therapeutic targeting and design. We therefore sought to characterize dendritic cell (DC) activation and antigen presentation following Streptococcus mutans exposure. We found that S. mutans up-regulated DC expression of co-stimulatory molecules and MHCII in vitro and that DCs effectively processed and presented exogenously administered antigen. These DCs effectively initiated T-cell proliferation, but this was abrogated by live bacteria. The in vitro DC activation effects were not mirrored in vivo, where DCs in draining lymph nodes did not mature following oral exposure to S. mutans. Analysis of these data provides a model for studying antigen uptake from the oral cavity and evidence that, in vitro, S. mutans activates dendritic cells, a critical event for initiating adaptive immunity.

Novel metabolic activity indicator in Streptococcus mutans biofilms.

Antimicrobial resistance of micro-organisms in biofilms requires novel strategies to evaluate the efficacy of caries preventive agents in actual biofilms. Hence we investigated fluorescence intensity (FI) in Streptococcus mutans biofilms constitutively expressing green fluorescent protein (GFP). Upon addition of glucose FI in these biofilms increased significantly to steady state levels. FI-increase could be inhibited by oral care products in a dose-responsive manner. Lactic acid produced in these biofilms was measured at the end of the FI-recording. A linear correlation with a coefficient of 0.96 (p<0.01) was observed between FI-increase and lactate production, irrespective of the inhibitor used. The viability of biofilm cells after chlorhexidine (CHX) titration was also examined. Reduction of FI-increase was observed at low concentrations of CHX whereas a loss in viability was only seen at high concentrations. In conclusion, GFP synthesis can be used as a metabolic activity indicator in S. mutans biofilms.

Caries-preventive agents induce remineralization of dentin in a biofilm model.

The aim was to study remineralization in dentin underneath a biofilm. This was done in a constant depth film fermentor (CDFF) which was modified so that two treatments can be applied simultaneously in one experiment. Forty-five Streptococcus mutans biofilms were grown in grooves in dentin. Growth medium (3.7 g/l BHI, 1.5 mM calcium and 25 mM PIPES) was administered alternately with 2% sucrose pulsing 4 x 30 min/day. Fluoride at 135 ppm as NaF only or in a mixture with 0.2% chlorhexidine was applied for 2 x 5 min/day. The treatments started 5 days after inoculation and lasted 15 days. Five specimens per group were removed at various time points. The biofilms were checked for viability (by plating) and acid content (by capillary electrophoresis). The dentin specimens were analysed for mineral loss and lesion depth (by transversal microradiography). Fluoride treatment had no effect on the viability but reduced lactic acid production by 75%. The mixture treatment reduced the viability by 80% and the lactic acid content by 93% on the first day and later reduced the two parameters to below the detection limits. Significant differences in changes in mineral loss and lesion depth were observed between the treatment groups. Partial remineralization but deeper lesions were observed in the fluoride group, while nearly complete remineralization was seen in the mixture group. In conclusion, the CDFF S. mutans biofilm model can be used as a de- and remineralization biofilm model, and the split mode is particularly suitable for testing caries-preventive agents.

A study of oral health condition in individuals with no oral hygiene and its association with plaque acidogenesis.

OBJECTIVE: To study the association of long-term deposited plaque, due to lack of oral hygiene, with acidogenesis of the plaque bacteria. METHODS: Seventy-seven subjects with poor oral hygiene were selected. Debris index (DI) and calculus index (CI) were recorded. Among them, 16 were DMFS > 8, and comprised the caries active (CA) group; 27 were caries free, and comprised the caries free (CF) group. Plaque fluids in both groups were analyzed for organic acids, phosphate, and inorganic cations by use of capillary electrophoresis, while pH was measured by microelectrodes. RESULTS: No differences were found on debris index (CF group measured 2.07-0.47, CA group measured 2.01-0.53) or calculus index (CF group measured 2.47-0.50, CA group measured 2.48-0.53) relative to carious status, although there was a positive relationship between DI and CI (r = 0.52, P < 0.001). The main finding in this study was that the quantity of lactic acid produced by sucrose exposure in these individuals with poor oral hygiene was much less (increased no more than 2 times, compared with content at rest) than in a previous report (increased 3 to 5 times, compared with content at rest) on subjects with good oral hygiene habits. CONCLUSIONS: Long-term deposited plaque due to lack of oral hygiene may have less cariogenic capability, although patients' susceptibility to periodontal disease would increase.

Demineralization of dentin by Streptococcus mutans biofilms grown in the constant depth film fermentor.

To develop a bacterial demineralization model, we grew Streptococcus mutans biofilms in a constant depth film fermentor (CDFF) and studied the effects of sucrose pulsing frequency (SPF) in time on dentin demineralization. S. mutans biofilms were grown in dentin specimens with grooves and on dentin surface specimens for 20 days. During the experiments, 2% sucrose was pulsed either 4 or 8 times per day for periods of 30 min. Diluted brain-heart infusion medium containing 25 mM PIPES buffer and 1.5 mM CaCl2 was pulsed as the alternative growth medium. Specimens with intact biofilms were taken out on days 5, 12 and 20. The model was assessed by viable counts of the biofilm, mineral loss and lesion depth in the dentin specimens (by transversal microradiography) and pH measurements in the groove (by pH microelectrode). The results showed that biofilms formed on the dentin surface specimens were constant in viable counts for the low SPF, while this parameter tended to increase with time under the high SPF. Lesions with intact surfaces were formed and the lesion size increased significantly over time and increased significantly with increasing SPF. Typical Stephan curves were found after sucrose pulsing. The pH inside the groove returned to neutral under low SPF, but remained below 6.5 under high SPF. With the CDFF S. mutans biofilm model, lesions can be created in dentin within reasonable experimental time periods, as a result of the presence of a biofilm and in response to carbohydrate challenges.