Difference in formation of a dental multi-species biofilm according to substratum direction.

OBJECTIVES: The aim of this study was to investigate the difference in dental biofilm formation according to substratum direction, using an artificial biofilm model. METHODS: A three-species biofilm, consisting of Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, was formed on saliva-coated hydroxyapatite (sHA) discs oriented in three directions: downward (the discs placed in the direction of gravity), vertical (the discs placed parallel to the direction of gravity), and upward (the discs placed in opposite direction of gravity). The biofilms at 22 h and 46 h of age were analyzed using microbiological and biochemical methods, fluorescence-based assays, and scanning electron microscopy to investigate difference in bacterial adhesion, early and mature biofilm formation. RESULTS: The biofilms formed in the upward direction displayed the most complex structure, with the highest number and biovolume of bacteria, as well as the lowest pH conditions at both time points. The vertical and downward directions, however, had only scattered and small bacterial colonies. In the 22-h-old biofilms, the proportion of S. oralis was similar to, or slightly higher than, that of S. mutans in all directions of substratum surfaces. However, in the 46-h-old biofilms, S. mutans became the dominant bacteria in all directions, especially in the vertical and upward directions. CONCLUSIONS: The direction of the substratum surface could impact the proportion of bacteria and cariogenic properties of the multi-species biofilm. Biofilms in an upward direction may exhibit a higher cariogenic potential, followed by those in the vertical and downward directions, which could be related to gravity.

The reciprocal interaction between fluoride release of glass ionomers and acid production of Streptococcus mutans biofilm.

Objectives: The aim of this study was to demonstrate the mode of action of glass ionomers (G-Is) against cariogenic biofilms in the slow fluoride release phase by analyzing the reciprocal interaction between fluoride release from G-Is and acid production of Streptococcus mutans biofilm. Methods: G-Is discs in the slow fluoride release phase were prepared and 51 h-old S. mutans biofilms were formed on these discs. The interrelationship between the acid production of the biofilm and the fluoride release of the G-Is discs was investigated by analyzing both factors simultaneously during the biofilm formation period. The composition of the 51 h-old biofilms was then examined using microbiological, biochemical, and confocal laser scanning microscopic methods. Results: Acid production by the cariogenic biofilm, particularly at < pH 5, promotes G-Is fluoride release. Conversely, G-Is fluoride release inhibits the acid production of the cariogenic biofilm. This reciprocal interaction results in the reduction of virulence such as extracellular polysaccharides formation and cariogenic biofilm bio-mass, which may reduce the potential of secondary caries development around G-Is. Conclusions: These results suggest that G-Is may play a role in preventing the development of secondary caries during the slow fluoride release phase.

Collagen Peptide in a Combinatorial Treatment with Lactobacillus rhamnosus Inhibits the Cariogenic Properties of Streptococcus mutans: An In Vitro Study.

Dental caries is caused by the formation of cariogenic biofilm, leading to localized areas of enamel demineralization. Streptococcus mutans, a cariogenic pathogen, has long been considered as a microbial etiology of dental caries. We hypothesized that an antagonistic approach using a prebiotic collagen peptide in combination with probiotic Lactobacillus rhamnosus would modulate the virulence of this cariogenic biofilm. In vitro S. mutans biofilms were formed on saliva-coated hydroxyapatite discs, and the inhibitory effect of a combination of L. rhamnosus and collagen peptide on S. mutans biofilms were evaluated using microbiological, biochemical, confocal imaging, and transcriptomic analyses. The combination of L. rhamnosus with collagen peptide altered acid production by S. mutans, significantly increasing culture pH at an early stage of biofilm formation. Moreover, the 3D architecture of the S. mutans biofilm was greatly compromised when it was in the presence of L. rhamnosus with collagen peptide, resulting in a significant reduction in exopolysaccharide with unstructured and mixed bacterial organization. The presence of L. rhamnosus with collagen peptide modulated the virulence potential of S. mutans via down-regulation of eno, ldh, and atpD corresponding to acid production and proton transportation, whereas aguD associated with alkali production was up-regulated. Gly-Pro-Hyp, a common tripeptide unit of collagen, consistently modulated the cariogenic potential of S. mutans by inhibiting acid production, similar to the bioactivity of a collagen peptide. It also enhanced the relative abundance of commensal streptococci (S. oralis) in a mixed-species biofilm by inhibiting S. mutans colonization and dome-like microcolony formation. This work demonstrates that food-derived synbiotics may offer a useful means of disrupting cariogenic communities and maintaining microbial homeostasis.

Relationship between sucrose concentration and bacteria proportion in a multispecies biofilm: Short title: Sucrose challenges to a multispecies biofilm.

Objective: The aim of this study was to evaluate the relationship between sucrose concentration and bacteria proportion in a multispecies biofilm model. Methods: Streptococcus mutans (S. mutans), Streptococcus oralis (S. oralis), and Actinomyces naeslundii (A. naeslundii) were chose to form a multispecies biofilm. Different concentration (0-40%) of sucrose was introduced to the multispecies biofilm 3 times per day (30 min per time). And then the bacteria proportion and acid production of the biofilms were analyzed. Results: Increasing sucrose level increased CFU count of S. mutans up to a certain concentration (5% sucrose), after which the number of S. mutans slightly decreased, but the CFU counts of S. oralis and A. naeslundii continually decreased with sucrose concentration increase, especially, from 5% sucrose, the reduction was significant, and S. mutans became the dominant species in the biofilms. Furthermore, the acid production ability of the multispecies biofilm gradually increased and slightly decreased with sucrose concentration increased, and the turning concentration was 5%. Conclusion: Our findings suggest that increasing sucrose level could increase the competitiveness of S. mutans in the multispecies biofilm, which may shift the biofilm to a more cariogenic one, and 5% sucrose formed a most cariogenic biofilm in this study.

Chronologic trends of cancer-related lymph node research in PubMed: informetrics analysis.

PURPOSE: Given the long history of investigation into cancer and its relevance to the lymph node (LN), it would be meaningful to plot the trends of research on cancer-related LN. METHODS: Queries such as "cancer," "lymph node," and "cancer and lymph node" were submitted to PubMed to collect articles on cancer and LN published between 1945 and 2017. The collected articles were then extracted by an automatic web crawler and examined through informetrics and linguistic analysis. RESULTS: The number of articles related to cancer was 2,795,476 and 127,897 articles (4.6%) were found to be relevant to LN. With regard to cancer types, breast cancer was the most studied (37%), followed by gastric cancer (17%). With regard to the subjects in which the surgeon is interested, LN metastasis (57%) was found to be the topic most discussed, followed by LN dissection (22%) and sentinel LN (17%). Publications on LN metastasis gradually increased over time from 1988 to 2017 although those on sentinel LN and LN dissection have stagnated since the early 2000s. CONCLUSION: Although research on cancer was abundant, only a small portion was dedicated to investigating its relevance to LN. Western countries had led the research on cancer-related LN, but Asian countries began to participate as major players, expanding their contributions. While LN metastasis, one of the major cancer-related LN topics, showed a steady increase, those involved in oncologic surgery such as LN dissection and sentinel LN did not.

The Effect of Probiotics on Halitosis: a Systematic Review and Meta-analysis.

Although several studies have evaluated the inhibitory effect of probiotics on halitosis, findings are inconsistent. This systematic review and meta-analysis of randomized clinical trials (RCT) was conducted to summarize the evidence on the effect of probiotics on halitosis. RCT on any type of probiotic treatment with at least 2-week duration were identified through electronic databases (PubMed, EMBASE, Web of Science, and the Cochrane Central Register of Controlled Trials) and hand searched between 1946 and January 17, 2017. Primary outcomes were organoleptic (OLT) scores and volatile sulfur compounds (VSC). Standardized mean difference (SMD) and 95% confidence interval (CI) were calculated. Meta-analysis was conducted to synthesize the evidence. Of the 153 articles identified, three met the inclusion criteria. Meta-analysis revealed that OLT scores (SMD = - 1.93, 95% CI - 2.85 to - 1.02, P < 0.0001) were significantly lower in subjects who received probiotics than in placebo groups, but no significant difference was observed at the VSC concentration (SMD = - 0.02, 95% CI - 2.12 to 2.07, P = 0.98). Current evidence is supportive of recommending probiotics for the management of halitosis. Based on this review, transient (average of 2 weeks) dosing with probiotics (mainly Lactobacillus strains) has a moderate effect on halitosis regarding OLT scores, but we could not confirm the effects of probiotics on the VSC reduction. The available evidence is quantitatively and qualitatively insufficient for further recommendations, especially with regard to administration strategies and pretreatment. Future studies should aim for longer follow-up and standardized administration methods to prove or refute the effect of probiotics on halitosis.

Chitosan-gold nanoparticles mediated gene delivery of c-myb facilitates osseointegration of dental implants in ovariectomized rat.

Osseointegration of dental implants is affected by osteoporosis. The purpose of this study was overcome the implant failure and facilitate the osseointegration of dental implants by c-myb in ovariectomized (OVX)-induced osteoporosis. c-myb is a transcription factor and supports bone formation. Plasmid DNA/c-myb conjugated with chitosan-gold nanoparticles (Ch-GNPs/c-myb) promoted osteogenesis and inhibited osteoclastogenesis in MC-3T3 E1 cells. Ch-GNPs/c-myb involved the reduction of the nuclear factor of activated T-cells 1, c-Fos, and tartrate-resistant acid phosphatase-positive multinucleated osteoclasts in receptor activator of nuclear factor-κB ligand (RANKL) stimulated bone marrow macrophages. In vivo results of rat mandibles demonstrated Ch-GNP/c-myb-coated titanium (Ti) implants increased the volume and density of newly formed bone and the osseointegration of dental implant with bone by micro computed tomography examination after OVX-induced osteoporosis. Immunohistochemical analysis showed increased c-myb expression and upregulation of bone morphogenic proteins, osteoprotegerin and EphB4, as well as the downregulation of RANKL by Ch-GNP/c-myb-coated Ti implants. Hematoxylin and Eosin staining expressed new bone formation by Ch-GNP/c-myb-coated Ti implants. Our findings indicated that c-myb delivered by Ch-GNPs supports osseointegration of dental implant even in osteoporotic condition. c-myb may be applicable to support dental implant integration and treatment in age-dependent bone destruction disease.

Sucrose challenges to Streptococcus mutans biofilms and the curve fitting for the biofilm changes.

The relationship between sugar level and development of dental caries has long been a main topic in dentistry. However, as a ubiquitous component of the modern diet, sucrose is mainly derived from three meals a day, rather than a long time exposure. In this study, various concentrations of sucrose were provided to Streptococcus mutans biofilms for 1 h per exposure (three times per day) to imitate a human meal pattern. And then the relationship between sucrose concentration and changes in the treated biofilms was determined. The results indicated that the components and acid production of the treated biofilms changed in a second-order polynomial curve pattern with sucrose concentration increase, which were confirmed by CLSM and SEM analyses. However, gene expression related to extracellular polysaccharides (EPS) formation, acid production and tolerance was up-regulated with sucrose concentration increase, which might have been due to compensation for the decrease in EPS formation and acid production by the biofilms at higher concentrations of sucrose. These findings suggest that sucrose in the range of 1%-5% can support the highest acid production and accumulation of S. mutans biofilms, which may further increase its cariogenic potential. However, additional studies are required to confirm the relationships in human cariogenic biofilms.

Association Between Breastfeeding and Childhood Breathing Patterns: A Systematic Review and Meta-Analysis.

OBJECTIVES: This systematic review aimed to evaluate the association between breastfeeding and breathing patterns in children. MATERIALS AND METHODS: We searched PubMed, EMBASE, Web of Science, CINAHL, and the Cochrane Central Register of Controlled Trials for publications from inception to October 1, 2017. The breastfeeding duration and period of exclusive breastfeeding were selected as primary outcomes. The odds ratio (OR) and 95% confidence interval (CI) were calculated. A meta-analysis was conducted to synthesize the evidence. The Newcastle-Ottawa Score was used for quality and comparability assessment. RESULTS: Of the 54 articles identified, three observational studies met the inclusion criteria for this meta-analysis, representing 1,046 participants. The results of the meta-analysis revealed that the prevalence rate of mouth breathing (OR = 2.04; 95% CI, 1.26-3.31; p = 0.004) was significantly higher in subjects who were breastfed for less than 6 months, but no significant difference was observed within the periods of exclusive breastfeeding (6 months or less) (OR = 1.27; 95% CI, 0.73-2.21; p = 0.40). CONCLUSION: We found only limited evidence about the association between breastfeeding and breathing patterns. However, the current evidence supports the association between breastfeeding and childhood breathing patterns. Based on this review, we found that the frequency of normal nasal respiration increases with the duration of breastfeeding. The methodological quality of the studies included was moderate. Thus, future studies should aim to correct the confounding factors related to breathing patterns, to use standardized diagnostic criteria of mouth breathing, and to conduct a prospective research to reduce the recall bias.

Trend Analysis of Studies on Water Fluoridation Related to Dental Caries in PubMed.

Water fluoridation has been cited as one of the top 10 public health achievements of the 20th century. Herein, we analyzed water fluoridation articles related to dental caries published in PubMed between 1950 and 2016 using informetrics and linguistic methods to investigate trends in the studies. To this aim, queries such as "dental caries and (water fluoridation or fluoridated water)," "dental caries and (fluoride or fluoridation)," and "dental caries" were submitted to PubMed to retrieve information about articles on water fluoridation within the area of dental caries and fluoride - their titles, abstracts, publication dates, author affiliations, and publication journals. This article information was then collected by an automatic web crawler and examined through informetrics and linguistic analyses. It was found that the number of articles concerned with water fluoridation and dental caries was 3,381 and declined over time after 1970. The articles were published by 750 journals - most notably, Community Dentistry and Oral Epidemiology and Caries Research. With regard to the geographic distribution of the authors, Europe and North America, especially the USA and UK, accounted for 59.9% of the articles published during the years 1987 to 2015, though there was a sharp increase in the number of authors in Oceania and Asia in recent years. In the titles and abstracts of the articles, "community" and "fluorosis" were mentioned more frequently than the other key terms selected in this study, regardless of the period examined. Our findings may allow one to assess how the research on water fluoridation has evolved over the past several decades.

Phelligridin D-loaded oral nanotube titanium implant enhances osseointegration and prevents osteolysis in rat mandible.

Poor bone quality and osteolysis are the major causes of implant failure in dentistry. Here, this study tested the effect of phelligridin D-loaded nanotubes titanium (Ti) for bone formation around the dental implants. The purpose of this study was to enhance osseointegration of phelligridin D-loaded implant into the bone for bone formation and prevention of osteolysis. Cell viability, crystal violet staining, Western blot, alizarin red S staining, alkaline phosphatase activity, tartrate-resistant acid phosphatase staining, micro-computed tromography (µ-CT), hematoxylin and eosin (H&E) and immunohistochemical staining were used in vitro and in vivo to test the biocompatibility of phelligridin D. Phelligridin D enhanced osteoblast differentiation and mineralization by increasing bone morphogenic protein-2/7 (BMP-2/7), Osterix, Runx-2, osteoprotegerin (OPG), alkaline phosphatase and inhibited osteoclast differentiation by decreasing receptor activator of nuclear factor kappa-B ligand (RANKL) in MC-3T3 E1 cells. Further, phelligridin D promoted bone regeneration around nanotube Ti implant surface by increasing the levels of BMP-2/7 and OPG in a rat model. Phelligridin D also inhibited osteolysis by suppressing the expression of RANKL. These findings strongly suggest that phelligridin D is a new compound representing a potential therapeutic candidate for implant failure caused by osteolysis and poor bone quality of teeth.

Difference in virulence and composition of a cariogenic biofilm according to substratum direction.

The aim of this study was to investigate the difference in composition and virulence of Streptococcus mutans biofilms according to substratum direction. S. mutans biofilms (46-h-old) were formed on three different saliva-coated hydroxyapatite (sHA) disc direction groups: downward (discs placed in the direction of gravity), vertical (discs placed parallel to gravity direction), and upward (discs placed opposite to gravity). The 46-h-old biofilms on sHA discs in the upward direction showed the highest biofilm accumulation, colony forming unit (CFU) count, and extracellular polysaccharide (EPS) amount, followed by those in the vertical and downward directions. In the confocal laser scanning microscopy (CLSM) study, the biofilms in the upward direction also showed the highest bacterial count (live or dead cells) and EPS biovolume. Scanning electron microscopy (SEM) analysis confirmed the microbiological and biochemical results. In addition, biofilm density and acid production were higher in the upward direction than those in the other directions. Our findings suggest that substratum direction, which might be related to gravity, strongly influences the formation and virulence of cariogenic biofilms and subsequent initiation of dental caries. Collectively, the differences in the formation and virulence of cariogenic biofilms are related to the direction of tooth surface (occlusal surfaces of mandibular teeth > proximal surfaces > occlusal surfaces of maxillary teeth).

Effect of brief periodic fluoride treatments on the virulence and composition of a cariogenic biofilm.

The present study investigated the effect of periodic 1-min fluoride treatments on Streptococcus mutans biofilms and then determined the relationship between anti-biofilm activity, treatment frequency, and fluoride concentration using a linear-fitting procedure. S. mutans biofilms were periodically treated (1-min/treatment) with fluoride during biofilm formation and analyzed using microbiological methods, confocal microscopy, and real-time PCR. The results indicated that reductions in the dry weight and acidogenicity of biofilms due to periodic fluoride treatment occurred in a concentration dependent manner. The reduction in dry weight without affecting bacterial cell viability was observed mainly due to the inhibitory effect of fluoride on gtfB and gtfC gene expression, which suppresses EPS production and avoids reduction of the pH below the critical point on the tooth surface. This study suggests that brief periodic exposure to appropriate fluoride concentrations through mouthwashes and toothpastes may affect the virulence and composition of cariogenic biofilms and subsequently prevent dental caries.

Nanoparticle mediated PPARγ gene delivery on dental implants improves osseointegration via mitochondrial biogenesis in diabetes mellitus rat model.

Diabetes mellitus (DM) has a detrimental effect on osseointegration, stability and longevity of implants due to osteoporosis. In this study, PPARγ-loaded dental implants were investigated for the improvement of osseointegration and peri-implantitis. Chitosan gold nanoparticles conjugated with PPARγ cDNA were introduced on titanium mini-implant surfaces for PPARγ release to rat mandibular. DM-induced rat mandible showed structural changes such as decreased bone mass and increased inflammatory molecules, and diminution of PPARγ expression and bone formation molecules compared to normal rats. PPARγ induced bone formation via reduction of inflammatory molecules even under glucose oxidative stress. Furthermore, PPARγ strongly activated mitochondrial biogenesis and cell viability via p-AMK and Wnt/ß-catenin signaling. Consequently, PPARγ gene delivery on regional dental implants contributed osseointegration, new bone formation and mineralization in DM-induced rats. This study demonstrates that PPARγ can be used as a therapeutic gene with dental implantation in diabetic patients since regional PPARγ expression enhances osseointegration and implant longevity.

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis.

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.

Recovery of Acid Production in Streptococcus mutans Biofilms after Short-Term Fluoride Treatment.

Fluoride is commonly used as an ingredient of topical oral hygiene measures. Despite the anti-acidogenic activities of fluoride against cariogenic biofilms, the recovery of the biofilms from fluoride damage is unclear. Herein, we investigated the recovery of acid production in Streptococcus mutans biofilms after short-term or during periodic 1-min fluoride treatments. For this study, 46-hour-old S. mutans biofilms were treated with fluoride (0-2,000 ppm F-) for 1-8 min and then incubated in saliva for 0-100 min. The 74-hour-old biofilms were also periodically treated with the fluoride concentration during biofilm formation (1 min/treatment). Changes in acidogenicity and viability were determined via pH drop and colony-forming unit assays, respectively. In this study, acid production after a 1-min fluoride treatment was recovered as saliva incubation time increased, which followed a linear pattern of concentration dependence (R = 0.99, R2 = 0.98). The recovery pattern was in a biphasic pattern, with an initial rapid rate followed by a second slow recovery. Furthermore, recovery from fluoride damage was retarded in a concentration-dependent manner as treatment time increased. In periodic 1-min fluoride treatments, acid production in the biofilms was not diminished during the non-fluoride treatment period; however, it was reduced in a concentration-dependent manner during the fluoride treatment period. The viability of the biofilm cells did not change, even at high fluoride concentrations. Collectively, our results suggest that brief fluoride treatment does not sustain anti-acidogenic activity against S. mutans in biofilms since the damage is recoverable with time.

Functional Relationship between Sucrose and a Cariogenic Biofilm Formation.

Sucrose is an important dietary factor in cariogenic biofilm formation and subsequent initiation of dental caries. This study investigated the functional relationships between sucrose concentration and Streptococcus mutans adherence and biofilm formation. Changes in morphological characteristics of the biofilms with increasing sucrose concentration were also evaluated. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs in culture medium containing 0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, or 40% (w/v) sucrose. The adherence (in 4-hour biofilms) and biofilm composition (in 46-hour biofilms) of the biofilms were analyzed using microbiological, biochemical, laser scanning confocal fluorescence microscopic, and scanning electron microscopic methods. To determine the relationships, 2nd order polynomial curve fitting was performed. In this study, the influence of sucrose on bacterial adhesion, biofilm composition (dry weight, bacterial counts, and water-insoluble extracellular polysaccharide (EPS) content), and acidogenicity followed a 2nd order polynomial curve with concentration dependence, and the maximum effective concentrations (MECs) of sucrose ranged from 0.45 to 2.4%. The bacterial and EPS bio-volume and thickness in the biofilms also gradually increased and then decreased as sucrose concentration increased. Furthermore, the size and shape of the micro-colonies of the biofilms depended on the sucrose concentration. Around the MECs, the micro-colonies were bigger and more homogeneous than those at 0 and 40%, and were surrounded by enough EPSs to support their structure. These results suggest that the relationship between sucrose concentration and cariogenic biofilm formation in the oral cavity could be described by a functional relationship.

The antibacterial activity of chlorhexidine digluconate against Streptococcus mutans biofilms follows sigmoidal patterns.

The aim of this study was to determine the pattern of the antibacterial activity of chlorhexidine digluconate (CHX) against mature Streptococcus mutans biofilms. Streptococcus mutans biofilms were formed on saliva-coated hydroxyapatite discs and then treated with 0-20% CHX, once, three times, or five times (1 min per treatment) during the period of mature biofilm formation (beyond 46 h). After the treatments, the colony-forming unit (CFU) counts of the treated biofilms were determined. The pH values of the spent culture medium were also determined to investigate the change in pH resulting from the antibacterial activity of CHX. The relationships between the concentration of CHX and the CFU counts and the concentration of CHX and culture medium pH, relative to the number of treatments performed, were evaluated using a sigmoidal curve-fitting procedure. The changes in CFU counts and culture medium pH followed sigmoidal curves and were dependent on the concentration of CHX (R2 = 0.99). The sigmoidal curves were left-shifted with increasing number of treatments. Furthermore, the culture-medium pH of the treated biofilms increased as their CFU counts decreased. The lowest CHX concentration to increase culture-medium pH above the critical pH also decreased as the number of treatments increased. These results may provide fundamental information for selecting the appropriate CHX concentrations to treat S. mutans biofilms.

Long-term anti-cariogenic biofilm activity of glass ionomers related to fluoride release.

OBJECTIVES: The aim of this study was to evaluate the difference between anti-cariogenic biofilm activities of glass ionomers (G-Is) during the initial and second fluoride release phases and to define relationships between the anti-biofilm activities and fluoride release. METHODS: Fluoride release of three commercially available G-Is in a buffer was evaluated for 770 h, and then 70-h-old Streptococcus mutans UA159 biofilms were formed on the G-Is that had been immersed in the buffer for 0, 100, 200, or 700 h. The dry weight, bacterial cell number, water-insoluble extracellular polysaccharides (EPSs), and accumulated fluoride concentration of the 70-h-old biofilms and fluoride release and acid production rates during biofilm formation were determined. Relationships between the experimental variables and fluoride release rate were also evaluated using linear regression analysis. RESULTS: In this study, fluoride release of the tested G-Is did not exhibit a biphasic pattern during biofilm formation. The release was sustained or did not rapidly decrease even over long immersion periods and was strongly correlated with an increase in accumulated fluoride concentration of the biofilms (R=0.99, R(2)=0.98) and reductions in dry weight, water-insoluble EPSs, and acid production rate of the biofilms (R=-0.99 to -0.96, R(2)=0.92-0.98). CONCLUSIONS: This study suggests that G-Is can effectively affect acid production, EPS formation, and accumulation of cariogenic biofilms even during the second fluoride release phase, and that the anti-cariogenic biofilm activity is strongly correlated with fluoride release, which may be enhanced by acid production of cariogenic biofilms. CLINICAL SIGNIFICANCE: G-Is can affect cariogenic biofilm formation even during the second fluoride release phase.

Anticancer activity of Ashwagandha against human head and neck cancer cell lines.

BACKGROUND: The aim of this study was to determine the apoptotic activity of methanol extract of Ashwagandha (MEAG) and in human head and neck squamous cell carcinoma (HNSCC) cells and to investigate the underlying mechanisms. METHODS: We investigated the effects of MEAG on programmed cell death in HNSCC cells using a Live/Dead assay, detection of nuclear morphologic changes, Mitotracker, siRNA knockdown, and RT-PCR. RESULTS: Treatment with MEAG showed dose-dependent growth-inhibitory activity that attribute to caspase-dependent apoptosis. Loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase 9 suggested that MEAG leads to activation of mitochondria-mediated apoptosis. MEAG selectively upregulated the expression of Bim protein at the transcriptional level and induced the translocation of Bim into the mitochondria. Knockdown of Bim by siRNA partially blocked MEAG-mediated apoptosis. MEAG also caused an increase in truncated Bid (t-Bid), cleaved caspase-8, and death receptor 5 (DR5). Interestingly, withaferin A (WA), a bioactive component of MEAG, clearly induced apoptosis accompanied by upregulation of Bim, t-Bid, caspase-8, and DR5 similar to the effects of MEAG. CONCLUSIONS: These suggest that MEAG and WA may be potential natural materials for the treatment of HNSCC.