Metabolomics reveals high fructose-1,6-bisphosphate from fluoride-resistant Streptococcus mutans.

BACKGROUND: Fluoride-resistant Streptococcus mutans (S. mutans) strains have developed due to the wide use of fluoride in dental caries prevention. However, the metabolomics of fluoride-resistant S. mutans remains unclear. OBJECTIVE: This study aimed to identify metabolites that discriminate fluoride-resistant from wild-type S. mutans. MATERIALS AND METHODS: Cell supernatants from fluoride-resistant and wild-type S. mutans were collected and analyzed by liquid chromatography-mass spectrometry. Principal components analysis and partial least-squares discriminant analysis were performed for the statistical analysis by variable influence on projection (VIP > 2.0) and p value (Mann-Whitney test, p < 0.05). Metabolites were assessed qualitatively using the Human Metabolome Database version 2.0 ( http://www.hmdb.ca ), or Kyoto Encyclopedia of Genes and Genomes ( http://www.kegg.jp ), and Metaboanalyst 6.0 ( https://www.metaboanalyst.ca ). RESULTS: Fourteen metabolites differed significantly between fluoride-resistant and wild-type strains in the early log phase. Among these metabolites, 5 were identified. There were 32 differential metabolites between the two strains in the stationary phase, 13 of which were identified. The pyrimidine metabolism for S. mutans FR was matched with the metabolic pathway. CONCLUSIONS: The fructose-1,6-bisphosphate concentration increased in fluoride-resistant strains under acidic conditions, suggesting enhanced acidogenicity and acid tolerance. This metabolite may be a promising target for elucidating the cariogenic and fluoride resistant mechanisms of S. mutans.

Possible interplay of diabetes mellitus and thyroid diseases in oral lichen planus: A pooled prevalence analysis.

The existence of a comorbidity between diabetes mellitus (DM) as well as between thyroid diseases (TD) and oral lichen planus (OLP), respectively, was substantially demonstrated. However, there is not enough attention to the concurrent status of both TD and DM in OLP patients. Herein, this short communication aimed to compare 1) the prevalence of DM when TD was concurrently investigated and that of DM when TD status was ignored; 2) the prevalence of TD when DM was concurrently investigated and that of TD when DM status was ignored in the studies. The pooled prevalence (9.86 %; 95 % confidence intervals [CI], 9.22-10.53 %) of DM when TD was concurrently investigated was significantly higher than that (8.13 %; 95%CI, 8.03-9.12 %) when TD status was not investigated in OLP patients. The pooled prevalence (12.48 %; 95%CI, 11.77-13.22 %) of TD when DM was concurrently investigated was significantly higher than that (10.45 %; 95%CI, 9.52-11.46 %) when DM status was not investigated in OLP patients. Thus, it is logical to presume for the first time that there is possible interplay of DM and TD in OLP occurrence. TD and DM should serve as important confounding factors each other in clinical investigation on OLP and associated comorbidities.

Chemopreventive efficacy of salvianolic acid B phospholipid complex loaded nanoparticles against experimental oral carcinogenesis: implication of sustained drug release.

Background: Although we have previously demonstrated that phospholipid complex loaded nanoparticles (PLC-NPs) encapsulating salvianolic acid B (SAB) can enhance anticancer activity in head and neck cancer and precancerous cells in vitro, the chemopreventive efficacy of SAB-PLC-NPs (nano-SAB) in vivo remains unclear. Here, we aimed to investigate the in vivo efficacy of nano-SAB against experimental oral carcinogenesis. Methods: Oral tongue carcinogenesis was induced in C57BL/6 mice through the administration of 4-nitroquinoline-N-oxide (4NQO, 100 µg/mL) in drinking water for 22 weeks. To preliminarily evaluate the effect of sustained drug release against oral carcinogenesis, free- or nano-SAB (16.6 mg/kg/d) was administered orally for 18 weeks, and the treatment was discontinued for the remaining 4 weeks. Results: Histological evaluation revealed a significant (P<0.05) decrease in the incidence of carcinoma in free-SAB-treated (16.7%) and nano-SAB-treated (10.0%) mice compared to mice exposed to 4NQO alone (34.3%). A decrease in carcinoma growth rate was also observed in free-SAB-treated (12.2%) and nano-SAB-treated (5.5%) mice compared to the 4NQO-exposed group (18.3%), even after drug withdrawal for 4 weeks. Immunohistochemical analysis revealed that nano-SAB treatment effectively suppressed Ki-67, proliferative cell nuclear antigen (PCNA), and cyclin D1 expression in high-risk dysplastic lesions compared to free-SAB-treated and 4NQO-exposed groups (all P<0.05). Importantly, nano-SAB maintained low levels of Ki-67, PCNA, and cyclin D1 expression even after drug withdrawal for 4 weeks. Conclusions: Together with our previous in vitro data, this in vivo study confirms that nano-SAB has superior chemopreventive efficacy by promoting more potent anti-proliferation and cell cycle arrest responses. These findings demonstrate the potential of SAB-PLC-NPs as promising chemopreventive agents for treating oral carcinogenesis.

Highly sensitive and portable mRNA detection platform for early cancer detection.

Pancreatic cancer, at unresectable advanced stages, presents poor prognoses, which could be prevented by early pancreatic cancer diagnosis methods. Recently, a promising early-stage pancreatic cancer biomarker, extracellular vesicles (EVs) related glypican-1 (GPC1) mRNA, is found to overexpress in pancreatic cancer cells. Current mRNA detection methods usually require expensive machinery, strict preservation environments, and time-consuming processes to guarantee detection sensitivity, specificity, and stability. Herein, we propose a novel two-step amplification method (CHAGE) via the target triggered Catalytic Hairpin Assembly strategy combined with Gold-Enhanced point-of-care-testing (POCT) technology for sensitive visual detection of pancreatic cancer biomarker. First, utilizing the catalyzed hairpin DNA circuit, low expression of the GPC1 mRNA was changed into amplification product 1 (AP1, a DNA duplex) as the next detection targets of the paper strips. Second, the AP1 was loaded onto a lateral flow assay and captured with the gold signal nanoparticles to visualize results. Finally, the detected results can be further enhanced by depositing gold to re-enlarge the sizes of gold nanoparticles in detection zones. As a result, the CHAGE methodology lowers the detection limit of mRNA to 100 fM and provides results within 2 h at 37 °C. Furthermore, we demonstrate the successful application in discriminating pancreatic cancer cells by analyzing EVs' GPC1 mRNA expression levels. Hence, the CHAGE methodology proposed here provides a rapid and convenient POCT platform for sensitive detection of mRNAs through unique probes designs (COVID, HPV, etc.).

Effect of TNF-α on the proliferation and osteogenesis of human periodontal mesenchymal stem cells.

The aim of the present study was to investigate the effect of tumor necrosis factor-α (TNF-α) on the proliferation and osteogenesis of human periodontal mesenchymal stem cells (hPDLSCs). Antigen expression in hPDLSCs was detected by flow cytometry. hPDLSCs were divided into four groups: A control group with no TNF-α treatment, and three experimental groups treated with 0.1, 1 and 10 ng/ml TNF-α, respectively. The effect of TNF-α on proliferation of hPDLSCs in vitro was detected using a Cell Counting Kit-8 assay. Differentiation into an osteogenic lineage was detected by alkaline phosphatase sand alizarin red staining, and the mRNA and protein expression levels of runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and type I collagen (Col-I) were detected using reverse transcription-quantitative PCR and western blot respectively. Following treatment with 10 ng/ml TNF-α, proliferation was significantly increased compared with an untreated control group (P<0.01). Additionally, there was a significant inhibition of alkaline phosphatase enzyme activity, alizarin red mineralization node size, and in the gene and protein expression levels of osteogenic differentiation markers, including Runx2, OCN and COL-I (all, P<0.05). Taken together, the results indicated that treatment with 10 ng/ml TNF-α promoted the proliferation of hPDLSCs in vitro and inhibited osteogenic differentiation of hPDLSCs, providing an experimental basis for regulation of hPDLSC-mediated periodontal tissue regeneration.

[Effect of different root canal file tapers on fracture strength of root canal wall].

PURPOSE: To compare the effects of different nickel-titanium rotary tapers on fracture strength of root canal and the application value of finite element analysis in root canal therapy. METHODS: Twenty four mandibular premolars were selected and all crowns were removed. All samples were randomly divided into 4 groups: A, B, C and D. Group A and C were instrumented with Z-K3 nickel titanium file as 0.04 taper for root canal preparation. Group B and D were instrumented with Z-K3 nickel titanium file as 0.06 taper. Group C and D were sliced into 2 mm thick slices 3 times after root canal preparation. Two teeth were selected from group A and group B and scanned with micro-CT. The model was imported into different softwares to optimize. The assembly model was built and imported into ANSYS for finite element analysis. Universal testing machine and force bar were used to apply 100 N force to samples until fracture occurring at any place of tooth sample. The fracture load was recorded and comparative analysis was conducted. RESULTS: The results of finite element analysis showed that the maximum stress of 0.04, 0.06 taper models was 138.88 MPa and 78.812 MPa, respectively. The average fracture load of group A, B was 490.12 N and 501.83 N, respectively. In group C and D, the maximum average fracture load was 93.61 N and 141.53 N, respectively. From the neck to the middle and then to the tip, the average fracture load of root canal decreased in turn. CONCLUSIONS: For normal form mandibular premolars, the fracture strength of the root instrumented with 0.06 taper nickel titanium rotary file was significantly higher than that instrumented with 0.04 taper. Three dimensional finite element analysis could guide dentists to choose a suitable taper file in root canal treatment to reduce the incidence of root fracture.

Comparison between two post-dentin bond strength measurement methods.

The push-out (PO) test and the diametral compression (DC) test were performed to compare the merits of two post-dentin bond strength measurement methods. Compared with the push-out test, the disk in DC provided post-dentin bond strength measurements that were more precise. The load-displacement curves from the DC test were much smoother and more linear up to the point of fracture when compared to those from the PO test. Compared to the PO test, DC is easier to perform for determining the bond strength between posts and dentin. No specimen alignment is needed in the DC test, and it produces a smaller standard deviation in the measured bond strength. The main disadvantage of the DC test, however, is that finite element analysis (FEA) is required to calculate the bond strength. The shear bond strength given by the PO test based on the simple formula is not valid, though, and the peak failure load is dependent on friction at the post-dentin interface.

Calibration of a lactic-acid model for simulating biofilm-induced degradation of the dentin-composite interface.

OBJECTIVE: To verify and calibrate a chemical model for simulating the degradation of the dentin-composite interface induced by multi-species oral biofilms in vitro. METHODS: Dentin-composite disks (5-mm dia.×2-mm thick) were made from bovine incisor roots and filled with either Z100™ (Z100) or Filtek™ LS (LS) composite. The disks, which were covered with nail varnish, but with one of the dentin-composite margins exposed, were immersed in lactic acid solution at pH 4.5 for up to 48h. Diametral compression was performed to measure the reduction in bond strength of the dentin-composite disks following acid challenge. Scanning electron microscopy (SEM) was used to examine decalcification of dentin and fracture modes of the disks. To better understand the degradation process, micro-computed tomography, in combination with a radiopaque dye (AgNO3), was used to assess interfacial leakage in 3D longitudinally, while SEM was used to determine the path of leakage. One-way analysis of variance (ANOVA) was used to analyze the results, with the level of statistical significance set at p<0.05. The results were compared with those obtained previously using multi-species biofilms for verification and calibration purposes. RESULTS: After 48h of acid challenge, the debonding load of both the LS- and Z100-filled disks reduced significantly (p<0.05). In the Z100-filled disks, debonding mostly occurred at the adhesive-dentin interface, while in the LS-filled disks, this happened at the adhesive-composite interface, instead. The degree of dentin demineralization, the reduction in debonding load and the modes of failure observed were very similar to those induced by multi-species oral biofilms found in the previous work. Leakage of AgNO3 occurred mainly along the hybrid layer. The specimens filled with Z100 had a thicker hybrid layer (∼6.5µm), which exhibited more interfacial leakage than those filled with LS. SIGNIFICANCE: The chemical model with lactic acid used in this study can induce degradation to the dentin-composite interface similar to those produced by multi-species biofilms. With appropriate calibration, this could provide an effective in vitro method for ageing composite restorations in assessing their potential clinical performance.

Prevalence of developmental odontogenic cysts in children and adolescents with emphasis on dentigerous cyst and odontogenic keratocyst (keratocystic odontogenic tumor).

OBJECTIVE: To investigate the incidence and prevalence of developmental odontogenic cysts in children and adolescents and compare the features of the two most common types, dentigerous cyst and keratocystic odontogenic tumor (KCOT). STUDY DESIGN: A retrospective review in a series of 369 patients with all histological diagnoses of developmental odontogenic cysts in children (≤12 years) and adolescents (13-18 years) was conducted. RESULTS: Among these, 361 (97.8%) patients were diagnosed as dentigerous cyst (n = 281) and KCOT (n = 80), with the male-to-female ratios of dentigerous cyst and KCOT both being 2:1. The average age of the patients with KCOT was older than that of those with dentigerous cyst (14.7 years vs 11.8 years, p < 0.001). Dentigerous cyst (59.1%) was more common in children, but KCOT (78.8%) was more common in adolescents (p < 0.001). Dentigerous cyst (57.6%) predominantly located on the maxilla, but KCOT (60.3%) predominantly located on the mandible (p = 0.010). CONCLUSIONS: Adolescent patients with lesions located on the mandible would favor KCOT over dentigerous cyst. This study aids in better knowledge of the prevalence of developmental odontogenic cysts in a large pediatric population, and shows that a well-supported early diagnosis is indispensable for a more adequate treatment.

An in vitro comparison of fluorescence-aided caries excavation and conventional excavation by microhardness testing.

OBJECTIVE: The aim of this in vitro study was to compare fluorescence-aided caries excavation with conventional excavation based on the Martens and Vickers hardness of dentin at the cavity floor after caries removal. MATERIALS AND METHODS: In total, 20 extracted human teeth with dentin caries were bisected through the lesion center into two halves, which were assigned to either the fluorescence-aided caries excavation group or the conventional excavation group. After the treatment, embedding, mounting, and polishing, a line of indentations from the dental pulp across the sound dentin to the cavity floor was made on each sample. The data were compared with Student's t and Mann-Whitney U tests. RESULTS: The calculated Vickers hardness of the sound dentin was 57 ± 10 kg/mm(2) in the fluorescence-aided caries excavation group and 59 ± 8 kg/mm(2) in the conventional excavation group, which is consistent with the previous studies. The absolute and relative Martens hardness measurements of the cavity floor were 224 ± 93 N/mm(2) and 46 ± 17%, respectively, in the fluorescence-aided caries excavation group and 412 ± 75 N/mm(2) and 81 ± 14%, respectively, in the conventional excavation group. Based on either the Martens or Vickers hardness, both the absolute and relative microhardness measurements of the cavity floor after fluorescence-aided caries excavation were significantly lower than the values obtained by conventional excavation. CONCLUSION: Fluorescence-aided caries excavation showed the tissue-preserving property and was more conservative than the conventional excavation in this in vitro study.

An in vitro study on the secondary caries-prevention properties of three restorative materials.

STATEMENT OF PROBLEM: Secondary caries is considered to be the most important and common reason for the replacement of all types of restorations. PURPOSE: The purpose of the study was to evaluate in vitro both the anticaries efficacy and marginal integrity of amalgam, glass ionomer cement, and composite resin restorations in a cariogenic condition. MATERIAL AND METHODS: Class II cavities were prepared on 60 extracted teeth, which were assigned to 3 groups and restored either with amalgam, glass ionomer, or composite resin. After thermal cycling and sterilization, the teeth were set in an artificial carious environment for 10 days. The bacteria from the margin of the restorations were then collected, and microleakage was evaluated by means of dyeing. According to the locations, the demineralization of tooth tissue around the restoration was divided into 3 parts: artificial outer lesion, artificial enamel wall lesion, and artificial dentin wall lesion. The depths of the artificial outer lesion and the areas of the artificial enamel and dentin wall lesions were investigated with a confocal laser scanning microscope after all the specimens had been stained with 0.1 mM Rhodamine B. Statistical analyses consisted of 1-way ANOVA and Kruskal-Wallis tests (α=.05). RESULTS: No significant difference was noted in terms of the number of bacteria around the restorations for the 3 materials; the microleakage of amalgam was the least among the 3 types of restorations; the outer enamel lesion depth around the glass ionomer cement was the lowest, and the wall lesion area around the amalgam was the smallest. CONCLUSIONS: No definite inhibitory effect of fluoride releasing restorative materials (glass ionomer) was apparent on Lactobacillus acidophilus in this study. The amalgam restorations showed the best marginal integrity and the smallest artificial wall lesion among the 3 types of restorations.

Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma.

Oral lichen planus (OLP) is a potentially malignant disorder associated with an increased risk of progression to oral squamous cell carcinoma (OSCC). The objective of this study was to determine protein expression of cancer stem cell factor Bmi1 in a longitudinal series of patients with OLP and evaluate the correlation between Bmi1 expression and the risk of progression to OSCC. In a retrospective study, Bmi1 expression was determined using immunohistochemistry in samples from 96 patients with OLP who received a mean follow-up of 54 months, including patients who did not progress to OSCC (n=87) and patients who had progressed to OSCC (n=9). Analysis of 10 cases of normal oral mucosa and 6 cases of postmalignant OSCC form previously diagnosed OLP was also performed. The results showed that Bmi1 expression was observed in 32 (36.8%) of 87 cases of nonprogressing OLP and in 8 (88.9%) of 9 cases of progressing OLP. Bmi1 was not expressed in normal oral mucosa, but it was positively expressed in the 6 (100%) cases of OSCC. Multivariate analysis revealed that the risk of malignant progression in the patients with Bmi1-positive expression was significantly higher than those with Bmi1 negativity (odds ratio, 20.75; 95% confidence interval, 2.21-194.57; P=.008). Collectively, Bmi1 expression was significantly associated with malignant transformation in a large series of patients with OLP who received a longitudinal observation. Our findings suggested that Bmi1 may serve as a useful marker for the identification of a high risk of malignant progression of OLP.

A retrospective clinicopathological study on oral lichen planus and malignant transformation: analysis of 518 cases.

OBJECTIVE: To investigate the epidemiological and clinical characteristics of a relatively large cohort of patients with oral lichen planus (OLP) from eastern China. STUDY DESIGN: A total of 518 patients with histologically confirmed OLP in a long-term follow-up period (6 months-21.5 years) were retrospectively reviewed in our clinic. RESULTS: Of the 518 patients, 353 females and 165 males were identified. The average age at diagnosis was 46.3 years (range 9-81 years) with the buccal mucosa being the most common site (87.8%). At initial presentation, white lichen and red lichen was seen in 52.3% and 47.7% patients, respectively. Of these, 5 (0.96%) patients previously diagnosed clinically and histopathologically as OLP developed oral cancer. All of them were the females with no a history of smoking or alcohol use. CONCLUSIONS: Clinical features of eastern Chinese OLP patients were elucidated. Notably, approximately 1% of OLP developed into cancer, which provides further evidence of potentially malignant nature of OLP.

Crystallization and preliminary X-ray analysis of S-ribosylhomocysteinase from Streptococcus mutans.

S-Ribosylhomocysteinase (LuxS) encoded by the luxS gene from Streptococcus mutans plays a crucial role in the quorum-sensing system. LuxS was solubly expressed in Escherichia coli with high yield. The purity of the purified target protein, which was identified by SDS-PAGE and MALDI-TOF MS analysis, was >95%. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as the primary precipitant. X-ray diffraction data were collected at Beijing Synchrotron Radiation Facility (BSRF). Diffraction by the crystal extended to 2.4 Å resolution and the crystal belonged to space group C222(1), with unit-cell parameters a = 55.3, b = 148.7, c = 82.8 Å.

Fatty-acid profiles and expression of the fabM gene in a fluoride-resistant strain of Streptococcus mutans.

OBJECTIVE: The aim of this study was to compare the membrane fatty acid profile and expression of the fabM gene in a fluoride-resistant Streptococcus mutans strain with those of its wild-type counterpart. Additionally, we aimed to investigate whether mutations were present in the fabM gene of the fluoride-resistant strain. DESIGN: A fluoride-resistant strain of S. mutans was obtained by step-by-step induction in vitro. The fluoride-resistant strain and its wild-type counterpart were grown anaerobically in a brain heart infusion broth, harvested and resuspended in a salt solution for an acid survival assay and a pH-drop experiment. The membrane fatty acid profile was determined by gas chromatography-mass spectrometry. Expression of the fabM gene was quantified by real-time PCR. The fabM gene was also sequenced. RESULTS: The ability of the fluoride-resistant strain to resist acid stress was greater than that of the wild-type strain. A significant difference in the amount of long-chain monounsaturated fatty acids between the fluoride-resistant strain and the wild-type strain was detected in acidic condition (P<0.01). In addition, the level of fabM mRNA in the fluoride-resistant strain was significantly higher than that of the wild-type strain in the acidic condition as well (P<0.01). However, the sequence of the fabM gene from the fluoride-resistant strain was 100% homologous with that from the wild-type strain. CONCLUSIONS: The membrane fatty acid profile and expression of the fabM gene in the fluoride-resistant strain were altered compared to the wild-type strain in acidic conditions, but no differences were found in the sequence of the fabM gene.

A clinicopathological study on verrucous hyperplasia and verrucous carcinoma of the oral mucosa.

BACKGROUND: Oral verrucous hyperplasia (VH) and verrucous carcinoma (VC) are two clinicopathologically distinctive oral verrucous lesions. The objective of this study was to investigate the clinicopathological features of the two verrucous lesions and estimate their relationship from China. METHODS: Retrospective review of two series of patients with histologically confirmed VH (n = 121) and VC (n = 56) between 1996 and 2009 in our hospital were conducted. RESULTS: The average age of VH was 58.5 years (ratio male:female = 1.37) with the tongue being the predominant site. The average age of VC was 64.3 years (ratio male:female = 1.15) with the lower lip being the predominant site. Multivariate analysis revealed that the elderly patient with verrucous lesion (≥60 years) was associated with 3.06-fold (P = 0.007) increased carcinoma risk compared with the non-elderly patient. The lesion located on lower lip was associated with 13.54-fold (P < 0.001) increased carcinoma risk compared with other sites. CONCLUSION: Clinicopathological features of VH and VC in China were elucidated. Elderly patient with oral verrucous lesion located on the lower lip correlates with higher risk of carcinoma.

[Expression and subcellular localization of XIAP and XAF1 in human normal oral keratinocytes and Tca8113 cells].

OBJECTIVE: To clarify expression and subcellular localization of XIAP and XAF1 protein in human normal oral keratinocytes (hNOK) and Tca8113 cells human tongue carcinoma cell line. METHODS: The hNOKs and Tca8113 cells were cultured in vitro. Expression and subcellular localization of XIAP and XAF1 protein were examined by combination of indirect immunofluorescence and confocal laser scanning microscopy. RESULTS: XIAP expression was weak in the hNOKs and fluorescence staining localized chiefly in the cytoplasm and perinuclear areas. In the Tca8113 cells, high level of XIAP protein could be detected in both the cytoplasm and the nucleus. In the hNOKs, XAF1 distributed mostly in the nucleus. Homogeneous nuclear and cytoplasmic distribution of XAF1 could be visualized in the Tca8113 cells. CONCLUSIONS: In cancerization of oral mucosa, XIAP protein could play an important antiapoptotic role by overexpression, while XAF1 protein does not appear to antagonize effectively the role of XIAP.