Sirtuin 3-activated superoxide dismutase 2 mediates fluoride-induced osteoblastic differentiation in vitro and in vivo by down-regulating reactive oxygen species.

Skeletal fluorosis is a chronic metabolic bone disease caused by long-term excessive fluoride intake. Abnormal differentiation of osteoblasts plays an important role in disease progression. Research on the mechanism of fluoride-mediated bone differentiation is necessary for the prevention and treatment of skeletal fluorosis. In the present study, a rat model of fluorosis was established by exposing it to drinking water containing 50 mg/L F-. We found that fluoride promoted Runt-related transcription factor 2 (RUNX2) as well as superoxide dismutase 2 (SOD2) and sirtuin 3 (SIRT3) expression in osteoblasts of rat bone tissue. In vitro, we also found that 4 mg/L sodium fluoride promoted osteogenesis-related indicators as well as SOD2 and SIRT3 expression in MG-63 and Saos-2 cells. In addition, we unexpectedly discovered that fluoride suppressed the levels of reactive oxygen species (ROS) and mitochondrial reactive oxygen species (mtROS) in osteoblasts. When SOD2 or SIRT3 was inhibited in MG-63 cells, fluoride-decreased ROS and mtROS were alleviated, which in turn inhibited fluoride-promoted osteogenic differentiation. In conclusion, our results suggest that SIRT3/SOD2 mediates fluoride-promoted osteoblastic differentiation by down-regulating reactive oxygen species.

Effect of formula factors on the properties of HPMC plant hollow capsule film.

In order to explore the effect of the hollow capsule material formulation on the capsule glue and film formation, this study used hydroxypropylmethylcellulose (HPMC), carrageenan, KCl and Tween 80 as raw materials to determine the production of HPMC hollow capsules suitable formula. The optimal process conditions are as follows: the proportions of HPMC, carrageenan, KCl and Tween 80 in the solvent (purified water) are 18% (m:V), 0.7% (m:V), 0.07% (m:V) and 0.018% (V:V), respectively. Under this condition, the viscosity of the resulting solution, glue solidification temperature and gel strength were medium. The resulting film has low hygroscopicity, good solubility, optical properties and mechanical properties. This research can provide data support for the precise formulation and industrial production of HPMC hollow plant capsules.

Phase 2 study of hyper-CMAD with liposomal vincristine for patients with newly diagnosed acute lymphoblastic leukemia.

Liposomal vincristine is designed to reduce neurotoxicity and increase dose intensity delivery, and has been approved as salvage therapy in relapsed/refractory acute lymphoblastic leukemia (ALL). Our aim was to evaluate the response rate, toxicities, and outcome of adults with newly diagnosed ALL who received liposomal vincristine, rather than regular vincristine in combination with intensive chemotherapy (Hyper-CMAD). In a single-center, phase 2 study, patients ≥18 years with newly-diagnosed B-cell ALL were eligible to receive hyper-CMAD alternating with high-dose methotrexate and cytarabine. Rituximab was administered in CD20 positive ALL. Tyrosine kinase inhibitors (imatinib or dasatinib) were added in Philadelphia chromosome-positive (Ph-positive) ALL. Thirty-one patients were enrolled, median follow-up of 59 months (0.3-70). Thirteen patients (42%) had CD20 positive ALL, and 21 (68%) had Ph-positive ALL. Thirty (97%) achieved complete remission (CR). All 26 patients with abnormal karyotype achieved complete cytogenetic response (CCyR), and 27/30 (90%) achieved negative minimal residual disease status by multicolor flow cytometry. Of 20 evaluable Ph-positive ALL patients, major molecular response (MMR) was achieved in 19 patients (95%); complete molecular response (CMR) in 14 (70%). Grade 3/4 peripheral neuropathy was observed in five (16%) with all grade peripheral neuropathy in 21 (68%). With a median follow-up of 59 months, 21 (68%) patients are alive. The 5-year CR duration and survival rates were 73% and 61%, respectively. Ten (32%) patients died: one, sepsis on C1D10; four, unknown; one, post-transplant complications; four, relapse. Hyper-CMAD with liposomal vincristine is safe and demonstrated high response and survival rates in newly diagnosed ALL.

Synergistic Lysosomal Activatable Polymeric Nanoprobe Encapsulating pH Sensitive Imidazole Derivative for Tumor Diagnosis.

Developing optical tumor imaging probes with minimal background noise is very important for its early detection of small lesions and accurate diagnosis of cancer. To overcome the bottleneck of low signal to noise ratio and sensitivity, it needs further improvement in fluorescent probe design and understanding of tumor development process. Recent reports reveal that lysosome's acidity in cancer cells can be below 4.5 with high Na+ /H+ exchange activity, which makes it an ideal target intracellular organelle for cancer diagnosis based on the variation of pH. Herein, a boron 2-(2'-pyridyl) imidazole complex derivative (BOPIM-N) is developed, with the ability to show a pH-activatable "OFF-ON" fluorescent switch by inhibiting twisted intramolecular charge transfer upon protonation at pH 3.8-4.5, which is studied for its selective viable cancer cell imaging ability in both in vitro and in vivo experiments. Interestingly, BOPIM-N can specifically emit green fluorescence in lysosomes of cancer cells, indicating its promising cancer cell specific imaging ability. More importantly, nanoformulated BOPIM-N probes can be specifically light-ON in tumor bearing site of nude mice with resolution up to cellular level, indicating its potential application in tumor diagnosis and precision medicine.

Effect of Probiotic Lactobacilli on the Growth of Streptococcus Mutans and Multispecies Biofilms Isolated from Children with Active Caries.

BACKGROUND The purpose of this investigation was to evaluate the effect of probiotic lactobacilli on Streptococcus mutans (MS) and multispecies biofilms isolated from children with severe caries. MATERIAL AND METHODS Twenty children with active caries (DMFS ≥6) were selected as the experimental group and Streptococcus mutans (MS) were isolated from their saliva. After identification the MS strains were mixed with lactobacilli at 37°C, following which viable MS colonies were counted. At the same time dental plaques from the children were mixed with lactobacilli in vitro to form biofilms, and the population of nine common strains in the biofilms was enumerated after 24 hours of growth. RESULTS Lactobacillus casei Shirota, L. casei LC01, L. plantarum ST-III and L. paracasei LPC37 all had strong inhibitory effects on the majority the MS isolated from children with active caries, with the inhibition rate reaching approximately 70-90% (p<0.05). L. casei Shirota, L. casei LC01, L. plantarum ST-III, L. paracasei LPC37 also significantly reduced the numbers of MS, Streptococcus spp., S. sanguinis and total bacteria in mixed biofilms compared with the control group (p<0.05). CONCLUSIONS The four strains of lactobacilli were able to inhibit the growth of Streptococcus mutans and had effects on the composition of bacterial biofilms in vitro. Ingestion of probiotics may be a promising method of caries prevention.

High-throughput sequencing identifies salivary microbiota in Chinese caries-free preschool children with primary dentition.

OBJECTIVES: The study aimed at identifying salivary microbiota in caries-free Chinese preschool children using high-throughput sequencing. METHODS: Saliva samples were obtained from 35 caries-free preschool children (18 boys and 17 girls) with primary dentition, and 16S ribosomal DNA (rDNA) V3-V4 hypervariable regions of the microorganisms were analyzed using Illumina MiSeq. RESULTS: At 97% similarity level, all of these reads were clustered into 334 operational taxonomic units (OTUs). Among these, five phyla (Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Candidate division TM7) and 13 genera (Streptococcus, Rothia, Granulicatella, Prevotella, Enterobacter, Veillonella, Neisseria, Staphylococcus, Janthinobacterium, Pseudomonas, Brevundimonas, Devosia, and Gemella) were the most dominant, constituting 99.4% and 89.9% of the salivary microbiota, respectively. The core salivary microbiome comprised nine genera (Actinomyces, Capnocytophaga, Gemella, Granulicatella, Lachnoanaerobaculum, Neisseria, Porphyromonas, Rothia,and Streptococcus). Analysis of microbial diversity and community structure revealed a similar pattern between male and female subjects. The difference in microbial community composition between them was mainly attributed to Neisseria (P=0.023). Furthermore, functional prediction revealed that the most abundant genes were related to amino acid transport and metabolism. CONCLUSIONS: Our results revealed the diversity and composition of salivary microbiota in caries-free preschool children, with little difference between male and female subjects. Identity of the core microbiome, coupled with prediction of gene function, deepens our understanding of oral microbiota in caries-free populations and provides basic information for associating salivary microecology and oral health.

Oral Microbiome Alterations Associated with Early Childhood Caries Highlight the Importance of Carbohydrate Metabolic Activities.

Globally, dental caries is the most prevalent chronic oral disease and affects roughly half of all children. The aim of this report was to use metagenomic analyses to investigate the relationship between the oral microbiome and caries in preschool children. A total of 25 preschoolers, aged 3 to 5 years old with severe early childhood caries (ECC), and 19 age-matched, caries-free children as controls were recruited. Saliva samples were collected from the participants and were subjected to metagenomic analyses, whereby the oral microbial communities were investigated. The metagenomic analyses revealed substantial microbiota differences between the two groups, indicating apparent shifts of the oral microbiome present in the ECC group. At the species level, the ECC-enriched microbes included Prevotella amnii, Shuttleworthia satelles, Olsenella uli, and Anaeroglobus geminatus Interestingly, Actinomyces odontolyticus and Actinomyces graevenitzii exhibited apparent differences at the strain level but not the species level between the ECC and control groups. Functional examination showed that the ECC group displayed extensive alterations in metabolic genes/pathways/modules, including enriched functions in sugar metabolism. Finally, an SVM (support vector machine) classifier comprising seven species was developed and generated a moderately good performance in predicting caries onset (area under the receiver operating characteristic curve [AUC] = 78.33%). Together, these findings indicate that caries is associated with considerable changes in the oral microbiome, some of which can potentially be exploited as therapeutic targets or diagnostic markers. (This study has been registered at ClinicalTrials.gov under registration no. NCT02341352.)IMPORTANCE Dental caries is a highly prevalent oral disease that can lead to severe dental damage and may greatly compromise the quality of life of the affected individuals. Previous studies, including those based on 16S rRNA gene, have revealed that the oral microbiota plays a prominent role in development of the disease. But the approach of those studies was limited in analyzing several key microbiome traits, including species- or strain-level composition and functional profile. Here, we performed metagenomic analyses for a cohort of preschool children with or without caries. Our results showed that caries was associated with extensive microbiota differences at various taxonomic and functional levels. Some caries-associated species had not been previously reported, some of which may have significant clinical implications. A microbiome gene catalogue from children with caries was constructed for the first time. The results demonstrated that caries is associated with alterations of the oral microbiome, including changes in microbial composition and metabolic functional profile.

Dynamic Alterations in Salivary Microbiota Related to Dental Caries and Age in Preschool Children With Deciduous Dentition: A 2-Year Follow-Up Study.

Dynamic alterations in oral microbiota are closely related to the development of dental caries;however, changes in salivary microbiota during this process have not been extensively studied. In addition, increasing evidence suggests that oral microbiome profiles differ according to dentition stages, but it is unclear whether they change with age during the same dentition, such as deciduous dentition. These two aspects were investigated in a 2-year follow-up study, and caries-free preschool children with complete deciduous dentition were enrolled. Saliva was collected and oral examination was conducted at the beginning of this trial, and then every subsequent 6 months for a total of five time points (T0, T1, T2, T3, and T4). Based on the clinical examination of teeth at the end of the trial, subjects were divided into health-to-health (H-H, N = 11) and health-to-caries (H-C, N = 12) groups at every time point. A total of 115 saliva samples from 23 subjects was detected by sequencing 16S rDNA V3-V4 hypervariable regions with the Illumina MiSeq platform to obtain microbiome profiles, and 100 samples finally passed quality control for further analyses. A total of 4,328,852 high-quality sequencing reads passed quality-control testing, representing 14 phyla, 27 classes, 43 orders, 67 families, and 127 genera. An α diversity analysis showed that salivary microbial diversity was similar in all groups, and a ß diversity analysis showed that salivary microbial community structure changed with dental caries. Linear discriminant analysis effect size (LEfSe) analysis revealed that the abundance of the genera Atopobium, Megasphaera, and Veillonella increased significantly, while that of the genera Shuttleworthia and Rothia decreased significantly with the development of dental caries. Megasphaera and Veillonella were enriched at the early stage of deciduous dentition whereas Peptococcus, Rothia, and Treponema were enriched at the later stage. The core microbiome in the H-H and H-C groups comprised 26 and 29 genera, respectively, with statistical differences observed in 11 shared core genera. These results provide new insights into variations in the salivary microbiome related to dental caries and age in the deciduous dentition period.

Profiling of Oral Microbiota in Early Childhood Caries Using Single-Molecule Real-Time Sequencing.

Background: Alterations of oral microbiota are the main cause of the progression of caries. The goal of this study was to characterize the oral microbiota in childhood caries based on single-molecule real-time sequencing. Methods: A total of 21 preschoolers, aged 3-5 years old with severe early childhood caries, and 20 age-matched, caries-free children as controls were recruited. Saliva samples were collected, followed by DNA extraction, Pacbio sequencing, and phylogenetic analyses of the oral microbial communities. Results: Eight hundred and seventy six species derived from 13 known bacterial phyla and 110 genera were detected from 41 children using Pacbio sequencing. At the species level, 38 species, including Veillonella spp., Streptococcus spp., Prevotella spp., and Lactobacillus spp., showed higher abundance in the caries group compared to the caries-free group (p < 0.05). The core microbiota at the genus and species levels was more stable in the caries-free micro-ecological niche. At follow-up, oral examinations 6 months after sample collection, development of new dental caries was observed in 5 children (the transitional group) among the 21 caries free children. Compared with the caries-free children, in the transitional and caries groups, 6 species, which were more abundant in the caries-free group, exhibited a relatively low abundance in both the caries group and the transitional group (p < 0.05). We conclude that Abiotrophia spp., Neisseria spp., and Veillonella spp., might be associated with healthy oral microbial ecosystem. Prevotella spp., Lactobacillus spp., Dialister spp., and Filifactor spp. may be related to the pathogenesis and progression of dental caries.

Effects of residual water on microtensile bond strength of one-bottle dentin adhesive systems with different solvent bases.

BACKGROUND: The wet-bonding technique is recommended for the one-bottle dentin adhesive systems, but the moisture concept varies widely among the instructions of manufacturers as well as among investigators. The aim of this study was to evaluate the effects of different dentin surface moisture on the microtensile bond strength(s) of an ethanol/water-based adhesive system and an acetone-based system to dentin. METHODS: Forty intact human premolars extracted for orthodontic reasons were used. Superficial occlusal flat dentin surfaces of these premolars were exposed, finished with wet 600-grit silicon carbide paper. Under four wet and dry conditions (overwet, blot dry, one-second dry and desiccated), resin composite was bonded to dentin by using Single Bond (SB) or Prime & Bond NT (PB) according to the manufacturers' instructions. The teeth were longitudinally sectioned in the "x" and "y" directions to obtain bonded beams with a cross-sectional area of 0.81 mm(2) with a slow-speed diamond saw. The bonded specimens were tested in tension at a crosshead speed of 1 mm/min until failure of the bonds. Failure modes were observed with a scanning electron microscope. The mean bond strengths were analyzed by one-way ANOVA and Turkey's test. RESULTS: The bond strength of the overwet/SB, blot dry/SB, one-second dry/SB and desiccated/SB groups was 10.87 MPa, 22.47 MPa, 24.91 MPa and 12.99 MPa, respectively. The bond strength of the overwet/PB, blot dry/PB, one-second dry/PB and desiccated/PB groups was 10.02 MPa, 20.67 MPa, 21.82 MPa and 10.09 MPa, respectively. For both SB and PB, the blot dry group and one-second dry group revealed significantly higher bond strengths than the overwet and desiccated groups (P < 0.05). CONCLUSIONS: In order to achieve the highest bond strength to dentin, keeping the dentin surface in an appropriately moist condition is critical for the one-bottle dentin adhesive systems with ethanol/water or acetone solvent.

[Microtensile bond strength and morphological evaluations of total-etch and self-etch adhesives to caries-affected dentin].

OBJECTIVE: To evaluate the microtensile bond strength and bond interface of total-etch or self-etch adhesives to normal dentin and caries-affected dentin. METHODS: A total of 20 molars with occlusal caries lesion were used. The caries-affected dentin was obtained by removing the caries-infected dentin under the guidance of the caries detector. Beyond the level of caries-affected dentin all the enamel and partial dentin were removed. The adhesive systems, two total-etch adhesives (All-Bond 2, Prime&Bond NT) and two self-etch adhesives (Clearfil SE Bond, Xeno III) were applied respectively under the instructions of manufacturers. A block of composite resin was build up superficially. All the teeth were sectioned to obtain bar-shaped specimens with bonded surface area about 0.9 mm x 0.9 mm. The specimens were divided into normal dentin group and caries-affected dentin group via stereomicroscope. The bond strength was tested in a microtensile tester with a crosshead speed of 1 mm/min. The mean values of bond strength were compared using two-way ANOVA. The bonding interface between the dentin and adhesives was qualitatively evaluated under the observation of scanning electron microscope (SEM). RESULTS: Two-way ANOVA revealed a significant influence of both the type of dentin and the adhesive systems tested on microtensile bond strength values. All the adhesives attained higher strength in normal dentin. In normal dentin, there was no significant difference between total-etch and self-etch adhesives. In caries-affected dentin, bond strength of Xeno III was significantly lower than the others. For SEM, the hybrid layer in caries-affected dentin was thicker but more porous than that in normal dentin. Compared with normal dentin, there was fewer resin tag exhibited in caries-affected dentin and no lateral branches were observed. CONCLUSIONS: The total-etch adhesive had higher bond strength than self-etch adhesive systems in caries-affected dentin.

[Influence of storage methods on microtensile bond strength of dentin adhesive system].

PURPOSE: To compare the effects of teeth stored with different methods and different durations of storage on the microtensile bond strength of the dentin adhesive system Single Bond. METHODS: 30 human first premolars were stored immediately after extraction in one of five commonly used methods respectively: 0.02% distilled water and thymol, 10% formalin, 1% chloramine, distilled water at 4 degrees C and were refrigerated at -20 degrees C. After 10 days, 90 days, the dentin adhesive system Single Bond and composite Z250 were applied to superficial occlusal flat dentin according to the manufacturers' instructions and microtensile bond strength measurements were evaluated, freshly extracted teeth being used as control. Stero-microscope and scanning electronic microscope were used to evaluate the fracture modes of the microtensile bond strength specimens. Two-way ANOVA was used for analysis of the microtensile bond strengths among different groups. RESULTS: The results showed that there was significant difference among five tooth storage methods on the microtensile bond strength of dentin bonding agent (P=0.01). Compared with freshly extracted teeth, two of five storage methods/media, 0.02% distilled water and thymol at 4 degrees C (P=0.008) and distilled water at 4 degrees C (P=0.024), resulted in significantly lower microtensile bond strengths. The duration of teeth storage had no effect on the microtensile bond strength of dentin bonding agent (P=0.279). The interaction of two factors was significant (P=0.000). Stero-microscope and SEM examination indicated that all fracture modes were adhesive failures. CONCLUSIONS: From this study, it can be concluded that the teeth storage methods/media can influence the microtensile bond strength of Single Bond adhesive system. If sufficient numbers of freshly extracted teeth are not available for bond strength test, the freezing teeth at -20 degrees C and teeth stored in 1% chloramine at 4 degrees C are preferred .