Knockout fth1b affects early mineralization of zebrafish pharyngeal teeth / 华西口腔医学杂志

OBJECTIVES@#A study was conducted to explore the expression pattern and function of ferritin heavy polypeptide gene (fth1b) in zebrafish pharyngeal teeth development and lay the foundation for subsequent research on teeth development and mineralization.@*METHODS@#The zebrafish embryos were harvested at 56, 72, 96, and 120 h after fertilization. The expression of fth1b in zebrafish pharyngeal teeth development was detected by whole embryo @*RESULTS@#The expression pattern of fth1b gene was very similar to that of the known zebrafish pharyngeal teeth marker dlx2b and was specifically expressed in the zebrafish pharyngeal teeth during development. After the specific knockout of the gene fth1b, the earliest gene that can be detect in zebrafish pharyngeal teeth-pitx2 was expressed normally during early development. The dlx2b expression was not significantly different from that of wild type zebrafish, but the mineralization of pharyngeal teeth in the mutant was weaker than that of wild type zebrafish.@*CONCLUSIONS@#The gene fth1b is specifically expressed in zebrafish pharyngeal teeth and acts on their early mineralization.

Research progress on the biomimetic remineralization of hard tooth tissues based on polyamide-amine dendrimer / 华西口腔医学杂志

Polyamide-amine (PAMAM) dendrimer, a new hyperbranched macromolecular polymer, is considered an "artificial protein" by many scholars on account of its excellent chemical and biological characteristics. PAMAM has internal cavities and a large number of reactive terminal groups. These structures allow the polymer to be used as a bionic macromoleculethat could simulate the biomimetic mineralization of the natural organic matrix on the surface of tooth tissue. Specifically, PAMAM can beused as an organic template to regulate mineral nucleation and crystal growth; thus, the polymerisa more ideal dental restoration material than traditional allogenic materials. This article reviews research progress on thePAMAM-induced biomimetic mineralization of hard tooth tissues.

Oral microbiological diversity in patients with salivary adenoid cystic carcinoma / 华西口腔医学杂志

OBJECTIVE@#The aim of this study was to identify the differences in microbial diversity and community in patients with salivary adenoid cystic carcinoma (SACC).@*METHODS@#Saliva was collected from 13 patients with SACC confirmed by histopathological diagnosis and 10 healthy control subjects. Total metagenomic DNA was extracted. The DNA amplicons of the V3-V4 hypervariable regions of the 16S rRNA gene were generated and subjected to high-throughput sequencing. Microbial diversity and community structure were analyzed with Mothur software.@*RESULTS@#A total of 16 genera of dominant bacteria in the SACC group were found, including Streptococcus (36.68%), Neisseria (8.55%), Prevotella_7 (7.53%), and Veillonella (6.37%), whereas 15 dominant bacteria in the control group were found, including Streptococcus (18.41%), Neisseria (18.20%), Prevotella_7 (8.89%), Porphyromonas (6.20%), Fusobacterium (5.86%) and Veillonella (5.82%). The statistically different phyla between the two groups were Firmicutes, Proteobacteria and Fusobacterium (P<0.05). The statistically different genera between the two groups were Streptococcus, Neisseria and Porphyromonas (P<0.05), and Capnocytophaga was only detected in patients with SACC.@*CONCLUSIONS@#Significant differences were observed in the oral microorganisms between the two groups.

Expression patterns of ectodysplasin and ectodysplasin receptor during early dental development in zebrafish / 华西口腔医学杂志

OBJECTIVE@#This study aims to study the expression patterns of ectodysplasin (EDA) and ectodysplasin receptor (EDAR) during the early development of zebrafish and provide a foundation for further research of the Eda signaling pathway in tooth development.@*METHODS@#Total RNA was extracted from zebrafish embryos at 48 hours postfertilization (hpf) and then reverse transcribed for cDNA library generation. The corresponding RNA polymerase was selected for the synthesis of the digoxin-labeled antisense mRNA probe of zebrafish pharyngeal tooth specific marker dlx2b and Eda signaling-associated genes eda and edar in vitro. The three sequences were ligated into a pGEMT vector with a TA cloning kit, and polymerase chain reaction (PCR) was applied to linearize the plasmid. The resultant PCR sequences were used as templates for synthesizing Dig-labeled mRNA probe dlx2b, eda, and edar. Zebrafish embryos were collected at 36, 48, 56, 60, 72, and 84 hpf, then whole mount in situ hybridization was performed for the detection of eda and edar expression patterns. Then, their expression patterns at 72 hpf were compared with the expression pattern of dlx2b.@*RESULTS@#The mRNA antisense probes of dlx2b, eda, and edar were successfully obtained. The positive signals of eda and edar were observed in zebrafish pharyngeal tooth region at 48-72 hpf and thus conform to the signals of dlx2b in the positive regions.@*CONCLUSIONS@#The ligand eda and edar, which are associated with the Eda signaling pathway, are strongly expressed only at the pharyngeal tooth region in zebrafish from tooth initiation to the morphogenesis stage. Thus, the Eda signaling pathway may be involved in the regulation of the early development of zebrafish pharyngeal teeth.

An in vitro study of the angiogenic effects of concentrate growth factor on human umbilical vein endothelial cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed to explore the effects of concentrate growth factor extracts (CGFe) on human umbilical vein endothelial cells (HUVECs) in vitro.</p><p><b>METHODS</b>Concentrate growth factor (CGF) were prepared from the peripheral blood of healthy donors, followed by CGFe. Four groups were designed based on cell culture medium, as follows: 2%CGFe, 5%CGFe, 10%CGFe, and control. The proliferation activity of HUVECs was detected by cell cycle and CCK-8 assays. The migration of HUVECs was detected by scratch assay. The mRNA expression levels of vascular endothelial growth factor (VEGF), chemokine receptor 4 (CXCR4), and platelet derived growth factor (PDGF) were examined by quantitative real time polymerase chain reaction (qRT-PCR).</p><p><b>RESULTS</b>Results of CCK-8 and cell cycle assays showed that CGFe promoted the proliferation capability of HUVECs in a dose-dependent manner, and the data had statistical significance among four groups (P<
0.05). The cell migration assay indicated that CGF accelerated wound closure in a dose-dependent manner after 12 h of culture (P<0.05). The results of qRT-PCR showed that CGF upregulated the expression levels of VEGF, CXCR4, and PDGF in HUVECs.</p><p><b>CONCLUSIONS</b>CGFe can promote the proliferation, migration, and angiogenic differentiation of HUVECs. Thus, CGF might be an appropriate cure for dental pulp revascularization.</p>

Construction of a red fluorescent shuttle vector controlled by recA operon promoter of Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To construct a red fluorescent shuttle vector controlled by recA operon promoter to transform Streptococcus mutans.</p><p><b>METHODS</b>The promoter of recA was amplified from Streptococcus mutans UA159, and connected to plasmid pDsRed2-N1 to construct pRred with a red fluorescent coding gene, which was then inserted into the shuttle vector pDL276 to construct pLRred.</p><p><b>RESULTS</b>pLRred was successfully constructed, and Escherichia coli transformed with the pLRred plasmid could express reporter gene DsRed.</p><p><b>CONCLUSIONS</b>The recombination plasmid pLRred can be used in the further research of the expression of cariogenic virulence factor gene by Streptococcus mutans in biofilm.</p>

Genetic diversity and mRNA expression of F-ATPase subunit uncG gene of streptococcus mutans clinical isolates / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the genetic diversity and the gene expression of membrane-bound proton-translocating ATPase (F-ATPase) subunit gene uncG derived from Streptococcus mutans (S. mutans) clinical isolates.</p><p><b>METHODS</b>38 S. mutans strains derived from caries-active and caries-free individuals including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncG was amplified with specific primers from S. mutans genomic DNA, then the PCR product was analyzed by RFLP and sequenced. The relative expression quantity of uncG gene against the housekeeping gene recA was determined by using RT-PCR method. A gel documentation system and QUANTITY ONES software were used to analyze the data results.</p><p><b>RESULTS</b>It was testified that four genotypes A, B, C and D of PCR-RFLP were revealed when respectively digested with Alu I and Bsr I, but the distributions of the four genotype strains showed no difference (P > 0.05). The differences of uncG gene transcript quantities derived from different genotype or different aciduranc strains had no significance (P > 0.05).</p><p><b>CONCLUSION</b>This study indicated that uncG gene of F-ATPase obviously displayed genetic diversity and existed polymorphism at mRNA expression level, while the Alu I-RFLP genotypes and the expression levels would not be responsive to different acid tolerance of S. mutans strains.</p>

Genetic diversity of F-ATPase subunits gene uncEBF amplified from Streptococcus mutans clinical isolates / 华西口腔医学杂志

<p><b>OBJECTIVE</b>The purpose of this research was to study the genetic diversity of F-ATPase subunit gene uncEBF derived from Streptococcus mutans (S. mutans) clinical isolates, furthermore to investigate the relationship between the genetic diversity of F-ATPase and S. mutans aciduric ability.</p><p><b>METHODS</b>38 S. mutans strains included 18 high acid tolerance strains and 20 low acid tolerance strains. Gene uncEBF of these isolates were amplified with specific primers from S. mutans genomic DNA, and the PCR products were analyzed by RFLP and sequenced. SPSS 11.0 statistic software assayed the results.</p><p><b>RESULTS</b>It was testified that two genotypes A and B of PCR-RFLP were revealed when digested with Alu I and Dde I digested fragments of uncEBF displayed two different patterns C and D. Fisher exact two-tail test showed that the distributions of A and B genotype strains with different acidurance were different (P < 0.05), and the proportion of A genotype strains from high acidurance group was higher than that from low acidurance one. Some of these amplified uncEBF genes from different genotype were sequenced and testified that there existed variation of Alu I and Dde I recognized sites.</p><p><b>CONCLUSION</b>This study indicated that uncEBF gene of S. mutans F-ATPase obviously exhibited genetic diversity.</p>

Dynamic study on saccharomyces albicans drug efflux pumps gene expression during the biofilm formation / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To observe the drug resistance and drug efflux pumps gene mRNA of Saccharomyces albicans, including CDR1 gene and MDR1 gene, at different stage of biofilm formation in chemostat, furthermore to analysis the relationship between the drug efflux pump gene expression and the biofilm related drug resistance.</p><p><b>METHODS</b>To form the mature biofilm in vitro in chemostat, then collect the biofilm strains at different development stages (2, 12, 24, 48 h) to semi-quantified mRNA amount of CDR1 gene and MDR1 gene by one step RT-PCR method. Using XTT reduction method to test the dynamic change of Saccharomyces albicans drug resistance in biofilm.</p><p><b>RESULTS</b>Antifungal resistance of biofilm-grown cells increased conjunction with the biofilm maturation. Compared with earth stage of biofiom strains, the amount of CDR1 mRNA gene in mature biofilm strains increased, while MDR1 gene did not.</p><p><b>CONCLUSION</b>There is positive correlation between drug resistance and biofilm maturation of Saccharomyces albicans. Biofilm related drug resistance appears to be partially associated with the upregulation of drug efflux pumps, although the variation is not shown coincidence. During the biofilm formation, CDR1 gene expression is actively up-regulated, but MDR1 gene expression is stable.</p>

Homology analysis of the extended-V region of the surface proteins in different serotype Streptococcus mutans / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To analysis the homology among the extended-V region of the surface proteins in different serotype Streptococcus mutans (c, f, d, g) and to find out it's significance in anti-caries vaccine.</p><p><b>METHODS</b>The DNA of the bacteria (standarded serotype c, d, f, g and partial serotype c clinicals) was extracted and the extended-V region (SrV+, 1 384-2 514 bp) was amplified using polymerase chain reaction (PCR). Then the products were assessed using restriction fragment length polymorphism (RFLP) by endonuclease Dde I. The genotypings were sequenced and analysised using the program of BLAST on NCBI Gene Bank database.</p><p><b>RESULTS</b>About 1.13 kb fragments were produced both in serotype c and f, the serotype d and g were failed. The RFLP results showed that five different patterns(A, B, C, D, E) among the 117 PCR products were reveled by Dde I. The ration of the genotypings A and B were the most among the strains, the C was lower, the D and E respectively was 1 and 3 strains per genotype. OMZ175 (serotype f) was belong to B genotype. Selected one of the A, B, C genotypings to sequenced and blasted. Then the results of the blastn showed that the identities of the gene sequence were 92%-98% between the serotype c and serotype f, part sequence of the serotype g was homology with the SrV+ of the serotype c, the protein sequence among serotype c, d, f, g were 77%-82%.</p><p><b>CONCLUSION</b>It is reasonable to use some putative pipetides to study the anti-caries vaccine among the extended-V regions of the surface proteins in different serotype (c, d, f, g) in S. mutans.</p>

Genetic diversity of F-ATPase subunits gene uncA amplified from Streptococcus mutans clinical isolates / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the genetic diversity of F-ATPase alpha subunit gene uncA derived from Streptococcus mutans (S. mutans) clinical isolates and to investigate the relationship between the genetic diversity of acidurance factor and S. mutans aciduric ability, also and the cariogenicity.</p><p><b>METHODS</b>Sixty-four S. mutans strains derived from 34 caries-active individuals and 30 caries-free individuals, including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncA was amplified with specific primers from S. mutans genomic DNA, then the PCR products were analyzed by RFLP and sequenced.</p><p><b>RESULTS</b>Two genotypes A and B of PCR-RFLP were revealed when digested with Hph I. Mbo II also produced two different pattern C and D. The distributions of A and B genotype strains with different caries-sensitivity groups were different (P < 0.05), and the proportion of A genotype strains from caries-activity group was higher than that from caries-free one. The distributions of C and D genotype strains with different acidurance strains were different (P < 0.05), and the proportion of C genotype strains from high acid tolerance group was higher than that from low acid tolerance group. These amplified uncA genes from different group were sequenced and there existed variation of Hph I and Mbo II recognized sites.</p><p><b>CONCLUSIONS</b>This study indicates that uncA gene of S. mutans F-ATPase obviously displayed genetic diversity. The different Hph I-RFLP and Mbo II-RFLP genotypes could be related to the cariogenicity and acid tolerance of S. mutans strains.</p>

Study on lactate dehydrogenase activity of Streptococcus mutans isolates derived from caries-active and caries-free individuals / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To preliminarily investigate the relationship between the Lactate dehydrogenase (LDH) activity of Streptococcus mutans (S. mutans) and dental caries initiation.</p><p><b>METHODS</b>100 S. mutans strains derived from caries-active and caries-free individuals were cultured in BHI medium supplemented with glucose. Cells were extracted and ruptured, and the extracted liquid protein was quantified with Coomassie brilliant blue G250 staining methods. LDH activity was assayed using the pyruvate-dependent oxidation of NADH-with and without FDP.</p><p><b>RESULTS</b>LDH activity of the two groups strains had no difference (P > 0.05), but the distribution of differ class enzyme activity strains in the two groups was different (P < 0.05).</p><p><b>CONCLUSION</b>LDH activity of S. mutans is correlated to the initiation of dental caries to some extent. The measurement methods in this study can be applied in preliminary quantitation of LDH activity and the screening of S. mutans strains.</p>