Objective To investigate the effects of miR-34a on proliferation and osteogenic differentiation of human periodontal stem cells.Methods Twenty healthy teeth that needed to be extracted for orthodontic treatment were collected.Human periodontal stem cells(hPDLSCs)were isolated and cultured in vitro,and miR-34a mimetics were constructed and transfected into hPDLSCs.The experimental groups were subsequently categorized into the mimics group(miR-34a overexpression group)and the mimics-NC group(control group without load).The transfection efficiency was assessed using real-time fluorescence quantitative PCR(qRT-PCR),while CCK-8 assays were used to evaluate the proliferation capacity of hPDLSCs post-transfection.Osteogenic differentiation of miR-34a-transfected hPDLSCs was induced,with samples being collected at day 0 and day 14 after the osteogenic induction.The expression level of Runx2-associated transcription factor 2(Runx2)was quantified via qRT-PCR,protein levels of Runx2-associated proteins were analyzed through Western blot,and mineralized nodule formation was examined using alizarin red staining.Results The expression level of miR-34a in the mimics group was significantly higher than that in the mimics-NC group(P<0.05).There was no significant difference in the value-added rate between the mimics group and the mimics-NC group on days 1～5(P>0.05),and the value-added rate between the mimics group and the mimics-NC group was significantly lower than that between the mimics-NC group and the mimics-NC group on days 5～11,and the difference was statistically significant.After the osteogenic induction,the mRNA expression level of Runx2 in the mimics group was higher than that in the mimics-NC group(P<0.05),and the expression level of Runx2 protein in the mimics group was also higher than that in the mimics-NC group(P<0.05),and there were more mineralized nodules in the mimics group than in the mimics-NC group after 14 days of osteogenic induction.Conclusion Under in vitro conditions,miR-34a inhibits the proliferative activity of hPDLSCs and promotes the osteogenic differentiation of human periodontal ligament stem cells.

Objective:To construct the chimeric virus ChimZIKV of Japanese encephalitis virus and Zika virus and evaluate whether the chimeric virus can be developed into a candidate vaccine strain against Zika virus.Methods:The infectious clone of chimeric virus ChimZIKV was constructed by replacement the prM/E of Japanese encephalitis virus vaccine strain with that of Zika virus. RNA of chimeric virus ChimZIKV had been transcribed in vitro and was electro-transfected into BHK-21 cells to rescue chimeric virus. Afterward, the chimeric virus was identified by plaque assay, immunofluorescence and gene sequencing. The growth was shown by the growth curve. The neurobirulence, viremia, neutralizing antibody production and immune protective effect of chimeric virus were tested by animal experiments.Results:The result of restriction enzyme digestion showed that the infectious clone of chimeric virus was successfully constructed, and the immunofluorescence assay and sequencing showed that chimeric virus ChimZIKV was successfully rescued. The cell proliferation activity test showed that the cell proliferation activity of the chimeric virus infected group was higher than that of the infected group. The result of animal experiments showed that chimeric virus showed very low neurovirulence to mice and suckling mice, and the result of viremia showed that chimeric virus had no obvious viremia in mice. Plaque reduction neutralization test (PRNT) showed 100% positive conversion of neutralizing antibody in sera of immunized mice, and the highest neutralizing antibody titer (GMT=85) was seen in the third week. The immune protection experiment showed that the protection rate against JE/ZIKV(MR766) was 30%.Conclusions:This study showed that the chimeric virus ChimZIKV is immunogenic and with low neurovirulence, and it may be further developed as a candidate vaccine strain against Zika virus.

Streptococcus mutans (S. mutans) is generally regarded as a major contributor to dental caries because of its ability to synthesize extracellular polysaccharides (EPS) that aid in the formation of plaque biofilm. The VicRKX system of S. mutans plays an important role in biofilm formation. The aim of this study was to investigate the effects of vicK gene on specific characteristics of EPS in S. mutans biofilm. We constructed single-species biofilms formed by different mutants of vicK gene. Production and distribution of EPS were detected through atomic force microscopy, scanning electron microscopy and confocal laser scanning microscopy. Microcosmic structures of EPS were analyzed by gel permeation chromatography and gas chromatography-mass spectrometry. Cariogenicity of the vicK mutant was assessed in a specific pathogen-free rat model. Transcriptional levels of cariogenicity-associated genes were confirmed by quantitative real-time polymerase chain reaction. The results showed that deletion of vicK gene suppressed biofilm formation as well as EPS production, and EPS were synthesized mostly around the cells. Molecular weight and monosaccharide components underwent evident alterations. Biofilms formed in vivo were sparse and contributed a decreased degree of caries. Moreover, expressional levels of genes related to EPS synthesis were down-regulated, except for gtfB. Our report demonstrates that vicK gene enhances biofilm formation and subsequent caries development. And this may due to its regulations on EPS metabolism, like synthesis or microcosmic features of EPS. This study suggests that vicK gene and EPS can be considered as promising targets to modulate dental caries.
Animals ; Biofilms ; Dental Caries ; Dental Plaque ; Rats ; Streptococcus mutans/genetics*

Objective: To investigate the effects of sodium butyrate on intestinal barrier of the severe scald mice and the related mechanism. Methods: Eighteen C57BL/6 female mice, aged eight to twelve weeks, were divided into sham scald group, pure scald group, and scald+ sodium butyrate group according to random number table, with 6 mice in each group. Back of each mouse in pure scald group and scald+ sodium butyrate group were immersed into 90 ℃ water for 9 s, causing full-thickness scald of 30% total body surface area, while back of each mouse in sham scald group were immersed into 37 ℃ water for 9 s, causing sham injury. All of the mice in 3 groups were intraperitoneally injected with 1 mL sterile lactated Ringer′s solution immediately after injury. Besides, mice in scald+ sodium butyrate group were intraperitoneally injected with 300 mg/kg sodium butyrate at 30 min before injury and immediately after injury, while mice in sham scald group and pure scald group were intraperitoneally injected with the same volume of sterile phosphate buffer solution. At post injury hour (PIH) 24, portal vein of mice in 3 groups was harvested, intestinal permeability was measured by fluorescin isothiocyanate-dextran fluorescence probe tracing method, then lileal tissue of mice in 3 groups was harvested, protein expressions of zonula occludens l (ZO-1), occludin, claudin-1, claudin-2, nucleotide-binding oligomerization domain-containing protein-like receptor family pyrin domain containing 3 (NLRP3), interleukin-1β (IL-1β), and IL-18 were detected by Western blotting, and distribution of ZO-1 in intestinal mucosa was observed by indirect immunofluorescence. Data were processed with one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) At PIH 24, the intestinal permeability of mice in sham scald group, pure scald group, and scald+ sodium butyrate group was 0.88±0.19, 2.62±0.48, 1.23±0.16, respectively. Compared with that in sham scald group, the intestinal permeability of mice in pure scald group was significantly elevated (P<0.01), while the intestinal permeability of mice in scald+ sodium butyrate group showed no obvious change (P>0.05). Compared with that in pure scald group, the intestinal permeability of mice in scald+ sodium butyrate group was significantly decreased (P<0.01). (2) At PIH 24, compared with those in sham scald group, the protein expressions of ZO-1, occludin, and claudin-1 of mice in pure scald group and scald+ sodium butyrate group were significantly decreased (P<0.05), while the protein expression of claudin-2 was significantly increased (P<0.05). At PIH 24, compared with those of pure scald group, the protein expressions of ZO-1 and occludin of mice in scald+ sodium butyrate group were significantly elevated (P<0.05), while the protein expression of claudin-2 was significantly decreased (P<0.05), the protein expression of claudin-1 showed no significant difference (P>0.05). (3) At PIH 24, compared with those in sham scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in pure scald group and scald+ sodium butyrate group were significantly increased (P<0.05). Compared with those of pure scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in scald+ sodium butyrate group were significantly decreased (P<0.05). (4) At PIH 24, ZO-1 in intestinal mucosa of mice in sham scald group was distributed smoothly, continuously and homogeneously along the membrane. ZO-1 in intestinal mucosa of mice in pure scald group was distributed unsmoothly with breaks. The distribution of ZO-1 in intestinal mucosa of mice in scald+ sodium butyrate group was ameliorated compared with that in pure scald group. Conclusions Sodium butyrate can inhibit the activation of NLRP3 inflammasome and decrease the production of IL-1β and IL-18 in intestinal mucosa of severe scald mice, which protects the intestinal barrier function by alleviating the alteration of tight junction protein expression and localization.

Objective:To explore the effect of D393E mutation on its intracerebral neurovirulence in mice after infectious clone of the chimeric Japanese encephalitis virus (JEV)/Zika virus (ZIKV) containing envelope protein D393E mutant was built and the virus was rescued.Methods:The full-length cDNA plasmid of JEV/ZIKV chimeric virus containing D393E mutation in the envelope protein was built by SOE PCR and gene cloning technology. The linearized fragment of the plasmid was used for in vitro transcription to obtain viral RNA, followed by electrotransfection into BHK-21 cells to rescue the mutant virus JEV/ZIKV(D393E). The plaque size and growth characteristics of JEV/ZIKV (D393E) and JEV/ZIKV were compared after BHK-21 cells were inoculated with the viruses. The intracerebral neurovirulence of the viruses was calculated after the viruses were inoculated in mice intracerebrally (i.c.).Results:Restriction enzyme digestion evaluation confirmed that the infectious clone of mutant virus was correctly built. The plaque assay demonstrated that the plaque diameter of JEV/ZIKV (D393E) was about 0.7±0.2 mm, which was smaller than that of JEV/ZIKV (1.3±0.2 mm). The LD 50 value of the mutant was 31.51 PFU when injected in mice via i. c. route, which was greater than 2.21 PFU of JEV/ZIKV. Conclusions:The D393E mutation of ZIKV envelope protein in JEV/ZIKA chimeric virus decreases the neurovirulence of the virus in mice.

OBJECTIVE:To determine the concentration of dehydroisoandrosterone sulfate (DHEAS) in human plasma by LC-MS.METHODS:With estrogen sulfate (ES) served as an internal standard,the plasma samples were deproteinized with acetonitrile,extracted by solid phase,hydrolyzed and derivatized.Then the concentration of DHEAS was determined by HPLC-MSD on Agilent SB C18 with column temperature kept at 40℃.The mobile phase consisted of acetonitrile-water (in gradient elution).Atmosphere pressure chemical ion source in negative ion detection model was employed.The ions selected for SIM (selected ion monitoring) quantitative analysis included m/z 490.0 (DHEAS ) and m/z 472.1(ES)[M-H]-.RESULTS:The linear range of DHEAS was 250.0～320.0 ng?mL-1(r=0.999 4).The extraction recovery of the simulated human albumin samples ranged from 71.1%～78.9% and its relative recovery ranged from 98.3%～101.4%.Both the intra-day RSD and inter-day RSD were less than 10%.The mean concentration of DHEAS in 15 health aged male volunteers was (981.6?353.4) ng?mL-1.CONCLUSION:The method is simple,practical,accurate and sensitive,and it is applicable for the determination of plasma concentration of DHEAS.