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Objective:To construct a recombinant herpes simplex virus 2 (HSV-2) expressing enhanced green fluorescent protein (EGFP) using clustered, regularly interspaced, short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology.Methods:Four strategies for inserting exogenous EGFP gene into HSV-2 genome using CRISPR/Cas9 technology were designed: (1) conventional homology-directed repair: circular two homology arm donor-mediated gene knock-in; (2) linearized single homology arm donor-mediated gene knock-in; (3) homology-independent targeted integration; (4) conventional homology-directed repair-mediated by cell lines stably expressing Cas9 and sgRNA.Results:The recombinant virus HSV-2-EGFP was successfully constructed based on the second, the third and the fourth strategies. The second strategy was the most efficient, followed by the third and the fourth strategies. The purified recombinant virus could stably express green fluorescent protein in seven passages and shared similar growth characteristics in Vero cells to the parental virus.Conclusions:Linearized single homology arm donor could increase the efficiency of gene knock-in, and cell lines stably expressing Cas9 and sgRNA could increase the efficiency of gene knock-in mediated by homology-directed repair.
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Objective:To investigate the effects of genomic location of a foreign gene in Shanghai-191 strain measles virus (MV) vector on gene expression and virus replication.Methods:The nucleotide sequence encoding S1 protein of SARS-CoV-2 was inserted at different positions in MV antigenome (the upstream of the N gene, between P and M genes, between H and L genes), and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and N, P, and L proteins, respectively. The transfected cells were lysed and the supernatants were used to infected Vero cells to harvest recombinant viruses. S1 proteins expressed by the recombinant viruses were identified by RT-PCR, indirect immunofluorescence assay, Western blot and ELISA. Growth kinetics of the recombinant viruses were analyzed.Results:Recombinant viruses were failed to be rescued when the S1 protein-coding sequence was cloned into the upstream of N gene. Two recombinant viruses, MV-M-S1 and MV-L-S1, were successfully rescued when cloning the S1 protein-coding sequence into the intergenic region between P and M genes, or H and L genes, and could express S1 protein. MV-M-S1 expressed more S1 protein than MV-L-S1, but the titer of MV-M-S1 was lower.Conclusions:Inserting a foreign gene at different positions in the MV genome might have different effects on gene expression and virus replication. This study provided reference for the subsequent construction of MV vector.
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<p><b>OBJECTIVE</b>This study evaluated the effect of different mechanical-chemical surface treatments on the characteristics, microstructure, and composition of zirconia ceramics and on the zirconia-dentin adhesion.</p><p><b>METHODS</b>The sintered commercial zirconia blocks (IPS e.max ZirCAD) were sectioned into 126 beams (6 mm×6 mm×5 mm) and randomly assigned to seven experimental groups (n=18). The zirconia block specimens were further treated as follows: (A) untreated, as control; (B) sandblasted with 50 μm Al2O3; (C) sandblasted with 50 μm Al2O3+30 μm silica powder; (D) sandblasted with 50 μm Al2O3+30% silica-sol coating; (E) sandblasted with 110 μm Al2O3; (F) sandblasted with 110 μm Al2O3+30 μm silica powder; and (G) sandblasted with 110 μm Al2O3+30% silica-sol coating. The surface roughness (Ra) of zirconia ceramics using X-ray diffraction (XRD), field emission scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) after seven surface treatments was analyzed. Seventy specimens of dentin surfaces were prepared. A dual-cure resin cement was applied into zirconia surfaces with its corresponding adhesive components to dentin. Shear bond strength (SBS) of each sample was measured using a universal testing machine. The data were analyzed by ANOVA using SPSS 17.0 software.</p><p><b>RESULTS</b>Ra of zirconia were significantly different compared with the control group (P<0.05). The crystalline transformation from tetragonal phase to monoclinic phase was observed after surface modification. Monoclinic volume content of the heat-treated group was highest than that in other groups. The content of element Si in the heat-treated group was higher than that in other treatment groups accompanied by a decrease in elements Zr, Y, and Hf after being treated by two silica-coating methods. Air abrasion significantly increased the micro-cracks in the ceramic surface and caused the grain boundaries to disappear. A serious shrinkage of the thin silica film can be observed after sintering procedure. This process induced cracks and the film to strip away slightly from the surfaces of Y-TZP substrate. The SBS values from large to small were F and G groups, C and D groups, B and E groups (P<0.05), and control group (P<0.05).</p><p><b>CONCLUSIONS</b>Alumina sandblasting, silica powder abrasion, and silica-sol coating affect the surface morphology, structure, and composition of the tooth. This effect can also achieve the improved micro-mechanical interlocking or chemical bonding and finally increase the bond strength between zirconia and tooth. Alumina sandblasting followed by silica coating is an effective technique to increase the bonding strength between the zirconia ceramic and dentin. .</p>
Subject(s)
Aluminum Oxide , Ceramics , Dentin , Materials Testing , Microscopy, Electron, Scanning , Resin Cements , Silicon Dioxide , Surface Properties , X-Ray Diffraction , ZirconiumABSTRACT
Objective To explore the clinical application of digital chair?side immediate all?ceramic restoration system,design and fabricate inlays and onlays to restore the large?defected molars,and to analyze and evaluate the efficacy of this clinical restoration. Methods Totally 84 inlays and 65 onlays were fabricated with IPS e.max CAD series ceramic blocks by using Cerec AC system to conduct immediate all?ceramic restorations of 149 teeth for 108 patients. The inlays and onlays were applied and cemented to the prepared teeth with dual?cured resin luting agent. The restored molars were re?examined by two dentists after six months. The analysis standard was based on the United States Public Health Service(USPHS criteria) and the results were statistically analyzed(α=0.05). Results There was no statistically significant difference in the case numbers of grade A and grade B for each index comparing the immediate condition to the condition after 6 months. Over 90 percent of ceramic inlays and onlays were quali?fied to be grade A. The restorations were well in the aspects of anatomic morphology,marginal integrity,color match,marginal discoloration,reten?tion,gingival health and secondary caries. Conclusion Computer aided design/computer aided manufacture(CAD/CAM)system combined with machinable glass ceramics can achieve a favorable result in restoration of large?defected teeth. Digital chair?side immediate all?ceramic restoration technique is an ideal solution for large?defected tooth restoration.