Three-dimensional reconstruction of systematic histological sections: application to observations on palatal shelf elevation / 国际口腔科学杂志·英文版

Normal mammalian secondary palate development undergoes a series of processes, including palatal shelf (PS) growth, elevation, adhesion and fusion, and palatal bone formation. It has been estimated that more than 90% of isolated cleft palate is caused by defects associated with the elevation process. However, because of the rapidly completed elevation process, the entire process of elevation will never be easy to clarify. In this article, we present a novel method for three-dimensional (3D) reconstruction of thick tissue blocks from two-dimensional (2D) histological sections. We established multiplanar sections of the palate and tongue in coronal and sagittal directions, and further performed 3D reconstruction to observe the morphological interaction and connection between the two components prior to and during elevation. The method completes an imaging system for simultaneous morphological analysis of thick tissue samples using both synthetic and real data. The new method will provide a comprehensive picture of reorientation morphology and gene expression pattern during the palatal elevation process.

Association of single nucleotide polymorphism of IL-6 gene with susceptibility to hepatitis B virus / 中华实验和临床病毒学杂志

Objective@#Leukocyte mediated IL-6 (IL-6) plays an important role in chronic viral hepatitis pathogenesis and related liver diseases, We did a large sample-size case-control study and clinical data analysis to find association between IL-6 single nucleotide polymorphism and HBV susceptibility, and to achieve the detection of the body on the expression of IL-6 for the prevention and treatment of HBV infection.@*Methods@#Totally 848 HBV patients and 894 healthy controls in Shenzhen were selected and rs1800796 genotypes were determined by TaqMan assays.@*Results@#The result showed that rs1800796 polymorphism was associated with susceptibility to HBV infection (P=0.0003, odds ratio (OR)=1.43, the difference was statistically significant.@*Conclusions@#The single nucleotide polymorphism of IL-6rs1800796 locus was associated with the susceptibility of HBV infection in Chinese population, and the rs1800796 G allele is a protective gene for HBV infection.

Construction of ARID1A gene mutants and identification of the overexpression in hepatocellular carcinoma cells / 中华实验和临床病毒学杂志

Objective@#To construct the mutants of ARID1A gene, which is an important component in human chromosomal remodeling complex Switch/Sucrose Non Fermentable (SWI/SNF), and identify their overexpression in liver cancer HepG2 cells.@*Methods@#Overlap PCR was used to construct domain truncated mutantss pcDNA6-ARID1A/ΔARID and pcDNA6-ARID1A/ΔD UF3518 based on wild type plasmids pcDNA6-ARID1A. Lipofectionmethod was used to transfect the wild type and mutants into HepG2. Real-time PCR and western blotting were used to confirm the overexpression of ARID1A and the mutants.@*Results@#SDS-PAGE and sequencingresult confirmed the successful construction of pcDNA6-ARID1A/ΔARID and pcDNA6-ARID1A/ΔDUF3518. Real-time PCR and western blottingresult confirmed the overexpression of both mRNA and protein of wild type ARID1A and ARID1A/ΔARID. The mRNA levels indicated that ARID1A/Δ DUF3518 were overexpressed, but the protein levels were quite low.@*Conclusions@#Functional domain truncated mutants of ARID1A were successfully constructed. Overexpression of wild type ARID1A and ARID1A/ΔARID in liver cancer HepG2 cells was successful. Loss of ARID1A/ΔDUF3518 protein suggest that DUF3518 may contribute to the protein structure stability.

Expression and identification of recombinant Clostridium difficile toxin B using Bacillus megaterium system / 中华传染病杂志

Objective To express and purify recombinant and biologically active Clostridium difficile toxin B (rTcdB). Methods The genes of TcdB were amplified by polymerase chain reaction (PCR) using chromosomal DNA from a toxigenic strain, and cloned into a shuttle vector pHis1522.The sequences of TcdB genes in the vector were verified by DNA sequencing. The construction was transformed into Bacillus megaterium protoplasts and the protein expression was driven by a xylose promoter. The purified protein was tested for biological activity. Results rTcdB was successfully purified from bacterial crude extracts. Approximately 5-10 mg of highly purified recombinant toxin was obtained from one liter of bacterial culture. The expressed rTcdB had molecular mass similar to the native toxin, and its biological activity was proved to be similar to its native counterpart after an extensive examination. Conclusion rTcdB with biological activities is successfully expressed in Bacillus megaterium.

The effect of Th17 cells on A(H1N1) influenza virus clearance / 中华传染病杂志

Objective To investigate the phenotype, frequency of Th17 cells and the association between Th17 cells and viral clearance in patients with H1N1 influenza A. Methods Three groups including 70 confirmed patients with H1N1 influenza A, 30 patients with seasonal influenza as well as 68 healthy subjects as controls were enrolled in this study. The percentages of Th1, Th2, Treg and Th17 lymphocytes in the peripheral blood were determined by intracellular staining and flow cytometry. The levels of interferon-γ (IFN-γ), transforming growth factor-beta (TGF-β),interleukin-6 (IL-6) in plasma and supernatant of the peripheral blood mononuclear cell (PBMC)culture were quantified by enzyme-linked immunosorbent assay (ELISA). Viral load in nasopharyngeal swabs was detected by real time quantitative reverse transcription-polymerase chain reaction (RTPCR). Data were analyzed by one way ANOVA and liner correlation analysis. Results The percentage of Th17 cells in H1N1 influenza A patients was (2. 740±0. 210)%, which the percentage of was significantly decreased compared to healthy subjects (3. 443 ±0. 154)% and seasonal influenza patients (3. 443±0. 277) % (F=4. 242, P＜0. 05); while the percentage of Thl, Th2 and Treg cells were not significantly different among these groups. Moreover, the TGF-β level in plasma of H1N1 influenza A patients was (10±8) ng/mL, which was significantly lower than healthy subjects (43 ±32 ) ng/mL and seasonal influenza patient ( 18 ± 10) ng/mL ( F= 17.72, P＜0.01 ). The TGF-β level in the supernatant of PBMC culture of H1N1 influenza A patients was (782 ± 736) pg/mL, which was significantly lower than healthy subjects (1462±315) pg/mL and seasonal influenza patients (1481 ±348) pg/mL (F=5. 730, P＜0.01). Additionally, the viral clearance period was inversely correlated with the percentage of Th17 cells (r=-0.38, P=0.02). Conclusions The proportion of Th17 cells in patients with H1N1 influenza A is significantly decreased, which is closely correlated with the level of TGF-β. This decrease may results in the delayed viral clearance.

Analysis on clinical features, viral load and viral shedding period of patients with mild or severe H1N1 influenza A virus infection / 中华传染病杂志

Objective To analyze clinical and laboratory features, viral load and viral shedding period of patients with mild or severe H1N1 influenza A infection. Methods Seventy mild cases and 16 severe cases with concurrent pneumonia were included from Shcnzhen area for analysis.Nasopharyngeal-swab specimens of patients were collected and viral load was detected by real-time quantitative polymerase chain reaction (PCR) assay during their hospitalization. The viral load and viral shedding period were compared between patients over 14 years old and less than 14 years old, and between 70 mild cases without pneumonia and 16 severe cases with pneumonia. The statistic analysis was performed using t test and chi square test. Results The most common symptoms and signs of the patients were fever, cough and enlargement of tonsils. However, the severe cases suffered more frequently from cough, dyspnea and high fever compared with the mild cases (x2 = 10. 9 and 14.3, respectively, t=3.65; both P＜0.01 ). The levels of white blood cell (WBC) count and alanine arninotransferase (ALT) of severe patients were both significantly higher than those of mild patients(t= 3.2, 2.4,respectively; both P＜0.05). The chest radiology of the severe cases showed interstitial pneumonia,mostly with ground glass image. The viral load of patients under 14 years was significantly higher than those over 14 years [(4.86± 1.23) lg vs (4. 17±0.89) lg; t=2.3, P＜0.05], and the viral shedding period of patients under 14 years was significantly longer than those over 14 years [(5.33±0. 49) d vs(3. 63±0.28) d; t=3.4, P＜0.01]. The severe patients also displayed significantly higher viral load and prolonged viral shedding period than the mild patients [(6. 36±1. 44) lg vs (4. 35±0.99) lg, t=6.1,P＜0.01; (5.75±1.77) d vs (4. 24±1. 96) d, t=3.2, P＜0.01]. Conclusion Age anddisease severity of patients with H1N1 influenza A infection are significantly associated with viral load and viral shedding period.

Development of a serological ELISA kit for detection of EV71 infection associated with hand-foot and mouth disease and its clinical application / 中华检验医学杂志

Objective To develop an ELISA(Enzyme-Linked Immunosorbent Assay)diagnostic kit for early rapid detection of sarum anti-EV71 antibody and evaluate its clinical application value.Methods Recombinant protein VP1 of EV71 were prepared and purified as an immobilized antigen for establishment of an indirect ELISA for detection of serum anti-EV71 IgM and anti-EV71 IgG.Compared with RT-PCR.isolation of EV71 and micro-neutralizing assay.the clinical application value of anti-EV71 IgM and anti-EV71 ISG in the diagnosis of EV71 disease was evaluated.Results In comparison with RT-PCR.the sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgM antibody were 83%,85%,81%and 87%,respectively.The sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgG antibody were 72%,74%,68%and 77%.respectively.Compared with viral isolation assay.the sensitivity and specificity of anti-EV71 IgM antibody were 85%and 97%,respectively.The sensitivity and specificity of anti-EV71 IsG antibody were 75%and 77%,respectively.In addition.the titers of anti-EV71 IgG antibody were significantly correlated with the titers of neutralizing antibody to EV71 by linear regression analysis(r=0.72,P＜0.05).Finally,the serum titers of anti-IgG from patients with EV71 associated hand food and mouth disease at convalescent stage exhibited significantly higher than that of the same patients at acute stage(P＜0.01),but the titers of anti-IgM had no significant difference(P＞0.05).Conclusions With VP1 recombinant protein used as an immobilized antigen,an indirect ELISA diagnostic kit was successfully develooed for detection of serum anti-human EV71 IgM and anti-human EV71 IgG antibodies.