Southern blot analysis with probe from mini-Tn5 gfp-km transposon indicated that 5 non-pathogenic mutants which were generated by insertion of mini-Tn5 gfp-km mutagenesis contained a single copy of the transposon. Using genomic DNA of each mutant as a template, TAIL-PCR was performed with seven arbitrary degenerate (AD) primers pairing with 3 nested specific primers designed based on the sequence of GFP toward outside in mini-Tn5 gfp-km. After 3-step PCR reactions, the flanking sequence of each mutant was obtained. The PCR product was ligated with pGEM-T EASY vector and then was transformed into E. coli DH5 alpha by electroporation. Positive clones were selected by white/blue colony and plasmid was isolated, then digested with EcoRI. Plasmid was sequenced if its insert was longer than 300 bp. Our results indicated that TAIL-PCR was proved to be a simple and efficient approach in identification of gene using insertion mutagenesis.
Base Sequence ; Cloning, Molecular ; methods ; DNA Transposable Elements ; DNA, Bacterial ; isolation & purification ; Genes, Bacterial ; Green Fluorescent Proteins ; Luminescent Proteins ; genetics ; Molecular Sequence Data ; Mutagenesis ; Polymerase Chain Reaction ; methods ; Xanthomonas campestris ; genetics ; pathogenicity

BACKGROUND: The size of artificial disc is not exactly identical to cervical vertebral endplate. However, the effect of implant size on cervical disc replacement has not yet been clearly identified. OBJECTIVE: To evaluate the effect of Bryan disc size on cervical artificial disc replacement. METHODS: Totally 71 patients with cervical degenerative disease underwent single-level Bryan disc replacement from December 2003 to December 2007 were enrolled. The average age was (45.90±8.12) years old, and all patients were followed up for more than 10 years. The artificial disc size was measured using lateral X-ray films, and expressed as (Bryan artificial disc's footprint sagittal diameter×2) / (upper endplate sagittal diameter + lower endplate's sagittal diameter)×100%. X-ray films were photographed to measure motion range of the treated segment before surgery, 3 months after surgery, and during final follow-up. Clinical outcomes, including Japanese Orthopaedic Association score, Neck Disability Index and Odom's scores, were evaluated before and after operation. Observational indexes of different disc sizes (≥95% and <95% groups) were compared and analyzed. RESULTS AND CONCLUSION: (1) There were 49 patients in the ≥95% group and 22 patients in the <95% group. (2) At 3-month follow-up, range of motion was significantly smaller in the <95% group than in the ≥95% group (P < 0.01). At the final follow-up, range of motion was significantly smaller in the <95% group than in the ≥95% group (P < 0.05). (3) At final follow-up, improvement rate of Japanese Orthopaedic Association score was (70.65±32.58)% in the <95% group and (68.83±38.85)% in the ≥95% group, and no significant difference was detected between the two groups (P > 0.05). (4) At final follow-up, Neck Disability Index decreased by (10.82±7.50)% in the <95% group, and (12.61±8.51)% in the ≥95% group, and no significant difference was detected between the two groups (P > 0.05). (5) At final follow-up, Odem's score results showed excellent in 10 cases (45%), good in 9 cases (41%) and average in 3 cases (14%) in the <95% group; and excellent in 26 cases (53%), good in 20 cases (41%) and average in 3 cases (6%) in the ≥95% group, and no significant difference was detected between the two groups (P > 0.05). (6) Results suggest that different sizes of artificial disc replacement have obtained good functional recovery, but small artificial disc may impact the range of motion. We should try to avoid placing the artificial disc smaller than 95%.

ObjectiveTo observe the thyroid status of pregnant women during the 1st and 2nd trimester of gestation,and its role in brain and neural development of their offspring's.MethodsFrom 2008 to 2009,pregnant women from nine townships of two counties in Wushi and Baicheng in Aksu prefecture of Xinjiang were selected as research subjects according to the survey standard.After informed consent signed,their urinary iodine,serum thyroid stimulating hormone(TSH) and free thyroxin(FT4) were analyzed.The value of thyroid hormone of normal pregnant women was used in diagnosis of subclinical hypothyroidism and hypothyroxinemia in pregnant women.From 2010 to 2011, The brain and neural development status among offspring born by those pregnant women were evaluated with DDST.In accordance with the results of Denver Development Screen Test (DDST) screening,pregnant women were classified into survey and control groups,and the survey group was the suspicious and abnormal of the result of DDST screening(delay),the control group was normal of the result.According to gestational age,pregnant women were divided into 4 gestation groups:G1(6 to 13 weeks),G2(14 to 18 weeks),G3 ( 19 to 23 weeks) and G4(24 to 28 weeks).ResultsA total of 396 cases of pregnant women during the 1st and 2nd trimester of gestation were investigated(survey group 102 cases,control group 294 cases).The median value of urinary iodine concentration among pregnant women in survey group was 152.4 μg/L The prevalence of subclinical hypothyroidism and hypothyroxinemia among pregnant women was 10.78％(11/102) and 3.93％(4/102),respectively.In control group,the median value of urinary iodine concentration was 180.0 μg/L The prevalence of subclinical hypothyroidism and hypothyroxinemia among pregnant women was 7.48％ (22/294) and 4.42％ (13/294),respectively.During the pregnant period from G1 to G3,the median serum TSH of pregnant women in DDST survey group (2.24,3.49,2.85 mU/L) was higher than that of DDST control group( 1.59,2.70,2.28 mU/L).Especially,the difference of TSH between the two groups during the period of G3 was statistically significant (t =4.906,P ＜ 0.05 ).ConclusionsHypothyroidism tendency of pregnant women during the period from gestation week 19th to 23rd may be an important factor in the development of brain abnormalities of their offsprings.

BACKGROUND: With the development of chemical synthesis technology, the application of anti-osteoporosis drugs incorporated into scaffolds to promote bone regeneration in osteoporotic bone defects has become a hot issue nowadays. OBJECTIVE: To introduce bone tissue engineering scaffolds, and to discuss and summarize the application of the scaffolds carrying anti-osteoporosis drugs for osteogenesis in osteoporotic bone defects. METHODS: PubMed, Web of Science, Springerlink, Medline, WanFang and CNKI databases were retrieved with "osteoporosis, bone defect, scaffold" as key words for relevant articles published from 2005 to 2017. Initially, 201 articles were retrieved, and finally 64 articles were selected for further analysis. RESULTS AND CONCLUSION: With the development of biochemical synthesis technology, various types of tissue-engineered bone scaffolds have been used for the treatment of osteoporotic bone defects. For example, the modified calcium phosphate cement, β-tricalcium phosphate, and hydroxyapatite all can promote bone regeneration. However, the ability of conventional scaffolds to promote bone regeneration is severely damaged under the state of osteoporosis. In this case, an anti-osteoporosis therapy becomes necessary. Considering severe adverse effects of systemic anti-osteoporosis drugs and limited local bone regeneration, the combination of scaffold implantation and local drug delivery can promote osteoporotic bone defect healing. Importantly, to incorporate bone regeneration inducers into scaffolds and steadily release with their biological activity is the key to success. Bisphosphonate, strontium, bone morphogenetic protein 2 and parathyroid hormone, traditional Chinese medicine and mesenchymal stem cells are the drugs commonly combined with tissue-engineered bone scaffolds. With the development of biotechnology, genetically modified stem cell-scaffold composites have gradually appealed to researchers.

Objective To study the effect of the 18kDa translocator protein (TSPO) on U251cells of human glioma. Methods U251 cell line was cultured in vitro conventionally.The specific ligand ofTSPO,pk11195,was used in 5 experimental groups respectively with concentrations of 100,50,25,12.5 and 6.25 μmol/L,in comparison with a control group.MTr colorimetry and trypan blue staining were used to detect cell proliferation.Hoechst33342 staining and flow cytometry were applied to detect cell apoptosis. Western blotting and immumofluorescence method were used to detect the expression level of TSPO. DCFH-DA probe and GSH kit were used to respectively detect the level of reactive oxygen species (ROS) and GSH level in cells.Jc-1 staining was applied to detect the mitochondrial membrane potential.Luciferase enzyme was used to detect the quantity of ATP in cells. Results MTT showed the survival of U251 cells was significantly higher in the groups of 50 and 25 μmol/L pk11195than in the control group (P＜0.05). Trypan blue staining showed the cell death rate was significantlylower in the group of 50 μmol/L pk11195 than in the control group (P＜0.05).The apoptosis rate,TSPO expression,ROS and GSH levels decreased significantly in the groups of 6.25 and 50 μmol/L pk11195,compared with the control group; the apoptosis rate was significantly lower in the group of 50 μmol/Lpk11195 than in the group of 6.25 μmol/L pk11195 (P＜0.05).The cell membrane potential and ATP quantity were significantly higher in the groups of 6.25 and 50 μmol/L pk11195 than in the control group,and those in the group of 50 μmol/L pk11195 were significantly higher than in the group of 6.25 μmol/Lpk11195 (P＜0.05). Conclusion TSPO may promote apoptosis of U251 cells in human glioma and inhibit proliferation of glioma cells,functioning similarly as a cancer suppressor gene.

OBJECTIVETo investigate the effect of intermittent fasting on metabolize and gut microbiota in obese presenium rats fed with high-fat-sugar-diet.

METHODSWe fed the Wistar rats with high-fat and high-sugar diet to induce adiposity, and the rats for intermittent fasting were selected base on their body weight. The rats were subjected to fasting for 72 h every 2 weeks for 18 weeks. OGTT test was performed and fasting blood samples and fecal samples were collected for measurement of TC, TG, HDL-C and LDL-C and sequence analysis of fecal 16S rRNA V4 tags using Illumina. Gut microbial community structure was analyzed with QIIME and LEfSe.

RESULTSAfter the intervention, the body weight of the fasting rats was significantly lower than that in high-fat diet group (P<0.01). OGTT results suggested impairment of sugar tolerance in the fasting group, which showed a significantly larger AUC than compared with the high-fat diet group (P<0.05). Intermittent fasting significantly reduced blood HDL-C and LDL-C levels (P<0.05) and partially restored liver steatosis, and improved the gut microbiota by increasing the abundance of YS2, RF32 and Helicobacteraceae and reducing Lactobacillus, Roseburia, Erysipelotrichaceae and Ralstonia. Bradyrhizobiaceae was found to be positively correlated with CHOL and HDL-C, and RF39 was inversely correlated with the weight of the rats.

CONCLUSIONIntermittent fasting can decrease the body weight and blood lipid levels and restore normal gut microbiota but can cause impairment of glucose metabolism in obese presenium rats.

Animals ; Body Weight ; Diet, High-Fat ; Fasting ; Fatty Liver ; microbiology ; physiopathology ; Gastrointestinal Microbiome ; Lipids ; blood ; Obesity ; microbiology ; physiopathology ; RNA, Ribosomal, 16S ; Rats ; Rats, Wistar

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.
Animals ; Antibodies ; Bacteria ; Chickens ; Hemagglutination ; HN Protein ; Homologous Recombination ; Immunoglobulin A, Secretory ; Immunoglobulin G ; Lymphocytes ; Methods ; Newcastle disease virus ; Newcastle Disease ; Plasmids ; Poultry ; Salmonella ; Suicide ; Vaccines

Objective To investigate the clinical effectiveness of magnetic resonance spectroscopy (MRS) combined with sodium fluorescein(FL) in the treatment of high grade gliomas(HGG). Methods From August 2013 to 2015 November,the clinical data of 72 supratentorial HGG(WHO grade Ⅲ-Ⅳ) patients who had received surgical treatment in our hospital were retrospectively studied,among whom 43 cases received MRS combined with intra-perative FL navigation(observation group),and 29 cases only received conventional surgery(control group). Post-operative radiotherapy and chemotherapy were applied for more than 3 months. Routine enhanced MRI were performed 24-48 hours after the operation to investigate the extent of tumor resection. Six months after the operation,the quality of life of patients was evaluated by using the Karnofsky score,and 1-year postoperative survival rate and progression-free survival(PFS) were observed. Results Postoperative MRI showed that the rate of gross total resection(GTR) in observation group was significantly higher than that in control group(72.09%vs.51.72%;χ=23.88,P=0.001),and the GTR rate of WHO grade Ⅳ tumors was significantly higher than that of WHO grade Ⅲ tumors in observation group(92.86% vs.62.07%;χ=6.06,P=0.042). The postoperative Karnofsky score in the observation group was significantly higher than that in control group(μ=2.34,P=0.021). The mean time of follow-up was(16.4±2.4) months(8-21 months) and there was no statistical significant difference between observation group and control group in 1-year survival rate(74.07% vs.77.50%;χ=4.90,P=0.165) and PFS [(13.2±1.2) months vs.(12.7±2.0) months;χ=7.26,P=0.067]. In observation group,the PFS of WHO grade Ⅳ patients was significantly higher than that in control group [(14.2±0.3) months vs.(10.0±1.1) months;χ=11.03,P=0.031]. There was also no statistical significant difference between WHO grade Ⅳ tumors in two groups in terms of 1-year survival rate(71.43% vs.72.54%;χ=5.33,P=0.089),and there was no statistical significant difference between WHO grade Ⅲ tumors in two groups in 1-year survival rate(75.86% vs. 72.22%;χ=3.78,P=0.250) and in PFS [(13.7±1.4) months vs.(12.4±0.8) months;χ=4.85,P=0.083]. Conclusions MRS combined with intraoperative FL navigation technology can improve the resection rate and improve survival quality of patients,and there is no evidence that MRS combined with intraoperative FL navigation prolong the overall survival of patients with high-grade gliomas. Different outcome may be found with longer follow-up and increased simple size.