In order to study the relationship between lengths of homologous fragments and chromsomic recombination rate in Saccharopolyspora erythraea, three homologous sequences, with mutant loci and different flanking sequences, (26bp + 27bp), (500bp + 576bp) and (1908bp + 1749bp), were synthesized by chemical reaction or PCR amplification, and cloned into pWHM3 to construct homologous recombination plasmids, pWHM1113, pWHM1116 and pWHM1119. When the plasmids were transformed into protoplast of Saccharopolyspora erythraea A226 under PEG mediated, on an average 30, 69 and 170 transformants grew on each plate for the three plasmids respectively, but chromosomic integration frequency were 0, 2% and 19% among corresponding transformants. Both pWHM1116 and pWHM1119 could take double crossover recombination, and exchange the mutant loci in the chromosome. It was concluded that when the flanking sequences were equal or more than (500bp + 576bp), they could take effective single and double recombination with Saccharopolyspora erythraea chromosome.
Chromosomes, Bacterial ; genetics ; Polymerase Chain Reaction ; Recombination, Genetic ; genetics ; Saccharopolyspora ; genetics

Objective To investigate the incidence and its secular trends of gastroschisis in Chinese perinatal infants.Methods Data on perinatal infants was collected at hospitals under surveillance program in Chinese Birth Defects Monitoring Network from 1996 to 2007.Data on incidence,trend and related factors of gastroschisis in perinatal infants were carried out.Both x2 test and Poisson regression model were used to test the differences between residential areas,sex and maternal age.Both x2 trends test and Poisson regression model were applied to analyze the trends.Results A total of 6 308 594 perinatal infants were monitored during 1996-2007,including 1601 infants with gastroschisis to show the incidence as 2.54 per 10 000 births.The overall prevalence of gastroschisis in China did not change remarkably during the period of our research.The incidence rates of gastroschisis were significantly different between urban and rural areas,between different sex and different maternal age groups.The incidence of gastroschisis was lower in urban area than in rural area (RR=0.58) and lower in female fetuses than in male fetuses (RR=0.76),highest in the group younger than 20 years of age,which was 11.43 times than incidence of the 30-34 age group (RR=11.432).Conclusion The overall prevalence of gastroschisis in China did not show remarkable change during 1996-2007 but the incidence of gastroschisis a bit increased in the area of study and significant differences were seen in different sex,regions and maternal age groups.Mothers aged younger than 20 years old appeared to be a significant risk factor for the occurrence of gastroschisis.

OBJECTIVEOsteopetrosis is a rare genetic disorder and the malignant infantile osteopetrosis (MIOP) is the worst subtype of this disease. Seventy percent of patients die in six years of life without proper treatment. Hematopoietic stem cell transplantation (HSCT) offers the only chance of cure for MIOP.

METHODRetrospective analysis was performed on 8 patients with MIOP who underwent HSCT in Beijing Children's Hospital during the period from 2006 to 2011.

RESULTEight cases (4 male and 4 female, mean age at HSCT 13.5 months) were diagnosed as malignant infantile osteopetrosis. Conditioning regimen included fludarabine, busulfan and cyclophosphamide. All patients received cyclosporin for prophylaxis of graft vs. host disease (GvHD). A UMD recipient underwent CD34(+) cell selection. ATG/ALG, mycophenolate mofetil (MMF) and methotrexate (MTX) used for recipients with unrelated cord donor (2) and recipients with haplo-identical donors (5). Average time for neutrophil engraftment was 15.7 day (9 - 36), platelet engraftment was 43.3 day (10 - 68). The patients were followed up from 47 days to 5 years, 1 patient died of post-transplant complications. Seven cases presented better in clinical manifestation. Acute GvHD I°-II° was observed in 6 patients, III°-IV° in 2 patients. It was controlled by anti-GvHD therapy.

CONCLUSIONNon-allogenic stem cell transplantation treatment of infantile MIOP showed high survival rate and restoration of hematopoiesis in haploid transplant patients, therefore, non-allogenic HSCT may be an option to treat MIOP in children.

Bone Marrow Transplantation ; adverse effects ; methods ; Child, Preschool ; Female ; Fetal Blood ; cytology ; Follow-Up Studies ; Genetic Predisposition to Disease ; Graft vs Host Disease ; drug therapy ; epidemiology ; prevention & control ; Haploidy ; Hematopoietic Stem Cell Transplantation ; adverse effects ; methods ; Humans ; Infant ; Male ; Osteopetrosis ; mortality ; therapy ; Retrospective Studies ; Survival Analysis ; Transplantation Conditioning ; methods ; Transplantation, Homologous ; Treatment Outcome

OBJECTIVETo evaluate the application of multi-detector row CT (MDCT) and CT angiography (CTA) for detecting early signs of acute bowel ischemia (ABI) in experimental porcine models.

METHODSTwelve pigs were assigned to four groups with 3 in each group. The digital subtraction angiography of superior mesenteric artery (SMA) and the embolization of branches of SMA with gelatin sponge and blood clot were performed by percutaneous transfemoral artery puncture and catheterization. MDCT pre- and post-contrast scanning in the arterial, venous and delay phase and CTA with three-dimensional reconstruction were carried out at pre-operation, 3 h, 6 h, 9 h, and 12 h after occlusion. The normal mesenteric vascular anatomy, arterial occlusion, mesentery and bowel changes, and dynamic change were evaluated.

RESULTSABI changes were identified pathologically in all the 12 experimental pigs, and the severity of ischemia increased over time after embolization. CTA showed all 57 embolized branches of SMA and 29 of 34 unoccluded arterial branches with 5 false-positive vessel occlusions. The sensitivity and specificity of CTA were 100% and 85.3%, respectively. Thin-slab maximum intensity projection (TSMIP) revealed the disappearance of distal comb-like vessel branches and brush-like vasa recta, which were clearly delineated in the normal bowel segments. Using this criterion, TSMIP correctly defined 23 of 24 ischemic bowel segments and all the 12 normal bowel segments with a sensitivity of 95.8% and a specificity of 100%.

CONCLUSIONSMDCT and CTA reliably define normal and occluded mesenteric vessels in the pig. It can easily detect ischemic bowel segment by identified early changes of ischemia. The early direct ischemic signs are occluded vessels, the disappearance of distal comb-like branches or brush-like vasa recta, and poor bowel enhancement. The early indirect sign is bowel dilatation with fluid collection.

Angiography ; methods ; Animals ; Female ; Intestinal Diseases ; diagnostic imaging ; etiology ; Ischemia ; diagnostic imaging ; etiology ; Mesenteric Arteries ; diagnostic imaging ; Mesenteric Vascular Occlusion ; complications ; diagnostic imaging ; Mesentery ; blood supply ; Swine ; Tomography, X-Ray Computed ; methods

"Periocular heat" acupuncture manipulation refers to the application of Zheng Kuishan "warming and unblocking acupuncture method" to treatment for eye diseases and the use of this method at point Fengchi(GB20) to produce a heat sensation and make it reach the periocular position. It is one of characteristic techniques from Gansu Zheng's acupuncture academic school. The main point of its performance is the cooperation between pressing hand and needle-holding hand. The pressing hand is valued to promote periocular arrival of needling sensation, unblock periocular meridian, replenish qi to improve eyesight and activate blood to dissipate stasis. It is clinically used to treat eye diseases such as myopia, amblyopia, xeroma and scleritis.

Objective To observe effects of Heyutai Fuzhu Jiangtang Tablets combined with metformin in insulin resistance (IR); To discuss its mechanism of action. Methods 6–7 week old male ZDF (fa/fa) rats were randomly divided into model group,metformin group,Heyutai Fuzhu Jiangtang Tablets group(Jiangtang Tablets group),and metformin combined with Heyutai Fuzhu Jiangtang Tablets group.ZDF(fa/+)rats were chosen as normal group.Each medication group was given relevant medicine for gavage for 6 weeks. Body weight, FBG, TG, TC, FFA, FINS, HOMA-IR, OGTT and HE staining were tested. HE staining was used to observe the pathological changes of skeletal muscle. RT-PCR and Western blot were used to detect skeletal muscle corresponding gene and protein expression. Results Compared with Jiangtang Tablets group and metformin group, TC, FFA, FBG, and HOMA-IR in metformin combined with Heyutai Fuzhu Jiangtang Tablets group decreased significantly (P<0.05, P<0.01). Blood glucose level and AUC significantly decreased at each time point in OGTT. HE staining of skeletal muscle fibers arranged in order; nucleus increased and internal movement was not significant, without obvious infiltration of inflammatory cells. Expressions of skeletal muscle InsR, Akt, and Glut4 mRNA expression increased (P<0.05, P<0.01). Expressions of skeletal muscle p-InsR, p-Akt, and Glut4 protein expression increased (P<0.05, P<0.01). Conclusion Heyutai Fuzhu Jiangtang Tablets combined with metformin can improve IR in type 2 diabetic rats, and the effect is better than single-application.

Objective:To detect the expression of HPV-DNA and P16 protein in laryngeal cancer,laryngeal benign lesion tissue,and vocal cord polyp tissue,and to explore the correlation between HPV infection and laryngeal cancer.Methods:The clinical data of 256 patients who had been treated in Department of Otorhinolaryngology & Head and Neck Surgery of Jinshan Hospital,Fudan University from May 2011 to July 2015 were collected.There were 34 cases of laryngeal squamous cell carcinoma,15 cases of benign laryngeal lesions (non-vocal cord polyps),207 cases of vocal cord polyps,184 males and 72 females,with a median age of 56 years.The levels of HPV-DNA in laryngeal squamous cell carcinoma tissues and benign laryngeal lesions were detected by real-time fluorescence PCR.Immunohistochemical staining was used to detect the expression of P16 protein in laryngeal cancer tissues,laryngeal benign lesions and vocal cord polyps.Results:Real time fluorescence PCR detection showed that the HPV DNA positive rate in 34 cases of laryngeal squamous cell carcinoma was 8.8％ (3/34),and it was all negative (0/15) in benign laryngeal lesions.There was no significant difference in the HPV-DNA positive rate between the two groups.Immunohistochemical staining showed that the positive expression rate of P16 protein in laryngeal squamous cell carcinoma,benign laryngeal lesions and vocal cord polyp tissues was 32.4％ (11/34),46.7％ (7/15),and 47.8％ (99/207).The positive rate of P16 protein in vocal cord polyp tissues was higher than that of laryngeal cancer tissues,and the difference was statistically significant (P=0.046).Conclusions:The positive rate of HPV-DNA in laryngeal cancer tissues is slightly higher than that in benign laryngeal lesions.The expression of P16 protein in laryngeal cancer tissues is slightly lower than that in benign laryngeal lesions,and is significantly lower than that in vocal cord polyps.

Objective To determine the efficient cut-off points of fasting fingertip blood glucose test for undiagnosed diabetes mellitus(DM), impaired glucose tolerance(IGT), and impaired fasting glucose(IFG)in community-based residents aged above 45 years old. Methods A cluster-randomized study was conducted from May 2008 to January 2009. A total of 3250 subjects aged above 45 years in two communities of Baoding city received questionnaire investigation and tested for fingertip blood glucose. Those subjects whose capillary blood glucose level ≥5.1 mmol/L were subjected to 75 g oral glucose tolerance test. Undiagnosed diabetes mellitus and pre- diabetes were identified by fasting plasma glucose and OGTT. In this study, the cut-off points of fasting capillary blood glucose for detecting undiagnosed diabetes and pre-diabetes were evaluated, using receiver operator characteristic curve(ROC). Results Of 1351 subjects that having had oral glucose tolerance test, 230 cases were diagnosed as diabetes mellitus(7.3%), 166 cases(5.2%)as IFG, and 204(6.7%)as IGT under fasting capillary blood glucose as test variable and state variables according to the following criteria.(1)FPG≥7.0 mmol/L or/and 2hPG≥11.1 mmol/L(2)FPG<5.6 mmol/L (3)FPG<7.0 mmol/L and 7.8 mmol/L≤2hPG≤ 11.1 mmol/L, areas under three ROC curves were 0.905, 0.633 and 0.719, respectively. The cut-offvalues of screening for undiagnosed DM, IGT and IFG were 6.0 mmol/L, 5.7 mmol/L, and 5.7 mmol/L, respectively. When cut-off value of screening for undiagnosed DM was 6.0 mmol/L, the maximal sensitivity was 78.0% and specificity was 89.3%.But there were both lower sensitivity and specificity in screening for IFG and IGT according to the best predicting value(5.7 mmol/L)from the ROC curves(50.3% and 28.0% vs. 60.8% and 28.0%). Conclusion Fasting capillary blood glucose with the lower cut-point of 6.0 mmol/L in screening for undiagnosed diabetes mellitus alone, was relatively reliable, whereas for both IFG and IGT the fasting fingertip blood glucose tests were fallible. It was convenient and could be used in screening the DM at the community level.

Objective The aim of this study is to investigate the macrolide resistance rate and molecular type withmultiple-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae of Beijing in 2016 in pediatric patients. Methods Real-time quantitative polymerase chain reaction (PCR) was used to identify M. pneumoniae, and MLVA was performed. The domain V of the 23S rRNA was sequenced to detect macrolide-resistant point mutations. We also investigated the activities of antibiotics against M. pneumoniae isolates in vitro. Results The PCR detection rate of M. pneumoniae in children in Beijing was 40％, and the macrolide resistance rate was 66％. The A2063G mutation in the 23S rRNA V region is the dominant mutation (137/146, 93.84％), whereas the A2064G mutation is rare (9/146, 6.16％). Seventy-three samples were typed successfully by MLVA typing, including 86.3％ (63/73) were MLVA type 4-5-7-2, and 13.7％ (10/73) were MLVA type 3-5-6-2. No other types were found. No strains were resistant to levofloxacin or tetracycline. Conclusion In 2016, a specific decrease in the macrolide resistance rate occurred in Beijing. The detection rate and macrolide resistance rate of outpatients are lower than those of inpatients. The A2063G mutants M. pneumoniae have high levels of resistance to erythromycin and azithromycin. The primary MLVA type is 4-5-7-2, followed by 3-5-6-2. No other MLVA types were detected. No strains resistant to tetracycline or levofloxacin were found in vitro.

Aim To observe the inhibitory effect of neferine(Nef)on the migration and invasion of non-small cell lung cancer(NSCLC)H1299 cells by blocking ROCK pathway.Methods H1299 cells were taken for in vitro culture, and treated with different concentrations of Nef.H1299 cell viability was measured by CCK-8 method to determine the dose of the experimental group.The migration and invasion abilities of H1299 cells were detected by cell scratch test and Transwell chamber test.The expression of matrix metalloproteinases MMP-2 and MMP-9 secreted from lung cancer cells was detected by enzyme linked immunosorbent assay(ELISA).The protein level of ROCK1 in H1299 cells was tested by real-time fluorescent quantitative PCR and Western blot; the binding mode and affinity between Nef and ROCK1 were stimulated by AutoDock semi flexible docking method.Results The doses of Nef in the experimental group were determined as 4, 6 and 10 μmol·L-1.These three concentrations of Nef could inhibit the migration and invasion of H1299 lung cancer cells to a certain degree in a dose-dependent manner.At the same time, Nef reduced the expression of MMP-2, MMP-9 and ROCK1 proteins related to the migration and invasion of the cancer cells.In addition, the affinity of Nef to ROCK1 was significantly higher than that of fasudil, an inhibitor of ROCK, and the binding force was stronger to A-chain of ROCK1.Conclusions As a potential natural anticancer compound, Nef can inhibit the migration and invasion of NSCLC by reducing the expression of MMP-2, MMP-9 and ROCK1 proteins related to the migration and invasion of the cancer cells.