ObjectiveTo summarize the nursing measures for patients with nasal facial soft tissue defect treated with transplantation of free foot skin flap.MethodThe clinical data of 13 patients with transplantation were reviewed to summarize the nursing measures.ResultAll skin flaps of the 13 patients survived.ConclusionEnough preoperative preparation,active psychological nursing and careful postoperative observation are critical for the success of the operation.

Objective To investigate the relationship between single nucleotide polymorphism (SNP) of transcription factor 7-like 2 (TCF7L2) at locus rs7903146,rs290487,rsl1196205,rs 12255372 and genetic susceptibility in women with gestational diabetes mellitus (GDM).Methods As a case-control study,100 pregnant women with GDM and 100 healthy pregnant women in the Maternal and Children Health Hospital of Jiangxi Province were recruited from January 2010 to July 2013.Clinical parameters,including body mass index (BMI),fasting insulin (FINS),fasting plasma glucose (FPG) and homeostatic model assessment-insulin resistance index (HOMA-IR) were measured after admission to hospital.Allelespecific PCR was used to analyze the SNP of TCF7L2 at locus rs7903146,rs290487,rs11196205,rs12255372.Results (1)The BMI,FPG,FINS and HOMA-IR in GDM group were(27.4±3.0) kg/m2,(5.6±1.0) mmol/L,(6.2±3.4) mU/L and 1.8± 1.0,and were (24.2±2.9) kg/m2,(5.3±0.8) mmol/L,(4.5±2.8) mU/L,1.2± 0.8 in the control group,respectively.The differences had statistically significance (P＜0.05).(2)The SNP of TCF7L2 gene,locus rs7903146 were CC,CT and TT genotype; the SNP of locus rs290487 were CC,CT and TT genotype; and the SNP of locus rs1 1196205 were GG and CC genotype; while the SNP of locus rs12255372 was GG genotype.(3)The distribution frequencies of genotype CC,CT and TT at locus rs7903146 in the GDM group were 40％ (40/100),36％ (36/100) and 24％ (24/100),respectively.While in the control group,they were 55％ (55/100),38％ (38/100) and 7％ (7/100),respectively.The frequencies of C and T allele of rs7903146 were 58％and 42％ in the GDM group,and in the control group they were 74％ (148/200) and 26％ (52/200).The differences of genotype distribution and C/T allele frequency of rs7903146 between the two groups were statistically significant (P＜0.05).(4)The distribution frequencies of genotype CC,CT and TT at locus rs290487 in the GDM group were 12 ％ (12/100),36 ％ (36/100) and 52％ (52/100),and were 16％ (16/100),34％ (34/100) and 50％ (50/100) in the control group.The frequencies of C and T allele of rs290487 were 30％ (60/200) and 70％ (140/200) in the GDM group,and were 33％ (66/200) and 67％ (134/200) in the control group.There was no difference of genotype distribution and C/T allele frequency of rs290487 between the two groups (P＞0.05).(5)The distribution frequencies of genotype GG and CC at locus rs1 1196205 in the GDM group were 99％ (99/100) and 1％ (1/ 100),while those in the control group were 100％(100/100) and 0％.The frequency of G and C allele of rs1 1196205 were 99％(198/200) and 1％(2/200) in the GDM group,while in the control group were 100％ (200/200) and 0.There was no difference of genotype distribution and G/C allele frequency of rs11196205 between the two groups (P＞0.05).(6)The distribution frequencies of genotype GG at locus rs12255372 were 100％(100/100) in both the GDM group and the control group.The frequencies of G allele of rs12255372 were 100％ (200/200) in both the GDM group and the control group.There was no difference of genotype distribution and G allele frequency of rs12255372 between the two groups (P＞0.05).(7)After adjusting for age,gestational age,BMI,FPG and FINS,pregnant women with TT genotype at locus rs7903146 were more likely to have hyperglycemia compared with the C allele carriers (OR=2.77,95％ CI:1.03-7.57,P＜0.05).Conclusions The polymorphism of locus rs7903146 in TCF7L2 gene may be associated with genetic susceptibility in women with GDM.TT genotype is likely to be risk factor in the pathogenesis of GDM.

Objective To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ ( StxⅡ) in shiga toxin-producing Escherichia coli ( STEC) infection. Methods A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-anti-body sandwich ELISA. The established ELISA system was used to detect StxⅡin the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated. Results Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-anti-body sandwich ELISA was set up. StxⅡand its variants rather than StxⅠwas detected in the culture super-natants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commer-cial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity. Conclusion The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential can-didate on the diagnosis and therapy of STEC infection.

Shiga toxin 2 (Stx2) toxoid produced by formaldehyde treatment of purified toxin was used to immunize BALB/c mice for monoclonal antibody (MAb) production.The neutralizing activities of positive clones against Stx2 were screened by in vitro cytotoxicity assay.The isotype and specificity of resultant clone was determined,and its efficacy to neutralize the activity of purified Stx2 was evaluated by in vitro and in vivo toxicity model.Lastly,its spectrum of activity against Stx2 variants was also accessed by mouse toxicity model.It was demonstrated that one of the 12 positive MAb clones against Stx2,designating $2C4 had neutralizing activity.S2C4 belongs to the immunoglobulin G1 subclass and has a K light chain,and it reacts with the A subunit of Stx2 and does not bind to Stx2 B subunit or to Stx1.S2CA could efficiently neutralize the cytotoxicity of Stx2 to Veto cells and mice.It also protected mice against lethal doses of Stx2 variants challenge including Stx2c and Stx2vha.S2C4 is a promising candidate molecule in preventing the progression of hemolytic-uremic syndrome (HUS) mediated mainly by Stx2 in Stx-producing Escherichia coli(STEC)infection.

Objective To investigate the eating problems of outpatient infants,preschool age children(1 to 7 years old) enrolled in the Department of Child Health Care,Nanjing Maternal and Child Health Hospital Affiliated to Nanjing Medical University,and to analyze its correlation with children's physical development,so as to establish strategies for preventing abnormal eating habit in children.Methods A toll of 2458 children met the criteria,and caregivers (mothers) completed the Children's Eating Behavior Questionnaires (CEBQ) in Department of Child Health Care,Nanjing Maternal and Child Health Hospital Affiliated to Nanjing Medical University were selected and the children's sociodemographic data and the morbidity of children eating problems were investigated.The correlation between children's body mass index(BMI) with children's eating problems was determined by using Chi-square test and multiple regression analysis.Results About 66.2％ (1627/2458 cases) had normal weight,and 10.8％ (257/2458 cases) and 8.5 ％ (210/2458 cases) were overweight (BMI ＞ P85-P95) or obese (BMI ≥ P95),respectively.The prevalence of eating behavior problems was detected during 25-36 months.For 1-to-7-year-old children,the highest detection rate of eating problems was inattention and eating at non-permanent locations,occupying 64.7％ (1590/2458 cases)and 50.5％ (1241/2458 cases),respectively;the prevalence rate of preferring to junk food was the lowest,accounting for 19.3％ (474/2458 cases).The children's eating problems had a high association with the children's BMI.Among them,children with eating problems,such as difficultly in accepting the varying food stuff[at the age of 12 month,odds ratio(OR)=11.50,95％ confidence interval(CI):1.84-72.16] and eating at non-permanent locations(at 25-36month,OR=1.77,95 ％ CI:1.11-2.83),were prone to be wasting away;children with eating problems,such as preferring to junk food (at 12 month,OR=5.08,95 ％ CI:1.43-18.00;13-18 month,OR=2.17,95 ％ CI:1.06-4.44),rarely eating vegetables or fruit (19-24 month,OR=4.06,95％CI:1.46-11.31) and inattention (12 month,OR=3.85,95 ％ CI:1.52-9.79),were associated with overweight or obesity (all P＜0.01).Conclusions There was a high prevalence of eating problems in children between 12-84 month(1-7 years old) in Nanjing.Improper children's eating behaviors can increase the risks of wasting away or children's overweight/obesity.

Objective To evaluate the practicability of using CRISPR/Cas9 genome editing tech-nology for inhibition of hepatitis B virus ( HBV) replication. Methods Two sgRNA targeting sites were de-signed for the S region of HBV genome. The CRISPR/Cas9 expression plasmids specific for HBV were con-structed and then transfected into a cell line expressing HBV genome(HepG2-N10). The cytotoxicity of cells transfected with different expression plasmids were detected by MTT assay. The levels of hepatitis B surface antigen ( HBsAg ) were determined by using chemiluminescent immunoassay ( CLIA ) . The expression of HBV at mRNA level was analyzed by quantitative real-time PCR ( qRT-PCR) . The qPCR was performed for the detection of extracellular and intracellular HBV DNA. The next-generation sequencing ( NGS) Illumina MiSeq Platform was used to analyze HBV genome editing. Results No significant cytotoxic effects were de-tected in HepG2-N10 cells transfected with different expression plasmids. Compared with the cells carrying pCas-Guide-GFP-Scramble, the levels of HBsAg in the supernatants of transfected cell culture harboring pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were decreased by 24. 2% (P<0. 05) and 16. 9% (P>0. 05), respectively. The levels of HBsAg in cells transfected with pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were respectively decreased by 16. 4% (P>0. 05) and 32. 1% (P>0. 05) as compared with that of pCas-Guide-GFP-Scramble transfected group. The expression of HBV at mRNA level was inhibited as indica-ted by the results of qRT-PCR. Moreover, the levels of extracellular HBV DNA were respectively suppressed by 23% (P>0. 05) and 35% (P<0. 05), and the levels of intracellular HBV DNA were respectively sup-pressed by 7. 2% (P>0. 05) and 18% (P>0. 05). Different types of insertion/deletion mutation were de-tected in HBV genome by high-throughput sequencing. Conclusion HBV-specific CRISPR/Cas9 system could inhibit the expression of HBV gene and the replication of virus. Therefore, the CRISPR/Cas9 genome editing technology might be used as a potential tool for the treatment of persistent HBV infection.

Objective To purify Shiga toxin Ⅱ (STX Ⅱ) of enterohaemorrhagic Escherichia coli (EHEC) O157: H7 by affinity chromatography, and characterize its biological function. Methods The immno-affinity chromatography column was prepared by STX Ⅱ A subunit-specific antibody S1D8 coupling to Sepharose 4B matrix. The purity and specificity of STX Ⅱ molecule secreted by EHEC O157:H7 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, respectively. The purified toxin was serially diluted and the toxic activities to Vero cell line and mice were observed. The 50% cytotoxic dose (CD50) for Vero cell line and 100% lethal dose (LD100) for mice were calculated. The protection effect of anti-STX Ⅱ polysera to the mice against the purified toxin challenge was also observed. Results STX Ⅱ was successfully purified from culture supernatant of EHEC O157:H7 using affinity chromatography scheme. The relative molecular weights of STX Ⅱ A and B subunits were 32 000 and 7 500 confirmed by SDS-PAGE, respectively. The purified toxin could react with monoclonal antibodies against STX Ⅱ A and B subunits, respectively.The toxin was cytotoxic to Vero cell with CD50 of 20 ng/L and lethal to mice with LD100 of 5 ng.The toxin could be neutralized by anti-STX Ⅱ polysera in vivo. Conclusion STX Ⅱ is successfully purified and its toxic effects are confirmed in both cell line and mouse model.

OBJECTIVETo understand the Escherichia coli O157:H7 carrier rate of host animals and the toxic gene of the strains in different areas in Jiangsu province.

METHODSSurveillance spots were set up in different areas, to collect feces of pigs, chickens, sheep, cattle to culture for O157:H7 with immunomagnetic separation as well as detection of toxic gene of the strain with MPCR were both carried out.

RESULTSOne hundred and seventy strains of O157:H7 were separated from 1 767 feces of different animals in six spots, with a overall positive rate 9.62%. The positive rates of cattle and sheep were 19.05% and 12.01% respectively. Among 85 strains SLT1, SLT2, eaeA and hly toxic genes were detected. In which, 56.47% of the strains were positive curturely while 79.17% of them carried SLT2, eaeA and hly gene simultaneously.

CONCLUSIONThe positive rate of O157:H7 in animals and the positive rates of strains were correlated to the incidence of the area. The highest rates were seen in areas where there had been O157:H7 epidemic, followed by the areas where there were only scattered cases identified while the lowest was in areas with no patients. Data indicated that it was important to enforce the surveillance of O157:H7 in animals to better predict and control of the disease.

Animals ; Cattle ; microbiology ; Chickens ; microbiology ; China ; Escherichia coli O157 ; isolation & purification ; Rabbits ; microbiology ; Sheep ; microbiology ; Swine ; microbiology ; Time Factors