Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.
Chromosomes, Bacterial ; genetics ; DNA ; Endonucleases ; metabolism ; Escherichia coli ; genetics ; Genetic Engineering ; methods ; Recombination, Genetic ; Sequence Deletion

Objective To investigate the involvement of mitogen-activated protein kinase(MAPK)and PI3K(phosphatidylinositol 3 kinase)/ Protein kinase B(Akt) signal transduction pathways in the mechanism of myocardium remodeling in patients with congestive heart failure(CHF).Methods Thirty nine patients of mitral valve disease with CHF were randomly selected and 30 cases of healthy persons were included as controls.Cardiac function parameters were measured by echocardiography.Concentration of AngⅡ in plasma and myocardial tissues was determined by radio immunoassay.Activity of PKC was determined by using competive prote in binding method,activity of MAPK was detected by the methods of immunoprecitipation.Immunoprecitipation was used to assay the protein expression and phosphorylation of PI3K and Akt(Protein kinase B),protein expression of C-FOS and ?-skeletal-actin in myocardial tissues.Results Pathological changes of myocardial tissues in CHF with valvular heart diseaseshowed typical myocardial remodeling.The hypertrophy was dominant at early stagy of CHF,while at end stage the characteristics include disordered alignament of the myocytes,the discontinuity and dissolving of cardiomyofibrills,destroyed subcellular organs,and the hyperplasia of interstitial tissue.AngⅡ concentration in plasm and myocardial tissues in patients with CHF was higher than those in the control group(P

Objective · To obtain the latest data on phospholipid composition of human milk in Shanghai and compare the differences in phospholipid composition at different lactation stages. Methods · Healthy postpartum women who delivered full-term infants in the Obstetrical Department of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine between April and July, 2016 were enrolled. The colostrum, transitional milk, and mature milk were collected at Day 3, 10, and 45 after delivering babies, respectively. Human milk fat was extracted with Folch's method and phospholipids were separated with solid phase extraction (SPE). The phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were quantitatively analyzed with HPLC/VWD. The differences in phospholipid composition at different lactation stages were compared with univariate analysis of variance and Games-Homell test. Results · One hundred women who provided at least one breast milk sample were enrolled. A total of 70 colostrum samples, 96 transitional milk samples, and 82 mature milk samples were collected. The total phospholipid content of mature milk [(281.93±118.54) μg/g] was significantly lower than that of colostrum [(381.99±205.90) μg/g]. At all lactation stages, the relative content of phosphatidylcholine was the highest (53.74%-59.36%), followed by sphingomyelin (28.12%-32.74%). The relative content of phosphatidylethanolamine was constant (P=0.617), the relative content of phosphatidylcholine gradually decreased (P=0.000), and that of sphingomyelin gradually increased (P=0.000) during the lactation. Conclusion · Sphingomyelin and phosphatidylcholine are major components of human milk phospholipids. The amount of phospholipids varies during the lactation. The total amount of phospholipids is lower in mature milk than in colostrum and transitional milk. The relative content of phosphatidylethanolamine is consistent at all lactation stages, the relative content of phosphatidylcholine gradually decreases, and that of sphingomyelin gradually increases.

Objective To determine the retest reliability and measurement error of walking energy consumption measurement in children with cerebral palsy in special school. Methods 13 children with cerebral palsy studying in Shanghai Pudong Special Education School from September to December in 2010 were enrolled in this study. They were asked to walk up and down continuously on a 50-meter-long walking pace in 6 minutes, while the distance of walking and the heart rate of both rest and walking were measured to calculate physical cost index (PCI). After 1 week, they took the 2nd measurement at the same time, the same location and the same condition. Then the results between the former and the later measurements were compared to identify the retest reliability and measurement error. Results The study showed a good retest reliability in heart rate of walking, distance of walking and PCI (ICC=0.77~0.83), but a low retest reliability in heart rate of rest (ICC=0.38). The study also showed a high measurement error in PCI. Conclusion PCI had good retest reliability and high measurement error, and was easily affected by the emotion of the children. So it was limited as a measurement of walking energy consumption in children with cerebral palsy.

Objective To investigate the relationship on the excision repair cross complementing gene 1(ERCC1)‐4533/8092 site single nucleotide polymorphisms(SNPs) and the susceptibility to hepatocellular carcinoma(HCC) in Guangxi Zhuang population . Methods Polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method was used to detect the ER‐CC1‐4533/8092 gene polymorphism in 88 cases with primary liver cancer and 82 cases of normal controls .Results There was no difference in the frequency distribution of ERCC1‐4533 in the case group and the control group ,the frequency distribution of the ERCC1‐8092 in the case group and the control group was different(P< 0 .05) .Compared with ERCC1‐8092 CC ,ERCC1‐C8092 CA/AA had higher risk of primary hepatocellular carcinoma(CA :OR=2 .556 ,95% CI:1 .345 -4 .855;AA :OR= 8 .667 ,95% CI:1 .000-75 .092) .ERCC1‐8092 C allele as a reference ,ERCC1‐8092 A allele can increase the risk of primary liver cancer (OR=2 .387 ,95% CI:1 .428-3 .992) .Conclusion The genetic polymorphisms of ERCC1‐8092 sites are associated with susceptibility to hepatocellular carcinoma in Guangxi Zhuang population .

Objective To analyze the efficacy and safety of DCF and XELOX regimens in the treatment of advanced gastric cancer and to explore the appropriate chemotherapy regimen for advanced gastric cancer.Methods 63 patients with advanced gastric cancer were divided into two groups.Group A (31 patients) was administered with DCF regimen,with docetaxel 60-75 mg/m2 on day 1,5-fluorouracil 500 mg/m2 on day 1 to day 5,cisplatin 75 mg/m2 on day 1,a total cycle of 21 days.Group B (32 patients) was performed with XELOX regimen,with oxaliplatin 130 mg/m2 on day 1,capecitabine 100 mg/m2 twice a day on day 1 to day 14.Results 63 cases were eligible to analyze the efficacy and adverse reactions.The efficient rate (PR+CR) of group A and B were 58.1％ and 62.5 ％,respectively.The median survival time were 10.9 months and 11.5 months,but there were no significant difference between the two groups (P ＞ 0.05).The patients in both groups showed the similar tolerance of adverse reaction.Bone marrow suppression above level 3 in group A (16.1％) was higher than that in group B (9.3 ％).Hair loss above level 2-3 in group A was higher (77.4 ％).Hand-foot syndrome in group B (68.8 ％) was higher than that in group A (9.6 ％).Mild liver function damage in group B (37.5 ％) was higher than that in group A (16.1％).Conclusion The DCF and XELOX schemes have the similar effect in the treatment of advanced gastric cancer with the tolerate side effect.

Objective To identify the reliability and validity of Chinese version of expanded and revised Gross Motor Function Classification System (GMFCS E&R). Methods 101 children with cerebral palsy aged 6～18 from 2 special schools in Shanghai and Guangzhou participated in this study. The interrater reliability was identified by analyzing the assessment results among different raters, including rehabilitation doctors, physical therapists, teachers and parents. Gross Motor Function Measure (GMFM) was used as the criterion to identify the parallel validity. Results GMFCS E&R had good interrater reliability (ICC=0.79～0.91) as well as the parallel validity (Spearman rank correlation coefficient is -0.46～-0.86). Conclusion Chinese version of GMFCS E&R has good reliability and validity. It is suitable for children with cerebral palsy as the tool of function classification in China.

Objective To explore the effect of colposcopy and LEEP in the treatment of cervical precancerous lesions. Methods A total of 96 patients with cervical precancerous lesions by biopsy directed under colposcopy were operated on by LEEP directed under colposcopy,the average operation time and the average blood loss,the diagnostic accuracy between biopsy directed under colposcopy and LEEP directed under colposcopy were compared. Results The average operation time was (6±0.9)min and the average blood loss was (11.3±2.6)mL.Among 96 cases,88 colposcopic biopsy cases had the same histopathology with LEEP biopsies. Among 96 cases who were followed up by TCT three months later,97.9% of the patients showed normal appearance,and it was up to 100% six months after operation. Conclusion Colposcopy combined LEEP has a high clinical value which has small trauma,less bleeding, short time and remove completely,and it is effectively avoided the excessive medical treatment and lack of treatment,so it is worth to popularize.

Objective To explore the influence factors on hepatitis B virus (HBV) relapse after nucleos(t)ide analogues (NA) withdrawal in the chronic hepatitis B (CHB) patients who met NA cessation criteria. Methods Eighty-one consecutive CHB patients were treated with NA, 38 with lamivudine (LAM), 25 with adefovir dipivoxil (ADV), 12 with entecavir (ETV), 6 with LAM +ADV. Among recruited patients, 40 were hepatitis B virus e antigen (HBeAg) positive, 41 were HBeAg negative, 67 of them were initial treatment, 14 were retreatment due to resistance to NA at baseline. The treatment was discontinued after meeting China therapeutic end-point criteria. HBV DNA, HBV serological markers, alanine aminotransferase (ALT) were measured respectively at baseline, every month before virological response, every 3 months after virological response, every month within first 6 months and every 2 months over 6 months after drugs withdrawal. Twelve probable influence factors on relapse which were sex, age, HBV family history, baseline HBV DNA,baseline HBeAg status, baseline ALT, virological response time, total duration of treatment, duration of additional treatment, the level of hepatitis B virus surface antigen (HBsAg) at cessation therapy,initial treatment or retreatment, drug category were analyzed with univariate, multivariate Cox regression modle and stratified analysis. The cumulative relapse was calculated by the Kaplan-Meier method. Results A total of 36 patients (44. 4%) relapsed within 1 year. Initial treatment or retreatment, HBV family history, virological response time, the level of HBsAg at cessation therapy were independent risk factors. The relapse rate of retreatment was higher than that of initial treatment (78.6% vs 37. 3% , χ2 = 7. 983, P = 0. 005) , those of patients with HBV family history higher than without family history (64. 5% vs 15.0%, χ2 =12. 096,P = 0.002), those of patients obtained virological response within 3 months lower than after 3 months(34. 0% vs 64. 3% , χ2 =6. 823,P=0. 009) , those of patients with HBsAg≤150 μg/L at cessation therapy lower than >150 μg/L(27. 6% vs 53. 8%, χ2=5. 199,P=0. 023). Conclusions Retreatment, HBV family history, later virological response and higher HBsAg level at cessation therapy are risk factors of relapse after NA withdrawal. Such patients should be treated with prolonged duration after meeting end-point criteria to strengthen the efficacy.

Objective To evaluate the efficacy and drug resistance profiles of nucleosides (NA) retreatment in NA experienced chronic hepatitis B (CHB) patients. Methods Totally 104 NA experienced CHB subjects were enrolled in this study.All these subjects had received at least 3 months NA monotherapy and stopped the treatment,and then received NA retreatment for at least one year.The subjects were divided into three groups according to the following criteria:reached the therapy endpoint of China guideline when they stopped NA-naive treatment (group A,n =39); did not reach the therapy endpoint when they stopped NA-naive treatment but hepatitis B virus (HBV) DNA＜1.0× 103 copy/mL (group B,n=33); and with HBV DNA＞1.0× 103 copy/mL (group C,n=32).The efficacy and drug resistance profiles of retreatment were compared among three groups. The effects of baseline alanine aminotransferase (ALT) levels,HBV DNA levels and HBeAg titers on the retreatment efficacies were analyzed. The mutations of HBV P gene were detected by nested polymerase chain reaction (PCR) and direct sequencing.The data were analyzd by Wilcoxon test and x2 test.Results The time to ALT normalization in patients with baseline ALT＜ 1.3 × upper limit normal (ULN) was shorter than that in patients with ALT≥1.3×ULN (2 months vs 4 months; Z=2.281,P=0.023).The time to virological response in patients with baseline HBV DNA＜5 lg copy/mL was shorter than that in patients with HBV DNA≥5 lg copy/mL (1 month vs 2 months; Z=2.054,P =0.040). The time to virological response and ALT normalization in baseline HBeAg negative were both shorter than those in patients with baseline HBeAg positive patents ( 1 month vs 3 months and 2 months vs 4.5 months,respectively; Z=2.580 and 2.304,respectively; both P＜0.05). The subjects in group A achieved virological response and HBeAg seroconversion after retreatment earlier compared to previous NA-naive therapy ([1.61 ± 1.76] months vs [3.48±4.066]months and [3.38 ± 3.34] months vs [9.92-11.22] months,respectively; Z=-2.854 and-1.094,respectively; both P＜0.05).The cumulative HBeAg seroconversion rate in group A was higher compared to those in group B and group C (80.0％ vs 36.8％ and 37.5％,respectively; x2 =4.368 and 5.100,respectively; both P＜0.05).Thirteen patients developed clinical resistance and four of them developed genotypic resistance proved by PCR direct sequencing.Among the patients retreated with the same regimen as previous in the C group,the cumulative resistance rate was highest compared to group A and B (44％ vs 9％ and 0,respectively; x2 =5.019 and 6.588,respectively;both P＜0.05).No resistance was detected in the 14 patients retreated with combined NA treatment without cross resistance.Conclusions For NA experienced CHB patients who fulfill the indication of antiviral therapy,the retreatment should be started as earlier as possible. Retreatment with NA combination without cross resistance can prevent (reduce) the risk of developing drug resistance.