Construction of seamless genome editing system for fast-growing Vibrio natriegens / 生物工程学报

Recently, fast-growing Vibrio natriegens, as the great potential chassis, has shown a wide application in synthetic biology. Genome editing is an indispensable tool for genetic modification in synthetic biology. However, genome editing tools with high efficiency and fidelity are still to be developed for V. natriegens synthetic biology. To deal with this problem, the physiological characteristics of 6 V. natriegens strains were evaluated, and CICC 10908 strain with fast and stable growth was selected as the host strain for genome editing study. Then, the natural transformation system of V. natriegens was established and optimized. The efficiencies of optimized natural transformation that integrates antibiotic resistance marker cat-sacB or Kan(R) onto the chromosome of V. natriegens could reach 4×10⁻⁵ and 4×10⁻⁴, respectively. Based on the optimized natural transformation, a double-selection cassette was used to achieve seamless genome editing with high efficiency and fidelity. The positive rates of four different types of genetic manipulation, including gene deletion, complementation, insertion and substitution, were 93.8%, 100%, 95.7% and 100%, respectively. Finally, transformation and elimination of the recombinant plasmid could be easily achieved in V. natriegens. This work provides a seamless genome editing system with high efficiency and fidelity for V. natriegens synthetic biology.

Directed evolution of tyrosine ammonia-lyase to improve the production of p-coumaric acid in Escherichia coli / 生物工程学报

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.

Association of neck circumference with waist circumference and body mass index in the elderly and its value in diagnosing obesity / 中华老年医学杂志

Objective To investigate the association between the neck circumference and central obesity in the elderly.Methods The neck circumference(NC),height,weight,waist circumference (WC) and hip circumference(HC) of 2820 subjects aged over 60 years were detected from some communities in Nanchang.All subjects were divided into different groups,according to the two different obesity indicators of WC and BMI.The relationship between NC and obesity was analyzed.A multiple linear regression analysis was conducted using the Enter method.An R()C curve analysis was employed to judge the tangent point of NC in central obesity.Results The height,weight and BMI were reduced along with age (all P＜0.05),and the waist hip ratio(WHR) was increased along with age(P＜0.01) in the elderly.The NC was decreased along with age in males(P＜0.01),and the WC was increased along with age in females(Pt＜0.01).The NC was longer in males than in females [(36.5±3.0) cm vs.(32.6±2.4) cm,P＜0.01].The NC was increased along with body weight,WC and BMI in the elderly:with each 1 kg increase in body weight,the NC was increased by 0.079 cm in males and 0.139 cm in females;with each 1cm increase in WC,the NC increased by 0.073 cm in males and 0.040 cm in females;with each 1 kg/m2 increase in BMI,the NC increased by 0.121 cm in males and 0.065 cm in females;with 1 year increase in age,the NC was decreased by 0.033 cm in males(all P＜0.05).when obesity was diagnosed according to both BMI and NC,the NC was longer in the obesity group than in the control group,(P ＜ 0.01).The area under receiver operating characteristic(ROC) curve of NC for diagnosing central obesity showed that the area was 0.802 and 0.743 in males and females,and the optimal cut-off value of NC was 36.85 cm and 31.75 cm in males and females,respectively.Conclusions NC is increased along with the increases of body weight,WC and BMI in the elderly.NC is significantly associated with central obesity.NC is more predictive of the central obesity when the value ＞36.85 cm in males and ＞31.75 cm in females.

Relationships between the neck circumference and cardiovascular risk factors of middle-aged and elder people in community / 中华内分泌代谢杂志

Objective To investigate the relationships between the neck circumference and cardiovascular risk factors of middle-aged and elder people in a community. Methods Altogether 9 665 community residents ( age≥40 years) were enrolled in the present study. The height, weight, neck circumference, waist circumference, blood pressure, total cholesterol, triglycerides, low density lipoprotein-cholesterol ( LDL-C ) , high density lipoprotein-cholesterol ( HDL-C) , blood glucose, glycosylated hemoglobin, and fasting insulin were measured, and documented in the questionnaire along with other basic information. After adjusted for age, Pearson correlation coefficient was used to analyze the relationship between the neck circumference and the binary variables of cardiovascular risk factors. SPSS16.0softwarewasusedforstatisticalanalysis. Results (1)Theneckcircumferenceswere(37.11±2.79)cm in male and (32. 79 ± 2. 31) cm in female;which were higher in males than females. In both males and females, after adjusted for age, the neck circumference was positively correlated to waist circumference, body mass index, blood pressure, plasma glucose, LDL-C, triglycerides, and insulin resistance, the neck circumference was negatively correlated to HDL-C(all P0. 05). (2) After adjusted for body mass index and the waist circumference, the neck circumference of males increased with the risk of diabetes ( OR=1. 14, 95% CI 1. 02-1. 28);while the higher neck circumference of females increased the risk of hypertension (OR=1.17,95% CI1.08-1.27),diabetes(OR=1.24,95% CI1.13-1.37),dyslipidemia(OR=1.17,95%CI 1.09-1.27),andinsulinresistance(OR=1.15,95% CI1.06-1.26). Theneckcircumferenceexertedhigher impact on women than it did on men. Conclusion The neck circumference is associated with the cardiovascular risk factors. This association in females is stronger than that in males. The neck circumference seems to be an independent indicator for the cardiovascular risk.

Relationship of parity and other factors with metabolic syndrome in middle-aged and elderly women in Nanchang community / 中华内分泌代谢杂志

Objective To investigate the relationship of parity and other reproduction-associated factors with metabolic syndrome(MS) in middle-aged and elderly women in Nanchang community.Methods Female permanent residents over 40 years old were sampled for a routine examination and a questionnaire survey covering basic information,5 894 subjects were enrolled and identified as effective cases.MS was diagnosed according to the standard of China Diabetes Society in 2004.Factors including age,education,profession,marital status,parity,child-bearing history of macrosomia,breastfeeding,menarche age,and menopausal age were analyzed in order to find their relationship with MS.The univariate logistic analysis was followed by binary logistic regression analysis if P＜0.05,and OR value and confidence interval were calculated.Results Univariate logistic regression analysis showed that age,education,profession,parity,breastfeeding,and menopausal age were influencing factors of MS (all P ＜ 0.05).There was no correlation to marital status,miscarriage history,child-bearing history of macrosomia,menarche age with MS(all P＞0.05).Binary Logistic regression analysis showed that the risk of MS increased with age:OR =1.51 (95 ％ CI 0.98-2.32) during 50-59 years,OR =2.10 (95 ％ CI 1.34-3.30) during 60-69 years,and OR =2.63 (95 ％ CI 1.61-4.31) in cases aged over 70 years,as compared to the group aged 40-49 years.The risk in non-manual workers increased about 34 percent OR =1.33 (95 ％ CI 1.04-1.70),compared with manual workers.The risk of MS increased with parity,being OR =1.46 (95％ CI 1.14-1.88),OR =1.78 (95％ CI 1.30-2.43),and OR =1.84 (95％ CI 1.24-2.72),with 2,3,and 4 child-bearings respectively as compared to cases with only one childbearing.The risk of MS was reduced with advancing menopausal age being about 6 percent reduction with each increased year of menopausal age(OR =0.94,95％ CI 0.90-0.99).There was no difference between education,breastfeeding and MS(P＞005).Conclusion Multiple parity,earlier menopausal age,less manual work,and aging seem to be the risk factors of MS in women.

Effects of Stromal Cell-Derived-Factor-1 on Endothelial Progenitor Cells of Peripheral Blood and Their Relationship with PI3K/AKT Signal Transduction Pathway in Patients with Diabetes / 天津医药

Objective To observe the effects of stromal cell-derived-factor-1(SDF-1) on the function of endotheli?al progenitor cells(EPCs)of peripheral blood in patients with diabetes, and to discuss the effects of PI3K/AKT signaling path?way on the role of SDF-1 in EPCs. Methods The peripheral blood samples (30 mL) were collected in 10 diabetes patients (DM group) and 10 healthy controls (HC group). (1) The 100μg/L SDF-1 was added in intervention group. EGM-2MV was added in non-intervention group. The Boyden chamber and in vitro angiogenesis kit were used to analyze the migration and in vitro angiogenesis of EPCs. (2) Cultured EPCs were divided into blank control group, 1μg/L SDF-1 group, 10μg/L SDF-1 group, 100μg/L SDF-1 group, pure AMD3100 group and 100μg/L SDF-1+AMD3100 group. AKT protein expression lev?els of endothelial progenitor cells were detected by Western blot assay in each group. Results (1) Without intervention with SDF-1, EPCs’migration and angiogenesis ability were lower in DM group than those in HC group. After intervention with SDF-1, the migration and angiogenesis ability were enhanced in two groups, but the increased level was higher in DM group than that of HC group. (2) Under the same concentration, AKT protein expression level was significantly lower in DM group than that in HC group (P<0.01). AKT protein expression levels were increased with the increased levels of SDF-1 in DM group and HC group (P<0.05). AKT protein expression was significantly lower in 100μg/L SDF-1+AMD3100 group than that of 100μg/L SDF-1 group (P<0.05). Conclusion SDF-1 can increase the chemotactic migration and angiogenesis ability of EPCs in peripheral blood, especially for patients with diabetes. The effects of SDF-1 on EPCs were related to the PI3K/AKT signaling pathway.

The on-site community investigation and analysis on lifestyle and the present situation of bone mineral density with middle aged and elderly people in Nanchang city / 中国实用护理杂志

Objective To get the facts on lifestyle and bone mineral density among the community residents who are above 40 years old in Nanchang,therefore to offer reference for prevention and treatment to the community residents with abnormal bone mineral density.Methods Use Ultrasound bone mineral density detection instrument to detect the calcaneus mineral density among 10 002 community residents who were over 40 years old.The results can be classified as normal,osteopenia and osteoporosis.A questionnaire survey was conducted among all interviewees.The questionnaire consisted of basic information,general situation,lifestyle,height,weight,waistline,hipline.The body mass index was calculated.The relationship between bone mineral density and smoking,drinking,milk,sodas,sports,etc was analyzed.Results As to osteoporosis,the morbidity of male patients was 6.8％,and that of female patients was 10.4％,as to osteopenia,the morbidity of male patients was 43.8％,and that of female patients was 39.7％.Male smokers' morbidity of abnormal bone mineral density was high,there were no significant differences among the groups,female smokers' morbidity of abnormal bone mineral density was low,there were significant differences among the groups.Drinkers' morbidity of abnormal bone mineral density was low,there were no significant differences among the groups;People taking milk and sodas,taking more exercises suffered from lower morbidity of abnormal bone mineral density,there were significant differences among the groups.Conclusions Female,the elderly,povertyin movement,low intake of milk result in high morbidity of abnormal bone mineral density,while the influence of drinking and smoking on abnormal bone mineral density requires further research.

Isolation,purification and identification of polysaccharide of Cistanche deserticola / 中草药

Object The polysaccharide (CDPS) was isolated, purified and identified from Cistanche deserticola Y. C. Ma, a Chinese materia medica. Methods The polysaccharide was extracted with hot water and precipitated by alcohol. Protein in the precipitates was removed by Sevag method. The products were further purified with column chromatography on DEAE-Sephadex A-50 and Sephacryl S-200. The CDPS was idendified by IR spectrum and UV (200-400 nm) scanning spectrum. Results IR spectrum indicates that there are typical characteristic absorption peaks of polysaccharides. UV scanning spectrum shows that there are no absorption peaks of protein and nucleic acid at point 280 nm and 260 nm. Conclusion The CDPS was identified as homogeneous one.