High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization

BACKGROUND: Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD. RESULTS: Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20 C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105. 81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression. CONCLUSIONS: After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.

Association between bullying and mental resilience in high grade pupils / 中国学校卫生

Objective@#To examine the status of bullying behaviors and psychological resilience of primary school students and the relationship between the two, and to provide references for related interventions.@*Methods@#Olweus bullying questionnaire and adolescent psychological resilience scale were used to select students in grades 4-6 for questionnaire survey.@*Results@#Primary school students who were bullied, bullied others, double-involved and uninvolved accounted 33.3%, 48.7%, 15.3%, and 2.7%. There were statistically significant differences in bullying in different dimensions of different genders, grades, and previous suicidal thoughts (P<0.05); there were statistically significant differences in the bullied status of only children, divorced parents, and different caregivers (t/F=3.64，2.65，6.62，P<0.05); The status of bullying behavior of students in the class was better than that of ordinary students (t=-2.18, P<0.05); senior students have a higher level of positive cognition (F=8.71, P<0.05); mental toughness of urban students better than rural areas (t=-2.20, P<0.05); students with divorced parents had poor levels of emotional control, family support, and psychological toughness (t=-4.60，-3.92，-3.44，P<0.05); class cadres focused on focus, positive cognition, family support, interpersonal assistance, and psychological resilience were better than ordinary students (t=5.19，6.43,5.64,4.13，6.58，P<0.05); students with parental care had better levels of target concentration, emotional control, family support, interpersonal assistance, and psychological resilience (t=4.59，8.68，8.76，12.35，13.45，P<0.05); the level of psychological resilience of students was low (t=15.90，P<0.05). Correlation analysis shows that there was a negative correlation between bullying behavior and psychological resilience of all pupils (P<0.01), regression analysis showed that bullying behavior may had a negative prediction of mental resilience (F=128.37, R2=0.12, P<0.01).@*Conclusion@#The current situation of bullying behavior and mental resilience of grade 4-6 students is worrisome, and it is urgent to improve the current situation of bullying and psychological resilience of primary school students.

Complete genome sequence of human pathogen Kosakonia cowanii type strain 888-76T

ABSTRACT Kosakonia cowanii type strain 888-76T is a human pathogen which was originally isolated from blood as NIH group 42. In this study, we report the complete genome sequence of K. cowanii 888-76T. 888-76T has 1 chromosome and 2 plasmids with a total genome size of 4,857,567 bp and C+G 56.15%. This genome sequence will not only help us to understand the virulence features of K. cowanii 888-76T but also provide us the useful information for the study of evolution of Kosakonia genus.

Effects of sevoflurane and isoflurane on blockade of adult type nicotinic acetylcholine receptors of skeletal muscle of rats by rocuronium / 中华麻醉学杂志

ObjectiveTo investigate the effects of sevotlurane and isoflurane on blockade of adult nicotinic acetylcholine receptor (ε-nAChR) of skeletal muscle of rats by rocuronium.MethodsHEK293 cell line was provided by Molecular Biology Laboratory,Chonqing Medical University and cultured in DMEM liquid culture medium containing 10％ bovine calf serum at 37 ℃in incubator filled with 5％ CO2.ε-nAChR was expressed heterologously in HEK293 cell using liposome transfection technology.The whole cell patch clamp technology was used to establish the concentration-effect curves for inhibition of acetylcholine (ACh)-induced current.The 5％ inhibitory concentration values ( IC5 ),25 ％ inhibitory concentration values ( IC25 ) and 50％ inhibitory concentration values (IC50) for sevoflurane,isoflurane and rocuronium were calculated.Firstly the inhibitory effect of rocuronium at a concentration of IC25 was studied in the presence of sevoflurane and isoflurane at concentrations of IC5,IC25 and IC50.Subsequently the inhibitory effect of rocuronium at concentrations of IC5,IC25 and IC50 was studied in the presence of sevoflurane and isoflurane at a concentration of IC25.Inhibition of ACh-induced current amplitude was recorded.ResultsAt the concentrations of IC5,IC25 and IC50 sevoflurane or isoflurane had synergistical inhibitory effect on the current amplitude of ε-nAChR with rocuronium at the concentrations of IC5 and IC25,while had addition inhibitory effect on the current amplitude of ε-nAChR with rocuronium at the concentration of IC50.The inhibitory effect of sevoflurane at the concentration of IC5 on the current amplitude of ε-nAChR with rocuronium at the concentration of IC25 was weaker than that of isoflurane,while the inhibitory effect of sevoflurane at the concentration of IC50 on the current amplitude of ε-nAChR with rocuronium at the concentration of IC25 was stronger than that of isoflurane.The inhibitory effect of sevoflurane at the concentration of IC25 on the current amplitude of ε-nAChR with rocuronium at the concentration of IC5 was weaker than that of isoflurane.ConclusionEither sevoflurane or isoflurane has synergistical blocking effect on ε-nAChR with low concentration of rocuronium,while has addition blocking effect with high concentration of rocuronium.Potentiation of blocking effect of rocuronium on ε-nAChR by low concentration of sevoflurane is weaker than that by isoflurane,while the potentiation by sevoflurane at high concentration is stronger than that by isoflurane.

Effects of hFRNK on human colon cancer cell invasion induced by gastrin / 中华消化内镜杂志

Objective To study the of effets of hFRNK gene transfected by adenoviral vector on human colon cancer cell invasion induced by gastfin.Methods The subjects were divided into three groups:control,G17 and hFRNK group.The G17 group was treated with 100 umol/L gastrin 17 for 12 h to induce Col0320WT cells.As to hFRNK group,Colo320WT cells were infected by pAdhFRNK(MOI:100)for 48 h after transient transfection of pCR3.1-CAR for 48 h and subsequently treated with gastrinl7 for 12 h,and the control group was untreated Colo320WT cells.Expression of phosphorylated FAK(PY397)were assayed by western blot.FAK(PY397)at lamellipoda was observed with a confocal microscope.The influence of hFRNK on formation of signal complex of FAK-Src-p130Cas-Dockl80 was assayed with coimnlunoprecipitation and immunity blotting.Activity of Rac-GTPase was determined by pull down.Results Phosphorylated FAKTyr397 drastically increased with the induction of gastrin.Compared with that of G17 group,FAK (PY397)expression decreased and little FAK(PY397)was found at lamellipoda,at the same time,the signal complex of FAK-Src-p130Cas-Dockl80 did not form,and the activity of Rac decreased.Conclusion hFRNK gene may block gastrin-induced FAK phosphorylation,prevent formation of the signal complex,prevention and treatment of tumor invasion and metastasis.