In modern biology and biotechnology research, recombinant gene expression has been the most popular method to obtain the target protein. In recent years, many foreign genes have been efficiently expressed in Escherichia coli. However, proteins encoded by animal, plant or mesophilic microbial genes often lose activities or become denatured within a few hours at regular growth temperatures for E. coli; some other target proteins are toxic to host cells and therefore difficult to be over-expressed. The new T-vector, pEXC-T, was constructed by combining TA cloning and cold-shock induction to obtain high expression levels with low costs. This paper reports the construction of pEXC-T and optimization of induction techniques for gene expression. Two instable proteins were tested and successfully expressed in soluble form by using pEXC vector. The development of pEXC-T offers a convenient technique for the preparations of recombinant proteins to be used in structure/function studies, or as diagnostic markers and medicinal proteins.
Biotechnology ; Cold Temperature ; Escherichia coli ; genetics ; Gene Expression ; Genetic Vectors ; Plasmids ; genetics ; Recombinant Proteins

Objective To investigate the role of hypoxia inducible factor-1α (HIF-1α) and von Hippel-Lindau (VHL) in murine endochondral ossification. Methods The knockout of HIF-1α or VHL gene in murine osteoblasts was accomplished by conditional knockout technique at 4th, 8th and 12th week, and the differences between wild-type group and knock-out group in endochondral ossification were detected by HE staining, micro-CT scanning, trabecular bone area measurement, calcium content measurement, tetracycline fluorescence labeling, Real-time PCR and Western blotting. Results After knockout of HIF-1α gene in osteoblasts, the expression of vascular endothelial growth factor ( VEGF) reduced, the rate of new bone formation stepped down, the content of calcium became less, and the trabecular bone volume decreased (P <0.05) . After knockout of VHL gene in osteoblasts, the expression of VEGF increased, the rate of new bone formation stepped up, the content of calcium became more, and the trabecular bone volume was promoted (P < 0.001). Conclusion During murine endochondral ossification, VHL/HIF-1α signal pathway promotes angiogenesis through the stimulation of VEGF expression, which subsequently accelerates osteogenesis.

Objective To review the diagnosis,monitoring,management and perinatal outcomes of monochorionic monoamniotic (MCMA) twin pregnancy.Methods From July 2010 to August 2013,there were 17 MCMA twin pregnancies diagnosed and delivered in Shanghai First Maternity and Infant Hospital.According to the MCMA management protocol,induced abortion,elective fetal reduction,and anticipant pregnancy were optional.For those anticipant pregnancies,fetal lung underwent maturation promotion at gestational weeks 28; hospitalization with 40 min/day continual fetal electronic monitoring and umbilical blood monitoring twice weekly at gestational weeks 28-30; 40 min continual fetal electronic monitoring twice daily and umbilical blood monitoring once every other day at gestational weeks 30-32; and 40 min continual fetal electronic monitoring three times daily and umbilical blood monitoring once daily at gestational weeks 32-34; and pregnancy ended on time.The presence of umbilical cord entanglement,congenital malformation,intrauterine fetal death,complications exclusive to monochorionic twins (e.g.selective fetal growth restriction,twin reversed arterial perfusion sequence and twin-twin transfusion syndrome) and the perinatal outcomes were retrospectively analyzed.Results Average maternal age of women with 17 MCMA twins was (29.0±2.7) years,and all were primiparas.They were diagnosed at (18.6± 5.5) weeks on average (11 +5-28+1 weeks).Umbilical cord entanglements were detected in all cases by ultrasonography and confirmed postnatally.There were three cases of complications specific to monochorionic twins,including two with selective fetal growth restriction and one with twin reversed arterial perfusion sequence.There were seven women with fetal congenital malformation; four of whom chose induced abortion; one case of anencephaly chose radiofrequency ablation fetal reduction,and the remaining two cases with congenital malformation and ten cases without chose anticipant pregnancy,and there were no abnormal ultrasonography signs during pregnancy.Among these 12 cases,intrauterine fetal death of both fetuses occurred in two cases at 16 and 21+1 weeks gestation and they were aborted.Intrauterine fetal death of a single fetus occurred in one case at 30+2 weeks gestation (another fetus was delivered by emergency cesarean section).There were a total of eleven live births delivered by cesarean section (four by emergency cesarean section) at a median gestational age of (32.7± 1.6) weeks.There were 20 live neonates with a mean birth weight of (1 850±496) g.Sixteen neonates were transferred to the neonatal intensive care unit and hospitalized for (37.9± 16.4) days.Nine neonates suffered from respiratory distress syndrome but were eventually cured.Conclusions MCMA twin pregnancy has high morbidity and mortality.Early ultrasonography helps to clarify the diagnosis and enhance the monitoring,thus improving the perinatal outcomes.

Adolescent ; Adult ; Child ; Elliptocytosis, Hereditary ; diagnosis ; genetics ; Female ; Humans ; Male

Objective To assess the contrast-enhanced ultrasound (CEUS) characteristics of pleomorphic adenoma (PA) and carcinoma ex pleomorphic adenoma (CXPA).Methods A retrospective analyse was made from the contrast-enhanced ultrasonic features of 42 PA and 8 CXPA.The features included tumor size,echotexture,boundary,shape and blood flow richness on conventional US imaging and rise time (RT),time to peak (TP),peak intensity(PI),time from peak to one half(HT),mean transit time (MTT),and area under the curve(AUC) on contrast-enhanced ultrasound.Results All of PA and CXPA were hypoechoic,but there was no difference between them in shape and blood flow richness (P ＞0.05).37.5％ CXPA has hetergeneous echotexture with the presence of anechoic area,but it appeared in only 4.8％ PA(P ＜0.05).The following findings were observed from a perfusion kinetics of CEUS in all of PA and CXPA:slow in,centripetal,heterogeneous enhancement,with no enhancement area.Well-defined margins were presented in 42 PA and in 7 CXPA(P ＜0.05).Their time-intensity curves showed RT,TP,HT and MTT in CXPA group were obviously higher than those in the PA group(P ＜0.05).However there was no statistical difference in PI and AUC between both groups (P ＞ 0.05).Conclusions Conventional US and CEUS perfusion patten provide limited diagnostic information for distinguishing PA from CXPA.CXPA could be diagnosed by ill defined margins,more internal anechoic area and poorly defined margins.But CEUS quantitative analysis can significantly promote the differentiation.

Objective To investigate the pathogen distribution and drug resistance of gram-negative bacteria causing lower respiratory tract infections in patients with acute exacerbation of chronic obstructive pulmonary disease ( AECOPD) .Methods A total of 742 sputum samples were collected from AECOPD patients with lower respiratory tract infections in Taizhou Hospital of Zhejiang Province during January 2013 and December 2014.Vitek 2 Compact system combined with disk diffusion was used for strains identification and drug susceptibility test.Modified Hodge test and ethylenediamine tetraacetic acid (EDTA) synergic test were used to screen carbapenem-resistant strains.Statistical analysis was performed using WHONET 5.6 and SPSS 20.0 software.Results A total of 593 strains of gram-negative bacteria were isolated , in which 367 strains were nonfermentative bacteria (61.89%), 220 strains were enterobacteriaceae (37.10%), and the rest 6 strains ( 1.01%) were other gram-negative bacteria.Acinetobacter baumannii ( 186 strains ), Pseudomonas aeruginosa (99 strains), Stenotrophomonas maltophilia (33 strains) and Burkholderia cepacia (22 strains ) were top 4 nonfermentative bacteria , and they all showed high resistance to ampicillin , ampicillin/sulbactam, cefazolin, ceftriaxone, cefotetan and nitrofurantoin with resistant rates of 67.20%-100.00%. Drug resistance rates to other β-lactams, fluoroquinolones, aminoglycoside, compound sulfamethoxazole and minocycline were significantly higher in carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa strains than those in non-carbapenem-resistant strains , and the differences were statistically significant (P <0.05 or <0.01).Stenotrophomonas maltophilia and Burkholderia cepacia strains were only sensitive to levofloxacin , compound sulfamethoxazole and minocycline . Klebsiella pneumonia (89 strains), Escherichia coli (80 strains) and Enterobacter cloacae (25 strains) were top 3 enterobacteriaceae , which were almost completely resistant to ampicillin , but were sensitive to piperacillin/tazobactam , cefoperazone/sulbactam , amikacin and carbapenems ( with resistant rates <15%) .More than 50%strains of extended spectrum β-lactamases ( ESBLs )-producing Klebsiella pneumonia and Escherichia coli were moderately or highly resistant to ampicillin/sulbactam , most cephalosporins ( except cefotetan and cefoperazone/sulbactam ), gentamycin, tobramycin, fluoroquinolones, aztreonam, compound sulfamethoxazole and minocycline , and the resistant rates were higher than those in non-ESBLs-producing strains (P <0.05 or <0.01).Conclusions Nonfermentative bacteria and enterobacteriaceae are the most prevalent gram-negative bacterial pathogens in lower respiratory tract infections in patients with AECOPD . Carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa strains, as well as ESBLs-producing Klebsiella pneumonia and Escherichia coli strains are highly resistant to most antibacterial agents .

In February, 2019, a patient with a defect of open dorsal cartilage and bone in the first metatarsal head, including the defects of soft tissue, tendon and joint capsule, was treated in our department. After multiple debridement, the vascularised medial femoral condyle osteochondral chimeric tissue flap was transferred to repair the composite tissue defect in the metatarsal head at the second stage. After 18 months of follow-up, the patient felt no pain in the foot and walking, and there was no sign of lameness and discomfort at donor sites. The postoperative functional recovery was satisfactory.

Objective To observe the effect of different intra-abdominal pressure and different time points on hemodynamics,ent-tidal CO_2(P_(ET)CO_2) and airway pressure(Paw) during the procedure of gynecological laparoscopic operations.Methods 60 cases undergoing gynecological laparoscopic operations were randomly divided into two groups:the intra-abdominal pressure was 1.3kPa in groupⅠ(30 cases) and 1.9kPa in groupⅡ(30 cases).ASAⅠgrade.In both groups,systolic blood pressure(SBP),diastolic blood pressure(DBP),mean arterial pressure(MAP), heart rates(HR).S-T.Paw and P_(ET)CO_2 were monitored and recorded before anesthesia(T_0),shortly after intubation (T_1),pre-pneumoperitoneum (T_2),5min after pneumoperitoneum (supine position) (T_3) and 5min (T_4),10min (T_5),20min(T_6),30min (T_7) after trendelenbury position (head down 200) and 5rain after deflation (T_8).Results In both groups SBP,DBP,MAP at time point T_3,T_4,T_5 were increased significantly compared with those of T_0 (P0.05),but there was significant difference in Paw and P_(ET)CO_2 in different time points within the same group and between the same time point in different groups after pneumoperitoneum(P

Objective To evaluate the effects of different doses of lidocaine on suppressing fentanyl-induced cough and determine a safe suppressing dose.Methods Two hundred patients undergoing general anesthesia were randomized to four groups evenly.The following medications were given within ten seconds:normal saline 10ml (groupⅠ,control group),lidocaine 1 mg/kg (groupⅡ),lidoeaine 1.5 mg/kg(groupⅢ),lidocaine 2mg/kg (groupⅣ).Toxic symptoms of lidocaine were recorded within lmin after the administration of lidocaine,then fentanyl 3?g/ kg was given intravenously within 5 seconds.Cough incidence and cough grade were recorded within 2rain after the administration of fentanyl.Systolic blood pressure (SBP),diastolic blood pressure (DBP),heart rates (HR),and satu- ration of pulse oximeter(SpO2) were recorded during different time points of induction,all recorded data were anal- ysed by the statistical software,P value

OBJECTIVETo observe the expression of phospholipid scramblase 1 (PLSCR1) in matrine (MAT) induced differentiation of all-trans retinoic acid (ATRA) resistant acute promyelocytic leukemia (APL) cells, and to explore its correlation to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal pathway.

METHODSNB4 (an APL cell line sensitive to ATRA) and NB4-R1 (a resistant strain of ATRA) were observed as subjects in this study. Effects of combined treatment of 0.1 mmol/L MAT and 1 [mol/L ATRA on the differentiation of two cell lines were detected using nitroblue tetrazolium (NBT) reduction test and flow cytometry (CD11b). Expressions of PML/RARot and PLSCR1 protein/gene were detected using Western blot and Real-time fluorescence quantitative PCR assay. Meanwhile, H89, PKA antagonist, was used to observe cell differentiation antigen and changes of aforesaid proteins and genes.

RESULTSMAT combined ATRA could significantly elevate positive rates of NBT and CD11 b in NB4-R1 cells, and significantly down-regulate the expression of PML/RARapha-fusion protein/gene (P < 0.05, P < 0.01). ATRA used alone could obviously enhance the expression of PLSCRI in NB4 cells at protein and mRNA levels (P < 0.01). But the expression of PLSCR1 was up-regulated in NB4-R1 cells, but with statistical.difference only at the protein level (P <0. 01). In combination of MAT, PLSCR1 protein expression was further elevated in the two cell lines (P < 0.01). Besides, there was statistical difference in mRNA expressions in NB4-R1 cells (P < 0.05). All these actions could be reversed by treatment of 10 micromol/L H89 (P < 0.05, P < 0.01).

CONCLUSIONMAT combined ATRA could significantly induce the differentiation of NB4-R1 cells, and inhibit the expression of PML/RARalpha fusion gene/protein, which might be associated with up-regulating PLSCR1 expression.

Alkaloids ; Antineoplastic Agents ; Cell Differentiation ; Cell Line, Tumor ; Down-Regulation ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; Phospholipid Transfer Proteins ; metabolism ; Quinolizines ; RNA, Messenger ; Signal Transduction ; Tretinoin ; Tumor Cells, Cultured ; Up-Regulation