Trypsin was purified from crude material of pig pancreas by AOT/isooctane reversed micellar system.The influence of the main operating parameters such as ethanol concentration in forward and backward extraction,pH,KCl and AOT concentration,temperature were investigated.Forward and backward extraction recovery of trypsin reached almost 90% and neared 100%,respectively.Finally,about 88% of total yield was obtained,and the specific activity of trypsin was increased to over 1800U/mg with purification factor of 5 folds more.In AOT-isooctane reverse micellar extraction system,denaturation or precipitation of proteins always occured due to strong electrostatic interaction between AOT-proteins molecules.It had been resolved by adding ethanol into reverse micellar system,and no denaturation was observed.Otherwise,the phase separation time was shortened significantly because of ethanol added.It was only 10 minutes or less to reach phases separation after forward and backward extraction.If this method can be applied in industry,efficiency will be greatly improved.

Objective To investigate the normal waveform,lantency and amplitude of auditory event-related potentials (ERPs) of exogenous components in children at different ages,and summarize the developmental rules and clinical perspectives of exogenous ERPs according to the International Federation of Clinical Neurophysiology (IFCN) Recommended Standards in 2009.Methods One hundred and thirty-six children aged from 3 to 17 years old (admitted in the departments from healthcare,orthopedics,general surgery,and respiratory and digestive diseases,and those recovered from respiratory infection and gastrointestinal infection),and healthy adults (from undergraduates of Chongqing medical university) were recruited in this study from 2008 to 2010.They were divided into 4 groups in accordance with their ages,that is,3 ～5,＞ 5 ～7,＞ 7 ～9,and ＞ 9 ～17 years old groups,and adult group (22 ～29 years old).ERPs were recorded from the midline site (Cz) with classical auditory Oddball paradigm.Each subject was tested twice or more to determine waveform repeatability.The overlapped graphs of ERPs under target and standard stimuli were obtained in the potentiometer.The exogenous ERPs,including P1,N1,P2 and N2,lantency and amplitude were measured.Results The latencies of exogenous ERPs under target and standard stimuli were shortened in a non-uniform manner with the increasing of age.No matter under target or standard stimuli,over 95％ of mean latencies of exogenous ERPs were within the range of 2SD.In addition to N1 component,the amplitude of P1,P2 and N2 components induced by target stimulus were in a trend of increased first and then decreased with the increasing of age.Compared with the children,the amplitude was much lower in the adults and adolescents (P ＜ 0.05).The N2 component induced by target stimulus was absent among 95％ healthy adults.The Standard deviations of amplitudes of exogenous components were great in all aged groups,indicating significant individual differences.Conclusion The latencies of exogenous ERPs are shortened with the increasing of age.Target stimulus will produce more stable results than the standard stimulus,and the ERPs under target stimulus can reflect the primary auditory cortex function of temporal lobe,and are independent on patients' cooperation.

Adult ; Betacoronavirus ; Clinical Laboratory Techniques ; Coronavirus Infections ; diagnosis ; epidemiology ; etiology ; prevention & control ; therapy ; Diagnosis, Differential ; Extracorporeal Membrane Oxygenation ; Humans ; Pandemics ; prevention & control ; Pneumonia, Viral ; epidemiology ; etiology ; prevention & control ; therapy ; Practice Guidelines as Topic ; Respiration, Artificial

Sixty patients with ulcerative colitis (UC),including 28 males and 32 females were enrolled in the study.Color Doppler ultrasonographic scans were performed and the disease activity was estimated base on the Sourtherland score index (DAI).On the sonography the colon with active inflammatory disease showed bowel wall thickening,alteration of layers structure or even discrimination and increased vascular signals.Using DAI as the gold standard,the sensitivity,specificity,positive predictive value,negative predictive value and accurate rate for ultrasonography in assessment of UC activity were 90.0％ (36/40),75.0％ ( 15/20),87.8％ (36/41),78.9％ (15/19),85.0％ (51/60),respectively.In conclusion,Color Doppler ultrasonography provides a convenient technique for assessment of bowel involvement and inflammatory activity in ulcerative colitis with high sensitivity and accuracy.

Objective To develop experimental animal model of the brain stem myoclonus,which more closely replicate clinic features of mechanism, behavior, neuroelectrophysiology and pharmacodynamics.Methods L-5-HTP (the precursor of L-5-HT)was microinjected into the dorsal pons of young guinea pig to induce myoclonus (electromyogram burst of myoclonus≤400 ms by synchronous recording).Some animals were pretreated with anticonvulsant VPA,CZP or CBZ at effective dose 50 (EC_(50)).Myoclonus was induced when the drug level was within their effective anticonvulsion concentration.The neuroelectrophysiological characteristics of myoclonus including latency,time of reaching its peak,duration of seizure peak,the maximum seizure frequency and total duration were detected.EMG and ictal electroencephalogram(EEG)were recorded synchronously.The origin of myoclonus and its correlation with epileptic discharges were further confirmed by jerk-locked back averaging(JLA).Results (1)L-5-HTP induced pure myoclonus from the dorsal pons of guinea pig permanently(8/every site,the rate of producing myoclonus is 100%).(2)The myoclonus presented bilaterally or as general myoclonus,which was sensitive to tactile and sound sensation.(3)The EMG duration of the myoclonus wag longer((208.75 ± 81.42)ms),and ictal EEG showed scattered and irregular spikes and sharp waves without time-locked correlation with EMG activities.(4)The synchronous ictal EEG of the myoclonus showed spike and sharp waves,but there was no time-locked EEG activity in JLA.(5)In the animals treated with anticonvulsant at EC_(50) concentrations:VPA and CZP decreased the maximum seizure frequency(there are 28.13±3.79 per minutes in VPA group and 37.17±4.67 perminutes in CZP group)and shortened the duration of peak time ((55.00±14.14)minutes in VPA group and(50.00±11.73)minutes in CZP group respectively)and total time(VPA group was(124.17±40.04)minutes and CZP group was(156.88±30.71)minutes)of myoclonus(F value were between 23.41 and 35.44,P＜0.01 or P＜0.05).CBZ increased duration of peak time((98.75±13.86)minutes)and total time((257.50±14.79)minutes)of myoclonus(P＜0.05 and 0.01).Conclusions The new model generates pure myoclonus originating from brain stem and also has a shorter duration of muscle construction(≤400 ms)and more sensitivity to tactile and sound sensation.Therefore,the model presents characteristics closer to the brain stem myoclonus in the clinic phenotype in respect of seizure behavior,pharmacodynamics and neuroelectrophysiology.

ObjectiveTo investigate the effect of SalB on bone marrow-derived mesenchymal stem cells (BMSCs) apoptosis induced by hypoxia and serum deprivation (hypoxia/SD) in the vitro. Methods BMSCs were cultured in the vitro and randomly divided into control group, hypoxia/SD group and SalB group.SalB group was composed by four groups and were pretreated by complete medium with 0.1、 1、 10、 100 mg/L SalB for 1 hour. And after that they were washed with phosphate buffer for 2 times, added by IMDM with 0.1、1、 10、 100 mg/L SalB and cultured with hypoxia/SD group together in the same condition of hypoxia/SD for 6hours. The control group was cultured for 6 hours in the condition of aerobic and enough serum. Apoptosis was detected by Hoechst33342 staining with inverted phase contrast, fluorescence microscope and Annexin V/PI dual-color flow cytometry. Results Significant apoptosis of BMSCs was induced by hypoxia/SD in the vitro.The early apoptosis of BMSCs induced by hypoxia/SD was significantly decreased by SalB of 0.1、 1、 10 mg/L(P＜0.05) . Conclusion0.1、 1、 10 mg/L SalB can decrease the early apoptosis of BMSCs induced by hypoxia/SD.

Objective To establish the rat overlap syndrome (OS) model of intermittent hypoxia (IH) and emphyse-ma, explore the systematic and endothelial inflammation status, and observe the changes of endothelial progenitor cell (EPC) level in peripheral blood. Methods Sixty male Wistar rats were randomly divided into four groups:normal oxygen control group (A), IH group (B), emphysema group (C) and OS group (D). The rat model of emphysema was established by smoke ex-posure for 16 weeks. From the 13-week, pre-programmed intermittent hypoxia/re-oxygenation (IH/ROX) exposure was giv-en in the meantime of smoke exposure. After exposure, ELISA method was used to detect values of tumor necrosis factor al-pha (TNF-α) and interleukin (IL)-6 in plasma and in the endothelium of right common carotid artery. Real-time-PCR assay was used to analyze RhoA mRNA level in the endothelium of right common carotid artery. The percentage of intima-media thickness (IMT) in the all wall of right carotid artery (C-IMT%) was measured. Flow cytometry was used to detect EPC levels. Results The values of TNF-α, IL-6, RhoA mRNA and C-IMT%were significantly higher in D group than those of A, B and C groups (P<0.05). The EPC levels were significantly lower in D group than those of A, B and C groups (P<0.05). Con-clusion OS rats had more serious vascular endothelial injury than that of emphysema or IH rats. Meanwhile, the repair ca-pacity of EPC for endothelium was worse, which increased the risk of cardiovascular diseases.

Objective To investigate the effect of emphysema and intermittent hypoxia (IH) on the hepatic oxidative stress and inflammatory injury in rats. Methods Sixty male Wistar rats were randomly assigned to 4 groups, control group (A), emphysema group (B), IH group (C) and emphysema+IH group (D). Group A was normally fed. Group B was exposed to smoke, 30 min per time, twice everyday. Group C was exposed to 5%O2 30 s/Air 90 s for 8 h/d. Group D was exposed to smoke twice, about 30 min each time, and exposed 5%O2 30 s/Air 90 s for 8 h/d. After continues exposure for 8 weeks, five rats in each group were randomly selected for arterial blood gas analysis. The tissue blocks of liver was obtained for pathologi-cal scoring and measurements of liver oxidative stress in the rest 10 rats of each group. HE staining was used to calculate the mean lining interval (MLI) and mean alveolar number (MAN). The hepatic inflammatory factor interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α), superoxide dismutase (SOD) activity, catalase (CAT) activity and malondialdehyde (MDA) con-centration were measured in four groups. Results Characteristics of emphysema were found in group B and group D. The values of MLI were significantly higher in Group B and group D than those of group A and group C (P<0.05). The values of MAN were significantly lower in group B and group D than those of group A and group C (P<0.05). The levels of SOD and CAT were significantly lower in group B, group C and group D than those of group A (P<0.05). And the levels of SOD and CAT were significantly lower in group D than those of group B and group C (P<0.05). The values of liver MDA were signifi-cantly higher in group B, group C and group D than those of group A, and the values were significantly higher in group D than those of group B and group C (P<0.05). The liver histological scores and the levels of IL-6 and TNF-αwere signifi-cantly higher in group B, group C and group D than those of group A, and the values were significantly higher in group D than those of group B and group C (P<0.05). Conclusion Emphysema and IH have synergistic action in causing hepatic oxidative stress and inflammation.

Objective To investigate the clinical effect of initiative content reduction surgery in the herniorraphy on the prevention and treatment of postoperative intra-abdominal hypertension of obese patients with giant ventral hernia.Methods The retrospective cross-sectional study was adopted.The clinical data of 62 obese patients with giant ventral hernias who were admitted to the Chao-Yang Hospital of Capital Medical University from January 2011 to December 2015 were collected.The initiative content reduction surgery was adopted during the herniorraphy of ventral hemia.Observation indices:(1) surgery situations:operation time,length of resected intestines,volume of intraoperative blood loss.(2) Postoperative recovery situations:change between preoperative and postoperative urethral bladder pressure,organ function of heart,lung,liver and kidney,time of gastrointestinal function recovery,time of drainage tube removal and duration of postoperative hospital stay.(3) Postoperative complications:incisional infection and intestinal fistula.(4) Follow-up situations.Follow-up using outpatient examination at 1 week,1 month,3 months and 6 months after surgery and using telephone interview at postoperative year 1 was conducted up to June 2016.The follow-up included hernia recurrence and chronic infection.Measurement data with normal distribution were represented as x ± s.Measurement data with skewed distribution were represented as M (range) and analyzed by rank-sum test.Results (1) Surgery situations:all the 62 patients received successful herniorraphy of giant ventral hemia combined with initiative content reduction surgery.Operation time,length of resected intestines and volume of intraoperative blood loss were (115 ±22)minutes,(207 ± 64)cm and (52 ± 35)mL.(2) Postoperative recovery situations:the preoperative urethral bladder pressure was 18 cmH2O (range,15-22 cmH2O,1 cmH2O =0.098 kPa) and postoperative urethral bladder pressure was 8 cmH2O (range,6-11 cmH2O),with a significantly statistical difference between before and after surgery (Z =-9.662,P ＜ 0.05).There was no abnormal function of heart,lung,liver and kidney after operation.The time of gastrointestinal function recovery,time of drainage tube removal and duration of postoperative hospital stay were 3.8 days (range,3.0-6.0) days,(3.8 ± 1.1) days and (14.5 ± 1.9) days.(3)Postoperative complications:of 62 patients,4 were complicated with incisional infection and improved after symptomatic treatment.There was no intestinal fistula.(4) Follow-up situations:all the 62 patients were followed up for (36 ± 19) months.During the follow-up,3 patients had incisional hernia recurrence and then were followed up without reoperation.No chronic infection occurred.Conclusion Initiative content reduction surgery is effective and feasible in the prevention and treatment of intraoperative intra-abdominal hypertension of obese patients with giant ventral hernia.

Objective To evaluate the effect of try psin on the proliferation,migration and adhesion of a skin squamous cell carcinoma cell line A431.Methods Cultured A431 cells were divided into several experimental groups treated with trypsin at concentrations of 0.1,1,10 and 100 nmol/L for 24,48 and 72 hours respectively,and a control group treated with DMEM complete medium only.Cell counting kit-8 (CCK8) assay was conducted to evaluate cellular proliferative activity to select the optimal concentration of trypsin.Then,some A431 cells treated with trypsin at the selected concentration for 24,48 and 72 hours respectively (or 48 hours only) served as the experimental groups (or group),and other A431 cells treated with DMEM complete medium served as the control group.Flow cytometry was performed to assess cell cycle distribution and proliferation index,fibronectin-based adhesion assay to estimate cell adhesive capacity,and wound healing assay and Transwell assay were conducted to evaluate the migratory capacity of cells in two-and three-dimensional space.Statistical analysis was carried out by using analysis of variance,paired samples t test and chi-square test.Results The proliferative activity of A431 cells increased along with the increase of trypsin concentrations,with the strongest increasing effect observed at 100 nmol/L.After treatment with 100 nmol/L trypsin,the experimental group showed a decrease in the percentage of G1-phase cells,but an increase in the percentage of S-phase cells,proliferation index,migratory and adhesive capacity compared with the control group (all P ＜ 0.05).Conclusion Trypsin can promote the proliferation,migration and adhesion of A431 cells.