Objective In order to further understand the bioactivity of sphingosine kinase, high efficient replication defective recombinant adenovirus carrying the wild type SPK gene (SPK WT ) and SPK mutant gene (SPK DN ) were constructed respectively. Methods The SPK genes of interest were subcloned into a shuttle vector pshuttle cmv respectively. Pshuttle cmv SPK WT and Pshuttle cmv SPK DN were linearized by PmeI and transformed respectively into the competent cells of E. coli BJ AD 1 cells which contain adenoviral backbone plasmid pAdEasy 1. The linearized recombinant plasmids were transfected into adenovirus 293 packaging cells respectively,and then recombinant adenoviruses were harvested. We performed the PCR, Western blot and enzyme assays to identify the recombinant adenoviruses. Results The recombinant adenoviruses containing the interest gene and being able to infect ECV 304 cells were obtained. Overexpression of wild type SPK gene in ECV 304 cells increased the endogenous SPK activity, whereas overexpression of mutant SPK (SPK DN ) inhibited intracellular SPK activity. Conclusion Recombinant adenoviral vectors can mediate interesting gene expression in cells.

OBJECTIVE:To study the influencing factors of protocol deviation in drug clinical trial implementation,and pro-vide reference for improving the quality of drug clinical trial. METHODS:Quality verification was conducted for the drug clinical trial projects in the First Affiliated Hospital of Chongqing Medical University during 2010-2016,and protocol deviations in each year were retrospectively studied,classified and analyzed. Category,frequency,international and domestic pilot projects and the differences of protocol deviation after full-time research nurse participating in trail management were explored,and the measures were put forward. RESULTS:27 drug clinical trials were implemented in our hospital during 2010-2016,including 949 cases,176 cases with protocol deviation,accounting for 18.55％. Deviation protocol in drug clinical trial was decreased year by year for 7 years. The categories were mainly incompleteness of observation/records (30.11％),checking omission/broaden the window(28.41％),adverse drug events and improper combined medication (14.20％) and omission in drug management (11.93％). The proportion of protocol deviation with full-time research nurse participated was lower than the projects without full-time research nurse(11.11％ vs. 28.67％,P<0.01),and proportion of deviation protocol in international multi-center project was lower than the domestic projects(6.60％ vs. 28.84％,P<0.01). CONCLUSIONS:It is suggested to pointedly strengthen the weak links of drug clinical trial. For example,clinical trial institutions should establish the clinical trial data retention system,electronic data should be timely backed up in a different places,etc. Besides,clinical trial institutions should equip professional full-time research nurses as much as possible,learn the rigorous scientific experimental design,standard operational procedures and the authenticity of data pro-cessing from the international multi-center clinical trial projects to effectively reduce the incidence of deviation protocol and im-prove the quality of drug clinical trials.

Acupuncture Therapy ; Acute Disease ; Fatal Outcome ; Hemorrhage/mortality* ; Humans ; Needles/adverse effects* ; Pharynx/injuries* ; Postoperative Complications ; Postoperative Hemorrhage/etiology*

ObjectiveTo assess the effect of PM2.5 on sympathetic innervation and the relationship with cardiac nerve growth factor in healthy rat hearts,identify the ability to the sympathetic nerve reconstruction,and explore the possible arrhythogenic mechanism of PM2.5.MethodsForty healthy SD rats were instilled into trachea with two different solutions twice per week for four weeks:control group with saline and experimental group with PM2.5 25 mg/kg.Then these rats were killed,and biatrial appendages,two ventricular anterior walls were left.Immune cytochemical staining of cardiac nerves was performed using anti-tyrosine hydroxylase antibodies and cardiac nerve growth factor was detected by western blotting.ResuitsCompared with the control group,both the density of sympathetic nerves and the expression of cardiac NGF protein in the experimental group were much higher in the left ventricular anterior wall ( P＜0.01 and P＜0.05) and in the right ventricular anterior wall ( P＜0.01 and P＜0.05).ConclusionsPM2.5 can induce regional sympathetic hyperinnervation in both ventricular anterior walls in healthy rats by stimulating the NGF protein secretion,and the sympathetic nerve reconstruction effect of PM2.5 was confirmed.

Objective To investigate the accuracy of careful clinical evaluation in hemodynamic status and guidance of PiCCO monitor in clinical treatment.Methods A total of 96 hemodynamic unstable cases were evaluated prior to the insertion of the PiCCO catheter.The attending physician in charge of the patient was required to complete a questionnaire to predict the range of key hemodynamic variables for CI,GEDI,SVRI and EVLWI.Additionally,the attending was also asked to indicate a plan for therapy based on the predicted hemodynamic profile and decide if the predicted therapy plan was altered after the the first measurement of hemodynamic variables.Results The accurate prediction of hemodynamic variables was CI (55.2％),GEDI(60.4％),SVRI(63.5％) 和 EVLWI (78.1％),among which EVLWI had a higher accuracy(P ＜ 0.05).49％ doctors altered their planned therapy according to the result of the PiCCO information.Doctors had more difficulty in accurately predicting hemodynamic values in critical patients which APACHE Ⅱ scored 15 ～25 (42.3％ vs 67.9％ and 42.3 ％ vs 75.0％,x2 =4.755,5.231,P ＜ 0.05).The prediction of patients with acute myocardial infarction was more accurate than those of without acute myocardial infarction,and less to alter the planned therapy(21.1％ vs 55.8％,x2 =7.382,P =0.007).The patients of impaired oxygenation had less accurate predictions and less therapy alterations(32.3％ vs 56.9％,x2 =5.110,P =0.024).Attending was able to predict the hemodynamic status more accurately(63.9％ vs 40％,x2 =5.152,P =0.023) and alter the predicted therapy less(39.3％ vs 65.7％,x2 =6.189,P =0.013) in patients who were enrolled later.Conclusions Clinical evaluation in hemodynamic status of critically ill patients had a lower accuracy,the information obtained by PiCCO often instruct clinical doctors to choose the optimal treatment.

Objective To evaluate the effect of heart rate control with esmolol on hemodynamics, inflammatory cytokines and clinical outcomes in patients with septic shock.Methods A prospective randomized controlled trial was conducted. The patients with septic shock admitted to Department of Critical Care Medicine of China-Japan Friendship Hospital from August 2014 to October 2016 were enrolled. After 24 hours of resuscitation and other therapy, they were randomly divided into two groups by sealed envelope. The patients in experimental group was treated with continuous intravenous esmolol infusion for 24 hours, initial dose was 0.05 mg·kg-1·h-1, and was titrated to decrease the heart rate by 20% as compared with the value at the time of enrollment or below 95 bpm, while isotonicsaline was given to control group through intravenous line at 3 mL/h for 24 hours. The differences in hemodynamic parameters at 0, 1, 4, 8, 12, 24 and 48 hours, as well as serum inflammatory cytokines and blood lactate (Lac) at 0, 12, and 24 hours, 28-day mortality were compared between the two groups.Results Seventy-six septic shock patients were admitted during the study, 12 were excluded forsuspicious acute myocardial infraction (AMI) or acute left heart failure or for the history of chronic obstructive pulmonary disease (COPD), 4 were quitted the study for being unable to tolerate the lowest dose of esmolol, giving up treatment, or death within 24 hours. Finally, 60 patients completed the study, 30 patients in experimental group, and 30 in control group. There were no differences in gender, age, acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score and infection source between two groups, indicating the general data between the two groups were balanced and comparable. The decrease in heart rate was more markedly in experimental group than that of control group at 1, 4, 48 hours after esmolol administration (bpm: 97.4±16.5 vs. 110.9±19.6, 95.2±15.3 vs. 105.1±17.9, 86.4±12.1 vs. 97.2±22.6, allP < 0.05), cardiac index (CI) at 8, 24, 48 hours was significantly increased(mL·s-1·m-2: 57.2±13.5 vs. 46.5±11.0, 57.7±15.7 vs. 48.7±14.7, 61.2±16.5 vs. 51.5±14.7, allP < 0.05), and stroke volume index (SVI) at 4, 8, 24 hours was significantly increased (mL/m2: 34.1±6.9 vs. 29.0±8.7, 35.0±6.1 vs. 28.8±9.6, 38.3±10.1 vs. 31.9±13.2, allP < 0.05). Interleukin-1β (IL-1β) at 24 hours in experimental group was significantly higher than that of control group (ng/L: 0.15±0.06 vs. 0.13±0.05,P < 0.01). There were no differences in mean arterial pressure (MAP), Lac, white blood cell (WBC), IL-6, IL-10, and tumor necrosis factor-α (TNF-α) between the two groups, and no difference in 28-day mortality between experimental group and control group was found (30.0% vs. 36.7%,χ2 = 0.300,P = 0.583).Conclusions It is efficient and safe to use esmolol for heart rate control in patients with septic shock after resuscitation. Esmolol can improve cardiac performance without affecting blood pressure and Lac, but has no effect on inflammatory cytokines and prognosis.

Objective To evaluate carotid intima-media thickness(IMT) and elasticity in patients with pregnancy-induced hypertension(PIH) using ultrasound radio-frequency data(RF-data) technology.Methods Twenty-seven PIH women(mean brachial blood pressure:108 mm Hg,1 mm Hg=0.133 kPa)were included and thirty age- and gestational week-matched normal pregnant women served as controls.Carotid IMT and stiffness were evaluated using quality IMT(QIMT) and quantitative artery stiffness(QAS)techniques with color Doppler.Results Carotid IMT and elasticity parameters,including pulse wave velocity,pressure at T1,arterial augmented pressure(AP) and arterial augmented pressure index(AIx) were significantly higher in PIH group than in the normal group[IMT:(466.84±118.50)μm vs (386.58±125.79)μm;PWV:(7.09±1.97)m/s vs (5.95±1.11) m/s;PT1:(127.50±14.29) mm Hg vs (105.89±11.02)mm Hg;AP:(5.14±3.39) mm Hg vs (1.98±2.19)mm Hg;AIx:(7.58±8.73)% vs (-4.79±7.92)%)], and there were significant differences(t=2.660,2.660,3.460,3.460,3.460,all P＜0.01).Conclusions PIH women have significantly increased carotid IMT and decreased elasticity compared with normal pregnant women.Radio-frequency technique could reliably reflect the changes of the carotid arterial structure and elasticity in patients with pregnancy-induced hypertension.

Objective To explore the value of quantitative measurement of carotid intima-media thickness and elasticity for evaluating the degree of coronary heart disease by radio frequency ultrasonography. Methods From February 2011 to June 2012, a total of 142 subjects in cardiovascular internal medicine of the fourth military medical university Tangdu hospital with suspected CAD were enrolled in this study and were divided into non CAD group (n=35), atherosclerosis group in which coronary stenosis rate was less than 50%and no hemodynamic changes were detected (n=33), single artery involved group in which coronary stenosis rate was more than 50% (n=36) and multi-artery involved group in which coronary stenosis rate was more than 50%(n=38) based on the results of the coronary angiography (CAG). Measure the left common carotid artery intima-media thickness (IMT) and blood vessel elasticity index including the left common carotid artery pulse wave velocity (PWV) and stiffness index (β). Receiver operating characteristic (ROC) curves were used to calculate and compare the degrees of coronary heart disease, area under the curve of the left carotid artery elasticity parameters, cut-off value,sensitivity, speciifcity and Youden's index. Results LIMT, LPWV and Lβhad statistical signiifcance differences among four groups (F=3.621, 4.769 and 4.756, all P<0.05). The maximum values of parameter area under ROC curve were all observed in multi-artery involved groups and were 0.759 (LIMT), 0.770 (LPWV) and 0.764 (Lβ). The maximum value of Youden's index of the left common carotid artery intima-media thickness was observed in the coronary atherosclerosis group and was 0.513. The maximum value of Youden's index of the left common carotid artery pulse wave velocity and stiffnessβwere both observed in the multi-artery involved group and were 0.524 and 0.469. For diagnosing multivessel lesions, the sensitivities of LIMT, LPWV and Lβwere 71.0%, 77.4%and 71.0%, and the speciifcities were 73.4%, 74.7%and 75.9%. Conclusions The carotid elastic parameters have better predictive value for multivessel lesions. LPWV and Lβhave higher predictive value for coronary arterial disease than LIMT.

Objective To evaluate the effect of hyperhomocysteinemia on ischemic diease in rats. Methods Thirty-two SD rats were randomized into two groups,control group(n=16,administered with tap water) and hyperhomocysteinemia group(HH group,n=16,administered with water containing L-methionine at 1 g/kg/d).At the 14thday of dietary modification,the left femoral artery and vein were excised,and the interventions continued for another 14 days.At the 15th day after operation,serum biochemical parameters as well as NOx and cGMP in ischemic tissues were tested,capillary vessel density of both hindlimbs were measured by histological analysis,and angiogenesis of ischemic hindlimb was observed by angiography. Results At the 15th day after operation,the level of high-density lipoprotein-cholesterol(HDL-C),blood urea nitrogen,uric acid and ratio of blood urea nitrogen to serum creatinine were significantly higher in HH group than those in control group,while the level of serum folic acid,VitB12,HDL-C and endogenous creatinine clearance rate were significantly lower in HH group than those in control group(P

OBJECTIVETo study the expression of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in children with tetralogy of Fallot (TOF), and to investigate the role of histone acetylation and acetylation-related enzymes in the pathogenesis of TOF.

METHODSMyocardial tissue samples in the TOF group were obtained from 46 children with TOF who underwent radical operation, and myocardial tissue samples in the control group were obtained from 16 children who suffered accidental deaths and had no cardiac anomalies as shown by autopsy. The acetylation of H3K9, H3K18 and H3K27 was evaluated by immunohistochemistry. The mRNA expression of HATs and HDACs in the myocardium was measured by real-time PCR. The correlation between mRNA expression of HATs and HDACs and histone acetylation was analyzed.

RESULTSCompared with the control group, the TOF group showed significantly increased acetylation of H3K9 (P=0.0165) and significantly decreased acetylation of H3K18 (P=0.0048) and H3K27 (P=0.0084). As to 4 HATs and 6 HDACs, the mRNA expression of EP300 and CBP was significantly higher in the TOF group than in the control group (P=0.025; P=0.017), and there was no significant difference in the mRNA expression of other HATs and HDACs between the two groups. The correlation analysis revealed a positive correlation between H3K9 acetylation and mRNA expression of EP300 (r=0.71, P<0.01) and CBP (r=0.72, P<0.01).

CONCLUSIONSUpregulated mRNA expression of EP300 and CBP may be associated with increased H3K9 acetylation, suggesting that EP300 and CBP might affect cardiac development by regulating H3K9 acetylation.

Acetylation ; E1A-Associated p300 Protein ; genetics ; Female ; Histone Acetyltransferases ; genetics ; Histone Deacetylases ; genetics ; Histones ; metabolism ; Humans ; Infant ; Male ; Myocardium ; metabolism ; Peptide Fragments ; genetics ; RNA, Messenger ; analysis ; Sialoglycoproteins ; genetics ; Tetralogy of Fallot ; metabolism