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.

Human bocavirus (HBoV) 1-4 have been detected both in respiratory and stool samples since the first HBoV was discovered in 2005. HBoV-1 is mostly associated with respiratory infection, while HBoV 2-4 are usually associated with intestinal tract infection. A variety of signs and symptoms have been described in patients with HBoV infection, including cough, wheezing, pneumonia, and diarrhea, but the research on pathogenic mechanism of HBoV is limited because HBoV cannot be cultured in vitro due to the lack of appropriate host cells. Three-dimensional epithelial cell culture, reverse genetics, and viral metagenomics are identified as novel tools that may promote the research on pathogenic mechanism of HBoV and the discovery of new viruses. This review summaries currently available diagnostic approaches such as electron microscopy, cell culture, PCR, and immunoassay in order to provide a method reference for indepth research on HBoV.
Animals ; Human bocavirus ; genetics ; growth & development ; isolation & purification ; pathogenicity ; Humans ; Parvoviridae Infections ; diagnosis ; virology ; Viral Proteins ; genetics ; metabolism ; Virology ; methods ; Virulence ; Virus Cultivation

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.

Objective To investigate the correlation between urinary 8-iso-prostaglandin F2α (8-iso-PGF2α) level and carotid atherosclerotic plaque stability in patients with acute ischemic stroke.Methods The patients with acute ischemic stroke were enrolled consecutively.According to the findings of carotid artery ultrasound,they were divided into either a non-plaque group or a plaque group,and then the plaque group was redivided into a stable plaque subgroup and an unstable plaque subgroup.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of urinary 8-iso-PGF2α.Multivariate logistic regression analysis and Spearman correlation analysis were used to investigate the relationship between 8-iso-PGF2α and carotid artery plaques.Results A total of 150 patients with acute ischemic stroke were enrolled,including 104 had carotid artery plaque (30 were stable plaques and 74 were unstable plaques) and 46 had no carotid artery plaque.The urinary 8-iso-PGF2α level (86.45 ± 6.20 ng/mmol creatinine vs.45.70 ±6.19 ng/mmol creatinine; t =37.136,P=0.001) and intima-media thickness (IMT) (2.89 ± 1.03 mm vs.0.86 ±0.53 mm; t =3.518,P =0.002) in the plaque group were significantly higher than those in the nonplaque group.Multivariate logistic regression analysis showed that urinary 8-iso-PGF2α level (odds ratio [OR] 1.183,95％ confidence interval [CI] 1.087-1.276; P=0.001) and IMT (OR 28.642,95％ CI 8.276-137.231; P =0.001) were the independent risk factors for carotid artery plaque.Pearson correlation analysis showed that there was no significant correlation between urinary 8-iso-PGF2α level and carotid artery IMT (r =0.075,P =0.264).The urinary 8-iso-PGF2α level of the unstable plaque subgroup was significantly higher than that of the stable plaque subgroup (97.30 ± 7.20 ng/mmol creatinine vs.69.17 ±9.25 ng/mmol creatinine; t =16.506,P =0.001).Multivariate logistic regression analysis showed that urinary 8-iso-PGF2α level (OR 4.652,95％ CI 1.732-12.643; P =0.001) was an independent risk factor for unstable plaque.Conelusions The urinary 8-iso-PGF2α level is associated with the existence of carotid atherosclerotie plaque and instability,its increased level is an independent risk factor for carotid plaque instability in patients with ischemic stroke.

Objective To observe and analyze the effect of B. Adolescentis and L. Acidophilus on the proportion of Th cell Th1/Th2 in peripheral blood of UC-mice in acute stage and recovery stage. Method 40 BABL/c mice were induced by 3% DSS water for 7 clays free drinking and then with distilled water for 10 days. They were randomly allocated in 4 groups: NS group, SASP group, BF0624 group and LT0637 group, also the fifth group-10 normal animals. The blood of mice were collected by removing their eyes at day 8 and day 18, and then the mononuclear cells were iso]atecl. The proportion of Th1/Th2 was analyze through flow eytometry,hy labeling the specific antibody ot Th cellular membrane with the CD4 antibody, and the cytoplastie antigen of Thl or Th2 with IIA antibody or IF'N-γ antibody. Result The proportion of Th1/Th2 in normal mouse was 0. 84 -0. 94,and it raised up in DSS-mice at both acute stage and recovery stage. It decreased unequally after 7 or 17 days'B, adoleseentis, L. Acidophilus and SASP treatment, but that of all three groups were lower than NS group (2.21±0. 83). Even the proportion got close to the normal animals after 17 days'L. Acidophilus -treated. Conclusion The proportion of Thl/Th2 increased at the acute stage and recovery stage of DSS-mice. Both B. Adoleseentis and L. Acidophilus had more effective than SASP on decreasing the proportion of Th1/Th2 at two stages,ospecially L. Acidophilus.

Objective To analyze the influence of age on the diagnosis of acute heart failure (AHF) with amino terminal brain natriuretic peptide (NT‐proBNP) ,and to explore the best cut‐off value of NT‐proBNP in the diagnosis of AHF .Methods Totally 457 inpatients suspected with AHF obtained a blood NT‐proBNP measurement within 24 hours when in charged ,including 194 patients with AHF .Patients were divided into three groups according to their age :≤50 years ,>50-70 years and >70 years . The relationship between NT‐proBNPand AHF was analyzed .The diagnostic value of NT‐proBNP was evaluated for AHF in differ‐ent age groups .Results The median NT‐proBNP levels of AHF in all age groups were significantly higher than those without (P<0 .01) .AUC of NT‐proBNP for AHF in ≤50 years ,>50-70 years and >70 years groups were 0 .95 ,0 .91 ,0 .88 ,the best diagnos‐tic cut‐off points were 1 100 ,1 800 ,3 000 pg/mL ,respectively .The diagnostic specificity was 88% ,76% and 73% ,respectively .O‐verall ,the specificity of NT‐proBNP cut‐off values for age group was higher than non‐age groups .Conclusion NT‐proBNP has a good value in the diagnosis of AHF in patients with different ages .According to the age group ,the different NT‐proBNP cut‐off values can be used to improve the specificity of diagnosis .

Objective To explore the application of adaptive statistical iterative reconstruction (ASIR)scanning technique in low dose pediatric cervical CT, especially in reducing the radiation dose for thyroid. Methods We retrospectivly analyzed 140 pediatric cervical CT. 70 cases of group A scaned with ASIR technology and preseting noise index 12 HU in Discovery CT 750 HD (GE Healthcare), and 70 cases of group B scaned with filtered back projection (FBP) technology and preseting noise index 14 HU in VCT 64 (64 Lightspeed VCT, GE Healthcare). The other scan parameters were same in 2 groups. The distribution characteristics of gender and age were completely consistent between groups. The minimum current, the maximum current, the current of thyroid on CT images and CT dose volume index (CTDIvol), scanning length and dose length product (DLP) on dose reports were all recorded for each child. The noise values of CT images were measured. 4 scores standard was used to evaluate the subjective image quality. The data were analyzed with SPSS 13, Kappa method for the test of consistency and t test for the differences of the measured data. Excel was used for mapping. Results The minimum current, the maximum current, the average current, the current for thyroid of the two groups were respectively (26.8 ± 4.2), (43.2 ± 15.4)mA;(35.8±9.6),(41.8±13.6)mA;(34.6±8.6), (41.0±13.2)mA;(36.8±12.3), (44.7±21.0)mA. The CTDIvol, DLP and the scanning length of the two groups were respectively(1.3 ± 0.3), (1.8 ± 0.6)mGy;(29.0 ± 8.3), (40.3 ± 15.6) mGy·cm and(157.5±20.2), (157.6±21.8)cm. The average current, the thyroid current, CTDI and DLP in group A were 15.8%, 17.9%, 27.3%and 28.4%lower than those in group B (t=-5.50—-0.02, P<0.01). The subjective image quality scores of the two doctors were respectively 3.4 ± 0.9, 3.3 ± 1.0, they were highly consistent (Kappa=0.834, P<0.01). The objective image noise of cancellous bone and neck muscles were respectively (32.5 ± 5.3),(32.9 ± 5.1)HU and (9.2 ± 2.3), (9.1 ± 2.0)HU, there were not statistically difference between the two groups (t=-0.47—0.24, P<0.05). Conclusion Compared with FBP, low dose pediatric cervical CT with 30%ASIR could provide dose reduction for thyroid and the whole neck while maintaining diagnostically acceptable images .

Objective To explore the significance of nuclear factor-?B(NF-?B)activation in peripheral blood mononuclear cells(PBMC)during acute Kawasaki disease(KD).Methods Peripheral blood was collected from children with acute KD(n=30)and healthy age-matched children(n=20).PBMC were cultured in vitro and divided into 3 groups:naturally cultured blank control group,protein kinase C(PKC)activator stimulated phorbol 12-myristate 13-acetate(PMA)group and PMA plus NF-?B inhibitor treated PMA plus pyrrolidine dithiocarbamate(PDTC)group.Percentages of NF-?B activation were detected by immunohistochemistry.Results Under natural culturing,the percentage of cells with activated NF-?B was significantly higher in acute KD blank control group than that in healthy blank control group.The percentage of cells with activated NF-?B was significantly higher in acute KD PMA group than that in acute KD blank group and that in normal control PMA group,respectively(Pa0.05).Conclusions NF-?B activation in PBMC during acute KD is markedly increased,which suggests that NF-?B activation plays an important role in the formation of vasulitis and CAL in this disease.NF-?B activation in PBMCs in children with KD is regulated by the PKC signaling pathway and PDTC obviously inhibits the activation of NF-?B.J Appl Clin Pediatr,2009,24(1):35-37

BACKGROUND:The etiology of anterior cruciate ligament injury remains unclear yet, and some researchers have pointed that interior and exterior factors both contribute to anterior cruciate ligament injury;additionally, the genetic factor interior factors stand out. Collagen genes COL1A1, COL5A1, and COL12A1 are reported to be associated with anterior cruciate ligament injury in Caucasian populations. OBJECTIVE:To investigate the association of polymorphisms of COL1A1, COL5A1 and COL12A1 genes with anterior cruciate ligament injury in Chinese Han population . METHODS:105 patients with anterior cruciate ligament injury were enrolled and 110 patients without history of anterior cruciate ligament injury were as controlls. The first intron rs1800012 in COL1A1, rs127722 and rs13946 in the 3'-UTR region of COL5A1 gene, rs970547 and rs240736 in the 65 and 29 regions of COL12A1 extron were detected and classified by restriction fragment length polymorphism and genetic sequencing technology. RESULTS AND CONCLUSION:rs1800012, rs12722 and rs13946 genotypes, phenotypes and haplotypes in COL1A1 and COL5A1 genes showed no significant differences between groups. rs970547 and rs240736 genotypes as well as phenotypes and haplotypes in COL12A1 also showed no significant differences between groups. However, there was a significant difference in rs970547 gene frequence in male patients between groups. In conclusion, the Sp1 binding site of COL1A1 rs1800012 is not the susceptibility locus of anterior cruciate ligament injury in Chinese Han population. COL5A1 genes rs12722 and rs13946 in COL5A1 are not closely related to anterior cruciate ligament injury. COL12A1 rs970547 and rs240736 have a certain association with anterior cruciate ligament injury in Chinese men. Male individuals with COL12A1 rs970547 A allelicgene and AA genotype are likely to be susceptible to anterior cruciate ligament injury in Chinese Han population.

Modern drying technology was used to explore suitable drying process to provide scientific basis for improving drying processing methods of Scrophulariae Radix. Controlled temperature and humidity drying, vacuum drying apparatus, microwave vacuum drying apparatus, short infrared drying device were used to gain samples for analyzing. The character appearance, concentration of main components and power consumption indicators were chosen for preliminary judging. Six major components, including iridoids and phenylpropanoids were analyzed by UPLC-MS/MS method. The contents of polysaccharides were determined by UV-visible spectrophotometry. The character appearance with controlled temperature and humidity drying and short infrared drying meet the pharmacopoeia standard (Ch. p, edition 2015), while samples with vacuum and microwave vacuum drying apparatus didn't. Compared to fresh sample, concentrations of harpagide, harpagoside, aucubin and catalpol were lower in the dried samples. Angoroside-C showed no significant change before and after drying. Concentration of acteoside increased after drying. Samples with controlled temperature (70 degrees C) and humidity (15% - 10%) drying had high content and short drying time. The better drying process of Scrophulariae Radix was controlled temperature and humidity drying. The method will provide the reference for the drying technology standard of roots medicine.
Desiccation ; methods ; Drugs, Chinese Herbal ; chemistry ; Plant Roots ; chemistry ; Quality Control ; Scrophularia ; chemistry