Objective To investigate the effects of berberine on basolateral calcium-activated potassium current I_K(Ca)and cAMP-activated potassium current I_K(cAMP)and its mechanism in treatment of secretory diarrhea.Methods The intact colonic crypt cells were isolated with EDTA solution.The effects of berberine(50,100,500?mol/L)on I_K(Ca)and I_K(cAMP)were detected by patch clamp technique under the conventional whole cell patch clamp mode.The solution of PSS was served as control.Results Berberine could significantly inhibite I_K(Ca)and I_K(cAMP)of rat colonic crypt cells(both P

Objective To construct cDNA entry library and cDNA expression library of Armillifer agkistrodontis (A.) nymphs and make a preliminary immunoscreening for the cDNA expression library.Methods The nymphs were collected from the Kunming mice infected experimentally with A.agkistrodontis eggs and the total RNA were extracted from the nymphs using TRIzol Reagent.After purifying the mRNA,the synthesized cDNAs were cloned into the donor vector pDONR222 by BP reaction of Gateway technology and the recombinants were transformed into the DH10B cells by electroporation,the cDNA entry library was obtained.Next,the expression vector pDEST17 was ligated with entry clones by LR reaction,and the recombinants were transformed into the BL21 (DE3) cells.Hence,the cDNA expression library was constructed.Then,the expression library was immunoscreened with the mixed sera of mice infected with A.agkistrodontis,and the insertions of positive clones were sequenced.After that,the open reading frame(ORF) of positive slone sequence,the homology of the screened genes and their encoded proteins were analyzed by Finder and BLAST (basic local alignment search tool) program of National Center of Biotechnology Information(NCBI),and the discovered new genes were submitted into the GenBank.Besides,the physico-chemical properties,secondary structure and B cell epitopes of encoded proteins were also analyzed by bioinformatics software.Results The average titer and total clones of the cDNA entry library were 1.45 × 105 CFU/ml(colony-forming unit,CFU) and 1.74 × 106 CFU,respectively,and the range of fragment length of the inserted cDNA was between 0.2-4.0 kb,with an average of 1.4 kb.The total clones of cDNA expression library were 1.00 × 105 CFU,and the fragment length of the inserted cDNA was between 0.3-2.2 kb,with an average of 1.0 kb.Five positive clones,coded S1,S5,A1,D1 and F1,respectively,were obtained through preliminary immunoscreening.The sequence and homology of the five positive clones were sequenced and analyzed by BLAST program.No significant similarities were found in pentastomida species,which meant that they were all novel genes of A.agkistrodontis.The gene sequences were submitted to GenBank,with the accession number from JQ180451 to JQ180455.Also,results obtained by bioinformatics software showed that the predictive encoding proteins were all potential to be valuable recombinant diagnostic antigens.Conclusions The cDNA library of A.agkistrodontis nymphs is successfully constructed,and five new genes of A.agkistrodontis are discovered.The establishment of cDNA library and the discovery of the new genes will lay a foundation for further studying the gene functions and screening the immunodiagnostic antigens.

The aims of microbiology experiment teaching are not only to cultivate the students’ capacity of basic operation,but also to expanse their knowledge scope.We applied the bacterial diversity on the teach-ing class to make the students understand the progress of microorganism genomics research.It is helpful to cultivate the students’ innovative spirit and ability.Easy to work,clear result and low cost facilitated the spread of this experiment in the university.

Objective To investigate the impact of paced QRS duration (pQRSd) on heart function in patients with right ventricular apical pacing. Methods Seventy-six patients with Ⅲ° atrioventricular block received pacemaker treatment were enrolled and randomized into group A (pQRSd < 190 ms, n = 52) and group B(pQRSd≥ 190 ms, n = 24). The concentration of brain natriuretic peptide (BNP),parameters of left atrial diameter (LAD), left ventricular ejection fracetion (LVEF), left ventricular end diastolic diameter (LVEDD), left ventricular end systolic dimension (LVEDD) were measured before operation, at 12 months and 24 months after implanting, respectively. The parameters of echocardiography assay, the concentration of BNP and the incidence of heart failure event after implantation were compared between two groups. Results At 12 months after implanting, LVEF of the patients in the group B decreased significantly compared with that of group A (P < 0.05). However, the echocardiography paramenters and the concentration of BNP were not significantly different between the two groups (P > 0.05). At 24 months after implanting, LAD、LVEDD、LVESD of group B increased significantly compared with those of group A [LAD,( 44.5 ± 6.2) mm vs (41.6 ± 5.1) mm, LVEDD, (52.7 ± 9.3) mm vs (48.2 ± 7.5) mm, LVESD, (37.5 ± 5.6) mm vs (33.8 ± 4.9)mm, each P < 0.05, respectively]. The concentration of BNP of group B increased significantly [(408.2 ± 102.1)ng / L vs (243.7 ± 92.8)ng / L, P < 0.001], and LVEF of the patients in group B decreased significantly compared with those of group A [(46.3 ± 6.8)% vs (51.6 ± 5.2)%, P < 0.001], respectively. No significant difference in the incidence of heart failure event (41.7% vs 26.9%, P > 0.05)between two groups during 24-month follow-up. Conclusion The prolonged paced QRS duration has a detrimental effect on long-term cardiac function during RVA pacing in patients with Ⅲ°atrioventricular block.

OBJECTIVETo evaluate the effects of prenatal stress on neurological functions after middle cerebral artery occlusion (MCAO) in adult offspring rats.

METHODSPregnant rats were randomly assigned to prenatal stress treatment, which was exposed to restraint three times daily in the last week of pregnancy, and no prenatal stress treatment. Adult male offspring rats were subjected to transient focal cerebral ischemia by MCAO. They were randomly divided into four groups: sham group, prenatal stress + sham group, MCAO group and prenatal stress + MCAO group (n = 10). After 24 hours of reperfusion, the neurological deficits were evaluated. The infarct size, cell apoptosis and expression of Caspase 3, cleaved Caspase 3 and Bcl-2 were detected.

RESULTSCompared with MCAO group, the neurological deficits, infarct size and apoptotic cells in prenatal stress + MCAO group were increased significantly (all P < 0.05). The expressions of Caspase 3 and cleaved Caspase 3 were much greater in prenatal stress + MCAO group than those of MCAO group, while the expression of Bcl-2 was significantly decreased in prenatal stress + MCAO group compared with MCAO group (all P < 0.05).

CONCLUSIONPrenatal stress might exacerbate neuroloeical deficits in the offspring rats after MCAO by increasing cell apoptosis.

Animals ; Apoptosis ; Caspase 3 ; metabolism ; Female ; Infarction, Middle Cerebral Artery ; physiopathology ; Ischemic Attack, Transient ; physiopathology ; Male ; Pregnancy ; Prenatal Exposure Delayed Effects ; physiopathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological

Objective:To explore the effect of probiotics on reducing ventilator-associated pneumonia (VAP) in sepsis patients.Methods:A total of 94 cases were randomly (random number) divided into the probiotic group ( n = 46) and the control group ( n = 48). All of the patients were given enteral nutrition therapy by nasogastric tube within 24-72 h after admission. And patients in the probiotic group were given live combined bifidobacterium, lactobacillus and enterococcus powder besides the regular therapy. The incidence of VAP, bacteremia, mortality, mechanical ventilation time and length of ICU stay were compared between the two groups. Results:Compared with the control group, the incidences of VAP and bacteremia in the probiotics group were significantly lower (χ 2=4.763, P=0.029; χ 2=4.438, P=0.035). There were no significant differences in 28-day mortality and the length of hospital stay between the two groups (χ 2=2.02, P=0.167; t=1.29, P=0.208). Mechanical ventilation time in the probiotics group was significantly shorter than that in the control group ( t=2.16, P=0.038). The Log-Rank test showed that the time of VAP-free in the probiotics group was significantly longer than that in the control group ( P < 0.05). After adjusting for APACHEⅡ score and age, COX proportional risk model analysis showed that the RR values of the probiotics group and the control group for 28-day VAP were 0.18 (95% CI: 0.12-0.74, P=0.025) and 0.21 (95% CI: 0.19-0.95, P=0.042), respectively. Conclusions:Probiotics treatment can reduce the incidence of VAP in sepsis patients.

In order to confirm the role that the 49th amino acid residue plays in enzymatic inactivity of Glutamine 49 phospholipase A2(Gln49-PLA2),site-directed mutagenesis of its 49th amino acid gene codon was conducted using PCR.Aspartic acid 49 phospholipase A2(Asp49-PLA2-Q49D-PLA2),the mutant of Gln49-PLA2 was expressed in E.coli with pET32a+ vector.The fusion protein,expressed as inclusion body,after being denatured,was on-column refolded and purified by immobilized metal affinity chromatography(IMAC),and then cleaved by Factor Xa.The mature Q49D-PLA2 mutant was obtained by Hitrap SP cation exchange and Superdex 75 gel filtration chromatography,with the recovery rate of 1.3%,and the specific activity of the mature Q49D-PLA2 mutant was 72 U/mg.It has been demonstrated that the 49th glutamine amino acid residue is the main reason in enzymatic inactivity of Gln49-PLA2 and the results are helpful for denatured protein refolding,especially in rich disulfide bonds conditions.

Objective To investigate the advantages of detection for EGFR gene mutations by denaturing high performance liquid chromatography (DHPLC) technology. Methods DHPLC was used to detect EGFR gene mutations at exon 19 and 21 in 49 cases of non-small cell lung cancer (NSCLC) patients,and the direct DNA sequencing was used to verify the accuracy of DHPLC detection. Results EGFR gene mutation was identified from 13 of 49 cases by DHPLC,including deletion mutation at exon 19 in 10 cases (76.92 %) and alternative mutations at exon 21 in 3 cases (23.08 %). Mutation results of DHPLC was consistent with DNA direct sequencing. The results of the direct DNA sequencing were the same as those of DHPLC. The sensitivity of mutation test by DHPLC was 100 %. Conclusion DHPLC technology can be used for large scale screening of EGFR gene mutation with rapid and accuracy.

To obtain higher potency and specificity, a series of 7-alkoxy analogues of illudalic acid was synthesized on the base of structure-activity relationship (SAR). All of these compounds exhibited submicromolar inhibition of the enzyme when tested against human leukocyte common antigen-related phosphatase (LAR) (for example, for 15e, IC50 = 180 nmol x L(-1)). They represent the most potent small-molecule inhibitors of LAR so far. These analogues also display excellent selectivity for LAR over other protein tyrosine phosphatases (PTPs) except for the highly homologous PTPsigma. The compound 15f is of 120-fold selectivity for LAR versus PTP-1B inhibition. The development of potent enzyme-specific inhibitors is so important that they may serve both as tools to study the role of LAR and as therapeutic agents for treatment of type II diabetes.