Objective To construct the recombinant plasmid with a human peptide antibiotic hPAB-? gene and to make it expressed in E. coli. Methods To replace the CNBr cleavage site in plasmid pFAST-hPAB-? (CNBr), a pair of primers containing the hydroxylamine cleavage site were designed, and the amplified PCR fragments were cloned into pFAST-HTa plasmid to produce pFAST-hPAB-? (HA), which was then transformed into E. coli DH10B. The constructed plasmid was identified by Ehe Ⅰ/Hind Ⅲ digestion and direct DNA sequencing. An Ehe Ⅰ/Hind Ⅲ digested fragment from pFAST-hPAB-? (HA) was subcloned into pQE32-CP to construct pQE32-CP-hPAB-?, which was transformed into E. coli JM109. The bacteria containing the expression plasmid were induced to express the fusion protein by IPTG. SDS-PAGE was carried out to analyze the molecular weight, expression quantity and expression form of the target fusion protein. After captured by Ni-NTA affinity column, the fusion protein was subjected to hydroxylamine cleavage analysis. Results An expected 230bp fragment was obtained by digesting pFAST-hPAB-? with Ehe Ⅰ/Hind Ⅲ. After this fragment was cloned into pQE32-CP, the recombinant plasmid was confirmed to contain the correct target sequence by DNA sequencing. The recombinant plasmid pQE32-CP-hPAB-? could express a desired protein with a relative molecular weight about 27kD, and its expression level reached 43 percent of the total bacterial proteins. The inclusion bodies were lysed by 8mol/L urea, and the fusion protein could then be captured by Ni-NTA column and cleaved by 2mol/L hydroxylamine at pH9.0. Conclusion The recombinant plasmid pQE32-CP-hPAB-? has been successfully constructed, and it can express the desired hPAB-? fusion protein in E. coli JM109 at high level. These results provide the foundation for future research.

Adenoma, Pleomorphic ; Aged ; Humans ; Male ; Nasal Cavity ; Nose Neoplasms

AIM: To determine the concentration of escitalopram in human plasma by HPLC-MS/MS and investigate the pharmacokinetics of escitalopram. METH-ODS: The method involved protein precipitation with methanol. The chromatographic separation was achieved within 6.0 min by using methanol-water with 15 mmol·L-1 ammonium acetate-formic acid (72:28:O.1, v/v/v) as mobile phase and a Lichrospher CN 150 mm×4.6 mm analytical column. The analytes were detected using an electrospray ionization tandem mass spectrometry in SRM mode. Detection of the ions was performed by monitoring the transitions of m/z 325.0 to 234.0 for escitalopram and m/z 409.1 to 238.1 for amlodipine (intemal standard), respectively. RESULTS:The standard curve was linear ( r = 0. 999) over the concentration range of 0.20 - 50.00 ng· ml- 1. Accuracy and precision were below the acceptance limits of 15%. The recoveries of escitalopram ranged from 96.0% to 103.6%. The lower limit of quantification for escitalopram was 0.20 ng· ml-1 using 200 μl plasma sample.The pharmacokinetic parameters of escitalopram after a single oral dosing of escitalopram oxalate tablet (10 rog)to ten healthy male volunteers were achieved. The Cmax, Tmax, AUC0-t, AUC0-∞, t1/2 and Ke of escitalopram were 9.21±2.10 ng·ml-1 , 3.75±1.04 h, 514.6±152.3 ng·h·ml-1 ,540.5±162.3 ng·h·ml-1 , 34.06±7.71 h and 0.021±0.004 h-1,respectively. CONCLUSION:The determination of concentration of escitalopram in human plasma by HPLC-MS/MS method was repid, sensitive and reliable. It can be used for clinical pharmacokinetic study of escitalopram.

Objective To explore the related infection factors of the internal jugular vein indwelling central venous catheter , and make prevention countermeasures according to the infection factors of central venous catheter infections .Methods A total of 564 patients admitted in liver and gallbladder surgical ward with external jugular vein indwelling central venous catheter were se‐lected ,extract the relevant hospital infection data in patients by the XingLin hospital infection real‐time monitoring system ,and SPSS15 .0 statistical analysis was conducted .Results The infection rate of 564 cases of patients was 4 .07% .The rate of gram‐neg‐ative bacteria infection was 43 .5% ,the gram positive bacteria infection accounted for 34 .8% ,fungi accounted for 21 .7% ,including multiple drug‐resistant bacteria infection accounted for 52 .1% .Catheter indwelling 14 d or more infection rate was 8 .5% ,14 d fol‐lowing infection rate was 2 .1% ,and infected patients for more advanced cancer and patients with severe acute pancreatitis .Pipe joint respectively with heparin cap and needle positive pressure infusion joint connections ,infection rate was statistically different (P<0 .05) .Conclusion The infection of internal jugular vein indwelling central venous catheter should not be ignored ,and the oc‐currence of catheter‐related infection of patients is closely related to state of an illness ,the time of catheter insertion ,and the joint device and so on .

Objective To assess the clinic application of a new-style automated immunoassay system HISCL ( high sensitivity chemiluminescent enzyme immunoassay ) with high sensitive chemiluminescence substrate CDP-Star?, bind-free separate technology and filter conversion technology.Methods The performance verification test evaluated HISCL′s specification including reproducibility, functional sensitivity, linearity, accuracy, and sensitivity for HBsAg seroconversion.To evaluate the specificity , 1 007 HBsAg-negative specimens , 82 potentially interfering specimens were from conventional specimens in West China Hospital , Sichuan University between October and December of 2014.At the same time, with these results to determine the rate of the specimens that their results are in negative grey zone.259 HBsAg-positive specimens and 27 weakly-positive specimens were tested for the comparison between the HISCL and ECLIA ( electrochemiluminescence immunoassay ) HBsAg quantification.A linear regression model , Pearson′s correlation and Bland-Altman analysis were used as statistical methods.Results The between day and within-lot variable coefficient of two level samples are 1.55%, 2.02%, 0.34%, 1.34%separately.The functional sensitivity of the HISCL HBsAg assay reaches 0.007 IU /ml.There is a good linearity (y=3.262x+0.082,r=0.994; y=2 303.608x-33.006,r=0.999) within range of 0-2 300 IU/ml.HISCL obtain the accurate results for the CAP control material , the agreement rate is 100%.The sensitivity for the detection of HBsAg seroconversion is high.The specificity is 99.91%.The negative grey zone rate is 1.66%.The results of these two methods have good correlation and uniformity , r=0.995, LOA:-0.3 -0.19 log10 IU/ml.Conclusions HISCL HBsAg detection system shows good detection performance.Be of the function of both qualitative and quantitative detection.And the negative “grey zone”specimen rate is low.It is wholly capable of wide linearity and quick assays for quantifying serum HBsAg levels.HISCL could contribute to improve HBsAg quantitative detection process for clinical laboratory.

Objectives: To design the mutants of peptide antibiotics hPAB ? based on its molecular structure. Methods: The three dimension structure of hPAB ? was constructed by protein homology modeling method. The mutant molecules were designed and generated by PCR and inserted into pQE CP4 expression plasmid. The recombinant plasmids were identified by PCR and DNA sequencing and then transformed into Escherichia coli JM109 to express target fusion proteins. Results:Peptide hPAB ? shows one ? helical and three ? sheet in its structure. Its ? helical regions seem play a key role in the formation of active oligomer. Aside from positioning Thr 7 and Lys 10 into contact positions, the orientation of the ? helix is conserved about the oligome core, forming a ridge around it. Additionally, the dipoles of the helices would overlap to create a positively charged region near the core. These dipoles may be offset, however, by the presence of Asp 4 at the base of the helix. Two mutant molecules, hPAB ? 38 and hPAB ? 34, were designed by deleting N or/and C terminal 2～5 amino acid residues based on hPAB ? structure. The recombinant plasmids containing the mutants gene can express interest fusion proteins in E. coli JM109 successfully. Conclusions: Design, cloning and expression of the mutants of peptide antibiotics hPAB ? lay down the foundation for screening of the mutant of shorter peptide chain and having high or same antimicrobial activity.

16M?) ). Using this method could not only avoid the interference of medium on the chromatographic behavior of Ralstonia solanacearum, but also keep the cell viability and cell surface properties.

The influence of separating effect of different chromatographic conditions of mouse gut flora by high performance ion exchange chromatography analysis was studied. The optimum chromatographic conditions for separating gut bacteria were determined. The sample was applied to the chromatography column packed with Toyopearl SuperQ-650c anion resin, equilibrated with 0.02mol/L piperazin-hydrochloric acid buffer (pH 8.0), and elution salt 1mol/L NaCl, eluted with the gradient of 0-50% NaCl/ 80 min, then 50%~75% NaCl/ 25 min at the flow rate 1ml/min, and injecting volume was 1ml.Under these conditions, intestinal flora were separated into several fractions. The establishment of HPLC analysis method will lay a foundation of further research on the components of mouse gut flora and their dynamic changes.

·Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are common outer retinal degenerative problems, which are also the predominant causes of most blinding retinal diseases. Retinal prosthesis is a promising solution for such photoreceptor degeneration diseases.Most of current concepts for a retinal prosthesis are based on neuronal electrical stimulation. In the past twenty years, retinal prosthesis has been developed in two different directions: epiretinal prosthesis and subretinal prosthesis. Each prosthesis technique has its advantages and disadvantages. For epiretinal prosthesis, it is easier to be implanted and has the advantage of keeping most of the electronics in the vitreous cavity, off the retinal surface, which greatly helps in dissipating the heat generated by the implant device. In this paper, a brief overview of retinal prostheses concepts is introduced. After that, several important aspects of epiretinal electrical stimulation will be discussed. Moreover, some practical epiretinal prosthesis devices developed by researchers in United States, Germany and Japan in the past have been reviewed. We hope that the devices will be used widely in the near future.

Objective:To screen and clone a carrier molecule for the expression of small bioactive peptides at high levels. Methods: A carrier molecule, PaP3.30, was screened out from the genome of Pseudomonas aeruginosa phage PaP3 and its gene was cloned by PCR method and inserted into pQE 32 expression plasmid, this recombinant plasmid was named pQE PaP30. The peptide antibiotics hPAB ? gene was then inserted into pQE PaP30 and induced to express the fusion protein in Escherichia coli . The ability of PaP3.30 to express other bioactive peptides was evaluated by fusing 6 different origins, varies in sizes and isoelectric points selected peptides to it. Results: After fused to PaP3.30, the peptide antibiotics hPAB ? could express as fusion protein above 30% of total bacterial proteins. Six selected peptides were also expressed by the level of 35%～44% total bacterial proteins when fused to carrier molecule, PaP3.30. Conclusion: The new carrier molecular, PaP3.30, is versatile in the expression of small bioactive peptides.