In response to problems from the development of multi-drug-resistant pathogenic bacteria,it is urgent to find new antimicrobials.Antimicrobial peptides(AMPs) are a kind of ideal new antimicrobials with the advantages including their potential for broad-spectrum activity,rapid bactericidal activity and low propensity for resistance development,and have a bright future.Alpha-helical antimicrobial peptides are a main kind of AMPs.The following features was reviewed and elucidated including the structure and activity relationship(SAR) from different aspects including the degree of helicity,hydrophobic moment,hydrophobicity,net positive charges and so on,and the application of SAR on the molecular design and improvement of AMPs.

The tandem expression vector can effectively increase the expression level and structure stability of the target peptides(protein)with small size of molecular,and avoid toxicity towards host cell from some toxic peptides product,therefore,this approach is widely applied in biotechnology.The four construction methods of tandem expression plasmid including asymmetric and complementary cohesive ends,directional adapter,isocaudarners,and tandem expression cassette were reviewed in terms of protocol,characteristic and applicable field.In addition,selection principles from various construction methods of tandem plasmid were reviewed in terms of efficiency,accuracy and product cleavage.

ObjectiveTo observe the clinical effect of unstable femoral intertrochanteric fracture treated with DHS. Methods Summarized 37 cases of unstable femoral intertrochanteric fracture treated by DHS,all cases were commented by fracture type,operation method and clinical effect. ResultsWe have followed up 37 cases from 0.8 to 4.7 years with average of 2.5 years.Conclusions DHS is one of the best implants due to its merits of strong fixation, little complication and early rehabilitation exercise.

OBJECTIVETo construct mouse enhanced green fluorecence protein (EGFP) -peroxisome proliferator-activated receptor (PPAR)gamma2, and to detect EGFP-PPARgamma2 expression in infected mouse bone marrow mesenchymal stem cells (BMSC).

METHODSCut the fragment of PPARgamma2 from the expression plasmid pcDNA flag PPARgamma2, then cloned the gene fragment into pEGFP-C1 and pEGFP-N1 vector. Subsequently, subclone the fragment EGFP-PPARgamma2 from pEGFP-C1-PPARgamma2 into the shuttle plasmid DC315. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid DC315-EGFP-PPARgamma2 and large adenovirus helper plasmid pBHGlox deltaE1, 3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus Ad-EGFP-PPARgamma2 was confirmed. Then it was propagated in HEK293 cells. After the BMSC were transfected for 72 h, adipogenic differentiation was demonstrated.

RESULTSHEK293 cells were transfected with the pEGFP-C1-PPARgamma2 or pEGFP-N1-PPARgamma2 in mediation of liposome. The former green fluorescence protein was better than the latter by fluorescence microscope. The recombinant plasmids were digested and identified. Western blot analysis showed the expression of EGFP-PPARgamma2 in vitro. EGFP-PPARgamma2 protein was detectable in the nucleus of BMSC.

CONCLUSIONThe recombinant adenovirus encoding EGFP-PPARgamma2 fusion protein was successfully constructed, which provided a basis for application of EGFP-PPARgamma2 gene to adenovirus-mediated gene therapy.

Adenoviridae ; Animals ; Bone Marrow Cells ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; HEK293 Cells ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Mice ; PPAR gamma ; metabolism ; Recombinant Proteins ; metabolism ; Transfection

OBJECTIVETo construct esat6-ppe68 fusion gene and its prokaryotic expression vector for expression in E. coli.

METHODSWith GeneSOEing method, a fusion gene was constructed by splicing esat6 gene and ppe68 gene and cloned into pGEX-4T-1 plasmid to construct the recombinant prokaryotic expression plasmid pGesat6-ppe68. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis of the plasmid, E. coli BL21 was transformed with the recombinant plasmid and induced with IPTG to obtain the expression of the fusion protein ESAT6-PPE68 with GST-tag (about 69 kD), which were purified with GST-fusion protein purification kit. The expression of esat6-ppe68 fusion gene was subsequently detected by SDS-PAGE and Western blot analysis.

RESULTSThe sequence of esat6 and ppe68 in the recombinant plasmid was consistent with that in GenBank report. The fusion protein was detected in the cytoplasm in soluble form and represented approximately 40% of the total bacterial protein of E. coli. After purification, the purity of the fusion protein reached 90%, and its antigenicity was confirmed by Western blotting.

CONCLUSIONThe prokaryotic expression vector pGesat6-ppe68 has been constructed and the fusion protein ESAT6-PPE68 obtained successfully, which provides an experimental basis for potential application of the recombinant ESAT6-PPE68 in the diagnosis of tuberculosis.

Antigens, Bacterial ; genetics ; isolation & purification ; metabolism ; Bacterial Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transformation, Genetic

To improve the after-sales service, a survey aimed at the after-serveis of 3 kinds of medical equipment is applied among 68 hospitals in Shanghai Area in 2008.The Stat. and analysis results are showed in the paper, which will certainly channel off suppliers to set up a harmonious market together.
China ; Consumer Behavior ; statistics & numerical data ; Data Collection ; Product Surveillance, Postmarketing

Objective To test the changes of the potassium(K+)and magnesium(Mg2+)concentrations in vitreous humor of rabbits along with postmortem interval(PMI)under different temperatures, and explore the feasibility of PMI estimation using mixed-effect model. Methods After sacrifice, rabbit carcasses were preserved at 5℃, 15℃, 25℃ and 35℃, and 80-100μL of vitreous humor was collected by the double-eye alternating micro-sampling method at every 12 h. The concentrations of K+and Mg2+in vitreous humor were measured by a biochemical-immune analyser. The mixed-effect model was used to perform analy-sis and fitting, and established the equations for PMI estimation. The data detected from the samples that were stoned at 10℃, 20℃ and 30℃ with 20, 40 and 65 h were used to validate the equations of PMI estimation. Results The concentrations of K+and Mg2+[f(x,y)] in vitreous humor of rabbits under different temperature increased along with PMI(x). The relative equations of K+and Mg2+concentration with PMI and temperature under 5℃~35℃ were fK+(x,y)=3.413 0+0.309 2 x+0.337 6 y+0.010 83 xy-0.002 47 x2 (P<0.000 1), and fMg2+(x,y)=0.745 6+0.006 432 x+0.033 8 y(P<0.000 1), respectively. It was proved that the time of deviation for PMI estimation by K+and Mg2+was in 10 h when PMI was between 0 to 40 h, and the time of deviation was in 21 h when PMI was between 40 to 65 h. Conclusion In the ambient temperature range of 5℃-35℃, the mixed-effect model based on temperature and vitreous humor sub-stance concentrations can provide a new method for the practical application of vitreous humor chemi-cals for PMI estimation.

Objective:To provide a simple and replicable method of building an atherosclerotic intracranial arterial stenosis model of rabbits .Methods:Twenty‐five New Zealand white rabbits were randomly divided into three groups .Five rabbits in control group were fed with normal diet and received sham arteriotomy of femoral artery .Ten rabbits in pure high‐fat diet group were fed with high‐fat diet and received sham arteriotomy of femoral artery .Ten rabbits in balloon‐injury group were fed with high‐fat diet and underwent endovascular balloon‐injury operation of intracranial vertebral artery through femoral artery .All the operations were performed after two weeks of adaptive feed .Each group was fed with previous diet after operation .Digital subtraction angiography (DSA) of target vertebral artery was performed two and four weeks after operation (four and six weeks after the scheduled diet) .Serum lipid level was test and the histopathologic examinations were performed . Results:No significant difference was found by DSA or pathological detection of blood vessel in target vertebral artery of control group and pure high‐fat diet group .DSA revealed the mild luminal stenosis in three cases and unsmooth inner wall in six cases at the target vertebral arteries in balloon‐injury group two weeks after operation .Also the DSA revealed the mild luminal stenosis in five cases ,moderate luminal stenosis in two cases and unsmooth inner wall in two cases in balloon‐injury group four weeks after operation .Detection of serum lipid level showed that the serum lipid level of white rabbits in pure high‐fat diet group and balloon‐injury group was significantly higher than that in control group (P< 0 .05) .No significant difference of serum lipid level was found between pure high‐fat diet group and balloon‐injury group .Histopathologic examination showed the disorder of endothelial structure in target vertebral arteries ,interruption of endothelial continuity and deposition of foam cell under intima in the high‐fat diet combined balloon‐injury group .Conclusions: The method of high‐fat diet combined with endovascular balloon‐injury is beneficial to successfully build a relatively stable model of rabbits atherosclerotic intracranial arterial stenosis ,and it provides the basis for further studies .

Laser induced breakdown spectroscopy ( LIBS ) was proposed to rapidly discriminate microbe species. Ten species of microbes were prepared in lab. Filter papers were selected as substrate for enriching bacteria and enhancing the quality of LIBS. The images of plasma were collected by ICCD camera and LIBS spectra were obtained by spectrometers. The results displayed that the images and spectra were different from 10 bacteria. It was demonstrated that this method was feasible to discriminate bacteria species by analyzing image and/or spectroscopy. Furthermore, nine smooth and multiple scattering correction ( MSC) were utilized to preprocess the LIBS full-spectrum data in the wavelength range of 200-420 nm and 560-680 nm. And principal component analysis ( PCA) and PCA-RF ( Random forest) were compared to validate the accuracy of discrimination. The investigation showed that the PCA-RF model coupled with suitable methods in preprocessing data could identify bacteria. The accuracy was 99. 6% for ten species of microbes by evaluating LIBS spectra in training set, and 96. 7% in predicting set. This report indicated that it is feasible to differentiate bacteria species by analyzing LIBS spectra.

Aim To investigate the effect of recombi-nant glycoprotein hormone β5/α2(rCGH)on lipolysis in 3T3-L1 adipocytes,and explore the underlying mechanism.Methods 3T3-L1 preadipocytes were cultured and induced to differentiate into mature adipo-cytes,then treated with different concentrations of rCGH for 24 h in vitro.Cell viability of 3T3-L1 adipo-cytes was evaluated by CCK-8 assay,the levels of in-tracellular triglyceride(TG)and glycerol in the culture supernatant were measured by enzymatic method,and the changes of lipid droplets were observed by oil red O staining.The expression levels of HSL and ATGL lipo-lytic proteins in adipocytes were detected by Western blot.To carry out the intervention experiment with dif-ferent concentrations of rCGH with or without the PKA inhibitor,H89,on the mature 3T3-L1 adipocytes,the cultured cells were divided into the control group,H89 pre treatment group,1 μmol·L-1 rCGH group,and(1 μmol·L-1 rCGH+H89)combined intervention group.The contents of intracellular TG and free glycer-ol were measured by enzymatic method,and the ex-pression of CREB and lipolysis-related proteins was de-tected using Western blot.Results Different concen-trations of rCGH(0.25,0.5,1,and 2 μmol·L-1)had no significant effect on the cell viability of adipo-cytes(P＞0.05).Compared with the control group,the treatment with rCGH significantly decreased the size of lipid droplets and intracellular TG content,while significantly elevated glycerol concentration in cell supernatant.rCGH treatment also stimulated the protein expression of p-HSL,ATGL,and p-PKA.In addition,the addition of a PKA inhibitor,H89,atten-uated the effects of rCGH on free glycerol level,intra-cellular TG content,and the expression of p-HSL,p-PLIN1,and p-CREB.Conclusions rCGH enhances the lipolysis of 3T3-L1 adipocytes by up-regulating the activities of HSL,ATGL and PKA,promoting glycerol release,inhibiting TG synthesis and lipid accumula-tion,and its mechanism of action is related to the acti-vation of cAMP/PKA/CREB signaling pathway.