Structure determination of an integral membrane protein at room temperature from crystals in situ

The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines

Analysis of Resistance to Clarithromycin and Virulence Markers in Helicobacter pylori Clinical Isolates from Eastern Taiwan

AbstractObjectiveLittle information is available concerning the relationships between clarithromycin resistance and virulence marker genes (iceA, cagA and vacA) in Helicobacter pylori isolated in Taiwan. The aim of this study was to evaluate the possible association between clarithromycin resistance and genotypes of the virulence markers on clarithromycin-resistant H. pylori isolates obtained in eastern TaiwanMaterials and MethodsThe genotypes of the virulence marker genes (iceA, cagA and vacA) were analyzed by PCR, and the 23S rDNA region from 18 clarithromycin-resistant clinical isolates of H. pylori was amplified by PCR and sequenced.ResultsPoint mutations were found to occur in all isolates. Two isolates had A2143G, six had T2182C, one had C2227T, six had A2143G plus T2182C, and three had heterozygous alleles. The latter included a wild-type allele (A2143) plus (i) an A2143G, (ii) an A2143G plus an A2223G, and (iii) an A2143G plus a T2182C. The prevalence of the marker genes cagA, iceA1, iceA2, and both iceA1 and iceA2, in the isolates was 95.5%, 66.9%, 7.5%, and 25.6%, respectively. The vacAs1 allele was detected in all isolates, whereas the m1 and m2 alleles were found in 44.4% and 55.6% of the isolates, respectivelyConclusionThere were no significant associations between clarithromycin resistance and the presence of the cagA gene, vacA allele mosaicism, and the iceA genotypes. The most notable finding of our study was that the C2227T single mutation in 23S rDNA could also be related to the high clarithromycin minimal inhibitory concentrations in clinical isolates from eastern Taiwan

La visite d’entreprise en Europe : un champ à explorer

La visite d’entreprise en Europe : un champ à explorer</p

SDAR: a practical tool for graphical analysis of two-dimensional data

<p>Abstract</p> <p>Background</p> <p>Two-dimensional data needs to be processed and analysed in almost any experimental laboratory. Some tasks in this context may be performed with generic software such as spreadsheet programs which are available ubiquitously, others may require more specialised software that requires paid licences. Additionally, more complex software packages typically require more time by the individual user to understand and operate. Practical and convenient graphical data analysis software in Java with a user-friendly interface are rare.</p> <p>Results</p> <p>We have developed SDAR, a Java application to analyse two-dimensional data with an intuitive graphical user interface. A smart ASCII parser allows import of data into SDAR without particular format requirements. The centre piece of SDAR is the Java class <it>GraphPanel</it> which provides methods for generic tasks of data visualisation. Data can be manipulated and analysed with respect to the most common operations experienced in an experimental biochemical laboratory. Images of the data plots can be generated in SVG-, TIFF- or PNG-format. Data exported by SDAR is annotated with commands compatible with the Grace software.</p> <p>Conclusion</p> <p>Since SDAR is implemented in Java, it is truly cross-platform compatible. The software is easy to install, and very convenient to use judging by experience in our own laboratories. It is freely available to academic users at <url>http://www.structuralchemistry.org/pcsb/</url>. To download SDAR, users will be asked for their name, institution and email address. A manual, as well as the source code of the <it>GraphPanel</it> class can also be downloaded from this site.</p

Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone

The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application

Co-Immobilization of Xylanase and Scaffolding Protein onto an Immobilized Metal Ion Affinity Membrane

Lignocellulosic biomass conversion technology seeks to convert agricultural waste to sugars through the use of various cellulases and hemicellulases. In practice, the application of free enzymes might increase the cost of the process due to difficulties with recovery of the enzymes and products. Immobilization might be an effective approach for recovering the hydrolysis products and improving the stability and reusability of the enzymes. In this study, we used a recombinant genetic engineering approach to construct a scaffold protein gene (CipA) and a xylanase gene (XynC) fused to a dockerin gene (DocT). After expressing CipA and XynC-DocT (XynCt) genes using E. coli hosts, the crude extracts were collected. An immobilized metal ion affinity membrane/Co2+ ion (IMAM-Co2+) system was prepared to adsorb CipA in its crude extract, thereby allowing simultaneous purification and immobilization of CipA protein. A similar approach was applied for the adsorption of XynCt protein, exploiting the interaction between the cohesin units in IMAM-Co2+-CipA and the dockerin unit in XynCt. The activity of the xylanase unit was enhanced in the presence of Co2+ for both the free XynCt enzymes and the immobilized CipA-XynCt. The heat resistance and stability over a wide range of values of pH of the immobilized CipA-XynCt were superior to those of the free XynCt. Furthermore, the immobilized CipA-XynCt retained approximately 80% of its initial activity after seven reaction cycles. The values of Km and &nu;max of IMAM-Co2+-CipA-XynCt (1.513 mg/mL and 3.831 U/mg, respectively) were the best among those of the other tested forms of XynCt

Assistive computer input device for muscular atrophy patients

This article is aimed at people who suffer from muscular dystrophy or amyotrophic lateral sclerosis. A webcam captures images of the user’s head, and the computer then processes these images to calculate and transform the swinging direction and distance of the user’s head into the position of the mouse cursor and into the action of clicking the mouse. To accurately and quickly recognize these features, this article used a round sticker with a diameter of 1 cm as an identification label, which is stuck on the user’s forehead. After the webcam captures the images, the program can recognize the positions of the identification label and move the mouse cursor. Through Windows API, this article determined the user’s intention and action of clicking the mouse on the basis of a series of movements to control the mouse cursor. As confirmed by the experimental results, the proposed assisted computer entry device for muscular atrophy patients is applicable and gives good results for the e-book reading program, use of the web browser, and the execution of the application software and mini-games developed by this system structure