Cell-free expression, purification and immunoreactivity assessment of recombinant Fasciola hepatica saposin-like protein-2.

Cell free protein synthesis has become a powerful method for the high-throughput production of proteins that are difficult to express in living cells. The protein SAP2 of Fasciola hepatica (FhSAP2), which has demonstrated to be both, an excellent vaccine candidate against experimental fascioliasis and a good antigen for serodiagnosis of human chronic fascioliasis, is a typical example of a molecule that is difficult to produce. This is mainly due to its tendency to get over-expressed in inclusion bodies by prokaryotes. FhSAP2 expressed in an Escherichia coli-based expression system is poorly glycosylated, insoluble and often undergoes improper folding leading it to reduced immunogenicity. In this work, FhSAP2 was expressed in vitro using the eukaryote cell free system, TNT T7 Quick coupled transcription/translation, that has been designed for the expression of PCR-generated DNA templates. FhSAP2 was expressed in micro-volumes and purified by an affinity chromatography method, which gave a protein yield of 500 µg/ml as determined by bicinchoninic acid assay method. Circular dichroism, Western blotting and enzyme-linked immunosorbent assay analysis were used to confirm the secondary structure, purity and integrity of protein. Results demonstrate that FhSAP2 can be expressed in a cell-free system retaining its main conformational and antigenic properties. The protein purified could be used in immunization experiments and immunodiagnostic techniques.

Qualimap: evaluating next-generation sequencing alignment data.

MOTIVATION: The sequence alignment/map (SAM) and the binary alignment/map (BAM) formats have become the standard method of representation of nucleotide sequence alignments for next-generation sequencing data. SAM/BAM files usually contain information from tens to hundreds of millions of reads. Often, the sequencing technology, protocol and/or the selected mapping algorithm introduce some unwanted biases in these data. The systematic detection of such biases is a non-trivial task that is crucial to drive appropriate downstream analyses. RESULTS: We have developed Qualimap, a Java application that supports user-friendly quality control of mapping data, by considering sequence features and their genomic properties. Qualimap takes sequence alignment data and provides graphical and statistical analyses for the evaluation of data. Such quality-control data are vital for highlighting problems in the sequencing and/or mapping processes, which must be addressed prior to further analyses. AVAILABILITY: Qualimap is freely available from http://www.qualimap.org.

Extending the Study of the 6,8 Rearrangement in Flavylium Compounds to Higher pH Values: Interconversion between 6-Bromo and 8-Bromo-apigeninidin.

The rearrangement between isomers 6- and 8-bromo-apigeninidin (6 and 8) was studied by pH jumps using stopped flow, UV/Vis, NMR, and HPLC analysis. The system constitutes a pH-dependent network of chemical reactions involving up to 18 different species. The dynamic network is equivalent to a single diprotic acid exhibiting two pKas, 2.55 and 5.4. Similar to other flavylium derivatives, the mole fraction of the species hemiketal and cis-chalcone in both multistate isomers are negligible at the equilibrium. At pH 1, the pure isomers are slowly converted in a mixture containing about 50 % of isomers 6 and 8, while at pH 4, the system evolves to mixture of 10 % trans-chalcone and 90 % of quinoidal bases. A series of pH jumps from pure isomer 6 at pH 1 to pH 6 and back to pH 1 leads to the same initial absorption spectra of the pure isomer 6. The same occurs for pure isomer 8, showing the lack of communication between the cis-chalcones, at least in the time scale of few minutes. A pH jump from the equilibrated mixture of the isomers at pH 1.0 to 5.8 permits to follow a very slow isomerization.

Duplicaçäo duodenal em adulto simulando pseudocisto de pancreas / Duodenal duplication first diagnosed as pancreas pseudocyst: case relate

Relata-se um paciente adulto com quadro clinico e de imagem de pseudocisto de pancreas em que na cirurgia foi diagnosticado duplicacao duodenal. Tendo em vista a raridade deste caso, os autores apresentam revisao da literatura e discutem o seu tratamento