TOPCONS: consensus prediction of membrane protein topology

TOPCONS (http://topcons.net/) is a web server for consensus prediction of membrane protein topology. The underlying algorithm combines an arbitrary number of topology predictions into one consensus prediction and quantifies the reliability of the prediction based on the level of agreement between the underlying methods, both on the protein level and on the level of individual TM regions. Benchmarking the method shows that overall performance levels match the best available topology prediction methods, and for sequences with high reliability scores, performance is increased by ∼10 percentage points. The web interface allows for constraining parts of the sequence to a known inside/outside location, and detailed results are displayed both graphically and in text format

Implantation of a colorectal stent as a therapeutic approach in the treatment of esophageal leakage

BACKGROUND: While the mortality of esophageal surgery has decreased due to technological advancements, there is still a complication rate of about 30%. One of the main complications is the anastomotic leakage associated with a significant rate of morbidity and mortality. To close the leakage the efficacy of self-expanding stents (SES) has been shown in different studies. However, the high rate of stent migration limits the use of commercial available stents. In our case we were faced with the problem that the diameter of all available stents was too small to attach tightly to the mucosal wall of the esophagogastric anastomosis. CASE PRESENTATION: We used, for the first time to our knowledge, a metal stent designed for colorectal application in an extensive anastomotic leak after esophageal resection in a patient with an esophageal cancer. After primary surgery with subtotal esohagectomy the anastomotic leak was stented endoscopically with a Polyflex self-expanding covered plastic stent after no response to intensive conventional management. Even though the stent was placed correctly, the diameter of the Polyflex stent was too small to attach onto the wall of the esophagogastric anastomosis. Again surgery was performed with a thoracal resection of the esophageal remnant and a hand made anastomosis. Unfortunately, again an anastomotic leak was detected soon after. To close the leak we decided to use a covered colorectal stent (Hanarostent) with an inner diameter of 30 mm. Sixteen weeks later the stent was extracted and complete mucosal healing of the esophageal leak was observed. CONCLUSION: The stent implantation with a large wide diameter offers a good chance to close more extensive leaks and prevent stent migration

Transmembrane protein topology prediction using support vector machines

Background: Alpha-helical transmembrane (TM) proteins are involved in a wide range of important biological processes such as cell signaling, transport of membrane-impermeable molecules, cell-cell communication, cell recognition and cell adhesion. Many are also prime drug targets, and it has been estimated that more than half of all drugs currently on the market target membrane proteins. However, due to the experimental difficulties involved in obtaining high quality crystals, this class of protein is severely under-represented in structural databases. In the absence of structural data, sequence-based prediction methods allow TM protein topology to be investigated.Results: We present a support vector machine-based (SVM) TM protein topology predictor that integrates both signal peptide and re-entrant helix prediction, benchmarked with full cross-validation on a novel data set of 131 sequences with known crystal structures. The method achieves topology prediction accuracy of 89%, while signal peptides and re-entrant helices are predicted with 93% and 44% accuracy respectively. An additional SVM trained to discriminate between globular and TM proteins detected zero false positives, with a low false negative rate of 0.4%. We present the results of applying these tools to a number of complete genomes. Source code, data sets and a web server are freely available from http://bioinf.cs.ucl.ac.uk/psipred/.Conclusion: The high accuracy of TM topology prediction which includes detection of both signal peptides and re-entrant helices, combined with the ability to effectively discriminate between TM and globular proteins, make this method ideally suited to whole genome annotation of alpha-helical transmembrane proteins

Superior Vena Cava Defibrillator Coils Make Transvenous Lead Extraction More Challenging and Riskier

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Characterization of Leishmania donovani Aquaporins Shows Presence of Subcellular Aquaporins Similar to Tonoplast Intrinsic Proteins of Plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani

The SAR11 Group of Alpha-Proteobacteria Is Not Related to the Origin of Mitochondria

Although free living, members of the successful SAR11 group of marine alpha-proteobacteria contain a very small and A+T rich genome, two features that are typical of mitochondria and related obligate intracellular parasites such as the Rickettsiales. Previous phylogenetic analyses have suggested that Candidatus Pelagibacter ubique, the first cultured member of this group, is related to the Rickettsiales+mitochondria clade whereas others disagree with this conclusion. In order to determine the evolutionary position of the SAR11 group and its relationship to the origin of mitochondria, we have performed phylogenetic analyses on the concatenation of 24 proteins from 5 mitochondria and 71 proteobacteria. Our results support that SAR11 group is not the sistergroup of the Rickettsiales+mitochondria clade and confirm that the position of this group in the alpha-proteobacterial tree is strongly affected by tree reconstruction artefacts due to compositional bias. As a consequence, genome reduction and bias toward a high A+T content may have evolved independently in the SAR11 species, which points to a different direction in the quest for the closest relatives to mitochondria and Rickettsiales. In addition, our analyses raise doubts about the monophyly of the newly proposed Pelagibacteraceae family

GO-PROMTO Illuminates Protein Membrane Topologies of Glycan Biosynthetic Enzymes in the Golgi Apparatus of Living Tissues

The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes