Bicycle: a bioinformatics pipeline to analyze bisulfite sequencing data.

Summary: High-throughput sequencing of bisulfite-converted DNA is a technique used to measure DNA methylation levels. Although a considerable number of computational pipelines have been developed to analyze such data, none of them tackles all the peculiarities of the analysis together, revealing limitations that can force the user to manually perform additional steps needed for a complete processing of the data. This article presents bicycle, an integrated, flexible analysis pipeline for bisulfite sequencing data. Bicycle analyzes whole genome bisulfite sequencing data, targeted bisulfite sequencing data and hydroxymethylation data. To show how bicycle overtakes other available pipelines, we compared them on a defined number of features that are summarized in a table. We also tested bicycle with both simulated and real datasets, to show its level of performance, and compared it to different state-of-the-art methylation analysis pipelines. Availability and implementation: Bicycle is publicly available under GNU LGPL v3.0 license at http://www.sing-group.org/bicycle. Users can also download a customized Ubuntu LiveCD including bicycle and other bisulfite sequencing data pipelines compared here. In addition, a docker image with bicycle and its dependencies, which allows a straightforward use of bicycle in any platform (e.g. Linux, OS X or Windows), is also available. Contact: ograna@cnio.es or dgpena@uvigo.es. Supplementary information: Supplementary data are available at Bioinformatics online.

A novel high-content analysis tool reveals Rab8-driven cytoskeletal reorganization through Rho GTPases, calpain and MT1-MMP.

Rab8 is a small Ras-related GTPase that regulates polarized membrane transport to the plasma membrane. Here, we developed a high-content analysis (HCA) tool to dissect Rab8-mediated actin and focal adhesion reorganization that revealed that Rab8 activation significantly induced Rac1 and Tiam1 to mediate cortical actin polymerization and RhoA-dependent stress fibre disassembly. Rab8 activation increased Rac1 activity, whereas its depletion activated RhoA, which led to reorganization of the actin cytoskeleton. Rab8 was also associated with focal adhesions, promoting their disassembly in a microtubule-dependent manner. This Rab8 effect involved calpain, MT1-MMP (also known as MMP14) and Rho GTPases. Moreover, we demonstrate the role of Rab8 in the cell migration process. Indeed, Rab8 is required for EGF-induced cell polarization and chemotaxis, as well as for the directional persistency of intrinsic cell motility. These data reveal that Rab8 drives cell motility by mechanisms both dependent and independent of Rho GTPases, thereby regulating the establishment of cell polarity, turnover of focal adhesions and actin cytoskeleton rearrangements, thus determining the directionality of cell migration.

Serum and tissue profiling in bladder cancer combining protein and tissue arrays.

Aiming at identifying biomarkers for bladder cancer, the serum proteome was explored in a pilot study through a profiling approach using protein arrays. Supervised analyses identified a panel 171 immunogenic proteins differentially expressed between patients with bladder cancer (n = 12) and controls without the disease (n = 10). The microanatomical expression patterns of novel immunogenic proteins, especially dynamin and clusterin, were found significantly associated with histopathologic variables and overall survival, as confirmed by immunohistochemistry using an independent series of bladder tumors contained in tissue microarrays (n = 289). Thus, the protein arrays approach has identified a panel of immunogenic candidates that may potentially play a role as diagnostic biomarkers, especially for muscle invasive disease. Moreover, the protein expression patterns of dynamin and clusterin in bladder tumors were shown to adjunct for histopathologic staging and clinical outcome prognosis.