Prophossi:automating expert validation of phosphopeptide-spectrum matches from tandem mass spectrometry

Motivation: Complex patterns of protein phosphorylation mediate many cellular processes. Tandem mass spectrometry (MS/MS) is a powerful tool for identifying these post-translational modifications. In high-throughput experiments, mass spectrometry database search engines, such as MASCOT provide a ranked list of peptide identifications based on hundreds of thousands of MS/MS spectra obtained in a mass spectrometry experiment. These search results are not in themselves sufficient for confident assignment of phosphorylation sites as identification of characteristic mass differences requires time-consuming manual assessment of the spectra by an experienced analyst. The time required for manual assessment has previously rendered high-throughput confident assignment of phosphorylation sites challenging

Fibroblast Growth Factor Receptor-1 Signaling in Pancreatic Islet β-Cells Is Modulated by the Extracellular Matrix

Maintenance of pancreatic β-cell mass depends on extracellular stimuli that promote survival and proliferation. In the islet, these stimuli come from the β-cell microenvironment and include extracellular matrix deposited by associated vascular endothelial cells. Fibroblast growth factor receptor-1 (FGFR1) has recently been implicated as a signaling pathway that is important for normal β-cell function. We would like to understand how extracellular matrix and FGFR1 signaling interact to promote β-cell survival and proliferation. To examine β-cell-specific receptor responses, we created lentiviral vectors with rat insulin promoter-driven expression of Venus fluorescent protein-tagged full-length (R1βv) and kinase-deficient (KDR1βv) FGFR1. Significant FGF-1-dependent activation of ERK1/2 was observed in βTC3 cells, dispersed β-cells, and β-cells in intact islets. This response was enhanced by R1βv expression and reduced by KDR1βv expression. Plating-dispersed β-cells on collagen type IV resulted in enhanced expression of endogenous FGFR1 that was associated with sustained activation of ERK1/2. Conversely, plating cells on laminin reduced expression of FGFR1, and this reduction was associated with transient activation of ERK1/2. Addition of neutralizing antibodies to inhibit β-cell attachment to laminin via α6-integrin increased high-affinity FGF-1-binding at the plasma membrane and resulted in sustained ERK1/2 activity similar to cells plated on collagen type IV. These data show that the FGF-stimulated β-cell response is negatively affected by α6-integrin binding to laminin and suggest regulation associated with vascular endothelial cell remodeling

Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells

Several cell-based immunotherapy strategies have been developed to specifically modulate T cell–mediated immune responses. These methods frequently rely on the utilization of tolerogenic cell–based antigen-presenting cells (APCs). However, APCs are highly sensitive to cytotoxic T-cell responses, thus limiting their therapeutic capacity. Here, we describe a novel bead-based approach to modulate T-cell responses in an antigen-specific fashion. We have generated killer artificial APCs (κaAPCs) by coupling an apoptosis-inducing α-Fas (CD95) IgM mAb together with HLA-A2 Ig molecules onto beads. These κaAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)–dependent fashion. T-cell depletion in cocultures is rapidly initiated (30 minutes), dependent on the amount of κaAPCs and independent of activation-induced cell death (AICD). κaAPCs represent a novel technology that can control T cell–mediated immune responses, and therefore has potential for use in treatment of autoimmune diseases and allograft rejection