Patients with MACE in the OCT guided arm of the CLI-OPCI study have more often an uncorrected stent deployment

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Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia

Copyright @ 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.Muscular Dystrophy Association (USA), the National Health and Medical Research Council (Australia), the Friedreich’s Ataxia Research Alliance (USA), the Brockhoff Foundation (Australia), the Friedreich Ataxia Research Association (Australasia), Seek A Miracle (USA) and the Victorian Government’s Operational Infrastructure Support Program

A targeted proteomics approach to amyloidosis typing

Background: Amyloidosis is a life threatening disease caused by deposition of various types of blood serum proteins in organs and tissues. Knowing the type of protein involved is the basis of a correct diagnosis and personalized medical treatment. While the classical approach uses immunohistochemistry, in recent years, laser micro-dissection, followed by high resolution LC-MS/MS, has been shown to provide superior diagnostic sensitivity and specificity. This techniques, however, is only available at major reference proteomics centers. // Objective: To perform clinical amyloid protein typing using low-resolution mass spectrometry and no laser micro dissection (LMD), we developed a targeted proteomics approach for the determination of both frequently encountered amyloid proteins (i.e., κ and -λ immunoglobulin light chains and transthyretin (TTR)) and specific reference proteins (i.e., actin (A) for cardiac muscle tissue, or fatty acid binding protein 4 (FBP4) for subcutaneous adipose tissue) in histologic specimens. // Method: Small tissue fragments and/or histological sections were digested to yield a protein mixture that was subsequently reduced, alkylated and trypsinized to obtain a peptide mixture. After SPE purification and LC separation, proteotypic peptides were detected by their MRM transitions. // Results: The method showed high specificity and sensitivity for amyloid protein proteotypic peptides. LODs were 1.0, 0.1, 0.2 picomoles in cardiac muscle tissue (CMT) and 0.1, 0.2, 0.5 picomoles in subcutaneous adipose tissue (SAT) for TTR, κ-, and λ-LC proteins, respectively. Amyloid to tissue-specific protein signal ratios correlated with the presence of amyloid deposits in clinical samples. // Conclusions: This targeted proteomics approach enables sensitive and specific discrimination of amyloidosis affected tissues for the purpose of clinical research

Eomes controls the development of Th17-derived (non-classic) Th1 cells during chronic inflammation

It is well accepted that Th17 cells are a highly plastic cell subset that can be easily directed towards the Th1 phenotype in vitro and also in vivo during inflammation. However, there is an ongoing debate regarding the reverse plasticity (conversion from Th1 to Th17). We show here that ectopic ROR-\u3b3t expression can restore or initiate IL-17 expression by non-classic or classic Th1 cells, respectively, while common pro-Th17 cytokine cocktails are ineffective. This stability of the Th1 phenotype is at least partially due to the presence of a molecular machinery governed by the transcription factor Eomes, which promotes IFN-\u3b3 secretion while inhibiting the expression of ROR-\u3b3t and IL-17. By using a mouse model of T cell-dependent colitis we demonstrate that Eomes controls non-classic Th1 cell development also in vivo and promotes their pathogenic potential. Eomes expression associates to a highly inflammatory phenotype also in patients with juvenile idiopathic arthritis. Indeed, it favors the acquisition of a cytotoxic signature, and promotes the development of IFN-\u3b3+ GM-CSF+ cells that have been described to be pathogenic in chronic inflammatory disorders

The Role of CyaY in Iron Sulfur Cluster Assembly on the E. coli IscU Scaffold Protein

Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S]2+ and [4Fe4S]2+ but directly affects enzymatic activity