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Siena, Italy

Moretti E.,University of Siena | Giannerini V.,University of Siena | Rossini L.,Siena Biotech | Matsuoka M.,Tokyo Medical University | And 2 more authors.
Fertility and Sterility | Year: 2010

We have discovered, by immunocytochemistry and immunoelectronmicroscopy, that humanin (HN) is expressed in human ejaculated sperm and testis. In sperm, the HN immunolabeling pattern depends on sperm morphology; in particular, HN is mainly localized in the midpiece of sperm in semen samples with normal morphology and in cytoplasmic residues and entire tail in those with abnormal morphology. We also found HN in the cytoplasm and nucleus of spermatocytes and spermatids and in experimentally uncoiled chromatin of mature ejaculated sperm. Because it has been established that HN has antiapoptotic properties, it is reasonably hypothesized that HN may play an important role in preventing apoptosis in human sperm and testis. Thus, the examination of the HN localization in normal and abnormal sperm could be proposed as an auxiliary test to better define sperm quality. Copyright © 2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

Incarnato D.,Human Genetics Foundation HuGeF | Incarnato D.,University of Siena | Neri F.,Human Genetics Foundation HuGeF | Diamanti D.,Siena Biotech | And 2 more authors.
Nucleic Acids Research | Year: 2013

The prediction of pairing between microRNAs (miRNAs) and the miRNA recognition elements (MREs) on mRNAs is expected to be an important tool for understanding gene regulation. Here, we show that mRNAs that contain Pumilio recognition elements (PRE) in the proximity of predicted miRNA-binding sites are more likely to form stable secondary structures within their 3′-UTR, and we demonstrated using a PUM1 and PUM2 double knockdown that Pumilio proteins are general regulators of miRNA accessibility. On the basis of these findings, we developed a computational method for predicting miRNA targets that accounts for the presence of PRE in the proximity of seed-match sequences within poorly accessible structures. Moreover, we implement the miRNA-MRE duplex pairing as a two-step model, which better fits the available structural data. This algorithm, called MREdictor, allows for the identification of miRNA targets in poorly accessible regions and is not restricted to a perfect seed-match; these features are not present in other computational prediction methods. © 2013 The Author(s). Published by Oxford University Press.

Castaldo C.,Siena Biotech | Ciambellotti S.,University of Florence | de Pablo-Latorre R.,University of Florence | Lalli D.,University of Florence | And 2 more authors.
PLoS ONE | Year: 2013

Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of 1H-15N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer's disease. © 2013 Castaldo et al.

Jimenez-Sanchez M.,University of Cambridge | Lam W.,University of Cambridge | Hannus M.,Cenix BioScience GmbH | Sonnichsen B.,Cenix BioScience GmbH | And 33 more authors.
Nature Chemical Biology | Year: 2015

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone α B-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development. © 2015 Nature America, Inc. All rights reserved.

Smith M.R.,University of California at Irvine | Syed A.,University of California at Irvine | Lukacsovich T.,University of California at Irvine | Purcell J.,University of California at Irvine | And 25 more authors.
Human Molecular Genetics | Year: 2014

Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntington's disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology inmodel organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophilamelanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selectivepharmacological inhibitionofDrosophila Sir2andmammalianSirT1usingthenovel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide)cansuppressHDpathologycausedby mutanthuntingtinexon1fragmentsinDrosophila,mammaliancellsandmice.WehavevalidatedSir2asthein vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease andmay also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for HD. © The Author 2014. Published by Oxford University Press. All rights reserved. Published by Oxford University Press. All rights reserved.

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