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Sotelo-Silveira J.R.,Institute Investigaciones Biologicas Clemente Estable IIBCE
Methods in molecular biology (Clifton, N.J.) | Year: 2011

The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demonstrate the importance of axon micro-dissection to obtain a whole mount perspective of mRNA distribution in the axonal territory. We describe a protocol to perform fluorescent ISH in isolated axons and guidelines for the preservation of structural and molecular integrity of cortical RNA-containing domains (e.g., Periaxoplasmic Ribosomal Plaques, or PARPs) in isolated axoplasm. Source

Binolfi A.,National University of Rosario | Valiente-Gabioud A.A.,National University of Rosario | Duran R.,Institute Pasteur Of Montevideo Ipm | Duran R.,Institute Investigaciones Biologicas Clemente Estable IIBCE | And 5 more authors.
Journal of the American Chemical Society | Year: 2011

The aggregation of α-synuclein (AS) is selectively enhanced by copper in vitro, and the interaction is proposed to play a potential role in vivo. In this work, we report the structural, residue-specific characterization of Cu(I) binding to AS and demonstrate that the protein is able to bind Cu(I) with relatively high affinity in a coordination environment that involves the participation of Met1 and Met5 residues. This knowledge is a key to understanding the structural-aggregation basis of the copper-catalyzed oxidation of AS. © 2010 American Chemical Society. Source

Botasini S.,Institute Quimica Biologica | Dalchiele E.A.,Institute Fisica | Benech J.C.,Institute Investigaciones Biologicas Clemente Estable IIBCE | Mendez E.,Institute Quimica Biologica
Journal of Nanoparticle Research | Year: 2011

The synthesis of silver non-spherical structures like nanotriangles, nanohexagons, and nanodisks, etc., follows a kinetic control that strongly depends on the nature and concentration of the reagents. By using sodium borohydride in a low molar ratio respect to the Ag+ source for working under kinetic control, it was possible to obtain different plane nanostructures which in turn could be stabilized by the use of the substituted mercaptopyrimidine 2-thiobarbituric acid. In addition, the use of this thiol allowed the stabilization of an unreported shape that could be an intermediate structure in the shape evolution of nanotriangles through nanodisks. This new particle, with 200-300 nm length and 6 nm height, is named "nanoheart" due to its heart-shaped resemblance. © Springer Science+Business Media B.V. 2010. Source

Rodriguez-Casuriaga R.,Institute Investigaciones Biologicas Clemente Estable IIBCE
Journal of visualized experiments : JoVE | Year: 2013

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses. Various strategies such as Staput, centrifugal elutriation, and flow cytometry (FC) have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates--which tend to form because of the syncytial nature of the seminiferous epithelium--and lack cell clumps . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones. On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types . The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting, and ulterior gene expression studies. Source

Carrau F.,University of the Republic of Uruguay | Gaggero C.,Institute Investigaciones Biologicas Clemente Estable IIBCE | Aguilar P.S.,Institute Pasteur Of Montevideo
Trends in Biotechnology | Year: 2015

Saccharomyces cerevisiae, the yeast used widely for beer, bread, cider, and wine production, is the most resourceful eukaryotic model used for genetic engineering. A typical concern about using engineered yeasts for food production might be negative consumer perception of genetically modified organisms. However, we believe the true pitfall of using genetically modified yeasts is their limited capacity to either refine or improve the sensory properties of fermented foods under real production conditions. Alternatively, yeast diversity screening to improve the aroma and flavors could offer groundbreaking opportunities in food biotechnology. We propose a 'Yeast Flavor Diversity Screening' strategy which integrates knowledge from sensory analysis and natural whole-genome evolution with information about flavor metabolic networks and their regulation. © 2015 Elsevier Ltd. Source

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