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North Bethesda, MD, United States

Yang Y.,McKusick Nathans Institute of Genetic Medicine | Chaerkady R.,McKusick Nathans Institute of Genetic Medicine | Chaerkady R.,Institute of Bioinformatics | Kandasamy K.,McKusick Nathans Institute of Genetic Medicine | And 10 more authors.
Molecular BioSystems | Year: 2010

Although the targets of most miRNAs have not been experimentally identified, microRNAs (miRNAs) have begun to be extensively characterized in physiological, developmental and disease-related contexts in recent years. Thus far, mainly computational approaches have been employed to predict potential targets for the large majority of miRNAs. Although miRNAs exert a major influence on the efficiency of translation of their targets in animals, most studies describing experimental identification of miRNA target genes are based on detection of altered mRNA levels. miR-143 is a miRNA involved in tumorigenesis in multiple types of cancer, smooth muscle cell fate and adipocyte differentiation. Only a few miR-143 targets are experimentally verified, so we employed a SILAC-based quantitative proteomic strategy to systematically identify potential targets of miR-143. In total, we identified >1200 proteins from MiaPaCa2 pancreatic cancer cells, of which 93 proteins were downregulated >2-fold in miR-143 mimic transfected cells as compared to controls. Validation of 34 of these candidate targets in luciferase assays showed that 10 of them were likely direct targets of miR-143. Importantly, we also carried out gene expression profiling of the same cells and observed that the majority of the candidate targets identified by proteomics did not show a concomitant decrease in mRNA levels confirming that miRNAs affect the expression of most targets through translational inhibition. Our study clearly demonstrates that quantitative proteomic approaches are important and necessary for identifying miRNA targets. © 2010 The Royal Society of Chemistry. Source

Zaritsky L.A.,Broadway Research Building | Dery A.,Broadway Research Building | Leong W.Y.,Broadway Research Building | Gama L.,Broadway Research Building | Clements J.E.,Broadway Research Building
Journal of Interferon and Cytokine Research | Year: 2013

Interferon alpha (IFNalpha) is a type I interferon that plays a major role in host defense. There are 13 different IFNalpha genes in humans, but much of the work concerning their role in viral defense has been limited to studying either subtype 2 or pan IFNalpha due to the inability to distinguish between highly similar genetic and amino acid sequences. Because of recent advances in molecular and biochemical techniques, it is possible to study the regulation of individual subtypes. It has been reported that HIV/SIV infection results in impaired IFNalpha responses in certain tissues. Using a pigtailed macaque SIV model, we examined the subtype response during acute infection in 3 tissues that are known to be infected with HIV/SIV, but whose IFNalpha subtype response has not been extensively studied: the brain, spleen, and lung. We found that the expression and regulation of specific subtypes occur in a tissue-specific manner. There was more limited IFNalpha subtype expression in the lung and brain, where predominantly macrophages are infected compared to the spleen, which contains both infected CD4+ lymphocytes and macrophages. Understanding the IFNalpha subtype response in tissues known to be infected with HIV/SIV can help tailor adjunctive treatment regimens to highly active antiretroviral therapy. © Copyright 2013, Mary Ann Liebert, Inc. Source

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