Methods in molecular biology
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Identification of phosphoproteins or phosphopeptides as cancer biomarkers is an emerging field in phosphoproteomics. Owing to the low stoichiometric nature of protein phosphorylation, phosphoproteins or phosphopeptides must be enriched prior to downstream mass spectrometry analysis. Titanium dioxide (TiO2) has been prevalently used to enrich phosphopeptides from complex proteome samples due to its high affinity for phosphopeptides, and the method is straightforward. ⋯ Phosphopeptides are eluted using an ammonia solution at high pH. Use of NH4Glu significantly reduces nonspecific bindings while a high recovery rate (84 %) of phosphopeptides is retained. The method is optimized for large-scale phosphoproteomic analysis and phosphoprotein biomarker discovery starting from sub-milligram or milligrams of proteome samples.
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Hypertrophic scar (HTS) represents the dermal equivalent of fibroproliferative disorders that occur after injury involving the deep dermis while superficial wounds to the skin heal with minimal or no scarring. HTS is characterized by progressive deposition of collagen that occurs with high frequency in adult dermal wounds following traumatic or thermal injury. Increased levels of transforming growth factor-β1 (TGF-β1), decreased expression of small leucine-rich proteoglycans (SLRPs), and/or fibroblast subtypes may influence the development of HTS. ⋯ Studying the characteristics of superficial dermal injuries that heal with minimal scarring will help us identify therapeutic approaches for tissue engineering and wound healing. In addition, our ability to develop novel therapies for HTS is hampered by limitations in the available animal models used to study this disorder in vivo. We also describe a nude mouse model of transplanted human skin that develops a hypertrophic proliferative scar consistent morphologically and histologically with human HTS, which can be used to test novel treatment options for these dermal fibrotic conditions.
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Peptide microarray technology can be used to identify substrates for recombinant kinases, to measure kinase activity and changes thereof in cell lysates and lysates from fresh frozen (tumor) tissue. The effect of kinase inhibitors on the kinase activities in relevant tissues can be investigated as well. The method for performing experiments on dynamic peptide microarrays with real-time readout is described, as well as the influence of assay parameters and suggestions for optimization of experiments.
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The search for potential drugs to treat neurodegenerative diseases has been intense in the last two decades. Among many candidates, erythropoietin (EPO) was identified as a potent protectant of neurons suffering from various adverse conditions. A wide array of literature indicates that endogenous or exogenous recombinant human erythropoietin and its variants activate cell signaling that initiates survival-promoting events in neurons and neuronal cells. ⋯ The signaling pathways involved in EPO are multiple; some are well known whereas others are still under intense investigation and few are observed in very specific cell types. It is important to note that neuronal signaling events triggered by EPO are still incomplete and require further research. Therefore, excellent review articles that explore specific EPO-signaling events are referenced.
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The isolation of embryonic stem cells (ESCs) has furthered our understanding of normal embryonic development and fueled the progression of stem cell derived therapies. However, the generation of ESCs requires the destruction of an embryo, making the use of these cells ethically controversial. In 2006 the Yamanaka group overcame this ethical controversy when they described a protocol whereby somatic cells could be dedifferentiated into a pluripotent state following the transduction of a four transcription factor cocktail. ⋯ The fast paced field of cellular reprogramming has recently produced protocols to generate iPSCs using non integrative techniques with an ever improving efficiency. These recent developments have brought us one step closer to developing a safe and efficient method to reprogram cells for clinical use. However, a lot of work is still needed before iPSCs can be implemented in a clinical setting.