Project Abstract The Bio-Techne Intracellular and Extracellular Protein Signaling Station (IEPSS) automates standard western blotting and immunoassay and produces reliable, reproducible quantitative data in a very short time. The IEPSS provides a wide array of capabilities, including single cell western blotting, identification of protein isoforms and post-translational modifications in a single cell, separation of proteins based on size and charge, measure four or more analytes in multiple samples. Thus, the IEPSS will provide immediate benefits for VAWNYHS investigators studying the underlying acute and chronic processes that result in physiological dysfunction, disease, and health decline, ultimately permitting the development of interventions to improve the quality of life in the Veteran population. These projects include: Dr. Troen’s research investigates physical performance, frailty, and healthspan during aging and is presently studying the benefits of high intensity interval training (HIIT) for skeletal muscle performance in aging veterans. The IEPSS will allow assessment of protein markers and posttranslational modifications associated with mitochondrial health and skeletal muscle metabolism. Dr. Fliesler’s projects investigate the underlying mechanism of progressive retinal degeneration and visual dysfunction associated with blast overpressure-induced polytrauma, and the impact of novel antioxidants as therapeutic agents to prevent, minimize, or slow the progression of the pathological processes. The IEPSS will afford assessment of biomarkers of inflammation, oxidative stress, and cell death. Dr. Sullivan’s study investigates the development of post-transcriptional gene silencing agents such as ribozymes as candidate therapeutics for orphan retinal degenerations and common age-related macular degeneration. The IEPSS will permit assessment of ribozyme rescue strategy and quantify target protein expression and post-translational modification in photoreceptor and retinal epithelial cells. Dr. Canty’s study investigates cellular remodeling during sudden cardiac arrest and cardiac stem cell mediated repair. The IEPSS will facilitate a proteomic profile of hematopoietic subtypes as well as macrophage subpopulations, also enable to study the chemokines/cytokines. In addition, it quantifies protein changes that arise in response to reversible ischemia. Dr. Feng’s study seeks to identify more efficient methods to understand how mutations of parkin cause the selective degeneration of human dopaminergic neurons and the ensuing Parkinson’s disease. The IEPSS will allow for characterization of protein expression profiles and posttranslational modifications. Dr. Russo’s research seeks to determine the role of penicillin binding protein (PBP) 7/8 as a novel anti- microbial target in XDR A. baumannii. The IEPSS will enable to resolve molecular mechanism of PBP 7/8 and develop receptor-targeted therapies by profiling various outer membrane proteins in single and cultured cells, identifying post-translational modifications in the presence or absence of PBP 7/8. Dr. Farkas’ research seeks to discover potential therapies to prevent and/or treat age-related macular degeneration (AMD) and inherited retinal dystrophies (IRDs). The IEPSS will enable validation of the effects of DNA methylation changes on corresponding protein expression and quantify the cell-to-cell variability of the retinitis pigmentosa 1 minor isoform in the mouse retina. Dr. Lang’s research seeks to determine the functional role of exosomes in stem cell-mediated cardiac repair. The IEPSS will enhance this work by performing multiple immunoassays on small quantities of initial sample and providing the ability to quantify cytokine panels in response to exosome therapy and measuring protein expression in stem cells and in murine cardiac tissue following myocardial infarction.

Project Narrative The Bio-Techne Intracellular and Extracellular Protein Signaling Station is a distinctive instrument that provides a wide array of capabilities, including: single cell western blotting; identification of protein isoforms and post-translational modifications in a single cell; detection of three different proteins with the same molecular weight in a run; measurement of four or more analytes in multiple samples; and utilization of very small sample volume to produce quantifiable results in few hours. Thus, this instrument will allow collection of critical data that will provide valuable insights into both normal and pathological cellular mechanisms, and also afford a means to assess the impact of potential therapeutic interventions. Ultimately, this device will accelerate bench-to-bedside translation of research that ultimately will lead to improvement of the quality of life for our veterans.