The overarching goal of this TPPG is to elucidate common pathogenic mechanisms of pulmonary arterial hypertension (PAH) and target these pathways with novel drug strategies. To that end, the initial TPPG funding period identified perturbations in several NO-based signaling pathways that contribute to PAH pathogenesis. Further, it identified and developed a number of promising candidate therapeutics. The current project focuses specifically on two candidate therapeutics - oral nitrite (NO2-) and nitro-oleic acid (NO2-OA) – and their ability to modulate NO signaling to prevent PAH progression and heart failure. Crucial to the success of this project is the ability to specifically and accurately measure NO, its metabolites, and its protein/lipid signaling targets. To that end, the Bioanalytical Core brings together existing infrastructure and expertise to support investigators across all three projects in detecting and quantifying NO/reactive species that participate in seminal signaling events in biological samples and in vivo models. Specifically, the core is comprised of three critical components that will synergize to comprehensively detect and quantify all pertinent reactive nitrogen (NO, nitrite, nitrate, S-nitrosothiol, nitrated lipids) and reactive oxygen (superoxide, hydrogen peroxide, lipid radicals) species in both bench and bedside projects. 1) The chemiluminescence core will utilize reductive chemistry in conjunction with chemiluminescence NO detection to measure NO and its metabolites in biological specimens and assess bacterial nitrite/nitrate reductase activity in the microbiome. 2) The EPR component will utilize cutting edge EPR technology with spin trapping to directly measure radicals in biological samples. Additionally, EPR technology will enable the differentiation of endogenous and 15-N labeled NO species in biological samples. 3) The Mass Spectroscopy component will enable the measurement of NO and oxo-modified lipid and protein biomolecules. All three components of the Bioanalytical Core are already optimized for human and animal samples and are directed by experts in the respective technologies. Collectively, the services offered by the Bioanalytical Core will greatly expand and refine the ability of researchers in all three research projects of this TPPG to elucidate novel NO pathways and develop unique therapeutics that work mechanistically through the modulation of NO signaling.