Project 2 Summary. Circulating Signals of ME/CFS Individuals with ME/CFS experience a multitude of disabling symptoms such as fatigue, pain, unrefreshing sleep, cognitive difficulties, orthostatic intolerance, and post-exertional malaise (PEM). PEM is the inability to increase physical or mental effort without symptom exacerbation and it greatly limits the quality of life of ME/CFS patients. In this project, we will learn more about the tissues and organs affected during and after exercise when ME/CFS patients are experiencing PEM. We have conducted a study in which ME/CFS and healthy sedentary control subjects have undergone two successive cardiopulmonary exercise tests (CPETs). As well as obtaining extensive clinical and exercise physiology data, we have collected blood samples from these subjects at four time points: before exercise (baseline), immediately after the first CPET, after a 24 hour recovery period, and immediately after the second CPET. We have already measured thousands of metabolites and proteins in these samples and measured gene expression in subpopulations of immune cells. In this project, using the same plasma samples from the longitudinal exercise study, we will examine novel types of signals that circulate in blood: Cell-free ribonucleic acid (RNA) and extracellular vesicles (EVs). Cell-free RNA is released into the blood from dying cells in circulation or from various tissues throughout the body. EVs are membrane-enclosed bodies that travel through the blood from different tissues and can deliver protein, RNA, and other signaling molecules. EVs provide information about tissues such as the brain that could otherwise not be obtained without invasive biopsy. We will determine which tissues released the cell-free RNA and the EVs into circulation. Learning about how the content and origin of these signals changes in ME/CFS patients compared to controls before and after exercise may reveal disruptions in pathways that lead to PEM and provide clues about additional tissues involved in PEM. We will also quantify the protein and RNA cargo inside EVs, which can inform us about the modulatory effect the EVs may have in recipient cells. Recent work also implicates disruption of the tissue that lines the inside of blood vessels (endothelium). We will culture endothelial cells with plasma and EVs from ME/CFS patients and controls at baseline to learn if molecules causing endothelial dysfunction in ME/CFS originate inside or outside EVs. The wealth of data we will have from the same subjects will be used to look for biomarkers to develop a diagnostic test for ME/CFS. We will also integrate these different types of data to see if we can define clinically relevant subsets of ME/CFS patients.