Deciphering the role of chemical signals in inflammation with open microfluidic functional assays

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Project Abstract Small molecule and protein signals provide a rich vocabulary for cellular communication, and upstream changes in RNA expression help drive the molecular dialogue. The downstream consequences of gene expression changes and signal molecule production are exquisitely sensitive and vary based on microenvironment, disease state (including temporal considerations), and individual heterogeneity. For example, gene expression changes vary temporally in response to disease flares or treatments. Dissecting the basic biology of cell signaling and molecular pathway changes in disease is challenging, and new methods are required to address fundamental questions: What is the downstream biological function of each signaling molecule? How is the biological function different when molecules are present in mixtures? How do signaling pathways vary across the human population and through time within an individual? To address these challenges, the parent R35 award has three stated goals: 1) Develop novel microscale co- and multi-culture platforms to study soluble factor signaling and use these tools to elucidate paracrine signaling mechanisms. 2) Develop and validate new readouts for inflammation (e.g., fibrosis, vasodilation) and apply these methods to identify key effector molecules and signaling pathways in inflammation. 3) Develop new analytical methods to stabilize, isolate, and study inflammatory signals. Under the parent R35 award Goal 3, we developed a novel platform that enables at-home blood sampling and RNA stabilization, homeRNA. The homeRNA kit contains a commercially available at-home blood collection device and a custom ‘stabilizer tube’ that our lab engineered to contain a stabilizing solution (e.g., RNAlater to stabilize RNA in blood). Leveraging our lab’s experience with microfluidics and utilizing passive forces (e.g., capillary flow, Laplace pressure), we engineered the ‘stabilizer tube’ with an integrated fluidic channel that prevents spillage of RNAlater while the user handles the kit, but enables the transfer of fluid (e.g., RNAlater solution) when the stabilizer tube is mated with the blood collection tube. Importantly, homeRNA enables longitudinal studies within an individual to capture temporal changes in gene expression signatures resulting from disease flares or in response to treatment. homeRNA enables evaluation of mechanistic hypotheses in human populations, providing a complement to our lab’s in vitro microfluidic platforms, which use cell lines or primary cells. In this supplement, we will use homeRNA to study the molecular mechanisms underlying inflammation in women of understudied, underrepresented, and underreported (U3) populations experiencing post-acute sequelae of SARS-CoV-2 (PASC, also called ‘long COVID’), ultimately enabling better diagnostic and therapeutic approaches for PASC. Further, we will establish homeRNA as a broadly applicable research tool for reaching women of U3 populations.