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

项目摘要 小分子和蛋白质信号为细胞通信和上游变化提供了丰富的词汇 帮助推动分子对话。基因表达的下游后果 变化和信号分子的产生是非常敏感的， 状态（包括时间因素）和个体异质性。例如，基因表达改变 随疾病发作或治疗而随时间变化。剖析细胞信号传导的基础生物学， 疾病的分子途径变化是具有挑战性的，需要新的方法来解决基本的 问题：每个信号分子的下游生物学功能是什么？生物功能如何 当分子以混合物的形式存在时会有什么不同呢？信号通路在人群中是如何变化的 在一个人的身体里为了应对这些挑战，母公司R35奖有三个声明 目标：1）开发新的微尺度共培养和多培养平台，以研究可溶性因子信号传导，并使用这些 阐明旁分泌信号机制的工具。2)开发并验证炎症的新读数（例如， 纤维化，血管舒张），并应用这些方法来鉴定关键的效应分子和信号通路， 炎症3)开发新的分析方法来稳定、分离和研究炎症信号。下 目标3，我们开发了一个新的平台，使在家里的血液采样和RNA 稳定化，homeRNA。homeRNA试剂盒包含市售的家用血液采集装置 以及我们实验室设计的包含稳定溶液的定制“稳定管”（例如，RNAlater稳定 血液中的RNA）。利用我们实验室在微流体方面的经验和利用被动力（例如，毛细流动， 拉普拉斯压力），我们设计的“稳定管”与集成的流体通道，防止溢出的 当使用者处理试剂盒时，RNA可以稍后传送，但是能够传送流体（例如，RNAlater溶液）时， 稳定器管与血液收集管匹配。重要的是，homeRNA使纵向研究 个体以捕获由疾病突发引起的基因表达特征的时间变化， 对治疗的反应homeRNA能够评估人类群体中的机制假设， 为我们实验室使用细胞系或原代细胞的体外微流体平台提供补充。在这 作为补充，我们将使用homeRNA来研究女性炎症的分子机制， 未充分研究、代表性不足和报告不足（U3）的人群发生急性后后遗症， SARS-CoV-2（PASC，也称为“长COVID”），最终能够更好地诊断和治疗 接近PASC。此外，我们将建立homeRNA作为一个广泛适用的研究工具， U3人口。

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.