During inflammation, flowing leukocytes tether to and roll on vascular surfaces through the association and dissociation of selectin/ligand bonds. The interactions of P- and L- selectins with their respective ligands exhibit catch-slip bonds, such that increasing force initially prolongs and then shortens bond lifetimes. In addition, catch-slip bonds have been shown to govern L-selectin-mediated cell rolling. Using a flow chamber and biomembrane force probe, we show a triphasic force dependence of E-selectin/ligand dissociation that initially behaves as slip bonds, then transitions to catch bonds, and finally transitions again to slip bonds as the force increases. These transitions govern the velocities of neutrophils, HL-60 cells, and Colo-205 cells rolling on E-selectin, as evidenced by the fact that their velocities exhibited a triphasic force dependence that inversely matched the triphasic lifetime-force relationship. At low forces, slip bonds may also precede catch bonds for interactions of P- and L-selectin with their ligands.
Selectin-ligand interactions mediate tethering and rolling of circulating leukocytes on the vessel wall during inflammation. Extensive study has been devoted to elucidating the kinetic and mechanical constraints of receptor-ligand-interaction-mediated leukocyte adhesion, yet many questions remain unanswered. Here, we describe our design of an inverted flow chamber to compare adhesions of HL-60 cells to E-selectin in the upright and inverted orientations. This new, to our knowledge, design allowed us to evaluate the effect of gravity and to investigate the mechanisms of flow-enhanced adhesion. Cell rolling in the two orientations was qualitatively similar, and the quantitative differences can be explained by the effect of gravity, which promotes free-flowing cells to tether and detached cells to reattach to the surface in the upright orientation but prevents such attachment from happening in the inverted orientation. We characterized rolling stability by the lifetime of rolling adhesion and detachment of rolling cells, which could be easily measured in the inverted orientation, but not in the upright orientation because of the reattachment of transiently detached cells. Unlike the transient tether lifetime of E-selectin-ligand interaction, which exhibited triphasic slip-catch-slip bonds, the lifetime of rolling adhesion displayed a biphasic trend that first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. We have developed a minimal mathematical model for the probability of rolling adhesion. Comparison of the theoretical predictions to data has provided model validation and allowed evaluation of the effective two-dimensional association on-rate, kon, and the binding affinity, Ka, of the E-selectin-ligand interaction. kon increased with the wall shear stress from 0.1 to 0.7 dyn/cm(2). Ka first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. Our results provide insights into how the interplay between flow-dependent on-rate and off-rate of E-selectin-ligand bonds determine flow-enhanced cell rolling stability.
Organizations that fund research to address global development challenges are increasingly interested in measuring the social and economic outcomes of research. However, traditional metrics for measuring research outputs are often insufficient for capturing the outcomes targeted by international assistance organizations. To address this, the Center for Development Research (CDR), part of the U.S. Global Development Lab at the United States Agency for International Development (USAID), has designed a new tool: the Program and Policy Change (PPC) framework for tracking and quantifying the influence of research on program and policy change in international development. The framework draws on existing conceptual frameworks of evidence uptake and the literature on policy change. This article describes the design of the PPC framework and presents the results of applying the framework to two USAID research programs. The benefits of the framework include applicability across research sectors, focus on evidence-informed policy at various levels of geographical influence, and inclusion of a numeric scoring system that enables quantification of outcomes.
OBJECTIVES/GOALS: Introduce students to programming and software development practices in the life sciences by analyzing standard clinical diagnostic bloodwork for differential immune responses. Including lectures and a semester project with the goal of enhancing undergraduate students’ education to prepare them for careers in translational science. METHODS/STUDY POPULATION: The educational content was taught for the first time as a component of the newly developed course BTEC 330 “Software Applications in the Life Sciences” in UMBC’s Translational Life Science Technology (TLST) Bachelor’s degree program at the Universities at Shady Grove campus. Eleven students took the course. All were beginners with no programming background. Lectures provided background on the diagnostic components of the CBC, criteria for differential diagnosis in the clinical setting, and introduction to hematology and flow cytometry, forming underpinnings for interpretation of the CBC results. Weekly computer lab practical sessions provided training fundamentals of R programming language, the R-studio integrated development environment (IDE), and the GitHub.com open-source software development platform. RESULTS/ANTICIPATED RESULTS: The graded assignment consisted of a coding project in which students were each assigned an individual parameter from the CBC results. These include, for example, relative lymphocyte count or hemoglobin readouts. Students each created their own R-language script using R-studio, with functional code which: 1) Read in data from a file provided, 2) Performed statistical testing, 3) Read out statistical results as text, and charts as image files, 4) “Diagnosed” individuals in the dataset as being inside or outside the clinical normal range for that parameter. Each student also registered their own GitHub account and published their open-source code. Grading was performed on code functionality by downloading each student repository and running the code with the instructor as an outside developer using the resource. DISCUSSION/SIGNIFICANCE OF IMPACT: In this curriculum, students with no background in programming learned to code a basic R-language script and use GitHub to automate interpretation of CBC results. With advanced automation now becoming commonplace in translational science, such course content can provide introductory level of literacy in development of clinical informatics software.
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