This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
Support from the NSF MRI-R2 program has allowed the purchase of a Beckman Coulter MoFlo XDP cell sorter, a BD Pathway 885 High-Content Bioimager, and a Miltenyi Gentle Macs Dissociator, and related equipment to support research focused on understanding the biodynamics of molecular interactions leading to cellular differentiation. This interdisciplinary effort integrates faculty, post-doctoral and student researchers in the disciplines of biochemistry, immunology, physiology, molecular cell biology, neurobiology and plant biology. Results of studies planned for this suite of instruments will allow specific subsets of T cells to be identified, separated, cultured, and their interactions with specific activating and inhibiting factors imaged in real time. The differential effects of growth factors on freshly isolated subpopulations of dorsal root ganglion neurons grown on carbon and magnetic nanotube substrates will be characterized. The uptake of plant lectins and fluorescent nanocrystals by pulmonary and immune cells in intact animals and the subsequent effects of this absorption on cellular molecular dynamics will be characterized. The equipment will also be used to support science education outreach to high school, undergraduate and graduate students. ASU has active NSF-funded REU, URM, GK-12, and Louis Stokes Minority Alliance programs, as well as a recently funded McNair project, that focus on recruiting, retaining and enhancing the educational experience of students of under-represented minorities. Many of these students are actively participating in research projects in the PIs labs. To further broaden potential impacts, training and applications seminars will be held in addition to integrating these technologies into our Molecular Biosciences PhD techniques courses in immunology and microscopy. Enhanced research capabilities will strengthen the University's effort to recruit and retain minority students, faculty, and collaborators who might otherwise choose other universities or other disciplines to study. Results from the studies will be published in peer-reviewed journals and presented by students and faculty at regional and national meetings.
This MRI-R2 grant enhances the instrumentation infrastructure to support research focused on understanding the biodynamics of molecular interactions leading to cellular differentiation. The grant specifically funded the purchase of a suite of equipments supporting cell imaging and immunological analyses that included a BD Pathway™ 885 High-Content Bioimager, a Miltenyi Gentle Macs™ Dissociator, an epMotion Liquid Handling System, and a BD FACSAria Cell Sorter. These instruments were installed in year one (2009-10) and have supported the four proposed research projects focused on testing the overall hypothesis that differential absorption by distinct cell populations linked with specific subcellular targeting mechanisms mediates differentiation and triggers unique developmental and immunological responses in vivo (Figure 1). Some specific impacts of each instrument on these projects are briefly described below. The BD Pathway Bioimager is a high-throughput fluorescence imaging system that functions as a confocal microscope enabling capture of cell images over time and with multiple replicates (Figure 2). For the project led by Dr. Malathi Srivatsan focused on nanomaterials for differentiation and regeneration of neurons, the BD Pathway enabled high-resolution imaging and quantification of neural growth. This research demonstrated the potential of carbon nanotubes and other nanomaterials to deliver different growth factors to enhance nerve growth and regeneration and to facilitate guided nerve regrowth. This work has significant implication for restoring nerve conduction and neural connections in wound healing. The research project of Drs. Dolan and Cramer focused on using plant lectins (sugar-binding proteins) to deliver vaccine antigens in a way that selectively directs either cell-mediated or antibody-mediated immunity. The BD Pathway enabled subcellular localization studies showing that modifying the lectin carrier could alter its subcellular destination, thereby potentially shifting immune "presentation" of associated antigens. This research could address a key challenge in vaccine development – triggering strong cell-mediated immunity using a simple "subunit" vaccine. The Gentle Macs™ Dissociator facilitated disruption of tissues yielding intact cells that can be analyzed directly or placed into cell culture for subsequent analyses (e.g., by cell sorting, flow cytometry, microscopy/imaging, or biochemistry). The instrument has become a "workhorse" in processing tissues for all of the projects. The Dissociater outperformed commonly used homogenizer/sieve technologies providing significantly greater yields of viable cells (up to 95% viability) and prep-to-prep consistency. The project led by Dr. Roger Buchanan focused on uptake and biodistribution of nanoparticles (‘quantum dots’) in mice following inhalational exposure. The Dissociater was key in providing intact lung cells for assessment of uptake and accumulation in different cell types and regions of the lung. The epMotion Liquid Handler (Figure 3) is a high-throughput liquid handling robot. It has been key in development and reproducibility of antibody-based quantification (e.g., ELISAs) and biochemical assays (e.g., enzyme kinetics). It has also been used to support high-throughput gene expression analyses (qRT-PCR) in studies assessing impacts of route of toxin exposure to cellular responses. These studies would not have been feasible without the epMotion System. The BD FACSAria Cell Sorter supports selective enrichment of specific mammalian cells from complex mixes enabling quantitative assessment of cell responses at the gene and protein levels for different cell types. This instrument was used extensively by Dr. Shiguang Yu, who leads research focused on understanding mechanisms of autoimmune diseases. The instrument enabled enrichment of very rare cells for functional assays. His data identified a subset of immune cells that are responsible for triggering thyrocyte proliferation linked with thyroid autoimmune disease. Broader Impacts. This instrumentation has significantly enhanced the cell biology capabilities at Arkansas State University in providing impacts well beyond the four proposed projects. The cell biology suite played an important role in recruiting new faculty and students, and is currently being used by faculty/students in four colleges (Sciences and Math; Agriculture and Technology; Nursing and Health Professions; Education). The technology has been integrated into teaching; supporting two graduate level techniques courses in immunology/cell biology (cell sorter and flow cytometry) and fluorescence microscopy (BD Pathway and confocal microscopy) and supporting demonstrations in multiple undergraduate cell biology and immunology courses. Research using these instruments has also been highlighted as part of the Arkansas Biosciences Institute’s STEM outreach program that brings nearly a thousand 6-12-graders/year on site for laboratory exercises and "science tours". Our outreach program is designed to bring these students into the laboratory setting where they not only perform experiments, but also see young and diverse researchers working at the bench who have opportunity to share their enthusiasm for science with student visitors. The MRI investment in research infrastructure is also driving significant innovation and collaboration. These instruments are now being used in research with applications in cancer biology and cell signaling, neurobiology and neuroregeneration, autoimmune diseases, vaccine development, mechanisms of nicotine addiction, protein trafficking in mammalian and plant cells, enzyme delivery for lysosomal storage diseases, plant metabolomics, and immune activation in mammalian, fish, and avian species.
