Host-Pathogen Interactions, Live Cell Imaging and Translation Research Shared Resources
With support from the NCRR COBRE Program Project and the Cystic Fibrosis Foundation Research Development Program, the Lung Biology Center offers the following core services.
Host-Pathogen Interaction Core
Director: George A. O'Toole, Ph.D.
Description: The goals of the Host-Pathogen Interaction Core are to:
1) Facilitate research on biological mechanisms that contribute to lung disease. The infrastructure and expertise of the Core supports basic science studies, including protein localization and trafficking, gene regulation and microbial co-culture experiments.
2) Accelerate therapeutic development. The Core allows investigators to build on basic studies to facilitate drug discovery and preclinical investigations, thereby directly enhancing the translational impact of COBRE-affiliated groups. The Core also provides expertise to biotechnology and pharmaceutical companies developing new therapies to treat lung disease.
1) A focus of the core is to provide access to physiologically relevant cell lines for mechanistic studies, with a focus on polarized airway epithelial cells. The Core facility provides access to validated CF patient-derived bronchial epithelial (CFBE) cell lines, as well as primary patient airway cells through a collaborative arrangement with the University of North Carolina. Facilities and support are provided for the culture, maintenance, treatment and analysis of cells, including electrophysiological studies of transepithelial chloride efflux (Ussing chamber), as well as support for siRNA and transfection protocols. The Core also provides cellular and molecular reagents to both the Live Cell Imaging Core and Translational Research Cores.
2) The core assists researchers with screening and design of target inhibitors as well as preclinical testing and evaluation of new therapeutics aimed at diseases of the lung, including targeting the chronic infections associated with Cystic Fibrosis.These resources are available to in-house research projects, as well as on a contract basis for pharmaceutical and biotechnology collaborators working on therapeutics relevant to the lung.
3) The Core also fulfills a critical function as a water-cooler, ensuring that as new state-of-the-art techniques in lung biology research are adopted by individual scientists or groups, they are rapidly distributed to the other members of the program. It also provides hands-on training for graduate students and post-doctoral fellows in the COBRE laboratories, and will also sponsor trainee participation in extramural workshops (e.g. Cold Spring Harbor, MBL). In addition, the Core partners with affiliated Departments to invite outside speakers with expertise, particularly in trafficking and drug-discovery.
Live Cell Imaging Core
Director: Bruce A. Stanton, Ph.D.
Description: The goal of the Live Cell Imaging Core is to facilitate research on biological mechanisms that contribute to lung disease and to accelerate efforts to identify candidate therapeutic targets. The infrastructure and expertise of the Core supports live cell imaging, as well as protein localization and host-microbe interaction studies.
1) Live cell imaging using a wide field Olympus fluorescent microscope or a Nikon swept-field confocal LSCM microscope. The Nikon LSC has 488, 563, and 633 nm laser lines for confocal microscopy. The system also can perform TIRF imaging using the 488 nm laser line, using an Apo TIRF 100X or 63X oil immersion 1.49 NA objectives. The Nikon LSC system is equipped with a Piezo Z stage, allowing rapid multi-position and 3D image capture. The Sweptfield Confocal system is also suited with Perfect Focus, an auto focus mechanism, critical to TIRF imaging, that permits highly accurate 3D imaging over time by memorizing the distance of the sample from the coverslip, thus preventing drift in the z axis. A full incubation chamber controlled for temperature, CO2 levels, and humidity maintains optimal conditions for cells during the experiment. The system is equipped with QuantEM:512sc camera and CoolSnapHQ Monochrome cameras (Photometrics, Tuscon, AZ). These cameras allow extremely rapid image acquisition of low intensity signals, while linearly amplifying signal to allow quantitation of detected fluorescence. This system employs Nikon Elements 2.2 Research Duo software with particle tracking module (Nikon, Inc.) to analyze images.Both live cell-imaging platforms are customized for the analysis of bacterial biofilms grown on abiotic or biotic surfaces (i.e., confluent, polarized human airway epithelial monolayers), for analyzing bacterial adhesion to biotic surfaces, and for conducting cell migration assays.
2) Confocal microscopy to monitor intracellular protein trafficking and localization.
3) Advanced imaging technique development, including the recent application of bimolecular fluorescent complementation to study protein-protein interaction and development of a fusion assay to study bacterial toxin delivery in live airway epithelia cells.
4) Technical support is provided by Dr. Qianru Yu. She assists investigators to obtain preliminary data for pilot projects and grant applications, with follow-up training to enable the investigator to perform his/her imaging.
5) Hands-on training by Dr. Yu and trainee participation in extramural workshops and short courses (e.g. Cold Spring Harbor, MDIBL and MBL). In addition, the Core partners with affiliated Departments to invite outside speakers with expertise in advanced imaging techniques.
Translational Research Core
Co-Directors: Deborah A. Hogan, Ph.D. and Alix Ashare, M.D./Ph.D.
Description: The goal of the Lung Biology Translational Core is to provide support to basic science investigators and physician-scientists focused on translational research who require access to clinical specimens from patients with lung disease to support their work and expand preliminary observations into human subjects. This Core facilitates ongoing studies of current and former COBRE Lung Biology investigators and investigators with pilot projects. The Translational Research Core coordinates with the Advanced Imaging and the Cell Biology and Target Discovery Cores to facilitate translational studies at the Geisel School of Medicine at Dartmouth. The Translational Core also provides assistance with IRB human subject study approvals, and feedback on project design and implementation.
1) Provide support for design and implementation of research studies involving human subjects, including IRB approval and study design.
2) Provide a comprehensive, collaborative, and efficient mechanism for obtaining and processing research specimens from subjects with lung disease.
3) Provide research coordinator support to obtain informed consent, interface with patients and families, and assist in obtaining and transporting clinical specimens and compiling approved patient information for research projects.
4) Provide a facility for processing of human samples for subsequent analysis.
5) Provide training to the members of the COBRE Lung Biology group in translational research methods and outcomes via the annual Translational Research Core Seminar, wherein the Core Co-Directors host a speaker working in translational research.