PI: Joe Lutkenhaus, Ph.D., Professor, Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center.
The administrative office of the COBRE is housed in the central administration office complex of the Department of Microbiology, Molecular Genetics and Immunology. Two work stations have been established for the accountant and the secretary, respectively, and will be situated in a 200 sq. ft. office. Each is computer-networked with investigators on the three campuses. All information related to the COBRE program is kept on file in this office. Functions 1)Conduct bi-monthly review of research programs of junior faculty on the three campuses. 2)Identify projects of individuals that have potential for COBRE support and provide guidance for the direction in which the research program should be pointed so as to increase its competitiveness or receiving COBRE support. 3)Provide oversight of selection of new faculty for COBRE support. 4)Insurance that COBRE funds are spent appropriately. 5)Chair annual review of progress of supported members. 6)Provide input to the IAC on the decision for termination of COBRE support of a particular candidate following his/her success in acquiring independent NIH funding. 7)Chair monthly seminar and/or Data-presentation sessions of supported faculty. 8) Chair an annual symposium at which COBRE members will make formal presentations to an audience comprised of the Outside Advisors, members of the IAC, and mentors.
PI: Todd Holyoak, Ph.D., Assistant Professor, Department of Biochemistry and Molecular Biology, University of Kansas Medical Center
Essential to the design of new therapies against emerging bacterial threats is the determination of the three-dimensional structure of potential protein targets. Macromolecular x-ray crystallography provides a powerful experimental tool that allows us to obtain that necessary structural information. The wealth of information contained within the structural data can subsequently be utilized in the determination of the important relationship between protein structure and function. Elucidation of the structure function relationship is essential to the understanding of enzyme mechanism and is a key step in the design of new inhibitors of protein function. A current interest in the lab is the study of highly specific proteases which are essential to pathogenic bacterial infection. Highly specific proteases are utilized by virulent bacteria at many stages throughout their life cycle and in certain instances allow the bacteria to circumvent the host’s innate immune response by targeting immunoglobins for proteolysis. Understanding the structural mechanisms which underlie how these specific proteases recognize their cognate substrates will allow for the rational design of new inhibitors that are specific for these enzymes and consequently provide new therapies against infection.
PI: Joe Lutkenhaus, Ph.D., Professor, Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center
One of the keys to identify inhibitors of proteins that have an essential role in microbial replication or pathogenesis is to have their crystal structures. Core C has been set up to provide the fermentation capacity for the production of large amounts of proteins for crystallographic studies. Several proteins have been overproduced and Core C is working closely with Core B to begin crystallization studies of several proteins, including inhibitors of bacterial cell division. A scientist has been hired who has experience in protein purification. Assistance is offered to all investigators, including doing the purification, offering advice and making the equipment readily available.
Core Director: Joyce Slusser, Ph.D., Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center
The KUMC flow cytometry core houses the Becton Dickinson Aria and the LSRII in the Department of Microbiology. Single cells are carried past 3 lasers in a fluid stream, fluorescence is measured via photomultiplier tubes which are placed inline with appropriate filters. Assistance is required with initial experiments to determine appropriate settings for data collection and interpretation as cells types vary in size, surface markers vary in density and therefore intensity, and investigators can select the combination of fluorochromes used; investigators will be able to work semi-independently in later appointments. Flow cytometry is especially powerful in that observations which can be made by microscopy will be made at an accelerated rate, in a quantitative manner, and on a single cell basis. Hundreds of corollary data points are collected to indicate cell size, granularity, and viability; when fluorescent dyes are added, activation of a cell can be measured by calcium flux or signal transduction by protein phosphorylation. Dyes can be used to quantitate proliferation or cell death, without radioactivity. Cells from mixed culture or tissue with specific properties can be separated and collected for additional study. A series of short topic based lectures is planned to introduce flow cytometry, flow applications, and software for data analysis.
Core Director: Shilpa Buch, Ph.D., Associate Professor, Department of Molecular and Integrative Physiology, University of Kansas Medical Center
Luminex technology provides the equivalent flow cytometric measurement of host response factors that are present in plasma, serum, or supernatant fluids of tissue cultures. The advantage of this technology is that a miniscule amount fluid can be analyzed rapidly for presence of multiple factors. These factors usually include cytokines, chemokines, and growth factors. The manufacturer usually sells kits of reagents consisting of markers for approximately six factors. In addition to variability in the constituents of the sets, each set has to be species specific. COBRE investigators will require sets that measure human, macaque, and mouse host-response factors. Having decided on a specific number of host-response factors or a specific species that is being used, the COBRE investigators will either perform the assay themselves or have the assay performed by the research assistant who is trained for this function. Prior to beginning any assays, Dr. Buch usually consults with the investigators to discuss particular needs and selection of factors.
Core Director: Christophe Nicot, Ph.D., Associate Professor, Department of Pathology and Laboratory Medicine, University of Kansas Medical Center
The purpose of this core is to provide technical assistance in designing, making vectors expressing fluorescent fusion proteins for intracellular localization and protein trafficking to investigators interested in studying cellular transduction and signaling pathways. Although the use of molecular biology is indispensable, cell biology studies offer a link that connect in vitro and in vivo data and is useful to confirm hypothesis or models proposed. Recent advances in microscopy methods have made it possible to visualize the dynamics of proteins and organelles in living cells. To do so, proteins can be covalently labeled with a fluorophore and injected into cells, or fluorescent tags can be genetically encoded. By far the most popular fluorescent label is the auto-fluorescent green fluorescent protein (GFP) from the jellyfish Aequorea victoria. GFP can be fused to any complementary DNA using standard cloning methods, and, as several spectrally distinct derivatives are available, multi-color observations are possible on several proteins simultaneously (RFP, CFP,YFP). Acquisition of a Nikon single molecule imaging system equipped for time lapse photography of single cells while under incubation at 37 OC will allow for imaging of live cells. Observations from studies with fluorescently tagged proteins typically show the steady state distribution of a protein, but they do not directly provide information about the kinetic properties of molecules. To determine the kinetic properties of proteins in vivo, the movement of the protein of interest must be made visible. The most commonly used technique for this is FRAP (fluorescence recovery after photobleaching). In this method, a small area of a cell is rapidly bleached using a high-intensity laser pulse. The movement of unbleached molecules from the neighboring areas is then recorded by time-lapse microscopy as the recovery of fluorescence in the bleached area. The microscope will also allow for time lapse movie caption to monitor fluorescently labeled protein movement in the cell in response to different drug treatment altering a specific pathway, ligand-receptor downstream targets and so on. In vivo molecular interactions could also be investigated by fluorescent energy transfer (FRET).
Core Director: Martha Montello, Ph.D., Associate Professor, Department of History and Philosophy of Medicine, University of Kansas Medical Center
Writing Development Seminars
4 teaching workshops, 2 hours each, will be offered to the faculty in the COBRE training program for each year of the grant. The workshops will emphasize the basic principles and methods of clear and effective professional and academic writing, focusing on strategies necessary for publishing articles and writing grants. The workshops have been developed from Professor Montello’s training at the University of Chicago’s program for Academic and Professional Writing and honed through her two years of previous teaching with the K-30 Clinical Scholars Program.
Individual editing services
Two hours of manuscript editing and individual consultation services scheduled with the Writing Consult Center will be offered for the faculty members working on the grant, up to a total of 25 hours. With advance notification and scheduling, editing of individual manuscripts will have a turnaround time of 48 hours.
This grant was made possible by NIH Grant Number P20 RR016443 from the COBRE program of the National Center for Research Resources.
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