Protein Identification and Localization Core
The Protein Identification and Localization (PIL) Core is a restructured core that has evolved from aspects of two previous cores, the Cell Biology and Cell Imaging Core which focused on cellular imaging and the Peptides and Proteomics Core which had a dual service of providing synthetic peptides and identifying proteins by mass spectrometry. While cell biological methods are now standard in most laboratories and peptide synthesis can be obtained both from University and commercial sources, what was unique about the two previous cores was their synergy in identifying and localizing proteins. These aspects are preserved and enhanced by the structure of the new PIL Core and build on the longstanding collaborative interactions between Drs. Williams, Andrews and Ernst. They also build on the fact that this type of research requires complex and expensive equipment not available in individual laboratories. The PIL Core is designed to provide information on the identity of proteins by mass spectrometry, either as individual proteins purified in an investigator’s laboratory and usually separated as a band or spot by gel electrophoresis or globally as a mixture of proteins in a protein complex or organelle. Information is also be provided on post-translational modifications of proteins and on quantitative changes in protein content. Once a protein is identified, the Core can provide information on the localization of the protein in live and fixed cells and their changes over time and in response to physiological and pathophysiological perturbation. It is centered around microscopic imaging and quantitative analysis of digital information obtained primarily by laser scanning confocal microscopy (LSCM) and multiwavelength fluorescence imaging, although traditional electron microscopy of fixed and embedded specimens is also available for fine structure analysis.
Modern biomedical research is characterized both by its interdisciplinary nature and by its dependence on increasingly sophisticated instrumentation and informatics. Although the goal is to look at a tissue, disease, or organism as a complex integrated system, we are still developing an understanding of the structure and function of the components which are often cell type dependent. With the mapping of the genome, attention has shifted to the more complex world of the proteome. Complete inventories of most mammalian protein complexes or organelles are yet to be completed and they often vary between cell types, during the cell cycle, and during various stages of development. Thus identification of proteins, their post-translational modifications, dynamic interactions and cellular localization, all in time and space and in a quantitative manner, is a daunting task that almost all laboratory scientists in the GI Peptide Center encounter in their research. These questions can be approached through the tools of the PIL Core, very often in concert with the Molecular Biology Core and national genomic resources such as the NCBI.
The new PIL Core is built around expertise in the director’s laboratories, and the availability of sophisticated instrumentation in three existing facilities, the Michigan Proteome Core, the Morphology and Imaging Laboratory (MIL) of the Department of Cell and Developmental Biology and the Michigan Diabetes Research and Training Center (MDRTC) which maintain facilities discussed in detail below for sophisticated mass spectrometry and high resolution microscopy. This arrangement is facilitated by the fact that Dr Andrews directs the Michigan Proteome Core, Dr Williams directs the Microscopy and Image Analysis Laboratory of the MDRTC and Dr Ernst has a long standing affiliation with the MIL. All three entities have established personnel and a recharge structure and are currently being use by GI Center members.
While the Protein Identification and Localization Core will make use of the available highly sophisticated instrumentation, it also is built around the over 90 years of proteomic, cell biology and imaging expertise of Drs. Williams, Andrews and Ernst which has been primarily devoted to GI Tissues. All Core personnel will demonstrate and teach techniques to GI Center investigators, trainees and technicians. Finally another mission of the Core is to initiate, implement and disseminate new and innovative techniques in proteomics and cell imaging.
In Vivo Studies Core
The objective of the In Vivo Studies Core is to provide a venue for focusing the human resources, facilities, new technology and equipment necessary for in vivo study of humans and animals. The Core serves to fully integrate investigations at the molecular, cellular and organ system level that are currently performed within the other Cores of the Center. The Core is organized to bring together expertise and in vivo techniques from different disciplines to investigate the role of peptide hormones in a wide variety of gastrointestinal functions. Toward these goals, the major services provided by the In Vivo Studies Core are as follows:
- To provide a variety of acute and chronic animal models for in vivo evaluation of the biological actions of peptide hormones.
- To examine the physiological changes in mouse models following transgene expression or gene mutation.
- To provide in vivo techniques and tests for the investigation of the physiology and pathophysiology of gastrointestinal peptides in humans.
- To facilitate innovations in methodology and to develop sophisticated new techniques for in vivo studies.
- To educate and consult on human clinical studies as well as the application of in vivo techniques so that basic physiological and biochemical questions pertaining to peptide hormones may be answered
Molecular Biology Core
The purpose of the Molecular Biology core is to facilitate the application of the tools of molecular genetics to the study of gastrointestinal peptides and their physiological functions. In all aspects, the Molecular Biology Core functions to provide essential services and serves as an educational resource to all Center Investigators. Although basic molecular techniques are widely available to Center investigators through commercial sources, there remains specific services that are best provided by pooled resources and by highly trained personnel such as those that can be provided by a Center Core facility. During the prior funding period, four Core programs were provided: the Transgenic Rodent Program, the Microarray Program, Viral Vector Program and the Molecular Techniques Program. The Microarray Gene Chip Program was added during the last competitive renewal and the Molecular Techniques Program was made available during the current funding cycle to provide education and training in those techniques that can be performed in the investigators own laboratory after receiving some theoretical and practical training. The latter is freely available as an educational resource for Center members interested in such techniques as chromatin structure analysis. Investigators can also obtain assistance with basic molecular techniques like quantitative RT-PCR as well as more sophisticated techniques like chromatin immunoprecipitation assays (ChIP) and ChIP-Seq.
Microbiome and Inflammation Core
The goal of the Microbiome & Inflammation Core is to provide researchers within the Michigan Gut Peptide Center (UM-MGPRC) access and training for state-of-the-art technologies in culture-independent and culture-based molecular tools to profile the complex community of microbes that exist in and on the human body or animals studied in relevant models of gastrointestinal disease. The different programs that will make up this Core will provide essential services, equipment and education for investigators who are interested in studying interactions between the host and the microbiome. These techniques require significant expertise and many are expensive and/or require sophisticated instrumentation typically unavailable in the laboratories of individual investigators. By offering a centralized source of expertise, instrumentation and resources, the UM-MGPRC gains a number of significant benefits by supporting this shared facility: (i) stable, highly-trained, and experienced personnel to train and oversee analyses, providing consistent performance standards and a high-level of quality control over time, (ii) an improved level of cost-effectiveness achieved by a larger scale of operation, and (iii) access to skilled investigators for consultation on experimental design and interpretation of results. This Core, and its directors, will be adapting to the evolving needs, accumulating knowledge and advancing technology related to the biology of the microbiome and regulation of inflammatory processes.
As currently envisioned, the Microbiome & Inflammation Core will consist of five programs and a bioinformatics interface, led by Dr. Pat Schloss, that also utilizes the resources of The University of Michigan Center for Computational Medicine and Biology (CCMB, http://www.ccmb.med.umich.edu/).
- Nucleic acid preparation - will facilitate the retrieval of nucleic acid, including genomic DNA and RNA, from human and animal tissue. This nucleic acid will be used for 16S and metagenomic analysis as well as host expression analysis. This program will also provide experimental design assistance to aid investigators in designing protocols that will permit analysis of the microbiome.
- 16S/metagenome analysis - will facilitate molecular analysis of complex microbial communities. A range of techniques for retrieval of small subunit rRNA-encoding gene sequence information will be available ranging from terminal restriction fragment length polymorphism (T-RFLP) analysis to the construction of 16S clone libraries and 16S variable region pyrosequencing. Assistance with metagenomic sequence analysis using massively-parallel DNA sequencing will also be available.
- Expression analysis - will facilitate the examination of host responses. Both ELISA-based and RT-PCR-based methods will be available.
- Microbial cultivation - will provide consultation expertise in traditional and novel methods for bacterial cultivation from host tissues and samples. Novel methods employ cultivation guided by nucleic acid sequence information to allow targeting of specific organisms of interest that are identified by molecular survey techniques, such as those provided by the 16S/metagenome analysis program.
- Gnotobiotic animal facility - will provide expertise in germ free mouse technology. The ability to re-derive various mouse strains in the germ free state and to selectively colonize with specific microbes will be available in this core facility. Investigators will be able to maintain strains in germ-free or gnotobiotic states and also perform experimental procedures on these animals.