Protein Localization, Identification and Folding Core
The Specific Aims of the Protein Localization, Identification and Folding (PLIF) core are as follows:
1) Provide state of the art protein localization and identification facilities. Core programs include: Imaging, Proteomics and Protein Folding Diseases Initiative resources.
2) Ensure delivery of high quality services and products and provide technical oversight of all PLIF services.
3) Train and educate members, associate members and pilot feasibility recipients in the application and use of protein identification techniques for the study of digestive and liver diseases.
The Protein Localization, Identification and Folding (PLIF) Core is a further evolution of our most recent Protein Identification and Localization (PIL) Core that combines proteomics and cell imaging. The included facilities serve as critical platforms to study many biological functions that contribute to the pathogenesis of a number of diseases in the gastrointestinal tract. This Core has been critical in facilitating member research programs in the past funding period with 76% of center members using the PLIF Core, contributing to 116 peer-reviewed publications since 2010. The selection of current Core services is based on past usage and recent member survey. The Core leadership has expanded previous facilities to include emerging techniques that will be provided by highly trained individuals. The importance of the PLIF Core is in part due to provision of access to specialized equipment and techniques not available in individual Center members’ laboratories, and also because of the services it provides are in high demand. It is built around more than 90 years of GI and Liver proteomic, cell biology and imaging expertise of Drs. Nusrat (our new Core co-director), Omary, and Williams. Dr. Nusrat was recently recruited to the University of Michigan and has previously directed a Digestive Disease Mini-Center image analysis and flow cytometry Core at Emory University. Our new Associate director Dr. Alexey Nesvizhskii has extensive experience in proteomics and bioinformatics. Also included are experienced Core staff Dr. Basrur and Mr. Nelson who will assist Center Investigators (as managers) and carry out critical Core procedures. Core leaders and managers will provide scientific advice by meeting with GI Center investigators and help with the planning, initiation, implementation and dissemination of new and innovative techniques in proteomics, cell imaging and protein folding.
The localization component of the Core is located in dedicated space in the Departments of Physiology and Medicine and utilizes microscopes in the Medical School’s Morphology and Image Analysis Laboratory (MIL) and in the Morphology and Image Analysis Core (MIAC) of the Michigan Diabetes Research Center (supported by an NIDDK P30 led by Dr. Martin Myers). Center members will have access to imaging, with use of laser scanning confocal microscopy (LSCM) and multi-wavelength fluorescence imaging. In addition, electron microscopy and immunogold labeling of fixed and embedded specimens is available for ultrastructural analysis. A new feature in this reapplication is the Aperio Digital Pathology system for high-resolution imaging of whole slides.
The proteomic resource facility (PRF) provides our Center members with high-end mass spectrometry instrumentation dedicated for proteomic analysis and highly skilled expertise in proteomics. The PRF has undertaken several important steps to provide state-of-the-art facilities. PRF is located in the Department of Pathology and houses 2 state-of-the-art mass spectrometers including the most recent acquisition of a high-resolution, ultra-fast and sensitive Q Exactive HF. This facility is headed by Dr. Alexey Nesviszhskii who serves as the PLIF Core Associate Director.
The protein folding facility uses University of Michigan Medical School Protein Folding Diseases (PFD) Initiative that was launched in 2013 with a $9.3M commitment from the Medical School (http://medicine.umich.edu/medschool/research/research-strengths/protein-folding-diseases-initiative/about-us). Dr. Omary serves on the Internal Advisory Committee of the PFD, and is a member of the PFD initiative investigator group together with 4 other members of our center. The PFD is one of two heavily invested initiatives (the second being the microbiome initiative which is now also linked in with our Center Microbiome and Metabolomics Core). The PFD initiative offers a ‘resource library’ that includes: a proteomics service that performs routine and highly specialized mass spectrometry with bioinformatics support, a mouse genetic model library, a rodent tissue bank, an antibody reagent library, and access to pilot awards.
In Vivo Animal and Human Studies Core
The Specific Aims of the In Vivo Animal and Human Studies (IVAHS) core are as follows:
1. To provide a variety of acute and chronic animal models as well as state-of-the-art in vivo technologies to enhance rapid translation of basic discoveries to physiological and clinical applications;
2. To develop and maintain an organoid and enteroid program in which these preparations grown from mucosa biopsies and induced pluripotent stem cells (iPSCs) may be used to study epithelial cell biology and provide therapeutic targets for clinical intervention;
3. To support a clinical study program which will provide consultation and technical support to Center investigators on design and statistical data analysis; and
4. To provide a user friendly, one-step service to access digestive disease-related biospecimens for Center members. These aims will be accomplished by: i) Continuing to ensure the fluid function and access to state-of-the-art technologies to Center members; ii) Providing education and technical advice, guidance and feedback on experimental design and the use of animal models; iii) expanding our clinical study service to include full statistics support and hiring of a dedicated study coordinator; iv) Integrating existing GI tissue collections and annotated data into a collaborative web-based organizational structure that is easily accessible by Center members.
The objective of the In Vivo Animal and Human Studies core is to provide a venue for focusing human resources, facilities, new technology and equipment necessary for the in vivo study of humans and animals. The core serves to fully integrate investigations at the molecular, cellular, and organ system levels that are currently being performed within other cores of the Center and enhance rapid translation of basic discoveries into clinical application. Since its inception in 1984, the In Vivo Animal and Human Studies core has evolved to meet the needs of Center investigators. Based on Center members’ feedback we reorganized the Core services into 4 programs. (i) We have streamlined our animal studies to include ones that are frequently utilized by Center investigators. These include mice endoscopy, in vivo cellular and molecular imaging studies in small animals, fluorescent-activated cell sorting (FACS) analysis, and electroporation for direct transfer of siRNA into targeted neuro-tissue or GI organs. (ii)The recent development of the Enteroid and Organoid core enables Center investigators to conduct research on in vitro models of human iPSCs, directed differentiation of human pluripotent stem cells (hPSCs), and 3-dimensional growth of primary human and mouse tissue delivered from the GI tract. These preparations may be used to study human GI development, homeostasis and disease in vitro. (iii) To enhance the rapid translation of basic science findings to clinical applications, the In Vivo Studies core has spent much of its efforts and resources to upgrade its infrastructure. The Clinical Design Program provides consultation regarding clinical study design, subject specification and sampling techniques, measurements and instruments, sample size estimation, power analysis, as well as data management and statistical analysis.(iv)The Biospecimen Banking service will provide support to enhance existing individual GI access to GI-related biobanks and utilization of GI biospecimens to translate basic research within the paradigm of human specimens. The In Vivo Animal and Human Studies Core has been critical to the execution of member research endeavors during the last funding period, with 60% of the 58 Center investigators using Core services resulting in 166 peer-reviewed publications.
Molecular Biology Core
The Specific Aims of the Molecular Biology (MB) core include:
1) To execute state of the art gene editing, gene transfer and gene profiling techniques in line with evolving member research needs.
2) To support highly trained personnel in the application of genetic technology, organized around four Core Programs: Transgenic Rodent, Genome Editing, Viral Vector and Integrated Genome Analysis.
3) To ensure delivery of high quality services and products and provide technical oversight of all Molecular Core services.
4) To train and educate members, associate members and pilot feasibility recipients in the application and use of molecular techniques for the study of digestive and liver diseases.
The overarching objectives of the University of Michigan Center for Gastrointestinal Research (UMCGR) Molecular Biology Core are to facilitate ACCESS TO and ADVANCE DEVELOPMENT of MOLECULAR GENETIC tools to study gastrointestinal biology and disease. The technological underpinnings of molecular
biology represent a perpetually changing landscape that members of the UMCGR consistently use to make seminal and impactful discoveries in gastrointestinal research. Towards this end the UMCGR Molecular Biology Core acquires and promotes new technologies to support Center member execution of cutting edge research in GI biology and disease. The value of the Molecular Core is in part due to access to specialized
techniques and equipment not available in individual Center members’ laboratories. The Molecular Core customizes and supports emerging and sophisticated techniques that are best provided by highly trained, wellestablished personnel using specialized and often expensive equipment, such as those provided in an Institutional Core facility. Thus, UMCGR investigator access to molecular technologies is enhanced through GIspecific
interfaces with Medical School Core facilities as well as through support of center-specific facilities. The Molecular Biology Core functions to provide a wide array of genetic techniques, to make access affordable, and to be an educational resource to all Center investigators. Specialized assistance with experimental design increases the rigor, reproducibility and ultimately success and impact of member projects.
During the prior funding period, The Molecular Biology Core included four Programs: the Transgenic Rodent Program; the Microarray Program; the Viral Vector Program; and the Molecular Techniques Program. These Programs were crucial to the execution of member research programs, with 82% of the UMCGR membership using the Molecular Biology Core, resulting in 132 peer-reviewed publications, of which 36 were
collaborative publications among two or more Center members and in successful grants.
Core programs have been reorganized to reflect advances in molecular techniques,
to serve evolving member research needs and to improve the quality of service delivery for GI research. The Molecular Techniques Program is discontinued due to low projected use. However the NextGen sequencing component of that Program was expanded to become a stand-alone program with microarray and enhanced
bioinformatics support. In addition a new full-service CRISPR/Cas9 genome-editing program has been developed. Finally to accelerate member research and to stimulate collaborations, GI databases of transgenic mouse strains, viral vectors/plasmids, and genomic sequence data are provided to enhance the GI-relevance of the Core.
In our current funding period four Molecular Core Programs will be offered. 1) The Transgenic Rodent Program continues due to the ongoing interest of UMCGR members to test genetic findings and molecular mechanisms in whole animals. 2) A new Genome Editing Program has been developed to reflect growing UMCGR
member interest in using genome-editing technology to create novel mouse strains (with the Transgenic Rodent Program), organoids (in collaboration with the In Vivo Core) and cell lines (with the Viral Vector Program). 3) Due to the popularity of in vitro methods to modulate gene expression in culture models, e.g., cell lines and now organoids, the Viral Vector Program continues in the current cycle, but updated to reflect refinements of the technologies and new featured reagents, e.g. Rabies Virus and CRISPER/Cas9 vectors. 4) The Microarray Gene Chip Program, supported for two cycles as a separate Program, is incorporated into an expanded Integrated Genomics Program to provide a comprehensive array of whole genome approaches, e.g., RNA-Seq, ChIP-Seq, genome-wide methylation.
Microbiome and Metabolomics Core
The Specific Aims for the UMCGR Microbiome and Metabolomics (MM) core include:
1) To provide the ability to retrieve nucleic acid, including genomic DNA and RNA, from human and animal tissue. This nucleic acid will be used for 16S, metagenomic and metatranscriptomic analysis for microbial communities. This material can also be exploited for expression analysis from host cells.
2) To facilitate the molecular analysis of the structure and function of complex microbial communities. The techniques for retrieval of small subunit rRNA-encoding gene sequence information will be available via the high-throughput generation of 16S variable region amplicon sequencing. Assistance with metagenomic sequence analysis using massively-parallel DNA sequencing will also be available. The generation and analysis of microbial genomes will be facilitated by the Core.
3) To 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. Investigators will be able to maintain strains in germ-free or gnotobiotic states and also perform experimental procedures on these animals.
4) To provide access to metabolomic analysis of human and animal samples. Through the Michigan Metabolomics and Obesity Center’s metabolomic Core, members of the UMCGR will have access to state-of-the-art methods for targeted and untargeted measurement of host and microbial-derived small molecules to assess function of the microbiome.
The past several years has seen a virtual explosion in interest into understanding the role of the indigenous microbiota and their surrounding environment (the microbiome) in health and disease. For the last funding cycle, we established a Microbiome and Metabolomics Core to facilitate the application of microbiome analysis on the part of members of the University of Michigan Center for Gastrointestinal Research (UMCGR). Because of the increased interest in the microbiome and the success of the newly established Core, we have modified and expanded the existing microbiome core to serve the needs of the UMCGR members.
Advances in analytic technology and the bioinformatic methods required to analyze the data from these new technologies have prompted us to strengthen services offered to UMCGR members. The overall goal of the Microbiome and Metabolomics Core is to provide researchers within the UMCGR 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. Assistance with experimental design will help ensure the rigor and transparency of the microbiologic and metabolomic aspects of the analysis.
In the previous funding period the Microbiome and Metabolomics Core has been very active. 27 digestive disease related grants were supported by the use of the core, and 38% of the center members used our core and this helped generate 105 publications.
The activities of the UMCGR Microbiome And Metabolomics Core are facilitated by the considerable investment and resources for this sort of activity on the University of Michigan Medical School. The Medical School has supported the establishment of the Host Microbiome Initiative Microbial Systems Lab, which is where the culture-independent analysis of microbial communities will take place. The University and NIH are providing support for the Michigan Metabolomics and Obesity Center, which is overseen by the UMCGR Microbiome and Metabolomics Core co-director Charles Burant. Finally, the University is also supporting the germ-free animal core. The presence of all of these facilities on the University of Michigan campus will permit the UMCGR Microbiome and Metabolomics Core to assist Center investigators in the utilization of these state-of-the-art facilities. Center members will have access to the latest technologies and analytic methods that will allow them to study the role of microbes in human health and disease within the gastrointestinal tract.