Core B: Research Support Core
The RSC (RSC) of the University of Kentucky Superfund Research Center (UK-SRC) provides vital access to expertise, research resources and state of the art instrumentation to researchers engaged in all aspects of the biomedical and environmental science research projects. The RSC enhances the productivity, quality and consistency of these projects by providing a formalized mechanism for access to expert investigators and professional staff and by enabling efficient use of expensive and sophisticated instrumentation that would otherwise be beyond the capabilities of individual laboratories to acquire and support. The RSC provides services in “Quantitative Biology” encompassing biostatistics support for experimental design and computational infrastructure for data management, analysis and sharing. Bioanalytical services provide technologies for quantitation and structural analysis of toxins, nutritional protectants and markers and mediators of oxidative stress and inflammation. The RSC is now also developing advanced mass spectrometry-based strategies for identification of biomarkers of PCB toxicity and nutritional protection from PCB exposure.
Establishment of a coordinated effort in the form of the Research Support Core maximizes both the research and cost effectiveness of our SRP investigators. This Core is divided into two major components directed by an expert in each area:
- Bioanalytical Component - Directed by Dr. Andrew Morris
- Quantitative Biology Component - Directed by Dr. Arnold Stromberg
The overall activity of the Research Support Core is managed by Dr. Morris in close collaboration with the coordinators of individual components, Dr. Hennig (the Director of UK-SRC) and the Administration Core, and the Project Leaders of all individual grants.
- Provide unified infrastructure for data management that can be used to share information between the component projects enabling integration of observations that will illuminate common pathways of PCB toxicity and nutritional protection.
- Provide expertise in biostatistics that is vitally necessary for the design and interpretation of experiments conducted by all of the participating investigators and to continue to expand and refine these capabilities to encompass informatics approaches to management of large data sets necessary for application and implementation of gene expression profiling and mass spectrometry.
- Provide access to state of the art mass spectrometry based methods for detection and quantitation of PCBs, nutritional protectants and other small molecules and mediators that are of shared importance to the participating investigators for calibration, validation and monitoring of PCB sensing and remediation technologies by the environmental science projects and for ensuring consistency and relevance to human exposure of PCB doses used in studies with cultured cell and animal models.
- Leverage recent institutional investments in advanced mass spectrometry instrumentation and research infrastructure for the further development of the metabolomics capabilities of the bioanalytical component of the RSC.
- Provide a conduit for synergistic interactions between the UK-SRC Center and Clinical and Translational researchers at the University of Kentucky who are engaged in studies of nutritional protection in case controlled clinical studies of cardiovascular and metabolic disease
The Research Support Core is directed by Dr. Andrew J. Morris, who also oversees the Bioanalytical Component. Dr. Morris is a Professor of Cardiovascular Medicine. Dr. Morris’s personal research program concerns studies of pathways of lipid metabolism involved in inflammation, cardiovascular and metabolic diseases. Professional staff of the Research Support Core includes Ms. Manjula Sunkara and and Dr. Sony Soman, who are responsible for day to day operations of the core. Drs. Sunkara and Soman have extensive backgrounds in analytical chemistry with experience in the design and implementation of tandem mass spectrometry assays for small molecules gained in both academia and industry. Dr. Arnold J. Stromberg directs the Quantitative Biology Component. Dr. Stromberg is the Chair of the Statistics Department and has published extensively in the area of computational analytical methods for large scale analysis of gene expression data.
- AB Sciex 4000 Q-Trap hybrid triple quadrupole linear ion trap mass spectrometers
- ABSciex 5600 “Triple TOF” hybrid quadrupole time of flight mass spectrometer
- Agilent 6890 gas chromatograph/ electron capture detector and Agilent 5975 inert mass selective detector
- Biorad Bioplex 200 suspension array reader and MAGPIX multiplex analyzer
- Nikon TE200/Nikon A1R resonance scanning confocal microscope
- LiCor Infra-Red Blot Imaging System
- HPLC systems with fluorescence and electrochemical detection capabilities
- Seahorse XF24 Extracellular Flux Analyzer
- Thermo LTQ Velos Orbitrap Mass Spectrometer with Electron Transfer Dissociation and Eksigent Nano LC system
- Bruker EMX ESR spectrometer
- Routine Assays, Services and Representative Collaborative Studies
- SRC-Wide PCB measurements to ensure inter project consistency and relevance to human exposure
- Quantitation of lipids, metabolites, small molecule toxins and therapeutics
- Targeted Metabolomics/Lipidomics
- Unbiased Metabolomics/Lipidomics for biomarker discovery
- Confocal Microscopy
- Suspension array analysis of inflammatory mediators
- Free Radical Biology Core Services
- Analysis of markers of oxidative and nitrosative stress
- Measurements of mitochondrial function and cellular phenotypes associated with oxidative stress
- Redox Proteomics
- Electron paramagnetic resonance (EPR) detection of free radicals
Intellectual Property (IP) Development and Technology Transfer
The University of Kentucky strives to provide an atmosphere where the spirit of inquiry flourishes and where this scholarly inquiry, in the form of research and other creative activity, ultimately leads to tangible benefits for society. The University of Kentucky Intellectual Property Development Guide is to assist all faculty, clinicians, staff and students who make a discovery or conceive or develop an innovation while at the University of Kentucky.
The Office of Intellectual Property Development and Technology Transfer has developed an updated version of UK’s IP Guide, which covers the following:
- how to make the IP office aware of an invention or innovation,
- a flowchart of UK’s IP disclosure and protection process,
- a commercialization pathway designed specifically for inventions and innovations that arise from clinical practice,
- information about patents, copyrights, trademarks, and trade secrets, and
- Intellectual Property Committee meeting dates and members,
- UK’s royalty structure.
The guide also contains FAQs and a table of whom to contact about various IP issues. The Website for the Office of Intellectual Property Development and Technology Transfer is http://www.research.uky.edu/ip/. The IP guide is also available here.
Routine Assays, Services and Representative Collaborative and Developmental Studies
SRC-Wide PCB measurements to ensure inter project consistency and relevance to human exposure.
- Quantitation of lipids, metabolites, small molecule toxins and therapeutics. We have developed methods for detection and quantitation of toxins, metabolites, and nutrients that are of broad importance to the SRC researchers including neutral lipids (sterols, cholesterol esters, oxysterols, mono- di and tri- glycerides), free fatty acids, prostaglandins and fatty acid derived mediators (Prostaglandin F1 alpha, Tetranor prostaglandin E metabolites, Thromboxane B2, 15-keto Prostaglandin E2, Prostaglandin D2, Prostaglandin E2, 6-Keto Prostaglandin F1 alpha, F and J type Isoprostanes, resolvin D1, resolvin D2, maresin, DiHDoHE, HETE, Lipoxin A4), all major classes of glycro- and sphingo- phospholipids, polyphenols and nutritional protectants (ECGC and Resveratrol) and PCBs and their metabolites including oxidized species and glucuronide conjugates.
- Targeted Metabolomics/Lipidomics. We are working with UK-SRC investigators to develop and apply mass spectrometry based methods for lipid and metabolite profiling that we hope could lead to the identification of biomarkers of PCB toxicity and the protective effects of nutrition and exercise.
- Unbiased Metabolomics/Lipidomics for biomarker discovery. This approach uses our AB Sciex 5600 instrument to provide unprecedented depth and coverage of analytes and is highly suitable for unbiased metabolite profiling and is being used to identify lipid and metabolite signatures associated with PCB exposure or protective effects of nutrients, diet and exercise.
- Confocal Microscopy. Confocal microscopy is of broad importance to the biomedical research projects for imaging proteins, markers and mediators of PCB toxicity and nutritional protection in cultured cells and tissues. For example, this includes studies of experimentally induced atherosclerosis in mice and the impact of PCBs and nutritional protectants on the organization and subcellular localization of proteins involved in PCB sensing and protection from oxidative stress in cultured vascular endothelial cells.
- Suspension array analysis of inflammatory mediators. Measurements of peptide and protein cytokines and inflammatory mediators in plasma (and some cases tissue samples) from human subjects and mice. The core personnel are responsible for instrument calibration and validation and can assist users with sample collection and preparation. Routinely measured analytes of direct relevance to the biomedical SRC investigators include IL-6, IL-1beta, IL-10, MCP-1, VEGF, TNFalpha, RANTEs, IFNgamma, G-CSF and adiponectin.
Free Radical Biology Core Services. A partnership with the Free Radical Biology Core (FRBC) of the University of Kentucky Markey Cancer Center provides the following capabitlies:
- Analysis of markers of oxidative and nitrosative stress is accomplished using immunological approaches coupled in some cases with direct measurements using HPLC methods with fluorescence or electrochemical detection that we are also working to conduct using HPLC MS/MS. Available assays include measurements of protein carbonyls and 3-nitrotyrosine and protein-bound 4-hydroxy-2-trans-nonenal, HNE as indices of protein and lipid oxidation. Measurements of 8-hydroxy-2 deoxy-guanosine or 8-hydroxy-2-guanosine as indices of DNA and RNA oxidation. Analyses of antioxidant enzyme activities and levels. Analyses of reduced and oxidized glutathione (NAD+/NADH, NADP+/NADPH).
- Measurements of mitochondrial function and cellular phenotypes associated with oxidative stress are accomplished by measurements of changes in oxygen consumption and pH in intact cells using the Seahorse flux analyzer, measurements and manipulation of the expression of antioxidant or redox-related proteins (including those that regulate the redox status of cells, scavenge free radicals, and repair oxidative/nitrosative damage) in cultured cells.
- Redox Proteomics. Identification of proteins with differential expression in systems of interest using standard proteomics approaches (protein separation, digestion, ESI-MS/MS sequence analysis of tryptic peptides using a Thermo LTQ-Orbitrap mass spectrometer whic can be extended to encompass so called “redox proteomics” in which proteins containing oxidized or nitrosylated residues or adducts formed by reaction with products of lipid oxidation are targeted for analysis either directly or after chemical derivatization.
- Electron paramagnetic resonance (EPR) detection of free radicals. EPR is the only technique that can directly detect and quantitate free radicals in live experimental systems. These services are provided using state-of-the-art equipment for detection and quantitation of free radicals, antioxidants, and pro-oxidants using spin trapping approaches.
Detailed Research Support Core Instrumentation Database
This instrumentation is contained in dedicated laboratory space directed by Dr. Morris and can be used for research conducted by members of the laboratory in accordance with the business operations of the facility core.
- Agilent 7890 GC/ 5975 mass selective detector. This instrument is configured with an electron impact ion source and dual inlets with the ability to run samples simultaneously in parallel on separate columns connected to the mass selective detector or an electron capture detector and controlled by a workstation computer running Agilent Chemstation software.
- Agilent 7890B Gas Chromatograph/ 7000C triple quadrupole mass spectrometer. This instrument is configured with an autosampler and multimode inlet capable of compressed air or liquid nitrogen assisted cooling and integrated microfluidics to enable column and inlet switching between electron capture, flame ionization and a triple quadrupole mass spectrometer detector and integration of gas selection and column backflushing into automated workflows. The mass spectrometer can be configured with electron impact or chemical ionization sources and is controlled by workstation computer running Agilent Mass Hunter Software.
- Two AB Sciex 4000 Q-Trap triple quadrupole linear ion trap mass spectrometer systems. Two completely separate ABSciex 4000 Q-Trap hybrid linear ion trap triple quadrupole mass spectrometers with ABSciex “Turbo V” electrospray and chemical ionization sources. One of these systems can also be operated with a vaccum MALDI ion source (ABSciex Flashquant). Each system can be operated with a Shimadzu HPLC system comprising an autosampler, column oven multiple pumps, switching valves and a controller and is connected to a workstation computer running AB Sciex Analyst and Multiquant Software for instrument control and data analysis.
- ABSciex 6500 plus Q-Trap triple quadrupole linear ion trap mass spectrometer system. This instrument is located at the Lexington VA Medical Center and available for use by university of Kentucky investigators with approval from that facility. The instrument is configured with a Shimadzu Nexera UPLC system including a rack changing autosampler and column oven and connected to a workstation computer running AB Sciex Analyst and Multiquant Software for instrument control and data analysis.
- ABSciex 5600 Quadrupole time of flight mass spectrometer. This instrument incorporates an AB Sciex electrospray and chemical ionization source and is configured for operation with either a complete automated Shimadzu HPLC system (of identical configuration to those used with the 4000 Q-Trap instruments), a or with a Eksigent microflow HPLC system that is configured for use with either capillary HPLC columns or perform automated direct infusion. This microflow system operates with a low dispersion electrode insert that provides some of the sensitivity benefits of nano flow electrospray ionization with increased robustness in comparison to glass emitter electrodes. This instrument can also be configured with an Advion Nanomate robotic chip based nano electrospray ionization source for sample analysis by direct infusion.
- Thermo Q-Exactive quadrupole orbitrap mass spectrometer. This is a quadrupole orbitrap mass spectrometer system that provides high sensitivity and high mass resolution for quantitative and qualitative analysis. The system is configured for use with a Thermo/Dionex U3000 UPLC chromatography system and is interfaced with a workstation computer running software for instrument control, data acquisition and analysis including Thermo Compound Discoverer and Lipid Search Software.
- Sample preparation. The laboratory contains two Gilson Gradient HPLC systems with absorbance, fluorescence and evaporative light scattering detectors. Other equipment in the laboratory for sample preparation for GC or LC MS includes two N-Evap N2 evaporator systems, three Biotage Turbo Vap evaporators (one configured for 4 ml vials, the other for 96 well plates), two LabConco centrifugal evaporator/concentrators, a Genevac Rocket Solvent Evaporator System, a Thermo/Dionex Accelerated Solvent Extraction System, two Supelco Solid Phase extraction systems, a Matrix Scientific Well Mate multiwell plate dispenser, a Gilson 215 Robotic Liquid Handling System configured for multiwell plate and cartridge based Solid Phase Extraction with workstation computer running Gilson Trilution Software, two multi sample vortexers, heating blocks for lipid phosphate analysis and chemical derivatization and a Biotek Multimode absorbance/fluorescence reader.
- Data analysis. In addition to the instrument control computers, the facility contains two workstation computers running instrument control software and software packages for data analysis that are available for off-line processing of data collected using the primary instrument control computers. Proprietary and open source software packages are available for compound identification, metabolomics and lipidomics. All of the computer systems in the laboratory have redundant hard drives and are networked for data transfer and backup.