Michael Stoskopf

The proposal is to apply advanced NMR-based metabolomics techniques developed in our laboratories to samples already collected during a traditional prospective crude oil and dispersant (Corexit) exposure trial to accomplish the objective of identifying useful biomarkers of low level oil exposure with potential to improve oil contamination diagnosis, evaluation of treatment protocols, and understanding of the mechanisms of oil toxicity.

This proposal seeks funding to use proton nuclear magnetic resonance techniques (1H-NMR) to analyze diagnostically and physiologically relevant samples that were collected as an adjunct to a well-designed, traditional exposure study of petroleum and dispersant kinetics using a hatchling loggerhead sea turtle (Caretta caretta) model developed in our laboratories. We have in hand tissue samples properly collected and stored for metabolomic analysis from 20 animals exposed at 3-days posthatching, the time expected for nest emergence, to a 96-hour cutaneous exposure to either crude oil (Gulf Coast ������������������ Mixed Crude Oil Sweet, CAS #8002-05-9, 0.833 mL/L), Corexit (Corexit 9500A, 0.083 mL/L), crude oil/Corexit 9500A combination, or unexposed aged seawater control in a traditional exposure study. Our objective is to better understand the mechanisms underlying the complex effects of crude oil and the dispersant, Corexit-9500A on the metabolism and physiology of sea turtles. Through these studies, we expect to identify biomarkers of crude oil exposure with the potential to improve our ability to diagnose oil contamination, assess outcomes of treatment protocols, and further our understanding of the mechanisms of oil toxicity in sea turtles through future prospective research. The hypothesis for our proposed first year of research was: the metabolome of hatchling loggerhead sea turtles exposed to crude oil will be altered compared to that of unexposed hatchlings in tissues suitable for future field assessment. During the first year of studies we characterized whole blood and skeletal muscle of sea turtle hatchlings in the oil treatment and control groups after refining techniques to achieve complete hemolysis of blood and optimized extraction for the muscle samples. Good spectra were achieved and 8 and 12 compounds identified in control spectra. No additional or missing metabolites were identified blood or muscle from oil exposed hatchlings. Routine PCA using uniform and smart binning did not identify markers of separation between treatment groups. In the 3 months remaining on the original first year schedule we are using a combination of key metabolite targeting techniques to focus on metabolic pathways in muscle published research suggests are affected by petroleum exposure (energy and osmoregulation). The hypothesis we propose to pursue in year 2: the metabolome of tissues and fluids known to have roles in detoxifying polycyclic aromatic hydrocarbons will be altered compared to that of unexposed hatchlings will be addressed by 1H-NMR study of archived hepatic and bile samples from the same hatchlings using the refinements in approaches to PCA we developed in our first year work. In the third year we propose to analyze the heart muscle to evaluate the hypothesis: the constant contractions of cardiac muscle will alter the metabolomic changes seen in energetics and oxidative pathways from exposure to crude oil from those detected in skeletal muscle. We will also be able to compare Corexit-9500A only and Corexit-9500A plus oil exposures to oil only and controls for all 5 tissues. The use of 1H-NMR techniques represents a rapidly emerging and new approach to the evaluation of petroleum exposure, which promises to generate new methods to detect, quantify, and document oil exposure in wildlife, even for chronic and very low-dose oil exposures, and to compile many useful biomedical health parameters of a species increasingly susceptible to oil exposure. The studies proposed will both generate and help validate novel methods to detect and quantify oil exposure in wildlife and document their effects including those that persist after release from treatment. The design of the proposed study also examines the impact of the application of Corexit and will allow us to directly assess the impacts of one chemical countermeasure on wildlife.

The bonnethead shark, Sphyrna tiburo, is a small member of the hammerhead shark family found in the wild in the warm waters of shallow estuaries and bays of both the Atlantic and Pacific coasts of the Americas. It is relatively abundant off North Carolina shores, which is near the northern extent of its range in the Western Atlantic. Considered one of the shark species with the highest population growth rates, the bonnethead is in many ways an ideal shark for development of captive self-sustaining populations for display and it is also a useful model for studies of shark physiology relevant to other less abundant species. There is an important need to develop better ways to monitor and assess the health of both captive specimens and wild populations of sharks. The proposed project will use advanced NMR metabolomics techniques to look at key metabolites of bonnethead sharks, investigating the impacts of different collection/fishing techniques. A number of shark species, including highly threatened conservation priority species such as sand tiger sharks, are actively fished both for sport (catch and release) and/or for display in public aquaria. Undesired mortalities following capture, and likely delayed mortality following release, are not uncommon, posing a substantial threat to these populations. The primary aim of the proposed investigation is to determine whether a gradual extended process of reeling in a hooked shark results in greater or lesser impact on the metabolomic processes of the animal than aggressive, shorter-duration reeling, and what impact this may have on morbidity and mortality. Baseline metabolomics information obtained from this research will also provide an important foundation for more advanced understanding of the nutritional needs and adequacy of diets of not only the bonnethead shark, but also other species of sharks maintained in captivity. This dataset will contribute to improved understanding of a relatively poorly documented syndrome of ���������������failure to thrive������������������ that has been observed in young and recently captured animals, creating difficulties in long term captive management. Failure to thrive syndrome (FTS) includes variable neurologic signs including abnormal swimming behavior sometimes referred to as ����������������skipping���������������, and in severe cases progresses to constant swimming in loops. The time course of the syndrome is variable with affected animals surviving a few days to quite long periods, but unfortunately effective therapeutic options are not established, and the condition is routinely fatal.

The Department of the Interior (DOI) is establishing a network of geographically dispersed DOI Regional Climate Science Centers (Regional Centers). Regional Centers will be based at host organizations that have suitable facilities, partnerships, and science capabilities, and will involve multiple active collaborators. Regional Centers will house up to twelve (12) USGS and DOI employees and will work in close partnership with the host institution with the goal to understand high priority science needs and to develop science information and tools that can help land, water, fish and wildlife, and cultural heritage resource managers develop strategies for responding to climate change. Objectives are to 1) Provide land, water, fish and wildlife, ocean, coastal, and cultural heritage resource managers with the tools and information to develop and execute strategies for successfully adapting to and mitigating the impacts of climate change. 2) Provide modeling and forecasting information and tools, integrate physical climate models with ecological models, assess climate change vulnerabilities, forecast changes, and develop standardized approaches. 3) Provide funding for researchers through cooperative agreements that involve climate change science as a major component.

Cold stun syndrome in sea turtles occurs when ocean temperatures suddenly drop below ~15C. Thousands of animals can be affected in a single event (over 4,500 in Florida in 2010). Many die despite clinical efforts by rehabilitation facilities. The underlying physiologic mechanism of cold stun syndrome is unknown. Knowledge of the detailed physiologic basis of the condition can improve therapeutic choices and efficacy. This project examines the physiologic basis of cold stun syndrome using advanced nuclear magnetic resonance (NMR) techniques to find ways to reduce mortality rates for affected endangered green (Chelonia mydas) and Kemp's ridley (Lepidochelys kempii) sea turtles, as well as threatened loggerhead (Caretta caretta) sea turtles. Routine blood panels from affected animals demonstrate multiple, often severe, derangements which while being complex do not give a complete picture of the metabolic state of the turtle. This has led to controversies over the appropriate treatment for these patients. We hypothesize that cold stun syndrome is precipitated by perturbations to key biochemical pathways, such as metabolism of glucose for energy and/or glutathione synthesis for antioxidant protection and other functions. Affected sea turtles also commonly develop pneumonia, skin lesions, and other problems during rehabilitation. We hypothesize that these problems are related to reperfusion injury and will be demonstrated by changes in associated pathways, such as xanthine to uric acid synthesis. Finally, the role of torpor or "hibernation" in sea turtles affected by this syndrome is unknown. Based on studies in other species, we hypothesize that if torpor is a component of this syndrome, turtles will have increases in compounds such as aspartic acid, taurine, and GABA with decreases in glutamic acid. Our preliminary studies have demonstrated the feasibility and potential for metabolomic analysis of blood, and other biofluids of sea turtles to refine therapeutic decision making in treatment of cold-stun patients. Our proposed project with establish baseline metabolic profiles for these biofluids in the three sea turtle species most commonly affected by cold stun syndrome and then compare them with the metabolic profiles of turtles affected by cold stun syndrome. By examining the metabolic perturbations caused by cold stun syndrome, we can determine specific treatments for affected individuals that can decrease morbidity and mortality due to this syndrome.

The NCSU CVM EMC faculty are interested in discovering and developing improved ways to deliver health care and management for aquarium collections as part of our research mission, and the education and training of aquatic veterinarians as a component of the education mission of the university.

This proposal requests funding to upgrade the current core Marine Magnetic Resonance Facility (MMRF) at the Center for Marine Sciences and Technology (CMAST) by installing a dual cryogen re-liquifier on the facility's 4.7T 40 cm bore Oxford magnet, to enhance sustainability and reduce environmental impact of advanced NMR/MRI research while significantly reducing the cost of operation of the facility. This will be accomplished by installation of state of the art cryogen recycling capability recently developed by CryoMech. The installation will also provide an important opportunity to gather data on the potential benefits of this approach for other cryogen cooled superconducting magnets operated in North Carolina. Superconducting magnets require cryogens, liquid helium and liquid nitrogen, to maintain the superconducting conditions necessary for the magnetic field. Although these magnets are well-insulated, continual ‘boil-off’ and loss of the helium and nitrogen cryogens to the atmosphere still occurs, requiring frequent refilling. Installation of the proposed equipment is predicted to reduce the annual MMRF consumption of liquid helium and liquid nitrogen by between 95 and 99 percent through recycling the gases that would otherwise evaporate out of the magnet into the air. This will generate significant operational savings that can lower base maintenance costs of the magnet. This in turn will help increase the availability of magnet time for exploratory and pilot research. The ability to re-liquify both helium and nitrogen for an individual magnet is a very new development, and this installation would be the first of its kind in North America, though the technology is based on the proven Cryomech PT410 Cryorefrigerator. Their single cryogen helium re-liquifiers have been installed around the world and are proving durable and effective. A unit similar to the proposed unit has been installed and is functioning to specifications in a facility in Sydney, Australia. The price of cryogens, and liquid helium in particular, has been subject to massive inflation driven by uncertainty about supply over the past decade. Based on current cryogen costs for the MMRF's 4.7 T Oxford research magnet without considering inflation, the proposed helium and nitrogen re-liquifier would completely recoup its purchase, installation and maintenance costs in between 4 and 5 years of operation solely based on cryogens cost savings. If projections by economic analysts for continued cryogen price inflation based on production limitations, increasing global demand and political uncertainties that may impact US access to supplies the unit would pay for itself in as little as 2 years.

This study will evaluate whether understanding the physiology of sea turtles can provide insight into the evolution of how animals and humans respond to sudden exposure to cold environmental conditions. We will compare results of metabolomic data already gathered from sea turtles naturally exposed suddenly to cold ocean conditions in the wild, and with normal unaffected individuals, to findings in the scientific literature on the physiologic responses of humans and other animals to exposure to cold and rewarming. We hope to identify metabolic changes that are either highly conserved or extremely divergent across the broad evolutionary history of the ancient sea turtles and mammals. Such findings could help us understand the best ways to reduce the harm of exposure to sudden extreme cold and recovery from such exposure.

NCSU CVM faculty and residents will provide harmonization and support for health care management of the animal collections of the NC Aquariums.

This proposal seeks funding to use proton nuclear magnetic resonance techniques (1H-NMR) to analyze diagnostically and physiologically relevant samples that were collected as an adjunct to a well-designed, traditional exposure study of petroleum and dispersant kinetics using a hatchling loggerhead sea turtle (Caretta caretta) model developed in our laboratories. Our objective is to better understand the mechanisms underlying the complex effects of crude oil and the dispersant, Corexit-9500A on the metabolism and physiology of sea turtles. Through these studies, we expect to identify biomarkers of crude oil exposure with the potential to improve our ability to diagnose oil contamination, assess outcomes of treatment protocols, and further our understanding of the mechanisms of oil toxicity in sea turtles through future prospective research.