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Page 71 PART II VALUE OF MARINE BIODIVERSITY TO BIOMEDICINE In Part I, threats to human health from the ocean were defined and identified as areas that would benefit from greater interaction between the oceanographic and medical communities. For example, research on the ecology of harmful algal blooms addresses problems relevent to both public health and environmental health. Sentinel species like marine mammals, fish, and birds provide initial indicators of environmental problems such as a harmful algal bloom. Tests to detect Florida red tide and ciguatera, developed for emergency room use, were used to identify red tide brevetoxin as the reason for manatee deaths in 1996. The cause of gannet and other sea bird deaths in 1995 was linked to paralytic shellfish poisoning, using techniques developed to study human nerve function. Using tests developed for molecular brain research, the lethal agent responsible for mortality of sea lions in Monterey Bay was identified as the algal toxin that causes amnesic shellfish poisoning. Animal model system work, funded by the National Institutes of Health (NIH), has provided answers to how toxins are accumulated and how these toxins affect nerves and metabolism. This knowledge provides clues to potential antidotes, therapies, and treatments. Hence, studies of marine toxins not only help prevent human illness and identify environmental problems but also provide insight into human biology and suggest new approaches for treating human diseases. In the second half of this report, research areas are defined that establish the basis for a unique partnershipa cross-fertilization that provides for an interactive approach to the ocean and human health. Ways in which issues common to different areas of marine research areas will help provide answers or approaches of value to the others are summarized in the table below:
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Page 72 TABLE PART II Interactive Elements of HABs, Marine Models, and Drug Discovery Harmful Algal Blooms Drug Discovery Programs Marine Models of Human Disease Environmental chemicals that affect man Known and emerging toxins Surveys for new agents Test system for identification, including sentinels High throughput assays Needed Available Receptor basis for development Mechanism of action Known but need detail Bioassay-guided System for deciphering Model system Need sensitive and specific models Models needed for specific actions Disease-based Therapeutics Need for each toxin type Potential for specific diseases In vivo system for development The research described in the following chapters falls under the category of marine biotechnology. Several previous reports have argued that the investment in marine biotechnology in the United States should be higher (NRC, 1994a; Zilinskas. 1995). For example, approximately one patent is issued for every $1.1 million spent on marine biotechnology, an indication of the high productivity of this research. Despite this success, it is estimated that only 1.1% of the nation's biotechnology budget was spent on research in marine biotechnology in 1992 (Zilinskas, 1995). Also, the absolute level of funds spent for marine biotechnology has been low compared to the investment made in other countries; in 1991, the U.S. spent between 7–10% of the amount spent in Japan on marine biotechnology (Zilinskas, 1995). The following chapters illustrate the potential of two areas of marine biotechnology, marine natural products and marine biomedical models. In Chapter 4, the principle of using marine biotoxins and other compounds from marine organisms as molecular probes in biomedical research is discussed in the larger context of how marine natural products provide a new source of molecular diversity that will be of value in developing new pharmaceuticals. Chapter 5 illustrates how basic research on marine organisms offers insights into disease processes that occur in humans.
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Page 73 4 Marine-Derived Pharmaceuticals and Related Bioactive Agents One of the major accomplishments of the 20th century is the development of modern pharmaceuticals. Since their emergence early in the 20th century, drugs such as penicillin, streptomycin, and vincristine, among others, have contributed significantly to the management of human disease. New drug therapies have extended human life span and improved the quality of life. In this regard, society has become more and more reliant upon the availability of safe and efficacious pharmaceutical products. Nature has been the traditional source of new pharmaceuticals. Today, over 50% of the marketed drugs are either extracted from natural sources or produced by synthesis using natural products as templates or starting materials. Since ancient times, early societies used natural medicines, generally as crude extracts from plants, to treat infection, inflammation, pain, and a variety of other maladies. Even today, in many parts of the world, natural medicines provide the only treatments available. Investigation of these ''natural" ethnobotanical preparations led to the isolation of compounds whose beneficial properties have provided the foundation of the current pharmaceutical industry. Of course, times have changed and science in both industrialized and developing nations has become much more sophisticated in its approach to drug discovery. Complex programs have been initiated to investigate diseases based on their fundamental biochemical and molecular causes. In some cases, it has been feasible to design drugs based on knowledge of an appropriate biochemical target. In other instances, new drugs have been discovered by modern high-through-put screening, a process in which thousands of natural or synthetic chemicals are tested in automated pharmacological bioassays. All of these approaches have a place in the discovery process, but without a natural product to lead science to its
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Page 74 biological target, many drugs would never have been developed. It appears that a diverse approach, involving all these methods, is the most effective approach. It is also clear that natural products, which have evolved over millions of years of selective pressures, provide one of the most important components of this process. There is a continual need for new therapeutic agents, especially to treat a large variety of diseases for which there are no effective therapies. Many forms of cancer, viral and fungal infections, inflammatory diseases, and neurodegenerative diseases cannot be treated successfully. Development of resistance of pathogenic microorganisms to antibiotics and cancer cells to antitumor drugs requires the compensatory generation of new drugs. The pathogens responsible for malaria and tuberculosis have developed resistance to most available drugs. Thus, heavy investment in the development of new antibiotics will be necessary in the next millennium. This chapter will address the issue of new drug discovery. As in the past, natural products will be an important source of new therapeutics, but it is necessary to identify sources of new biological activities and chemical structural diversity. As described below, the biological diversity of the ocean offers great promise as a source of drugs for the future. The Marine Environment as a Source of Chemical Diversity Representatives of every phylum are found in the sea; twelve phyla are exclusively marine. The ocean contains more than 200,000 described species of invertebrates and algae (Winston, 1988), however, it is estimated that this number is but a small percentage of the total number of species that have yet to be discovered and described. Conservative estimates suggest that oceanic subsurface bacteria could constitute as much as 10% of the total living biomass carbon in the biosphere (Parkes et al., 1994). From a relatively small number of these species that have been studied to date, thousands of chemical compounds have been isolated (Ireland et al., 1993). Moreover, only a small percentage of these compounds has been tested in clinically relevant bioassays. The ocean represents a virtually untapped resource for discovery of novel chemicals with pharmaceutical potential. Marine plants, animals, and microbes produce compounds that have potential as pharmaceuticals. These "secondary metabolites," chemicals that are not needed by the organism for basic or primary metabolic processes, are believed to confer some evolutionary advantage. Because many of these plants and animals live in densely populated habitats (Plate X), are non-motile, and have only primitive immune systems, they have evolved chemical compounds to help defend against predators (Paul, 1992), to attract or inhibit other organisms from settling
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Page 75 or growing on them (Pawlik, 1993), and to provide chemical cues to synchronize reproduction among organisms that expel their eggs and sperm into the water (Morse, 1991). The mechanisms by which they prevent encroachment or predation interact with the same or similar enzymes and receptors that are involved in human disease processes. For example, many natural products have been identified that inhibit cell division, the process that is the primary target of many anti-cancer drugs. In most cases, there is a greater understanding of the effect of the natural product on human disease processes than of the function in the marine organism from which it was isolated. The marine environment became a focus of natural products drug discovery research because of its relatively unexplored biodiversity compared to terrestrial environments. The potential of marine natural products as pharmaceuticals was introduced by the pioneering work of Bergmann in the 1950s (Bergmann and Feeney, 1951; Bergmann and Burke, 1955), which led to the only two marine-derived pharmaceuticals that are clinically available today. The anticancer drug, Ara-C, is used to treat acute myelocytic leukemia and non-Hodgkin's lymphoma. The antiviral drug, Ara-A, is used for the treatment of herpes infections (McConnell et al., 1994). Both are derived from nucleosides isolated from a shallow-water marine sponge collected off the coast of Florida. Marine sponges are among the most prolific sources of diverse chemical compounds with therapeutic potential (Plate XI). Of the more than 5000 chemical compounds derived from marine organisms, more than 30% have been isolated from sponges (Ireland et al., 1993). Sponges occur in every marine environment, from intertidal to abyssal regions, in all the world's oceans, and they produce a greater diversity of chemical structures than any other group of marine invertebrates. Other marine sources of bioactive molecules with therapeutic potential are bryozoans, ascidians, molluscs, cnidarians, and algae. Several strains of phytoplankton, especially cultured species of diatoms, have been described as exhibiting antibacterial and antifungal activity (Viso et al., 1987). However, the levels of activity are low and hence the active compounds have not yet been isolated or characterized. The Discovery and Development of Marine Pharmaceuticals: Current Status Since the mid-1970s, academic, government, industrial, and private research laboratories have devoted varying levels of effort to the discovery of marine-derived pharmaceuticals. The major emphasis has been on the discovery of anti-cancer compounds, due in large part to the availability of funding to support marine-based drug discovery. The National Cancer Institute (NCI) has led this effort through its aggressive programs to support both single-investigator and
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Page 76 multi-institutional cancer drug discovery research. As a result, several marine-derived compounds are in clinical trials for the treatment of cancer. Bryostatin, isolated from the bryozoan Bugula neritina, is a polyketide with both anticancer and immune modulating activity (Kalechman et al., 1982; Pettit et al., 1996; Philip et al., 1993; Suffness et al., 1989). Its mechanism of action is through activation of protein kinase C mediation of cell signal transduction pathways. This compound is currently in Phase II clinical trials for non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and multiple myeloma through a Cooperative Research and Development Agreement (CRADA) between the NCI and Bristol-Myers Squibb. Ecteinascidin 743, a complex alkaloid derived from the ascidian Ecteinascidia turbinata (Rinehart et al., 1990; Wright et al., 1990) and licensed by the University of Illinois to PharmaMar S.A. is in Phase I clinical trials for ovarian cancer and other solid tumors in the United States and Europe. Discodermolide, a polyketide isolated from deep-water sponges of the genus Discodermia (Plate XIIa; Gunasekera et al., 1990) is a potent immunosuppressive and anticancer agent which inhibits the proliferation of cells by interfering with the cell's microtubule network (Plate XIIb and c; Longley et al., 1991a and b; ter Haar et al., 1996). This compound may be effective against breast and other types of cancer that have become resistant to other microtubule disrupting drugs. Discodermolide has been licensed by Harbor Branch Oceanographic Institution (Fort Pierce, FL) to Novartis Pharmaceutical Corporation, and is in advanced preclinical trials. Another promising sponge metabolite in advanced preclinical trials at the NCI is halichondrin B, derived from a New Zealand deep water sponge, Lissodendoryx sp. (Hirata and Uemura, 1986; Litaudon et al., 1994). Like discodermolide, halichondrin B blocks cell division by disruption of microtubule structure. In each of these cases, however, bulk supply of the chemicals for continued clinical development is a problem. It is often neither economically nor ecologically feasible to rely on large-scale collections of the source organisms from their natural habitats for supply of marine drug candidates. Whether a drug company decides to support the clinical development of a new drug is dependent on identifying an adequate supply of the source material. This could be through culture of the organism or through synthesis of the compound using an economically feasible, industrial-scale process. Research is in progress on options for biological supply (e.g., aquaculture, cell culture, microbial fermentation, and genetic engineering) to address this critical issue in the development of chemicals from natural sources. Unfortunately, neither the NIH nor must drug companies are prepared to invest funds in basic research to develop general models for biological supply of marine natural products with therapeutic potential. Despite the emphasis on identifying new anticancer compounds, marine natural products have also been found to have other biological activities, including
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Page 77 mediation of the inflammatory response. The pseudopterosins are glycosides derived from the Caribbean soft coral Pseudopterogorgia elisabethae (Plate XIII; Look et al., 1986; Roussis et al., 1990). These are in advanced preclinical trials as anti-inflammatory and analgesic drugs. A number of marine-derived compounds have been discovered with antiviral and antifungal activity. Indeed, one of the two clinically-available marine-derived drugs is used for the treatment of herpes infections. Although no marine natural products are currently in clinical trials as treatments of infectious diseases, there is high potential for future development. Marine Microorganisms as a Novel Resource for New Drugs Since the discovery of penicillin in the late 1920s, terrestrial, mainly soil-derived microorganisms have provided the single most important resource for discovery of new drugs. Over 120 microbially-produced drugs are in clinical use today to treat infectious diseases, cancer, and to facilitate organ transplantation by suppression of the immune response. Examples of these highly used drugs are the antibiotics, such as the penicillin, cephalosporines, streptomycin, and vancomycin, the cancer drugs actinomycin and mitomycin, and the immunosuppressant drug cyclosporin. Not only have microorganisms been a tremendous source of biodiversity and chemical diversity, but their capacities to produce highly complex molecules from common nutrients in fermentation culture have led to their widespread use in the economic, industrial-scale production of drugs. Today, the pharmaceutical industries worldwide (but particularly in the United States and in Japan) continue to rely on microorganisms as the single most useful source for natural product drugs. But there are growing concerns about the continuance of this historic approach to drug discovery. Over the past 50 years, soil-derived microorganisms have been extensively investigated from virtually all terrestrial regions of the Earth. Literally millions of isolated microbial strains have been extensively evaluated in a large variety of pharmacological bioassays. Although these microorganisms were at one time a rich source of novel chemical compounds, much of their chemical diversity has been exploited. New antibiotics are not being developed as before. Anticancer drugs, possessing radically new chemical formulations, are not being found at the rate once observed. Depending upon the area of investigation, it has been observed that greater than 95% of the "active" molecules identified are actually molecules discovered in the past. This extensive duplication of discovery has required the development of sophisticated "dereplication" schemes, which are designed to quickly discard well-known substances that frequently recur. More important, the costs of new discoveries have escalated. These costs are so high that the pharmaceutical industry has invested in a more cost efficient process, "high-throughput
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Page 78 screening," which allows literally hundreds of thousands of samples to be evaluated in weeks to months, rather than years. Most of the Earth's microbial diversity is found in the ocean. In addition to the typical organisms found on land, many classes of microorganisms exist only in the sea. The photosynthetic microorganisms alone comprise over 12 plant divisions. With the inclusion of the known unique adaptations of microorganisms to high salt environments and high hydrostatic pressure, the immense diversity of the microorganisms in marine habitats becomes apparent. However, the pharmaceutical industries have not taken advantage of this enormous resource. The reason rests with the tendency of researchers to explore familiar and more readily accessible microorganisms. In the 1950s, it was reported that fewer than 5% of the marine bacteria present in environmental samples could be cultured. This led to the widespread belief that marine microorganisms were simply unculturable. This, together with the traditional lack of cross-disciplinary interaction between marine and medical microbiology, resulted in a great hesitation to embark on an aggressive marine-oriented program. With no guarantees that any new drugs will be found, the risk was difficult to justify. However, times have changed and marine microorganisms can now be cultivated successfully (Davidson, 1995; Fenical, 1993; Kobayashi and Ishibashi, 1993; Okami, 1993). The microbial diversity of the marine environment has thus become available for scientific study, and new programs are emerging worldwide. In the United States and Japan, intense focus on coastal sediments and organisms associated with marine invertebrates has resulted in a growing number of papers documenting the production of novel, bioactive metabolites. Both bacteria and fungi are now the target of biomedical study, and fascinating reports of novel metabolites are becoming more and more common. Bacterial samples from coastal sediments, when grown under saline conditions, have yielded new antibiotics, antitumor, and anti-inflammatory compounds (Pathirana et al., 1992; Trischman et al., 1994a, b). Similarly, when the surfaces of marine plants and the internal tissues of invertebrates have been sampled, bacteria and fungi have been discovered that also produce bioactive compounds with pharmaceutical potential. Marine fungi, in particular, seem to be the focus of increasing interest (Belofsky et al., 1998; Cheng et al., 1994; Kakeya et al., 1995; Numata et al., 1992; Takahashi et al., 1995). A unique source in the world's oceans, the deep ocean and the geothermal vents, is now becoming the focus of considerable microbiological interest. Microbiological studies of the deep sea environment have shown the presence of obligate barophiles, bacteria which require pressures as high as 600 atmospheres for growth to occur (Yayanos, 1995). These highly adapted marine microorganisms represent a biomedical resource of unknown magnitude, but great promise, as demonstrated recently when unusual compounds produced by bacteria retrieved from deep sea drilling cores were shown to inhibit colon tumor cell growth and prevent replication of HIV, the AIDS virus (Gustafson et al., 1989).
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Page 79 The Marine Environment as a Source of Molecular Probes One important application of the many bioactive compounds derived from the marine environment is their use as molecular probes, molecules broadly defined as non-drug substances which can be used to probe the foundations of important biochemical events. Molecules such as the potent marine neurotoxins, tetrodotoxin, saxitoxin, conotoxin, lophotoxin, and others, have been instrumental in defining the functions and overall structures of the membrane channels which facilitate nerve transmission. Knowledge of the function of these neurotoxins has allowed drugs to be designed and targeted to those sites of nerve transmission. Other examples are the discovery that the dinoflagellate toxin, okadaic acid, shows potent and selective inhibition of phosphatases; the utilization of the anti-inflammatory agent, monoalide, as a selective inhibitor of the inflammation enzyme phospholipase A2 (Glaser and Jacobs, 1986); the use of latrunculin A, jaspamide (jasplakinolide), and swinholide A as selective binding agents to the intracellular actin network (Bubb et al., 1995; Matthews et al., 1997; Senderowicz et al., 1995); and the recent discovery of the unique sponge metabolite, adociasulfate-2, which selectively inhibits the intracellular molecular motor protein kinesin (HHMI, 1998). The importance of molecular probes in resolving the complexities of diseases and cellular processes has often outweighed any value that they would have as commercial drugs. Marine natural products have not only contributed probes for studying specific cellular proteins and enzymes, but they have also provided visual markers for proteins specified by antibodies, for cellular events mediated by calcium, and for elucidating mechanisms of tissue-specific gene expression. Antibodies are an indispensable tool of molecular biology and biomedicine because they can be used to identify specific biomolecules. However, they must be coupled to a reporter molecule, usually an enzyme with a colorimetric substrate or a fluorescent compound. Phycoerythrin, a fluorescent protein isolated from red algae, is crosslinked to antibodies for use as an indicator in many immunological assays. In the algae, phycoerythrin functions in light harvesting during photosynthesis. For this role, the protein shows optimization of both absorption and fluorescence resulting in high quantum yield while showing minimal dependence on pH or ionic conditions. (Glazer, 1988; 1989) These properties make it ideal for assays requiring high sensitivity (quantum yield is 30–100 times greater than the chemical dyes fluoresein and rhodamine); therefore phycoerythrin-conjugated antibodies are a favored reagent for use in flow cytometry, a common clinical diagnostic procedure (Roederer et al., 1997; Sohn and Sautter, 1991). Aequorin, a compound isolated from a bioluminiscent jellyfish Aequora victoria has been used extensively in cell biology because it has the unique property of emitting light in the presence of calcium. For example, aequorin has been
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Page 80 used to illuminate the calcium wave during sea urchin egg fertilization (Eisen and Reynolds, 1985) a phenomenon described in greater detail in Chapter 5. The photoprotein component of aequorin has been cloned into gene expression vectors, and is currently used to monitor calcium in the cytoplasm and organelles of tissue culture cells (Badminton and Kendall, 1998; Badminton et al., 1995; Brini et al., 1994; Montero et al., 1995; Rutter et al., 1993). Recently, there has been an exciting new development of another product derived from the bioluminescent jellyfish A. victoria, the cloning of green fluorescent protein (GFP) (Chalfie et al., 1994). In jellyfish, GFP absorbs the blue light produced by aequorin and fluoresces green light. GFP has been developed for use as a reporter gene in numerous studies on the regulation of gene expression (Plate XIV). Because GFP fluoresces in living tissues, it is now possible to monitor gene expression continuously, a property of particular value in the study of differentiation in both embryos and tissue culture cells. There are many other marine products that have contributed to basic and clinical research including enzymes for molecular biology, most notably the Vent DNA polymerase used in the polymerase chain reaction (PCR). PCR, a technique used to amplify minute amounts of DNA or RNA, requires the use of enzymes that are stable at high temperature. A marine microorganism isolated from the deep sea hydrothermal vents yielded the Vent DNA polymerase which is used in high fidelity PCR reactions common to both diagnostic procedures and the gene mapping studies of the Human Genome Project. Marine bacteria have also provided many unique restriction enzymes used in the cloning of DNA. Since we have only begun to investigate marine biodiversity, it is reasonable to expect that marine molecular probes will continue to advance the frontiers of molecular and cellular biology. The Ocean as a Source of New Nutritional Supplements Docosahexonoic acid (DHA) and arachidonic acid (ARA) are the most abundant polyunsaturated fatty acids (PUFAs) in breast milk, and are the predominant structural fatty acids in brain gray matter. High dietary levels of these PUFAs are believed to result in higher levels in the brain, and they have been recommended as nutritional supplements for infants. A marine microalgal species has been discovered that produces large quantities of the fatty-acid docosahexenoic acid (DHA). It is used in an infant formula supplement Formulaid® (Martek Biosciences, Columbia, MD). Marine-derived nutritional supplements, or "nutriceuticals," present a new opportunity for research in the application of marine natural products to human health issues.
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Page 81 Conclusions The Discovery and Development of Marine Pharmaceuticals: Needs for the 21st Century • Marine organisms as a source of pharmaceuticals The successes to date in the discovery of novel chemicals from marine organisms that have demonstrated potential as new treatments for cancer, infectious diseases, and inflammation, suggest that there needs to be a greater focus on the development of drugs from marine sources. Exploration of unique habitats, such as deep sea environments, and the isolation and culture of marine microorganisms offer two underexplored opportunities for discovery of novel chemicals with therapeutic potential. The successes to date based on a very limited investigation of both deep sea organisms and marine microorganisms suggests a high potential for continued discovery of new drugs. Marine microorganisms are particularly attractive because they fit in with the traditional pharmaceutical "model" of a natural product drug source. Moreover, supply of bulk amounts of a microbially-derived drug can be addressed by large-scale fermentation of bioactive marine microorganisms. • Expand marine drug discovery beyond cancer to include other diseases Programs such as the Natural Products National Cancer Drug Discovery Groups (NPNCDDGs) at the National Cancer Institute have been tremendously successful in interfacing non-traditional drug sources, such as marine organisms, with the screening and development potential of major pharmaceutical companies. Similarly, the Small Business Innovative Research (SBIR) grants have fostered interactions on a smaller scale. Other institutes within the NIH should consider developing programs for marine-based drug discovery for diseases that desperately need new therapies, such as neurodegenerative, cardiovascular, and infectious diseases. In particular, there needs to be a more organized approach to the development of antibiotics from marine sources. The increasingly limited effectiveness of currently available drugs has dire consequences for public health, although the consequences have not yet been felt by the public or the medical community. The United States is faced with the serious threat of re-emerging infectious diseases, such as tuberculosis, indicating that a radical and aggressive approach needs to be taken to control these multiple-drug-resistant pathogens. • New interdisciplinary research and education programs Few researchers have training in both medicine and marine science. Cross-training in both disciplines could provide innovative approaches to marine-based biomedical research. For example, marine organisms have already demonstrated their utility as biomedical models, the results of which have been applied to understanding normal and disease processes in humans (cf. Chapter 5). Marine
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Page 82 organisms also offer the potential to understand and develop treatments for disease based on the normal physiological role of their secondary metabolites. In some systems, e.g., Conus toxins (Hart, 1997; Hopkins et al., 1995), the mechanisms of action of the metabolites are well-known and can be applied to the development of new classes of drugs. In most systems, however, the natural functions of bioactive secondary metabolites are poorly understood. The respective expertise of marine and medical scientists should be optimized and the cross-training of marine and biomedical scientists should be encouraged to study the role of these compounds in nature, to determine how chemical interactions in the ocean can be applied to the development of new drugs, and then to design appropriate bioassays to test their effectiveness against human diseases. One possible way to achieve this goal would be to develop a new graduate student training initiative. The purpose of the program would be to educate graduate students in both marine and medical sciences and to facilitate the development of a strong interface between medicine and marine sciences.
Representative terms from entire chapter: