Collaborative Research: Pacific Research Center for Marine Biomedicine

太平洋海洋生物医学研究中心（PRCMB）的重建将是夏威夷大学、斯坦福大学和杰克逊州立大学之间的合作。 研究活动将集中在两个领域：（1）热带海洋沃茨中的病原体和（2）海洋毒素和药物先导发现。这项新的研究是在PRCMB资助第一阶段取得的成就和发现的逻辑基础上进行的。病原体和病原体指标工作将侧重于两条排入海洋的溪流，一条是热带溪流（奥廷加岛的Kaelepulu），另一条是温带溪流（加州的圣佩德罗溪）。每月在两条溪流进行采样，使用培养依赖性和非培养依赖性（基于PCR）方法表征病原体指标和病原体（细菌和病毒）浓度的季节性变化。这些研究将辅之以风暴和干旱天气条件下的高频采样（10分钟和每小时），以提供有关空间和时间变异性的见解。将使用病原体和病原体指示剂浓度作为输入，开发健康风险模型（定量微生物风险评估）。海洋毒素工作将涉及雪卡毒素和β-N-甲基氨基-L-丙氨酸（BMAA），这两者都是最近PRCMB研究的重点。利用（1）改进的检测方法（n2 a），现在能够检测鱼组织中的雪卡毒素，其浓度低于雪卡毒素生物学阈值的十倍，以及（2）与夏威夷休闲渔民的合作，PRCMB科学家将从鱼组织中提取并浓缩足够的雪卡毒素，以获得分子结构，然后，与华盛顿大学的科学家合作，致力于开发一种基于抗体的雪卡毒素检测方法。雪卡毒素的研究还将涉及（1）基于n2 a细胞系产生一氧化氮的n2 a测定的探索性改进和（2）触发分类学上定义的甲藻Gambierdiscus菌株产生雪卡毒素的环境条件的调查。BMAA的工作将建立在初步研究的基础上，以确定BMAA在海洋蓝细菌中的流行程度以及这种毒素向更高营养级的转移。药物研究将利用PRCMB培养物中尚未筛选生物活性的2，500多种微生物分离株。这些培养物的提取物将用于细胞和分子测定，以确定它们是否影响微生物病原体（白色念珠菌、大肠杆菌、金黄色葡萄球菌、多重耐药链球菌）的生长。金黄色葡萄球菌和万古霉素抗性屎肠球菌）或它们是否对人腺癌细胞、蛋白激酶C或丝裂原活化蛋白激酶发挥作用。将通过生物测定指导的分级分离和光谱分析（例如，质谱法、高场核磁共振光谱法）拟议工作的更广泛影响分为几类。一个是公共卫生终点：（i）可用于确定沿海沃茨是否可安全用于娱乐用途的可靠方法，（ii）对鱼类中的雪卡毒素进行简单、廉价的测定，（iii）更好地了解与BMAA通过海洋食物链移动有关的对人类健康的威胁，和（iv）发现新的化合物并应用于治疗人类健康问题。第二，这是一个与历史上的黑人大学（杰克逊州立大学）和一位来自斯坦福大学的女性首席研究员的合作提案。最后，PRCMB将继续培训学生，并与其他机构和海洋与人类健康中心合作。

This accomplishment-based renewal of the Pacific Research Center for Marine Biomedicine (PRCMB) will be a collaboration between the University of Hawaii, Stanford, and Jackson State University. Research activities will be focused on two areas: (1) pathogens in tropical marine waters and (2) marine toxins and pharmaceutical lead discovery. This new research follows logically from accomplishments and discoveries made during the first phase of PRCMB funding. The pathogen and pathogen indicator work will focus on two streams that discharge to the ocean, one tropical (Kaelepulu on the island of Oahu) and the other temperate (San Pedro Creek in California). Monthly sampling at the two streams will be used to characterize seasonal variability in pathogen indicators and pathogen (bacterial and viral) concentrations using both culture-dependent and culture-independent (PCR-based) methods. These studies will be complemented by high-frequency sampling (ten-minute and hourly) during storm and dry-weather conditions to provide insights concerning spatial and temporal variability. Health-risk models (Quantitative Microbial Risk Assessment) will be developed using pathogen and pathogen indicator concentrations as input. The marine toxin work will concern ciguatoxin and beta-N-methylamino-L-alanine (BMAA), both of which were the focus of recent PRCMB studies. Taking advantage of (1) the refinement of an assay (n2a) that is now capable of detecting ciguatoxin in fish tissue at concentrations ten times below the threshold associated with ciguatera symptomology and (2) a collaboration with Hawaiian recreational fishermen, PRCMB scientists will extract and concentrate sufficient ciguatoxin from fish tissue to obtain a molecular structure and will then, in collaboration with scientists at the University of Washington, work toward the development of an antibody-based assay for ciguatoxin. Ciguatoxin research will also involve (1) exploratory refinement of the n2a assay based on generation of nitric oxide by the n2a cell line and (2) an investigation of environmental conditions that trigger ciguatoxin production by taxonomically defined strains of the dinoflagellate Gambierdiscus. The BMAA work will build on initial studies to determine the prevalence of BMAA in marine cyanobacteria and the transfer of this toxin to higher trophic levels. The pharmaceutical studies will take advantage of the more than 2,500 microbial isolates in the PRCMB culture collection that have not yet been screened for bioactivity. Extracts of these cultures will be used in cell-based and molecular assays to determine if they affect the growth of microbial pathogens (Candida albicans, Escherichia coli, Staphlyococcus aureus, multi-drug resistant S. aureus, and vancomycin resistant Enterococcus faecium) or if they exert effects on human adenocarcinma cells, on protein kinase C, or on mitogen-activated protein kinase. Identification of the compounds responsible for reproducible bioactivity in these extracts will be accomplished through bioassay-guided fractionation and spectroscopic analysis (e.g., mass spectrometry, high-field nuclear magnetic resonance spectrometry)The broader impacts of the proposed work fall into several categories. One is certainly the public health endpoints: (i) credible methods that can be used to determine whether coastal waters are safe for recreational use, (ii) a simple, inexpensive assay for ciguatoxin in fish, (iii) a better appreciation of the threat to human health associated with the movement of BMAA through marine food chains, and (iv) the discovery of novel compounds with application to the treatment of human health problems. Second, this is a collaborative proposal with an historically black university (Jackson State University) and a female principal investigator from Stanford. Finally, the PRCMB will continue to train students and to collaborate with other institutions and Ocean and Human Health centers.