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Current anti-doping testing is primarily conducted in urine and blood. Recently, due to confounding factors with urine and blood collections such as invasiveness, cost, and stringent shipping conditions, there has been a push for the use of alternative sample matrices to ameliorate these issues. Gaining support within the anti-doping field is the use of oral fluid, and more recently exhaled breath, as viable alternative or complementary matrices to traditional urine and blood for drug testing. Thus, we designed a first-in-field study with the purpose of investigating the utility of oral fluid and exhaled breath testing, and the preference of athlete participants, comparative to conventional anti-doping methods of urine testing. To accomplish this, 521 total matched samples, consisting of exhaled breath, oral fluid, and urine samples, were collected and analyzed, and the results compared across matrices. Participants in this study preferred the exhaled breath collection (rated 4.90 ± 0.34 out of 5, mean ± SD) over the oral fluid collection procedure (4.29 ± 0.85), and most preferred both over urine collections. Exhaled breath resulted in the shortest collection time (2.58 ± 1.00 min, mean ± SD), followed by urine (3.08 ± 1.50 min), and finally oral fluid (4.14 ± 1.94 min). Prohibited substances from the drug categories of stimulants, narcotics, cannabinoids, diuretics, glucocorticoids, beta-blockers, and others, were analyzed in this study for a comparison of testing efficacy. Of the total findings 49% were detectable in only urine, 38% in urine + oral fluid, and 9% in all three matrices. Of the unique findings 3% were detectable in only oral fluid, 1% in oral fluid + breath, and 0% of unique findings were present only in exhaled breath. The findings from this study provide a strong foundation for the future use of oral fluid and exhaled breath as viable alternative or complementary matrices for in-competition anti-doping testing.

Defined experimental regimes were used to determine the effects of nutrient limitation on the toxicity of Alexandrium peruvianum in batch culture. Subsamples for cell counts and spiroimine analysis at six day intervals were used to investigate the concentrations and composition of these compounds throughout growth. An erythrocyte lysis assay for hemolytic activity was performed on cell pellets and supernatants also collected every six days over the entire growth period from all treatments. From the data, growth rates, cellular spiroimine quotas and effective concentration-fifty (EC50s) for cellular and supernatant associated hemolytic activity were calculated. Phosphate limitation was identified as a key regulator of toxicity in this species, yielding maximum values of 54.1 pg cell−1 for 13-desmethyl spirolide C, 96.4 pg cell−1 for 12-methylgymnodimine and a potent hemolytic EC50 value of 7.1 × 103 cells. The concentrations of spiroimines detected in A. peruvianum among various treatments, in addition to a unique profile of paralytic shellfish poisoning toxins, is unique in the body of microalgal literature. Because of the multiple toxin arsenal produced by this organism, the evaluation of a single toxin clearly would have underestimated the potential virulence and significance of this clone. This study provides the first evidence that growth and toxin production of A. peruvianum are influenced by altered nutrient ratios.

Routine sampling of the water quality stations in the New River Estuary (Jacksonville, North Carolina, USA) during November 2004 revealed the presence of a previously unidentified dinoflagellate. Preliminary observations of its morphology suggested it to be consistent with that of Alexandrium peruvianum (Balech et Mendiola) Balech et Tangen. Observations using brightfield, epifluorescence and scanning electron microscopy confirmed the diagnostic thecal plates to be those of A. peruvanium . Clonal cultures established from cells isolated from the New River Estuary samples were also used for further studies of morphology and for the presence of toxins. Thecal morphology was consistent with that described by Balech clearly separating it from the sister species Alexandrium ostenfeldii . Three classes of toxins were detected from these cultures. An erythrocyte lysis assay (ELA) was used to confirm the presence of hemolytic toxins in A. peruvianum cultures. A cellular EC 50 for lysis was 1.418 × 10 4 cells, well within the range the maximal cells densities found in the New River and more potent when compared on a cellular basis with Prymnesium parvum . Another toxin class detected in A. peruvianum cultures was the fast acting 13-desmethy C and D spirolides also produced by the sister species A. ostenfeldii . The last toxin type detected in the A. peruvianum cultures was the paralytic shellfish toxins, GTX 2, 3, B1, STX and C1,2. These findings expand the geographic range of occurrence for A. peruvianum in the U.S. to be much greater than previously considered. The morphological characters agreed with previously reported molecular data in separating A. peruvianum from A. ostenfeldii . It is also the first confirmed report that this species produces PSP toxins, spirolides and naturally occurring hemolytic substances. In light of these findings additional attention is needed for the detection of Alexandrium species in all coastal waters of the U.S. This added effort will enhance the evaluation of the relative impacts of the species to shellfish safety and bloom surveillance.

Green plant-origin electrophilic compounds are a newly-recognized class of neuroprotective compounds that provide neuroprotection through activation of the Nrf2/ARE pathway. Electrophilic hydroquinones are of particular interest due to their ability to become electrophilic quinones upon auto-oxidation. Although marine organisms frequently produce a variety of electrophilic compounds, the detailed mechanisms of action of these compounds remain unknown. Here, we focused on the neuroprotective effects of strongylophorine-8 (STR8), a para-hydroquinone-type pro-electrophilic compound from the sponge Petrosia (Strongylophora) corticata. STR8 activated the Nrf2/ARE pathway, induced phase 2 enzymes, and increased glutathione, thus protecting neuronal cells from oxidative stress. Microarray analysis indicated that STR8 induced a large number of phase 2 genes, the regulation of which is controlled by the Nrf2/ARE pathway. STR8 is the first example of a neuroprotective pro-electrophilic compound from marine organisms.

The novel gymnodimine congener 12-methylgymnodimine was isolated from the dinoflagellate Alexandrium peruvianum from the New River in North Carolina. In addition, the presence of 13-desmethylspirolide C was confirmed by NMR characterization. This marks the first time these two classes of cyclic imines have been found in the same organism, providing further evidence for a direct genetic relationship between the biosynthetic pathways for gymnodimines and spirolides suggested by comparison of their structures.