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Ocean ecosystems are in decline, yet we also have more ocean data, and more data portals, than ever before. To make effective decisions regarding ocean management, especially in the face of global environmental change, we need to make the best use possible of these data. Yet many data are not shared, are hard to find, and cannot be effectively accessed. We identify three classes of challenges to data sharing and use: uploading, aggregating, and navigating. While tremendous advances have occurred to improve ocean data operability and transparency, the effect has been largely incremental. We propose a suite of both technical and cultural solutions to overcome these challenges including the use of natural language processing, automatic data translation, ledger-based data identifiers, digital community currencies, data impact factors, and social networks as ways of breaking through these barriers. One way to harness these solutions could be a combinatorial machine that embodies both technological and social networking solutions to aggregate ocean data and to allow researchers to discover, navigate, and download data as well as to connect researchers and data users while providing an open-sourced backend for new data tools. [ABSTRACT FROM AUTHOR]

Abstract: Saxitoxin (STX) contaminates seafood and freshwater catchments worldwide. Conjugation of STX with biotin would enable new biochemical methods to quantitate STX and its analogues as well as diversify its utility as a research tool. We conjugated biotin at the region of the toxin normally occupied by a carbamoyl and this conjugate could concurrently bind both avidin/streptavidin and saxiphilin. Increasing the length of the linker between biotin and the STX portion of the semisynthetic analogue increased potency of saxiphilin binding of the STX moiety. [Copyright &y& Elsevier]

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C4 plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 423 ± 0.8 μM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C4 plant but no effect on a model C3 plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants. [ABSTRACT FROM AUTHOR]

Abstract: Several radioreceptor assays using tritiated saxitoxin ([3H]STX) were developed to identify a suitable primary screening method for the detection and characterization of soluble saxitoxin binding proteins from biological extracts. Assays using anion and cation exchange, protein binding, and traditional charcoal radioreceptor methods were compared with two previously reported formats. A protein binding assay incorporating filters of mixed cellulose esters (MCE) outperformed all other assay strategies with maximal signal, low background, exceptional reproducibility, minimal matrix effects, and high throughput. Binding site titrations verified that an increase in total protein in the assay led to a concomitant linear increase in the amount of specifically bound [3H]STX within the range of 1–90μg total protein. Saturation binding experiments demonstrated that the binding sites were saturable and that nonspecific binding was linear. The MCE assay was unaffected by 600mM NaCl and 500mM KCl. Likewise, minimal variation of specific binding was observed between pH 5 and pH 9, but inhibition was observed below pH 5. [Copyright &y& Elsevier]

Five healthy adult female first-generation hybrid tree frogs were produced by interspecific breeding of closely related tree frogs Litoria splendida and L. caerulea in a cage containing large numbers of males and females of both species. Phylogenetic analysis of mitochondrial DNA sequences established the female parent to be L. splendida. The peptide profile of the hybrid frogs included the neuropeptide caerulein, four antibiotics of the caerin 1 family and several neuronal nitric oxide synthase inhibitors of the caerin 1 and 2 classes of peptides. The skin secretions of the hybrids contained some peptides common to only one parent, some produced by both parental species, and four peptides expressed by the hybrids but not the parental species. [ABSTRACT FROM AUTHOR]

Abstract: The skin secretion of the Dainty Green Tree Frog Litoria gracilenta contains 16 peptides, which protect the animal from predators, both large and small. A combination of negative and positive ion electrospray mass spectrometry together with Lys-C enzymic digest and Edman sequencing identifies three new wide-spectrum caerin 1 antibiotics, namely Caerin 1.17: [GLFSVLGSVAKHLLPHVAPIIAEKL-NH2], Caerin 1.18: [GLFSVLGSVAKHLLPHVVPVIAEKL-NH2], and Caerin 1.19: [GLFKVLGSVAKHLLPHVAPIIAEKL-NH2], and a narrow spectrum antibiotic Caerin 3.5: [GLWEKVKEKANELVSGIVEGVK-NH2]. [Copyright &y& Elsevier]

We report for the first time, the presence of saxitoxin (STX) in a common cephalopod, Octopus (Abdopus) sp. 5, collected from Cooke Point on the northern coastline of Western Australia. Sodium channel and saxiphilin based radio-receptor assays detected saxitoxin-like binding in octopi extracts. Further analysis by liquid chromatography-fluorescence detection (LC-FLD) identified STX as the major contributing toxin in these samples. The presence of STX was confirmed by LC-mass spectrometry and comparison of fragmentation patterns with an authentic STX standard. LC-FLD quantitation and conversion of the Octopus sp. 5 extracts revealed toxin concentrations as high as 246μg STX/100g tissue, more than three times the US, European and Australian regulatory limit for human consumption of shellfish of 80μg STX/100g tissue. There was no evidence of tetrodotoxin or other paralytic shellfish toxin derivatives. This level and distribution of STX in octopi poses a potential public health risk, particularly when routine toxin screening of wild catch is not regulated. [Copyright &y& Elsevier]

Host-defence peptides secreted from the skin glands of Australian frogs and toads, are, with a few notable exceptions, different from those produced by anurans elsewhere. This review summarizes the current knowledge of the following classes of peptide isolated and characterized from Australian anurans: neuropeptides (including smooth muscle active peptides, and peptides that inhibit the production of nitric oxide from neuronal nitric oxide synthase), antimicrobial and anticancer active peptides, antifungal peptides and antimalarial peptides. Other topics covered include sex pheromones of anurans, and the application of peptide profiling to (i) recognize particular populations of anurans of the same species and to differentiate between species, and (ii) investigate evolutionary aspects of peptide formation. [Copyright &y& Elsevier]

Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva -1 and as high as 0.2 s larva -1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species.

Sponge diseases have increased dramatically, yet the causative agents of disease outbreaks have eluded identification. We undertook a polyphasic taxonomic analysis of the only confirmed sponge pathogen and identified it as a novel strain of Pseudoalteromonas agarivorans. 16S ribosomal RNA (rRNA) and gyraseB (gyrB) gene sequences along with phenotypic characteristics demonstrated that strain NW4327 was most closely related to P. agarivorans. DNA-DNA hybridization and in silico genome comparisons established NW4327 as a novel strain of P. agarivorans. Genes associated with type IV pili, mannose-sensitive hemagglutinin pili, and curli formation were identified in NW4327. One gene cluster encoding ATP-binding cassette (ABC) transporter, HlyD and TolC, and two clusters related to the general secretion pathway indicated the presence of type I secretion system (T1SS) and type II secretion system (T2SS), respectively. A contiguous gene cluster of at least 19 genes related to type VI secretion system (T6SS) which included all 13 core genes was found. The absence of T1SS and T6SS in nonpathogenic P. agarivorans S816 established NW4327 as the virulent strain. Serine proteases and metalloproteases of the classes S8, S9, M4, M6, M48, and U32 were identified in NW4327, many of which can degrade collagen. Collagenase activity in NW4327 and its absence in the nonpathogenic P. agarivorans KMM 255(T) reinforced the invasiveness of NW4327. This is the first report unambiguously identifying a sponge pathogen and providing the first insights into the virulence genes present in any pathogenic Pseudoalteromonas genome. The investigation supports a theoretical study predicting high abundance of terrestrial virulence gene homologues in marine bacteria.

The most detailed dataset of ciguatera intensity is that produced by the South Pacific Epidemiological and Health Information Service (SPEHIS) of the Secretariat of the Pacific Community. The SPEHIS fish poisoning database has been previously analysed yielding statistically significant correlations between the Southern Oscillation Index (SOI) and ciguatera case numbers in several countries raising concerns this affliction will increase as oceans warm. Mapping of the SPEHIS records and other data hints at ciguatera not only being restricted to warm waters but that the Indo-Pacific Warm Pool, a body of water that remains hot throughout much of the year, may inhibit ciguatera prevalence. A qualitative assessment of ciguatera intensity and sea surface temperature (SST) behaviour within the EEZ of selected South Pacific nations supported the notion that ciguatera intensity was highest when SST was between an upper and lower limit. Many more climate and SST indices beyond the SOI are now available, including some that measure the abovementioned phenomenon of oceanic warm pools. Statistically significant, positive and negative cross-correlations were obtained between time series of annual ciguatera case rates from the SPEHIS dataset and the Pacific Warm Pool Index and several ENSO related indices which had been lagged for up to 2 years before the ciguatera time series. This further supports the possibility that when considering the impact of climate change on ciguatera, one has to consider two thresholds, namely waters that remain warm enough for a long enough period can lead to ciguatera and that extended periods where the water remains too hot may depress ciguatera case rates. Such a model would complicate projections of the effects of climate change upon ciguatera beyond that of a simple relationship where increased SST may cause more ciguatera., (Crown Copyright 2009. Published by Elsevier Ltd. All rights reserved.)

Saxitoxin (STX), tetrodotoxin (TTX) and their many chemical relatives are part of our daily lives. From killing people who eat seafood containing these toxins, to being valuable research tools unveiling the invisible structures of their pharmacological receptor, their global impact is beyond measure. The pharmacological receptor for these toxins is the voltage-gated sodium channel which transports Na ions between the exterior to the interior of cells. The two structurally divergent families of STX and TTX analogues bind at the same location on these Na channels to stop the flow of ions. This can affect nerves, muscles and biological senses of most animals. It is through these and other toxins that we have developed much of our fundamental understanding of the Na channel and its part in generating action potentials in excitable cells.

Molecular descriptors and their mathematical combination have been used for predictive toxicology and risk assessments of environmental pollutants and pharmaceutical leads. However, this approach has not yet been used for natural toxins and may contribute to health and environmental risk assessments of newly discovered toxins without having to undertake whole animal toxicology. To explore this approach, over 3000 descriptors were calculated for each of the 30 saxitoxins for which mouse toxicities have been reported. This dataset was reduced to only 87 descriptors by firstly eliminating descriptors that were the same for all toxins or could not be calculated for all 30 toxins, and then removing those descriptors that did not have a statistically significant linear relationship with toxicity values. From the remaining 87 descriptors, a subset of seven descriptors was identified upon which various mathematical approaches were assessed for their ability to fit the dataset both with and without leave-one-out cross-validation. K-nearest neighbours and support vector machine regression along with various combinations of these seven descriptors fit the toxicity data almost perfectly and also achieved high predictability as measured by leave-one-out cross-validation. Of these seven descriptors, five incorporated weighting by estimates of atomic polarizability and electronic states. Predicted toxicities of several saxitoxins of unknown toxicity bore similarities to the pattern of known potencies of these toxins on various isoforms of the voltage-gated sodium channel. Some of these predicted toxicity values however would not be expected if there was a direct relationship between mammalian sodium channel affinity of the saxitoxins and whole animal toxicity.

Cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2) is a potent venom component of the spider Cupiennius salei. Cupiennin 1a shows multifaceted activity. In addition to known antimicrobial and cytolytic properties, cupiennin 1a inhibits the formation of nitric oxide by neuronal nitric oxide synthase at an IC50 concentration of 1.3 +/- 0.3 microM. This is the first report of neuronal nitric oxide synthase inhibition by a component of a spider venom. The mechanism by which cupiennin 1a inhibits neuronal nitric oxide synthase involves complexation with the regulatory protein calcium calmodulin. This is demonstrated by chemical shift changes that occur in the heteronuclear single quantum coherence spectrum of 15N-labelled calcium calmodulin upon addition of cupiennin 1a. The NMR data indicate strong binding within a complex of 1 : 1 stoichiometry.

Plants using the C(4) photosynthetic pathway are highly represented among the world's worst weeds, with only 4 C(4) species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C(4) acid cycle operating as a biochemical appendage of C(3) photosynthesis, the additional enzymes involved in C(4) photosynthesis represent an attractive target for the development of weed-specific herbicides. The rate-limiting enzyme of this metabolic pathway is pyruvate orthophosphate dikinase (PPDK). PPDK, coupled with phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide-malate dehydrogenase, was used to develop a microplate-based assay to detect inhibitors of enzymes of the C(4) acid cycle. The resulting assay had a Z' factor of 0.61, making it a high-quality assay able to reliably identify active test samples. Organic extracts of 6679 marine macroscopic organisms were tested within the assay, and 343 were identified that inhibited the 3 enzyme-coupled reaction. A high confirmation rate was achieved, with 95% of these hit extracts proving active again upon retesting. Sequential addition of phosphoenolpyruvate and oxaloacetate to the assay facilitated identification of 83 extracts that specifically inhibited PPDK.

Gymnodinium catenatum is one of several dinoflagellates that produce a suite of neurotoxins called the paralytic shellfish toxins (PST), responsible for outbreaks of paralytic shellfish poisoning in temperate and tropical waters. Previous research suggested that the bacteria associated with the surface of the sexual resting stages (cyst) were important to the production of PST by G. catenatum. This study sought to characterise the cultivable bacterial diversity of seven different strains of G. catenatum that produce both high and abnormally low amounts of PST, with the long-term aim of understanding the role the bacterial flora has in bloom development and toxicity of this alga. Sixty-one bacterial isolates were cultured and phylogenetically identified as belonging to the Proteobacteria (70%), Bacteroidetes (26%) or Actinobacteria (3%). The Alphaproteobacteria were the most numerous both in terms of the number of isolates cultured (49%) and were also the most abundant type of bacteria in each G. catenatum culture. Two phenotypic (functional) traits inferred from the phylogenetic data were shown to be a common feature of the bacteria present in each G. catenatum culture: firstly, Alphaproteobacteria capable of aerobic anoxygenic photosynthesis, and secondly, Gammaproteobacteria capable of hydrocarbon utilisation and oligotrophic growth. In relation to reports of autonomous production of PST by dinoflagellate-associated bacteria, PST production by bacterial isolates was investigated, but none were shown to produce any PST-like toxins. Overall, this study has identified a number of emergent trends in the bacterial community of G. catenatum which are mirrored in the bacterial flora of other dinoflagellates, and that are likely to be of especial relevance to the population dynamics of natural and harmful algal blooms.

The paralytic shellfish poison family has been recently extended by the discovery of several analogues possessing a hydoxybenzoate moiety instead of the carbamoyl group one finds in saxitoxin, the parent molecule of this toxin family. We have investigated the potency of these new analogues on a representative isoform of the pharmacological target of these toxins, the voltage gated sodium channel. These toxins were found to have K1's in the low nanomolar range, only slightly less potent than saxitoxin. The hydroxybenzoate group may increase the lipophilicity of these toxins and improve their ability to pass through epithelia and therefore its uptake and elimination in both intoxication victims and animals that bioaccumulate paralytic shellfish toxins.

The peptide frenatin 3 is a major component of the skin secretion of the Australian giant tree frog, Litoria infrafrenata. Frenatin 3 is 22 amino acids in length, and shows neither antimicrobial nor anticancer activity. It inhibits the production of nitric oxide by the enzyme neuronal nitric oxide synthase at a micromolar concentration by binding to its regulatory protein, Ca2+ calmodulin, a protein known to recognize and bind amphipathic alpha-helices. The solution structure of frenatin 3 has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. In trifluoroethanol/water mixtures, the peptide forms an amphipathic alpha-helix over residues 1-14 while the C-terminal eight residues are more flexible and less structured. The flexible region may be responsible for the lack of antimicrobial activity. In water, frenatin 3 exhibits some alpha-helical character in its N-terminal region., (Copyright 2003 Wiley Periodicals, Inc.)

A novel microtiter plate assay was developed to determine the total peroxyl radical-trapping activity of antioxidants extracted from marine organisms by measuring the inhibition rate of dye-substrate oxidation. We compared use of dihydrorhodamine-123, dihydrofluorescein, and dichlorodihydrofluorescein as reduced substrates for oxidation by peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride. The oxidation products of these highly reactive substrates are intensely colored dyes that absorb maximally in the wavelength region, lambda(max) = 489 to 512 nm, and their concentrations were determined photometrically using a 96-well, microtiter plate reader. The microtiter plate method provides for concurrent multisample analysis with automated data storage, regression analyses, and calculation of oxidation inhibition rates. Dihydrorhodamine was selected as the preferred substrate for screening crude extracts, and typical assay results are presented. Novel lead antioxidants are selected from active extracts by chromatographic analysis with electrochemical detection.

Two neuropeptides have been isolated and identified from the secretions of the skin glands of the Stony Creek Frog Litoria lesueuri. The first of these, the known neuropeptide caerulein 1.1, is a common constituent of anuran skin secretions, and has the sequence pEQY(SO3)TGWMDF-NH2. This neuropeptide is smooth muscle active, an analgaesic more potent than morphine and is also thought to be a hormone. The second neuropeptide, a new peptide, has been named lesueurin and has the primary structure GLLDILKKVGKVA-NH2. Lesueurin shows no significant antibiotic or anticancer activity, but inhibits the formation of the ubiquitous chemical messenger nitric oxide from neuronal nitric oxide synthase (nNOS) at IC(50) (16.2 microm), and is the first amphibian peptide reported to show inhibition of nNOS. As a consequence of this activity, we have tested other peptides previously isolated from Australian amphibians for nNOS inhibition. There are three groups of peptides that inhibit nNOS (IC(50) at microm concentrations): these are (a) the citropin/aurein type peptides (of which lesueurin is a member), e.g. citropin 1.1 (GLFDVIKKVASVIGGL-NH(2)) (8.2 microm); (b) the frenatin type peptides, e.g. frenatin 3 (GLMSVLGHAVGNVLG GLFKPK-OH) (6.8 microm); and (c) the caerin 1 peptides, e.g. caerin 1.8 (GLFGVLGSIAKHLLPHVVPVIAEKL-NH(2)) (1.7 microm). From Lineweaver-Burk plots, the mechanism of inhibition is revealed as noncompetitive with respect to the nNOS substrate arginine. When the nNOS inhibition tests with the three peptides outlined above were carried out in the presence of increasing concentrations of Ca(2+) calmodulin, the inhibition dropped by approximately 50% in each case. In addition, these peptides also inhibit the activity of calcineurin, another enzyme that requires the presence of the regulatory protein Ca(2+) calmodulin. It is proposed that the amphibian peptides inhibit nNOS by interacting with Ca(2+)calmodulin, and as a consequence, blocks the attachment of this protein to the calmodulin domain of nNOS.