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Pyrodinium bahamense var compressum is a harmful dinoflagellate that produces saxitoxin, which causes paralytic shellfish poisoning (PSP) that is deadly to humans. A non-axenic culture of P. bahamense was established using f/2 media from samples collected from Sepanggar Bay, Kota Kinabalu, Sabah. Toxin analyses of cultures harvested on days 60, 120, 180, and 360 were performed using high-performance liquid chromatography with a fluorescence detector and compared with samples collected at the same location during the bloom in 2021. The highest cell toxin content was found in the bloom sample (86.2 fmole/ cell), and no toxin was detected in the culture 60 days old. In addition, cell toxin content for the P. bahamense culture was low (9.4-16.5 fmole/cell). Based on the toxin profile, P. bahamense comprises 84-98% of gonyautoxin 4. In summary, the current findings add to the existing knowledge of the toxin profiling of P. bahamense, a toxic, harmful algal bloom species, thus, leading to better toxin management. [ABSTRACT FROM AUTHOR]

Marine phycotoxins are organic compounds synthesized by some species of microalgae, which accumulate in the tissues of filter-feeder organisms such as bivalve mollusks. These toxins can cause acute intoxication episodes in humans, a severe threat to aquaculture and fisheries. In the State of Pará, Brazil, oyster farming has community, artisanal and sustainable bases, using mangroves as cultivation environment and seed banks. In small-scale production, there are often no established methods of safeguarding the health of consumers elevating the potential risks of shellfish poisoning outbreaks. Our study evaluated the presence of phycotoxins in oysters cultivated in five municipalities in the region of the Atlantic Amazon (Pará, Brazil) assessing the quality of the final product. We further evaluated the microalgae, water quality, and the spatio-temporal variation of physicochemical factors in the same area. Diatoms dominated the microalgae composition, followed by dinoflagellates, some of which are reported to be potentially toxic and producers of paralytic shellfish toxins. For the first time, we describe the occurrence of the potentially toxic dinoflagellate Ostreopsis sp. in the Amazon region. Furthermore, for the first time, toxins were detected in oyster farming in the northeast of the State of Pará, namely GTX2,3, STX, and dc-STX nevertheless, with nontoxic values. The identified toxins represent a potential threat to shellfish consumers. [ABSTRACT FROM AUTHOR]

Nucleic acids with G-quadruplex (G4) structures play an important role in physiological function, analysis and detection, clinical diagnosis and treatment, and new drug research and development. Aptamers obtained using systematic evolution of ligands via exponential enrichment (SELEX) screening technology do not always have the best affinity or binding specificity to ligands. Therefore, the establishment of a structure-oriented experimental method is of great significance. To study the potential of surface-enhanced Raman spectroscopy (SERS) in aptamer optimization, marine biotoxin gonyautoxin (GTX)1/4 and its G4 aptamer obtained using SELEX were selected. The binding site and the induced fit of the aptamer to GTX1/4 were confirmed using SERS combined with two-dimensional correlation spectroscopy. The intensity of interaction between GTX1/4 and G4 was also quantified by measuring the relative intensity of SERS bands corresponding to intramolecular hydrogen bonds. Furthermore, the interaction between GTX1/4 and optimized aptamers was analyzed. The order of intensity change in the characteristic bands of G4 aptamers was consistent with the order of affinity calculated using microscale thermophoresis and molecular dynamics simulations. SERS provides a rapid, sensitive, and economical post-SELEX optimization of aptamers. It is also a reference for future research on other nucleic acid sequences containing G4 structures. [ABSTRACT FROM AUTHOR]

Marine phycotoxins are organic compounds synthesized by some species of microalgae, which accumulate in the tissues of filter-feeder organisms such as bivalve mollusks. These toxins can cause acute intoxication episodes in humans, a severe threat to aquaculture and fisheries. In the State of Pará, Brazil, oyster farming has community, artisanal and sustainable bases, using mangroves as cultivation environment and seed banks. In small-scale production, there are often no established methods of safeguarding the health of consumers elevating the potential risks of shellfish poisoning outbreaks. Our study evaluated the presence of phycotoxins in oysters cultivated in five municipalities in the region of the Atlantic Amazon (Pará, Brazil) assessing the quality of the final product. We further evaluated the microalgae, water quality, and the spatio-temporal variation of physicochemical factors in the same area. Diatoms dominated the microalgae composition, followed by dinoflagellates, some of which are reported to be potentially toxic and producers of paralytic shellfish toxins. For the first time, we describe the occurrence of the potentially toxic dinoflagellate Ostreopsis sp. in the Amazon region. Furthermore, for the first time, toxins were detected in oyster farming in the northeast of the State of Pará, namely GTX2,3, STX, and dc-STX nevertheless, with nontoxic values. The identified toxins represent a potential threat to shellfish consumers.

Nucleic acids with G-quadruplex (G4) structures play an important role in physiological function, analysis and detection, clinical diagnosis and treatment, and new drug research and development. Aptamers obtained using systematic evolution of ligands via exponential enrichment (SELEX) screening technology do not always have the best affinity or binding specificity to ligands. Therefore, the establishment of a structure-oriented experimental method is of great significance. To study the potential of surface-enhanced Raman spectroscopy (SERS) in aptamer optimization, marine biotoxin gonyautoxin (GTX)1/4 and its G4 aptamer obtained using SELEX were selected. The binding site and the induced fit of the aptamer to GTX1/4 were confirmed using SERS combined with two-dimensional correlation spectroscopy. The intensity of interaction between GTX1/4 and G4 was also quantified by measuring the relative intensity of SERS bands corresponding to intramolecular hydrogen bonds. Furthermore, the interaction between GTX1/4 and optimized aptamers was analyzed. The order of intensity change in the characteristic bands of G4 aptamers was consistent with the order of affinity calculated using microscale thermophoresis and molecular dynamics simulations. SERS provides a rapid, sensitive, and economical post-SELEX optimization of aptamers. It is also a reference for future research on other nucleic acid sequences containing G4 structures.

This study aimed to optimize the detection conditions for surface-enhanced Raman spectroscopy (SERS) of single-stranded DNA (ssDNA) in four different buffers and explore the interaction between gonyautoxin (GTX1/4) and its aptamer, GO18. The influence of the silver colloid solution and MgSO4 concentration (0.01 M) added under four different buffered conditions on DNA SERS detection was studied to determine the optimum detection conditions. We explored the interaction between GTX1/4 and GO18 under the same conditions as those in the systematic evolution of ligands by exponential enrichment technique, using Tris-HCl as the buffer. The characteristic peaks of GO18 and its G-quadruplex were detected in four different buffer solutions. The change in peak intensity at 1656 cm−1 confirmed that the binding site between GTX1/4 and GO18 was in the G-quadruplex plane. The relative intensity of the peak at 1656 cm−1 was selected for the GTX1/4–GO18 complex (I1656/I1099) to plot the ratio of GTX1/4 in the Tris-HCl buffer condition (including 30 μL of silver colloid solution and 2 μL of MgSO4), and a linear relationship was obtained as follows: Y = 0.1867X + 1.2205 (R2 = 0.9239). This study provides a basis for subsequent application of SERS in the detection of ssDNA, as well as the binding of small toxins and aptamers.

• Centrodinium punctatum prefers warm waters and an oceanic habitat for its growth. • Toxin contents of Centrodinium punctatum do not change significantly with temperature. • A salinity of 40 can decrease the toxin contents of Centrodinium punctatum. To understand environmental effects affecting paralytic shellfish toxin production of Centrodinium punctatum, this study examined the growth responses, and toxin contents and profiles of a C. punctatum culture exposed to drastic changes of temperature (5-30 °C) and salinity (15–40). C. punctatum grew over a temperature range of 15–25 °C, with an optimum of 20 °C., and over a salinity range of 25–40, with optimum salinities of 30–35. This suggests that C. punctatum prefers relatively warm waters and an oceanic habitat for its growth and can adapt to significant changes of salinity levels. When C. punctatum was cultivated at different temperature and salinity levels, the PST profile included four major analogs (STX, neoSTX, GTX1 and GTX4, constituted >80 % of the profile), while low amounts of doSTX and traces of dc-STX and dc-GTX2 were also observed. Interestingly, though overall toxin contents did not change significantly with temperature, increases in the proportion of STX, and decreases in proportions in GTX1 and GTX4 were observed with higher temperatures. Salinity did not affect either toxin contents or profile from 25 to 35. However, the total toxin content dropped to approximately half at salinity 40, suggesting this salinity may induce metabolic changes in C. punctatum. [ABSTRACT FROM AUTHOR]

A rapid screening method is described for the determination of paralytic shellfish toxins (PST), in fresh marine vectors (bivalves and gastropods), at levels ranging from 0.05 to 5.0 mg STX-eq kg−1. PST are extracted from marine vector homogenates with acetic acid according to the Pre-COX-LC-FLD method. At the same time, the obtained extract is oxidised simultaneously in hydrogen peroxide and periodate oxidate to determine PST, non-N-hydroxylated and N-hydroxylated toxins, respectively. Then, they are analysed using a microplate fluorometer (Ex: 335 nm/Em: 405 nm). All the samples were compared with the liquid chromatography post-column oxidation method. Recoveries of PST added to fresh and processed marine vectors averaged 93.9% with a coefficient of variation of 6.1%. Both methods showed a good linear regression (r2 = 0.97). The method shows good intra- and inter-day precisions with a relative coefficient of variation of ≈ 3.8% and 5.7%, respectively. The limit of quantification of the rapid screening fluorescence method was ≈ 0.082 mg STX-eq kg−1, with ≤5% false positives. The established rapid screening fluorescence methods offer highly effective and verifiable pre-analyses of PST contamination in marine vectors and can be used for routine screening of the PST in seafood before formal identification by confirmatory methods (Pre-COX LC-FLD method, Lawrence method). [ABSTRACT FROM AUTHOR]

Paralytic shellfish poisoning is caused by saxitoxin and its analogues. The paralytic shellfish toxins (PSTs) are produced by marine dinoflagellates and can be accumulated in filter feeding shellfish, such as mussel, clam, oyster and ark shell. The worldwide regulatory limits for PSTs in shellfish are set at 80 μg STX eq./100 g meat and this is widely accepted as providing adequate public health protection. In this study, we have determined five individual PSTs (STX, GTX1, GTX2, GTX3 and GTX4) in shellfish using LC-MS/MS and assessed the human acute and chronic exposures to PSTs through shellfish consumption. Food consumption data was obtained from the Korea National Health and Nutrition Examination Survey ( KNHANES 2010–2015 ). The acute exposure using a large portion size of 88 g/day (95th percentile for consumers only) with maximum toxin level of 198.7 μg/kg was 0.30 μg/kg bw. Even though we estimated the acute exposure with a conservative manner, it was below the ARfDs (0.5 or 0.7 μg STX eq./kg bw) proposed by the international organizations, representing 43–60% of the ARfDs. The chronic exposures using mean consumption data for whole population with mean concentration of PSTs were ranged from 0.002 to 0.026 μg STX eq./kg bw/day. For consumers only, the chronic exposures were in the range of 0.012–0.128 μg STX eq./kg bw/day. [ABSTRACT FROM AUTHOR]

SUMMARY: Harmful algal bloom (HAB) is a proliferation of algae, which naturally produce biotoxins and cause harmful effects to humans, the environment and organisms associated with it. Paralytic shellfish poisoning (PSP) was reported for the first time in Kuantan Port, Pahang, Malaysia, in November 2013, followed by a second episode in August 2014. The toxicity level reported during the second event was as high as 3500 μg of STX equiv./100 g shellfish. Ten people were hospitalized with PSP symptoms after consuming contaminated shellfish. This study was conducted at Kuantan Port to identify the organisms responsible for these events. Water samples were collected monthly for a period of 12 months beginning in September 2014. HAB species were identified based on their morphology using light and fluorescence microscopes, and their classification was supported by molecular evidence based on internal transcribed spacer (ITS) sequences. Monthly cell abundance of Alexandrium tamiyavanichii was measured at four sampling stations. Toxin production by three strains isolated from the area was determined using HPLC. Our results revealed the presence of several HAB species, including the PSP‐producing species A. tamiyavanichii. The highest cell density of A. tamiyavanichii was 840 cells L−1. The presence of GTX components was detected in these strains. However, other toxin components could not be determined. This study reported, for the first time, the presence of PSP‐producing A. tamiyavanichii on the Pahang coast of east Peninsular Malaysia and confirmed that the PSP events in Kuantan Port were attributable to this species. The presence of this species further indicates that several safety measures need to be considered to safeguard public health, particularly in Pahang coastal waters. [ABSTRACT FROM AUTHOR]

Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. Symptoms can range from minor neurological dysfunction to respiratory distress and death. There are over 40 different chemical congeners of saxitoxin and its analogs, many of which are toxic and many of which have low toxicity or are non-toxic. This makes accurate toxicity assessment difficult and complicates decisions regarding whether or not shellfish are safe to consume. In this study, we describe a new antibody-based bioassay that is able to detect toxic congeners (saxitoxin, neosaxitoxin, and gonyautoxins) with little cross-reactivity with the low or non-toxic congeners (decarbamoylated or di-sulfated forms). The anti-saxitoxin antibody used in this assay detects saxitoxin and neosaxitoxin, the two most toxic congers equally well, but not the relatively highly toxic gonyautoxins. By incorporating an incubation step with L-cysteine, it is possible to convert a majority of the gonyautoxins present to saxitoxin and neosaxitoxin, which are readily detected. The assay is, therefore, capable of detecting the most toxic PSP congeners found in commercially relevant shellfish. The assay was validated against samples whose toxicity was determined using standard HPLC methods and yielded a strong linear agreement between the methods, with R2 values of 0.94−0.96. As ELISAs are rapid, inexpensive, and easy-to-use, this new commercially available PSP ELISA represents an advance in technology allowing better safety management of the seafood supply and the ability to screen large numbers of samples that can occur when monitoring is increased substantially in response to toxic bloom events

Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, Anabaena circinalis (TA04), that are primarily produced during the early and middle stages in the biosynthetic pathway to produce STX. These findings allowed us to propose a putative biosynthetic pathway responsible for STX production based on the structures of these intermediates. In the present study, we identified 12β-deoxygonyautoxin 3 (12β-deoxyGTX3), a novel STX analogue produced by A. circinalis (TA04), by comparing the retention time and MS/MS fragmentation pattern with those of synthetic standards using LC−MS. The presence of this compound in A. circinalis (TA04) is consistent with stereoselective enzymatic oxidations at C11 and C12, and 11-O-sulfation, during the late stage of STX biosynthesis, as proposed in previous studies.

Trichodesmium is an enigmatic bloom forming, non-heterocystous cyanobacterium reported most frequently in the coastal waters of India. However, the toxigenic potential of this globally significant N 2 fixing cyanobacterium has not been characterized. In this study, we report for the first time the presence of potent multi-class neurotoxins such as Anatoxin-a, Saxitoxins, Gonyautoxin and hepatotoxins like MC-LR, MC-YA from a bloom material of Trichodesmium sp. MBDU 524 collected at the Gulf of Mannar region. Toxins were determined using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis of HPLC purified aqueous and solvent fractions. Molecular phylogenetic analysis through 16S rRNA gene sequencing showed the close relationship with Trichodesmium erythraeum clade. The toxigenic potential was validated through brine shrimp toxicity assay and showed 100% mortality after 48 h of incubation. The results suggest the potential toxigenic and environmental impacts of Trichodesmium bloom sample from the Gulf of Mannar region. [ABSTRACT FROM AUTHOR]

Paralytic shellfish toxins (PSTs), including gonyautoxins and saxitoxins, are produced by multiple species of microalgae and dinoflagellates, and are bioaccumulated by shellfish and other animals. Human exposure to PSTs typically occurs through ingestion of recreationally harvested contaminated shellfish and results in nonspecific symptomology. Confirmation of exposure to PSTs has often relied on the measurement of saxitoxin, the most toxic congener; however, gonyautoxins (GTXs), the sulfated carbamate derivatives of saxitoxin, may be present in shellfish at higher concentrations. To improve identification of PST exposures, our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography method to identify GTX1-4 in human urine with tandem mass spectrometry. The reportable range varied for each analyte, with all falling within 0.899 and 250 ng/mL in urine with precision <15% and >85% accuracy as determined for all quality control samples. This new online method quantitates GTX1-4 following exposures to PSTs, supporting the work of public health authorities. [ABSTRACT FROM AUTHOR]

An analytical method using liquid chromatography-tandem mass spectrometry has been developed for the determination of 5 paralytic shellfish poisoning (PSP) toxins (saxitoxin, gonyautoxin1-4) in shellfish and tunicate. The tested parameters included extraction solvent, clean-up method, mobile phase and mass spectrometric detection parameters including multiple reaction monitoring (MRM) conditions. The PSP toxins were extracted with 0.1 N hydrochloric acid and cleaned up using C18 SPE cartridges. Chromatographic separation of the 5 PSP toxins was carried out on a TSK-gel Amide-80 column with gradient elution. The developed method was validated according to the requirements of SANCO 12571/2013. The average recoveries of the PSP toxins ranged from 82.7% to 110.7%, the intra-day precisions were in the range of 0.3–10.9%, the inter-day precisions were in the range of 0.8–10.0% and the limits of quantification (LOQs) were in the range of 25–100 μg/kg, depending on the analytes and matrices. The validated method was successfully applied for the determination of 5 PSP toxins in real shellfish and tunicate samples, including mussel, oyster, ark shell, sea squirt and styela clava. [ABSTRACT FROM AUTHOR]

Improvements in pain management techniques in the last decade have had a major impact on the practice of total knee arthroplasty (TKA). Gonyautoxin are phycotoxins, whose molecular mechanism of action is a reversible block of the voltage-gated sodium channels at the axonal level, impeding nerve impulse propagation. This study was designed to evaluate the clinical efficacy of Gonyautoxin infiltration, as a long acting pain blocker in TKA. Fifteen patients received a total dose of 40 μg of Gonyautoxin during the TKA operation. Postoperatively, all patients were given a standard painkiller protocol: 100 mg of intravenous ketoprofen and 1000 mg of oral acetaminophen every 8 hours for 3 days. The Visual Analog Scale (VAS) pain score and range of motion were recorded 12, 36, and 60 hours post-surgery. All patients reported pain of 2 or less on the VAS 12 and 36 hours post-surgery. Moreover, all scored were less than 4 at 60 hours post-surgery. All patients achieved full knee extension at all times. No side effects or adverse reactions to Gonyautoxin were detected in the follow-up period. The median hospital stay was 3 days. For the first time, this study has shown the effect of blocking the neuronal transmission of pain by locally infiltrating Gonyautoxin during TKA. All patients successfully responded to the pain control. The Gonyautoxin infiltration was safe and effective, and patients experienced pain relief without the use of opioids. [ABSTRACT FROM AUTHOR]

Gonyautoxins are potent natural neurotoxic analogues of saxitoxin. Due to their biological activity and submilligram lethal dose for man, fast and efficient methods are required for their characterization. Recent advances in ion mobility–mass spectrometry (IM-MS) showed that differentiation of isomers could be achieved using specific experimental conditions involving particular buffer gases as well as cationic species. In this work, IM-MS experiments were carried out using alkali metal ions (Met = Li + , Na + or K + ) and different buffer gases (N 2 , N 2 O and CO 2 ) to improve the differentiation of gonyautoxin isomers. The separation of [GTX+Met] + ion was achieved for GTX2/3 and GTX1/4 from their Na or K adducts. For dcGTX2/3, the ion mobility separation can only be obtained with peak to peak resolution (R p–p ) close to 1 from the [GTXs+Met−H 2 O] + species. To understand the gas phase conformation of the different diastereomers, Density Functional Theory (DFT) calculations were performed. IM separation, and collision cross-sections on [GTXs+Met] + are reported for the first time. [ABSTRACT FROM AUTHOR]

Highlights: [•] The V genes of an antibody for a shellfish toxin gonyautoxin were cloned using phage display. [•] An open sandwich ELISA was designed with the cloned antibody fragment. [•] The assay had more than three orders of magnitude working range while keeping subnanomolar sensitivity. [ABSTRACT FROM AUTHOR]

Abstract: The responses of cell growth and paralytic shellfish poison (PSP) content and composition of a strain of the marine dinoflagellate Alexandrium tamarense to the availability of nitrate, ammonium, and phosphate were tested with a gradient of N:P ratios in a semi-continuous batch culture. The N:P ratios ranged from 4 to 64 and, were prepared with either a fixed phosphorus concentration at 1.56μM-P or a fixed nitrate or ammonium concentrations at 25μM-N. These ratios were applied to study the responses of cell growth and PSP. Cell growth was correlated with the concentrations of nitrogen and phosphorous but was correlated with not high concentrations of ammonium, which depressed the growth due to ammonium toxicity. When the phosphate concentration was held at 1.56μM, the growth rate increased hyperbolically as the N:P ratio increased but decreased logarithmically at ammonium concentrations higher than 25μM. When nitrogen was held at 25μM, the growth rate decreased hyperbolically with increasing N:P ratio. The PSP consisted of C2 toxin and gonyautoxins, including GTX4, GTX3, and GTX1, in order of decreasing magnitude. The total cellular toxicities of PSP were estimated from the measurements of cellular content and the composition of C2 toxin and gonyautoxins. The cellular contents of PSP increased hyperbolically with the nitrate concentration as a result of saturated growth, and logarithmically with the ammonium concentration as a result of depressed growth (p <0.001). PSP contents decreased logarithmically with increasing phosphorus concentration (p <0.001). The cellular contents of PSP increased significantly with increasing N:P ratios regardless of whether nitrate or ammonium was used (p <0.05). The most dominant toxin was C2 (about 80%), whereas the least dominant was GTX1 (less than 8%). The highest abundance of GTX was observed at an N:P ratio of 16, which usually corresponded to the transition between the initial increase and saturated phases of growth for the nitrogen variable experiment and vice versa for the phosphate variable experiment, or corresponded to the beginning of a decline in growth due to ammonium toxicity. The present study suggests that the cellular toxicities of PSP in the present strain of the dinoflagellate A. tamarense can be estimated from the N:P ratios, ranging from 4 to 64. The high total cellular toxicities of PSP observed at high N:P ratios should be considered in addition to the conventional consideration of P-limitation for further regulation of nutrients by provincial governments to reduce the aquatic damage caused by the toxic dinoflagellates in coastal waters. [Copyright &y& Elsevier]

Outbreaks of human intoxication by toxic crabs have been reported in various parts of the world. Sporadic outbreaks of intoxication in humans and domestic animals due to ingestion of the toxic crab have especially occurred in the Ryukyu Islands, Japan. Using a screening test for toxic crabs, 1000 specimens of 72 species in the family Xanthidae were tested for toxicity and Zosimus aeneus, Atergatis floridus and Platypodia granulosa were found to be toxic. The toxin was confirmed to be identical to saxitoxin (STX). It was later confirmed that there are great individual and regional variations in toxicity and the toxin varies with exposure of the toxic crab to Paralytic Shellfish Poisoning (PSP) toxin and/or tetrodotoxin (TTX). Edible crabs 'Togekurigani' ( Telmessus acutidens) and 'Ishigani' ( Charybdis japonica) were contaminated with PSP for a short time in 2003, in Japan. The mechanism of PSP intoxication was shown to be the food chain by feeding PSP-contaminated bivalves. However, in the case of toxic xanthid crabs, which are not plankton feeders, the conclusions were uncertain. This review describes the occurrence, causative agents and intoxication mechanisms for food poisoning by toxic crabs containing PSP or TTX or other marine toxins, either alone or mixed. [ABSTRACT FROM AUTHOR]

The paralytic shellfish poisoning toxins (PSTs) were, as their name suggests, discovered as a result of human poisoning after consumption of contaminated shellfish. More recently, however, the same toxins have been found to be produced by freshwater cyanobacteria. These organisms have worldwide distribution and are common in our sources of drinking water, thus presenting another route of potential human exposure. However, the regulatory limits for PSTs in drinking water are considerably lower than in shellfish. This has increased the need to find alternatives to the mouse bioassay, which, apart from being ethically questionable, does not have a limit of detection capable of detecting the PSTs in water at the regulated concentrations. Additionally, the number of naturally occurring PSTs has grown substantially since saxitoxin was first characterised, markedly increasing the analytical challenge of this group of compounds. This paper summarises the development of chromatographic, toxicity, and molecular sensor binding methodologies for detection of the PSTs in shellfish, cyanobacteria, and water contaminated by these toxins. It then summarises the advantages and disadvantages of their use for particular applications. Finally it recommends some future requirements that will contribute to their improvement for these applications. [ABSTRACT FROM AUTHOR]

Saxitoxin and gonyautoxin 2 and 3 are among the most toxic components of the Paralytic Shellfish Poison from red tides. Being small molecules, they often require to be immobilized in order to be handled experimentally. Here is presented a methodology for covalently binding the toxins to carboxilatemodified surfaces. Both toxins were successfully bound to magnetic beads and saxitoxin was additionally bound to a modified golden surface in order to perform a surface plasmon resonance analysis. Success of binding to magnetic beads was evaluated through a standard immune-based toxin assay. Despite the different methods used for each toxin, the maximum binding yield for both toxins occurred when using concentration of 120 μM. [ABSTRACT FROM AUTHOR]

This study aimed to optimize the detection conditions for surface-enhanced Raman spectroscopy (SERS) of single-stranded DNA (ssDNA) in four different buffers and explore the interaction between gonyautoxin (GTX1/4) and its aptamer, GO18. The influence of the silver colloid solution and MgSO4 concentration (0.01 M) added under four different buffered conditions on DNA SERS detection was studied to determine the optimum detection conditions. We explored the interaction between GTX1/4 and GO18 under the same conditions as those in the systematic evolution of ligands by exponential enrichment technique, using Tris-HCl as the buffer. The characteristic peaks of GO18 and its G-quadruplex were detected in four different buffer solutions. The change in peak intensity at 1656 cm−1 confirmed that the binding site between GTX1/4 and GO18 was in the G-quadruplex plane. The relative intensity of the peak at 1656 cm−1 was selected for the GTX1/4–GO18 complex (I1656/I1099) to plot the ratio of GTX1/4 in the Tris-HCl buffer condition (including 30 μL of silver colloid solution and 2 μL of MgSO4), and a linear relationship was obtained as follows: Y = 0.1867X + 1.2205 (R2 = 0.9239). This study provides a basis for subsequent application of SERS in the detection of ssDNA, as well as the binding of small toxins and aptamers. [ABSTRACT FROM AUTHOR]

Abstract: During surveillance of the distribution of the paralytic shellfish poison (PSP)-producing dinoflagellate in 2003, 2004 and 2005 along the coastlines of the Seto Inland Sea, Hiroshima Prefecture, Japan, some species of toxic phytoplankton were isolated from the eastern coasts, Bingo-Nada, the central regions of the Seto Inland Sea. It was rather unexpectedly revealed from the basis of the morphological characteristics that they were unambiguously identified as Alexandrium tamiyavanichii and Alexandrium catenella. Two strains (ATY041106, ATY051018) of A. tamiyavanichii showed a specific toxicity of 38.7×10−6 and 111.5×10−6 MU/cell, respectively. These values seemed to be several times or much higher than that of A. catenella (AC030816, AC040614), having a specific toxicity of 4.5×10−6 and 4.1×10−6 MU/cell, respectively, isolated in the same area. From the results of HPLC-furuorometric analysis, it revealed that the toxins in ATY041106 exist almost exclusively as β-epimers (C2, GTX3, GTX4), which accounted for 72.7mol%. The toxin profiles of this strain are featured by the presence of a large amount of GTX3 (59.1mol%) and a small amount (20.6%) of C1 and 2 in comparison with the PSP compositions of A. tamarense, which is isolated as the main responsible species in Hiroshima Bay, a western part of coastal sea in Hiroshima Prefecture. On the other hand, it revealed that the toxin profiles of two strains (AC030816, AC040614) of A. catenella exist almost exclusively as β-epimers (C2, GTX3, GTX4), which accounted for 81.8 and 56.5mol%, as the same manner. The toxin profiles of these two strains are featured by the presence of a large amount of C2 (80.5 and 46.3mol%) in comparison with the PSP compositions of A. tamiyavanichii. To our knowledge, this is the first record to show the distribution and harmful influence of A. tamiyavanichii and A. catenella in Bingo-Nada in Hiroshima Prefecture. Though contamination of bivalves with these PSP-producing planktons in this area has not occurred yet so far, attention should be paid to this species as well as the other causative dinoflagellate from the stand point of public health and food hygiene. [Copyright &y& Elsevier]

Objective The use of gonyautoxin has been reported to be safe and effective in healing acute and chronic anal fissures. This study was designed to show better efficacy in healing patients with chronic anal fissure by increasing the frequency of toxin injection. Method Twenty-three chronic anal fissure patients were treated with doses of 100 units of gonyautoxin, which was intrasphincteric, infiltrated. The frequency of injection was every 4 days. Anorectal manometries were performed before and 4 min after infiltration. Results Total remissions were achieved within 7–14 days. The patients healed with a mean time of 8.2 ± 2.4 days. No relapsed were observed during the 10-month follow up. Neither faecal incontinence nor other side effects were observed. All patients showed immediate sphincter relaxation detected by clinical examination. The maximum anal resting pressures, recorded 4 min after injections decreased to 62.9 ± 27.7 mmHg, being 65.3 ± 29.6% of baseline. Immediately after infiltration, patients reported anaesthetic effect with a fall down of the postdefecatory pain. Conclusion Although, gonyautoxin anal fissures treatment recently published proved to be safe and effective, this study shows a better protocol for anal fissure treatment, showing better efficacy by shorting the healing time with better perception of healing by patients. Gonyautoxin anal sphincter infiltration proves to be safe and effective, reducing discomfort and healing time, advantageously comparing with alternative therapeutic approaches for chronic anal fissure. [ABSTRACT FROM AUTHOR]

Abstract: We briefly report here the occurrence of toxic blooms in the eutrophic reservoir Billings, São Paulo city, Brazil. Water samples were collected in May 2004, during a cyanobacterial bloom. The presence of toxic species was confirmed by using PCR amplifications of a fragment region of genes encoding microcystin synthetase-mcyB. The determination of toxins was performed by liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS analyses of the toxins from the bloom revealed variants of microcystins (MC), such as MC-LR, MC-RR and MC-YR. HPLC-FLD was used to determine the paralytic shellfish poisoning (PSP) saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 (GTX2) and 3 (GTX3). GTX2, GTX3 and NEO were detected for the first time in a natural sample from Billings reservoir. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, specifically in São Paulo. [Copyright &y& Elsevier]

Abstract: Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques—mouse bioassay, ELISA, HPLC and mass spectrometry—we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6±12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6±0 5MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry. [Copyright &y& Elsevier]

Abstract: Paralytic toxicity was detected by tetrodotoxin (TTX) bioassay in 41 specimens of four species of coral reef crabs collected from Kenting National Park, southern Taiwan from January 2003 to March 2004. The frequency of toxicity in Zosimus aeneus, Xanthias lividus, Actaeodes tomentosus (Xanthidae family) and Camposcia retusa (Majidae family) specimens was 93.3%, 100%, 46.7% and 66.7%, respectively. The average toxicity of crab specimens was 483±225 (mean±SD) mouse units (MU) for Z. aeneus, 51±32MU for X. lividus, 6±5MU for A. tomentosus, and 18±11MU for C. retusa. Each toxin of four species of crabs was extracted with acidic methanol, cleansed using a C18 solid-phase extraction column, filtered through a microcentrifuge filter and analyzed by HPLC, LC-MS and GC-MS. The toxins of Z. aeneus and X. lividus contained TTX (90%) and a small amount of gonyautoxins (10%), whereas those of A. tomentosus and C. retusa all mainly contained TTX, but no paralytic shellfish poison. Except for Z. aeneus and X. lividus, two species A. tomentosus and C. retusa were first recorded as toxic in Taiwan. [Copyright &y& Elsevier]

Abstract: We report here a rapid detection method for paralytic shellfish poisoning (PSP) toxins using a cultured neuroblastoma cell line, modified from the bioassay system previously established by Manger et al. [Manger, R.L., Leja, L.S., Lee, S.Y., Hungerford, J.M., Kirkpatrick, M.A., Yasumoto, T., Wekell, M.M., 2003. Detection of paralytic shellfish poison by rapid cell bioassay: antagonism of voltage-gated sodium channel active toxins in vitro. J. AOAC Int. 86 (3), 540–543]. In the present study, we made two major modifications to the previous method. The first is the use of maitotoxin, a marine toxin of ciguatera fish poisoning, which enables the incubation period to be reduced to 6h when applied to the microplate 15min prior to the end of the incubation. The second is the use of WST-8, a dehydrogenase detecting water-soluble tetrazolium salt for determining the target cell viability, which permits the omission of a washing step and simplifies the counting process. In addition, we attempted to reduce the required materials as much as possible. Thus, our modified method should be useful for screening the PSP-toxins from shellfish. [Copyright &y& Elsevier]

PURPOSE: The mayor symptoms of chronic anal fissure are permanent pain, intense pain during defecation that lasts for hours, blood in the stools, and sphincter cramps. It is subsequent to formation of fibrosis infiltrate that leads to an increased anal tone with poor healing tendency. This vicious circle leads to fissure recurrence and chronicity. This study was designed to show the efficacy of gonyautoxin infiltration in healing patients with anal fissures. METHODS: Gonyautoxin is a paralyzing phytotoxin produced by dinoflagellates. Fifty recruited patients received clinical examination, including proctoscopy and questionnaire to evaluate the symptoms. Anorectal manometries were performed before and after toxin injection. Doses of 100 units of gonyautoxin in a volume of 1 ml were infiltrated into both sides of the anal fissure in the internal anal sphincter. RESULTS: Total remission of acute and chronic anal fissures were achieved within 15 and 28 days respectively. Ninety-eight percent of the patients healed before 28 days with a mean time healing of 17.6 ± 9 days. Only one relapsed during 14 months of follow-up. Neither fecal incontinence nor other side effects were observed. All patients showed immediate sphincter relaxation. The maximum anal resting pressures recorded after two minutes decreased to 56.2 ± 12.5 percent of baseline. CONCLUSIONS: Gonyautoxin breaks the vicious circle of pain and spasm that leads to anal fissure. This study proposes gonyautoxin anal sphincter infiltration as safe and effective alternative therapeutic approach to conservative, surgical, and botulinum toxin therapies for anal fissures. [ABSTRACT FROM AUTHOR]

During surveillance on the toxicity of invertebrates such as bivalves inhabiting the coasts of Hiroshima Bay in 2001 and 2002, the carnivorous gastropod rapa whelk Rapana venosa collected in the estuary of Nikoh River, was found to contain toxins which showed paralytic actions in mice; the maximum toxicities (as paralytic shellfish poison, PSP) were 4.2 MU/g (May 2001) and 11.4 MU/g (April 2002). Their total toxicities were 224 and 206 MU/viscera of one specimen throughout the monitoring period. Attempts were made to identify the toxic principle in the gastropod. The viscera were extracted with 80% ethanol acidified with acetic acid, followed by defatting with dichloromethane. The aqueous layer obtained was treated with activated charcoal and then applied to a Sep-Pak C18 cartridge. The unbound toxic fraction was analyzed by high-performance liquid chromatography techniques. The gastropod toxin was rather unexpectedly identified as PSP. It was comprised of high toxic component (gonyautoxin-3; GTX3, GTX2, saxitoxin; STX) as the major components, which accounted for approximately 91 mol% of all components along with C1 and C2, which are N-sulfocarbamoyl derivatives.Judging from their toxin patterns, it is suggested that the PSP toxification mechanism of the gastropod that PSP toxins produced by phytoplankton such as Alexandrium tamarense, are transferred to and accumulated in plankton feeders such as the short-necked clam, and then transfered to this carnivorous rapa whelk R. venosa through predation. [Copyright &y& Elsevier]

Toxic dinoflagellates are important in natural ecosystems and are ofglobal economic significance because of the impact of toxic blooms onaquaculture and human health. Both the organisms and the toxins they producehave potential for biotechnology applications. We investigated autotrophicgrowth of a toxic dinoflagellate, Alexandrium minutum, inthree different high biomass culture systems, assessing growth, productivityandtoxin production. The systems used were: aerated and non-aerated2-L Erlenmeyer flasks; 0.5-L glass aerated tubes; anda 4-L laboratory scale alveolar panel photobioreactor. A range ofindicators was used to assess growth in these systems. Alexandriumminutum grew well in all culture conditions investigated, with amarked increase in both biomass and productivity in response to aeration. Thehighest cell concentration (4.9 × 105 cellsmL−1) and productivity (2.6 ×104cells mL−1d−1) was achieved inthe aerated glass culture tubes. Stable growth of A.minutum in the laboratory scale alveolar panel photobioreactor wasmaintained over a period of five months, with a maximum cell concentration of3.3 × 105 cells mL−1, a meanproductivity of 1.4 × 104 cells mL−1d−1, and toxin production of approximately 20μg L−1 d−1 with weeklyharvesting. [ABSTRACT FROM AUTHOR]

A novel sulfotransferase (O-ST), which transferred the sulfate group of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to O-22 of 11-α,β-hydroxy saxitoxin (STX) and produced GTX2 + 3, was purified to homogeneity from the cytosolic fraction of clonal-axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum GC21V. After four purification steps, including affinity chromatography and anion exchange chromatography, the enzyme was purified 500-fold and the yield was 4%. On affinity chromatography with a PAP-agarose column, O-ST was observed in the bound fraction, and N-ST specific to N-21 of STX and GTX2 + 3 was found in the unbound fraction. The molecular mass of the purified enzyme was determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be 65 kDa. Gel filtration chromatography showed a native molecular mass of 67 kDa, indicating that O-ST is a monomeric enzyme. The enzyme was optimally active at pH 6.0 and 35°C. O-ST did not require metal cations for its activity. O-ST required PAPS as the sole source of sulfate. O-ST transferred a sulfate group from PAPS to only O-22 of 11-α,β-hydroxy STX and not to N-21 of these toxins. These observations suggested that two ST, N-ST and O-ST, participate in the sulfation of PSP toxins. [ABSTRACT FROM AUTHOR]

Paralytic toxicity was detected by tetrodotoxin (TTX) bioassay in four and five specimens of new toxic xanthid crab Xanthia lividus collected from Lanyu, south-east Taiwan in April 1999 and Hsiaoliuchiu south-west Taiwan in November 1999, respectively. The average toxicity of crab specimens collected from Lanyu and Hsiaoliuchiu was 230 ± 94 (mean ± SD) mouse units (MU) and 241 ± 114 MU, respectively. The toxin was partially purified by YM-1 membrane ultrafiltration and Bio-Gel P-2 column chromatography. Electrophoresis, high-performance liquid chromatography and gas chromatography–mass spectroscopy analyses showed that the crab toxin was composed mainly of tetrodotoxin (83%), as well as a small amount of gonyautoxin 1-4 (17%). [ABSTRACT FROM AUTHOR]

SUMMARY To reconsider whether toxin profile could be used as a marker for populations from different geographical areas, clonal isolates of the toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and Alexandrium catenella (Whedon et Kofoid) Balech from Ofunato Bay (Iwate Prefecture), Atsumi Bay (Aichi Prefecture), Tanabe Bay (Wakayama Prefecture), Harima-Nada (Kagawa Prefecture), Uranouchi Bay (Kochi Prefecture), Hiroshima Bay (Hiroshima Prefecture) and Yamakawa Bay (Kagoshima Prefecture), which were identified on the basis of morphotaxonomy, immunological and molecular biological techniques, were subjected to analysis of paralytic shellfish poisoning toxins by high performance liquid chromatography-fluorometric method. All the isolates except A. tamarense OF152 from Ofunato Bay contained mainly N-sulfocarbamoyl toxins (C1 + 2) with various amounts of derivatives, and a typical north-to-south trend of decreasing toxicity was observed. In both A. tamarense and A. catenella, toxin profiles were rather constant within a geographical area and divergent among different geographical areas. The toxin profiles of A. tamarense from Harima-Nada were well conserved among different bloom years. Toxin profile showed that isolates of A. tamarense from Ofunato Bay, A. tamarense from Harima-Nada isolated in 1988 and A. catenella from Uranouchi Bay were heterogeneous. However, only two or three groups of isolates with different toxin profiles were observed in a geographical region, suggesting that several representative isolates express the genotype in a given region. These observations confirmed that toxin composition could be used as a marker to discriminate different geographical populations of these species. [ABSTRACT FROM AUTHOR]

SUMMARY:The activity of bacterial extracts to transform gonyautoxins to saxitoxins was found to be due to glutathione. This activity was also found in another thiol compound, 2-mercaptoethanol. These thiol compounds did not reduce 1-N-OH of gonyautoxin 1 and 4. Interestingly, the stable intermediate conjugates of paralytic shellfish toxins and thiol compounds were formed in the course of the reaction, which demonstrates that this is a two-step reaction consisting of formation of intermediate and degradation of the intermediate to saxitoxins. [ABSTRACT FROM AUTHOR]