The Experts below are selected from a list of 87 Experts worldwide ranked by ideXlab platform
Luis M Botana - One of the best experts on this subject based on the ideXlab platform.
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Evidence of Methylobacterium spp. and Hyphomicrobium sp. in azaspiracid toxin Contaminated Mussel tissues and assessment of the effect of azaspiracid on their growth.
Toxicon : official journal of the International Society on Toxinology, 2011Co-Authors: Judith Kouassi Nzoughet, John T. G. Hamilton, Luis M Botana, Irene R. Grant, Paulo A. Prodöhl, Christopher T. ElliottAbstract:A flagellar protein belonging to the genus Methylobacterium or Agrobacterium was previously observed by proteomics in azaspiracids (AZA) toxic Mussels. Here, we report the isolation of two different Methylobacterium spp. (NTx1 and Tx1) from non-toxic and AZA toxic Mussels, respectively, which when co-cultured with AZA exhibited significantly different growth responses - isolate Tx1 growth rate was enhanced, whereas growth of isolate NTx1 was adversely affected, compared to non-AZA supplemented control cultures. A Hyphomicrobium sp. (Tx2) also isolated from the toxic Mussels achieved greater cell density in AZAs supplemented cultures.
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Palytoxins specific and dynamic detection by in vitro microplate assay with human neuroblastoma cells
Bioscience Reports, 2008Co-Authors: Begoña Espiña, Eva Cagide, M. Carmen Louzao, Maria Martinez-fernandez, Mercedes R Vieytes, Panagiota Katikou, Adriano Villar, David Jaen, Luz Maman, Luis M BotanaAbstract:Palytoxin is one of the most complex and biggest molecules known showing extreme acute toxicity. During the last years, the dinoflagellate Ostreopsis spp, the producer organism of palytoxin, has shown a world-wide distribution thus making palytoxin an emerging toxin. Rat derived hepatocytes Clone 9 and BE (2)-M17 human neuroblastoma were used to test palytoxin or palytoxin -like compounds by measuring cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity as low as 0.2 ng/ mL. This method was useful for toxin detection in Ostreopsis extracts and naturally Contaminated Mussel samples. A comparative study testing toxic Mussel extracts by LC-MS/MS, MBA, hemolysis neutralization assay and cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.
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Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells.
Bioscience reports, 2008Co-Authors: Begoña Espiña, Eva Cagide, M. Carmen Louzao, Mercedes R Vieytes, Panagiota Katikou, Adriano Villar, David Jaen, Luz Maman, María Del Mar Fernández, Luis M BotanaAbstract:Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally Contaminated Mussel samples. A comparative study testing toxic Mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds. Abbreviations: CE, collision energy; DP, declustering potential; EMEM, Eagle's minimum essential medium; ESI, electrospray ionization; HNA, haemolysis neutralization assay; LC, liquid chromatography; LOD, limit of detection; MBA, mouse bioassay; MS/MS, tandem MS; MU, mouse unit; OA, okadaic acid; PLT, palytoxin
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Extraction and cleaning methods to detect yessotoxins in Contaminated Mussels.
Analytical biochemistry, 2007Co-Authors: Carmen Alfonso, Takeshi Yasumoto, Anna Milandri, Roberto Poletti, Mercedes R Vieytes, Amparo Alfonso, María-josé Pazos, Luis M BotanaAbstract:Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from Mussels that does not interfere with this detection method. YTX concentrations were measured in spiked Mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction-cleaning-detection methods were applied to a naturally Contaminated Mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85-90% recovery.
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Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP).
Toxicon : official journal of the International Society on Toxinology, 2000Co-Authors: F. Leira, Mercedes R Vieytes, Juan M. Vieites, Luis M BotanaAbstract:Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157-9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked Mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-Contaminated Mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.
Brian Quinn - One of the best experts on this subject based on the ideXlab platform.
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The determination of pharmaceutical residues in cooked and uncooked marine bivalves using pressurised liquid extraction, solid-phase extraction and liquid chromatography-tandem mass spectrometry.
Analytical and bioanalytical chemistry, 2013Co-Authors: Gillian L. Mceneff, Leon Barron, Brian P. Kelleher, Brett Paull, Brian QuinnAbstract:An optimised and validated method for the determination of pharmaceutical residues in blue Mussels (Mytilus spp.) is presented herein, as well as an investigation of the effect of cooking (by steaming) on any potential difference in human exposure risk. Selected pharmaceuticals included two non-steroidal anti-inflammatory drugs (diclofenac and mefenamic acid), an antibiotic (trimethoprim), an anti-epileptic (carbamazepine) and a lipid regulator (gemfibrozil). An in vivo exposure experiment was set up in the laboratory in which Mussels were exposed either directly by injection (10 ng) or daily through spiked artificial seawater (ASW) over 96 h. In liquid matrices, pharmaceutical residues were either determined using liquid chromatography–tandem mass spectrometry (LC-MS/MS) directly, or in combination with solid-phase extraction (SPE) for analyte concentration purposes. The extraction of pharmaceuticals from Mussel tissues used an additional pressurised liquid extraction step prior to SPE and LC-MS/MS. Limits of quantification of between 2 and 46 ng L−1 were achieved for extracted cooking water and ASW, between 2 and 64 μg L−1 for ASW in exposure tanks, and between 4 and 29 ng g−1 for Mussel tissue. Method linearities were achieved for pharmaceuticals in each matrix with correlation coefficients of R2 > 0.975. A selection of exposed Mussels was also cooked (via steaming) and analysed using the optimised method to observe any effect on detectable concentrations of parent pharmaceuticals present. An overall increase in pharmaceutical residues in the Contaminated Mussel tissue and cooking water was observed after cooking.
Takeshi Yasumoto - One of the best experts on this subject based on the ideXlab platform.
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Extraction and cleaning methods to detect yessotoxins in Contaminated Mussels.
Analytical biochemistry, 2007Co-Authors: Carmen Alfonso, Takeshi Yasumoto, Anna Milandri, Roberto Poletti, Mercedes R Vieytes, Amparo Alfonso, María-josé Pazos, Luis M BotanaAbstract:Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from Mussels that does not interfere with this detection method. YTX concentrations were measured in spiked Mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction-cleaning-detection methods were applied to a naturally Contaminated Mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85-90% recovery.
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a protein phosphatase 2a inhibition assay for a fast and sensitive assessment of okadaic acid contamination in Mussels
Toxicon, 1996Co-Authors: Aurelia Tubaro, Chiara Florio, E Luxich, R Della Loggia, Silvio Sosa, Takeshi YasumotoAbstract:The specific inhibitory activity exerted by okadaic acid on protein phosphatase 2A was used to assess the presence of okadaic acid in Mussels, using a commercially available protein phosphatase 2A preparation. Under the conditions used, okadaic acid inhibits the enzymatic activity dose-dependently, with an IC50 = 0.26 ng/ml (0.32 nM). The assay is accurate and reproducible. Okadaic acid was detected in concentrations as low as 0.063 ng/ml in aqueous solutions and 2 ng/g in Mussel digestive glands. Thirty naturally Contaminated Mussel samples were submitted to the protein phosphatase 2A inhibition assay as well as to an ELISA assay and to a MTT cytotoxicity assay, with similar results. The proposed assay is sensitive, rapid and does not require expensive equipment. These characteristics make it a good candidate for employment in the routine assessment of okadaic acid shellfish contamination.
Paula Vanninen - One of the best experts on this subject based on the ideXlab platform.
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optimization of sample preparation for the identification and quantification of saxitoxin in proficiency test Mussel sample using liquid chromatography tandem mass spectrometry
Toxins, 2015Co-Authors: Kirsi Harju, Marjaleena Rapinoja, Marcandre Avondet, Werner Arnold, Martin Schar, Stephen Burrell, Werner Luginbuhl, Paula VanninenAbstract:Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in Mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of Mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized Mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally Contaminated Mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic Mussel extracts. The results were within the z-score of ±1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD).
Mercedes R Vieytes - One of the best experts on this subject based on the ideXlab platform.
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Palytoxins specific and dynamic detection by in vitro microplate assay with human neuroblastoma cells
Bioscience Reports, 2008Co-Authors: Begoña Espiña, Eva Cagide, M. Carmen Louzao, Maria Martinez-fernandez, Mercedes R Vieytes, Panagiota Katikou, Adriano Villar, David Jaen, Luz Maman, Luis M BotanaAbstract:Palytoxin is one of the most complex and biggest molecules known showing extreme acute toxicity. During the last years, the dinoflagellate Ostreopsis spp, the producer organism of palytoxin, has shown a world-wide distribution thus making palytoxin an emerging toxin. Rat derived hepatocytes Clone 9 and BE (2)-M17 human neuroblastoma were used to test palytoxin or palytoxin -like compounds by measuring cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity as low as 0.2 ng/ mL. This method was useful for toxin detection in Ostreopsis extracts and naturally Contaminated Mussel samples. A comparative study testing toxic Mussel extracts by LC-MS/MS, MBA, hemolysis neutralization assay and cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.
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Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells.
Bioscience reports, 2008Co-Authors: Begoña Espiña, Eva Cagide, M. Carmen Louzao, Mercedes R Vieytes, Panagiota Katikou, Adriano Villar, David Jaen, Luz Maman, María Del Mar Fernández, Luis M BotanaAbstract:Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally Contaminated Mussel samples. A comparative study testing toxic Mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds. Abbreviations: CE, collision energy; DP, declustering potential; EMEM, Eagle's minimum essential medium; ESI, electrospray ionization; HNA, haemolysis neutralization assay; LC, liquid chromatography; LOD, limit of detection; MBA, mouse bioassay; MS/MS, tandem MS; MU, mouse unit; OA, okadaic acid; PLT, palytoxin
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Extraction and cleaning methods to detect yessotoxins in Contaminated Mussels.
Analytical biochemistry, 2007Co-Authors: Carmen Alfonso, Takeshi Yasumoto, Anna Milandri, Roberto Poletti, Mercedes R Vieytes, Amparo Alfonso, María-josé Pazos, Luis M BotanaAbstract:Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from Mussels that does not interfere with this detection method. YTX concentrations were measured in spiked Mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction-cleaning-detection methods were applied to a naturally Contaminated Mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85-90% recovery.
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Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP).
Toxicon : official journal of the International Society on Toxinology, 2000Co-Authors: F. Leira, Mercedes R Vieytes, Juan M. Vieites, Luis M BotanaAbstract:Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157-9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked Mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-Contaminated Mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.