Influence of Different Shellfish Matrices on the Separation of PSP Toxins Using a Postcolumn Oxidation Liquid Chromatography Method

The separation of PSP toxins using liquid chromatography with a post-column oxidation fluorescence detection method was performed with different matrices. The separation of PSP toxins depends on several factors, and it is crucial to take into account the presence of interfering matrix peaks to produce a good separation. The matrix peaks are not always the same, which is a significant issue when it comes to producing good, reliable results regarding resolution and toxicity information. Different real shellfish matrices (mussel, scallop, clam and oyster) were studied, and it was seen that the interference is not the same for each individual matrix. It also depends on the species, sampling location and the date of collection. It was proposed that separation should be accomplished taking into account the type of matrix, as well as the concentration of heptane sulfonate in both solvents, since the mobile phase varies regarding the matrix. Scallop and oyster matrices needed a decrease in the concentration of heptane sulfonate to separate GTX4 from matrix peaks, as well as dcGTX3 for oysters, with a concentration of 6.5 mM for solvent A and 6.25 mM for solvent B. For mussel and clam matrices, interfering peaks are not as large as they are in the other group, and the heptane sulfonate concentration was 8.25 mM for both solvents. Also, for scallops and oysters, matrix interferences depend not only on the sampling site but also on the date of collection as well as the species; for mussels and clams, differences are noted only when the sampling site variesThe research leading to these results received funding from the following FEDER cofunded-grants: CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01 and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX; from CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD; and from the European Union’s Seventh Framework Programme managed by REA – Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEAS

Determination of Gonyautoxin-4 in Echinoderms and Gastropod Matrices by Conversion to Neosaxitoxin Using 2-Mercaptoethanol and Post-Column Oxidation Liquid Chromatography with Fluorescence Detection

Paralytic Shellfish Toxin blooms are common worldwide, which makes their monitoring crucial in the prevention of poisoning incidents. These toxins can be monitored by a variety of techniques, including mouse bioassay, receptor binding assay, and liquid chromatography with either mass spectrometric or pre- or post-column fluorescence detection. The post-column oxidation liquid chromatography with fluorescence detection method, used routinely in our laboratory, has been shown to be a reliable method for monitoring paralytic shellfish toxins in mussel, scallop, oyster and clam species. However, due to its high sensitivity to naturally fluorescent matrix interferences, when working with unconventional matrices, there may be problems in identifying toxins because of naturally fluorescent interferences that co-elute with the toxin peaks. This can lead to erroneous identification. In this study, in order to overcome this challenge in echinoderm and gastropod matrices, we optimized the conversion of Gonyautoxins 1 and 4 to Neosaxitoxin with 2-mercaptoethanol. We present a new and less time-consuming method with a good recovery (82.2%, RSD 1.1%, n = 3), requiring only a single reaction stepThis research was partially funded by the Portuguese Fundation of Science and Technology (FCT) project UID/Multi/04423/2013 and by the projects MARBIOTECH (reference NORTE-07-0124-FEDER-000047) within the Scientific Resaerch and Technological Development (SR&TD) Integrated Program. MARVALOR—Building research and innovation capacity for improved management and valorizationof marine resources, supported by the Programa Operacional Regional do Norte (ON.2-O Novo Norte) and NOVOMAR (reference 0687-NOVOMAR-1-P), supported by the European Regional Development Fund. Marisa Silva also acknowledges FCT for the grant SFRH/BD/73269/2010. The spanish research leading to these results has received funding from the following European Fund for Economic and Regional Development (FEDER) cofunded-grants. From Centro para el Desarrollo Tecnológico Industrial (CDTI) and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under India&Spain Innovating Program (ISIP) Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEAS

Liquid Chromatography with a Fluorimetric Detection Method for Analysis of Paralytic Shellfish Toxins and Tetrodotoxin Based on a Porous Graphitic Carbon Column

The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, Xunta de Galicia Axencia Galega de Innovación, ITC-20133020 SINTOX, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEAS

Yessotoxin, a promising therapeutic tool

Yessotoxin (YTX) is a polyether compound produced by dinoflagellates and accumulated in filter feeding shellfish. No records about human intoxications induced by this compound have been published, however it is considered a toxin. Modifications in second messenger levels, protein levels, immune cells, cytoskeleton or activation of different cellular death types have been published as consequence of YTX exposure. This review summarizes the main intracellular pathways modulated by YTX and their pharmacological and therapeutic implicationsThe research leading to these results has received funding from the following FEDER cofunded grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R; Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016; CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD; and the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 312184 PHARMASEAS

How Safe Is Safe for Marine Toxins Monitoring?

Current regulation for marine toxins requires a monitoring method based on mass spectrometric analysis. This method is pre-targeted, hence after searching for pre-assigned masses, it identifies those compounds that were pre-defined with available calibrants. Therefore, the scope for detecting novel toxins which are not included in the monitoring protocol are very limited. In addition to this, there is a poor comprehension of the toxicity of some marine toxin groups. Also, the validity of the current approach is questioned by the lack of sufficient calibrants, and by the insufficient coverage by current legislation of the toxins reported to be present in shellfish. As an example, tetrodotoxin, palytoxin analogs, or cyclic imines are mentioned as indicators of gaps in the system that require a solid comprehension to assure consumers are protectedThe research leading to these results has received funding from the following FEDER cofunded-grants. From Centro Desarrollo Tecnológico e Industrial (CDTI), supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 312184 PHARMASEAS

LC–MS/MS Analysis of the Emerging Toxin Pinnatoxin-G and High Levels of Esterified OA Group Toxins in Galician Commercial Mussels

The occurrence of marine harmful algae is increasing worldwide and, therefore, the accumulation of lipophilic marine toxins from harmful phytoplankton represents a food safety threat in the shellfish industry. Galicia, which is a commercially important EU producer of edible bivalve mollusk have been subjected to recurring cases of mussel farm closures, in the last decades. This work aimed to study the toxic profile of commercial mussels (Mytilus galloprovincialis) in order to establish a potential risk when ingested. For this, a total of 41 samples of mussels farmed in 3 Rías (Ares-Sada, Arousa, and Pontevedra) and purchased in 5 local markets were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS). Chromatograms showed the presence of okadaic acid (OA), dinophysistoxin-2 (DTX-2), pectenotoxin-2 (PTX-2), azaspiracid-2 (AZA-2), and the emerging toxins 13-desmethyl spirolide C (SPX-13), and pinnatoxin-G (PnTX-G). Quantification of each toxin was determined using their own standard calibration in the range 0.1%–50 ng/mL (R2 > 0.99) and by considering the toxin recovery (62–110%) and the matrix correction (33–211%). Data showed that OA and DTX-2 (especially in the form of esters) are the main risk in Galician mollusks, which was detected in 38 samples (93%) and 3 of them exceeded the legal limit (160 µg/kg), followed by SPX-13 that was detected in 19 samples (46%) in quantities of up to 28.9 µg/kg. Analysis from PTX-2, AZA-2, and PnTX-G showed smaller amounts. Fifteen samples (37%) were positive for PTX-2 (0.7–2.9 µg/kg), 12 samples (29%) for AZA-2 (0.1–1.8 µg/kg), and PnTX-G was detected in 5 mussel samples (12%) (0.4 µg/kg–0.9 µg/kg). This is the first time Galician mollusk was contaminated with PnTX-G. Despite results indicating that this toxin was not a potential risk through the mussel ingestion, it should be considered in the shellfish safety monitoring programs through the LC–MS/MS methods.This research has received funding from the following FEDER co-funded grants. From Conselleria de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830, RTC-2016-5507-2, and ITC-20161072. From the European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX. This work was also supported by the program “Juan de la Cierva 2016” from the Spanish Government. Paz Otero is recipient of a Postdoctoral Funding (Ref. IJCI-2016-27774)S

First Detection of Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the Dinoflagellate Prorocentrum minimum

During official shellfish control for the presence of marine biotoxins in Greece in year 2012, a series of unexplained positive mouse bioassays (MBA) for lipophilic toxins with nervous symptomatology prior to mice death was observed in mussels from Vistonikos Bay–Lagos, Rodopi. This atypical toxicity coincided with (a) absence or low levels of regulated and some non-regulated toxins in mussels and (b) the simultaneous presence of the potentially toxic microalgal species Prorocentrum minimum at levels up to 1.89 × 103 cells/L in the area’s seawater. Further analyses by different MBA protocols indicated that the unknown toxin was hydrophilic, whereas UPLC-MS/MS analyses revealed the presence of tetrodotoxins (TTXs) at levels up to 222.9 μg/kg. Reviewing of official control data from previous years (2006–2012) identified a number of sample cases with atypical positive to asymptomatic negative MBAs for lipophilic toxins in different Greek production areas, coinciding with periods of P. minimum blooms. UPLC-MS/MS analysis of retained sub-samples from these cases revealed that TTXs were already present in Greek shellfish since 2006, in concentrations ranging between 61.0 and 194.7 μg/kg. To our knowledge, this is the earliest reported detection of TTXs in European bivalve shellfish, while it is also the first work to indicate a possible link between presence of the toxic dinoflagellate P. minimum in seawater and that of TTXs in bivalves. Confirmed presence of TTX, a very heat-stable toxin, in filter-feeding mollusks of the Mediterranean Sea, even at lower levels to those inducing symptomatology to humans, indicates that this emerging risk should be seriously taken into account by the EU to protect the health of shellfish consumersThe research leading to USC results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01 and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX. In addition from the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 315285 CIGUATOOLS and 312184 PHARMASEA. Inés Rodriguez is supported by a fellowship from Subprograma de Formación de Personal Investigador (AGL2012-40185-CO2-01), Spain. In depth investigation of the toxic episodes leading to the results and publication of the present work was undertaken by the Greek National Reference Laboratory of Marine Biotoxins (NRLMB) to fulfill the requirements of EU Regulation 178/2002/EC (Articles 6 and 7) regarding risk analysis and communication and scientific information needed for risk assessment and EU Regulation 882/2004/EC (article 7) with regard to transparency and information to the public. Collaboration of all the staff of the NRLMB is greatly appreciated. Thanks are also expressed to all the Greek regional veterinary services for their contribution to the shellfish samplings and for provision of the seawater analyses results for the presence of potentially toxic microalgae. The use of cell counts’ data within the period 2006–2009 and 2012 regarding P. minimum presence in seawater, derived from the Greek “National Programme for Monitoring of Bivalve Molluscs’ Production Areas for the presence of Marine Biotoxins” and conducted by the Laboratory Unit of Toxic Marine Microalgae (LUTMM), Department of Biology, Aristotle University of Thessaloniki (scientific coordinator: G. Nikolaidis (until February 2010) and M. Arsenakis (March 2010–to date)), as well as the restrictions of LUTMM regarding the use of data produced within 2013–2015 due to contract terms and ISO 17025 requirements are acknowledgedS

Cyclophilins A, B, and C Role in Human T Lymphocytes Upon Inflammatory Conditions

Cyclophilins (Cyps) are a group of peptidyl-prolyl cis/trans isomerases that play crucial roles in regulatory mechanisms of cellular physiology and pathology in several inflammatory conditions. Their receptor, CD147, also participates in the development and progression of the inflammatory response. Nevertheless, the main function of Cyps and their receptor are yet to be deciphered. The release of CypA and the expression of the CD147 receptor in activated T lymphocytes were already described, however, no data are available about other Cyps in these cells. Therefore, in the present work intra and extracellular CypA, B and C levels were measured followed by induced inflammatory conditions. After activation of T lymphocytes by incubation with concanavalin A, both intra and extracellular Cyps levels and the CD147 membrane receptor expression were increased leading to cell migration towards circulating CypA and CypB as chemoattractants. When CypA was modulated by natural and synthetic compounds, the inflammatory cascade was avoided including T cell migration. Our results strengthen the relationship between CypA, B, and C, their receptor, and the inflammatory process in human T lymphocytes, associating CypC with these cells for the first timeThe research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, Spain, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, Spain, ISCIII/PI19/01248, ISCIII/PI19/00879. In European Union, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX. SG was supported by a fellowship from FIDIS, SpainS

Biological Activities of Cyclic and Acyclic B-Type Laxaphycins in SH-SY5Y Human Neuroblastoma Cells

Laxaphycins are a family of non-ribosomal lipopeptides that have been isolated from several cyanobacteria. Some of these compounds have presented cytotoxic activities, but their mechanism of action is poorly understood. In this work, the already described laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria Anabaena torulosa. Moreover, two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycins B and B3, were purified from the herviborous gastropod Stylocheilus striatus, with this being the first description of biotransformed laxaphycins. The structure of these new compounds was elucidated, together with the absolute configuration of acyclolaxaphycins B and B3. The bioactivities of the six peptides were determined in SH-SY5Y human neuroblastoma cells. Laxaphycins B and B3 were cytotoxic (IC50: 1.8 and 0.8 µM, respectively) through the induction of apoptosis. In comparison, acyclic laxaphycins did not show cytotoxicity but affected mitochondrial functioning, so their effect on autophagy-related protein expression was analyzed, finding that acyclic peptides affected this process by increasing AMPK phosphorylation and inhibiting mTOR. This work confirms the pro-apoptotic properties of cyclic laxaphycins B and is the first report indicating the effects on autophagy of their acyclic analogs. Moreover, gastropod-derived compounds presented ring opening and amino-acids deletion, a biotransformation that had not been previously describedThe research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2, ITC-20161072. From European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOXS

Evaluation of the impact of mild steaming and heat treatment on the concentration of okadaic acid, dinophysistoxin-2 and dinophysistoxin-3 in mussels

This study explores the effect of laboratory and industrial steaming on mussels with toxin concentrations above and below the legal limit. We used mild conditions for steaming, 100 ˝C for 5 min in industrial processing, and up to 20 min in small-scale laboratory steaming. Also, we studied the effect of heat on the toxin concentration of mussels obtained from two different locations and the effect of heat on the levels of dinophysistoxins 3 (DTX3) in both the mussel matrix and in pure form (7-O-palmitoyl okadaic ester and 7-O-palmytoleyl okadaic ester). The results show that the loss of water due to steaming was very small with a maximum of 9.5%, that the toxin content remained unchanged with no concentration effect or increase in toxicity, and that dinophysistoxins 3 was hydrolyzed or degraded to a certain extent under heat treatment. The use of liquid-certified matrix showed a 55% decrease of dinophysistoxins 3 after 10 min steaming, and a 50% reduction in total toxicity after treatment with an autoclave (121 ˝C for 20 min)This work could not have been done without the kind collaboration of Pescados Marcelino. The research leading to these results has received funding from the following FEDER cofunded grants from CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, Xunta de Galicia Axencia Galega de Innovación, ITC-20133020 SINTOX, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016; from CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD; from the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 312184 PHARMASEAS