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The dinoflagellate Gymnodinium catenatum is considered the primary cause of recurrent paralytic shellfish toxins (PSTs) in shellfish on the Moroccan Mediterranean coasts. The impacts of key environmental factors on the growth, cell yield, cell size and PST content of G. catenatum were determined. Results indicated that increasing salinity from 32 to 39 and nitrate concentrations from 441 μM to 1764 μM did not significantly (ANOVA, P-value >0.63) modify the growth rate of the studied species. Gymnodinium catenatum exhibited the highest growth rate at 24 °C. Cells arrested their division at 15 °C and at ammonium concentration above 441 μM, suggesting that this nitrogen form is toxic for G. catenatum. Furthermore, G. catenatum was unable to assimilate urea as a nitrogen source. In G. catenatum cells, eight analogues of saxitoxin were detected, belonging to the N-sulfocarbamoyl (C1-4, B1 and B2) and decarbamoyl (dc-GTX2/3) toxins. C-toxins contributed 92 % to 98 % of the molar composition of the PSTs. During the exponential growth, C2 tended to dominate, while C3 prevailed during the stationary phase. Toxin content per cell (ranging from 5.5 pg STXeq.cell -1 to 22.4 pg STXeq.cell -1 ) increased during the stationary growth phase. Cell toxin content increased with the concentrations of nitrate, ranging from 12.1 pg STXeq.cell -1 at 441 μM to 22.4 pg STXeq.cell -1 at 1764 μM during the stationary growth phase. The toxin content of G. catenatum showed the highest values measured at the highest tested temperatures, especially during the stationary phase, where toxicity reached 17.8 pg STXeq.cell -1 and 16.4 pg STXeq.cell -1 at 24 °C and 29 °C, respectively. The results can help understand the fluctuations in the growth and PST content of G. catenatum in its habitat in response to changing environmental variables in the Mediterranean Sea when exposed to increases in warming pressure and eutrophication., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Consumption of seafood contaminated by phycotoxins produced by harmful algae is a major issue in human public health. Harmful algal blooms are driven by a multitude of environmental variables; therefore predicting human dietary exposure to phycotoxins based on these variables is a promising approach in health risk management. In this study, we attempted to predict the human health risks associated with Vulcanodinium rugosum and its neurotoxins, pinnatoxins (PnTXs), which have been regularly found in Mediterranean lagoons since their identification in 2011. Based on environmental variables collected over 1 year in four Mediterranean lagoons, we developed linear mixed models to predict the presence of V. rugosum and PnTX G contamination of mussels. We found that the occurrence of V. rugosum was significantly associated with seawater temperature. PnTX G contamination of mussels was highest in summer but persisted throughout the year. This contamination was significantly associated with seawater temperature and the presence of V. rugosum with a time lag, but not with dissolved PnTX G in seawater. By using the contamination model predictions and their potential variability/uncertainty, we calculated the human acute dietary exposures throughout the year and predicted that 25% of people who consume mussels could exceed the provisional acute benchmark value during the warmest periods. We suggest specific recommendations to monitor V. rugosum and PnTX G., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Vulcanodinium rugosum is an emerging benthopelagic neuro-toxic dinoflagellate species responsible for seasonal Pinnatoxins and Portimines contaminations of shellfish and marine animals. This species is challenging to detect in the environment, as it is present in low abundance and difficult to be identified using light microscopy. In this work, we developed a method using artificial substrates coupled with qPCR (AS-qPCR) to detect V. rugosum in a marine environment. This sensitive, specific and easy-to-standardize alternative to current techniques does not require specialized expertise in taxonomy. After determining the limits and specificity of the qPCR, we searched for the presence of V. rugosum in four French Mediterranean lagoons using artificial substrates collected every two weeks for one year. The AS-qPCR method revealed its occurrences in summer 2021 in every studied lagoon and detected cells in more samples than light microscopy. As V. rugosum development induces shellfish contamination even at low microalga densities, the AS-qPCR method is accurate and relevant for monitoring V. rugosum in a marine environment.

Mediterranean waters have undergone environmental changes during the last decades leading to various modifications of the structure of phytoplankton populations, especially Harmful Algal Blooms (HABs) species. Monitoring of the potentially toxic phytoplankton species was carried out biweekly in the western Mediterranean coast of Morocco from March 2018 to March 2019. Lipophilic Shellfish Toxins (LSTs) using LC-MS/MS and Domoic Acid (DA) using HPLC-UV were measured in the exploited mollusks, the cockle Acanthocardia tuberculata and the smooth clam Callista chione. We also determined the prevailing environmental factors in four surveyed sites (M'diq bay, Martil, Kaa Asras, and Djawn) selected to cover a variety of coastal ecosystems. Results showed that Pseudo-nitzschia spp. a DA producer species, was abundant with a pick of 50 × 10 3  cells l -1 on October 2018 in Djawn. Dinophysis caudata was the dominate Dinophysis species and showed a maximum density of 2200 cells l -1 on July in Djawn. Prorocentrum lima, an epibenthic dinoflagellate, appeared rarely in the water column with densities <80 cells l -1 . Gonyaulax spinifera and Protoceratium reticulatum were found occasionally with a maximum density of 160 cells l -1 . Karenia selliformis was detected only five times (<80 cells l -1 ) throughout the survey period. LC-MS/MS analyses revealed the presence of OA/DTX3, PTX-2, PTX-2 sa, and PTX-2 sa epi in the cockle at concentrations of up to 44.81 (OA/DTX-3+PTXs) ng g -1 meat. GYM-A was detected in the clam at concentrations of up to 4.22 ng g -1 meat. For the first time, AZAs and YTXs were detected in the southwestern Mediterranean with maximum values of 2.49 and 10.93 ng g -1 meat of cockle, respectively. DA was detected in moderate concentrations not exceeding 5.65 μg g -1 in both mollusks. Results showed that the observed toxic algae in the water column were responsible from the analysed toxins in the mollusks. It is likely that the southwestern Mediterranean waters could see the development of emergent species producing potent toxins (YTXs, AZAs, GYM-A). These dinoflagellates have to be isolated, ribotyped, and their toxin profiles determined., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pinnatoxins (PnTX) and Portimines (Prtn), two toxins produced by the benthic dinoflagellate Vulcanodinium rugosum , are known to be lethal to mice after intraperitoneal or oral administration. They are also known to accumulate in shellfish such as mussels and clams, but their effect on fish and the upper food chain remains unknown. In this work, juveniles of the fish Liza ramada (Mullet) were exposed to a strain of V. rugosum producing PnTX G and Prtn A. The fishes' viability and contamination were recorded at times interval. Results showed that L. ramada juveniles were able to feed on V. rugosum and that their tissues could be contaminated by PnTX G and Prtn A without impact on fish viability. Furthermore, the microalgae temporary cysts survived and germinated after fish gut passage. This study showed the potential of L. ramada to transfer PnTX and Prtn toxins to the upper food chain and to disseminate V. rugosum in environment.

Context: In June 2019, a paralytic shellfish poisoning (PSP) case related to the consumption of mussels contaminated by saxitoxins at a concentration below the regulatory threshold came to the attention of the French Agency for Food, Environmental and Occupational Health and Safety (ANSES). This pointed to probable undetected human cases of poisoning by neurotoxic phycotoxins., Methods: We conducted a retrospective study of poisoning cases by bivalve shellfish (oysters, mussels and scallops) recorded by the French Poison Control Centres (PCC) from 2012 to 2019. All medical records were reviewed by a toxicologist.Cases that could be related to neurotoxic phycotoxins were selected and described. Diagnosis was based on symptoms compatible with ingestion of contaminated shellfish and on contamination data for the shellfish production area (analysed by the French Research Institute for Exploitation of the Sea, Ifremer), or notifications to the European Rapid Alert System for Food and Feed when the origin of the shellfish was known., Results: Among the 619 shellfish poisoning cases recorded by the PCCs from 2012 to 2019, 22% ( n  = 134) had reported at least one neurological symptom (headache, dizziness or paraesthesia). Review of medical records for the 134 patients led to suspicion of 14 cases of PSP and one case of amnesic shellfish poisoning. Five patients experienced persistent neurological symptoms. Marine toxins were not tested for in the blood or urine of these patients., Conclusion: This retrospective identification of cases strongly suspected of being related to neurotoxic phycotoxins led ANSES, PCCs and Ifremer to develop a specific questionnaire and to recommend actions to take when neurological symptoms related to shellfish consumption are reported to a PCC. Daily monitoring of shellfish poisoning cases registered in the national PCCs database was also implemented in order to rapidly detect any suspicious cases, alert the competent authorities, and warn the general population.

French Mediterranean lagoons are frequently subject to shellfish contamination by Diarrheic Shellfish Toxins (DSTs) and Paralytic Shellfish Toxins (PSTs). To predict the effect of various environmental factors (temperature, salinity and turbidity) on the abundance of the major toxins producing genera, Dinophysis and Alexandrium, and the link with shellfish contamination, we analysed a 10-year dataset collected from 2010 to 2019 in two major shellfish farming lagoons, Thau and Leucate, using two methods: decision trees and Zero Inflated Negative Binomial (ZINB) linear regression models. Analysis of these decision trees revealed that the highest risk of Dinophysis bloom events occurred at temperature <16.3°C and salinity <27.8, and of Alexandrium at temperature ranging from 10.4 to 21.5°C and salinity >39.2. The highest risk of shellfish contaminations by DSTs and PSTs occurred during the set of conditions associated with high risk of bloom events. Linear regression prediction enables us to understand whether temperature and salinity influence the presence of Alexandrium and affect its abundance. However, Dinophysis linear regression could not be validated due to overdispersion issues. This work demonstrates the tools which could help sanitary management of shellfish rearing areas., (Copyright © 2022. Published by Elsevier B.V.)

In recent decades, more than 130 potentially toxic metabolites originating from dinoflagellate species belonging to the genus Karenia or metabolized by marine organisms have been described. These metabolites include the well-known and large group of brevetoxins (BTXs), responsible for foodborne neurotoxic shellfish poisoning (NSP) and airborne respiratory symptoms in humans. Karenia spp. also produce brevenal, brevisamide and metabolites belonging to the hemi-brevetoxin, brevisin, tamulamide, gymnocin, gymnodimine, brevisulcenal and brevisulcatic acid groups. In this review, we summarize the available knowledge in the literature since 1977 on these various identified metabolites, whether they are produced directly by the producer organisms or biotransformed in marine organisms. Their structures and physicochemical properties are presented and discussed. Among future avenues of research, we highlight the need for more toxin occurrence data with analytical techniques, which can specifically determine the analogs present in samples. New metabolites have yet to be fully described, especially the groups of metabolites discovered in the last two decades (e.g tamulamides). Lastly, this work clarifies the different nomenclatures used in the literature and should help to harmonize practices in the future.

Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum ) were surveyed in six French production areas over 2.5-3 month periods (2018-2019). Concentrations of TTX ranged from 'not detected' to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex.

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis , but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.

Alexandrium minutum and Alexandrium pacificum are representatives of the dinoflagellate genus that regularly proliferate on the French coasts and other global coastlines. These harmful species may threaten shellfish harvest and human health due to their ability to synthesize neurotoxic alkaloids of the saxitoxin group. However, some dinoflagellates such as A. minutum, and as reported here A. pacificum as well, may also have a beneficial impact on the environment by producing dimethylsulfoniopropionate-DMSP, the precursor of dimethylsulfur-DMS and sulfate aerosols involved in climate balance. However, environmental conditions might influence Alexandrium physiology towards the production of harmful or environmentally friendly compounds. After assessing the influence of two salinity regimes (33 and 38) relative to each species origin (Atlantic French coast and Mediterranean Lagoon respectively), it appears that DMSP and toxin content was variable between the three experimented strains and that higher salinity disadvantages toxin production and tends to favor the production of the osmolytes DMSP and glycine betaine. Hence, this key metabolite production is strain and species-dependent and is influenced by environmental conditions of salinity which in turn, can diversely affect the environment. Widespread coastal blooms of A. minutum and A. pacificum, although being a risk for seafood contamination with toxins, are also a DMSP and DMS source that potentially contribute to the ecosystem structuration and climate. Regarding recent advances in DMSP biosynthesis pathway, 3 dsyB homologs were found in A. minutum but no homolog of the diatom sequence TpMMT., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This is the first study on the dinoflagellate cysts in Algerian waters and in Mellah Lagoon (South Western Mediterranean), located within a protected reserve. In total, 42 species of dinocysts belonging to 7 orders, 12 families and 23 genera, were identified in the 26 superficial sediment samples from Mellah Lagoon. The distribution of dinocysts in the sediment of this lagoon is heterogeneous. Indeed, their abundance oscillates between 1 and 315 cysts g -1 dry sediment (DS). Cyst morphotype assemblages were dominated by a few numbers of species: Alexandrium minutum (15.87%), Gonyaulax verior (9.81%), Protoperidinium spp. (7.74%), Alexandrium affine (7.05%), Scrippsiella trochoidea (6.67%), and Alexandrium pseudogonyaulax (6.19%). There is a positive correlation between the density of cysts and the depth (r = 0.61; p < 0.05), organic matter (r = 0.70; p < 0.05), water content (r = 0.71; p < 0.05), and the fine fraction of sediment (r = 0.74; p < 0.05). Surprisingly, although the Mellah Lagoon is almost semi-closed, it holds an important specific richness in dinocysts (42 species) higher than others observed in Mediterranean lagoons. However, cyst abundances are low compared to other lagoons in the Mediterranean Sea. Finally, the presence of dinocysts of Alexandrium catenella/tamarense, A. minutum, and Gymnodinium catenatum associated to paralytic shellfish toxins, A. pseudogonyaulax which produces goniodomin A, also Protoceratium reticulatum and Gonyaulax spinifera complex which produce yessotoxins, needs to implement a monitoring program to prevent a potential human intoxication due to the consumption of contaminated sea products by these potent neurotoxins.

Pinnatoxins (PnTXs) are a group of emerging marine biotoxins produced by the benthic dinoflagellate Vulcanodinium rugosum, currently not regulated in Europe or in any other country in the world. In France, PnTXs were detected for the first time in 2011, in mussels from the Ingril lagoon (South of France, Mediterranean coast). Since then, analyses carried out in mussels from this lagoon have shown high concentrations of PnTXs for several months each year. PnTXs have also been detected, to a lesser extent, in mussels from other Mediterranean lagoons and on the Atlantic and Corsican coasts. In the French data, the main analog is PnTX G (low levels of PnTX A are also present in some samples). No cases of PnTXs poisoning in humans have been reported so far in France or anywhere else in the world. In mice, PnTXs induce acute neurotoxic effects, within a few minutes after oral administration. Clinical signs of toxicity include decreased mobility, paralysis of the hind legs, tremors, jumps and breathing difficulties leading to death by respiratory arrest at high doses. The French agency for food safety (ANSES) recently conducted a review of the state of knowledge related to PnTXs and V. rugosum. Based on (i) the clinical signs of toxicity in mice, (ii) the mode of action of PnTXs as nicotinic acetylcholine receptor competitive antagonists and (iii) knowledge on drugs and natural toxins with PnTX-related pharmacology, potential human symptoms have been extrapolated and proposed. In this work, a provisional acute benchmark value for PnTX G of 0.13 μg/kg bw per day has been derived from an oral acute toxicity study in mice. Based on this value and a large shellfish meat portion size of 400g, a concentration lower than 23 μg PnTX G/kg shellfish meat is not expected to result in adverse effects in humans. ANSES recommends taking into account PnTXs in the French official monitoring program for shellfish production and identified data gaps to refine health risk assessment., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The authors’ declarations of interests are made public via the ANSES website (www.anses.fr)., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Physiological plasticity gives HABs species the ability to respond to variations in the surrounding environment. The aim of this study was to examine morphological and physiological variability in Alexandrium pacificum R.W. Litaker (Group IV) (former Alexandrium catenella) blooming in Annaba bay, Algeria. Monoclonal cultures of up to 30 strains of this neurotoxic dinoflagellate were established by the germination of single resting cysts from the surface sediment of this southern Mediterranean marine ecosystem. Ribotyping confirmed formally for the first time that A. pacificum is developing in Eastern Algerian waters. Toxin analyses of A. pacificum strains revealed substantial intraspecific variability in both the profile and toxin amount. However, the toxin profile of most strains is characterized by the dominance of GTX6 (up to 96 mol %) which is the less toxic paralytic molecule. The toxin concentrations in the isolated strains varied widely between 3.8 and 30.82 fmol cell -1 . We observed an important variation in the growth rate of the studied A. pacificum strains with values ranging from 0.05 to 0.33 d -1 . The lag time of the studied strains varied widely and ranged from 4 to 20 days. The intraspecific diversity could be a response to the selection pressure which may be exerted by different environmental conditions over time and which can be genetically and in turn physiologically expressed. This study highlights, for the first time, that the sediment of a limited area holds an important diversity of A. pacificum cysts which give when germinate populations with noticeable physiological plasticity. Consequently, this diversified natural populations allow an exceptional adaptation to specific environmental conditions to outcompete local microalgae and to establish HABs which could explain why this dinoflagellate is successful and expanding worldwide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The spatial and temporal variation of phytoplankton communities including HAB species in relation to the environmental characteristics was investigated in the protected meso-oligotrophic Mellah lagoon located in the South Western Mediterranean. During 2016, a biweekly monitoring of phytoplankton assemblages and the main abiotic factors were realized at three representative stations. Taxonomic composition, abundance, and diversity index were determined. In total, 227 phytoplankton species (160 diatoms and 53 dinoflagellates) were inventoried. There was a clear dominance of diatoms (62.9%) compared with dinoflagellates (36.8%). Diatoms dominated in spring and dinoflagellates developed in summer and early autumn in Mellah showing a marked seasonal trend. Data showed that the dynamic of the phytoplankton taxa evolving in the lagoon was mainly driven by temperature and salinity. For the first time, a number of potentially toxic species have been identified, including 2 diatoms (Pseudo-nitzschia group delicatissima, Pseudo-nitzschia group seriata) and 5 dinoflagellates (Alexandrium minutum, Alexandrium tamarense/catenella, Dinophysis acuminata, Dinophysis sacculus, Prorocentrum lima). These harmful species could threat the functioning of the Mellah lagoon and human health and require the establishment of a monitoring network. Finally, our study suggests that the observed decrease of the phytoplankton diversity between 2001 and 2016 could result from the reduction in water exchanges between the lagoon and the adjacent coast following the gradual clogging of the channel.

Pinnatoxins (PnTXs) are emerging neurotoxins that were discovered about 30 years ago. They are solely produced by the marine dinoflagellate Vulcanodinium rugosum , and may be transferred into the food chain, as they have been found in various marine invertebrates, including bivalves. No human intoxication has been reported to date although acute toxicity was induced by PnTxs in rodents. LD 50 values have been estimated for the different PnTXs through the oral route. At sublethal doses, all symptoms are reversible, and no neurological sequelae are visible. These symptoms are consistent with impairment of central and peripheral cholinergic network functions. In fact, PnTXs are high-affinity competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Moreover, their lethal effects are consistent with the inhibition of muscle nAChRs, inducing respiratory distress and paralysis. Human intoxication by ingestion of PnTXs could result in various symptoms observed in episodes of poisoning with natural nAChR antagonists. This review updates the available data on PnTX toxicity with a focus on their mode of action on cholinergic networks and suggests the effects that could be extrapolated on human physiology.

Vibrio species have been associated with recurrent mass mortalities of juvenile oysters Crassostrea gigas threatening oyster farming worldwide. However, knowledge of the ecology of pathogens in affected oyster farming areas remains scarce. Specifically, there are no data regarding (i) the environmental reservoirs of Vibrio populations pathogenic to oysters, (ii) the environmental factors favoring their transmission, and (iii) the influence of oyster farming on the persistence of those pathogens. This knowledge gap limits our capacity to predict and mitigate disease occurrence. To address these issues, we monitored Vibrio species potentially pathogenic to C. gigas in 2013 and 2014 in the Thau Lagoon, a major oyster farming region in the coastal French Mediterranean. Sampling stations were chosen inside and outside oyster farms. Abundance and composition of phyto-, microzoo-, and mesozooplankton communities were measured monthly. The spatial and temporal dynamics of plankton and Vibrio species were compared, and positive correlations between plankton species and vibrios were verified by qPCR on isolated specimens of plankton. Vibrio crassostreae was present in the water column over both years, whereas Vibrio tasmaniensis was mostly found in 2013 and Vibrio aestuarianus was never detected. Moreover, V. tasmaniensis and V. crassostreae were found both as free-living or plankton-attached vibrios 1 month after spring mortalities of the oyster juveniles. Overall, V. crassostreae was associated with temperature and plankton composition, whereas V. tasmaniensis correlated with plankton composition only. The abundance of Vibrio species in the water column was similar inside and outside oyster farms, suggesting important spatial dispersion of pathogens in surrounding areas. Remarkably, a major increase in V. tasmaniensis and V. crassostreae was measured in the sediment of oyster farms during cold months. Thus, a winter reservoir of pathogenic vibrios could contribute to their ecology in this Mediterranean shellfish farming ecosystem.

Vulcanodinium rugosum, a dinoflagellate developing in Ingril Lagoon (Mediterranean, France) is responsible for shellfish intoxications due to the neurotoxin pinnatoxin G. A one year survey (March 2012-April 2013) was conducted in this oligotrophic shallow lagoon and key environmental parameters were recorded (temperature, salinity and nutrients). The spatio-temporal distribution of V. rugosum in water column and on macrophytes was also determined. Planktonic cells of V. rugosum were observed at all sampling stations, but in relatively low concentrations (maximum of 1000 cell/L). The highest abundances were observed from June to September 2012. There was a positive correlation between cell densities and both temperature and salinity. Non-motile cells were detected on macrophytes, with a maximum concentration of 6300 cells/g wet weight. Nitrite and ammonium were negatively related to V. rugosum abundance whereas total nitrogen, total phosphorus and phosphates showed a positive correlation. Altogether, in situ results suggest that V. rugosum is rather thermophilic and that organic nutrients should be considered when studying the nutrition requirements for this noxious expanding dinoflagellate., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study investigated the genetic diversity of phytoplankton communities in six shallow lagoons located on the French coast of the northwestern Mediterranean Sea that represented a trophic gradient ranging from oligotrophic to hypereutrophic. The phytoplankton communities were sampled once a month from spring (May) to the beginning of autumn (September/early October) in 2012 and fractionated by size. Metabarcodes were generated from cDNAs by targeting the D1-D2 region of the 28S rRNA gene and pyrosequenced using Roche 454 technology. Examination of the annotated barcodes revealed harmful algal species not previously documented in these lagoons. Three ichthyotoxic species belonging to Pfiesteriaceae were detected: Luciella masanensis was relatively widespread and abundant in many samples, whereas Pfiesteria piscicida and Stoeckeria changwonensis were found as single barcode sequences. Furthermore, a phylogenetic analysis of barcodes annotated as belonging to Pfiesteriaceae suggested the existence of two previously undescribed clades. The other toxic or potentially harmful dinoflagellates detected through rare barcodes were Dinophysis acuminata, Vulcanodinium rugosum, Alexandrium andersonii and A. ostenfeldii. The two most abundant dinoflagellate taxa were Gymnodinium litoralis and Akashiwo sanguinea with respect to sequence numbers. Four diatom species from the genus Pseudo-nitzschia that potentially produce domoic acid were identified (P. galaxiae, P. delicatissima, P. brasiliana and P. calliantha). These observations are discussed in terms of the literature and monitoring records related to the identified taxa in this Mediterranean area., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: It has been suggested that differences in health technology assessment (HTA) processes among countries, particularly within Europe, have led to inequity in patient access to new medicines., Objectives: To provide an up-to-date snapshot analysis of the present status of HTA and reimbursement systems in select European countries, and to investigate the implications of these processes, especially with regard to delays in market and patient access., Methods: HTA and reimbursement processes were assessed through a review of published and gray literature, and through a series of interviews with HTA experts. To quantify the impact of differences among countries, we conducted case studies of 12 products introduced since 2009, including 10 cancer drugs., Results: In addition to the differences in HTA and reimbursement processes among countries, the influence of particular sources of information differs among HTA bodies. The variation in the time from the authorization by the European Medicines Agency to the publication of HTA decisions was considerable, both within and among countries, with a general lack of transparency as to why some assessments take longer than others. In most countries, market access for oncology products can occur outside the HTA process, with sales often preceding HTA decisions., Conclusions: It is challenging even for those with considerable personal experience in European HTA processes to establish what is really happening in market access for new drugs. We recommend that efforts should be directed toward improving transparency in HTA, which should, in turn, lead to more effective processes., (Copyright © 2017 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.)

Harmful benthic dinoflagellates, usually developing in tropical areas, are expanding to temperate ecosystems facing water warming. Reports on harmful benthic species are particularly scarce in the Southern Mediterranean Sea. For the first time, three thermophilic benthic dinoflagellates ( Ostreopsis cf. ovata , Prorocentrum lima and Coolia monotis ) were isolated from Bizerte Bay (Tunisia, Mediterranean) and monoclonal cultures established. The ribotyping confirmed the morphological identification of the three species. Maximum growth rates were 0.59 ± 0.08 d -1 for O. cf. ovata , 0.35 ± 0.01 d -1 for C. monotis and 0.33 ± 0.04 d -1 for P. lima. Toxin analyses revealed the presence of ovatoxin-a and ovatoxin-b in O. cf. ovata cells. Okadaic acid and dinophysistoxin-1 were detected in P. lima cultures. For C. monotis , a chromatographic peak at 5.6 min with a mass m / z = 1061.768 was observed, but did not correspond to a mono-sulfated analogue of the yessotoxin. A comparison of the toxicity and growth characteristics of these dinoflagellates, distributed worldwide, is proposed., Competing Interests: The authors declare no conflict of interest.

Passive samplers (solid phase adsorption toxin tracking: SPATT) are able to accumulate biotoxins produced by microalgae directly from seawater, thus providing useful information for monitoring of the marine environment. SPATTs containing 0.3, 3, and 10 g of resin were deployed at four different coastal areas in France and analyzed using liquid chromatography coupled to high resolution mass spectrometry. Quantitative targeted screening provided insights into toxin profiles and showed that toxin concentrations and profiles in SPATTs were dependent on the amount of resin used. Between the three amounts of resin tested, SPATTs containing 3 g of resin appeared to be the best compromise, which is consistent with the use of 3 g of resin in SPATTs by previous studies. MassHunter and Mass Profiler Professional softwares were used for data reprocessing and statistical analyses. A differential profiling approach was developed to investigate and compare the overall chemical diversity of dissolved substances in different coastal water bodies. Principal component analysis (PCA) allowed for spatial differentiation between areas. Similarly, SPATTs retrieved from the same location at early, medium, and late deployment periods were also differentiated by PCA, reflecting seasonal variations in chemical profiles and in the microalgal community. This study used an untargeted metabolomic approach for spatial and temporal differentiation of marine environmental chemical profiles using SPATTs, and we propose this approach as a step forward in the discovery of chemical markers of short- or long-term changes in the microbial community structure.

The European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases convened a task force of experts in rheumatoid arthritis (RA) and clinical trial methodology to comment on the new draft 'Guideline on clinical investigation of medicinal products for the treatment of RA' released by the European Medicines Agency (EMA). Special emphasis was placed by the group on the development of new drugs for the treatment of early RA. In the absence of a clear definition of early RA, it was suggested that clinical investigations in this condition were conducted in disease-modifying antirheumatic drugs naïve patients with no more than 1 year disease duration. The expert group recommended using an appropriate improvement in disease activity (American College of Rheumatology (ACR) or Simplified/Clinical Disease Activity Index (SDAI/CDAI) response criteria) or low disease activity (by any score) as primary endpoints, with ACR/European League Against Rheumatism remission as a secondary endpoint. Finally, as compelling evidence showed that the Disease Acrivity Score using 28-joint counts (DAS28) might not provide a reliable definition of remission, or sometimes even low disease activity, the group suggested replacing DAS28 as a measurement instrument to evaluate disease activity in RA clinical trials. Proposed alternatives included SDAI, CDAI and Boolean criteria., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Vulcanodinium rugosum, a recently described species, produces pinnatoxins. The IFR-VRU-01 strain, isolated from a French Mediterranean lagoon in 2010 and identified as the causative dinoflagellate contaminating mussels in the Ingril Lagoon (French Mediterranean) with pinnatoxin-G, was grown in an enriched natural seawater medium. We tested the effect of temperature and salinity on growth, pinnatoxin-G production and chlorophyll a levels of this dinoflagellate. These factors were tested in combinations of five temperatures (15, 20, 25, 30 and 35 °C) and five salinities (20, 25, 30, 35 and 40) at an irradiance of 100 µmol photon m(-2) s(-1). V. rugosum can grow at temperatures and salinities ranging from 20 °C to 30 °C and 20 to 40, respectively. The optimal combination for growth (0.39 ± 0.11 d(-1)) was a temperature of 25 °C and a salinity of 40. Results suggest that V. rugosum is euryhaline and thermophile which could explain why this dinoflagellate develops in situ only from June to September. V. rugosum growth rate and pinnatoxin-G production were highest at temperatures ranging between 25 and 30 °C. This suggests that the dinoflagellate may give rise to extensive blooms in the coming decades caused by the climate change-related increases in temperature expected in the Mediterranean coasts.

The multifactorial etiology of massive Crassostrea gigas summer mortalities results from complex interactions between oysters, opportunistic pathogens and environmental factors. In a field survey conducted in 2014 in the Mediterranean Thau Lagoon (France), we evidenced that the development of the toxic dinoflagellate Alexandrium catenella, which produces paralytic shellfish toxins (PSTs), was concomitant with the accumulation of PSTs in oyster flesh and the occurrence of C. gigas mortalities. In order to investigate the possible role of toxic algae in this complex disease, we experimentally infected C. gigas oyster juveniles with Vibrio tasmaniensis strain LGP32, a strain associated with oyster summer mortalities, after oysters were exposed to Alexandrium catenella. Exposure of oysters to A. catenella significantly increased the susceptibility of oysters to V. tasmaniensis LGP32. On the contrary, exposure to the non-toxic dinoflagellate Alexandrium tamarense or to the haptophyte Tisochrysis lutea used as a foraging alga did not increase susceptibility to V. tasmaniensis LGP32. This study shows for the first time that A. catenella increases the susceptibility of Crassostrea gigas to pathogenic vibrios. Therefore, in addition to complex environmental factors explaining the mass mortalities of bivalve mollusks, feeding on neurotoxic dinoflagellates should now be considered as an environmental factor that potentially increases the severity of oyster mortality events.

Vulcanodinium rugosum, a recently described dinoflagellate species producing a potent neurotoxin (pinnatoxin G), has been identified in French Mediterranean lagoons and was responsible for recurrent episodes of shellfish toxicity detected by mouse bioassay. Until now, the biology and physiology of V. rugosum have not been fully investigated. We studied the growth characteristics and toxicity of a V. rugosum strain (IFR-VRU-01), isolated in the Ingril lagoon in June 2009 (North-Western French Mediterranean Sea). It was cultivated in Enriched Natural Sea Water (ENSW) with organic (urea) and inorganic (ammonium and nitrate) nitrogen, at a temperature of 25 °C and irradiance of 100 μmol/m²·s(-1). Results showed that ammonium was assimilated by cells more rapidly than nitrate and urea. V. rugosum is thus an osmotrophic species using urea. Consequently, this nitrogen form could contribute to the growth of this dinoflagellate species in the natural environment. There was no significant difference (Anova, p = 0.856) between the growth rate of V. rugosum cultivated with ammonium (0.28 ± 0.11 day(-1)), urea (0.26 ± 0.08 day(-1)) and nitrate (0.24 ± 0.01 day(-1)). However, the production of chlorophyll a and pinnatoxin G was significantly lower with urea as a nitrogen source (Anova, p < 0.027), suggesting that nutritional conditions prevailing at the moment of the bloom could determine the cellular toxicity of V. rugosum and therefore the toxicity measured in contaminated mollusks. The relatively low growth rate (≤0.28 day(-1)) and the capacity of this species to continuously produce temporary cysts could explain why cell densities of this species in the water column are typically low (≤20,000 cells/L).

The neurotoxin BMAA (β-N-methylamino-l-alanine) and its isomer DAB (2,4-diaminobutyric acid) have been detected in seafood worldwide, including in Thau lagoon (French Mediterranean Sea). A cluster of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease associated with BMAA, has also been observed in this region. Mussels, periphyton (i.e. biofilms attached to mussels) and plankton were sampled between July 2013 and October 2014, and analyzed using HILIC-MS/MS. BMAA, DAB and AEG (N-(2-aminoethyl)glycine) were found in almost all the samples of the lagoon. BMAA and DAB were present at 0.58 and 0.83, 2.6 and 3.3, 4.0 and 7.2 μg g(-1) dry weight in plankton collected with nets, periphyton and mussels, respectively. Synechococcus sp., Ostreococcus tauri, Alexandrium catenella and eight species of diatoms were cultured and screened for BMAA and analogs. While Synechococcus sp., O. tauri and A. catenella did not produce BMAA under our culture conditions, four diatoms species contained both BMAA and DAB. Hence, diatoms may be a source of BMAA for mussels. Unlike other toxins produced by microalgae, BMAA and DAB were detected in significant amounts in tissues other than digestive glands in mussels., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Osteoarthritis (OA), a disease affecting different patient phenotypes, appears as an optimal candidate for personalized healthcare. The aim of the discussions of the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO) working group was to explore the value of markers of different sources in defining different phenotypes of patients with OA. The ESCEO organized a series of meetings to explore the possibility of identifying patients who would most benefit from treatment for OA, on the basis of recent data and expert opinion. In the first meeting, patient phenotypes were identified according to the number of affected joints, biomechanical factors, and the presence of lesions in the subchondral bone. In the second meeting, summarized in the present article, the working group explored other markers involved in OA. Profiles of patients may be defined according to their level of pain, functional limitation, and presence of coexistent chronic conditions including frailty status. A considerable amount of data suggests that magnetic resonance imaging may also assist in delineating different phenotypes of patients with OA. Among multiple biochemical biomarkers identified, none is sufficiently validated and recognized to identify patients who should be treated. Considerable efforts are also being made to identify genetic and epigenetic factors involved in OA, but results are still limited. The many potential biomarkers that could be used as potential stratifiers are promising, but more research is needed to characterize and qualify the existing biomarkers and to identify new candidates.

Despite the near concurrent publication by influential scientific organizations, there are important differences in interpretation of the evidence base and the conclusions derived from the recent Osteoarthritis Research Society International (OARSI) guidelines for the management of knee osteoarthritis, the American College of Rheumatology (ACR) guidelines (concerning also hip and hand osteoarthritis) and the algorithm recommendations by the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO). This is particularly evident for the drug class of symptomatic slow-acting drugs in osteoarthritis. In this paper, we highlight these differences and try to understand where they derive from, proposing an evidence-based interpretation.

Marine biotoxins are algal metabolites that can accumulate in fish or shellfish and render these foodstuffs unfit for human consumption. These toxins, released into seawater during algal occurrences, can be monitored through passive sampling. Acetone, methanol and isopropanol were evaluated for their efficiency in extracting toxins from algal biomass. Isopropanol was chosen for further experiments thanks to a slightly higher recovery and no artifact formation. Comparison of Oasis HLB, Strata-X, BondElut C18 and HP-20 sorbent materials in SPE-mode led to the choice of Oasis HLB, HP-20 and Strata-X. These three sorbents were separately exposed as passive samplers for 24 h to seawater spiked with algal extracts containing known amounts of okadaic acid (OA), azaspiracids (AZAs), pinnatoxin-G (PnTX-G), 13-desmethyl spirolide-C (SPX1) and palytoxins (PlTXs). Low density polyethylene (LDPE) and silicone rubber (PDMS) strips were tested in parallel on similar mixtures of spiked natural seawater for 24 h. These strips gave significantly lower recoveries than the polymeric sorbents. Irrespective of the toxin group, the adsorption rate of toxins on HP-20 was slower than on Oasis HLB and Strata-X. However, HP-20 and Strata-X gave somewhat higher recoveries after 24 h exposure. Irrespective of the sorbent tested, recoveries were generally highest for cyclic imines and OA group toxins, slightly lower for AZAs, and the lowest for palytoxins. Trials in re-circulated closed tanks with mussels exposed to Vulcanodinium rugosum or Prorocentrum lima allowed for further evaluation of passive samplers. In these experiments with different sorbent materials competing for toxins in the same container, Strata-X accumulated toxins faster than Oasis HLB, and HP-20, and to higher levels. The deployment of these three sorbents at Ingril French Mediterranean lagoon to detect PnTX-G in the water column showed accumulation of higher levels on HP-20 and Oasis HLB compared to Strata-X. This study has significantly extended the range of sorbents for passive sampling of marine toxins. In particular, sorbents were included that had previously been evaluated for polyhalogenated contaminants, pharmaceuticals, phytochemicals or veterinary residues. Moreover, this study has for the first time demonstrated the usefulness of the polymeric Oasis HLB and Strata-X sorbents in laboratory and field studies for various microalgal toxins., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

β-N-methylamino-L-alanine (BMAA) is a neurotoxic non-protein amino acid suggested to be involved in neurodegenerative diseases. It was reported to be produced by cyanobacteria, but also found in edible aquatic organisms, thus raising concern of a widespread human exposure. However, the chemical analysis of BMAA and its isomers are controversial, mainly due to the lack of selectivity of the analytical methods. Using factorial design, we have optimized the chromatographic separation of underivatized analogues by a hydrophilic interaction chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) method. A combination of an effective solid phase extraction (SPE) clean-up, appropriate chromatographic resolution and the use of specific mass spectral transitions allowed for the development of a highly selective and sensitive analytical procedure to identify and quantify BMAA and its isomers (in both free and total form) in cyanobacteria and mollusk matrices (LOQ of 0.225 and 0.15 µg/g dry weight, respectively). Ten species of cyanobacteria (six are reported to be BMAA producers) were screened with this method, and neither free nor bound BMAA could be found, while both free and bound DAB were present in almost all samples. Mussels and oysters collected in 2009 in the Thau Lagoon, France, were also screened, and bound BMAA and its two isomers, DAB and AEG, were observed in all samples (from 0.6 to 14.4 µg/g DW), while only several samples contained quantifiable free BMAA.

To better understand the effect of Paralytic Shellfish Toxins (PSTs) accumulation in the digestive gland of the Pacific oyster, Crassostrea gigas, we experimentally exposed individual oysters for 48 h to a PSTs producer, the dinoflagellate Alexandrium catenella. In comparison to the effect of the non-toxic Alexandrium tamarense, on the eight apoptotic related genes tested, Bax and BI.1 were significantly upregulated in oysters exposed 48 h to A. catenella. Among the five detoxification related genes tested, the expression of cytochrome P450 (CYP1A) was shown to be correlated with toxin concentration in the digestive gland of oysters exposed to the toxic dinoflagellate. Beside this, we observed a significant increase in ROS production, a decrease in caspase-3/7 activity and normal percentage of apoptotic cells in this tissue. Taken together, these results suggest a feedback mechanism, which may occur in the digestive gland where BI.1 could play a key role in preventing the induction of apoptosis by PSTs. Moreover, the expression of CYP1A, Bax and BI.1 were found to be significantly correlated to the occurrence of natural toxic events, suggesting that the expression of these genes together could be used as biomarker to assess the biological responses of oysters to stress caused by PSTs.

Introduction: Amyotrophic lateral sclerosis (ALS) is the most common motor neurone disease. It occurs in two forms: (1) familial cases, for which several genes have been identified and (2) sporadic cases, for which various hypotheses have been formulated. Notably, the β-N-methylamino-L-alanine (L-BMAA) toxin has been postulated to be involved in the occurrence of sporadic ALS. The objective of the French BMAALS programme is to study the putative link between L-BMAA and ALS., Methods and Analysis: The programme covers the period from 1 January 2003 to 31 December 2011. Using multiple sources of ascertainment, all the incident ALS cases diagnosed during this period in the area under study (10 counties spread over three French regions) were collected. First, the standardised incidence ratio will be calculated for each municipality under concern. Then, by applying spatial clustering techniques, overincidence and underincidence zones of ALS will be sought. A case-control study, in the subpopulation living in the identified areas, will gather information about patients' occupations, leisure activities and lifestyle habits in order to assess potential risk factors to which they are or have been exposed. Specimens of drinking water, food and biological material (brain tissue) will be examined to assess the presence of L-BMAA in the environment and tissues of ALS cases and controls., Ethics and Dissemination: The study has been reviewed and approved by the French ethical committee of the CPP SOOM IV (Comité de Protection des Personnes Sud-Ouest & Outre-Mer IV). The results will be published in peer-reviewed journals and presented at national and international conferences., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Alexandrium catenella (group IV) and Alexandrium tamarense (group III) (Dinophyceae) are two cryptic invasive phytoplankton species belonging to the A. tamarense species complex. Their worldwide spread is favored by the human activities, transportation and climate change. In order to describe their diversity in the Mediterranean Sea and understand their settlements and maintenances in this area, new microsatellite markers were developed based on Thau lagoon (France) samples of A. catenella and A. tamarense strains. In this study twelve new microsatellite markers are proposed. Five of these microsatellite markers show amplifications on A. tamarense and ten on A. catenella. Three of these 12 microsatellite markers allowed amplifications on both cryptic species. Finally, the haplotypic diversity ranged from 0.000 to 0.791 and 0.000 to 0.942 for A. catenella and A. tamarense respectively., (Copyright © 2014. Published by Elsevier Ltd.)

Environmental conditions ultimately leading to blooms of the toxic dinoflagellate Alexandrium catenella/tamarense were investigated at two Mediterranean sites (Annaba Bay, Algeria and Thau lagoon, France). Three years were examined in details: 1992 (a pre-Alexandrium period), 2002 (a year with the first bloom in Annaba) and 2010 (a year with a major bloom in Annaba). Most conditions were similar, but ammonium concentrations were much higher in Annaba (up to 100μM) than in Thau (up to 10μM). First records of A. catenella/tamarense were in 1995 for Thau and 2002 for Annaba, and coincided with soluble reactive phosphorus (SRP) decreasing below a concentration of about 1μM. No other environmental variable could be related to those blooms. Thus, it is likely that the large reductions in SRP at both sites led to phosphorus limitation of a certain number of phytoplankton species and favored the development of A. catenella/tamarense., (Copyright © 2013 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Background: Dietary exposure to the cyanotoxin BMAA is suspected to be the cause of amyotrophic lateral sclerosis in the Western Pacific Islands. In Europe and North America, this toxin has been identified in the marine environment of amyotrophic lateral sclerosis clusters but, to date, only few dietary exposures have been described., Objectives: We aimed at identifying cluster(s) of amyotrophic lateral sclerosis in the Hérault district, a coastal district from Southern France, and to search, in the identified area(s), for the existence of a potential dietary source of BMAA., Methods: A spatio-temporal cluster analysis was performed in the district, considering all incident amyotrophic lateral sclerosis cases identified from 1994 to 2009 by our expert center. We investigated the cluster area with serial collections of oysters and mussels that were subsequently analyzed blind for BMAA concentrations., Results: We found one significant amyotrophic lateral sclerosis cluster (p = 0.0024), surrounding the Thau lagoon, the most important area of shellfish production and consumption along the French Mediterranean coast. BMAA was identified in mussels (1.8 µg/g to 6.0 µg/g) and oysters (0.6 µg/g to 1.6 µg/g). The highest concentrations of BMAA were measured during summer when the highest picocyanobacteria abundances were recorded., Conclusions: While it is not possible to ascertain a direct link between shellfish consumption and the existence of this ALS cluster, these results add new data to the potential association of BMAA with sporadic amyotrophic lateral sclerosis, one of the most severe neurodegenerative disorder.

Following a review of official control data on shellfish in France, Ingril Lagoon had been identified as a site where positive mouse bioassays for lipophilic toxins had been repeatedly observed. These unexplained mouse bioassays, also called atypical toxicity, coincided with an absence of regulated toxins and rapid death times in mice observed in the assay. The present study describes pinnatoxin G as the main compound responsible for the toxicity observed using the mouse bioassay for lipophilic toxins. Using a well-characterised standard for pinnatoxin G, LC-MS/MS analysis of mussel samples collected from 2009 to 2012 revealed regular occurrences of pinnatoxin G at levels sufficient to account for the toxicity in the mouse bioassays. Baseline levels of pinnatoxin G from May to October usually exceeded 40 μg kg(-1) in whole flesh, with a maximum in September 2010 of around 1200 μg kg(-1). These concentrations were much greater than those at the other 10 sites selected for vigilance testing, where concentrations did not exceed 10 μg kg(-1) in a 3-month survey from April to July 2010, and where rapid mouse deaths were not typically observed. Mussels were always more contaminated than clams, confirming that mussel is a good sentinel species for pinnatoxins. Profiles in mussels and clams were similar, with the concentration of pinnatoxin A less than 2% that of pinnatoxin G, and pteriatoxins were only present in non-quantifiable traces. Esters of pinnatoxin G could not be detected by analysis of extracts before and after alkaline hydrolysis. Analysis with a receptor-binding assay showed that natural pinnatoxin G was similarly active on the nicotinic acetylcholine receptor as chemically synthesized pinnatoxin G. Culture of Vulcanodinium rugosum, previously isolated from Ingril lagoon, confirmed that this alga is a pinnatoxin G producer (4.7 pg cell(-1)). Absence of this organism from the water column during prolonged periods of shellfish contamination and the dominance of non-motile life stages of V. rugosum both suggest that further studies will be required to fully describe the ecology of this organism and the accumulation of pinnatoxins in shellfish., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Laboratory experiments were designed to study the toxin content and profile of the Alexandrium catenella strain ACT03 (isolated from Thau Lagoon, French Mediterranean) in response to abiotic environmental factors under nutrient-replete conditions. This dinoflagellate can produce various paralytic shellfish toxins with concentrations ranging from 2.9 to 50.3 fmol/cell. The toxin profile was characterized by carbamate toxins (GTX3, GTX4 and GTX5) and N-sulfocarbamoyl toxins (C1, C2, C3 and C4). C2 dominated at 12-18 °C, but only for salinities ranging from 10 to 25 psu, whereas GTX5 became dominant at temperatures ranging from 21 to 30 °C at almost all salinities. There was no significant variation in the cellular toxin amount from 18 °C to 27 °C for salinities ranging between 30 and 40 psu. At salinities of 10 to 25 psu, the toxin concentrations always remained below 20 fmol/cell. Toxin content was stable for irradiance ranging from 10 to 70 μmol photons/m2/s then slightly increased. Overall, the toxin profile was more stable than the toxin content (fmol/cell), except for temperature and/or salinity values different from those recorded during Alexandrium blooms in Thau Lagoon.

The European Commission issued on March 17, 2011, a marketing authorization valid throughout the European Union (EU) for eribulin (Halaven; Eisai Limited). The decision was based on the favorable opinion of the Committee for Medicinal Products for Human Use recommending a marketing authorization for the treatment of patients with locally advanced or metastatic breast cancer who have progressed after at least 2 chemotherapeutic regimens for advanced disease. Eribulin mesylate is a structurally simplified synthetic analogue of halichondrin B, which is a natural product isolated from the marine sponge Halichondria okadai (ATC code L01XX41). Eribulin is a nontaxane, microtubule dynamics inhibitor belonging to the halichondrin class of antineoplastic agents. Eribulin inhibits the growth phase of microtubules without affecting the shortening phase and sequesters tubulin into nonproductive aggregates leading to G(2)-M cell-cycle block, disruption of mitotic spindles, and, ultimately, apoptotic cell death after prolonged mitotic blockage. The recommended dose of eribulin is 1.23 mg/m(2) (equivalent to 1.4 mg/m(2) eribulin mesylate) to be administered intravenously over 2 to 5 min on days 1 and 8 of a 3-week cycle. In the pivotal trial, eribulin was associated with increased overall survival in patients with locally advanced or metastatic breast cancer who received at least 2 prior chemotherapy lines for advanced disease (median overall survival was 13.2 months in the eribulin arm vs. 10.6 months in the control arm; HR = 0.805; 95% confidence interval, 0.677-0.958; P = 0.014). The most common side effects are asthenia or fatigue and neutropenia. The objective of this article is to summarize the scientific review of the application leading to approval in the EU. The detailed scientific assessment report and product information, including the summary report and product information, including product characteristics, are available on the European Medicines Agency website., (©2012 AACR.)

Glycaemic control is an inadequate surrogate marker of cardiovascular event reduction in patients with type 2 diabetes. Clinical trials to date have been unsuccessful in identifying a therapeutic approach that addresses the underlying problem in diabetes (glycaemic control) and reduces cardiovascular risk. The potential for some agents to increase the risk of cardiovascular events has led to substantial changes in regulatory requirements for new anti-diabetic therapies. These requirements, while key to ensuring the cardiovascular safety of new agents, fail to emphasize the need to show clinical benefits, such as less visual impairment, less need for dialysis, or fewer cardiovascular events and deaths. Changes in test results such as glycaemic control, serum creatinine, micro-albuminuria, or retinopathy are inadequate surrogates. Regulators should consider the potential advantages of offering extended patent protection in order to encourage companies to conduct long-term trials in diabetes and many other chronic medical conditions. Cooperative efforts among physicians, clinical trialists, regulators, and sponsors are needed to address unresolved issues including re-defining therapeutic targets that are meaningful to patients with diabetes, determining the appropriate length of follow-up for future trials, and considering the ethical and operational challenges of non-inferiority designs.

On March 17, 2011 the European Commission issued a marketing authorization valid throughout the European Union for Jevtana® (Sanofi-Aventis, Paris, France) for the treatment of patients with hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing regimen. The active substance of Jevtana® is cabazitaxel acetone solvate, an antineoplastic agent that acts by disrupting the microtubular network in cells. The recommended dose of cabazitaxel is 25 mg/m2 administered as a 1-hour i.v. infusion every 3 weeks in combination with oral prednisone or prednisolone, 10 mg, administered daily throughout treatment. In the main study submitted for this application, a 2.4-month longer median overall survival time and a 30% lower risk for death were observed for cabazitaxel, compared with mitoxantrone. The most common side effects with cabazitaxel were anemia, leukopenia, neutropenia, thrombocytopenia, and diarrhea. This paper summarizes the scientific review of the application leading to approval in the European Union. The detailed scientific assessment report and product information, including the summary of product characteristics, are available on the European Medicines Agency Web site (http://www.ema.europa.eu).

On 13 July 2011 the European Commission issued a marketing authorisation valid throughout the European Union (EU) for ipilimumab for the treatment of advanced (unresectable or metastatic) melanoma in adults who have received prior therapy. Ipilimumab is a monoclonal antibody that specifically blocks the inhibitory signal of cytotoxic T lymphocyte antigen 4 (CTLA-4), resulting in T cell activation, proliferation and lymphocyte infiltration into tumours, leading to tumour cell death. The recommended induction regimen of ipilimumab is 3mg/kg administered intravenously over a 90 min period every 3 weeks for a total of four doses. In a phase 3 trial in patients with advanced melanoma, median overall survival for ipilimumab was 10 months versus 6 months for gp100, an experimental melanoma vaccine (Hazard ratio (HR) 0.66; 95% confidence interval (CI): 0.51, 0.87; p = 0.0026). Ipilimumab was most commonly associated with adverse reactions resulting from increased or excessive immune activity. Most of these, including severe reactions, resolved following initiation of appropriate medical therapy or withdrawal of ipilimumab. The most common side-effects (affecting more than 10% of patients) were diarrhoea, rash, pruritus, fatigue, nausea, vomiting, decreased appetite and abdominal pain. The objective of this paper is to summarise the scientific review of the application leading to approval in the EU. The detailed scientific assessment report and product information, including the summary of product characteristics (SmPC), are available on the European Medicines Agency (EMA) website (www.ema.europa.eu)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The European Medicines Agency (EMA) in London is responsible for the Regulatory review of new medicinal products for Marketing Authorisation, through which pharmaceutical companies may obtain first Marketing Authorisation and subsequent Variations valid throughout the EU and EFTA. The qualification opinion of novel methodologies is a new procedure where applicants can obtain scientific advice on new methodologies for regulatory clinical trials of efficacy of new compounds. It will help benefit/risk assessment of the CHMP. The definition of prodromal AD is acceptable. The "Dubois Criteria" as criteria to define the population must be validated in full at the time of the submission of the dossiers. Including a positive CSF biomarker profile is considered predictive for the evaluation of the AD-dementia type. However, although high CSF tau and low CSF Aβ42 are predictive of Alzheimer's disease, the criterion "positive CSF tau/Aβ42 ratio" is not well defined. The qualification of biomarkers in the pre-dementia stage of Alzheimer's disease will allow better inclusion criteria of patients in pre-dementia trials in which the benefit/risk is higher for treatment with these novel compounds., (Copyright © 2011 Elsevier B.V. and ECNP. All rights reserved.)

On June 14, 2010, the European Commission issued a conditional marketing authorization valid throughout the European Union for pazopanib for the treatment of advanced renal cell carcinoma. Pazopanib is an antineoplastic agent that inhibits multiple receptor tyrosine kinases. The recommended oral dose is 800 mg once daily. The benefit of pazopanib is an increased progression-free survival. In the pivotal trial VEG105192, the median progression-free survival was 9.2 months (95% confidence interval, 7.4-12.9) in the pazopanib arm compared with 4.2 months (95% confidence interval, 2.8-4.2) in the placebo arm. The most common side effects include diarrhea, hair color change, hypertension, nausea, fatigue, anorexia, vomiting, dysgeusia, elevated alanine aminotransferase, elevated aspartate aminotransferase, and abdominal pain. The objective of this article is to summarize the scientific review of the application that led to approval in the European Union., (©2011 AACR)

On 11(th) March 2010, the European Commission issued a marketing authorization valid throughout the European Union for Revolade for the treatment of adult chronic immune (idiopathic) thrombocytopenic purpura. Revolade is an orphan medicinal product indicated for splenectomized patients with immune (idiopathic) thrombocytopenic purpura who are refractory to other treatments (e.g. corticosteroids, immunoglobulins) and as second-line treatment for non-splenectomized patients where surgery is contraindicated. The active substance of Revolade is eltrombopag (ATC code B02BX05). Eltrombopag increases platelet production through activation of the thrombopoietin receptor. The recommended oral dose is 50 mg once daily to achieve and maintain a platelet count of the 50×10(9)/L or more necessary to reduce or prevent the risk of bleeding. The benefit of Revolade is a durable response in maintaining platelet levels. The most common side effects include headache, nausea, hepatobiliary toxicity, diarrhea, fatigue, paresthesia, constipation, rash, pruritus, cataract, arthralgia and myalgia. The decision to grant the marketing authorization was based on the favorable recommendation of the Committee for Medicinal Products for Human Use of the European Medicines Agency. The objective of this paper is to describe the data submitted to the European Medicines Agency and to summarize the scientific review of the application. The detailed scientific assessment report and product information, including the summary of product characteristics, are available on the European Medicines Agency website (www.ema.europa.eu).