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Melanoma is an aggressive tumor with invasive and metastatic potential, frequently exhibiting multidrug resistance mechanisms. In our continuous search for antimelanoma molecules, we have identified some effective marine compounds capable of not only inducing cell death, but also of sensitizing chemoresistant tumor cells to clinically used anticancer drugs. In this report, the cryptophyte Rhodomonas salina (Wislouch) D.R.A.Hill & R.Wetherbee, Pyrenomonadaceae, was chemically investigated in order to identify pigments efficiently inhibiting melanoma cells proliferation. All pharmacological tests were performed on A2058 cells expressing the oncogenic BRAF V600E mutation and resistant to dacarbazine treatment. Flash chromatography of R. salina ethanol extract led to purification of alloxanthin and crocoxanthin, which showed significant antiproliferative activity against A2058 cells, exhibiting IC50 = 29 and 50 μM, respectively. These carotenoids promoted growth inhibition, decreased cell migration, and induced apoptosis and sub-G1 cells accumulation after 72 h of treatment. In addition, alloxanthin potentiated the cytotoxic activity of vemurafenib (a BRAF inhibitor) and restored the sensitivity of A2058 cells to dacarbazine treatment.

A bibliographic database of scientific papers published by authors affiliated worldwide, especially focused in Europe and in the European Atlantic Area, and containing the keywords “microalga(e)” or “phytoplankton” was built. A corpus of 79,020 publications was obtained and analyzed using the Orbit Intellixir software to highlight the evolution of the research domain. Publication rates from 1960 to 2019, organization of the research, collaboration networks between countries and organizations, emerging and fading research concepts, major studied species, and associated concepts, as well as journals publishing microalgae research were considered. As a result, of the 79,020 papers published worldwide, 26,137 included authors from Europe (33% of world production) and 6989 from the European Atlantic Area (AA) (27% of European production, 9% of world production). The main worldwide scientific research topics found in this study were phytoplankton, community, bloom, diatoms, distribution, ecosystem, coastal, chlorophyll, zooplankton, photosynthesis, and primary production. At the European scale, the most studied topics were related to the environment, food, chemicals, pigments, protein, feed, and drugs. The highest scientific trends and market opportunities analysis identified bioplastics and biostimulants as top emerging concepts at the European level and agricultural, animal feed, and blue biotechnology at the European AA level.

Tisochrysis lutea (T. lutea, ex T. Isochrysis galbana or T-Iso) is a marine haptophyte that was first isolated from Tahiti seawater. Because of its high content in lipids, this tropical species is commonly used in aquaculture to feed fishes, crustaceans and molluscs larvae. It is also a rich source of fucoxanthin with a high potential for nutraceutical, cosmetic and pharmaceutical applications. The purpose of the present study was to detail the pigment composition of T. lutea and to develop an efficient process to recover highly purified fucoxanthin. Using ultra performant liquid chromatography coupled to diode arrays and high-resolution mass spectrometry detectors (UPLC-DAD-MS/MS), we demonstrated for the first time the presence of echinenone, 3-hydroxy-echinenone and chlorophyll c2-monogalactosyldiacylglycerol [18:4/14:0] in unstressed cultures of T. lutea. The chemotaxonomic relevance of this updated pigment composition was discussed in relation to the Haptophyta phylum. A two-step purification of fucoxanthin was then optimized using centrifugal partition chromatography coupled to flash chromatography. This process allowed the efficient isolation of fucoxanthin (purity > 99%), that was further assessed as a low-toxicity antineoplastic and chemosensitizing natural product in human chemoresistant melanoma cells. This carotenoid exerted an antiproliferative activity in A2058 melanoma cells and reversed in vitro their chemoresistance to dacarbazine, a DNA-alkylating agent clinically used for the treatment of metastatic melanoma.

Phytoplankton, with an estimated 30 000 to 1 000 000 species clustered in 12 phyla, presents a high taxonomic and ecophysiological diversity, reflected by the complex distribution of pigments among the different algal classes. High performance liquid chromatography is the gold standard method for qualitative and quantitative analysis of phytoplankton pigments in seawater and culture samples, but only a few pigments can be used as robust chemotaxonomic markers. A major challenge is thus to identify new ones, characteristic of a strain, species, class or taxon that cannot be currently identified on the basis of its pigment signature. Using an optimized extraction process coupled to a HPLC de-replication strategy, we examined the pigment composition of 37 microalgae strains, representative of the broad taxonomic diversity of marine and freshwater species (excluding cyanobacteria). For each species, the major pigments already described were unambiguously identified. We also observed the presence of several minor unidentified pigments in each chromatogram. The global analysis of pigment compositions revealed a total of 124 pigments, including 98 pigments or derivatives unidentified using the standards. Absorption spectra indicated that 35 corresponded to chlorophyll/porphyrin derivatives, 57 to carotenoids and six to derivatives having both spectral signatures. Sixty-one of these unidentified or new carotenoids and porphyrin derivatives were characteristic of particular strains or species, indicating their possible use as highly specific chemotaxonomic markers capable of identifying one strain out of the 37 selected. We developed a graphical analysis using Gephi software to give a clear representation of pigment communities among the various phytoplankton strains, and to reveal strain-characteristic and shared pigments. This made it possible to reconstruct the taxonomic evolution of microalgae classes, on the basis of the conservation, loss, and/or appearance of pigments.

International audience; A fast and high-resolution UPLC-MSE analysis was used to identify phytoplankton pigments in an ethanol extract of Porphyridium purpureum (Pp) devoid of phycobiliproteins. In a first step, 22 standard pigments were analyzed by UPLC-MSE to build a database including retention time and accurate masses of parent and fragment ions. Using this database, seven pigments or derivatives previously reported in Pp were unequivocally identified: β,β-carotene, chlorophyll a, zeaxanthin, chlorophyllide a, pheophorbide a, pheophytin a, and cryptoxanthin. Minor amounts of Divinyl chlorophyll a, a chemotaxonomic pigment marker for prochlorophytes, were also unequivocally identified using the database. Additional analysis of ionization and fragmentation patterns indicated the presence of ions that could correspond to hydroxylated derivatives of chlorophyll a and pheophytin a, produced during the ethanolic extraction, as well as previously described galactosyldiacylglycerols, the thylakoid coenzyme plastoquinone, and gracilamide B, a molecule previously reported in the red seaweed Gracillaria asiatica. These data point to UPLC-MSE as an efficient technique to identify phytoplankton pigments for which standards are available, and demonstrate its major interest as a complementary method for the structural elucidation of ionizable marine molecules.

Understanding microalgal lipid accumulation under nitrogen starvation is of major interest for biomass feedstock, food and biofuel production. Using a domesticated oleaginous algae Tisochrysis lutea , we performed the first comparative proteomic analysis on the wild type strain and a selected lipid over-accumulating mutant. 2-DE analysis was made on these strains cultured in two metabolic conditions, with and without nitrogen deprivation, which revealed significant differences in proteomes according to both strain and nitrogen availability. Mass spectrometry allowed us to identify 37 proteins that were differentially expressed between the two strains, and 17 proteins regulated by nitrogen starvation concomitantly with lipid accumulation. The proteins identified are known to be involved in various metabolic pathways including lipid, carbohydrate, amino acid, energy and pigment metabolisms, photosynthesis, protein translation, stress response and cell division. Four candidates were selected for possible implication in the over-accumulation of lipids during nitrogen starvation. These include the plastid beta-ketoacyl-ACP reductase protein, the coccolith scale associated protein and two glycoside hydrolases involved in biosynthesis of fatty acids, carbon homeostasis and carbohydrate catabolism, respectively. This proteomic study confirms the impact of nitrogen starvation on overall metabolism and provides new perspectives to study the lipid over-accumulation in the prymnesiophyte haptophyte T. lutea . Biological significance This paper study consists of the first proteomic analysis on Tisochrysis lutea , a non-model marine microalga of interest for aquaculture and lipids production. Comparative proteomics revealed proteins putatively involved in the up-accumulation of neutral lipids in a mutant strain during nitrogen starvation. The results are of great importance for future works to improve lipid accumulation in microalgae of biotechnological interest for biofuel production. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Azadinium spinosum (Elbrächter and Tillmann), a small marine dinoflagellate, has been recently described as a de novo producer of azaspiracid-1 and -2 (AZA1 and -2) diarrhoeic toxins. A culture of A. spinosum was established in our laboratory and optimised for pilot-scale production of this organism, to evaluate and understand AZA1 and -2 accumulation and biotransformation in blue mussels (Mytilus edulis) fed with A. spinosum. Adult mussels were continuously exposed to A. spinosum over 1 week in 160 L cylindrical conical tanks. Three different diets were tested for contamination: 5000, 10 000 cells mL−1 of A. spinosum and a mixture of 5000 cells mL−1 of A. spinosum with 5000 cells mL−1 of Isochrysis aff. galbana (T-Iso, CCAP 927/14). During the subsequent period of detoxification (2 weeks), contaminated mussels were continuously fed with 5000 cells mL−1 of T-Iso. Kinetics of accumulation, detoxification and biotransformation were evaluated, as well as the toxin distribution and the effect of A. spinosum on mussel digestive gland tubules. M. edulis fed on A. spinosum in the three tested conditions; this finding confirmed our recent experiments feeding A. spinosum to mussels. The original algal toxins AZA1 and -2, as well as mussel metabolites AZA3 to 12, -17, -19, -21 and -23 were found during these trials. After as little as 6 h, azaspiracid contents in mussels reached the EU regulatory limit, and metabolites were observed in all conditions at approximately 25% of the total AZA content. This fraction exceeded 50% after 24 h, and continued to increase until the end of the study. AZA17 and -19 were found to be the main metabolites, with AZA17 concentrations estimated in the same order of magnitude as that of the main algal toxin, AZA1. Highlights ► Azadinium spinosum was cultured and fed to blue mussels (Mytilus edulis). ► Mussels accumulated azaspiracids in less than 6 h to greater than legal limit. ► Biotransformation of algal toxins into shellfish metabolites was also rapid with >25% metabolites observed after 6 h. ► Detoxification speed was comparable with other lipophilic toxins (half-life ca. 11 days). ► Azaspiracids-6, -17 and -19 should also be considered in legislation.

MALDI Mass Spectrometry Imaging (MALDI-MSI) is a two-dimensional MALDI mass spectrometric technique used to visualize the spatial distribution of a large variety of biological molecules (from small metabolites to large proteins) without extraction, purification, separation or labelling of biological samples. Since its introduction by Caprioli et al in 1997, it has become one of the most important molecular histology methods for biomarker hunting and for understanding the spatial distribution of biomolecules in various tissues. MALDI-MSI is thus ideal for complementing the expanding field of metabolomics. Despite the fact that MALDI-MSI have been very successfully applied in divers studies (even in clinical applications) its use remains far from routine, and there is still a need to adapt protocols to suit specific tissues4 or metabolites. Here we describe the methodology of MALDI-MSI with examples taken from applications in plant (Cichorium intybus) and in seashell (Mytilus edulis).

Over the past seven years, methods available for the detection of phycotoxins have been extensively reviewed in a number of international expert committees, such as the consultations organised by FAO/IOC/WHO and EFSA, as well as by individual scientists. These reviews have shown that the methods available have severe limitations for the use in official control, either due to their limited scope and detection capability or due to a lack of calibration standards, reference materials and validation efforts. The present review focuses on recent developments in the detection of phycotoxins in several areas of applied research. Not being able to exhaustively describe all recent developments, the review focussed on three areas of interest to the authors: (i) detection of ultra-trace amounts of toxins, (ii) metabolism of toxins and their localisation in biological tissues, and (iii) approaches to detect unknown toxins or analogues of known toxins. Miniaturisation in combination with physico-chemical techniques appears to be a very efficient approach to detect low trace amounts of individual toxin analogues. In particular, the detection of azaspiracids and okadaic acid and analogues, using micro-filtration and on-line pre-concentration techniques, has shown to be useful for the characterisation of various algal and shellfish species. In the area of interactions of toxins with shellfish and mammalian systems, it is noted that several studies on biomarkers reveal either protein biomarkers of exposure to toxins or potential pathways of metabolism of the toxins themselves. A particular focus is given to recent findings in the areas of brevetoxin metabolism and biomarkers as well as azaspiracid localisation and metabolism. Finally, the detection of novel compounds is a particularly challenging area. The interest in this area has risen over the past years following cases of unexplained mouse toxicity such as the UK cockle toxicity and the French atypical toxicity in mussels and oysters from the Atlantic and Mediterranean coasts. Some attention is given to immuno-, functional and cellular bio-assays for the identification of bioactive agents in shellfish., Durant les sept dernières années, les méthodes disponibles pour la détection des phycotoxines ont été abondamment revues dans de nombreux groupes internationaux d’expertises, tels que les consultations organisées par FAO/IOC/WHO et EFSA ainsi que dans de nombreux articles scientifiques de synthèse. Ces revues ont démontré que, vis-à-vis des systèmes de régulations officiels, les méthodes disponibles présentent des limites d’utilisation importantes. Soit elles ont un périmètre réduit et des limites de détection trop élevées, soit il y a un manque d’étalons, de matériaux de référence ou d’efforts de validation. La présente revue focalise sur les développements récents dans la détection de phycotoxines en recherche appliquée. Sans vouloir décrire de manière exhaustive tous les développements récents, la revue examine trois domaines d’intérêt pour les auteurs : (i) la détection de quantités ultra-traces de toxines, (ii) la métabolisation et la localisation des toxines dans des tissus biologiques, et (iii) les approches pour la détection de toxines non-répertoriées ou des analogues de toxines connues. La miniaturisation en combinaison avec les techniques physico-chimiques constitue apparemment une approche efficace pour la détection de faibles traces d’analogues individuels des toxines. En particulier, la micro-filtration et des techniques de pré-concentration en ligne se sont montrées utiles pour la détection des azaspiracides et des toxines du groupe de l’acide okadaïque dans la caractérisation de diverses espèces d’algues et de coquillages. Dans le domaine des interactions des toxines avec les coquillages et des systèmes vivants de mammifères, nous avons noté que plusieurs études sur les bio-marqueurs révèlent soit des marqueurs protéiniques d’exposition aux toxines, soit des marqueurs du métabolisme des toxines elles-mêmes. Un intérêt spécifique a été trouvé dans les résultats d’étude sur le métabolisme des brévétoxines et des azaspiracides. La détection des composés bioactifs non-répertoriés constitue un défi particulièrement difficile. Ce domaine a trouvé plus d’intérêt dû à plusieurs cas de toxicités inexpliquées dans le test souris telles que les toxicités observées dans les coques en provenance de Grande-Bretagne ou encore dans les moules et huîtres des côtes méditerranéennes et atlantiques françaises. Une attention particulière a été consacrée à l’identification d’agents bioactifs par les essais cellulaires ou fonctionnels ou basés sur la détection immuno-chimique.

International audience; The 2-ethyhexyl nitrate (2-EHN) is currently added to diesel oil to improve ignition and boost cetane number. The biodegradability of this widely used chemical needed to be assessed in order to evaluate the environmental impact in case of accidental release. In aerobic liquid cultures, biodegradation of 2-EHN was assessed in biphasic liquid cultures using an inert non-aqueous phase liquid such as 2,2,4,4,6,8,8-heptamethylnonane (HMN) as solvent for the hydrophobic substrate. 2-EHN was found to be biodegradable by microbial communities from refinery wastewater treatment plants, but was recalcitrant to those of urban wastewater treatment facilities. Out of eighteen hydrocarbon-polluted or non-polluted soil samples, six microbial populations were also able to degrade 2-EHN. However, strain isolation from these microbial populations was rather difficult suggesting close cooperation between members of the microbial communities. Specific axenic bacterial strains selected for their ability to catabolize recalcitrant-hydrocarbons were also tested for their capacity to degrade 2-EHN. In liquid cultures with HMN phase as non-aqueous phase liquid, some Mycobacterium austroafricanum strains were found to degrade and mineralize 2-EHN significantly.

International audience; 2-Ethyhexyl nitrate (2-EHN) is a synthetic chemical used as a diesel fuel additive, which is recalcitrant to biodegradation. In this study, the enzymes involved in 2-EHN degradation were investigated in Mycobacterium austroafricanum IFP 2173. Using two-dimensional gel electrophoresis and a shotgun proteomic approach, a total of 398 proteins appeared to be more abundant in cells exposed to 2-EHN than in acetate-grown cells. This set of proteins includes multiple isoenzymes of the beta-oxidation pathway, two alcohol and one aldehyde dehydrogenase, as well as four cytochromes P450, including one CYP153 which functions as an alkane hydroxylase. Strain IFP 2173 was also found to contain two alkB-like genes encoding putative membrane-bound alkane hydroxylases. RT-PCR experiments showed that the gene encoding the CYP153 protein, as well as alkB genes, were expressed on 2-EHN. These findings are discussed in the light of a recently proposed 2-EHN degradation pathway involving an initial attack by an alkane hydroxylase and one turn of beta-oxidation, leading to the accumulation of a gamma-lactone as a dead-end product.

Zeaxanthin, an abundant carotenoid present in fruits, vegetables and algae was reported to exert antiproliferative activity and induce apoptosis in human uveal melanoma cells. It also inhibited uveal melanoma tumor growth and cell migration in nude mice xenograft models. Here we report that zeaxanthin purified from the rhodophyte Porphyridium purpureum (Bory) K.M.Drew & R.Ross, Porphyridiaceae, promotes apoptosis in the A2058 human melanoma cell line expressing the oncogenic BRAF V600E mutation. Zeaxanthin 40 μM (IC50) induced chromatin condensation, nuclear blebbing, hypodiploidy, accumulation of cells in sub-G1 phase, DNA internucleosomal fragmentation and activation of caspase-3. Western blot analysis revealed that zeaxanthin induced up-regulation of the pro-apoptotic factors Bim and Bid and inhibition of NF-κB transactivation. Additionally, zeaxanthin sensitized A2058 melanoma cells in vitro to the cytotoxic activity of vemurafenib, a BRAF inhibitor widely used for the clinical management of melanoma, suggesting its potential interest as dietary adjuvant increasing melanoma cells sensitivity to chemotherapy. Keywords: Cancer, Carotenoid, Melanoma, Microalgae, Phytoplankton, Zeaxanthin

Depuis plus de 40 ans, les microalgues suscitent un immense intérêt du fait de leur fonction écologique majeure dans les écosystèmes aquatiques et grâce à leurs nombreuses applications en biotechnologie et pharmacologie marine Un champ d’étude très actif concerne la recherche dans ces micro organismes de molécules présentant un intérêt pour la santé humaine, pour la prévention, le diagnostic et le traitement des maladies Avec un nombre estimé de 30 000 à 1 000 000 d’espèces regroupées en 12 phyla les microalgues se sont adaptées à une grande diversité d’environnements photiques et de conditions de croissance et présentent donc une chimiodiversité pigmentaire très large en fonction des groupes taxonomiques considérés Si la fonction biologique de ces pigments dans la cellule algale est relativement bien connue, leur vaste potentiel d’applications en santé humaine reste à explorer Les équipes du LIENSs et de l’IFREMER Centre Atlantique collaborent à explorer ce réservoir d’innovation autour de la thématique « Pigments de microalgues pour la santé » Plusieurs projets et travaux ont ainsi été réalisés, portant notamment sur la recherche d’activités anticancéreuses, la purification et l’identification de pigments cytotoxiques et pro apoptotiques et la recherche de pigments phototoxiques pour des applications en photothérapie dynamique des tumeurs et photothérapie antibactérienne Plus particulièrement, ces travaux ont notamment permis de développer des technologies innovantes d’extraction de pigments sous micro ondes, de réaliser des études chimiotaxonomiques basées sur l’analyse des communautés de pigments, de purifier des caroténoïdes pro apoptotiques sur des cellules de mélanomes métastatiques chimiorésistantes, et de breveter l’utilisation d’un extrait pigmentaire de microalgues pour le traitement dermocosmétique de l’acné

Microalgae are photosynthetic microorganisms that have an extraordinary diversity which biotechnological potential is just beginning to be explored. Microalgae in biotechnology are presently the focus of an unprecedented surge in interest and investment worldwide. Understanding microalgal lipid accumulation under nitrogen starvation is of major interest for feedstocks, food and biofuel production. Although studies have been performed in model species, to this day it is critical to understand through particular mechanisms of biotechnological species. Tisochrysis lutea is traditionally used for aquaculture feed and presents advantages for lipid production. The wild type strain and a lipid over-accumulating selected mutant were used to perform the first comparative proteomic analysis about domesticated oleaginous micoalgae. 2-DE analysis was performed on two metabolic conditions for both strains and revealed significant differences in proteoms between the two strains and depending on nitrogen starvation. MS analysis allowed us to identify proteins. Four candidates were selected for their potential implication in lipid over accumulation during nitrogen starvation. They include proteins involved in biosynthesis of fatty acids, carbon homeostasis and carbohydrate catabolism. This proteomic study confirms the impacts of nitrogen starvation on global metabolism and provides insights on regulation of lipid metabolism in the oleaginous haptophyta Tisochrysis lutea.