Detection of Anatoxin-a and Three Analogs in Anabaena spp. Cultures: New Fluorescence Polarization Assay and Toxin Profile by LC-MS/MS

Anatoxin-a (ATX) is a potent neurotoxin produced by several species of Anabaena spp. Cyanobacteria blooms around the world have been increasing in recent years; therefore, it is urgent to develop sensitive techniques that unequivocally confirm the presence of these toxins in fresh water and cyanobacterial samples. In addition, the identification of different ATX analogues is essential to later determine its toxicity. In this paper we designed a fluorescent polarization (FP) method to detect ATXs in water samples. A nicotinic acetylcholine receptor (nAChR) labeled with a fluorescein derivative was used to develop this assay. Data showed a direct relationship between the amount of toxin in a sample and the changes in the polarization degree of the emitted light by the labeled nAChR, indicating an interaction between the two molecules. This method was used to measure the amount of ATX in three Anabaena spp. cultures. Results indicate that it is a good method to show ATXs presence in algal samples. In order to check the toxin profile of Anabaena cultures a LC-MS/MS method was also developed. Within this new method, ATX-a, retention time (RT) 5 min, and three other molecules with a mass m/z 180.1 eluting at 4.14 min, 5.90 min and 7.14 min with MS/MS spectra characteristic of ATX toxin group not previously identified were detected in the Anabaena spp. cultures. These ATX analogues may have an important role in the toxicity of the sampleThe research leading to these results has received funding from the following FEDER cofunded-grants: From Ministerio de Ciencia y Tecnología, Spain: AGL2009-13581-CO2-01, AGL2012-40485-CO2-01. From Xunta de Galicia, Spain: 10PXIB261254 PR. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency http://ec.europa.eu/research/rea (FP7/2007-2013) under grant agreement Nos. 211326—CP (CONffIDENCE), 265896 BAMMBO, 265409 µAQUA, and 262649 BEADS, 315285 Ciguatools and 312184 PharmaSea. From the Atlantic Area Programme (Interreg IVB Trans-national): 2008-1/003 (Atlantox) and 2009-1/117 (Pharmatlantic)S

Bio essaies pour la détection et identification d'imines cycliques toxiaues

International audienceMarine neurotoxins move-up fast in the food chain through shellfish that filter-feed on toxic dinoflagellate species constituting a serious hazard for Public Health. Monitoring of regulated marine toxins is performed by physicochemical analyses. It is essential however to develop functional bioassays as an alternative to mouse bioassay for high-throughput detection of novel and emergent marine toxins. Cyclic imine toxins are very-fast acting neurotoxins highly lethal to mice acting as potent antagonists of nicotinic acetylcholine receptors. Although not regulated, chronic exposition to cyclic imine toxins is of concern given their high affinity for neuronal and muscle nicotinic receptors and their capacity to cross the intestinal and the brain blood barriers. A series of bioassays based on the mode of action of cyclic imine toxins are here reviewed.Proceedings of the 18th International Conference on Harmful Algae 21-26 October 2018 Nantes, France

Isolation, purification and functional characterization of alpha-BnIA from Conus bandanus venom.

International audienceWe report the isolation and characterization by proteomic approach of a native conopeptide, named BnIA, from the crude venom of Conus bandanus, a molluscivorous cone snail species, collected in the South central coast of Vietnam. Its primary sequence was determined by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry using collision-induced dissociation and confirmed by Edman's degradation of the pure native fraction. BnIA was present in high amounts in the crude venom and the complete sequence of the 16 amino acid peptide was the following GCCSHPACSVNNPDIC*, with C-terminal amidation deduced from Edman's degradation and theoretical monoisotopic mass calculation. Sequence alignment revealed that its -C1C2X4C3X7C4- pattern belongs to the A-superfamily of conopeptides. The cysteine connectivity of BnIA was 1-3/2-4 as determined by partial-reduction technique, like other α4/7-conotoxins, reported previously on other Conus species. Additionally, we found that native α-BnIA shared the same sequence alignment as Mr1.1, from the closely related molluscivorous Conus marmoreus venom, in specimens collected in the same coastal region of Vietnam. Functional studies revealed that native α-BnIA inhibited acetylcholine-evoked currents reversibly in oocytes expressing the human α7 nicotinic acetylcholine receptors, and blocked nerve-evoked skeletal muscle contractions in isolated mouse neuromuscular preparations, but with ∼200-times less potency

Detection of Anatoxin-a and Three Analogs in Anabaena spp. Cultures: New Fluorescence Polarization Assay and Toxin Profile by LC-MS/MS

Anatoxin-a (ATX) is a potent neurotoxin produced by several species of Anabaena spp. Cyanobacteria blooms around the world have been increasing in recent years; therefore, it is urgent to develop sensitive techniques that unequivocally confirm the presence of these toxins in fresh water and cyanobacterial samples. In addition, the identification of different ATX analogues is essential to later determine its toxicity. In this paper we designed a fluorescent polarization (FP) method to detect ATXs in water samples. A nicotinic acetylcholine receptor (nAChR) labeled with a fluorescein derivative was used to develop this assay. Data showed a direct relationship between the amount of toxin in a sample and the changes in the polarization degree of the emitted light by the labeled nAChR, indicating an interaction between the two molecules. This method was used to measure the amount of ATX in three Anabaena spp. cultures. Results indicate that it is a good method to show ATXs presence in algal samples. In order to check the toxin profile of Anabaena cultures a LC-MS/MS method was also developed. Within this new method, ATX-a, retention time (RT) 5 min, and three other molecules with a mass m/z 180.1 eluting at 4.14 min, 5.90 min and 7.14 min with MS/MS spectra characteristic of ATX toxin group not previously identified were detected in the Anabaena spp. cultures. These ATX analogues may have an important role in the toxicity of the sample

Antigen discovery: a postgenomic approach to leprosy diagnosis.

International audienceLeprosy is an infectious, neurodegenerative disease of humans caused by Mycobacterium leprae. Despite effective control programs, the incidence of leprosy remains stubbornly high, suggesting that transmission may be more common than expected. The rationale of this work was to use bioinformatics and comparative genomics to identify potentially antigenic proteins for diagnostic purposes. This approach defined three classes of proteins: those restricted to M. leprae (class I), those present in M. leprae with orthologues in other organisms besides mycobacteria (class II), and exported or surface-exposed proteins (class III). Twelve genes (two class I, four class II, and six class III proteins) were cloned in Escherichia coli, and their protein products were purified. Six of these proteins were detected in cell extracts of M. leprae by immunoblotting. The immunogenicity of each recombinant protein was then investigated in leprosy patients by measuring the reactivity of circulating antibody and gamma interferon (IFN-gamma) responses in T-cell restimulation assays. Several class II and class III proteins were recognized by circulating antibodies. Importantly, most class II proteins elicited IFN-gamma responses that were significantly stronger than those produced by previously identified antigens. Among them, two class II proteins, ML0308 and ML2498, showed marked humoral and cellular immunogenicity, therefore providing promising candidates for the diagnosis of both tuberculoid and lepromatous forms of leprosy

Antigen discovery: a postgenomic approach to leprosy diagnosis

Leprosy is an infectious, neurodegenerative disease of humans caused by Mycobacterium leprae. Despite effective control programs, the incidence of leprosy remains stubbornly high, suggesting that transmission may be more common than expected. The rationale of this work was to use bioinformatics and comparative genomics to identify potentially antigenic proteins for diagnostic purposes. This approach defined three classes of proteins: those restricted to M. leprae (class I), those present in M. leprae with orthologues in other organisms besides mycobacteria (class II), and exported or surface-exposed proteins (class III). Twelve genes (two class I, four class II, and six class III proteins) were cloned in Escherichia coli, and their protein products were purified. Six of these proteins were detected in cell extracts of M. leprae by immunoblotting. The immunogenicity of each recombinant protein was then investigated in leprosy patients by measuring the reactivity of circulating antibody and gamma interferon (IFN-gamma) responses in T-cell restimulation assays. Several class II and class III proteins were recognized by circulating antibodies. Importantly, most class II proteins elicited IFN-gamma responses that were significantly stronger than those produced by previously identified antigens. Among them, two class II proteins, ML0308 and ML2498, showed marked humoral and cellular immunogenicity, therefore providing promising candidates for the diagnosis of both tuberculoid and lepromatous forms of leprosy