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Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin. These findings identify an unanticipated role for ciguatoxin in the regulation of homeostatic plasticity in central neurons involving NaV channels and raise the possibility that some of the neurological symptoms of ciguatera might be explained by these compensatory mechanisms.

Background: Zebrafish (Danio rerio) is a model organism that has emerged as a tool for cancer research, cancer being the second most common cause of death after cardiovascular disease for humans in the developed world. Zebrafish is a useful model for xenotransplantation of human cancer cells and toxicity studies of different chemotherapeutic compounds in vivo. Compared to the murine model, the zebrafish model is faster, can be screened using high-throughput methods and has a lower maintenance cost, making it possible and affordable to create personalized therapies. While several methods for cell proliferation determination based on image acquisition and quantification have been developed, some drawbacks still remain. In the xenotransplantation technique, quantification of cellular proliferation in vivo is critical to standardize the process for future preclinical applications of the model. Methods: This study improved the conditions of the xenotransplantation technique - quantification of cellular proliferation in vivo was performed through image processing with our ZFtool software and optimization of temperature in order to standardize the process for a future preclinical applications. ZFtool was developed to establish a base threshold that eliminates embryo auto-fluorescence and measures the area of marked cells (GFP) and the intensity of those cells to define a 'proliferation index'. Results: The analysis of tumor cell proliferation at different temperatures (34 °C and 36 °C) in comparison to in vitro cell proliferation provides of a better proliferation rate, achieved as expected at 36°, a maintenance temperature not demonstrated up to now. The mortality of the embryos remained between 5% and 15%. 5- Fluorouracil was tested for 2 days, dissolved in the incubation medium, in order to quantify the reduction of the tumor mass injected. In almost all of the embryos incubated at 36 °C and incubated with 5-Fluorouracil, there was a significant tumor cell reduction compared with the control group. This was not the case at 34 °C. Conclusions: Our results demonstrate that the proliferation of the injected cells is better at 36 °C and that this temperature is the most suitable for testing chemotherapeutic drugs like the 5-Fluorouracil. Fil: Cabezas-Sainz, Pablo. Universidad de Santiago de Compostela; España Fil: Guerra-Varela, Jorge. Universidad de Santiago de Compostela; España Fil: Carreira, María J.. Universidad de Santiago de Compostela; España Fil: Mariscal, Javier. Universidad de Santiago de Compostela; España Fil: Roel, María. Universidad de Santiago de Compostela; España Fil: Rubiolo, Juan Andrés. Universidad de Santiago de Compostela; España Fil: Sciara, Andres Angel. Universidad Nacional de Rosario; Argentina. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET) - Facultad de Bioquímica y Ciencias Farmacéuticas, ; Argentina Fil: Abal, Miguel. Universidad de Santiago de Compostela; España Fil: Botana, Luis M.. Universidad de Santiago de Compostela; España Fil: López, Rafael. Universidad de Santiago de Compostela; España Fil: Sánchez, Laura. Universidad de Santiago de Compostela; España

In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INa Inés Rodríguez is supported by a fellowship from Subprograma de Formación de Personal Investigador MINECO (AGL2012-40185-CO2-01), Spain. The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA – Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA. SI

// Maria Roel 1 , Juan A. Rubiolo 1 , Jorge Guerra-Varela 2 , Siguara B. L. Silva 3, 4 , Olivier P. Thomas 3, 5 , Pablo Cabezas-Sainz 2 , Laura Sanchez 2 , Rafael Lopez 6 , Luis M. Botana 1 1 Department of Pharmacology, Universidade de Santiago de Compostela, Campus Lugo, 27002 Lugo, Spain 2 Department of Genetics, Universidade de Santiago de Compostela, Campus Lugo, 27002 Lugo, Spain 3 Geoazur, UMR Universite Nice Sophia Antipolis-CNRS-IRD-OCA, 06560 Valbonne, France 4 Laboratoire de Pharmacognosie, UMR CNRS 8076 BioCIS, LabEx LERMIT, Universite Paris-Sud, Faculte de Pharmacie, 92290 Châtenay-Malabry, France 5 School of Chemistry, Marine Biodiscovery, National University of Ireland Galway, SW4 Galway, Ireland 6 Translational Medical Oncology, Health Research Institute of Santiago (IDIS), Complexo Hospitalario Universitario de Santiago de Compostela (SERGAS), 15706 Santiago de Compostela, Spain Correspondence to: Luis M. Botana, email: luis.botana@usc.es Keywords: crambescidins, cell cycle inhibition, apoptosis, zebrafish xenograft model, cancer treatment Received: March 13, 2016 Accepted: September 27, 2016 Published: November 04, 2016 ABSTRACT The marine environment constitutes an extraordinary resource for the discovery of new therapeutic agents. In the present manuscript we studied the effect of 3 different sponge derived guanidine alkaloids, crambescidine-816, -830, and -800. We show that these compounds strongly inhibit tumor cell proliferation by down-regulating cyclin-dependent kinases 2/6 and cyclins D/A expression while up-regulating the cell cyclin-dependent kinase inhibitors -2A, -2D and -1A. We also show that these guanidine compounds disrupt tumor cell adhesion and cytoskeletal integrity promoting the activation of the intrinsic apoptotic signaling, resulting in loss of mitochondrial membrane potential and concomitant caspase-3 cleavage and activation. The crambescidin 816 anti-tumor effect was fnally assayed in a zebrafish xenotransplantation model confirming its potent antitumor activity against colorectal carcinoma in vivo . Considering these results crambescidins could represent promising natural anticancer agents and therapeutic tools.

The Mediterranean marine sponge Crambe crambe is the source of two families of guanidine alkaloids known as crambescins and crambescidins. Some of the biological effects of crambescidins have been previously reported while crambescins have undergone little study. Taking this into account, we performed comparative transcriptome analysis to examine the effect of crambescin-C1 (CC1) on human tumor hepatocarcinoma cells HepG2 followed by validation experiments to confirm its predicted biological activities. We report herein that, while crambescin-A1 has a minor effect on these cells, CC1 protects them against oxidative injury by means of metallothionein induction even at low concentrations. Additionally, at high doses, CC1 arrests the HepG2 cell cycle in G0/G1 and thus inhibits tumor cell proliferation. The findings presented here provide the first detailed approach regarding the different effects of crambescins on tumor cells and provide a basis for future studies on other possible cellular mechanisms related to these bioactivities FEDER cofunded-grants. CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01 and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and by Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX, IN852A 2013/16-3 MYTIGAL. CDTIunderISIP Programme, Spain, IDI-20130304 APTAFOOD. European Union’s 7th Framework Programme managed by Research Executive Agency (FP7/2007-2013) under grant agreement Nos. 265409 µAQUA, 315285 CIGUATOOLS and 312184 PHARMASEA SI