Targeting cancer stem cell OXPHOS with tailored ruthenium complexes as a new anti-cancer strategy.

BACKGROUND: Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking. METHODS: The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action. RESULTS: We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration. CONCLUSIONS: Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.

Non-synonymous variation and protein structure of candidate genes associated with selection in farm and wild populations of turbot (Scophthalmus maximus).

Non-synonymous variation (NSV) of protein coding genes represents raw material for selection to improve adaptation to the diverse environmental scenarios in wild and livestock populations. Many aquatic species face variations in temperature, salinity and biological factors throughout their distribution range that is reflected by the presence of allelic clines or local adaptation. The turbot (Scophthalmus maximus) is a flatfish of great commercial value with a flourishing aquaculture which has promoted the development of genomic resources. In this study, we developed the first atlas of NSVs in the turbot genome by resequencing 10 individuals from Northeast Atlantic Ocean. More than 50,000 NSVs where detected in the ~ 21,500 coding genes of the turbot genome, and we selected 18 NSVs to be genotyped using a single Mass ARRAY multiplex on 13 wild populations and three turbot farms. We detected signals of divergent selection on several genes related to growth, circadian rhythms, osmoregulation and oxygen binding in the different scenarios evaluated. Furthermore, we explored the impact of NSVs identified on the 3D structure and functional relationship of the correspondent proteins. In summary, our study provides a strategy to identify NSVs in species with consistently annotated and assembled genomes to ascertain their role in adaptation.

A continuous in silico learning strategy to identify safety liabilities in compounds used in the leather and textile industry.

There is a widely recognized need to reduce human activity's impact on the environment. Many industries of the leather and textile sector (LTI), being aware of producing a significant amount of residues (Keßler et al. 2021; Liu et al. 2021), are adopting measures to reduce the impact of their processes on the environment, starting with a more comprehensive characterization of the chemical risk associated with the substances commonly used in LTI. The present work contributes to these efforts by compiling and toxicologically annotating the substances used in LTI, supporting a continuous learning strategy for characterizing their chemical safety. This strategy combines data collection from public sources, experimental methods and in silico predictions for characterizing four different endpoints: CMR, ED, PBT, and vPvB. We present the results of a prospective validation exercise in which we confirm that in silico methods can produce reasonably good hazard estimations and fill knowledge gaps in the LTI chemical space. The proposed protocol can speed the process and optimize the use of resources including the lives of experimental animals, contributing to identifying potentially harmful substances and their possible replacement by safer alternatives, thus reducing the environmental footprint and impact on human health.

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis.

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.

Manganese(I) tricarbonyl complexes as potential anticancer agents.

The antiproliferative activity of [Mn(CO)3(N^N)Br] (N^N = phendione 1, bipy 3) and of the two newly synthesized Mn complexes [Mn(CO)3(acridine)(phendione)]OTf (2) and [Mn(CO)3(di-triazole)Br] (4) has been evaluated by MTS against three tumor cell lines A2780 (ovarian carcinoma), HCT116 (colorectal carcinoma), HCT116doxR (colorectal carcinoma resistant to doxorubicin), and in human dermal fibroblasts. The antiproliferative assay showed a dose-dependent effect higher in complex 1 and 2 with a selectivity toward ovarian carcinoma cell line 21 times higher than in human fibroblasts. Exposure of A2780 cells to IC50 concentrations of complex 1 and 2 led to an increase of reactive oxygen species that led to the activation of cell death mechanisms, namely via intrinsic apoptosis for 2 and autophagy and extrinsic apoptosis for 1. Both complexes do not target DNA or interfere with cell cycle progression but are able to potentiate cell migration and neovascularization (for 2) an indicative that their application might be directed for initial tumor stages to avoid tumor invasion and metastization and opening a new avenue for complex 2 application in regenerative medicine. Interestingly, both complexes do not show toxicity in both in vivo models (CAM and zebrafish).

Brains in Metamorphosis: Temporal Transcriptome Dynamics in Hatchery-Reared Flatfishes.

Metamorphosis is a captivating process of change during which the morphology of the larva is completely reshaped to face the new challenges of adult life. In the case of fish, this process initiated in the brain has traditionally been considered to be a critical rearing point and despite the pioneering molecular work carried out in other flatfishes, the underlying molecular basis is still relatively poorly characterized. Turbot brain transcriptome of three developmental stages (pre-metamorphic, climax of metamorphosis and post-metamorphic) were analyzed to study the gene expression dynamics throughout the metamorphic process. A total of 1570 genes were differentially expressed in the three developmental stages and we found a specific pattern of gene expression at each stage. Unexpectedly, at the climax stage of metamorphosis, we found highly expressed genes related to the immune response, while the biological pathway enrichment analysis in pre-metamorphic and post-metamorphic were related to cell differentiation and oxygen carrier activity, respectively. In addition, our results confirm the importance of thyroid stimulating hormone, increasing its expression during metamorphosis. Based on our findings, we assume that immune system activation during the climax of metamorphosis stage could be related to processes of larval tissue inflammation, resorption and replacement, as occurs in other vertebrates.

N-Heterocyclic Carbene Iron Complexes as Anticancer Agents: In Vitro and In Vivo Biological Studies.

Cisplatin and its derivatives are commonly used in chemotherapeutic treatments of cancer, even though they suffer from many toxic side effects. The problems that emerge from the use of these metal compounds led to the search for new complexes capable to overcome the toxic side effects. Here, we report the evaluation of the antiproliferative activity of Fe(II) cyclopentadienyl complexes bearing n-heterocyclic carbene ligands in tumour cells and their in vivo toxicological profile. The in vitro antiproliferative assays demonstrated that complex Fe1 displays the highest cytotoxic activity both in human colorectal carcinoma cells (HCT116) and ovarian carcinoma cells (A2780) with IC50 values in the low micromolar range. The antiproliferative effect of Fe1 was even higher than cisplatin. Interestingly, Fe1 showed low in vivo toxicity, and in vivo analyses of Fe1 and Fe2 compounds using colorectal HCT116 zebrafish xenograft showed that both reduce the proliferation of human HCT116 colorectal cancer cells in vivo.

Conservation of Zebrafish MicroRNA-145 and Its Role during Neural Crest Cell Development.

The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3'UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expression.

Crambescin C1 Acts as A Possible Substrate of iNOS and eNOS Increasing Nitric Oxide Production and Inducing In Vivo Hypotensive Effect.

Crambescins are guanidine alkaloids from the sponge Crambe crambe. Crambescin C1 (CC) induces metallothionein genes and nitric oxide (NO) is one of the triggers. We studied and compared the in vitro, in vivo, and in silico effects of some crambescine A and C analogs. HepG2 gene expression was analyzed using microarrays. Vasodilation was studied in rat aortic rings. In vivo hypotensive effect was directly measured in anesthetized rats. The targets of crambescines were studied in silico. CC and homo-crambescine C1 (HCC), but not crambescine A1 (CA), induced metallothioneins transcripts. CC increased NO production in HepG2 cells. In isolated rat aortic rings, CC and HCC induced an endothelium-dependent relaxation related to eNOS activation and an endothelium-independent relaxation related to iNOS activation, hence both compounds increase NO and reduce vascular tone. In silico analysis also points to eNOS and iNOS as targets of Crambescin C1 and source of NO increment. CC effect is mediated through crambescin binding to the active site of eNOS and iNOS. CC docking studies in iNOS and eNOS active site revealed hydrogen bonding of the hydroxylated chain with residues Glu377 and Glu361, involved in the substrate recognition, and explains its higher binding affinity than CA. The later interaction and the extra polar contacts with its pyrimidine moiety, absent in the endogenous substrate, explain its role as exogenous substrate of NOSs and NO production. Our results suggest that CC serve as a basis to develop new useful drugs when bioavailability of NO is perturbed.

Inhibition of Mitochondrial Dynamics Preferentially Targets Pancreatic Cancer Cells with Enhanced Tumorigenic and Invasive Potential.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors, partly due to its intrinsic aggressiveness, metastatic potential, and chemoresistance of the contained cancer stem cells (CSCs). Pancreatic CSCs strongly rely on mitochondrial metabolism to maintain their stemness, therefore representing a putative target for their elimination. Since mitochondrial homeostasis depends on the tightly controlled balance between fusion and fission processes, namely mitochondrial dynamics, we aim to study this mechanism in the context of stemness. In human PDAC tissues, the mitochondrial fission gene DNM1L (DRP1) was overexpressed and positively correlated with the stemness signature. Moreover, we observe that primary human CSCs display smaller mitochondria and a higher DRP1/MFN2 expression ratio, indicating the activation of the mitochondrial fission. Interestingly, treatment with the DRP1 inhibitor mDivi-1 induced dose-dependent apoptosis, especially in CD133+ CSCs, due to the accumulation of dysfunctional mitochondria and the subsequent energy crisis in this subpopulation. Mechanistically, mDivi-1 inhibited stemness-related features, such as self-renewal, tumorigenicity, and invasiveness and chemosensitized the cells to the cytotoxic effects of Gemcitabine. In summary, mitochondrial fission is an essential process for pancreatic CSCs and represents an attractive target for designing novel multimodal treatments that will more efficiently eliminate cells with high tumorigenic potential.

Morphological Abnormalities and Gene Expression Changes Caused by High Incubation Temperatures in Zebrafish Xenografts with Human Cancer Cells.

Published studies show that most of the human cancer xenograft studies in zebrafish embryos have used incubation temperatures in the range of 32-34 °C for 3-6 days post-injection, trying to find a compromise temperature between the zebrafish embryos (28 °C) and the human injected cells (37 °C). While this experimental setup is widely used, a question remains: is possible to overcome the drawbacks caused by a suboptimal temperature for the injected cells? To clarify the effect of temperature and injected cells on the host, in this study, we analyzed the development and health of the last in response to different temperatures in the presence or absence of injected human cancer cells. Comparing different incubation temperatures (28, 34 and 36 °C), we determined morphological abnormalities and developmental effects in injected and non-injected embryos at different time points. Besides this, the expression of selected genes was determined by qPCR to determine temperature affected metabolic processes in the embryos. The results indicate that an incubation temperature of 36 °C during a period of 48 h is suitable for xenotransplantation without morphological or metabolic changes that could be affecting the host or the injected cells, allowing them to proliferate near their optimal temperature.

The hemoglobin Gly16ß1Asp polymorphism in turbot (Scophthalmus maximus) is differentially distributed across European populations.

Turbot is an important flatfish widely distributed along the European coasts, whose fishery is centered in the North Sea. The commercial value of the species has boosted a successful aquaculture sector in Europe and China. Body growth is the main target of turbot breeding programs and is also a key trait related to local adaptation to temperature and salinity. Differences in growth rate and optimal growth temperature in turbot have been shown to be associated with a hemoglobin polymorphism reported more than 50 years ago. Here, we identified a Gly16Asp amino acid substitution in the ß1 globin subunit by searching for genetic variation in the five functional globin genes within the whole annotated turbot genome. We predicted increased stability of the turbot hemoglobin by the replacement of the conserved Gly with the negative charged Asp residue that is consistent with the higher rate of αß dimer assembly in the human J-Baltimore Gly16ß->Asp mutant than in normal HbA. The turbot Hbß1-Gly16 variant dominated in the northern populations examined, particularly in the Baltic Sea, while the Asp allele showed elevated frequencies in southern populations and was the prevalent variant in the Adriatic Sea. Body weight did not associate with the Hbß1 genotypes at farming conditions (i.e., high oxygen levels, feeding ad libitum) after analyzing 90 fish with high growth dispersal from nine turbot families. Nevertheless, all data at hand suggest that the turbot hemoglobin polymorphism has an adaptive significance in the variable wild conditions regarding temperature and oxygen availability.

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells.

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7-9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.

New insights into the Manila clam - Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges.

The Manila clam (Ruditapes philippinarum) is the bivalve species with the highest global production from both fisheries and aquaculture, but its production is seriously threatened by perkinsosis, a disease caused by the protozoan parasite Perkinsus olseni. To understand the molecular mechanisms underlying R. philippinarum-P. olseni interactions, we analysed the gene expression profiles of in vitro challenged clam hemocytes and P. olseni trophozoites, using two oligo-microarray platforms, one previously validated for R. philippinarum hemocytes and a new one developed and validated in this study for P. olseni. Manila clam hemocytes were in vitro challenged with trophozoites, zoospores, and extracellular products from P. olseni in vitro cultures, while P. olseni trophozoites were in vitro challenged with Manila clam plasma along the same time-series (1 h, 8 h, and 24 h). The hemocytes showed a fast activation of the innate immune response, particularly associated with hemocyte recruitment, in the three types of challenges. Nevertheless, different immune-related pathways were activated in response to the different parasite stages, suggesting specific recognition mechanisms. Furthermore, the analyses provided useful complementary data to previous in vivo challenges, and confirmed the potential of some proposed biomarkers. The combined analysis of gene expression in host and parasite identified several processes in both the clam and P. olseni, such as redox and glucose metabolism, protease activity, apoptosis and iron metabolism, whose modulation suggests cross-talk between parasite and host. This information might be critical to determine the outcome of the infection, thus highlighting potential therapeutic targets. Altogether, the results of this study aid understanding the response and interaction between R. philippinarum and P. olseni, and will contribute to developing effective control strategies for this threatening parasitosis.

In vivo toxicity assays in zebrafish embryos: a pre-requisite for xenograft preclinical studies.

The human cancer cell xenograft in zebrafish embryos has become a very useful preclinical tool in oncology research. While many anticancer drugs have been assayed with this model, few studies regarding the toxicity limits of these drugs for the host have been addressed. Here, we evaluated the acute toxicity of five approved and routinely used human anticancer drugs embracing different mechanism action types: Carboplatin (CarboPt), Irinotecan (IT), Doxorubicin (DOX), Paclitaxel (PT) and Chloroquine (CQ). They were tested in zebrafish embryos using the Fish Embryo Acute Toxicity (FET) test at 0 and 72 hours per fertilization (hpf). Additionally, we compared those results with in vitro toxicity assays and could find notable differences between both models. Our results indicate that the toxicity data of a compound evaluated in vitro and in a FET test at 0 hpf do not guarantee a reliable toxicity determination for performing xenografts in zebrafish, being necessary additional toxicity studies using 72 hpf embryos, the starting point of drug treatment in this kind of preclinical assays.

Transcriptomic Analysis of Ciguatoxin-Induced Changes in Gene Expression in Primary Cultures of Mice Cortical Neurons.

Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin.

Improving zebrafish embryo xenotransplantation conditions by increasing incubation temperature and establishing a proliferation index with ZFtool.

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.

Gene expression analysis of Ruditapes philippinarum haemocytes after experimental Perkinsus olseni zoospore challenge and infection in the wild.

The production of Manila clam (Ruditapes philippinarum) is seriously threatened by the protistan parasite Perkinsus olseni. We characterized and compared gene expression of Manila clam haemocytes in response to P. olseni in a time-course (10 h, 24 h, 8 d) controlled laboratory challenge (LC), representing the first step of infection, and in a more complex infection in the wild (WI), using a validated oligo-microarray containing 11,232 transcripts, mostly annotated. Several immune-genes involved in NIK/NF-kappaB signalling, Toll-like receptor signalling and apoptosis were activated at LC-10 h. However, down-regulation of genes encoding lysozyme, histones, cathepsins and heat shock proteins indicated signals of immunodepression, which persisted at LC-24 h, when only down-regulated genes were detected. A rebound of haemocyte activity occurred at LC-8 d as shown by up-regulation of genes involved in cytoskeleton organization and cell survival. The WI study showed a more complex picture, and several immune-relevant processes including cytoskeleton organization, cell survival, apoptosis, encapsulation, cell redox- and lipid-homeostasis were activated, illustrating the main mechanism of host response. Our results provide useful information, including potential biomarkers, to develop strategies for controlling Manila clam perkinsosis.

Differential gene expression and SNP association between fast- and slow-growing turbot (Scophthalmus maximus).

Growth is among the most important traits for animal breeding. Understanding the mechanisms underlying growth differences between individuals can contribute to improving growth rates through more efficient breeding schemes. Here, we report a transcriptomic study in muscle and brain of fast- and slow-growing turbot (Scophthalmus maximus), a relevant flatfish in European and Asian aquaculture. Gene expression and allelic association between the two groups were explored. Up-regulation of the anaerobic glycolytic pathway in the muscle of fast-growing fish was observed, indicating a higher metabolic rate of white muscle. Brain expression differences were smaller and not associated with major growth-related genes, but with regulation of feeding-related sensory pathways. Further, SNP variants showing frequency differences between fast- and slow-growing fish pointed to genomic regions likely involved in growth regulation, and three of them were individually validated through SNP typing. Although different mechanisms appear to explain growth differences among families, general mechanisms seem also to be involved, and thus, results provide a set of useful candidate genes and markers to be evaluated for more efficient growth breeding programs and to perform comparative genomic studies of growth in fish and vertebrates.

The association of bacterial C9-based TTX-like compounds with Prorocentrum minimum opens new uncertainties about shellfish seafood safety.

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.