Toxin Variability Estimations of 68 Alexandrium ostenfeldii (Dinophyceae) Strains from The Netherlands Reveal a Novel Abundant Gymnodimine

Alexandrium ostenfeldii is a toxic dinoflagellate that has recently bloomed in Ouwerkerkse Kreek, The Netherlands, and which is able to cause a serious threat to shellfish consumers and aquacultures. We used a large set of 68 strains to the aim of fully characterizing the toxin profiles of the Dutch A. ostenfeldii in consideration of recent reports of novel toxins. Alexandrium ostenfeldii is known as a causative species of paralytic shellfish poisoning, and consistently in the Dutch population we determined the presence of several paralytic shellfish toxins (PST) including saxitoxin (STX), GTX2/3 (gonyautoxins), B1 and C1/C2. We also examined the production of spiroimine toxins by the Dutch A. ostenfeldii strains. An extensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed a high intraspecific variability of spirolides (SPX) and gymnodimines (GYM). Spirolides included 13-desMethyl-spirolide C generally as the major compound and several other mostly unknown SPX-like compounds that were detected and characterized. Besides spirolides, the presence of gymnodimine A and 12-Methyl-gymnodimine A was confirmed, together with two new gymnodimines. One of these was tentatively identified as an analogue of gymnodimine D and was the most abundant gymnodimine (calculated cell quota up to 274 pg/ cell, expressed as GYM A equivalents). Our multi-clonal approach adds new analogues to the increasing number of compounds in these toxin classes and revealed a high strain variability in cell quota and in toxin profile of toxic compounds within a single population

Toxin Variability Estimations of 68 Alexandrium ostenfeldii (Dinophyceae) Strains from The Netherlands Reveal a Novel Abundant Gymnodimine

Alexandrium ostenfeldii is a toxic dinoflagellate that has recently bloomed in Ouwerkerkse Kreek, The Netherlands, and which is able to cause a serious threat to shellfish consumers and aquacultures. We used a large set of 68 strains to the aim of fully characterizing the toxin profiles of the Dutch A. ostenfeldii in consideration of recent reports of novel toxins. Alexandrium ostenfeldii is known as a causative species of paralytic shellfish poisoning, and consistently in the Dutch population we determined the presence of several paralytic shellfish toxins (PST) including saxitoxin (STX), GTX2/3 (gonyautoxins), B1 and C1/C2. We also examined the production of spiroimine toxins by the Dutch A. ostenfeldii strains. An extensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed a high intraspecific variability of spirolides (SPX) and gymnodimines (GYM). Spirolides included 13-desMethyl-spirolide C generally as the major compound and several other mostly unknown SPX-like compounds that were detected and characterized. Besides spirolides, the presence of gymnodimine A and 12-Methyl-gymnodimine A was confirmed, together with two new gymnodimines. One of these was tentatively identified as an analogue of gymnodimine D and was the most abundant gymnodimine (calculated cell quota up to 274 pg cell(-1), expressed as GYM A equivalents). Our multi-clonal approach adds new analogues to the increasing number of compounds in these toxin classes and revealed a high strain variability in cell quota and in toxin profile of toxic compounds within a single population.Peer reviewe

Cauda Equina Enhancing Lesion in an HIV-Infected Patient. Case Report and Literature Review.

We report the case of an HIV-infected young men with neuro-toxoplasmosis localized in the spinal cord. The patient received chemotherapy and immunotherapy for Burkitt lymphoma one year before. At the time of the diagnosis of toxoplasmosis, he was on prophylaxis with trimethoprim and sulfamethoxazole and in complete remission of Burkitt lymphoma. The CD4+ T cell count was 270/μl and the HIV viremia was undetectable. These findings suggest that in this patient, the immunodeficiency promoting the neurologic toxoplasmosis arose more from previous immuno-chemotherapy than from the HIV-infection itself. On the whole, this case highlights that the risk stratification for opportunistic infections of HIV-infected patients should carefully consider their previous medical history and therapies received

Improving in vitro ciguatoxin and brevetoxin detection: selecting neuroblastoma (Neuro-2a) cells with lower sensitivity to ouabain and veratridine (OV-LS).

Abstract Marine biotoxins accumulating in seafood products pose a risk to human health. These toxins are often potent in minute amounts and contained within complex matrices; requiring sensitive, reliable, and robust methods for their detection. The mouse neuroblastoma (Neuro-2a) cytotoxicity assay (N2a-assay) is a sensitive, high-throughput, in vitro method effective for detecting sodium channel-specific marine biotoxins. The N2a-assay can be conducted to distinguish between specific effects on voltage-gated sodium (NaV) channels, caused by toxins that activate (e.g., ciguatoxins (CTXs), brevetoxins (PbTxs)) or block (e.g., tetrodotoxins, saxitoxins) the target NaV. The sensitivity and specificity of the assay to compounds activating the NaV are achieved through the addition of the pharmaceuticals ouabain (O) and veratridine (V). However, these compounds can be toxic to Neuro-2a cells and their application at insufficient or excessive concentrations can reduce the effectiveness of this assay for marine toxin detection. Therefore, during growth incubation, Neuro-2a cells were exposed to O and V, and surviving cells exhibiting a lower sensitivity to O and V (OV-LS) were propagated. OV-LS Neuro-2a cells were selected for 60–80% survival when exposed to 0.22/0.022 mM O/V during the cytotoxicity assay. At these conditions, OV-LS N2a cells demonstrated a 3.5-fold higher survival rate 71% ± 7.9 SD (n = 232), and lower sensitivity to O/V, compared to the original Neuro-2a cells 20% ± 9.0 SD (n = 16). Additionally, OV-LS N2a cells were 1.3–2.6-fold more sensitive for detecting CTX3C 1.35 pg/ml, CTX1B 2.06 pg/ml, and PbTx-3 3.04 ng/ml compared to Neuro-2a cells using 0.1/0.01 mM O/V. Detection of CTX3C in a complex fish matrix using OV-LS cells was 0.0048 pg CTX3C/mg fish tissue equivalent. This work shows the potential for a significant improvement in sensitivity for CTX3C, CTX1B, and PbTx-3 using the OV-LS N2a-assay

The sxt Gene and Paralytic Shellfish Poisoning Toxins as Markers for the Monitoring of Toxic Alexandrium Species Blooms

Paralytic shellfish poisoning (PSP) is a serious human illness caused by the ingestion of seafood contaminated with saxitoxin and its derivatives (STXs). These toxins are produced by some species of marine dinoflagellates within the genus Alexandrium. In the Mediterranean Sea, toxic Alexandrium spp. blooms, especially of A. minutum, are frequent and intense with negative impact to coastal ecosystem, aquaculture practices and other economic activities. We conducted a large scale study on the sxt gene and toxin distribution and content in toxic dinoflagellate A. minutum of the Mediterranean Sea using both quantitative PCR (qPCR) and HILIC-HRMS techniques. We developed a new qPCR assay for the estimation of the sxtA1 gene copy number in seawater samples during a bloom event in Syracuse Bay (Mediterranean Sea) with an analytical sensitivity of 2.0 × 10° sxtA1 gene copy number per reaction. The linear correlation between sxtA1 gene copy number and microalgal abundance and between the sxtA1 gene and STX content allowed us to rapidly determine the STX-producing cell concentrations of two Alexandrium species in environmental samples. In these samples, the amount of sxtA1 gene was in the range of 1.38 × 105 − 2.55 × 108 copies/L and the STX concentrations ranged from 41−201 nmol/L. This study described a potential PSP scenario in the Mediterranean Sea.Versión del editor5,228

Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-κB

BACKGROUND: Palytoxin and, likely, its analogues produced by the dinoflagellate genus Ostreopsis, represent a class of non-proteinaceous compounds displaying high toxicity in animals. Owing to the wide distribution and the poisonous effects of these toxins in humans, their chemistry and mechanism of action have generated a growing scientific interest. Depending on the exposure route, palytoxin and its Ostreopsis analogues may cause several adverse effects on human health, including acute inflammatory reactions which seem more typical of cutaneous and inhalation contact. These observations have led us to hypothesize that these toxins may activate pro-inflammatory signalling cascades. METHODOLOGY AND PRINCIPAL FINDINGS: Here we demonstrate that palytoxin and a semi-purified Ostreopsis cf. ovata toxin extract obtained from a cultured strain isolated in the NW Adriatic Sea and containing a putative palytoxin and all the ovatoxins so far known--including the recently identified ovatoxin-f--significantly increase the levels of mRNAs encoding inflammation-related proteins in immune cells, i.e. monocyte-derived human macrophages, as assessed by Real-Time PCR analysis. Western immunoblot and electrophoretic mobility shift assays revealed that nuclear transcription factor -κB (NF-κB) is activated in cells exposed to toxins in coincidence with reduced levels of the inhibitory protein IκB-α. Moreover, Mitogen-Activated Protein Kinases (MAPK) were phosphorylated in response to palytoxin, as also reported by others, and to the Ostreopsis toxin extract, as shown here for the first time. By using specific chemical inhibitors, the involvement of NF-κB and p38 MAPK in the toxin-induced transcription and accumulation of Cycloxigenase-2, Tumor Necrosis Factor-α, and Interleukin-8 transcripts has been demonstrated. CONCLUSIONS AND SIGNIFICANCE: The identification of specific molecular targets of palytoxin and its Ostreopsis analogues, besides contributing to expand the still limited knowledge of the intracellular signalling cascades affected by these toxins, may have important implications in setting up focused pharmacological interventions, replacing currently used symptomatic treatments

Identification of a Sorbicillinoid-Producing Aspergillus Strain with Antimicrobial Activity Against Staphylococcus aureus: a New Polyextremophilic Marine Fungus from Barents Sea

The exploration of poorly studied areas of Earth can highly increase the possibility to discover novel bioactive compounds. In this study, the cultivable fraction of fungi and bacteria from Barents Sea sediments has been studied to mine new bioactive molecules with antibacterial activity against a panel of human pathogens. We isolated diverse strains of psychrophilic and halophilic bacteria and fungi from a collection of nine samples from sea sediment. Following a full bioassay-guided approach, we isolated a new promising polyextremophilic marine fungus strain 8Na, identified as Aspergillusprotuberus MUT 3638, possessing the potential to produce antimicrobial agents. This fungus, isolated from cold seawater, was able to grow in a wide range of salinity, pH and temperatures. The growth conditions were optimised and scaled to fermentation, and its produced extract was subjected to chemical analysis. The active component was identified as bisvertinolone, a member of sorbicillonoid family that was found to display significant activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 30 μg/mL. © 2018, Springer Science+Business Media, LLC, part of Springer Nature

Toxin Levels and Profiles in Microalgae from the North-Western Adriatic Sea—15 Years of Studies on Cultured Species

The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported

Analisi di biotossine marine in mitili del mar Mediterraneo e dei loro organismi produttori

L’attività di ricerca che ho svolto durante i tre anni di dottorato presso il Dipartimento di Chimica delle Sostanze Naturali, ha riguardato la chimica delle biotossine marine, filone di ricerca da anni investigato dal gruppo del prof. Fattorusso. Tale tematica risulta di notevole rilevanza sia dal punto di vista igienico-sanitario che economico poichè ogni anno, l'industria ittica subisce repentine interruzioni della propria attività a causa dell'insorgere di tossicità da biotossine nei mitili. Il mio lavoro si è focalizzato sugli aspetti analitici del problema, infatti, con particolare riferimento alla tecnica LC-ESI-ion trap- MS e LC- TIS- triple quad- MS, mi sono occupata dello studio di tossine appartenenti alle classi delle DSP (Diarrhetic Shellfish Poisoning), ASP (Amnesic Shellfish Poisoning) e degli Spirolidi (Fast Acting Toxins). Ho dunque analizzato non solo campioni di mitili contaminati (responsabili del trasferimento della tossicità all'uomo) ma anche campioni di cellule (reali sintetizzatori delle tossine in questione) sia in retinata che in coltura. Ho messo a punto una metodica HILIC-MS basata sull’accoppiamento della cromatografia liquida ad interazioni idrofiliche e la spettrometria di massa, che permette la rivelazione e quantizzazione contemporanea, anche al di sotto dei limiti previsti dalla legge, di Acido Domoico (tossina amnesica, ASP) e delle tossine paralitiche (PSP), tenendo conto dell’effetto matrice, sempre correlato alla rivelazione LC-MS. Attraverso questa metodica ho messo in luce, per la prima volta nei mitili coltivati nel mar Adriatico, la presenza di acido domoico, che complica ulteriormente il profilo tossicologico dei mitili coltivati nei nostri mari. Ciò rende necessario l’inserimento dell’acido domoico nel panorama di tossine da monitorare nei Mytilus galloprovincialis italiani. Inoltre ho contribuito all’identificazione di una nuova specie tossica della diatomea Pseudonitzschia, la Pseudonitzschia galaxiae del golfo di Napoli, che, grazie alla sensibilità del metodo su menzionato, è risultata produttrice di acido domoico. Mi sono occupata anche dell’indagine LC-MS di un ceppo del dinoflagellato Alexandrium ostenfeldii proveniente dal mar Adriatico che si è rivelato produttore di una tossina mai prima d’ora riscontrata nei mari italiani: lo spirolide 13-desMe-C, appartenente ad una classe di tossine ad azione rapida. Inoltre, via LC-MS ho evidenziato dallo stesso ceppo, la presenza di 3 nuovi spirolidi, riscontrati però in tracce, che non hanno consentito la completa elucidazione strutturale. Ho poi analizzato ancora attraverso la tecnica LC-MS, colture del dinoflagellato Protoceratium reticulatum cresciuto in diverse condizioni di salinità, indagando gli effetti che tale parametro induce sulla produzione e rilascio nel mezzo di coltura di yessotossine (tossine cardiotossiche) evidenziando una diminuzione della YTX rilasciata nel mezzo, all’aumentare della salinità. Infine ho analizzato via LC-MS un ceppo del dinoflagellato Prorocentrum lima raccolto dall’Adriatico mettendo in luce la presenza dell’acido okadaico (tossina diarroica e tumor-promoter) e della dynofisistossina-1 insieme ai quali ho rivelato una serie di derivati esterei, tra cui alcuni noti ed altri sconosciuti