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  • Open access
  • 138 Reads
Recombinase Polymerase Amplification for Gambierdiscus and Fukuyoa detection: a step further in the ciguatera risk management
Published: 14 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Ciguatera fish poisoning is one of the most relevant seafood-borne illnesses worldwide. It is caused by the ingestion of fish contaminated by ciguatoxins (CTXs). Primary producers of CTXs are dinoflagellates of the genera Gambierdiscus and Fukuyoa.

This study is focused on the development of bioanalytical tools for the detection of Gambierdiscus and Fukuyoa. To achieve this objective, Recombinase Polymerase Amplification (RPA), which consists of an isothermal DNA amplification during a short period (30 minutes), was combined with an enzyme-linked oligonucleotide assay (ELONA).

To evaluate the specificity of the RPA-ELONA, firstly primers for the genera Gambierdiscus/Fukuyoa were exposed to genomic DNA of different species (G. australes, G. excentricus, G. belizeanus, G. balechi and F. paulensis) and other microalgae used as controls (O. cf. ovata, P. lima and C. monotis). The same genomic DNA pools were also tested with species-specific primers for Gambierdiscus australes and Gambierdiscus excentricus. Finally, DNA was extracted from single cells of the previously mentioned genera and species, and tested with all the primer sets. For both the experiments, detection was achieved only when combining capture probes with their target RPA product, and no significant responses were observed in the presence of non-target DNA.

Obtained results demonstrate the ability of the system to discriminate not only the genus Gambierdiscus/Fukuyoa from other microalgae, but also G. australes and G. excentricus species from their congeners. Furthermore, the limit of detection is as low as a single cell.

Acknowledgments: The research has received funding from the Ministerio de Ciencia, Innovación y Universidades through the CIGUASENSING (BIO2017-87946-C2-2-R) project. The authors acknowledge support from CERCA Programme/Generalitat de Catalunya. G. Gaiani acknowledges IRTA-Universitat Rovira i Virgili-Banco Santander for her PhD grant (2018PMF-PIPF-19).

  • Open access
  • 73 Reads
The antitumoral potential of Pllans-II, an acidic PLA2 from Porthidium lansbergii lansbergii snake venom on human cervical carcinoma cells.
Published: 14 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Pllans-II, an Asp49-type acidic phospholipase A2 from Porthidium lansbergii lansbergii snake venom, displayed for the first time antitumoral potential against the squamous epithelial cell line of cervical cancer Ca Ski. Pllans–II presented a dose-dependent cytotoxic effect on cancer cells and an insignificant effect on healthy endothelial cells HUVEC. Pllans–II also inhibited the adhesion and migration ability of cancer cells and induced cell cycle arrest in the G2/M phase and apoptosis on Ca Ski cells. Transcriptomic analysis revealed that cell death was related to endoplasmic reticulum stress by interfering with α5 and β1–containing integrins. These results demonstrate that Pllans–II has an antitumor potential on cervical cancer and represents a possible biotechnological tool for designing anticancer prototypes.

  • Open access
  • 268 Reads
Cyclodextrins as capture agents of lipophilic marine toxins
Published: 14 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Seafood contamination with marine toxins due to harmful algal blooms (HAB’s) is a global public health issue on the rise. Most Countries have monitoring programs for the detection of toxins in shellfish of toxic phytoplankton in seawater to prevent consumer intoxifications. The use of solid phase adsorbent and toxin tracking (SPATT) technology as toxin detection straight from the aquatic environment could complement the labour-intensive traditional monitoring methods. In this work, several types of cyclodextrins (cyclic oligomers with a conical structure and an internal cavity) have been evaluated as novel materials for SPATT. Cyclodextrins were tested at Masnou harbour (Catalonia, NW Mediterranean) during a Dinophysis sp. bloom. The cyclodextrins and the commercial Diaion (HP-20) were deployed twice for a 1-week period at five different locations of Masnou harbour. At the time of the experiment, Dinophysis sp. reached abundances as high as 91 341 cells /L.

Successful accumulations of the lipophilic marine toxins; okadaic acid (OA) and pectenotoxin-2 (PTX2) were quantified by Liquid Chromatography- tandem Mass Spectrometry (LC-MS/MS).

Higher levels of PTX2 were found in all cyclodextrins whereas OA and PTX2 contents were similar in the commercial resin. Accumulation of OA was higher in the commercial resin than in cyclodextrins, but these proved best for PTX2 adsorption. A clear correlation between cell abundance and toxin accumulation was observed.

The research has received funding from the Ministerio de Ciencia, Innovación y Universidades (MICINN), the Agencia Estatal de Investigación (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER) through the CIGUASENSING project (BIO2017-87946-C2-1-R and BIO2017-87946-C2-2-R). The authors acknowledge support from CERCA Programme/Generalitat de Catalunya.

  • Open access
  • 126 Reads
Engineering the NADPH specificity of DepB, a novel aldo-keto reductase involved in the detoxification of the agroeconomic mycotoxin deoxynivalenol (DON)
Published: 14 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Deoxynivalenol (DON) is a toxic fungal secondary metabolite produced by Fusarium graminearum which causes Fusarium Head Blight and Pink Ear Rot disease in wheat and corn respectively. DON is a predominant contaminant in cereal grain crops with outbreaks costing the North American cereal grain industry millions of dollars annually. There is a growing need for effective DON mitigation strategies due to DON’s inherent toxicity which affects the performance of livestock fed contaminated grain.

Current DON management strategies involve physical decontamination or marginally effective chemical treatments; however, a holistic and targeted approach via the incorporation of DON detoxifying enzymes is a promising strategy. Previous studies demonstrated that D. mutans 17-2-E-8, a soil bacterium, epimerizes DON to the less toxic 3-epi-DON via the intermediate, 3-keto-DON. The process involves two enzymes, DepA, a PQQ-dependent dehydrogenase, and DepB, an NADPH-dependent aldo-keto reductase (AKR). The strict requirement for the expensive cofactor, NADPH, poses a significant impediment to the practical application of these enzymes. Protein engineering approaches can address this issue – by ‘switching’ DepB’s cofactor preference to the cheaper co-factor, NADH. DepB was found to catalyze the transformation of 3-keto DON to 3-epi DON with Km and kcat values of 563.9 µM and 2.49s-1, respectively, using NADPH as a cofactor. Secondly, the enzyme’s Kd for NADPH was determined to be 44.23 µM using fluorescence enhancement assays. Using the solved crystal structure of DepB, docking experiments with DepB revealed that Arg-289, Gln-293, and Lys-216 may be important for NADPH specificity. Therefore, site-specific mutagenesis was performed to replace these residues to enable the enzyme to utilize NADH. The catalytic efficiencies for these designed mutants will next be determined and compared to catalytic efficiencies of the wild type DepB.

  • Open access
  • 156 Reads
Free-binding energies and molecular interactions of yessotoxin in the voltage-gated sodium channel NaV1.5: an in silico approach
Published: 14 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Several marine dinoflagellates produce unique secondary metabolites with intriguing biological activities, eliciting anticancer, antiepileptic, anti-inflammatory, or anti-microbial responses in various cell types. Among these known compounds are phycotoxins, such as the linear and cyclic polyethers considered as potential therapeutants due to their complex alternative mode of action as ion-channel effectors or enzyme inhibitors capable of modifying diverse intracellular signaling pathways. Yessotoxin (YTX) and analogs are polyketide-derived polycyclic toxins produced by certain species of marine dinoflagellates, Protoceratium reticulatum, Lingulodinium polyedra, and Gonyaulax spinifera, and are structurally related to ciguatoxins and brevetoxins with potent ion-channel activity. The over-expression or aberrant function of ion channels is considered a channelopathy, and critical pathologies in different intracellular signaling pathways that involve receptors such as voltage-gated ion-channels, particularly voltage-gated sodium channels (NaV), are exhibited in response to exposure to such toxins. In the search for innovative therapeutants, this study aimed to evaluate the affinity of YTX for the NaV1.5 channel, using in silico modelling tools. This approach allowed for identification of these interactions and determination of the respective free-binding energies. Our results showed significant interactions and low binding free energies (ΔG), between -6.79 and -10.32 Kcal mol-1 for YTX in the NaV1.5 protein model. Certain amino acid residues in Domains I and II were reached, indicating that this toxin is a potential NaV 1.5 modulator. This study constitutes the first approach to in silico exploration of polyketide-derived dinoflagellate toxins in pursuit of evaluating their therapeutic potential.

  • Open access
  • 92 Reads
Production of Monoclonal Antibodies (mAcs) Purified Anti-metalloprotease from the Venom of the Serpent Bitis arietans

The African snake Bitis arietans is of great medical importance and is found in sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. It contributes significantly to the epidemiology of snakebites in humans and animals. Poisoning is characterized by local and systemic effects, involving inflammation and hemostatic and cardiovascular disorders. Permanent disabilities and deaths are common. The lack of specific antivenoms aggravates this situation. Identifying toxins, knowing their toxic properties and developing antitoxins are the goals of emerging projects. This project aims to develop monoclonal antibodies (mAcs) anti-metalloprotease from the toxin of Bitis arietans poison. The toxin will be purified by the team and used to produce mAcs.

The main strategies used in the development of the project were: (1) use of established knowledge on the molecular and cellular mechanisms involved in the differentiation of B, T lymphocytes and memory plasma cells, in addition to carrying out preliminary experiments aimed at identifying points in the immunization protocols suitable for stimulating memory plasma cells. The results confer a bias of traditional biotechnology; (2) Isolate and characterize relevant toxins, develop antitoxin mAcs, isolate hypervariable regions of these mAcs, identify and sequence CDRs, model scFv antitoxins.

This project aims to develop specific mAcs for epitopes related to the toxic domains of the snake venom Bitis arietans, which will serve as sources of CDRs.

Expectations: Obtaining populations of anti-SVMP mAcs with high titers and high affinity with different anti-toxic potencies.

  • Open access
  • 162 Reads
A closer look at the functional diversity of cytotoxins from Southeast Asian cobras: Biomedical and clinical significance
Published: 21 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

The monocled cobra (Naja kaouthia) and equatorial spitting cobra (Naja sumatrana) are two medically important venomous snakes in Southeast Asia. Proteomics and toxicity studies established that alpha-neurotoxins are invariably the principal lethal toxins in their venoms. The cytotoxins (cardiotoxins, CTX), on the other hand, are much complex functionally and structurally. We investigated the venom gene complexity of the cobras through de novo venom-gland transcriptomics, and showed that cytotoxin genes were the most abundantly and diversely expressed. The CTX were classified into P-type or S-type CTX based on the presence of Pro31 or Ser28 amino acid in the sequence accordingly. To further characterize their pharmacological properties, the CTX were purified from the venoms through sequential high-performance liquid chromatography, validated with nano-liquid chromatography-tandem mass spectrometry, and investigated for their cytotoxic effects in vitro and in vivo. The P-type and S-type CTX exhibited differential cytotoxicity, consistent with the variable degree of hydrophobicity in the membrane-binding loop of the toxin molecule. Protein antigenicity was, nevertheless, conserved among the cobra cytotoxins, and this enabled cross-reactivity and cross-neutralization activity of cobra antivenom. The cobra cytotoxins, however, were phylogenetically and immunologically divergent from cytotoxin-like proteins of the Asiatic coral snakes (Calliophis spp.), whose cytotoxins form a distinct clade of three-finger toxins with distinct evolutionary implications. Furthermore, the cobra cytotoxins demonstrated high anti-proliferative activities in breast, prostate and lung cancer cell lines, with promising selectivity noted in the latter. Future studies should aim to unleash the anticancer potentials of the cytotoxins.