Task leader – Isabel Cunha

Task leader – Filipe Castro

The objective of the task is to develop a multiplex non-target specific cell-based transactivation assay, to detect peroxisome proliferator-activated receptors (PPARs) agonists in cyanobacteria exudates, crude extracts, fractions and sub-fractions.

Development of a transactivation assay where the ligand binding domain (LBD) of the target Nuclear Receptors (NR), PPARa, PPARb and PPARg, will be cloned into a pBIND plasmid. Translation will result in a phusion protein, with a GAL DNA-binding domain fused with the NR-LBD. An immortalized mammal cell-line will be transfected with pBIND-NRs plasmids and the reporter plasmid pGL4.31, the latter containing a luciferase gene fused to a GAL4 responsive element. After exposure to the extracts to screen, luciferase emission will be evaluated in a microplate reader.

Task leader – Goreti Sales

The objective of the task is to develop multiplex biosensors based on aptamers to detect the cyanotoxins most frequently reported in Portugal, both in fresh and marine waters, prone to cause human health problems and economic impacts. Those will be:

• domoic acid, associated with amnesic shellfish poisoning – ASP;
• okadaic acid, associated with diarrheic shellfish poisoning – DSP, and its dinophysistoxins analogues (DTX1, DTX2);
• saxitoxin, associated with paralytic shellfish poisoning – PSP;
• b-methylamino-L-alanine (BMAA - a neurotoxin, possibly associated with amyotrophic lateral sclerosis and Alzheimer’s Disease.

The first 5 mentioned toxins are routinely analysed by IPMA, the Portuguese Institution responsible to monitor and decide to set or lift bans on shellfish harvest sector. Due to small size (< 1000 Da) and low concentrations (often in the nM range) of such targets, their detection is mostly based on liquid chromatography coupled to mass spectrometry, which are complex, costly, and time-consuming methods. We are trying to overcome this situation using aptasensors as a preliminary approach. Aptamer-target binding affinity is comparable to that of monoclonal antibodies, are cheaper to produce, more stable, have a longer shelf life, and return to their active state after being denatured by pH or temperature. Unfortunately, their use is not yet incorporated in toxins’ detection and quantification standard methodologies.

Regarding emergent toxins, cylindrospermopsin, tetrodotoxin and ciguatoxin that have been recently reported in Portuguese waters, will be considered. Most of the toxins have aptamers already developed that we will use. In the case of BMAA and ciguatoxin, aptamers are not available, for which Aptamers SELEX (Systematic Evolution of Ligands by Exponential Enrichment) evolution will be outsourced to an international player, based on the cyanotoxin provided by CIIMAR. Aspects related to platform type, signal transduction, signal detection system and readouts quality will be investigated.

Task leader – Nádia Ferreira

In this task, the sensors developed in tasks 2 and 3 will be characterize and validate under laboratorial and real conditions. In the case of the aptasensors, accuracy and specificity will be determined using appropriate cyanotoxin standards to compare aptasensor readouts to those obtained through conventional chromatographic methodology, either GC-MS or HPLC-MS, as appropriate. Parameters such us as lower limit of detection (LOD), linear range of detection (LRD), affinity (Kd), reproducibility and variability of the results will be determined in the various matrices analysed. Cyanobacteria exudates, crude extracts and fractions will be analysed to validate sensor in real conditions.

Cell-based sensor performance and sensitivity will be tested using known PPARa, b and g ligands (standard agonists and antagonists).

Upon biosensors’ characterization and validation those will be used in tasks 5 and 6.

Task leader – Inês Páscoa

The objective of this task is to use the sensors developed on task 2 and 3 and validated on task 4, to screen the Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC) for PPARs ligands and real samples for cyanotoxins.

A minimum of 20 LEGE-CC strains will be cultivated, harvested and biomass extracted to characterize according to the presence of PPAR ligands (cell sensor - biosensor 1) and cyanotoxins (aptasensors - biosensor 2). Possibly much more strains may be analysed if the biosensors perform well with the matrices to analyse, since the techniques are very high throughput. Cyanobacteria culture media with respective exudates, methanolic extracts of cyanobacteria’s biomass, and fraction of the crude extract obtained through HPLC in an acetonitrile/water gradient, will be analysed. Samples will be tested in concentration ranges to detect the presence of the target compounds in the conditions set at task 4. Culture media will give mainly positive result to compounds that are hydro soluble, and those that cell excrete to the medium. Those that are liposoluble and those that the cell keeps in their membrane, will mostly be detected in the biomass extracted with solvents. The fractions show positive with sensor 1 will be further analysed on the following task 6.

Task leader – Marco Preto

The objective of this task is to identify compounds that activate PPARs a, b and g and elucidate the structure of those compounds. In the case the new compounds identified are new molecules never described, or molecules already know but which the capacity to activate PPARs was unknown, their use as possible therapeutic targets acting via PPAR activation will be subject to a European patenting process.

Cyanobacteria extracts and fractions that gave a confirmed positive hit during the preceding task 5, will be further analysed to determine which compound(s) are responsible for the hit. A biosensor-guided screening assay will be followed, to select active fractions of the crude extract or exudates for further fractioning, based on a range of polarity solvents, until the final identification of the bioactive compounds by HPLC-MS and elucidation by MNR.

Large volumes (300-400 L) of the cyanobacteria strains that gave positive hits will be produced, and biomass harvested, freeze dried, and extracted, to obtain enough bioactive compound for elucidation. Hundred-litre polypropylene transparent bags with Z8 culture media, adequate LED illumination and temperature, and aeration with compressed air, will be used to grow cultures for 1,5-2 months. Biomass will be harvested through centrifugation or filtration and freeze dried. Large amounts of the specific fraction where the active compound was identified, will be used for it separation through HPLC-MS, tested again with the cell-biosensor and its structure elucidated.

Through NMR we will acquire HR-ESI-MSn data to obtain the molecular formula and key fragmentations, 1H and 13C NMR data, as well as 2D NMR data (at least HSQC, HMBC, COSY, NOESY). These data will be used to construct fragments that will be connected on the basis of chemical logic, MS data and/or NOESY data. Once the planar structure of the compound is found, any existing chiral centres will be resolved, ideally with chiral chromatography, to compare with available standards, computational prediction or with circular dichroism techniques, allowing for the separation of enantiomeric compounds due to chiral molecules which are bound to the stationary phase.

The results of this task will feed task 7.

Task leader – Manuela Frasco

The objective of this task is to develop a method to separate cyanotoxins and bioactive compounds detected on Task 6 using Molecular Imprinted Polymers (MIPs). The objective is to obtain enough compound with minimal effort, cost and ecological footprint to perform other biochemical assays, e.g. characterization of its mode of action.

Solid-phase columns filled with MIPs will be used to separate the target compounds, either cyanotoxins or PPARs’ ligands. MIPs will be produced by radical polymerization of suitable vinyl monomers, where the target molecule is first incubated with the monomers and cross-linkers, achieving a pre-monomeric arrangement. The polymerization is then initiated by adding a radical compound into the system (e.g. benzoyl peroxide). Once the polymerization is completed, the polymer is crushed into small particles that will be packed latter into the separation columns and evaluated.

Classical monomers to be used herein include methacrylic acid, vinyl pyridine, ethylene glycol dimethylacrylate, vynilbenzene, or divynilbenzene. Overall, these monomers shall have chemical functions that display a great affinity for the specific chemical features of the target compound. In addition to these, other monomers leading to materials that respond to external stimulus (temperature or hydration) are considered. Examples of these include N-isopropylacrylamide and N,N-methylene-bis-acrylamide or different chitosan hydrogel mixtures.

MIPs columns performance will be determined using standards and spiked samples, and different chromatographic techniques depending on the chemical nature of the compounds involved, either HPLC-MS or GC-MS. The retention capacity of the column and its specificity will be accessed, as well as the efficiency of different solvents to elute the target compounds, for the different polymers used. To the materials offering thermos-responsive response, temperature will be employed as a stimulus to remove the rebound template.

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