Method for quantifying NSAIDs and clofibric acid in aqueous samples, lumpfish (Cyclopterus lumpus) roe, and zebrafish (Danio rerio) eleutheroembryos and evaluation of their bioconcentration in zebrafish eleutheroembryos.

Pharmaceuticals such as nonsteroidal anti-inflammatory drugs (NSAIDs) and lipid regulators are being repeatedly detected at low concentrations (pg · mL-1-ng · mL-1) in the environment. A large fraction of these compounds are ionizable. Ionized compounds show different physico-chemical properties and environmental behavior in comparison to their neutral analogs; as a consequence, the quantification methods currently available, based on the neutral molecules, might not be suitable to detect the corresponding ionized compounds. To overcome this problem, we developed a specific analytical method to quantify NSAIDs and lipid regulators (i.e., ibuprofen, diclofenac, naproxen, and clofibric acid) and their ionized compounds. This method is based on three steps: (1) the extraction of the organic compounds with an organic solvent assisted with an ultrasonic probe, (2) the cleaning of the extracts with a dispersive SPE with C18, and (3) the determination of the chemical compounds by GC-MS (prior derivatization of the analytes). We demonstrated that the proposed method can successfully quantify the pharmaceuticals and their ionized compounds in aqueous samples, lumpfish eggs, and zebrafish eleutheroembryos. Additionally, it allows the extraction and the cleanup of extracts from small samples (0.010 g of wet weight in pools of 20 larvae) and complex matrixes (due to high lipid content) and can be used as a basis for bioaccumulation assays performed with zebrafish eleutheroembryos in alternative to OECD test 305.

Zebrafish dives into food research: effectiveness assessment of bioactive compounds.

Zebrafish have been traditionally used in ecotoxicology and developmental biology. However, due to the advances in available methodologies and the similitude with mammals, it has been increasingly used in other fields. One of the most recent fields using zebrafish is food research, being the focus of this review. Most relevant and recent publications including food component toxicity and key metabolic effects together with effectiveness on some zebrafish disease models have been reviewed. This model is a good intermediate tool between in vitro and rodent models, because it provides information from a complete organism in a fast and cost-effective manner. Definitely, in the near future, we will see this model being used by the ingredient suppliers and scientists in order to show the potential impact on health of several compounds.

Bioconcentration of ionic cadmium and cadmium selenide quantum dots in zebrafish larvae.

The concern related to the use of nanomaterials is growing nowadays, especially the risk associated with their emission or exposure. One type of nanomaterials that has attracted much attention is quantum dots (QDs). QDs incorporation in consumer goods increases the probability of their entering in the environment and then into living organisms and human. In order to evaluate their potential to be bioconcentrated, zebrafish larvae have been exposed to SeCd/ZnS QDs, after performing an exhaustive characterization of these nanoparticles under the assay conditions. These data were compared with those obtained when zebrafish larvae were exposed to ionic cadmium. Finally, distribution of ionic Cd and QDs in exposed zebrafish larvae have been evaluated by Laser Ablation ICP-MS.

Comparison of bioconcentration of ionic silver and silver nanoparticles in zebrafish eleutheroembryos.

The production of silver nanoparticles has reached nowadays high levels. Bioconcentration studies, information on persistence and toxicity are fundamental to assess their global risk and thus necessary to establish legislations regarding their use. Previous studies on silver nanoparticle toxicity have determined a clear correlation between their chemical stability and toxicity. In this work, experimental conditions able to assure silver nanoparticles stability have been optimized. Then, zebrafish (Danio rerio) eleutheroembryos were exposed to ionic silver and to Ag NPs for comparison purposes. A protocol alternative to the OECD 305 technical guideline was used. To determine silver concentration in both the eleutheroembryos and the exposure media, an analytical method consisting in ultrasound assisted extraction, followed by inductively coupled plasma mass spectrometry and graphite furnace atomic absorption spectrometry, was developed. Then, bioconcentration factors were calculated. The results revealed that ionic silver was more accumulative for zebrafish eleutheroembryos than nanoparticles at the levels tested.

Zebrafish (Danio rerio) larvae as a system to test the efficacy of polysaccharides as immunostimulants.

The present study was carried out to examine the use of zebrafish (Danio rerio) as a preliminary screening model for testing the effect of potential immunostimulant substances on the innate immune system. ß-Glucan, a polysaccharide used widely as an immunostimulant, was used as a representative molecule and tested on zebrafish embryos and larvae. The efficacy of the molecule was evaluated by determining the differential expression of some selected genes related to the immune system by RT-qPCR. Larvae from 72 hours post fertilization were found at the optimal developmental stage for assessing the expression of the selected genes. To verify if the ß-glucan entered the larvae and therefore was responsible for the effects produced, the molecule was labeled fluorescently to check its localization by using microscopy. For estimating the effects of ß-glucan on gene expression, zebrafish embryos and larvae were immersed in three different concentrations of ß-glucan (50, 100, and 150 µg/mL) using five different exposure times. A stronger gene induction was observed when longer times of exposure and older larvae were used. The most evident effects of ß-glucan were the overexpression of the genes TNFα, MPO, TRF, and LYZ. Moreover, slight changes in MPO expression were detected using a transgenic line of zebrafish (MPO::GFP), and a temporal increase in resistance against Vibrio anguillarum was found after ß-glucan immersion. The assay used in this study permits the testing potential of immunostimulants in a simple and cost-effective way.

Saccharomyces uvarum, a proper species within Saccharomyces sensu stricto.

Saccharomyces uvarum is proposed as a proper species within the complex Saccharomyces sensu stricto. Molecular characteristics including the similarity of the restriction profile of the non-transcribed spacer 2 (NTS2) and of the D1/D2 sequences of the rDNA, as well as other genotypic and phenotypic characteristics confirm that this group of strains is highly homogeneous and distinguishable from other species of the Saccharomyces sensu stricto group.

Saccharomyces uvarum, a distinct group within Saccharomyces sensu stricto.

A natural subgroup (that we refer to as Saccharomyces uvarum) was identified, within the heterogeneous species Saccharomyces bayanus. The typical electrophoretic karyotype, interfertility of hybrids between strains, distinctive sugar fermentation pattern, and uniform fermentation characteristics in must, indicated that this subgroup was not only highly homogeneous, but also clearly distinguishable from other species within the Saccharomyces sensu stricto group. Investigation of the S. bayanus type strain and other strains that have been classified as S. bayanus, confirmed the apparent lack of homogeneity and, in some cases, supported the hypothesis that they are natural hybrids.

AFLP fingerprinting for analysis of yeast genetic variation.

Amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in commercial strains, type strains and winery isolates from a number of yeast species. AFLP was shown to be effective in discriminating closely related strains. Furthermore, sufficient similarity in the fingerprints produced by yeasts of a given species allowed classification of unknown isolates. The applicability of the method for determining genome similarities between yeasts was investigated by performing cluster analysis on the AFLP data. Results from two species, Saccharomyces cerevisiae and Dekkera bruxellensis, illustrate that AFLP is useful for the study of intraspecific genetic relatedness. The value of the technique in strain differentiation, species identification and the analysis of genetic similarity demonstrates the potential of AFLP in yeast ecology and evolutionary studies.

Electrophoretic profile of hybrids between cryotolerant and non-cryotolerant Saccharomyces strains.

The chromosomal DNAs of cryotolerant Saccharomyces bayanus, non-cryotolerant Saccharomyces cerevisiae strains and their intra and interspecific hybrids were separated by pulsed field electrophoresis (PFGE). The cryotolerant and non-cryotolerant strains gave distinctly different electrophoretic profiles. The hybrids cryotolerant x cryotolerant and non-cryotolerant x non-cryotolerant were fertile and they gave the same electrophoretic karyotype as the respective parents. The cryotolerant x non-cryotolerant hybrids were sterile and gave electrophoretic karyotypes which showed both the bands the parents have in common and those they do not share.

Karyotyping of Saccharomyces strains with different temperature profiles.

This study examined the karyotype, the fermentation performance and the optimum growth temperature (Topt) of 28 yeast strains all identified as species belonging to Saccharomyces sensu stricto. The strains were isolated from fermented musts, which had not been inoculated, at two temperature ranges: 20-40 degrees C and approximately 0-6 degrees C. The results demonstrated a correlation between the Topt and the chromosome organization. In particular, strains with Topt of less than 30 degrees C showed only two bands in the region between 365 and 225 kb, while those with a Topt greater than 30 degrees C had three bands in this size range. From a taxonomic viewpoint, the Topt is a better indicator for the Saccharomyces sp. than the ceiling temperature of 37 degrees C currently used to differentiate cryotolerant Saccharomyces bayanus and S. pastorianus from non-cryotolerant S. cerevisiae and S. paradoxus strains.