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Mycotoxins are among the most concerning natural toxic food contaminants. Over the years, significant efforts have been made to characterize the risk associated with their exposure. However, assessing their toxicity has so far been elusive due to the lack of adequate models that closely mimic the physiological conditions of human cells in vivo. Here, we present the SpheroFlow Device (SFD), an efficient microfluidic platform designed, manufactured, and validated to evaluate mycotoxin-induced cytotoxicity under dynamic and continuous exposure in 3D culture settings. In the present study, we integrated human neuroblastoma SH-SY5Y spheroids into the SFD to assess the acute toxicity induced by the mycotoxins ochratoxin A (OTA) and patulin (PAT). The developed system enabled qualitative and quantitative measurements of concentration–response relationships and provided accurate control over the culture microenvironment. Our findings show that by enhancing 3D culture model by applying dynamic flow, SH-SY5Y spheroids exhibited different sensitivities to OTA and PAT compared to conventional static SH-SY5Y spheroids, confirming the critical role of culture models in mycotoxin toxicity assessment. This is the first study assessing the neurotoxicity of OTA and PAT on 3D neuroblastoma spheroids considering the contribution of fluid flow.

Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by Aspergillus and Penicillium genera present mainly in fruit, cereals and nuts. This study compares the cytotoxicity produced by CPA after 24, 48 and 72 h of exposure using both monolayers and 3D spheroids in human neuroblastoma SH-SY5Y cells. Furthermore, CPA toxicokinetics was evaluated using in silico models. Cytotoxicity increased dose- and time-dependently, as shown by the MTT assay. The lowest CPA IC50 values were found in the monolayer study compared to the 3D spheroids at all exposure times (24 h: 864.01 vs. 1132; 48 h: 437 vs. 1069; 72 h: 392 vs. 567 nM). The CPA exposure on SH-SY5Y spheroid organization and morphology was also studied. Morphological changes, including spheroid disaggregation, were observed after mycotoxin exposure. The in silico methods, SwissADME and admetSAR, were used for short and full ADMEt profiles of CPA. The ADMEt predictive profile shows high gastrointestinal absorption and ability to penetrate the blood–brain barrier. Including in silico studies emphasizes the comprehensive approach to understanding mycotoxin toxicity and risk assessment. By combining in vitro 3D spheroid models with computational simulations, this study aims to provide a holistic perspective on the effects of CPA, enhancing the accuracy and relevance of our findings.

The Fusarium fungi is found in cereals and feedstuffs and may produce mycotoxins, which are secondary metabolites, such as the T-2 toxin (T-2). In this work, we explored the hepatotoxicity of T-2 using microfluidic 3D hepatic cultures. The objectives were: (i) exploring the benefits of microfluidic 3D cultures compared to conventional 3D cultures available commercially (Aggrewell plates), (ii) establishing 3D co-cultures of hepatic cells (HepG2) and stellate cells (LX2) and assessing T-2 exposure in this model, (iii) characterizing the induction of metabolizing enzymes, and (iv) evaluating inflammatory markers upon T-2 exposure in microfluidic hepatic cultures. Our results demonstrated that, in comparison to commercial (large-volume) 3D cultures, spheroids formed faster and were more functional in microfluidic devices. The viability and hepatic function decreased with increasing T-2 concentrations in both monoculture and co-cultures. The RT-PCR analysis revealed that exposure to T-2 upregulates the expression of multiple Phase I and Phase II hepatic enzymes. In addition, several pro- and anti-inflammatory proteins were increased in co-cultures after exposure to T-2.

Mycotoxins are secondary metabolites produced by filamentous fungi associated with a variety of acute and chronic foodborne diseases. Current toxicology studies mainly rely on monolayer cell cultures and animal models, which are undeniably affected by several limitations. To bridge the gap between the current in vitro toxicology approach and the in vivo predictability of the data, we here investigated the cytotoxic effects induced by the mycotoxins sterigmatocystin (STE), ochratoxin A (OTA) and patulin (PAT) on different 2D and 3D cell cultures. We focused on human tumours (neuroblastoma SH-SY5Y cells and epithelial breast cancer MDA-MB-213 cells) and healthy cells (bone marrow-derived mesenchymal stem cells, BM-MSC, and umbilical vein endothelial cells, HUVECs). The cytotoxicity of STE, OTA, and PAT was determined after 24, 48 and 72 h of exposure using an ATP assay in both culture models. Three-dimensional spheroids’ morphology was also analysed using the MATLAB-based open source software AnaSP 1.4 version. Our results highlight how each cell line and different culture models showed specific sensitivities, reinforcing the importance of using more complex models for toxicology studies and a multiple cell line approach for an improved and more comprehensive risk assessment.

The bioaccessibility of mycotoxins is an important factor that has to be considered when assessing the risk they pose to human health. Bioactive compounds like phenolics could play a protective role against the toxic effects of contaminants. In this work, the bioaccessible fraction of the T-2 toxin (T-2) contained in breakfast cereals and its effect on the viability of Caco-2 cells were investigated. Furthermore, the effect of tyrosol (a polyphenol abundant in EVOO) on T-2-induced cytotoxicity was evaluated in the same cell line. After standardized in vitro gastrointestinal digestion, the T-2 toxin was released from T-2-spiked breakfast cereals and further quantified by UHPLC-MS/MS. The bioaccessible fraction of T-2 was 51 ± 4%. The cell viability study was performed by pre-treating the cells for 24 h with tyrosol (25, 50 and 100 µM) and subsequently adding T-2 at 15 nM or by treating the cells with a combination of tyrosol and T-2. In the simultaneous treatment, 25 µM tyrosol prevented the toxic effects produced by the exposure to T-2 at 15 nM; however, cytotoxic effects were observed for the other combinations tested. The pre-treatment of Caco-2 cells with tyrosol did not attenuate the cytotoxic effects caused by exposure to T-2. These results suggest that tyrosol at low concentrations (25 µM) could exert a cytoprotective effect on Caco-2 cells against 15 nM T-2 when administered simultaneously with T-2. However, more studies are required to corroborate this hypothesis.

Fish side streams are an environmental and economic problem. In this work, pulsed electric fields (PEF) extraction was optimized and used as a new method for their valorization. Sea bass head, skin, viscera, and backbone were used. PEF technology (123–300 kJ/kg, 1–3 kV/cm) improved the extraction of proteins and antioxidant compounds from head and skin, but was not successful for viscera. SDS-PAGE showed that the protein molecular weight distribution was affected by the extraction process, revealing differences between the control and PEF extraction conditions. In addition, the extraction of macro-minerals and micro-minerals were also evaluated. The effect of PEF differed according to the matrix and the mineral studied. Heavy metals were also taken into account, studying the presence of As, Cd, Hg, and Pb in the extracts. PEF pre-treatment reduced the presence of As in skin, viscera, and backbone, ranging from 18.25 to 28.48% according to the matrix evaluated. The analysis of potential antioxidant bioactive peptides showed that the treatment of the sample directly influenced their variety. Additionally, the extracts obtained from the head were found to increase cell viability when tested on SH-SY5Y cells. In conclusion, PEF extraction can be a useful tool for the valorization of fish side streams.

This work explores the efficiency of supercritical fluid extraction (SFE) to recover minerals, pigments, and antioxidant compounds from the spirulina microalgae. Moreover, the fatty acids and phenolic profiles of the extracts obtained were also investigated, and the effect of the extracts on SH-SY5Y cell viability was tested. The extraction of phycocyanin was improved by SFE compared to conventional extraction, from 2.838 ± 0.081 mg/g dry matter (DM) (control) to 6.438 ± 0.411 mg/g DM (SFE). SFE treatment also improved chlorophyll a and carotenoid recoveries increasing from 5.612 ± 0.547 to 8.645 ± 0.857 mg/g DM and from 0.447 ± 0.096 to 0.651 ± 0.120 mg/g DM, respectively. Regarding minerals, the SFE improved Mg recovery with 77% more than the control extraction. Moreover, palmitoleic, stearic, γ-linolenic, eicosadienoic and eicosatrienoic acids recovery was improved by SFE. Phenolic profiles were identified via triple-TOF-LC-MS-MS. Considering heavy metals, a higher rate of Pb extraction was observed for the SFE extract, while no significant differences were observed for Hg between both extractions. Finally, SFE extract improved cell viability compared to the control extract. Thus, SFE constitutes an interesting tool to sustainably extract high-added-value compounds; however, potential contaminants such as Pb need to be controlled in the resulting extracts.

Current investigations in the field of toxicology mostly rely on 2D cell cultures and animal models. Although well-accepted, the traditional 2D cell-culture approach has evident drawbacks and is distant from the in vivo microenvironment. To overcome these limitations, increasing efforts have been made in the development of alternative models that can better recapitulate the in vivo architecture of tissues and organs. Even though the use of 3D cultures is gaining popularity, there are still open questions on their robustness and standardization. In this review, we discuss the current spheroid culture and organ-on-a-chip techniques as well as the main conceptual and technical considerations for the correct establishment of such models. For each system, the toxicological functional assays are then discussed, highlighting their major advantages, disadvantages, and limitations. Finally, a focus on the applications of 3D cell culture for mycotoxin toxicity assessments is provided. Given the known difficulties in defining the safety ranges of exposure for regulatory agency policies, we are confident that the application of alternative methods may greatly improve the overall risk assessment.

The population decrease of bees that has been observed in recent years due to the Varroa destructor parasite may endanger the production of bee-products whose demand is on the rise. To minimize the negative effects caused by this parasite, the pesticide amitraz is commonly used by beekeepers. Based on these, the objectives of this work are to determine the toxic effects caused by amitraz and its metabolites in HepG2 cells, as well as its determination in honey samples and the study of its stability with different heat treatments commonly used in the honey industry and its relationship with the amount of 5-hydroxymethylfurfural (HMF) produced. Amitraz significantly decreased cell viability by MTT assay and total protein content (PC) assay, being more cytotoxic than its metabolites. Amitraz and its metabolites caused oxidative stress by Lipid Peroxidation (LPO) production and Reactive Oxygen Species (ROS) generation. Residues of amitraz and/or its metabolites were found in analyzed honey samples, with 2,4-Dimethylaniline (2,4-DMA) being the main metabolite confirmed by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-QTOF HRMS). Amitraz and its metabolites resulted as unstable even at moderate heat treatments. Additionally, a positive correlation in terms of HMF concentration in samples and the severity of heat treatment was also observed. However, quantified amitraz and HMF were within the levels set in the regulation.

The Varroa destructor parasite is responsible for varroasis in honeybees worldwide, the most destructive disease among parasitic diseases. Thus, different insecticides/acaricides have been widely used within beehives to control these parasitic diseases. Namely, amitraz is the most used acaricide due to its high efficacy shown against Varroa destructor. However, pesticides used for beehive treatments could be incorporated into the honey and accumulate in other hive products. Hence, honeybee health and the impairment of the quality of honey caused by pesticides have gained more attention. Amitraz and its main metabolites, N-(2,4-dimethylphenyl) formamide (2,4-DMF) and 2,4-dimethylaniline (2,4-DMA), are known to be potent neurotoxicants. In this research, the cytotoxicity of amitraz and its metabolites has been assessed by MTT and PC assays in HepG2 cells. In addition, possible target receptors by in silico strategies have been surveyed. Results showed that amitraz was more cytotoxic than its metabolites. According to the in silico ADMEt assays, amitraz and its metabolites were predicted to be compounds that are able to pass the blood–brain barrier (BBB) and induce toxicity in the central and peripheral nervous systems. The main target class predicted for amitraz was the family of A G protein-coupled receptors that comprises responses to hormones and neurotransmitters. This affects, among other things, reproduction, development, locomotion, and feeding. Furthermore, amitraz and its metabolites were predicted as active compounds interacting with diverse receptors of the Tox21-nuclear receptor signaling and stress response pathways.

The T-2 toxin (T-2), a mycotoxin produced by several species of Fusarium which belongs to group A of trichothecenes, is rapidly metabolized, and its main metabolites are HT-2, Neosolaniol (Neo), T2-triol and T2-tetraol. In this work, the antioxidant defense system of HepG2 cells against oxidative stress induced by T-2 and its metabolites was evaluated. The results obtained demonstrated that there is an overall decrease in glutathione (GSH) levels after all mycotoxins exposure. Moreover, the GSH levels and the enzymatic activities related to GSH (GPx and GST) increased with NAC pre-treatment (glutathione precursor) and decreased with BSO pre-treatment (glutathione inhibitor). The GPx activity is increased by T2-tetraol. The GST activity increased after T-2 and T2-triol exposure; however, T2-tetraol decreased its activity. Furthermore, CAT activity increased after T-2 and T2-triol; nevertheless, Neo decreased its activity. Finally, SOD activity is increased by all mycotoxins, except after T-2 exposure. So, the damage associated with oxidative stress by T-2 and its metabolites is relieved by the antioxidant enzymes system on HepG2 cells.

Earth’s climate is undergoing adverse global changes as an unequivocal result of anthropogenic activity. The occurring environmental changes are slowly shaping the balance between plant growth and related fungal diseases. Climate (temperature, available water, and light quality/quantity; as well as extreme drought, desertification, and fluctuations of humid/dry cycles) represents the most important agroecosystem factor influencing the life cycle stages of fungi and their ability to colonize crops, survive, and produce toxins. The ability of mycotoxigenic fungi to respond to Climate Change (CC) may induce a shift in their geographical distribution and in the pattern of mycotoxin occurrence. The present review examines the available evidence on the impact of CC factors on growth and mycotoxin production by the key mycotoxigenic fungi belonging to the genera Aspergillus, Penicillium, and Fusarium, which include several species producing mycotoxins of the greatest concern worldwide: aflatoxins (AFs), ochratoxins, and fumonisins (FUMs).

Red beans contain human bioactive compounds such as polyphenols. Several in vitro studies have proposed the natural compounds as an innovative strategy to modify the toxic effects produced by mycotoxins. Hence, in this work, a complete investigation of the polyphenolic fraction of red beans was performed using a Q-Orbitrap high-resolution mass spectrometry analysis. Notably, epicatechin and delphinidin were the most detected polyphenols found in red bean extracts (3.297 and 3.108 mg/Kg, respectively). Moreover, the red bean extract was evaluated against the T-2 toxin (T-2) induced cytotoxicity in hepatocarcinoma cells (HepG2) by direct treatment, simultaneous treatment, and pre-treatment assays. These data showed that T-2 affected the cell viability in a dose-dependent manner, as well as observing a cytotoxic effect and a significant increase in ROS production at 30 nM. The simultaneous treatment and the pre-treatment of HepG2 cells with red bean extract was not able to modify the cytotoxic T-2 effect. However, the simultaneous treatment of T-2 at 7.5 nM with the red bean extract showed a significant decrease in ROS production, with respect to the control. These results suggest that the red bean extract could modulate oxidative stress on HepG2 cells.

Many studies report the potent antioxidant capacity for fish protein hydrolysates, including radical scavenging activity and inhibition ability on lipid peroxidation (LPO). In this study, the in vitro cytotoxicity of protein hydrolysates from different salmon, mackerel, and herring side streams fractions was evaluated in the concentration range from 1 to 1:32 dilution, using cloned human colon adenocarcinoma cells TC7 (Caco-2/TC7) by MTT and PT assays. The protein hydrolysates’ antioxidant capacity and oxidative stress effects were evaluated by LPO and reactive oxygen species (ROS) generation, respectively. The antioxidant capacity for pure and bioavailable hydrolysate fraction was also evaluated and compared. Additionally, mycotoxin levels were determined in the fish protein hydrolysates, and their cytoprotective effect against T-2 toxin was evaluated. Both hydrolysates and their bioavailable fraction induced similar cell viability rates. The highest cytoprotective effect was obtained for the salmon viscera protein hydrolysate (HSV), which increased the cell viability by 51.2%. ROS accumulation induced by H2O2 and LPO was suppressed by all pure hydrolysates. The cytoprotective effect of hydrolysates was observed against T-2. Moreover, the different fish fraction protein hydrolysates contain variable nutrients and unique bioactive peptide composition showing variable bioactivity, which could be a useful tool in developing dietary supplements with different target functional properties.

Pulsed electric fields (PEF) is a promising technology that allows the selective extraction of high-added value compounds by electroporation. Thus, PEF provides numerous opportunities for the energy efficient isolation of valuable microalgal bioactive substances (i.e., pigments and polyphenols). The efficiency of PEF-assisted extraction combined with aqueous or dimethyl sulfoxide (DMSO) solvents in recovering pigments and polyphenols from microalgae Tetraselmis chuii (T. chuii) and Phaeodactylum tricornutum (P. tricornutum) was evaluated. Two PEF treatments were applied: (1 kV/cm/400 pulses, 3 kV/cm/45 pulses), with a specific energy input of 100 kJ/kg. The total antioxidant capacity (TAC) was positively influenced by the use of DMSO. The highest TAC in the T. chuii culture was achieved at a lower extraction time and electric field than for P. tricornutum. The use of DMSO only improved the polyphenols′ extraction for P. tricornutum, whereas the PEF and extraction time were more important for T. chuii. Carotenoids and chlorophyll a were more efficiently extracted using DMSO, while chlorophyll b levels were higher following aqueous extraction for both microalgae. In P. tricornutum, the TAC and pigment extraction efficiency were in general higher at lower extraction times. It can be concluded that PEF may be a promising alternative for the enhancement of the selective extraction of antioxidant bioactive compounds from microalgae.

During fish production processes, great amounts of by-products are generated, representing ≈30–70% of the initial weight. Thus, this research study is investigating 30 lactic acid bacteria (LAB) derived from the sea bass gastrointestinal tract, for anti-fungal activity. It has been previously suggested that LAB showing high proteolitic activity are the most suitable candidates for such an investigation. The isolation was made using a MRS (Man Rogosa Sharpe) broth cultivation medium at 37 ºC under anaerobiosis conditions, while the evaluation of the enzymatic activity was made using the API® ZYM kit. Taking into account the selected bacteria, a growing research was made fermenting two kinds of broths: (i) by-products (WB), and (ii) meat (MB). Both were fermented at three different times (24, 48 and 72 h). Then, the antifungal activities of both fermented by-products and meat broths were determined qualitatively and quantitatively in solid and liquid medium against two different strains of the genera Penicillium, Aspergillus and Fusarium. After the experiments, a total of 30 colonies were isolated, observing a proteolytic activity in 7 of the isolated strains, which belong to Lactobacillus genus, and the two more active strains were identified by polymerase chain reaction (PCR) as L. plantarum. Several strains evidenced antifungal activity showing an inhibition halo and Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) values between 1–32 g/L and 8–32 g/L, respectively. In conclusion, the isolated bacteria of sea bass had the ability to promote the antifungal activity after the fermentation process, thus being a useful tool to give an added value to fish industry by-products.

The aim of the present research was to obtain antioxidant compounds through the fermentation of fish byproducts by bacteria isolated from sea bass viscera. To that purpose, bacteria from sea bass stomach, intestine, and colon were isolated. With the selected bacteria, growing research was undertaken, fermenting different broths prepared with sea bass meat or byproducts. After the fermentation, the antioxidant activity, phenolic acids, and some proteins were evaluated. The main phenolic acids obtained were DL-3-phenyl-lactic acid and benzoic acid at a maximum concentration of 466 and 314 ppb, respectively. The best antioxidant activity was found in the extracts obtained after the fermentation of fish byproducts broth by bacteria isolated from the colon (6502 μM TE) and stomach (4797 μM TE). Moreover, a positive correlation was found between phenolic acids obtained after the fermentation process and the antioxidant activity of the samples. It was also concluded that the lactic acid bacteria isolated from sea bass had an important proteolytic capacity and were able to synthesize phenolic acids with antioxidant capacity. This work has shown the relevance of fermentation as a useful tool to valorize fish byproducts, giving them an added economic value and reducing their environmental impact.

Mycotoxins are produced by a number of fungal genera spp., for example, Aspergillus, Penicillium, Alternaria, Fusarium, and Claviceps. Beauvericin (BEA) and Ochratoxin A (OTA) are present in various cereal crops and processed grains. This goal of this study was to determine their combination effect in HepG2 cells, presented for the first time. In this study, the type of interaction among BEA and OTA through an isobologram method, cell cycle disturbance by flow cytometry, and genotoxic potential by in vitro micronucleus (MN) assay following the TG 487 (OECD, 2016) of BEA and OTA individually and combined in HepG2 cells are presented. Cytotoxic concentration ranges studied by the MTT assay over 24, 48, and 72 h were from 0 to 25 µM for BEA and from 0 to 100 µM for OTA, while BEA + OTA combinations were at a 1:10 ratio from 3.4 to 27.5 µM. The toxicity observed for BEA was higher than for OTA at all times assayed; additive and synergistic effects were detected for their mixtures. Cell cycle arrest in the G0/G1 phase was detected for OTA and BEA + OTA treatments in HepG2 cells. Genotoxicity revealed significant effects for BEA, OTA, and in combinations underlining the importance of studying real exposure scenarios of chronic exposure to mycotoxins.

Reversed-phase liquid chromatography (RPLC) relies on a non-polar stationary phase and a more polar hydro-organic mobile phase, where compound retention is primarily governed by hydrophobicity, with more hydrophobic compounds being retained longer. The introduction of secondary equilibria in the chromatographic system through additives, such as anionic surfactants and ionic liquids (ILs), was proposed to mitigate ionic interactions between positively charged analytes and the anionic free silanol groups in non-endcapped stationary phases, thereby preventing increased retention and peak tailing. Additionally, the combined hydrophobic and ionic interactions between cationic analytes and the ions in these additives was demonstrated to create mixed retention mechanisms that influence retention and selectivity. In this regard, this study investigates aqueous chromatographic systems incorporating both the anionic surfactant sodium dodecyl sulfate (SDS) and the IL 1-hexyl-3-methylimidazolium chloride as mobile phase reagents. This combination of reagents modulates the retention, eliminating the need for organic solvents and resulting in highly sustainable HPLC procedures. The chromatographic behavior was assessed using two different C18 columns (Zorbax Eclipse and XTerra-MS). The strength of solute interactions was estimated by calculating equilibrium parameters and the contributions of hydrophobic and ionic interactions through simple mathematical models. Focusing on the retention of six basic drugs (β-adrenoceptor antagonists), the study highlighted the significant role of ionic interactions. The results demonstrate the feasibility of using aqueous systems combining SDS and an IL for the efficient separation of moderately polar basic compounds without the use of organic solvents.

Citrinin (CIT) is a mycotoxin commonly found in grains, fruits, herbs, and spices. Its toxicity primarily affects the kidney and liver. Meanwhile, food industry by-products, particularly from fishing and aquaculture, contribute significantly to environmental concerns but can also serve as valuable sources of nutrients and bioactive compounds. Additionally, microalgae like spirulina (Arthrospira platensis) offer interesting high-added-value compounds with potential biological and cytoprotective properties. This study aims to reduce CIT’s toxicity on SH-SY5Y cells using natural extracts from the microalgae spirulina and fish processing by-products (sea bass head). The combination of these extracts with CIT has shown increased cell viability up to 15% for fish by-products extract and about 10% for spirulina extract compared to CIT alone. Furthermore, a notable reduction of up to 63.2% in apoptosis has been observed when fish by-products extracts were combined with CIT, counteracting the effects of CIT alone. However, the extracts’ effectiveness in preventing CIT toxicity in the cell cycle remains unclear. Overall, considering these nutrient and bioactive compound sources is crucial for enhancing food safety and mitigating the harmful effects of contaminants such as mycotoxins. Nevertheless, further studies are needed to investigate their mechanisms of action and better understand their protective effects more comprehensively.

Geography is a significant element of social innovation. This paper focuses on exploring differences and similarities in the characteristics and contributions towards impact of Work Integration Social Enterprises (WISEs), a form of social innovation which provides otherwise unmet services and opportunities to people at risk of social and economic exclusion and distant from the labour market, in rural and urban areas of Ireland. To do so, we use data from 336 surveys from urban (213) and rural (123) WISEs and conduct an exploratory and spatially sensitive analysis to compare the characteristics, in terms of organisational age, legal and governance form, multiplicity of activities, revenue diversification; and contributions towards impact, in terms of geographical focus/reach, employment, volunteers, and income generation. Our analysis shows that WISEs in urban and rural areas present rather similar organisational characteristics and ways of functioning (legal structure, multiactivity, multiple sources of funding), but their contributions to socioeconomic impact differ according to their spatial location, with urban WISEs generating significantly more employment and income than their rural counterparts. Our study illustrates that socially innovative organisations are spatially sensitive, and that context influences their capacity to create sustainable employment opportunities and contribute to the local economy.

The T-2 toxin (T-2) is a type A trichothecene found in cereals. The formation of metabolites is a frequent cause of mycotoxin-induced toxicity. In this work, the conversion of T-2 during biotransformation reactions in HepG2 cells was evaluated. For this, HepG2 cells were exposed to 30 (IC50/2) and 60 (IC50) nM of T-2 for 0, 1, 2, 3, 6, 8 and 24 h, and the concentrations of T-2 and its metabolites HT-2, T2-triol, T2-tetraol and neosolaniol were determined in both the cell fraction and culture medium through liquid chromatography coupled to high-resolution mass spectrometry–time of flight (LC-Q-TOF MS). Results showed a fast metabolization of T-2 (>90%) during the first 2 h, with HT-2 as its main (>95%) biotransformation product. The cell fraction showed higher levels (p < 0.05) of HT-2 (39.9 ± 2.1 nM) compared to the culture medium (12.53 ± 2.4 nM). This trend was also observed for the identified metabolites. T2-triol reached its maximum concentration (1.7 ± 0.4 nM) at 2 h, and at later times a time-dependent increase in the T2-tetraol and neosolaniol concentrations was observed. The identification of T-2 metabolites shows the need to continue combined toxicity studies of mycotoxins for a correct risk characterization of these natural contaminants.

West Nile Virus (WNV) is a mosquito vector-borne zoonosis with an increasing incidence in Europe that has become a public health concern. In Spain, although local circulation has been known for decades, until 2020, when a large outbreak occurred, West Nile Virus cases were scarce and mostly occurred in southern Spain. Since then, there have been new cases every year and the pathogen has spread to new regions. Thus, monitoring of circulating variants and lineages plays a fundamental role in understanding WNV evolution, spread and dynamics. In this study, we sequenced WNV consensus genomes from mosquito pools captured in 2022 as part of a newly implemented surveillance program in southern Spain and compared it to other European, African and Spanish sequences. Characterization of WNV genomes in mosquitoes captured in 2022 reveals the co-circulation of two WNV lineage 1 variants, the one that caused the outbreak in 2020 and another variant that is closely related to variants reported in Spain in 2012, France in 2015, Italy in 2021–2022 and Senegal in 2012–2018. The geographic distribution of these variants indicates that WNV L1 dynamics in southern Europe include an alternating dominance of variants in some territories.

Emerging infectious diseases are one of the most important global health challenges because of their impact on human and animal health. The vector-borne West Nile virus (WNV) is transmitted between birds by mosquitos, but it can also infect humans and horses causing disease. The local circulation of WNV in Spain has been known for decades, and since 2010, there have been regular outbreaks in horses, although only six cases were reported in humans until 2019. In 2020, Spain experienced a major outbreak with 77 human cases, which was followed by 6 additional cases in 2021, most of them in the Andalusian region (southern Spain). This study aimed to characterize the genomes of the WNV circulating in wild-trapped mosquitoes during 2020 and 2021 in Andalusia. We sequenced the WNV consensus genome from two mosquito pools and carried out the phylogenetic analyses. We also compared the obtained genomes with those sequenced from human samples obtained during the outbreak and the genomes obtained previously in Spain from birds (2007 and 2017), mosquitoes (2008) and horses (2010) to better understand the eco-epidemiology of WNV in Spain. As expected, the WNV genomes recovered from mosquito pools in 2020 were closely related to those recovered from humans of the same outbreak. In addition, the strain of WNV circulating in 2021 was highly related to the WNV strain that caused the 2020 outbreak, suggesting that WNV is overwintering in the area. Consequently, future outbreaks of the same strain may occur in in the future.

Hydrophilic interaction liquid chromatography (HILIC) is able to separate from polar to highly polar solutes, using similar eluents to those in the reversed-phase mode (RPLC) and a polar stationary phase, where water is adsorbed onto its surface. It is widely accepted that multiple modes of interaction take place in the HILIC environment, which can be far more complex than the interactions in an RPLC column. The behaviour in HILIC should be adequately modelled to predict the retention with optimisation purposes and improve the understanding on retention mechanisms, as is the case for RPLC. In this work, the prediction performance of several retention models is studied for seven HILIC columns (underivatised silica, and silica containing diol, amino and sulfobetaine functional groups, together with three columns recently manufactured with neutral, anionic, and cationic character), using uracil and six polar nucleosides (adenosine, cytidine, guanosine, thymidine, uridine, and xanthosine) as probe compounds. The results in HILIC are compared with those that were offered by the elution of several polar sulphonamides and diuretics analysed with two C18 columns (Chromolith Speed ROD and Zorbax Eclipse XDB). It is shown that eight retention models, which only consider partitioning or both partitioning and adsorption, give similar good accuracy in predictions for both HILIC and RPLC columns. However, the study on the elution strength behaviour, at varying mobile phase composition, reveals similarities (or differences) between RPLC and HILIC columns of diverse nature. The particular behaviour for the HILIC and RPLC columns was also revealed when the retention, in both modes, was fitted to a model that describes the change in the elution strength with the modifier concentration.

The objective of the present study was to evaluate whether gender determination in two amphibian species (Kaloula pulchra and Xenopus laevis) can be reliably carried out by means of magnetic resonance imaging (benchtop magnetic resonance imaging; BT-MRI) or ultrasound (high-resolution ultrasound; HR-US) techniques. Two species of healthy, sexually mature anurans have been used in the present study. Eight Kaloula (blind study) and six Xenopus were used as controls. Magnetic resonance imaging experiments were carried out on a low-field (1 Tesla) benchtop-MRI (BT-MRI) system. HR-US examination was performed with high-resolution equipment. Low-field BT-MRI images provided a clear and quantifiable identification of all the sexual organs present in both genders and species. The HR-US also allowed the identification of testes and ovaries in both species. Results indicate that BT-MRI allowed a very precise sex identification in both anuran species, although its use is limited by the cost of the equipment and the need for anesthesia. HR-US allowed an accurate identification of ovaries of both species whereas a precise identification of testes is limited by the ultrasonographer experience. The main advantages of this technique are the possibility of performing it without anesthesia and the higher availability of equipment in veterinary and zoo institutions.

The earlier activation of the catalytic converters in internal combustion engines is becoming highly challenging due to the reduction in exhaust gas temperature caused by the application of CO2 reduction technologies. In this context, the use of pre-turbine catalysts arises as a potential way to increase the conversion efficiency of the exhaust aftertreatment system. In this work, a small-sized oxidation catalyst consisting of a honeycomb thin-wall metallic substrate was placed upstream of the turbine to benefit from the higher temperature and pressure prior to the turbine expansion. The change in engine performance and emissions in comparison to the baseline configuration are analyzed under driving conditions. As an individual element, the pre-turbine catalyst contributed positively with a relevant increase in the overall CO and HC conversion efficiency. However, its placement produced secondary effects on the engine and baseline aftertreatment response. Although small-sized monoliths are advantageous to minimize the thermal inertia impact on the turbocharger lag, the catalyst cross-section is in trade-off with the additional pressure drop that the monolith causes. As a result, the higher exhaust manifold pressure in pre-turbine pre-catalyst configuration caused a fuel consumption increase higher than 3% while the engine-out CO and HC emissions did around 50%. These increments were not completely offset despite the high pre-turbine pre-catalyst conversion efficiency (>40%) because the partial abatement of the emissions in this device conditioned the performance of the close-coupled oxidation catalyst.

In reversed-phase liquid chromatography (RPLC), positively charged basic compounds yield broad and asymmetric peaks, as a result of ionic interactions with free silanols that remain on conventional silica-based columns. Diverse solutions have been proposed to mask the silanophilic activity, which is translated to an improved peak shape. In this work, the chromatographic performance of hydrophilic interaction liquid chromatography (HILIC) was evaluated as an alternative to the addition of an ionic liquid (IL) to the aqueous-organic mobile phase used with RPLC columns, for the analysis of eight β-adrenoceptor antagonists. ILs change the behavior of RPLC stationary phases owing to adsorption on their surface. Meanwhile, in HILIC, a layer of adsorbed water is formed on the stationary phase surface. The association of cationic basic compounds with the adsorbed additive ions, hydrophilic partitioning on the HILIC columns, and other interactions, give rise to complex retention mechanisms. The chromatographic behavior was examined in terms of retention, elution strength, selectivity, peak shape and resolution, using acetonitrile-water mobile phases buffered at pH 3. Both chromatographic modes, RPLC with added IL and HILIC, proved to be a viable solution to the problem of poor peak shape for basic compounds.

The presence of free silanols on alkyl-bonded reversed-phase stationary phases is responsible for broad and asymmetrical peaks when basic drugs are chromatographed with conventional octadecylsilane (C18) columns due to ionic interactions. In the last few years, ionic liquids (ILs) have attracted attention to reduce this undesirable silanol activity. ILs should be considered as dual modifiers (with a cationic and anionic character), which means that both cations and anions are able to adsorb on the stationary phase, creating a positively or negatively charged layer, depending on the relative adsorption. The accessibility of basic compounds to the silanols is prevented by both the IL cation and anion, improving the peak profiles. A comparative study of the performance of six imidazolium-based ILs, differing in their cation/anions, as modifiers of the chromatographic behavior of a group of ten β-adrenoceptor antagonists, is addressed. Mobile phases containing cationic amines (triethylamine and dimethyloctylamine) were used as a reference for the interpretation of the results. Using a mathematical model based on two chemical equilibria, the association constants between the solutes and modified stationary phase as well as those between solutes and the additive in the mobile phase were estimated. These values, together with the changes in retention and peak shape, were used to obtain conclusions about the retention mechanism, changes in the nature of the chromatographic system, and silanol suppression effect.

The aim of this research is to study the mediating role of the learning process in explaining the relationships between certain psychological and biographical characteristics and entrepreneurial intention. The findings suggest that the effect of psychological and biographical aspects on entrepreneurial intention depend on the extent to which students are able to take advantage of their personal capabilities in order to develop an effective learning process. Learning process factors (information about course guides, student effort and educational processes) mediate the relationships between specific psychological factors (achievement need, internal control and autonomy) and entrepreneurial intention. With regard to biographical factors, we find no mediating effect on entrepreneurial intention through the learning process. The present study provides a better understanding of the antecedents of entrepreneurial intention, helping to fill the gap in the literature.