Development and validation of an analytical method based on QuEChERS followed by UHPLC-ToF-MS for the determination of tropane alkaloids in buckwheat (Fagopyrum esculentum L.) and buckwheat products.

A method was developed for the determination of tropane alkaloids (TAs), including atropine, scopolamine, anisodamine and homatropine in buckwheat and related products. This work presents an optimised methodology based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction procedure followed by ultra-high performance liquid chromatography combined with time-of-flight mass spectrometry for the determination of TAs (atropine, scopolamine, anisodamine and homatropine) in buckwheat samples. The analytical methodology was successfully validated, demonstrating good linearity, low limit of quantification, repeatability (RSDr < 15%), inter-day precision (RSDR < 19%) and recovery (74-113%). Finally, 13 commercial samples of buckwheat were analysed and the results demonstrated that they were in compliance with the current European regulations regarding TAs.

Molecular characterization of cetacean poxviruses along the coast of mainland Portugal.

Cetacean poxvirus (CePV) is the causative agent of tattoo skin disease (TSD) in dolphins, porpoises and whales, a condition characterized by pinhole, ring-like lesions or generalized tattoo-like skin lesions. This study genetically characterized cetacean poxviruses from stranded animals along mainland Portugal. Samples from skin lesions compatible with TSD were obtained from 4 odontocete species (Delphinus delphis, Stenella coeruleoalba, Phocoena phocoena, and Tursiops truncatus) and analyzed using a conventional PCR assay targeting the DNA polymerase gene partially. Among the positive samples (n = 29, 65.9%), a larger DNA polymerase gene fragment was obtained, allowing a robust phylogenetic analysis. Nineteen samples (43.2%) were successfully amplified and sequenced using Sanger sequencing. By combining 11 of these sequences with those from public databases, a maximum likelihood phylogenetic tree was constructed, revealing high heterogeneity within the group. These findings contribute to a better understanding of the genetic diversity, epidemiology, phylogenetics, and evolution of CePV.

Pyrrolizidine Alkaloids in Foods, Herbal Drugs, and Food Supplements: Chemistry, Metabolism, Toxicological Significance, Analytical Methods, Occurrence, and Challenges for Future.

Consumers are increasingly seeking natural alternatives to chemical compounds, including the use of dried aromatic plants as seasonings instead of salt. However, the presence of pyrrolizidine alkaloids (PAs) in food supplements and dried plants has become a concern because of their link to liver diseases and their classification as carcinogenic by the International Agency for Research on Cancer (IARC). Despite European Union (EU) Regulation (EU) 2023/915, non-compliance issues persist, as indicated by alerts on the Rapid Alert System for Food and Feed (RASFF) portal. Analyzing PAs poses a challenge because of their diverse chemical structures and low concentrations in these products, necessitating highly sensitive analytical methods. Despite these challenges, ongoing advancements in analytical techniques coupled with effective sampling and extraction strategies offer the potential to enhance safety measures. These developments aim to minimize consumer exposure to PAs and safeguard their health while addressing the growing demand for natural alternatives in the marketplace.

Snapshot of the Phylogenetic Relationships among Avian Poxviruses Circulating in Portugal between 2017 and 2023.

Avipoxvirus (APV), a linear dsDNA virus belonging to the subfamily Chordopoxvirinae of the family Poxviridae, infects more than 278 species of domestic and wild birds. It is responsible for causing avian pox disease, characterized by its cutaneous and diphtheric forms. With a high transmission capacity, it can cause high economic losses and damage to the ecosystem. Several diagnostic methods are available, and bird vaccination can be an effective preventive measure. Ten APV-positive samples were analyzed to update the molecular characterization and phylogenetic analysis of viruses isolated in Portugal between 2017 and 2023. A P4b gene fragment was amplified using a PCR, and the nucleotide sequence of the amplicons was determined using Sanger sequencing. The sequences obtained were aligned using ClustalW, and a maximum likelihood phylogenetic tree was constructed. With this study, it was possible to verify that the analyzed sequences are distributed in subclades A1, A2, B1, and B3. Since some of them are quite similar to others from different countries and obtained in different years, it is possible to conclude that there have been several viral introductions in Portugal. Finally, it was possible to successfully update the data on Avipoxviruses in Portugal.

Ergot Alkaloids on Cereals and Seeds: Analytical Methods, Occurrence, and Future Perspectives.

Ergot alkaloids are secondary metabolites resulting from fungi of the genus Claviceps that have proven to be highly toxic. These mycotoxins commonly infect cereal crops such as wheat, rye, barley, and oats. Due to the increase worldwide consumption of cereal and cereal-based products, the presence of ergot alkaloids in food presents a concern for human safety. For this reason, it is essential to develop several analytical methods that allow the detection of these toxic compounds. This review compiles and discusses the most relevant studies and methods used in the detection and quantification of ergot alkaloids. Moreover, the decontamination techniques are also addressed, with special attention to sorting, cleaning, frying, baking, peeling, and ammonization methods, as they are the only ones already applied to ergot alkaloids.

The potential of citrus by-products in the development of functional food and active packaging.

Food by-product valorization has become an important research area for promoting the sustainability of the food chain. Citrus fruits are among the most widely cultivated fruit crops worldwide. Citrus by-products, including pomace, seeds, and peels (flavedo and albedo), are produced in large amounts each year. Those by-products have an important economic value due to the high content on bioactive compounds, namely phenolic compounds and carotenoids, and are considered a valuable bio-resource for potential applications in the food industry. However, green extraction techniques are required to ensure their sustainability. This chapter addresses the main components of citrus by-products and their recent applications in food products and active food packaging, towards a circular economy. In addition, the concern regarding citrus by-products contamination (e.g. with pesticides residues and mycotoxins) is also discussed.

Roles of the interaction with children and families in mediating the association between digital health literacy and well-being of early childhood teachers in Portugal: A cross-sectional study.

AIM: To analyze the associations between early childhood education (ECE) teachers´ digital health literacy (DHL) and well-being, and to determine whether the ECE teachers´ interaction with children and family mediated these associations. METHODS: A total of 853 early childhood teachers, predominantly women (99.4%) participated in this cross-sectional study. The participants had a mean age of 39.9 years (standard deviation SD = 8.2). Data was collected through an online survey. DHL was assessed using five subscales adapted to the new coronavirus context, and the professionals´ well-being was measured using the WHO-5 well-being scale. Binary logistic regression and mediation analysis were used to analyze the data. RESULTS: ECE teachers' DHL in dimensions of "information searching" and "determining relevance" had both direct and indirect effect on their well-being. Specifically, higher DHL in these dimensions was associated with better well-being. The dimension "evaluating reliability" had an indirect positive effect on well-being by promoting interaction with children. CONCLUSION: These findings suggest that improving ECE teachers´ DHL could have a positive effect on their well-being and their interactions with children. Therefore, it is recommended to develop health promotion practices aimed at enhancing DHL among ECE teachers. Additionally, integrating DHL contents and competencies more prominently into the qualification, further education and training of ECE teachers may help equip them with the necessary skills to access and apply health information effectively. This, in turn, can enhance interactions with children and contribute to their overall well-being.

Reopening ECEC Services for Children Under Three Amidst the Pandemic: Investigating the Association of Health Measures with Pedagogical Practices and Children's Well-being.

In Portugal, early childhood education and care services for children under-three were the first educational services to reopen after periods of lockdown. COVID-19 prevention and control measures had to be implemented nationwide, but no knowledge was yet produced on their impact in educational settings. This study aimed to map the implementation of COVID-19 prevention and control measures and examine associations among prevention and control measures, perceived changes to pedagogical practices and children's well-being in early childhood education and care services for children under three. In this study, 1098 early childhood education and care professionals from all districts completed an online survey during January and February 2021. Results indicated that prevention and control measures were widely implemented. Furthermore, early childhood education and care professionals who started to implement prevention and control measures more frequently were more likely to perceive a reinforcement of their pedagogical practices at the level of adult-child interaction, emotional climate, and interaction with families, and reported higher levels of children's well-being. Findings highlighted the potential role of pedagogical practices in mitigating the effects of COVID-19 in early childhood education and care services for children under-three.

The Bright and Dark Sides of Herbal Infusions: Assessment of Antioxidant Capacity and Determination of Tropane Alkaloids.

Herbal infusions are highly popular beverages consumed daily due to their health benefits and antioxidant properties. However, the presence of plant toxins, such as tropane alkaloids, constitutes a recent health concern for herbal infusions. This work presents an optimized and validated methodology based on the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction procedure followed by Ultra-High Performance Liquid Chromatography combined with Time-of-Flight Mass Spectrometry (UHPLC-ToF-MS) for the determination of tropane alkaloids (atropine, scopolamine, anisodamine, and homatropine) in herbal infusions, in accordance with criteria established by Commission Recommendation EU No. 2015/976. One of the seventeen samples was contaminated with atropine, exceeding the current European regulation regarding tropane alkaloids. In addition, this study evaluated the antioxidant capacity of common herbal infusions available on Portuguese markets, indicating the high antioxidant capacity of yerba mate (Ilex paraguariensis), lemon balm (Melissa officinalis), and peppermint (Mentha x piperita).

Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021.

In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species.

Multiresidues Multiclass Analytical Methods for Determination of Antibiotics in Animal Origin Food: A Critical Analysis.

Veterinary drugs are widely used to prevent and treat diseases. The European Union has forbidden the use of antibiotics as growth promoters since 2006. Its abusive use leads to the presence of antibiotic residues (AR) in foods of animal origin which is associated with antibiotic resistance. The monitoring of AR in food intended for human consumption is of utmost importance to assure Food Safety. A systematic bibliographic review was carried out on the analytical methodologies, published in 2013, for the determination of AR in foods of animal origin. The food processing effect in the AR detected in animal products is also addressed. However, there is a preference for multiresidues multiclass methods, i.e., methodologies that allow determining simultaneously different classes of antibiotics, which is still a challenge for researchers. The wide diversity of physico-chemical properties of these drugs is an obstacle to achieving excellent analytical performance for a vast number of molecules analyzed concurrently. New techniques in sample preparation continue to be developed in order to obtain a compromise between good recoveries and extracts without interferences (clean extracts). The most widely used analytical methodology for the determination of AR is liquid chromatography coupled with mass spectrometry. However, the current trend is focused on the use of powerful high-resolution MS detectors such as Time of Flight and Orbitrap with modern chromatographic systems. Cooking time and temperature control are the key processing conditions influencing the reduction of AR in foods.

Mycotoxins Contamination in Rice: Analytical Methods, Occurrence and Detoxification Strategies.

The prevalence of mycotoxins in the environment is associated with potential crop contamination, which results in an unavoidable increase in human exposure. Rice, being the second most consumed cereal worldwide, constitutes an important source of potential contamination by mycotoxins. Due to the increasing number of notifications reported, and the occurrence of mycotoxins at levels above the legislated limits, this work intends to compile the most relevant studies and review the main methods used in the detection and quantification of these compounds in rice. The aflatoxins and ochratoxin A are the predominant mycotoxins detected in rice grain and these data reveal the importance of adopting safety storage practices that prevent the growth of producing fungi from the Aspergillus genus along all the rice chain. Immunoaffinity columns (IAC) and QuECHERS are the preferred methods for extraction and purification and HPLC-MS/MS is preferred for quantification purposes. Further investigation is still required to establish the real exposition of these contaminants, as well as the consequences and possible synergistic effects due to the co-occurrence of mycotoxins and also for emergent and masked mycotoxins.

Bagaza Virus in Wild Birds, Portugal, 2021.

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.

West Nile virus transmission potential in Portugal.

It is unclear whether West Nile virus (WNV) circulates endemically in Portugal. Despite the country's adequate climate for transmission, Portugal has only reported four human WNV infections so far. We performed a review of WNV-related data (1966-2020), explored mosquito (2016-2019) and land type distributions (1992-2019), and used climate data (1981-2019) to estimate WNV transmission suitability in Portugal. Serological and molecular evidence of WNV circulation from animals and vectors was largely restricted to the south. Land type and climate-driven transmission suitability distributions, but not the distribution of WNV-capable vectors, were compatible with the North-South divide present in serological and molecular evidence of WNV circulation. Our study offers a comprehensive, data-informed perspective and review on the past epidemiology, surveillance and climate-driven transmission suitability of WNV in Portugal, highlighting the south as a subregion of importance. Given the recent WNV outbreaks across Europe, our results support a timely change towards local, active surveillance.

Kynurenine inhibits melanogenesis in human melanocyte-keratinocyte co-cultures and in a reconstructed 3D skin model.

Kynurenine (KYN), the most abundant metabolite of tryptophan, is classically associated with immune tolerance and tumor immune escape. In the last years, KYN is in the spotlight in other biological processes. Here, we showed that KYN inhibited tyrosinase expression and melanin content in primary human melanocyte and keratinocyte co-cultures. Furthermore, KYN decreased melanosome content in a 3D human skin reconstruction model. In these experiments, we used tyrosine + NH4 Cl to induce pigmentation. We compared the inhibitory effect of KYN on melanogenesis with the already known inhibitory effect promoted by IFN-γ. Since increased KYN production depends on the IFN-γ-inducible enzyme indoleamine-2,3-dioxygenase (IDO), we propose that part of the effect of IFN-γ on melanogenesis involves KYN production. From that, we tested if, during melanogenesis, changes in tryptophan metabolism would occur. For this purpose, we measured tryptophan, KYN and downstream products along with pigmentation. There were no significant changes in Trp metabolism, except for the high consumption of kynurenic acid. Our data identify the skin as a potential target for the action of KYN relevant for skin physiology and pigmentation. The results are discussed concerning the high production of KYN in skin inflammatory disorders and cancer.

The Future of Uncertainty Factors With In Vitro Studies Using Human Cells.

New approach methodologies (NAMs), including in vitro toxicology methods such as human cells from simple cell cultures to 3D and organ-on-a-chip models of human lung, intestine, liver, and other organs, are challenging the traditional "norm" of current regulatory risk assessments. Uncertainty Factors continue to be used by regulatory agencies to account for perceived deficits in toxicology data. With the expanded use of human cell NAMs, the question "Are uncertainty factors needed when human cells are used?" becomes a key topic in the development of 21st-century regulatory risk assessment. M.D., PhD, the coauthor of an article detailing uncertainty factors within the U.S. EPA, and L.E., PhD., Executive Vice President, Science, Emulate, who is involved in developing organ-on-a-chip models, debated the topic. One important outcome of the debate was that in the case of in vitro human cells on a chip, the interspecies (animal to human) uncertainty factor of 10 could be eliminated. However, in the case of the intraspecies (average human to sensitive human), the uncertainty factor of 10, additional toxicokinetic and/or toxicodynamic data or related information will be needed to reduce much less eliminate this factor. In the case of other currently used uncertainty factors, such as lowest observable adverse effect level to no-observed adverse effect level extrapolation, missing important toxicity studies, and acute/subchronic to chronic exposure extrapolation, additional data might be needed even when using in vitro human cells. Collaboration between traditional risk assessors with decades of experience with in vivo data and risk assessors working with modern technologies like organ chips is needed to find a way forward.

O envelhecer na sétima arte: o capitão américa e seus retratos biopsicossociais / Aging in the seventh art: captain america and his biopsychosocial portraits

Este artigo propõe uma análise das representações sociais da velhice, do lazer e dos aspectos biopsicossociais relacionados ao processo de envelhecimento, tendo como base as idades cronológica, biológica, psicológica e social do indivíduo, a partir da trajetória do Capitão América - personagem de histórias em quadrinho criado nos anos 1940. Os resultados desta pesquisa apontam que as temáticas relativas à longevidade necessitam de rearranjos de ideias e de valores, no sentido de assegurar um envelhecimento ativo e socialmente inserido, tendo o entretenimento e as produções cinematográficas um potencial para auxiliar nesse movimento.

This article proposes an analysis of the social representations of old age, leisure, entertainment and biopsychosocial aspects related to the aging process, based on the individual's chronological, biological, psychological and social ages, based on the trajectory of Captain America - character of comic books created in the 1940s. The results of this research show that the themes related to old age need rearrangements of ideas and values, in order to ensure an active and socially inserted aging, with leisure and film productions a potential for assist in this movement.

Mycotoxins in Pistachios (Pistacia vera L.): Methods for Determination, Occurrence, Decontamination.

The consumption of pistachios (Pistacia vera L.) has been increasing, given their important benefit to human health. In addition to being an excellent nutritional source, they have been associated with chemical hazards, such as mycotoxins, resulting in fungal contamination and its secondary metabolism. Aflatoxins (AFs) are the most common mycotoxins in pistachio and the most toxic to humans, with hepatotoxic effects. More mycotoxins such as ochratoxin A (OTA), fumonisins (FBs), zearalenone (ZEA) and trichothecenes (T2, HT2 and DON) and emerging mycotoxins have been involved in nuts. Because of the low levels of concentration and the complexity of the matrix, the determination techniques must be very sensitive. The present paper carries out an extensive review of the state of the art of the determination of mycotoxins in pistachios, concerning the trends in analytical methodologies for their determination and the levels detected as a result of its contamination. Screening methods based on immunoassays are useful due to their simplicity and rapid response. Liquid chromatography (LC) is the gold standard with new improvements to enhance accuracy, precision and sensitivity and a lower detection limit. The reduction of Aspergillus' and aflatoxins' contamination is important to minimize the public health risks. While prevention, mostly in pre-harvest, is the most effective and preferable measure to avoid mycotoxin contamination, there is an increased number of decontamination processes which will also be addressed in this review.