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- Bioaccessibility and cytotoxicity of arsenic and mercury in cooked seafood : implications for food safety and human health riskPublication . Ventura, Marta; Assunção, Ricardo; Matos, Beatriz; Coelho, Inês; Delgado, Inês; Gueifão, Sandra; Santiago, Susana; Castanheira, Isabel; Martins, MartaSeafood is an important component of healthy dietary patterns. However, it can also serve as a dietary source of toxic elements that bioaccumulate in aquatic environments and biomagnify through the food web. This study quantified arsenic (As) and mercury (Hg) in cooked seafood, assessed their bioaccessibility, and evaluated the cytotoxic potential of the bioaccessible fractions using the human intestinal HT-29 cell line. The highest As concentrations were found in bivalves (3.7 ± 0.2 mg kg−1), and the highest Hg concentrations in scabbardfish (0.57 ± 0.01 mg kg−1). The bioaccessibility of As was substantially higher (ranging from 91 ± 2% to 127 ± 6%) than that of Hg (ranging from 4.8 ± 0.3% to 24 ± 8%). The highest bioaccessibility was observed in fresh tuna, 127 ± 6% and 24 ± 8% for As and Hg, respectively. Cytotoxicity assays at 24 h, 48 h, and 72 h indicated that bioaccessible Hg concentrations remained below cytotoxic thresholds. These results highlight the importance of integrating bioaccessibility and toxicological indicators into the evaluation of seafood safety.
- Bioengineered baculovirus-derived extracellular vesicles loaded with of γ-carboxylated Gla-rich protein : dual modulation of inflammation and vascular calcificationPublication . Viegas, Carla; Pichard, Simon; Carreira, Joana; Ova, Adélia; Troffer-Charlier, Nathalie; Maia, Teresa M.; Edelweiss, Evelina; Macedo, Anjos L.; Matos, António; Faria, Tiago Q.; Calado, Sofia M.; Monico, Carina; Devos, Simon; Impens, Francis; Schaeffer-Reiss, Christine; Cianférani, Sarah; Peixoto, Cristina; Poterszman, Arnaud; Simes, DinaChronic inflammation and ectopic calcification are interrelated processes driving major chronic inflammatory diseases such as cardiovascular and chronic kidney diseases. Gla-rich protein (GRP), a vitamin K–dependent protein (VKDP) with dual anti-inflammatory and anti-calcific properties, has emerged as a promising therapeutic molecule. However, its biomedical development has been limited by difficulties in producing the γ-carboxylated (cGRP) form and by its poor solubility at physiological pH, constraining formulation and delivery. To address these challenges, we established a baculovirus expression vector system (BEVS) designed to couple GRP post-translational maturation with its secretion in extracellular vesicles (EVs). Co-expression of GRP with γ-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR), and the convertase Furin enabled efficient γ-carboxylation, propeptide removal, and secretion of mature cGRP. GGCX and VKOR were essential for γ-carboxylation, while Furin mediated propeptide processing. EVs were isolated by differential ultracentrifugation into 30 K and 100 K fractions and characterized by NTA, TEM, Western blot, ELISA, and proteomics. All vesicles displayed physical and molecular features resembling mammalian EVs, including canonical EV markers and distinct proteomic profiles, with GRP, GGCX, VKOR, and Furin preferentially enriched in the 30 K population. Functional assays demonstrated that the resulting EVs associated with human THP-1 macrophages and vascular smooth muscle cells (VSMCs) without inducing cytotoxicity, and both cGRP-EVs and uncarboxylated GRP-EVs reduced pro-inflammatory cytokine release while exerting dual anti-inflammatory and anti-mineralizing effects. This study establishes the first bioengineered platform capable of generating functional γ-carboxylated GRP and its vesicular formulation, providing a dual innovation for VKDP research and therapeutic biomaterial development.
- Clonal and plasmid-mediated dissemination of mcr-1 in Escherichia coli strains at the human–companion animal interface : genomic characterisation of colistin resistance plasmidsPublication . Menezes, Juliana; Silva, Joana Moreira da; Fernandes, Laura; Amaral, Andreia J.; Pomba, ConstançaThe global emergence of plasmid-mediated colistin resistance (mcr-1) gene poses a critical threat to human and animal health due to its ability for horizontal dissemination. While the role of food-producing animals is well recognised, the contribution of companion animals and household environments to the persistence and circulation of mcr-1 remains poorly understood. In this study, we investigated the genetic relatedness of mcr-1-positive Escherichia coli strains and their associated plasmids from dogs and their cohabiting humans in Portugal (2018–2020). Whole-genome sequencing, was performed on 17 strains, including repeated sampling from the same hosts over time. Core genome SNP analysis revealed clonal relatedness among several strains from the same host and between household members (≤6 SNPs). A total of 14 mcr-1-harbouring plasmids were identified and classified into three major incompatibility groups: IncX4 (n = 2), IncHI2 (n = 5), and IncI2 (n = 7). IncX4 plasmids were detected in clonally related strains from the same human host and were identical, indicating maintenance within a persistent lineage. A subset of IncI2 plasmids formed a closely related cluster (1–6 SNPs) across genetically distinct hosts, supporting the possibility of horizontal dissemination. IncHI2 plasmids displayed greater structural diversity and carried multiple antimicrobial and metal resistance determinants. Notably, chromosomal integration of mcr-1 was identified in three strains, suggesting a potential pathway for stabilistion of colistin resistance. Overall, these findings highlight the combined role of clonal expansion and plasmid circulation in shaping the epidemiology of mcr-1 genes in community settings, reinforcing the importance of genomic surveillance within a One Health framework.
- Comparative ex vivo responses of ovine and bovine polymorphonuclear neutrophils induced by Neospora caninum tachyzoitesPublication . Gutiérrez-Expósito, D.; Silva, L. M. R.; Wagner, H.; Gärtner, U.; Hermosilla, C.; Taubert, A.; Conejeros 1stNeospora caninum infection causes reproductive failure in ruminants with a traditionally higher incidence of bovine neosporosis compared to the ovine system. Differences in innate immune response could explain this observation. We here focused our study on the comparison of the N. caninum-induced response on bovine and ovine polymorphonuclear neutrophils (PMN) that might play a key role in the pathogenesis of neosporosis in both small and large ruminants. We here examined and compared in parallel PMN responses of healthy adult sheep (n = 6) and cows (n = 6) after exposure to N. caninum tachyzoites (ratio 1:4). PMN activation was evaluated by induction of NETosis, determined by immunofluorescence and scanning electron microscopy (SEM), extracellular reactive oxygen species (ROS) production via Amplex Red assays, and oxygen consumption rates (OCR) and proton efflux rates (PER) quantified by Seahorse XF technology. N. caninum tachyzoite-driven percentage of NETotic PMN was higher in the bovine system (24.4%) when compared to sheep (11.3%). For both, SEM analyses confirmed PMN activation and the formation of NET structures upon N. caninum tachyzoite exposure. The increase in tachyzoite-mediated ROS production proved higher in cattle than in sheep and these data were in line with significantly higher PER on bovine PMN indicating a differential glycolytic activity upon N. caninum exposure. Overall, this study documents early (minutes) and mid-late (hours) ovine and bovine PMN reactions after being exposed to N. caninum tachyzoites. The fundamental information here given contributes to the understanding of neosporosis in cows and sheep that should be complemented with in vivo studies.