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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.
- Bench-press performed with a velocity- and tempo-based approach : are there differences in volume load, time under tension, and metabolic demands?Publication . Fitas, Afonso; Miras-Moreno, Sérgio; Oliveira, João Henriques; Cidrais, Margarida; Pezarat-Correia, Pedro; Schoenfeld, Brad J.; Mendonça, Gonçalo V.Background: Velocity-based training (VBT) is a resistance training approach that uses lifting velocity to determine training load and track strength progress. This study determined the impact of a VBT versus a tempo-based training (TBT) approach on volume load and time under tension during a single set of submaximal bench press performed to failure. Hypothesis: VBT would result in larger volume load and similar time under tension as TBT. Study Design: Randomized-crossover design. Level of Evidence: Level 3. Methods: A total of 14 healthy men (24.1 ± 5.8 years) performed free-weight bench-press exercise at low intensities (12%, 16%, 20%, and 24% of 1-repetition maximum [1RM]) with oxygen uptake (V.O2) measurements. V.O2 was then extrapolated to a set performed at 70% 1RM to failure and the accumulated O2 deficit was calculated together with the relative energy contribution of aerobic and anaerobic metabolism. Mechanical data were collected with a linear encoder. Results: Despite the lack of differences between conditions for total time under tension (P > 0.05), VBT achieved a higher volume load at set failure (P < 0.05). Moreover, the VBT condition resulted in a larger total V.O2 from set initiation to termination (P < 0.01). Conversely, the accumulated O2 deficit did not differ between conditions (P > 0.05). Compared with TBT, VBT elicited a higher relative contribution of aerobic energy (VBT, ~41%; TBT, 33%) and a lower relative contribution of anaerobic energy (VBT, ~59; TBT, 67%) during exercise (P < 0.01). Conclusion: VBT is an effective strategy to enhance volume load during bench-press performed to failure at 70% 1RM. This effect occurs without compromising time under tension. These findings are associated with a higher contribution of aerobic energy supply to exercise. Clinical Relevance: VBT may be beneficial for athletes aiming to maximize volume load in response to resistance exercise.
- Evaluating reporting completeness in oral health clinical guidelines : a meta-research studyPublication . Lancry, P.; Lyra, P.; Mendes, José João; Nascimento, G. G.; Machado, Vanessa; Botelho, JoãoBackground: To assess the adherence to clinical practice guidelines (CPGs) reporting guidelines for oral health. Methods: A literature search was carried out in PubMed, Embase and Web of Science from March 2016 to December 2023, selecting CPGs related to oral health. The study selection and data extraction were conducted independently by 2 researchers. Guidelines were cross-checked against the 23-item Appraisal of Guidelines, Research, and Evaluation (AGREE). The results were then collected, and the overall adherence and adherence to each AGREE item and section were calculated. Regression analyses were performed considering journal characteristics, such as quartile, year, and publishing options in journals’ guideline endorsement. There were no language restrictions. Results: Twenty-one CPGs were included in this study. The mean overall AGREE adherence was 48.7%. The results showed considerable variability in the rates of compliance with the reporting guidelines. Three areas appear to have (much) higher levels of compliance than the others, notably “Clarity of Presentation” (83.6%, 95% confidence interval [CI]: 75.4%–91.8%), “Scope and Purpose” (74.0%, 95% CI: 67.1%–80.9%), and “Stakeholder Involvement” (54.5%, 95% CI: 43.4%–65.7%). The lowest level of agreement was found in “Applicability,” with a level of agreement of 18.5% (95% CI: 11.4%–25.6%). Four of the 6 domains had a complete lack (0.0%) of adherence. Journal quartiles were significant, as guidelines published in the second- (B = −27.3%; standard error [SE] = 6.1) and third-quartile (B= −22.8%; SE = 10.6) impact factor journals displayed a lower overall adherence than those published in first-quartile journals. Guideline endorsement by journals was also a significant variable (B = −20.9%, SE = 5.9). Conclusion: Reporting completeness in dental/oral CPGs is suboptimal and is associated with the journal’s quartile and guideline endorsement. Increasing awareness of CPG reporting guidelines and ensuring their rigorous application are decisive toward better adherence.
- The impact of positive childhood experiences : a systematic review focused on children and adolescentsPublication . Sousa, Marta; Machado, Ana Beatriz; Pinheiro, Marina; Pereira, Bárbara; Caridade, Sónia; Almeida, Telma Catarina; Cruz, Ana Rita; Cunha, OlgaChildhood and adolescence are crucial stages of life, characterized by significant changes that profoundly influence overall development. While positive childhood experiences (PCEs) can help mitigate the effects of adverse events during these formative years, they have not been as thoroughly researched. Then, this systematic review aims to address this gap by organizing the existing literature on PCEs and examining their impact on both positive and negative outcomes in children and adolescents. A search through databases such as B-On, PsycINFO, PubMed, SCOPUS, and Scielo, as well as supplementary searches, identified 30 studies that met the inclusion criteria. The results indicate that most studies were published in the last 4 years, primarily in the USA, and focused on community populations with mixed samples. In addition, the results reveal that among children and adolescents, higher levels of PCEs were associated with better mental health outcomes (e.g., reduced depressive symptoms, anxiety, self-harm, substance use, and suicidal ideation), improved psychosocial outcomes (e.g., enhanced adult functioning and future orientation), better academic achievement (e.g., reduced absenteeism and fewer academic difficulties), and some improvements in physical health (e.g., reduced chronic pain). However, the relationship between PCEs and behavioral outcomes showed mixed results. Strengthening efforts to promote PCEs and resources that support child and adolescent resilience is crucial. Further research involving diverse samples is needed to gain a deeper understanding of the role of PCEs.
- Nebulised heparin as a treatment for lung diseases : formulation challenges and pulmonary drug delivery strategiesPublication . Miranda, Margarida; Brown, Marc; Haren, Frank M. P. van; Brown, Beth; Page, Clive P.The COVID-19 pandemic further emphasized the global demand for heparin and its expanding clinical relevance, indicating that even one of the oldest drugs in medicine continues to reveal new therapeutic horizons. Traditionally recognized for its anticoagulant and antithrombotic activities, heparin is increasingly being explored for its versatile therapeutic potential in the treatment of a range of pulmonary diseases, including respiratory infections (e.g. COVID-19), Acute Respiratory Distress Syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. In all of these diseases, inhaled unfractionated heparin (UFH) therapy has been investigated in a number of clinical trials that have demonstrated promise for this drug when administered directly to the lungs. However, using heparin by this “off label” route of administration, poses a number of technical challenges: the physicochemical properties of heparin at therapeutic doses often results in highly viscous formulations, causing device blockage and drug sorption during nebulization. These limitations underscore the need for innovative formulation strategies to improve aerosol flow, reduce dosing inefficiencies, and enable reliable pulmonary administration. Advancing heparin formulations for delivery to the lung could therefore unlock significant benefits for a wide spectrum of respiratory disorders, marking a new chapter in the long medical history of this drug as discussed below.
- Preclinical evaluation of Asparagus stipularis in a rat model of metabolic syndrome and development of its nanoencapsulated formPublication . Adouni, Khaoula; Zouaoui, Olfa; Brandão, Pedro; Rijo, Patrícia; Lima, Sofia A. Costa; Reis, Salette; Achour, LotfiContext: Asparagus stipularis Forssk decoction (ASD) has shown potential metabolic and antioxidant benefits, yet its effects on pancreatic dysfunction associated with metabolic syndrome remain insufficiently explored. Objective: The aim of this work was to assess the pancreatic protective properties of ASD in high-fructose diet (HFrD)-fed rats and to characterize ASD-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) as a delivery system to enhance its therapeutic potential. Methods: Rats were fed an HFrD and treated with ASD at two dose levels. Serum α-amylase and lipase activities were measured to assess digestive enzyme modulation. Pancreatic lipid peroxidation was quantified using thiobarbituric acid reactive substances (TBARS), while antioxidant enzyme activities, including superoxide dismutase, catalase, and glutathione peroxidase, were determined. Histopathological examination was performed to evaluate structural alterations in pancreatic tissues. ASD was encapsulated into PLGA NPs, and particle size, polydispersity index (PdI), zeta potential (ZP), and encapsulation efficiency (EE) were analyzed. Results: ASD significantly reduced serum α-amylase activity to 2285.3 ± 256.6 U/L (low dose) and 1846.4 ± 82.8 U/L (high dose) compared to HFrD controls. Serum lipase activity decreased by 13% and 18% at the respective doses. TBARS levels were markedly reduced, and antioxidant enzyme activities were restored to near-control levels. Histological analysis revealed improved β-cell morphology and reduced acinar degeneration. ASD-loaded PLGA NPs exhibited a mean size of 248 ± 5 nm, PdI of 0.13 ± 0.01, ZP of −24.7 ± 1.3 mV, and an EE of 75.5 ± 3.2%. Conclusion: ASD demonstrates significant pancreatic protective effects, and nanoencapsulation enhances its therapeutic promise for metabolic disorders.
- Boletim da Academia das Ciências de Lisboa 2025Publication . Academia das Ciências de Lisboa; Academia das Ciências de Lisboa
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