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Background: This study assesses the impact of having a surgical trainee performing a carotid endarterectomy (CEA) procedure on the postoperative rates of stroke and death., Methods: In this observational retrospective study, consecutive patients, who underwent CEA between May 01, 2016, and July 31, 2022, were entered into a retrospectively collected database. Patients were stratified into 2 categories - consultant-led cases and trainees-led cases. Primary outcomes were 30-day stroke rate, and 30-day morbimortality. A sub analysis was performed after grouping the patients in whether there was a neurological event in the previous 6 months - symptomatic or asymptomatic., Results/conclusions: Trainees-led cases had significantly longer clamping times and higher rates of stroke in asymptomatic patients compared with consultant-led cases. Patient's safety should be our top priority. Any practice leading to a significantly increased rate of postoperative stroke must be discontinued. Training protocols and adequate supervision must ensure that trainees possess the necessary skills and knowledge to safely and effectively perform CEA procedures, thereby prioritizing patient safety., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Despite a good knowledge of cadmium accumulation in Gammarus fossarum, studies to date have focused on Cd accumulated via the dissolved pathway, leaving aside the trophic pathway. The aim of this study was to assess cadmium organotropism and bioaccumulation processes following a trophic exposure of the species Gammarus fossarum. Adult male gammarids were fed with 109 Cd contaminated alder leaves discs for 6 days and then with clean alder leaves for 12 days. During both phases, some gammarids were collected and dissected, and intestines, hepatopancreas, cephalons, gills and remaining tissues were separated to measure their Cd concentrations. Their relative proportions of Cd and their respective BMFs were estimated. The ingestion rate (IR) measured during the exposure phase was divided by 3 between days 2 and 6, indicating that gammarids reduced their feeding activity and therefore the exposure pressure. A multi-compartments TK model was developed, and an iterative inference process was run to select the most parsimonious model that best fits all organ datasets simultaneously. The results showed that: i) intestine and hepatopancreas bioconcentrate Cd the most; ii) no cadmium was quantified in gills, meaning that they do not appear to play a role in Cd storage or elimination with a trophic exposure; iii) Cd elimination occurs only through the intestine; and iv) the general pattern of Cd fate in gammarids, obtained here after dietary highlights once again the importance of the intestine and hepatopancreas, as for the dissolved pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Population-based studies suggest an inverse relationship between the dietary share of ultra-processed foods (UPF), as defined by NOVA classification, and the overall dietary nutritional quality. However, few studies have evaluated the impact of ultra-processed foods on the fatty acid profile of the diet. The aim of this study was to assess the association between consumption of UPF and the fatty acids profile of the diet in Portugal., Methods and Results: Cross-sectional data from IAN-AF 2015-2016 were used for this study. Food consumption data were collected through two 24-h food recalls, and food items were classified according to the NOVA system. For both adults and elderly, the contents of total fatty acids (TFA), saturated fatty acids (SFA) and trans fatty (TRFA) were higher in the fraction of UPF, compared to the other three NOVA groups, while [monounsaturated fatty acids (MUFA)+, polyunsaturated fatty acids (PUFA)/SFA] ratio was lower. The UPF population attributable fraction (PAF) demonstrated that if the dietary contribution of UPF was reduced to levels observed in the first quintile, statistically significant reductions in the prevalence of inadequate intakes of fatty acids would be observed for adults [TRFA (PAF 98.37%, 95% CI 87.27-99.79) and SFA (PAF 37.26%, 95% CI 25.46-47.19)] and for elderly [TRFA (PAF 94.61%, 95% CI 77.59-98.71) and PUFA (PAF 98.28, 95% CI 48.22-99.94)]., Conclusions: In this study the consumption of UPF was associated with a worse fatty acids profile in the Portuguese diet, adding evidence regarding the negative impact of UPF on diet quality., Competing Interests: Conflicts of interest All authors declare that they do not have any conflict of financial interests or personal relationships that could cause bias in this work., (Copyright © 2024 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Ultra-processed foods (UPF) consumption has been associated with unhealthy outcomes. However, the literature lacks robust longitudinal studies considering its cumulative effect, particularly in young populations. This study aimed to evaluate the relationship between UPF consumption patterns throughout childhood with growth and adiposity trajectories., Methods: Generation XXI population-based birth cohort (Porto, Portugal) participants were included. Food frequency questionnaire items at 4, 7 and 10 years were classified according to the processing degree using NOVA. UPF consumption patterns based on total quantity were identified using a probabilistic Gaussian mixture model using participants with complete data and predicting for the total sample (n = 8647). To assess whether the outcome trajectories from 4 to 13 years [body weight (kg), height (cm), body mass index (BMI) z-score, waist circumference (WC) (cm) and fat mass (FM) (%)] depend on UPF patterns, a mixed-effects model with linear and quadratic terms for age adjusted for confounders was used. Participants with at least 2 measurements at 4, 7, 10 or 13 years were included in this study (n range: 5885-6272)., Results: Four UPF consumption patterns were identified: constantly lower consumption (15.4%), constantly intermediate consumption (56.4%), transition from low to high consumption (11.2%), and constantly higher consumption (17.1%). Compared to the constantly lower UPF consumption, the constantly higher consumption pattern was associated with greater acceleration in body weight (β: 0.119; 95%CI: 0.027; 0.212), BMI z-score (β: 0.014; 95%CI: 0.004; 0.023), WC (β: 0.232; 95%CI: 0.144; 0.319) and FM% (β: 0.200; 95%CI: 0.092; 0.308) and with lower acceleration in height (β: -0.063; 95%CI: -0.111;-0.015). The constantly intermediate UPF consumption pattern was associated with greater acceleration in body weight (β: 0.123; 95%CI: 0.043; 0.203), WC (β: 0.120; 95%CI: 0.045; 0.195) and FM% (β: 0.146; 95%CI: 0.054; 0.238)., Conclusion: Constantly higher and constantly intermediate UPF consumption throughout childhood were associated with worse growth and adiposity trajectories until adolescence., Competing Interests: Conflict of Interest statement None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: To adapt four dietary patterns for children and adolescents, and study their associations with sustainability (greenhouse-gas emissions-GHGE and land use-LU), and health (body mass index-BMI) indicators., Research Methods & Procedures: Dietary intake of children (3-9y) and adolescents (10-17y) from the National Food, Nutrition and Physical Activity Survey 2015-2016, Portugal (n = 1153) was assessed through two non-consecutive interviews: one-day food diaries (children), and 24-h recalls (adolescents), using an automated multiple-pass method, including a picture book for portion sizes estimation. Adherence to the Eat-Lancet and World Health Organization (WHO) recommendations, Mediterranean and Atlantic diets were evaluated using adapted versions for pediatrics of the World Index for Sustainability and Health (WISH), Diet Quality Index (DQI), Mediterranean Diet Score (MDS) and Southern-European Atlantic Diet (SEAD), respectively. Diet-related GHGE and LU were estimated using the SHARP-Indicators database. BMI (measured) z-scores were classified according to WHO criteria. A standardized weighted health-sustainability composite index was created (BMI + 0.5*GHGE+0.5*LU). Adjusted linear regression models were computed., Results: WISH, DQI, MDS, and SEAD had weighted mean scores (range) of 50 (0-130), 24 (9-36), 20 (8-32), and 17 (8-32), respectively. All dietary patterns were associated with lower environmental impact, particularly in children, but not with BMI. Only MDS explained both health and sustainability indicators in childhood (composite index: sd.β = -0·223,95%CI:-0·347,-0·072,R 2  = 25.1 %), and adolescence (composite index: sd.β = -0·159,95%CI:-0·315,-0·003,R 2  = 31.3 %)., Conclusion: The Mediterranean diet was the most related to (higher) environmental sustainability and (lower) BMI. However, in children (not adolescents), the WISH, DQI, and SEAD showed then same associations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Increased cardiometabolic risk is associated with abnormalities in blood biomarkers profile and adiposity measurements. Some substances found in the food matrix and the environment, called endocrine-disrupting chemicals, may impair cardiometabolic health in the early and later stages of life. Bisphenol A (BPA) is a food contaminant that migrates from food contact materials and may act as an endocrine disruptor, negatively affecting human health. The present work aims to longitudinally assess the association between BPA exposure and cardiometabolic outcomes, considering data from Portuguese population-based birth cohort Generation XXI. Blood insulin (0.06stdβ; 95 %CI:0.03,0.09) and insulin resistance (0.05stdβ; 95 %CI:0.02,0.08) presented a significant longitudinal association with BPA daily exposure after adjustment for important variables and energy. The same findings were observed for fat mass (0.03stdβ; 95 %CI 0.01,0.06) and waist circumference (0.06stdβ; 95 %CI:0.04,0.08). For z-BMI, a significant cross-sectional (0.03stdβ; 95 %CI:0.01,0.04) and longitudinal (0.02stdβ; 95 %CI:0.00,0.04) association was found. This was the first study assessing the association between BPA exposure and health outcomes from childhood to adolescence. We found an association between BPA exposure and increased blood insulin level, insulin resistance, fat mass percentage, waist circumference and z-BMI. Our results point to the need to reduce exposure to BPA in the early stages of life., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Bisphenol A (BPA), an endocrine disruptor widely used in food contact materials, has been linked to a worse health profile. This study intends to estimate the association between BPA exposure and cardiometabolic patterns at adolescence., Methods and Results: Data from the Portuguese population-based birth cohort Generation XXI at the age of 13 were used (n = 2386 providing 3-day food diaries and fasting blood samples). BPA exposure was measured in 24-h urine from a subsample (n = 206) and then predicted in all participants using a random forest method and considering dietary intake from diaries. Three cardiometabolic patterns were identified (normal, modified lipid profile and higher cardiometabolic risk) using a probabilistic Gaussian mixture model. Multinomial regression models were applied to associate BPA exposure (lower, medium, higher) and cardiometabolic patterns, adjusting for confounders. The median BPA exposure was 1532 ng/d, corresponding to 29.4 ng/kg/d. Adolescents higher exposed to BPA (compared to medium and lower levels) had higher BMI z-score (kg/m 2 ) (0.68 vs. 0.39 and 0.52, respectively; p = 0.008), higher levels of body fat (kg) (16.3 vs. 13.8 and 14.6, respectively; p = 0.002), waist circumference (76.2 vs. 73.7 and 74.9, respectively; p = 0.026), insulinemia (ug/mL) (14.1 vs. 12.7 and 13.1, respectively; p = 0.039) and triglyceridemia (mg/dL) (72.7 vs. 66.1 and 66.5, respectively; p = 0.030). After adjustment, a significant association between higher BPA and a higher cardiometabolic risk pattern was observed (OR: 2.55; 95%CI: 1.41, 4.63)., Conclusion: Higher BPA exposure was associated with a higher cardiometabolic risk pattern in adolescents, evidencing the role of food contaminants in health., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The accumulation of microplastics (MP) by marine species of ecological and commercial interest represents a major concern, particularly for those present in human diet. This study analysed the accumulation of MP in three species of coastal pelagic fish with high commercial value, European sardine (Sardina pilchardus), European anchovy (Engraulis encrasicolus) and horse mackerel (Trachurus trachurus), collected along the Western coast of the Iberian Peninsula. The gastrointestinal tract (GT), gills and muscle were analysed and a total of 504 particles were observed. MP were found in all target tissues of the studied species. Horse mackerel exhibited significantly higher concentrations of microplastics in GT compared to other tissues. On the other hand, anchovies and sardines had significantly lower microplastic concentrations in their muscle tissue. The accumulation of microplastics in the gills showed a significant difference between species, with anchovy having significantly higher concentrations compared to horse mackerel. Horse mackerel had the highest percentage of individuals with microplastics in their GT (92 %), followed by sardine (75 %) and anchovy (50 %). Horse mackerel was also the species that registered the highest percentage of individuals with particles in the muscle (63 %), followed by anchovy (40 %) and finally sardine (39 %). MP in the gills of European sardines and anchovies were similar to those found in water samples. The majority of MP found measured <0.5 mm and were blue fibers. Furthermore, the presence of MP in the GT showed a weak and moderated significant negative correlation with the Fulton Condition Index in horse mackerel and European sardine. Our study confirms the ubiquitous extent of MP contamination in the ocean and provides baseline evidence of MP tissue distribution in three small pelagic fish species with distinct feeding behaviour, while correlating this with the presence of MP in water. Importantly, the results of this study contribute to improve the understanding of biological partitioning of MP in open sea fish species with high commercial relevance, and the potential deleterious effects of our increasingly MP contaminated world., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The therapeutic use of monoclonal antibodies (mAbs) ranges from cancer treatment to immune-mediated conditions, covering infectious and cardiovascular disorders, among others. The development of improved methods for therapeutic antibody discovery has accelerated the identification of numerous mAbs: a discovery campaign can be deeply mined, resulting in hundreds, even thousands, of potential antibody leads for a given target of interest. High throughput mAb expression and purification methods are required for the rapid validation of those leads. In this work, we describe the implementation of a Protein-A coated membrane plate system, the Purexa™ AHT membrane plate, for robust preparative purification of hundreds of recombinant mAbs, without the need for automation. The high efficiency (>80%) recovery generated sufficient mAb for downstream screening analyses such as ELISA and surface plasmon resonance (SPR). This new system allows the functional validation of hundreds of lead antibodies from discovery campaigns in a timely manner regardless of operational size., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests which may be considered as potential competing interests: Camila Leal-Lopes, Andre A. R. Teixeira, received financing from Specifica a Q(2) Solution Company. Sara D’Angelo, M. Frank Erasmus, Andrew R. M. Bradbury, and Fortunato Ferrara are employed at Specifica Inc a Q(2) Solution Company. Graham Temples and Jinxiang Zhou are employed at Purilogics, a Donaldson Brand., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Mola mola is the largest teleost inhabiting our ocean and the presence of microplastics (MP) in this flagship species was, before this study, never described. Thus, this investigation focused on analysing MP ingestion in 53 ocean giant sunfish in the Northeast Atlantic Ocean. A total of 116 MP were found in 79% of the specimens, with a median of 1 MP.ind -1 , ranging from 0 to 11 MP.ind -1 . Seasonal differences were observed, with more fibers registered in specimens caught in autumn. Among the different size classes observed, the smallest category (<300 μm) was the most frequent (43%). Blue (43%) was the most prevalent color, followed by green (29%) and black (10%). The majority of fragments were styrene acrylic copolymer (53%), while most fibers were rayon (78%). These findings emphasize that the ocean sunfish population crossing the southern waters of Portugal is exposed to microplastic pollution and highlight the need for effective management policies to address plastic pollution in marine ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microplastics (<5 mm) are a ubiquitous marine pollutant which are highly bioavailable to marine organisms across all trophic levels. Marine predators are especially vulnerable to microplastic pollution through direct and indirect ingestion (e.g., trophic transfer) due to their high trophic position. In particular, oceanic islands are more susceptible to plastic accumulation, increasing the relative number of microplastics in the environment that are available for consumption. The dynamics of microplastic accumulation in marine predators inhabiting remote islands, however, is sparsely documented. Here we describe microplastic exposure in the Critically Endangered Mediterranean monk seal (Monachus monachus) from the Madeira archipelago (Northeast Atlantic) using scat-based analysis. Microplastics were recovered from 18 scat samples collected between 2014-2021 and were characterized to the polymer level using Fourier-Transform Infrared (u-FTIR) spectroscopy. A total of 390 microplastic particles were recovered, ranging between 0.2-8.6 particles g -1 dry weight (mean 1.84 ± 2.14 particles g -1 ) consisting mainly of fragments (69 %) of various sizes and polymer composition (e.g., PE, PET, PS). Microplastic prevalence (100 % of samples analysed) was higher than what has been previously recorded using scat-based analysis in other pinniped species. Our results suggest that the levels of microplastic pollution in the coastal food-web in the Madeira archipelago are relatively high, placing higher-trophic level organisms at increased risk of microplastic consumption, including humans. This study provides the first insights into microplastic exposure to Madeira's monk seals that may contribute to future management decisions for the species and their long-term survival., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objectives: The objective of this study was to evaluate the expression of several cell membrane markers in oral squamous cell carcinomas (OSCC) and to examine their prognostic influence., Study Design: We analyzed the immunohistochemical expression of claudin-1 (CLDN-1), claudin-4 (CLDN-4), claudin-5 (CLDN-5), claudin-7 (CLDN-7), occludin (OCLN), and E-cadherin (CDHE) in 60 patients with OSCC treated in a central hospital Center of Oporto. The prognostic significance of these biomarkers in cancer-specific survival and recurrence-free survival were evaluated using multivariate analysis., Results: Claudin-1 was observed in the membrane of tumor cells in 51 cases (89.5%), CLDN-4 in 36 cases (63.2%), and CLDN-7 in 48 cases (80%). Claudin-5 was detected in the cytoplasm of tumor cells in 46 cases (78%) and OCLN in 40 cases (70.2%). In a multivariate analysis, the combined evaluation of OCLN and CLDN-1 revealed a significant and independent association with cancer-specific survival and recurrence-free survival. We found a low extent score for OCLN and a high intensity score for CLDN-1, presenting the hazard ratios of 15.48 (P = .014) and 9.446 (P = .012), respectively., Conclusion: The CLDN-1 and OCLN proteins could be involved in tumor progression of OSCC. Their combined deregulated expression showed an adverse effect on survival and therefore they could be regarded as important prognostic biomarkers in OSCC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background & Aims: Ultra-processed foods (UPF) consumption has increased greatly over the last decades but its impact on health remains unclear. This study aimed to evaluate the prospective effect of different degrees of food processing on children's cardiometabolic profile., Methods: Children from the Portuguese population-based birth cohort Generation XXI evaluated at 7 and 10 years of age (y), with dietary information at 7 y and anthropometric measurements at 10 y, were included in the present study (n = 3034). Dietary data were collected by 3-day food diaries and all reported food items were classified according to the degree of processing using the NOVA classification system. The daily consumption of the NOVA classification groups was calculated (in grams, as a proportion of total grams and total energy intake). The cardiometabolic outcomes included were serum markers [fasting glucose, insulin, blood lipids], anthropometric measurements [body mass index (BMI) and waist circumference (WC)], body composition [fat-free mass (FFM) and fat mass (FM)] and blood pressure (BP). Age- and sex-specific sample z-scores were obtained for all outcomes. Principal Component Analysis was used to identify a potential cluster of cardiovascular risk factors. Generalized linear models were fitted to evaluate the association between the consumption according to the processing degree (as absolute gram intake) and the individual cardiometabolic risk factors and cluster, adjusted for child's sex, maternal age and education and remaining NOVA groups. A mediation role of children's BMI in the previous analysis were tested., Results: At 7 y, the daily median consumption of un/minimally processed, processed and UPF was 1210 g, 113 g and 433 g (68%, 6% and 25% of the total grams, as well as 51%, 15% and 31% of the total energy intake, respectively). After adjustment, an increase of 100 g in the consumption of un/minimally processed at 7 y was associated with a lower BMI (βˆ = -0.028; 95%CI: -0.043; -0.014), WC (βˆ = -0.020; 95%CI: -0.032; -0.008), FM (βˆ = -0.023; 95%CI: -0.035; -0.011), insulin (βˆ = -0.022; 95%CI: -0.036; -0.008) and BP (systolic BP: βˆ = -0.014; 95%CI: -0.025;-0.004; diastolic BP: βˆ = -0.013; 95%CI: -0.020;-0.005), at 10 y. BMI was a mediator between the consumption of un/minimally processed foods and BP. Positive associations were found between consumption of processed foods and the cluster 'higher blood pressure', while an inverse association was described between un/minimally processed and the cluster 'higher blood pressure'. No significant associations were found between the UPF consumption and any cardiometabolic outcomes., Conclusions: High consumption of unprocessed or minimally processed foods has a favourable effect on later children's cardiometabolic health, namely lower body weight and body fat, lower waist circumference, blood pressure insulin serum levels., Competing Interests: Conflict of Interest None., (Copyright © 2022 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

The R-package rbioacc allows to analyse experimental data from bioaccumulation tests where organisms are exposed to a chemical (exposure) then put into clean media (depuration). Internal concentrations are measured over time during the experiment. rbioacc provides turnkey functions to visualise and analyse such data. Under a Bayesian framework, rbioacc fits a generic one-compartment toxicokinetic model built from the data. It provides TK parameter estimates (uptake and elimination rates) and standard bioaccumulation metrics. All parameter estimates, bioaccumulation metrics and predictions of internal concentrations are delivered with their uncertainty. Bioaccumulation metrics are provided in support of environmental risk assessment, in full compliance with regulatory requirements required to approve market release of chemical substances. This paper provides worked examples of the use of rbioacc from data collected through standard bioaccumulation tests, publicly available within the scientific literature. These examples constitute step-by-step user-guides to analyse any new data set, uploaded in the right format., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: This study aimed to assess kinetics and predictive variables of humoral immune response to mRNA SARS-CoV-2 vaccine administration., Methods: We collected blood samples before (T0) and 15, 90, and 180 days after vaccination (T1, T2, and T3, respectively). The Quant SARS-CoV-2 Immunoglobulin (IgG) II Chemiluminescent Microparticle Immunoassay was used to determine anti-spike IgG., Results: In almost 3000 healthcare-collected blood samples at the three time points, we found the following: at 15 days postvaccination, 97.6% of subjects presented a robust IgG anti-spike response (>4160 AU/ml); then, at three and six months, it decreased in median 6.5-fold to 35.0% and 3.0-fold to 3.3%, respectively. A linear mixed-effects model supported that female gender, younger age groups, and being seropositive prevaccination maintained higher antibody titers. Curves became tighter with time progression, although titers from seropositive subjects decrease at a slower rate than seronegative ones., Conclusion: These findings strengthen the case for a steep decrease of anti-SARS-CoV-2 antibodies up to six months, suggesting that serological evaluation might guide the need for periodic booster vaccinations in specific groups prone to lower antibody titers., (Copyright © 2022. Published by Elsevier Ltd.)

Potassium hydroxide (KOH) digestion protocols are currently applied to separate microplastics from biological samples, allowing efficient digestion with minor degradation of polymers in a time- and cost-effective way. For biota samples with high-fat content, KOH reacts with triglycerides generating an overlying soap layer, making difficult the digestion and solubilization and subsequent microplastics extraction. Here we studied the addition of Tween-20 in different concentrations to evaluate the effect on the soap layer of post-digested samples. Addition of 10 % of Tween-20 presented higher flow rate during filtration, being set as optimal value. Incorporation of Tween-20 in the extraction procedure increased recovery rates of LDPE, PC and PET and appears to have a protective effect on PC and PET degradation. Tween-20 did not interfere in FTIR spectrum of polymers available in the marine environment. Being low-toxic, makes addition of Tween-20 a simple and economical way to optimize KOH digestion protocols for microplastics extraction., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Rationale: Sputum biomarkers hold promise as a direct measure of inflammation within the cystic fibrosis (CF) lung, but variability in study design and sampling methodology have limited their use. A full evaluation of the reliability, validity and clinical relevance of individual biomarkers is required to optimise their use within CF clinical research., Objectives: A biomarker Special Interest Working Group was established within the European Cystic Fibrosis Society-Clinical Trials Network Standardisation Committee, to perform a review of the evidence regarding sputum biomarkers in CF., Methods: From the 139 included articles, we identified 71 sputum biomarkers to undergo evaluation of their clinimetric properties, responsiveness, discriminant, concurrent and convergent validity., Results: Current evidence confirms the potential of sputum biomarkers as outcome measures in clinical trials. Inconsistency in responsiveness, concurrent and convergent validity require further research into these markers and processing standardisation before translation into wider use. Of the 71 biomarkers identified, Neutrophil Elastase (NE), IL-8, TNF-α and IL-1β, demonstrated validity and responsiveness to be currently considered for use in clinical trials. Other biomarkers show future promise, including IL-6, calprotectin, HMGB-1 and YKL-40., Conclusion: A concerted international effort across the cystic fibrosis community is needed to promote high quality biomarker trial design, establish large population-based biomarker studies, and work together to create standards for collection, storage and analysis of sputum biomarkers., Competing Interests: Conflict of Interest Statement AL, KH, LD, MF, OE, TL, CL, & DGD have no conflicts of interest to declare; CA has received honoraria from Gilead & is Member of the British Thoracic Society Board; P-RB has received grants from Vertex and GSK and consulting fees from Astra-Zeneca, Boehringer Ingelheim, Chiese, Insmed, Novartis, Pfizer, Teva, Vertex and Zambon; SN is currently an employee of Vertex Pharmaceuticals Inc. and, as such, may own stock or stock options in that company, the submitted work was performed by SN whilst a research fellow at the French Institute for Health and Medical Research (INSERM, Paris, France) and had no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript; SB has received consulting fees from Vertex and Zambon; IS has received grant and consulting fees from Vertex pharmaceuticals., (Copyright © 2021 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

A biomonitoring approach based on a single model species cannot be representative of the contaminations impacts on the ecosystem overall. As part of the Interreg DIADeM program ("Development of an integrated approach for the diagnosis of the water quality of the River Meuse"), a study was conducted to establish the proof of concept that the use of a multispecies active biomonitoring approach improves diagnostic of aquatic systems. The complementarity of the biomarker responses was tested in four model species belonging to various ecological compartments: the bryophyte Fontinalis antipyretica, the bivalve Dreissena polymorpha, the amphipod Gammarus fossarum and the fish Gasterosteus aculeatus. The species have been caged upstream and downstream from five wastewater treatment plants (WWTPs) in the Meuse watershed. After the exposure, a battery of biomarkers was measured and results were compiled in an Integrated Biomarker Response (IBR) for each species. A multispecies IBR value was then proposed to assess the quality of the receiving environment upstream the WWTPs. The effluent toxicity was variable according to the caged species and the WWTP. However, the calculated IBR were high for all species and upstream sites, suggesting that the water quality was already downgraded upstream the WWTP. This contamination of the receiving environment was confirmed by the multispecies IBR which has allowed to rank the rivers from the less to the most contaminated. This study has demonstrated the interest of the IBR in the assessment of biological impacts of a point-source contamination (WWTP effluent) but also of the receiving environment, thanks to the use of independent references. Moreover, this study has highlighted the complementarity between the different species and has emphasized the interest of this multispecies approach to consider the variability of the species exposition pathway and sensibility as well as the mechanism of contaminants toxicity in the final diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Increased Rare earth elements (REE) usage culminates in discharges into the environment. Mussels have been chosen as models in biomonitoring, hence, REE concentrations in Mytilus galloprovincialis from six locations on the Portuguese coast were accessed to determine natural concentrations and possible linkage to local ecosystem characteristics and temporal variations, by determining them in distinct seasons (autumn and spring). Samples from Porto Brandão (located on the south bank of the Tagus estuary) exhibited the highest REE concentrations, while mussels from Aljezur (the southernmost point on the Portuguese coast) exhibited the lowest, in both seasons. Overall, ∑REE concentration was greater in the spring. LREE enrichment relative to HREE occurs and a negative Ce and Eu anomaly was observed. This study constitutes the first assessment of REE composition on this model species in the Portuguese coast, in two distinct seasons and contributes to a better understanding of REE uptake for future biomonitoring studies., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background/aims: There is a consistent body of evidence on the association between single nutrients and cognition, but the role of a healthful dietary pattern on cognition in children has been seldomly studied. This study aims to assess the association between dietary patterns at 4 years (y) and cognitive abilities at 10-13y and examine whether adiposity mediated these associations., Methods: This study used data from a sub-sample of the population-based birth cohort Generation XXI, with complete information on diet and cognition (n = 3575). At 4y, data on dietary intake was collected by a validated food frequency questionnaire and dietary patterns were derived by latent class analysis, namely Energy-dense food (EDF) pattern, Snacking pattern and Healthier pattern (Reference). At 10-13y, the Portuguese Version of the Wechsler Intelligence Scale for Children®-Third Edition was administered by trained psychologists and age-adjusted composite scores were computed: a Full-Scale Intelligence Quotient (IQ), plus a Verbal IQ, Performance IQ and Processing Speed IQ. Age- and sex-specific body mass index (BMI) z-scores, body fat percentage from bioimpedance, and waist-to-weight ratio and waist-to-hip ratio were used as measures of adiposity. Regression coefficients and 95% confidence intervals (CI) were computed using linear regression models (adjusted for maternal age and education, pre-pregnancy BMI, smoking and alcohol intake during pregnancy, child's sex, birthweight, exclusive breastfeeding duration and having siblings at 4y). Mediation analysis was conducted using path analysis., Results: After adjustment, children classified in the EDF or a Snacking patterns at 4y were more likely to have lower scores on total IQ (β = -0.116; 95%CI:-0.192,-0.039 and β = -0.148; 95%CI -0.252,-0.044, respectively), Verbal IQ (β = -0.104; 95%CI -0.177, -0.031 and β = -0.163; 95%CI -0.262,-0.064, respectively) and Performance IQ (β = -0.116 95%CI -0.193,-0.040 and β = -0.147; 95%CI -0.250,-0.042, respectively) at 10-13y, when compared to those classified in the Healthier pattern. None of the adiposity measures seemed to explain the associations between dietary patterns and IQ., Conclusion: This study supports that early unhealthy dietary patterns were associated with lower child's cognitive ability, but this effect did not seem to be mediated by adiposity., Competing Interests: Conflict of interest None., (Copyright © 2021 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background and Aim: To evaluate the association of Glycemic Load (GL) with glucose metabolism and blood lipids among young adults., Methods and Results: This study included 1538 participants (51% females), evaluated at 21 years of age as part of the EPITeen cohort. The GL of each individual was obtained from the assessment of their dietary intake by using a 86-item semi-quantitative food frequency questionnaire. The evaluation included anthropometric measurements and a fasting blood sample was used to measure glucose, insulin, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Insulin resistance was calculated based on the homeostasis model method (HOMA-IR). The association between the GL and the biochemical parameters was evaluated by linear regression models using β and 95% confidence intervals (95% CI), stratified by sex and adjusted for body mass index (BMI), energy and fiber intake, and self-perceived social class. No association was found between GL and the glucose metabolism parameters after adjustment. Regarding blood lipids, a positive association was found with LDL-C (β = 1.507, 95% CI 0.454; 2.561 for females; β = 0.216, 95% CI -0.587; 1.020 for males) and a negative association with HDL-C (β = -0.647, 95% CI -1.112; -0.181 for females; β = -0.131, 95% CI -0.422; 0.160 for males)., Conclusions: Our results suggest that, in healthy young subjects, a high GL diet may have a negative impact on lipid profile., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2021 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Wild fish (Cyprinus carpio, Mugil cephalus, Platichthys flesus) from an estuary of the NE Atlantic coast were investigated for plastic contamination (N = 128). From the 1289 particles recovered from fish samples, 883 were plastics. Among these, 84% were fibres and 97% were microplastics. Thirty-six polymers were identified. The number of microplastics (mean ± SD) per individual fish (MP/fish) was 8 ± 6 in C. carpio, 10 ± 9 in M. cephalus and 2 ± 2 in P. flesus. The means of MP/fish per body site were 6 ± 7 in gastrointestinal tract, 0.5 ± 1.1 in gills, 0.3 ± 0.7 in liver and 0.6 ± 1.2 in muscle samples. A few large fibres in liver (≤ 4841 μm) and muscle (≤ 5810 μm) samples were found. The results evidence the existence of high fish contamination by microplastics and reinforce the need of further research on plastic pollution in estuaries., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study comprises the first record of a juvenile Giant Devil Ray specimen for Rio de Janeiro, Southeastern Brazil, and its metal and metalloid contents. A scientometric assessment was also performed for the Manta and Mobula genera. Only five records were found, and only As, Cd, Pb, Hg, Pt, Pd and Rh have been assessed. All studies but one concerned human consumption. A significant knowledge gap on metal and metalloid ecotoxicology for mobulid rays is noted, indicating the emergence of a new field of research that th may be applied for wildlife conservation and management in response to anthropogenic contamination. Our study is also the first to provide Al, Cr, Cu, Fe, Mn, Sr, Ti, V and Zn contents for muscle, liver, brain and kidney for a mobulid ray and one of the scarce reports concerning As, Cd, Hg and Pb in muscle, liver and kidney., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cumulative and continuing human emissions of greenhouse gases to the atmosphere are causing ocean warming. Rising temperature is a major threat to aquatic organisms and may affect physiological responses, such as acid-base balance, often compromising species fitness and survival. It is also expected that warming may influence the availability and toxicological effects of pollutants, including Rare Earth Elements. These are contaminants of environmental emerging concern with great economic interest. This group comprises yttrium, scandium and lanthanides, being Lanthanum (La) one of the most common. The European eel (Anguilla anguilla) is critically endangered and constitutes a delicacy in South East Asia and Europe, being subject to an increasing demand on a global scale. Considering the vulnerability of early life stages to contaminants, we exposed glass eels to 1.5 μg L -1 of La for five days, plus five days of depuration, under a present-day temperature and warming scenarios (△T = +4 °C). The aim of this study was to assess the bioaccumulation, elimination and specific biochemical enzymatic endpoints in glass eels (Anguilla anguilla) tissues, under warming and La. Overall, our results showed that the accumulation and toxicity of La were enhanced with increasing temperature. The accumulation was higher in the viscera, followed by the head, and ultimately the body. Elimination was less effective under warming. Exposure to La did not impact acetylcholinesterase activity. Moreover, lipid peroxidation peaked after five days under the combined exposure of La and warming. The expression of heat shock proteins was majorly suppressed in glass eels exposed to La, at both tested temperatures. This result suggests that, when exposed to La, glass eels were unable to efficiently prevent cellular damage, with a particularly dramatic setup in a near-future scenario. Further studies are needed towards a better understanding of the effects of lanthanum in a changing world., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Microplastics (MP) pollution has received increased attention over the last few years. However, while the number of studies documentating the ingestion of microplastics by fish has increased, fewer studies have addressed the toxicological effects derived from the ingestion of these small items in wild conditions. Here, MP contamination and effect biomarkers were investigated in three commercially important fish species from the North East Atlantic Ocean. From the 150 analysed fish (50 per species), 49 % had MP. In fish from the 3 species, MP in the gastrointestinal tract, gills and dorsal muscle were found. Fish with MP had significantly (p ≤ 0.05) higher lipid peroxidation levels in the brain, gills and dorsal muscle, and increased brain acetylcholinesterase activity than fish where no MP were found. These results suggest lipid oxidative damage in gills and muscle, and neurotoxicity through lipid oxidative damage and acetylcholinesterase induction in relation to MP and/or MP-associated chemicals exposure. From the 150 fish analysed, 32 % had MP in dorsal muscle, with a total mean (± SD) of 0.054 ± 0.099 MP items/g. Based on this mean and on EFSA recommendation for fish consumption by adults or the general population, human consumers of Dicentrachus labrax, Trachurus trachurus, Scomber colias may intake 842 MP items/year from fish consumption only. Based on the mean of MP in fish muscle and data (EUMOFA, NOAA) of fish consumption per capita in selected European and American countries, the estimated intake of microplastics through fish consumption ranged from 518 to 3078 MP items/year/capita. Considering that fish consumption is only one of the routes of human exposure to microplastics, this study and others in the literature emphasize the need for more research, risk assessment and adoption of measures to minimize human exposure to these particles. Thus, MP pollution and its effects should be further investigated and addressed according to the WHO 'One Health' approach., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background & Aims: Diet is usually represented as single foods or dietary patterns not related with a specific outcome, and its cardiometabolic effects at early ages are not clearly understood. This study aims to assess whether dietary patterns derived at 7 years of age have an effect on cardiometabolic health at 10 year-old., Methods: This study uses data from the Generation XXI birth cohort (northern Portugal, 2005-2006). Dietary data were collected by a validated food frequency questionnaire at 7 year-old and dietary patterns were previously derived through partial least squares (PLS), principal component analysis and latent class analysis. At 10 year-old, systolic (SBP) and diastolic (DBP) blood pressure were measured, and blood samples were drawn to analyze cardiometabolic parameters. Linear regression coefficients and 99% confidence intervals [βˆ (99% CI)] were computed (covariates: birth weight, gestational age, physical activity, maternal age and education) (n = 3350)., Results: A dietary pattern that explains the BMI z-score at 10 year-old (PLS-1, characterized by the intake of processed meat, energy-dense foods and low in vegetables) was significantly associated with SBP (βˆ = 0.052, 99% CI:0.022; 0.082), DBP (βˆ = 0.043, 99% CI: 0.022; 0.065), triglycerides (βˆ = 0.065, 99% CI: 0.026; 0.104), HDL-cholesterol (βˆ = -0.059, 99% CI: -0.099; -0.019), LDL-cholesterol (βˆ = 0.040, 99% CI: 0.001; 0.080) and HOMA-IR (βˆ = 0.110, 99% CI: 0.071; 0.149). After further adjustment for BMI at 10 year-old, the magnitude of the associations was weakened. No associations were observed between the other derived dietary patterns and cardiometabolic health., Conclusions: Adherence at 7 years of age to a dietary pattern rich in energy-dense foods, processed meat and low in vegetables, may increase several cardiometabolic parameters at 10 years of age. BMI at 10 year-old explained part of these effects., (Copyright © 2019 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Purpose: To prospectively evaluate the relation of free sugars intake and glycemic load with adiposity and insulin resistance, from adolescence into early adulthood., Methods: Data from the population-based cohort EPITeen (Porto, Portugal) at 13 and 21 years old was used (n = 1034). At both ages, dietary assessment was obtained by food frequency questionnaires (FFQ). Added sugar of each food item was estimated based on a systematic methodology described by Louie et al. Free sugars (FS) were defined according to World Health Organization. To each food item of the FFQ, a calculated mean value of FS and glycemic index was assigned. Dietary glycemic load (GL) was determined for each participant. Sex- and age-specific body mass index z-score (BMIz) and waist-to-weight ratio (WWr) were used as measures of adiposity, and the homeostasis model assessment (HOMA-IR) as a measure of insulin resistance. A cross-lagged path analysis was performed to examine causal relationships between FS intake or dietary GL with BMIz, WWr or HOMA-IR., Results: No significant association was found between intake of FS and dietary GL at 13 years with BMIz, WWr or HOMA-IR at 21. A significant inverse association was found between BMIz at 13 and FS (β = -0.595,95%CI -0.830, -0.359) and dietary GL (β = -0.687,95%CI -0.937,-0.437) at 21 years. Intake of FS, dietary GL, as well as BMIz, WWr and HOMA-IR tracked from 13 to 21 years., Conclusion: No significant association was found between consumption of FS and GL at 13 years with cardiometabolic features at 21 years. Dietary intake and cardiometabolic outcomes tracked from adolescence into early adulthood., (Copyright © 2020 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.)

Optimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP β-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Toxicokinetic (TK) models are relevant and widely used to predict chemical concentrations in biological organisms. The importance of dietary uptake for aquatic invertebrates has been increasingly assessed in recent years. However, the model parameters are estimated on limited specific laboratory data sets that are bounded by several uncertainties. The aim of this study was to implement a Bayesian framework for simultaneously estimating the parameters of a generic TK model for benthic invertebrate species from all data collected. We illustrate our approach on the bioaccumulation of PCB153 by two species with different life traits and therefore exposure routes: Chironomus riparius larvae exposed to spiked sediment for 7 days and Gammarus fossarum exposed to spiked sediment and/or leaves for 7 days and then transferred to a clean media for 7 more days. The TK models assuming first-order kinetics were fitted to the data using Bayesian inference. The median model predictions and their 95% credibility intervals showed that the model fit the data well. From a methodological point of view, this paper illustrates that simultaneously estimating all model parameters from all available data by Bayesian inference, while considering the correlation between parameters and different types of data, is a real added value for TK modeling. Moreover, we demonstrated the ability of a generic TK model considering uptake and elimination routes as modules to add according to the availability of the data measured. From an ecotoxicological point of view, we show differences in PCB153 bioaccumulation between chironomids and gammarids, explained by the different life traits of these two organisms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bacillus licheniformis I89 is a non-pathogenic, Gram-positive bacterium, frequently found in soil. It has several biotechnological applications as producer of valuable compounds such as proteases, amylases, surfactants, and lantibiotics. Herein, it is reported the identification of the polar lipidome of B. licheniformis I89 during the different growth phases (lag, exponential and stationary) at 37 °C. The analytical approach relied on hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry (HILIC-ESI-MS), accurate mass measurements and tandem mass spectrometry (MS/MS). In the lipidome of B. licheniformis I89 were identified four phospholipid classes: phosphatidylethanolamine, phosphatidylglycerol, lysyl-phosphatidylglycerol, and cardiolipin; two glycolipid classes: monoglycosyldiacylglycerol and diglycosyldiacylglycerol; and two phosphoglyceroglycolipid classes: mono-alanylated lipoteichoic acid primer and lipoteichoic acid primer. The same lipid species were identified at the different growth phases, but there were significant differences on the relative abundance of some molecular species. There was a significant increase in the 30:0 lipid species and a significant decrease in the 32:0 lipid species, between exponential and stationary phases, when compared to lag phase. No differences were observed between exponential and stationary phases. The lipidomic-based approach used herein is a very promising tool to be employed in the study of bacterial lipid composition, which is a requirement to understand its metabolism and response to growth conditions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This work explored a novel approach to enhance the antibacterial activity of commercial Gutta-percha (GP) cones, the most commonly used core filling materials used in endodontic treatment. The reported procedure involved an argon (Ar) plasma treatment (PT) of the GP cone surface, followed by the deposition of a ZnO thin film by magnetron sputtering. The resulting surfaces were evaluated for surface topography, antibacterial activity against Enterococcus faecalis and Staphylococcus aureus, and cytocompatibility with human osteoblastic cells. GP cones treated with NaOCl, a routine chair-side protocol, were also tested as reference. The deposition of a ZnO film on pristine GP cones increased its antibacterial activity. Cones pre-treated with Ar-plasma (PT) and coated with the ZnO thin film presented significantly higher antibacterial activity than that observed on the pristine and, also, compared to the ZnO coated cones. The higher antibacterial activity of PT + ZnO cones appears related to the major effects induced by the PT pre-treatment on the cone surface endowing the deposited ZnO film with a homogeneous nanostructured topography that greatly improved surface reactivity. The modified GP cones maintained an appropriate cytocompatibility with human cells. This novel approach provides ready-to-use cones with enhanced antibacterial activity, improving a strict asepsis protocol during endodontic treatment and preventing secondary endodontic infections., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Aims: Diet comprises factors with anti and pro-inflammatory potential that can contribute to modulate obesity-induced inflammation. We aimed to assess the association between food intake and high-sensitivity C-reactive protein (hsCRP) levels in adolescents., Methods and Results: A cross-sectional analysis of 991 adolescents aged 13 years old was conducted as part of the EPITeen cohort, Porto, Portugal. Food intake was assessed by a food frequency questionnaire and thirteen food groups were defined. Anthropometric assessment was performed and serum hsCRP was measured in a fasting blood sample. hsCRP concentrations above the 75th percentile were considered high. Logistic regression was fitted to estimate the association between the intake frequency of the food groups and hsCRP, stratified by BMI and adjusted for sex, parental education and total energy intake. Median (25-75th percentiles) hsCRP concentrations increased with increasing values of BMI [normal weight: 0.20 (0.10-0.50); overweight: 0.40 (0.20-0.80); obese: 1.10 (0.40-2.15) mg/l, p < 0.001]. After adjustment for sex, parental education and total energy intake, no statistically significant associations were found amongst normal weight and overweight participants. However, among obese individuals, having as reference the first frequency category (<1 per day), a higher frequency of vegetables/legumes intake showed a decreased odds of high hsCRP levels (OR: 0.10, 95%CI 0.03-0.38, 1-3 per day; and OR: 0.14, 95%CI 0.04-0.52, >3 per day)., Conclusion: Among participants with obesity-induced higher hsCRP levels, a higher frequency of vegetable/legume intake was inversely related to hsCRP., (Copyright © 2018 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Background & Aims: A possible relationship between children's dietary intake and certain aspects of eating behaviours has been documented, but most studies are cross-sectional and do not consider the complexity of the diet. The aim of this study was to quantify the association between dietary patterns established at 4 years old and appetite-related eating behaviours identified at 7 years old., Methods: Participants are children from the Generation XXI population-based birth cohort. Trained interviewers collected data at birth, 4 and 7 years old on socio-demographics, health and lifestyles, and anthropometrics. At 4 years old, diet was assessed by a Food Frequency Questionnaire and three dietary patterns were identified by Latent Class Analysis: 'Healthier', 'Snacking' and 'Energy Dense Foods' (EDF). A Portuguese version of the original Children's Eating Behaviour Questionnaire (CEBQ) was self-completed by mothers at 7 year-old. This version has previously shown good psychometric properties and the 8 CEBQ sub-domains were combined into two wider dimensions: Appetite Restraint and Appetite Disinhibition. Generalized linear models were used to estimate the associations after adjustment for maternal characteristics (n = 4358). Interaction effects were tested., Results: Children belonging to the 'Snacking' (β = 0.329, 95%CI: 0.265; 0.393) and to the 'EDF' (β = 0.138, 95%CI: 0.098; 0.179) dietary patterns at 4 years old scored increasingly higher, respectively, on Appetite Restraint and Appetite Disinhibition dimensions at 7 years old, comparatively to children in the 'Healthier' dietary pattern. Maternal BMI before pregnancy modified the 'Snacking' pattern associations; they were stronger in children from underweight/normal weight mothers for Appetite Restraint and present only among overweight/obese mothers for Appetite Disinhibition., Conclusions: This study suggests that children following less healthy dietary patterns early in life have more often disordered eating behaviours in later childhood. Maternal weight status may influence these associations., (Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4 knockdown was performed in order to address its role in endothelial cells (EC) behavior. Normal EC and shRNA-Syn4-EC cells were studied comparatively using complementary confocal, super-resolution and non-linear microscopic techniques. Confocal and super-resolution microscopy revealed that Syn4 knockdown alters the level and arrangement of essential proteins for focal adhesion, evidenced by the decoupling of vinculin from F-actin filaments. Furthermore, Syn4 knockdown alters the actin network leading to filopodial protrusions connected by VE-cadherin-rich junction. shRNA-Syn4-EC showed reduced adhesion and increased migration. Also, Syn4 silencing alters cell cycle as well as cell proliferation. Moreover, the ability of EC to form tube-like structures in matrigel is reduced when Syn4 is silenced. Together, the results suggest a mechanism in which Syndecan-4 acts as a central mediator that bridges fibronectin, integrin and intracellular components (actin and vinculin) and once silenced, the cytoskeleton protein network is disrupted. Ultimately, the results highlight Syn4 relevance for balanced cell behavior., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Chronic kidney disease (CKD) is a common progressive condition described in dogs and cats, involving several non-specific morphological and histological lesions. Recently, renal interstitial lipid accumulation was reported in cats with CKD; however, to date, little is known about this condition and its pathogenesis. The aim of this study was to investigate the occurrence and to characterize renal interstitial lipid deposits in dogs and cats. A total of 49 animals (27 cats and 22 dogs) with CKD were included in the study. Interstitial lipid accumulation was found exclusively in cats, affecting both males and females. In 55.6% of the cases, the extent of the lesion was not equally distributed in right and left kidneys. The lesion was always found in the cortical region, associated with an inflammatory reaction. Lipid macrovacuoles were also observed in the tubular epithelium, as well as in areas of tubulorrhexis. The amount of lipid deposited was variable, being more extensive in older animals. Data from this study suggest that interstitial lipid accumulation may be related to tubular lipidosis (typical of feline kidneys) associated with epithelial degeneration and lysis, and to tubular basement membrane fragmentation. Extended studies on this condition are necessary, as it appears to be involved in the progression of CKD and may, therefore, have repercussion in the clinical management of the disease and in the development of new approaches to delay its advance., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Differentiating between medical procedures performed antemortem, perimortem or postmortem in skeletal remains can be a major challenge. This work aims to present evidence of procedures to treat rhinosinusitis (RS) and mastoiditis, suggest criteria for the diagnosis of frontal sinus disease, and frame the individuals described in their medical historical context. In the International Exchange collection, the skull (878) of a 24-year-old male, who died in 1933 due to frontal sinusitis and meningitis, presents evidence of a trepanation above the right frontonasal suture, and micro/macroporosity on the superciliary arches. The available Coimbra University Hospitals archives (1913-1939) reported that 46 females and 59 males (aged 15 months-84 y.o., x̄=35.33) underwent surgery to treat RS, primarily by trepanation (94.3%). In a search for similar evidence in the collection, the skull of a 42-year-old female (85), who died in 1927 due to sarcoma in the abdomen, shows four quadrangular holes located above the right supraorbital notch, right and left maxilla, and left mastoid process. The number/location of the holes and cut marks point to postmortem medical training (possible dissection). This paper discusses the value of information from historical contexts to differentiate between surgery and medical training in the paleopathological record., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: The association of myasthenia gravis (MG) and inflammatory myopathy is rare and often only one of the diseases is diagnosed. Thymus pathology may be in the origin of such disease association., Methods: We described four patients with both MG and inflammatory myopathy., Results: These cases correspond to 2.3% of our MG cohort. Case 1: MG, polymyositis and thymolipoma; case 2: MG and necrotizing myopathy without thymic pathology on a background of scleroderma, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia (CREST); case 3: MG and dermatomyositis without thymic pathology; case 4: MG and dermatomyositis with type C thymoma., Discussion: The recognition of these neuromuscular co-morbidities contributes to (i) understanding their pathogenic mechanisms, (ii) developing better management approaches and (iii) further improving disease outcomes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cancer patients often show a wasting syndrome for which there are little therapeutic options. Dietary polyphenols have been proposed for treating this syndrome, but their usefulness in cases associated with human papillomavirus (HPV)-induced cancers is unknown. We characterized HPV16-transgenic mice as a model of cancer cachexia and tested the efficacy of long-term oral supplementation with polyphenols curcumin and rutin. Both compounds were orally administered to six weeks-old HPV16-transgenic mice showing characteristic multi-step skin carcinogenesis, for 24weeks. Skin lesions and blood, liver and spleen inflammatory changes were characterized histologically and hematologically. Hepatic oxidative stress, skeletal muscle mass and the levels of muscle pro-inflammatory transcription factor NF-κB were also assessed. Skin carcinogenesis was associated with progressive, severe, systemic inflammation (leukocytosis, hepatitis, splenitis), significant mortality and cachexia. Curcumin and rutin totally suppressed mortality while reducing white blood cells and the incidence of splenitis and hepatitis. Rutin prevented muscle wasting more effectively than curcumin. Preservation of muscle mass and reduced hepatic inflammation were associated with down-regulation of the NF-κB canonical pathway and with reduced oxidative stress, respectively. These results point out HPV16-transgenic mice as a useful model for studying the wasting syndrome associated with HPV-induced cancers. Dietary NF-κB inhibitors may be useful resources for treating this syndrome., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Growing evidence suggests a possible association between low vitamin D levels and increased cardiovascular risk. However, research regarding the period of adolescence is scarce. We aimed to evaluate the association of vitamin D, intake and serum 25(OH)D levels, with cardiometabolic risk factors in 13-year-old adolescents., Methods: We conducted a cross-sectional analysis of 1033 adolescents evaluated at 13years old as part of the population-based cohort EPITeen. Vitamin D intake was assessed by a food frequency questionnaire. Serum 25(OH)D levels were assessed for a subsample of 514 participants. Metabolic syndrome (MetS) features were defined according to the National Cholesterol Education Program Adult Treatment Panel III definition modified for age. Logistic regression was fitted to estimate the association between vitamin D status and cardiometabolic risk factors, adjusting for sex, parental education, BMI, physical activity and season., Results: Mean (SD) vitamin D levels, 4.61 (2.50)μg for intake and 16.52 (5.72)ng/mL for serum, were below the recommendations. The prevalence of MetS was 13.2%. Total cholesterol and LDL levels significantly decreased with 25(OH)D serum increase. After adjustment, no association was found between vitamin D levels and MetS. Regarding MetS features, an increased odds of high BMI was observed for those with a lower intake (OR 1.87 95% CI 1.04-3.35)., Conclusions: Although a significant increase in total and LDL cholesterol was observed for lower 25(OH)D levels, and an increased odds of high BMI was observed for those with a lower vitamin D intake, no significant association was observed between vitamin D levels and metabolic syndrome., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to the expression of mutant huntingtin. Bioenergetic dysfunction has been described to contribute to HD pathogenesis. Thus, treatment paradigms aimed to ameliorate energy deficits appear to be suitable candidates in HD. In previous studies, we observed protective effects of insulin growth factor-1 (IGF-1) in YAC128 and R6/2 mice, two HD mouse models, whereas IGF-1 and/or insulin halted mitochondrial-driven oxidative stress in mutant striatal cells and mitochondrial dysfunction in HD human lymphoblasts. Here, we analyzed the effect of IGF-1 versus insulin on energy metabolic parameters using striatal cells derived from HD knock-in mice and primary cortical cultures from YAC128 mice. STHdh(Q111/Q111) cells exhibited decreased ATP/ADP ratio and increased phosphocreatine levels. Moreover, pyruvate levels were increased in mutant cells, most probably in consequence of a decrease in pyruvate dehydrogenase (PDH) protein expression and increased PDH phosphorylation, reflecting its inactivation. Insulin and IGF-1 treatment significantly decreased phosphocreatine levels, whereas IGF-1 only decreased pyruvate levels in mutant cells. In a different scenario, primary cortical cultures derived from YAC128 mice also displayed energetic abnormalities. We observed a decrease in both ATP/ADP and phosphocreatine levels, which were prevented following exposure to insulin or IGF-1. Furthermore, decreased lactate levels in YAC128 cultures occurred concomitantly with a decline in lactate dehydrogenase activity, which was ameliorated with both insulin and IGF-1. These data demonstrate differential HD-associated metabolic dysfunction in striatal cell lines and primary cortical cultures, both of which being alleviated by insulin and IGF-1., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Organic and inorganic pollutant levels were determined for the most discarded species from trawlers operating in Great Sole and Spanish coastal fishing grounds. Results for heavy metals indicated that Cd can reach values higher than legal limits for some species and tissues, while Hg and Pb concentrations are below established values. No significant variation was noticed with fishing grounds, but both season influences in the case of Pb and interspecies variation for Hg and Cd have been detected. Valorization recommendations could be therefore established according to the levels found in the different species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Contaminant effects on species are generally assessed with single-species bioassays. As a consequence, interactions between species that occur in ecosystems are not taken into account. To investigate the effects of contaminants on interacting species dynamics, our study describes the functioning of a 2-L laboratory microcosm with two species, the duckweed Lemna minor and the microalgae Pseudokirchneriella subcapitata, exposed to cadmium contamination. We modelled the dynamics of both species and their interactions using a mechanistic model based on coupled ordinary differential equations. The main processes occurring in this two-species microcosm were thus formalised, including growth and settling of algae, growth of duckweeds, interspecific competition between the two species and cadmium effects. We estimated model parameters by Bayesian inference, using simultaneously all the data issued from multiple laboratory experiments specifically conducted for this study. Cadmium concentrations ranged between 0 and 50 μg·L(-1). For all parameters of our model, we obtained biologically realistic values and reasonable uncertainties. Only duckweed dynamics was affected by interspecific competition, while algal dynamics was not impaired. Growth rate of both species decreased with cadmium concentration, as well as competition intensity showing that the interspecific competition pressure on duckweed decreased with cadmium concentration. This innovative combination of mechanistic modelling and model-guided experiments was successful to understand the algae-duckweed microcosm functioning without and with contaminant. This approach appears promising to include interactions between species when studying contaminant effects on ecosystem functioning., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: This study compared 2 types of recombinant follicle stimulating hormone (rFSH): diluted and diluted/dynamized, on in vitro development of ovine follicles., Methods: In experiment 1, ovarian fragments were cultured for 1 or 7 days in α-MEM(+) in the absence or presence of different concentrations of diluted rFSH to determine the best concentration. In experiment 2, the effect of diluted and diluted/dynamized rFSH (rFSH 6 cH--ultradiluted and succussioned), alone or in combination, was studied., Results: In experiment 1, compared to control, 50ng/mL of diluted rFSH induced higher rates of follicular survival after 7 days of culture and higher percentages of growing follicles at day 1 of culture (P<0.05). In experiment 2, compared to control, diluted/dynamized rFSH induced higher follicular diameter and survival rate after 7 days and early follicle activation at day 1 of culture (P<0.05). Compared to diluted rFSH, diluted/dynamized rFSH induced higher rates of follicle activation at day 1 of culture (P<0.05)., Conclusion: In conclusion, compared to the control medium, diluted/dynamized rFSH promoted survival and early activation of follicles, while diluted rFSH promoted higher activation later in the culture. Thus, diluted/dynamized rFSH may be used as an alternative to diluted rFSH for the in vitro culture of ovine preantral follicles., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: Male Breast Cancer (MBC) remains a poor understood disease. Prognostic factors are not well established and specific prognostic subgroups are warranted., Patients/methods: Retrospectively revision of 111 cases treated in the same Cancer Center. Blinded-central pathological revision with immunohistochemical (IHQ) analysis for estrogen (ER), progesterone (PR) and androgen (AR) receptors, HER2, ki67 and p53 was done. Cox regression model was used for uni/multivariate survival analysis. Two classifications of Female Breast Cancer (FBC) subgroups (based in ER, PR, HER2, 2000 classification, and in ER, PR, HER2, ki67, 2013 classification) were used to achieve their prognostic value in MBC patients. Hierarchical clustering was performed to define subgroups based on the six-IHQ panel., Results: According to FBC classifications, the majority of tumors were luminal: A (89.2%; 60.0%) and B (7.2%; 35.8%). Triple negative phenotype was infrequent (2.7%; 3.2%) and HER2 enriched, non-luminal, was rare (≤1% in both). In multivariate analysis the poor prognostic factors were: size >2 cm (HR:1.8; 95%CI:1.0-3.4 years, p = 0.049), absence of ER (HR:4.9; 95%CI:1.7-14.3 years, p = 0.004) and presence of distant metastasis (HR:5.3; 95%CI:2.2-3.1 years, p < 0.001). FBC subtypes were independent prognostic factors (p = 0.009, p = 0.046), but when analyzed only luminal groups, prognosis did not differ regardless the classification used (p > 0.20). Clustering defined different subgroups, that have prognostic value in multivariate analysis (p = 0.005), with better survival in ER/PR+, AR-, HER2-and ki67/p53 low group (median: 11.5 years; 95%CI: 6.2-16.8 years) and worst in PR-group (median:4.5 years; 95%CI: 1.6-7.8 years)., Conclusion: FBC subtypes do not give the same prognostic information in MBC even in luminal groups. Two subgroups with distinct prognosis were identified in a common six-IHQ panel. Future studies must achieve their real prognostic value in these patients., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Evidence of the association between parental child-feeding practices and the child's body mass index (BMI) is controversial, and bidirectional effects have been poorly studied., Objective: We aimed to examine bidirectional associations between parental child-feeding practices and BMI at 4 and 7 y of age., Design: This study included 3708 singleton children from the Generation XXI birth cohort with data on parental child-feeding practices and BMI at 4 and 7 y old. Feeding practices were assessed through a self-administered questionnaire by combining the Child Feeding Questionnaire and the Overt/Covert Control scale and then adapting it to Portuguese preschool children. Weight and height were measured according to standardized procedures, and age- and sex-specific BMI z scores were computed based on the WHO Growth References. Linear regression models were used to estimate the bidirectional associations between each practice and BMI z score. Crosslagged analyses were performed to compare the directions of those associations (the mean score of each practice and BMI z score at both ages were standardized to enable effect size comparisons)., Results: After adjustments, pressure to eat and overt control at 4 y of age were associated with a lower BMI z score 3 y later (β: -0.05; 95% CI: -0.08, -0.03 and β: -0.05; 95% CI: -0.09, -0.01, respectively). Regarding the opposite direction of association, a higher BMI z score at 4 y of age was significantly associated with higher levels of restriction and covert control at 7 y of age (β: 0.06; 95% CI: 0.03, 0.08 and β: 0.06; 95% CI: 0.04, 0.08, respectively) and with lower levels of pressure to eat (β: -0.17; 95% CI: -0.20, -0.15). The only bidirectional practice, pressure to eat, was more strongly influenced by the BMI z score than the reverse (βstandardized: -0.17 compared with βstandardized: -0.04; likelihood ratio test: P < 0.001)., Conclusions: We found that parents both respond to and influence the child's weight; thus, this child-parent interaction should be considered in future research., (© 2016 American Society for Nutrition.)

The Cystic Fibrosis p.Ile1234Val missense mutation actually creates a new dual splicing site possibly used either as a new acceptor or donor. Here, we aimed to test the accuracy of in silico predictions by comparing them with in vitro and ex vivo functional analyses of this mutation for an accurate CF diagnosis/prognosis. To this end, we applied a new in vitro strategy using a CFTR mini-gene which includes the complete CFTR coding sequence plus intron 22 (short version) which allows the assessment of alternatively spliced mRNA levels as well as the properties of the resulting abnormal CFTR protein regarding processing, intracellular localization and function. Our data demonstrate that p.Ile1234Val leads to usage of the alternative splicing donor (but not acceptor) resulting in alternative CFTR transcripts lacking 18 nts of exon 22 which produce a truncated CFTR protein with residual Cl- channel function. These results recapitulate data from native tissues of a CF patient. In conclusion, the existing in silico prediction models have limited application and ex vivo functional assessment of mutation effects should be made. Alternatively the in vitro strategy adopted here can be applied to assess the disease liability of mutations for an accurate CF diagnosis/prognosis., (Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Objective: To examine the relationship between probe tip size and force readings of mechanical nociceptive thresholds (MTs) to identify appropriate probes for horses., Study Design: Randomized, crossover study., Animals: Eight adult, mixed-breed horses aged 5-10 years, weighing 268-460 kg., Methods: Four probe configurations (PCs) were used in random sequence: 1.0 mm diameter (SHARP); 3.2 mm (BLUNT); spring-mounted 1.0 mm (SPRING), and 3 × 2.5 mm (3PIN). A remote-controlled unit on the horse increased force (1.2 N second(-1)) in a pneumatic actuator on the metacarpus. Mean MT for each PC was calculated from 10 readings for each horse. Data were log-transformed for analysis using mixed-effects anova/linear regression (p < 0.05). Variability of data for each PC was assessed using the coefficient of variation (CV)., Results: Mean ± standard deviation MTs were: SHARP, 5.6 ± 2.3 N; BLUNT, 11.4 ± 3.4 N; 3PIN, 9.6 ± 4.6 N, and SPRING 6.4 ± 1.8 N. Mean MT for SHARP was significantly lower than for BLUNT (p < 0.001) and 3PIN (p < 0.001), but not different from SPRING (p > 0.05). Mean MT was significantly higher for BLUNT than for 3PIN (p < 0.05) and SPRING (p < 0.001). Mean MT for 3PIN was significantly higher than for SPRING (p < 0.001). Larger contact area PCs produced higher MTs than smaller PCs, but the relationship was not linear. BLUNT (area: 10-fold greater) gave a MT two-fold higher than SHARP. 3PIN (area: 20-fold greater) produced more variable MTs, less than two-fold higher than SHARP. SPRING was similar to SHARP. CVs were: SHARP, 22.9%; BLUNT, 72.3%; 3PIN, 44.2%, and SPRING, 28.7%., Conclusions and Clinical Relevance: The PC has nonlinear effects on MT. Therefore, it is important to define PC when measuring MT. Smaller probe tips may be preferable as MT data are less variable., (© 2015 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia.)