Loss of Filamin A leads to heart failure in zebrafish

Dilated Cardiomyopathy (DCM) is frequently leading to heart failure. Familial analysis revealed 30% - 60 % of cases to be of genetic cause, predominantly occurring in genes encoding for structural proteins. The underlying mechanisms and signalling events that translate DCM causing mutations into the clinical phenotype are poorly understood. Previously, our group identified the Core- Binding Factor beta (CBFb) to be an essential factor for maintaining the sarcomeric Z-disc structure of the heart muscle. CBFb is mainly located in the sarcomere and is retained in the cytoplasm via the interaction with Filamin A (FLNA). However it is hypothesized that under stress conditions, CBFb is translocated to the nucleus, where it regulates the expression of several genes. To analyze the contribution of FLNA to the development of heart failure we created a transient and a stable loss-of-function model of FLNA in zebrafish. FLNA transient knockdown (MO-flna) resulted in systolic dysfunction, defects in re-absorption of cardiac matrix, a dilated atrium, blood regurgitation and maturation defects shown by uncompleted heart looping. We observed a significantly reduced heart rate (MO-control vs MO-flna; mean±SD: 148±6.9 vs 108±25.1 beats/min, respectively; p<0.05) and fractional shortening (MO-control vs MO-flna; mean±SD: 64.7±4.0 vs 19.3±10.8 beats/min, respectively; p<0.0001). We further established the CRISPR-CAS9 mediated FLNA stable knockout (FLNA KO). Indeed, in F0 embryos we observed a significantly reduced heart rate (wildtype vs FLNA KO; mean±SD: 144±17.6 vs 106±12.3 beats/min, respectively; p<0.0001) and fractional shortening (wildtype vs FLNA KO; mean±SD: 64±8.9 % vs 43±8.8 %, respectively; p<0.0001), as observed for the FLNA knockdown. F0 mosaic embryos were grown to adulthood and outcrossed with wildtype fish. Two FLNA KO lines (with a predicted stop codon on flna exon 4) were selected for propagation. Heterozygous adults from both lines showed a significantly reduced ejection fraction (wildtype vs FLNA KO; mean±SD: 62±6.9 % vs 41±12.2 %, respectively; p<0.01) and increased ventilation frequency (wildtype vs FLNA KO; mean±SD: 7±4.2 vs 15±7.0 buccal movements, respectively; p<0.001). Compound heterozygous, with both lines mutations, developed a pericardial edema with a dilated heart, stressing the potential role of FLNA in maintaining heart function. Homozygous embryos from one of the lines, did not show a heart failure phenotype. In accordance, flnb was shown to be up-regulated in FLNA KO adults, illustrating possible compensatory mechanisms activation. In regard to CBFb translocation mechanism, for the first time, beta-adrenergic stress was identified as a trigger for CBFb translocation to the nucleus. In conclusion, FLNA knockdown and knockout in zebrafish leads to a heart failure phenotype. By deeply investigating FLNA regulation (such as cleavage, phosphorylation, response to external cellular stress, etc) and its interacting partners (such as CBFb), we will further our understanding of the genetic pathogenic pathways involved in heart failure

Fate map of zebrafish left-right organizer

The organizer is a ciliated signalling transient organ, responsible for the patterning of embryo tissues during embryonic development. In higher vertebrates, such as mouse and chick, this organizer (the node and the Hensen’s node, respectively) performs dorsalventral and anteriorposterior axis definition, as well as left-right patterning of the internal organs. In lower vertebrates, such as frog and zebrafish, there is a separate specialized organ for left-right purposes called the Gastrocoel Roof Plate (GRP) and Kupffer’s Vesicle (KV), respectively. It is known that mouse and chick organizer cells give rise to structures like floor plate, notochord, hypochord and somites. Frog GRP originates all these but floor plate. In zebrafish, at 13-14 somite stage (ss) the KV finished its left-right patterning but what happens to this organizer’ cells is still poorly studied. This research attempts to understand the fate and behaviour of the KV cells. We followed the fate of KV cells by live imaging and by tight time-courses with fixed larvae. We assessed in detail their proliferative and death profile, as well as cilia length progression from 9-10 ss until 29-30 ss. We conclude that the KV cells mostly follow the evolutionarily conserved fates described for other organizers. These cells mainly incorporate the notochord and hypochord; few cells incorporate the floor plate and the somites. As a novelty, it is also hypothesized that the hypural cell fate may be among the KV cell fates

Patient-physician discordance in assessment of adherence to inhaled controller medication: a cross-sectional analysis of two cohorts

We aimed to compare patient's and physician's ratings of inhaled medication adherence and to identify predictors of patient-physician discordance.(SFRH/BPD/115169/2016) funded by Fundação para a Ciência e Tecnologia (FCT); ERDF (European Regional Development Fund) through the operations: POCI-01-0145-FEDER-029130 ('mINSPIRERS—mHealth to measure and improve adherence to medication in chronic obstructive respiratory diseases—generalisation and evaluation of gamification, peer support and advanced image processing technologies') cofunded by the COMPETE2020 (Programa Operacional Competitividade e Internacionalização), Portugal 2020 and by Portuguese Funds through FCT (Fundação para a Ciência e a Tecnologia).info:eu-repo/semantics/publishedVersio

Genomic structural variations lead to dysregulation of important coding and non-coding RNA species in dilated cardiomyopathy

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non-coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome-wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non-protein-coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non-coding RNAs and 1,756 protein-coding transcripts. 65.3% of the SV-eQTLs do not harbor a significant SNV-eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein-coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike

Morphological and Postural changes in the foot during pregnancy and puerperium : a longitudinal study

The aim of this study is to observe the morphological and postural changes to the foot that take place during pregnancy and the puerperium. Method: In this descriptive, observational, longitudinal study, we analysed 23 pregnant women, with particular attention to morphological and postural aspects of the foot, at three time points during and after pregnancy: in weeks 9-13 of gestation, weeks 32-35 of gestation and weeks 4-6 after delivery. The parameters considered were changes in foot length, the Foot Posture Index (FPI) and the Hernández Corvo Index, which were analysed using a pedigraph and taking into account the Body Mass Index (BMI). The same procedure was conducted in each review. Results: The statistical analyses obtained for each foot did not differ significantly between the three measurement times. A pronator-type footprint was most frequently observed during the third trimester of pregnancy; it was predominantly neutral during the postpartum period. Statistically significant differences between the measurement times were obtained in the right foot for cavus vs. neutral foot type (between the first and third trimesters and also between the first trimester and the puerperium) (in both cases, p < 0.0001). Conclusions: Foot length increases in the third trimester and returns to normal in the puerperium. According to FPI findings, the third trimester of pregnancy is characterised by pronation, while the posture returns to neutrality during the postpartum period. During pregnancy, the plantar arch flattens, and this persists during the puerperium. The incidence of cavus foot increases significantly in the third trimester and in the puerperium

Identification of clusters of asthma control: A preliminary analysis of the inspirers studies

This work was funded by ERDF (European Regional Development Fund) through the operations: POCI- -01-0145-FEDER-029130 (“mINSPIRERS—mHealth to measure and improve adherence to medication in chronic obstructive respiratory diseases - generalisation and evaluation of gamification, peer support and advanced image processing technologies”) co-funded by the COMPETE2020 (Programa Operacional Competitividade e Internacionalização), Portugal 2020 and by Portuguese Funds through FCT (Fundação para a Ciência e a Tecnologia).© 2020, Sociedade Portuguesa de Alergologia e Imunologia Clinica. All rights reserved. Aims: To identify distinct asthma control clusters based on Control of Allergic Rhinitis and Asthma Test (CARAT) and to compare patients’ characteristics among these clusters. Methods: Adults and adolescents (≥13 years) with persistent asthma were recruited at 29 Portuguese hospital outpatient clinics, in the context of two observational studies of the INSPIRERS project. Demographic and clinical characteristics, adherence to inhaled medication, beliefs about inhaled medication, anxiety and depression, quality of life, and asthma control (CARAT, >24 good control) were collected. Hierarchical cluster analysis was performed using CARAT total score (CARAT-T). Results: 410 patients (68% adults), with a median (percentile 25–percentile 75) age of 28 (16-46) years, were analysed. Three clusters were identified [mean CARAT-T (min-max)]: cluster 1 [27(24-30)], cluster 2 [19(14-23)] and cluster 3 [10(2-13)]. Patients in cluster 1 (34%) were characterised by better asthma control, better quality of life, higher inhaler adherence and use of a single inhaler. Patients in clusters 2 (50%) and 3 (16%) had uncontrolled asthma, lower inhaler adherence, more symptoms of anxiety and depression and more than half had at least one exacerbation in the previous year. Further-more, patients in cluster 3 were predominantly female, had more unscheduled medical visits and more anxiety symp-toms, perceived a higher necessity of their prescribed inhalers but also higher levels of concern about taking these inhalers. There were no differences in age, body mass index, lung function, smoking status, hospital admissions or specialist physician follow-up time among the three clusters. Conclusion: An unsupervised method based on CARAT--T, identified 3 clusters of patients with distinct, clinically meaningful characteristics. The cluster with better asthma control had a cut-off similar to the established in the validation study of CARAT and an additional cut-off seems to distinguish more severe disease. Further research is necessary to validate the asthma control clusters identified.publishersversionpublishe

Efeitos dos filtros UV em espermatozoides e adultos de Mytilus galloprovincialis

Multiple stressors are leading to unprecedent pressures in coastal systems namely pollutants such as personal care products and climate change such as ocean warming. Avobenzone (AVO) is one of the most used ultraviolet (UV) filters in personal care products which use is progressively higher. UV filters are persistent and can be accumulated in the environment whereas their impacts in marine organisms, in particular bivalves, are poorly studied. Bivalves represent a significant proportion of aquaculture production, being highly abundant in European coastal zones and having socioeconomic importance. Mussels are considered sentinel organisms and are often used in ecotoxicological research. Due to recreational activities at the beach, UV filters reach higher peaks during summer in aquatic bodies, sometimes coincident with the spawning period of mature sedentary mussels. For this reason, this study aims to evaluate the toxic effects of a range of AVO concentrations (0.1, 1.0, 10.0 μg/L) and investigate in which way warming (21 °C) may change the effects caused at different biological levels of the Mediterranean mussel species Mytilus galloprovincialis (Lamarck, 1819): sperm (in vitro 30 min-exposure) and adults (in vivo 28 days-exposure). Effects were assessed in sperm cells (oxidative status, viability, genotoxicity, motility and kinetics) and adults (energy reserves, metabolic capacity, biological defence mechanisms, cell damages and neurotoxicity). Cellular damages occurred only in sperm exposed to warming and their susceptibility to warming increased significantly when combined with AVO, since this UV filter alone did not exert any effects. AVO concentration range, warming and AVO with higher temperature led to oxidative stress in sperm. A decrease of viability was observed in sperm exposed to the highest AVO concentrations and warming. All AVO concentrations induced genotoxicity and warming increased sperm sensitivity to AVO. Respiration rate increased significantly in sperm exposed to 0.5 μg/L AVO, warming and AVO with warming. AVO concentration range and AVO with higher temperature induced a decrease of motility and the highest AVO concentration caused a complete immobility. A decrease of metabolism was observed at the lowest AVO concentration and an increase occurred with warming in adults. Energy reserves and antioxidant defences’ activity increased significantly in adults at the highest AVO concentration. Results suggest a higher sensitivity of sperm regarding AVO and warming, a higher susceptibility to the interactive effect of both stressors and to the highest AVO concentration, with possible impairment of male reproductive functions and disruption of M. galloprovincialis populations. This study highlights the importance of further research on the toxic impacts resulting from the interaction of emergent pollutants and climate change towards bivalves, considering their ecological and economic relevance. Pollution levels in coastal systems tend to increase and mussels’ responses can change under different scenarios, compromising the maintenance of the population, reinforcing the need for more research.Stressores múltiplos estão a levar a pressões sem precedentes nos sistemas costeiros, nomeadamente poluentes como os produtos de cuidado pessoal e as alterações climáticas como o aquecimento dos oceanos. Avobenzona (AVO) é um dos filtros ultravioleta (UV) mais utilizados em produtos de cuidado pessoal cujo uso é progressivamente crescente. Os filtros UV são persistentes e podem acumular-se no meio ambiente enquanto os seus impactos nos organismos marinhos, em particular nos bivalves, são pouco conhecidos. Os moluscos bivalves representam uma proporção significativa da produção aquícola, sendo abundantes nas zonas costeiras da Europa e importantes a nível socioeconómico. Os mexilhões são considerados sentinelas e são frequentemente utilizados em trabalhos de investigação em ecotoxicologia. Devido a atividades recreativas nas praias, os filtros UV atingem concentrações mais elevadas nas massas de água durante o verão, por vezes coincidentes com o período de desova dos mexilhões maturos que são sedentários. Por esta razão, este estudo tem como principal objetivo avaliar os efeitos tóxicos de uma gama de concentrações de AVO (0,1, 1,0, 10,0 μg/L) e os efeitos AVO (0,5 μg/L) e investigar de que forma o aumento da temperatura (21 °C) poderá alterar os efeitos causados a diferentes níveis biológicos da espécie de mexilhão do Mediterrâneo Mytilus galloprovincialis (Lamarck, 1819): espermatozoides (exposição in vitro 30 min) e adultos (exposição in vivo 28 dias). Os efeitos foram avaliados em espermatozoides (estado oxidativo, viabilidade, genotoxicidade, motilidade e cinética) e nos adultos (reservas energéticas, capacidade metabólica, mecanismos de defesa biológica, danos nas células e neurotoxicidade). Ocorreram danos celulares apenas em espermatozoides expostos à temperatura mais alta, cuja suscetibilidade ao aquecimento aumentou significativamente quando em conjunto com AVO, uma vez que este filtro UV por si só não despoletou quaisquer danos celulares. A gama de concentrações da AVO, temperatura mais alta e a combinação de ambas levaram a stress oxidativo nos espermatozoides. Foi observada uma diminuição da viabilidade nos espermatozoides expostos às maiores concentrações da AVO e à temperatura mais elevada. Todas as concentrações de AVO induziram genotoxicidade e o aquecimento aumentou a sensibilidade dos gâmetas a AVO. A taxa de respiração aumentou significativamente nos espermatozoides expostos a 0,5 μg/L AVO, à temperatura mais alta e ao conjunto de ambas. A gama de concentrações da AVO e a combinação com o aumento de temperatura induziram uma diminuição da motilidade, tendo-se verificado uma inibição total deste parâmetro na concentração mais elevada. Verificou-se uma diminuição do metabolismo na concentração mais baixa de AVO e um aumento do mesmo quando na presença de temperatura mais alta nos adultos. As reservas de energia e a atividade das defesas antioxidantes aumentaram significativamente nos adultos após exposição à concentração mais alta de AVO. Os resultados obtidos sugerem uma maior sensibilidade dos espermatozoides em relação à AVO e ao aquecimento, e uma maior suscetibilidade ao efeito interativo que ambos os stressores exercem e à maior concentração de AVO, podendo comprometer as funções reprodutivas masculinas e levar à consequente rutura das populações de M. galloprovincialis. Tendo em conta os resultados obtidos, é fundamental investigar os impactos tóxicos resultantes da interação de poluentes emergentes e das alterações climáticas em bivalves, tendo em conta o seu valor ecológico e económico. Com o aumento dos níveis de poluição nos sistemas costeiros as respostas dos mexilhões podem mudar em diferentes cenários, comprometendo a manutenção da população, reforçando assim a necessidade de uma investigação mais abrangente e aprofundada.Mestrado em Biologia Marinha Aplicad

Caso clínico dermatológico

A previously healthy 15-year-old girl was sent to the Pediatric Emergency Department by the general practitioner due to suspicion of a hematological condition. The girl presented with ecchymotic lesions on the anterior region of the thorax and antero-proximal region of the upper limbs, which had apparently worsened in the last 24 hours. She denied trauma or impact sports, fever or recent infections, and blood loss or other symptoms. Laboratory evaluation showed no alterations and urine toxicology screening was negative. When confronted with normal laboratory evaluation, the girl confessed intentional self-inflicted injuries following a discussion with her mother. Parents were made aware of the situation, and a pediatric psychiatry consultation was recommended. After discharge, follow-up in the girl’s home country was assured. With this report, the author intend to raise awareness of the importance of maintaining a high index of suspicion to non-suicidal self-injury, identifying alarm signs and establishing early preventive strategies.info:eu-repo/semantics/publishedVersio

Púrpura de Henoch-Schönlein na prática clínica – proposta de abordagem e orientação

A Púrpura de Henoch-Schönlein (PHS) é a vasculite sistémica mais comum na infância. Na maioria das crianças/ adolescentes, o prognóstico é excelente com resolução espontânea dos sinais e sintomas. No entanto, uma pequena percentagem de doentes pode desenvolver complicações, nomeadamente nefrite, que dita o prognóstico. Embora a apresentação clínica e o diagnóstico sejam facilmente reconhecidos, o tratamento e a orientação durante o seguimento em consulta vão depender da evolução da doença e, por isso, não estão absolutamente definidos. Esta revisão pretende oferecer uma visão geral da PHS, incluindo fisiopatogenia, clínica e diagnósticos diferenciais e propor procedimentos a adotar na abordagem e orientação destes doentes, na tentativa de otimizar a terapêutica e o seguimento em consulta desta doença