Repositório RCAAP

Engineering brain organoids from human induced pluripotent stem cells (hiPSCs) is a powerful tool for modeling brain development and neurological disorders. Rett syndrome (RTT), a rare neurodevelopmental disorder, can greatly benefit from this technology, since it affects multiple neuronal subtypes in forebrain sub-regions. We have established dorsal and ventral forebrain organoids from control and RTT patient-specific hiPSCs recapitulating 3D organization and functional network complexity. Our data revealed a premature development of the deep-cortical layer, associated to the formation of TBR1 and CTIP2 neurons, and a lower expression of neural progenitor/proliferative cells in female RTT dorsal organoids. Moreover, calcium imaging and electrophysiology analysis demonstrated functional defects of RTT neurons. Additionally, assembly of RTT dorsal and ventral organoids revealed impairments of interneuron's migration. Overall, our models provide a better understanding of RTT during early stages of neural development, demonstrating a great potential for personalized diagnosis and drug screening.

Major depressive disorder (MDD) is the foremost cause of global disability, being responsible for enormous personal, societal, and economical costs. Importantly, existing pharmacological treatments for MDD are partially or totally ineffective in a large segment of patients. As such, the search for novel antidepressant drug targets, anchored on a clear understanding of the etiological and pathophysiological mechanisms underpinning MDD, becomes of the utmost importance. The adenosinergic system, a highly conserved neuromodulatory system, appears as a promising novel target, given both its regulatory actions over many MDD-affected systems and processes. With this goal in mind, we herein review the evidence concerning the role of adenosine as a potential player in pathophysiology and treatment of MDD, combining data from both human and animal studies. Altogether, evidence supports the assertions that the adenosinergic system is altered in both MDD patients and animal models, and that drugs targeting this system have considerable potential as putative antidepressants. Furthermore, evidence also suggests that modifications in adenosine signaling may have a key role in the effects of several pharmacological and non-pharmacological antidepressant treatments with demonstrated efficacy, such as electroconvulsive shock, sleep deprivation, and deep brain stimulation. Lastly, it becomes clear from the available literature that there is yet much to study regarding the role of the adenosinergic system in the pathophysiology and treatment of MDD, and we suggest several avenues of research that are likely to prove fruitful.

Background: Circular RNAs (circRNAs) are known to participate in lung cancer. However, their role in spinal metastasis (SM) of lung adenocarcinoma remains elusive. In this study, we determined that hsa_circ_0006571 serves as a sponge for miR-138, which targets sirtuin 1 (Sirt1) in the development of SM. Methods: A human circRNA microarray was performed to compare SM and lung adenocarcinoma samples. The expression of hsa_circ_0006571 and miR-138 was determined using quantitative polymerase chain reaction (qPCR) in vitro and in vivo. Cell proliferation was performed by Cell Counting Kit-8 (CCK-8) and apoptosis was analyzed by Annexin V/PI staining. RNA-pulldown and RNA immunoprecipitation (RIP) were used to analyze the interaction between hsa_circ_0006571. Tumor metastasis was determined through a xenograft experiment in vivo. Results: Hsa_circ_0006571 was observed to be significantly upregulated in SM tissues through circRNA microarray and qPCR. We detected a lower expression of miR-138 in SM tissues compared with lung adenocarcinoma. Hsa_circ_0006571 silencing suppressed lung cancer cell proliferation and migration while promoting apoptosis. Hsa_circ_0006571 interacted with miR-138 to promote expression of Sirt1, leading to activation of epithelial-mesenchymal transition (EMT). Xenograft experiments showed that downregulation of hsa_circ_0006571 delayed the SM of lung adenocarcinoma cells via the miR-138-Sirt1 axis. Conclusions: Hsa_circ_0006571 promoted tumor cell migration and invasion via the miR-138/Sirt1 pathway. Our observations indicate that circRNAs are possible novel therapeutic targets for SM of lung adenocarcinoma.

Two young males with refractory epilepsy of unknown aetiology were referred for vagus nerve stimulation (VNS). Sleep disturbances emerged following VNS parameter changes. In Patient 1, video-polysomnogram (PSG) disclosed snoring and catathrenia in non-REM sleep. Central apnoea also occurred, but more rarely. In Patient 2, video-PSG showed mixed apnoea with desaturation and episodes of stridor followed by a catathrenia-like sound. A drug-induced sleep endoscopy (DISE) revealed, during VNS OFF time, glossoptosis, "trap door" of the epiglottis, and paresis of the left side of the larynx and ipsilateral vocal cords. During ON time, there were periods of pharyngeal collapse, in which video-PSG revealed patterns suggestive of both obstructive and central sleep apnoea. All these sleep-related phenomena were coincident with VNS ON time. In the first patient, VNS parameter adjustment was sufficient to successfully reverse all the symptoms, whereas the other patient required concomitant treatment with continuous positive airway pressure. The data broaden our knowledge about sleep disorders related to VNS, in particular stridor and catathrenia. We suggest that central sleep apnoea may be associated with laryngeal occlusion. DISE may be considered in selected cases as a valuable clinical tool to evaluate, in a single session, the effectiveness of multiple VNS parameter changes on respiration and laryngeal side effects.

Este artigo centra-se na ética e na responsabilidade social como elementos indispensáveis da educação em ciências. Apresenta-se o estudo de casos como metodologia adequada: a) à identificação, análise e discussão dos dilemas éticos suscitados pela ciência e pela tecnologia contemporâneas; e b) ao desenvolvimento do raciocínio moral.

Discovering antibiotic molecules able to hold the growing spread of antimicrobial resistance is one of the most urgent endeavors that public health must tackle. The case of Gram-negative bacterial pathogens is of special concern, as they are intrinsically resistant to many antibiotics, due to an outer membrane that constitutes an effective permeability barrier. Antimicrobial peptides (AMPs) have been pointed out as potential alternatives to conventional antibiotics, as their main mechanism of action is membrane disruption, arguably less prone to elicit resistance in pathogens. Here, we investigate the in vitro activity and selectivity of EcDBS1R4, a bioinspired AMP. To this purpose, we have used bacterial cells and model membrane systems mimicking both the inner and the outer membranes of Escherichia coli, and a variety of optical spectroscopic methodologies. EcDBS1R4 is effective against the Gram-negative E. coli, ineffective against the Gram-positive Staphylococcus aureus and noncytotoxic for human cells. EcDBS1R4 does not form stable pores in E. coli, as the peptide does not dissipate its membrane potential, suggesting an unusual mechanism of action. Interestingly, EcDBS1R4 promotes a hemi-fusion of vesicles mimicking the inner membrane of E. coli. This fusogenic ability of EcDBS1R4 requires the presence of phospholipids with a negative curvature and a negative charge. This finding suggests that EcDBS1R4 promotes a large lipid spatial reorganization able to reshape membrane curvature, with interesting biological implications herein discussed.

Objectivo: As fluoroquinolonas são, actualmente, antibióticos largamente prescritos. Têm emergido preocupações de segurança relativamente ao seu uso – suportadas por estudos in-vitro, animais e epidemiológicos – quanto a potenciais efeitos nocivos em estruturas de colagénio, provocando tendinopatias e descolamento da retina. Mais recentemente, um associação de risco tem sido estabelecida quanto a aneurismas e dissecções/ruturas da aorta (AA ou AD/AR), ambas condições graves ou mesmo potencialmente fatais. Assim, elaborámos uma revisão sistemática para avaliar o efeito do uso de fluoroquinolonas no risco de desenvolvimento de AA ou AD/AR. Métodos: As bases de dados MEDLINE, Web of Science Core Collection e Cochrane Central Register of Controlled Trials (Março 2019) foram pesquisadas por estudos longitudinais avaliando o uso de fluoroquinolonas comparado com o uso de outros antibióticos ou com não-tratamento, em doentes co m AA ou AD/AR. O risco de viés foi determinado através da ferramenta ROBINS-I. Realizámos uma meta-análise de efeitos aleatórios para estimar os agregados odds ratio com IC 95%, e a heterogeneidade foi avaliada usando a estatística I2. Resultados: Sete estudos observacionais, contemplando designs variados, avaliando aproximadamente 1 024 984 doentes com AA ou AD/AR, foram incluídos na meta-análise. O uso de fluoroquinolonas foi associado com um maior risco de AA ou AD/AR, em comparação com a intervenção de não-tratamento (OR=2.26; 95% CI 1.93 to 2.65; I2=30%) e em comparação com a intervenção de uso de beta-lactâmico (OR=1.56; 95% CI 1.37 to 1.79; I2=0%). O efeito lesivo do uso de fluoroquinolonas permaneceu positivo e estatisticamente significativo quando foram agregados os resultados para o desfecho AD/AR apenas e agregando por design do estudo. Foi considerado que os desfechos dos estudos que utilizaram comparação com beta-lactâmicos têm risco de viés moderado, enquanto que foi considerado que os restantes têm pelo menos um grave risco de viés. Todos os desfechos avaliados têm uma muito baixa qualidade de evidência e força de recomendação (GRADE). Conclusão: O uso de fluoroquinolonas foi associado com um risco significativo de desenvolvimento de AA ou AD/AR.

Background: Parkinsonian variant of multiple system atrophy is a neurodegenerative disorder frequently misdiagnosed as Parkinson's disease. No early imaging biomarkers currently differentiate these disorders. Methods: Simple visual imaging analysis of the substantia nigra and locus coeruleus in neuromelanin-sensitive magnetic resonance imaging and nigrosome 1 in susceptibility-weighted sequences was performed in thirty patients with parkinsonian variant of multiple system atrophy fulfilling possible/probable second consensus diagnostic criteria. The neuromelanin visual pattern was compared to patients with Parkinson's disease with the same disease duration (n = 10) and healthy controls (n = 10). Substantia nigra semi-automated neuromelanin area/signal intensity was compared to the visual data. Results: Groups were similar in age, sex, disease duration, and levodopa equivalent dose. Hoehn & Yahr stage was higher in parkinsonian multiple system atrophy patients, 69% of whom had normal neuromelanin size/signal, significantly different from Parkinson's disease patients, and similar to controls. Nigrosome 1 signal was lost in 74% of parkinsonian multiple system atrophy patients. Semi-automated neuromelanin substantia nigra signal, but not area, measurements were able to differentiate groups. Conclusions: In patients with parkinsonism, simple visual magnetic resonance imaging analysis showing normal neuromelanin substantia nigra and locus coeruleus, combined with nigrosome 1 loss, allowed the distinction of the parkinsonian variant of multiple system atrophy from Parkinson's disease and healthy controls. This easy and widely available method was superior to semi-automated measurements in identifying specific imaging changes in substantia nigra and locus coeruleus.

Plasmodium parasites kill 435 000 people around the world every year due to unavailable vaccines, a limited arsenal of antimalarial drugs, delayed treatment, and the reduced clinical effectiveness of current practices caused by drug resistance. Therefore, there is an urgent need to discover and develop new antiplasmodial candidates. In this work, we present a novel strategy to develop a multitarget metallic hybrid antimalarial agent with possible dual efficacy in both sexual and asexual erythrocytic stages. A hybrid of antimalarial drugs (chloroquine and primaquine) linked by gold(I) was synthesized and characterized by spectroscopic and analytical techniques. The CQPQ-gold(I) hybrid molecule affects essential parasite targets, it inhibits β-hematin formation and interacts moderately with the DNA minor groove. Its interaction with PfTrxR was also examined in computational modeling studies. The CQPQ-gold(I) hybrid displayed an excellent in vitro antimalarial activity against the blood-stage of Plasmodium falciparum and liver-stage of Plasmodium berghei and efficacy in vivo against P. berghei, thereby demonstrating its multiple-stage antiplasmodial activity. This metallic hybrid is a promising chemotherapeutic agent that could act in the treatment, prevention, and transmission of malaria.

Since the discovery of gd T cells, this rare and unique component of the immune system has been recognized for its potential in cancer immunology and immunotherapy. In the mid-1980s, it became clear that a major component of adaptive immune responses is the ability of T cell receptors (TCR) to undergo somatic recombination in order to recognize multiple antigens. TCRs consisting of either ab and gd chains were discovered in rapid succession (1–6). An important observation was made in these initial studies: gd T cells stimulated through their TCR are able to kill cancer cells (2). Over these past decades, researchers have learned that gd T cells share many similarities with ab T cells, as well as major differences. However, discoveries in gd T cell biology have failed to keep the same pace as ab T cell biology. The molecular targets of gdTCRs and functions of these cells have largely eluded researchers, partly because gd T cell recognition of cancer cells and their response kinetics are very different to ab T cells (7, 8). Recent years have seen major advances in gd T cell biology and established the non-redundancy of this lymphocyte subset, particularly in the context of cancer (9–11). gd T cells are being used as cellular vehicles to target tumors and prognostic indicators of cancer progression. The aim of the articles collected in this Research Topic is to describe new developments and approaches to enhance the anti-tumor functions of gd T cells, and to discuss how expression of their ligands can assist with prognosis of cancer patients.

Inflammation can support or restrain cancer progression and the response to therapy. Here, we searched for primary regulators of cancer-inhibitory inflammation through deep profiling of inflammatory tumor microenvironments (TMEs) linked to immune-dependent control in mice. We found that early intratumoral accumulation of interferon gamma (IFN-γ)-producing natural killer (NK) cells induced a profound remodeling of the TME and unleashed cytotoxic T cell (CTL)-mediated tumor eradication. Mechanistically, tumor-derived prostaglandin E2 (PGE2) acted selectively on EP2 and EP4 receptors on NK cells, hampered the TME switch, and enabled immune evasion. Analysis of patient datasets across human cancers revealed distinct inflammatory TME phenotypes resembling those associated with cancer immune control versus escape in mice. This allowed us to generate a gene-expression signature that integrated opposing inflammatory factors and predicted patient survival and response to immune checkpoint blockade. Our findings identify features of the tumor inflammatory milieu associated with immune control of cancer and establish a strategy to predict immunotherapy outcomes.

Introduction. The Cape Verdean bryophyte flora comprises representatives of various floristic elements including Afrotropical, Neotropical, Mediterranean and Asiatic elements, together with some endemic taxa. However, the knowledge of Cape Verdean bryophytes lags far behind that of vascular plants. This study contributes to increase the knowledge of bryophytes in the Cape Verde archipelagos. Methods. A total of seven sites were sampled during 2016 in the Cape Verde archipelago, one on São Vicente and six on Santo Antão. Results and conclusions. Five species are reported new to Cape Verde, Cheilolejeunea rigidula (Nees ex Mont.) R.M.Schust., Riccia trabutiana Steph., Lewinskya acuminata (H.Philib.) F.Lara, Garilleti & Goffinet, Lindbergia patentifolia Dixon and Timmiella cameruniae Broth. The presence of Porella canariensis (F.Weber) Underw in the archipelago is also confirmed. Additionally, Frullania spongiosa Steph., Bryum dichotomum Hedw., Cryptoleptodon longisetus (Mont.) Enroth and Didymodon hastatus(Mitt.) R.H.Zander are reported for the first time from Santo Antão island. Cheilolejeunea rigidula, Lindbergia patentifolia and Timmiella cameruniae are new to Macaronesia and a description of the last taxon is presented.

Este estudo examinou como uma intervenção em Inteligência Emocional sobre estudantes de medicina consegue aumentar os níveis de IE e PsyCap nos alunos. Oitenta e um estudantes da Faculdade de Medicina da Universidade de Lisboa, voluntariaram-se para participar neste estudo e foram testados inicialmente quanto aos seus níveis de IE e PsyCap, através da Escala de Inteligência Emocional de Wong & Law (WLEIS) e do Psychological Capital Questionnaire (PCQ-12), respetivamente. Foi aplicada uma intervenção ao longo de um mês e meio, estruturada em 8 sessões, e analisados posteriormente os mesmos níveis de IE e PsyCap. As análises de resultados comprovaram as nossas hipóteses de que a intervenção potencia o aumento dos níveis de IE e PsyCap nos estudantes. Esta pesquisa salienta a importância dos construtos de IE e PsyCap no ensino superior e no contexto particular dos estudantes de medicina e a necessidade de estes dois recursos serem fomentados e desenvolvidos ao longo do curso.

A adolescência é um período em que ocorrem transformações importantes nas relações dos adolescentes com a sua família, o que que pode ter impacto no seu bem-estar. Este estudo transversal teve como objectivo principal analisar a associação entre as variáveis da coesão familiar, relação com a mãe, relação com o pai e o bem-estar na adolescência, assim como examinar as diferenças em função do sexo e da idade dos adolescentes. Os participantes neste estudo foram 135 adolescentes portugueses com idades compreendidas entre os 10 e os 19 anos. As medidas utilizadas neste estudo foram as versões portuguesas das Escalas de Relacionamento Positivo com os Pais para adolescentes (PRP) para avaliar a relação dos adolescentes com ambos os pais, a Escala de Ambiente Familiar (FES) para avaliar a coesão familiar e a Escala de Bem-estar de Ryff para Crianças e Adolescentes (RWS) para avaliar o bem-estar dos adolescentes. As análises realizadas foram as correlações de Pearson, a análise de regressão múltipla hierárquica, e testes t para amostras independentes. Os resultados demonstraram uma associação positiva entre a relação com a mãe, a relação com o pai, coesão familiar e o bem-estar, sendo que a coesão familiar se revelou um preditor significativo do bemestar dos adolescentes. Foram ainda encontradas diferenças significativas em função da idade: os adolescentes mais novos reportaram níveis mais elevados de coesão familiar, de relação com a mãe, de relação com o pai e de bem-estar. Contudo, não foram encontradas diferenças significativas em função do sexo dos participantes. Estes resultados sugerem que a coesão familiar, a relação com os pais têm impacto no bem-estar dos adolescentes.

Hydrogen peroxide (H2O2) functions as an important signaling molecule in plants during biotic interactions. However, the extent to which H2O2 accumulates during these interactions and its implications in the development of disease symptoms is unclear. In this work, we provide a step-by-step optimized protocol for in situ quantification of relative H2O2 concentrations in wheat leaves infected with the pathogenic bacterium Pseudomonas syringae pv. atrofaciens (Psa), either alone or in the presence of the beneficial bacterium Herbaspirillum seropedicae (RAM10). This protocol involved the use of 3-3'diaminobenzidine (DAB) staining method combined with image processing to conduct deconvolution and downstream analysis of the digitalized leaf image. The application of a linear regression model allowed to relate the intensity of the pixels resulting from DAB staining with a given concentration of H2O2. Decreasing H2O2 accumulation patterns were detected at increasing distances from the site of pathogen infection, and H2O2 concentrations were different depending on the bacterial combinations tested. Notably, Psa-challenged plants in presence of RAM10 accumulated less H2O2 in the leaf and showed reduced necrotic symptoms, pointing to a potential role of RAM10 in reducing pathogen-triggered H2O2 levels in young wheat plants.

Durante os últimos anos tem-se verificado um interesse crescente pela utilização de e-portefólios em contexto educativo, em resultado das potencialidades que lhes são atribuídas na promoção e avaliação de competências. Esta investigação interpretativa, de tipo qualitativo, pretendeu estudar as potencialidades e limitações da utilização de portefólios electrónicos em escolas do 1º Ciclo do Ensino Básico e propor modelos de e-portefólios adequados à realidade deste nível de ensino. Procedeu-se: 1) à análise global dos e-portefólios produzidos no Distrito de Santarém no âmbito do Projecto CBTIC@EB1 - Projecto de Competências Básicas em TIC - durante o ano lectivo de 2005-2006; e 2) à construção de três estudos de caso, onde se analisa e discute a forma como professores, alunos e monitores se envolveram na dinamização de e-portefólios. Com base nos resultados obtidos, procedeu-se à concepção de dois modelos de e-portefólio que procuram adequar-se à realidade específica das escolas do 1º Ciclo portuguesas, potenciando as vantagens educativas desta metodologia e ultrapassando as dificuldades detectadas no estudo empírico prévio.

Introduction: The presymptomatic phase of neurodegenerative disease can last many years, with sustained cognitive function despite progressive atrophy. We investigate this phenomenon in familial frontotemporal dementia (FTD). Methods: We studied 121 presymptomatic FTD mutation carriers and 134 family members without mutations, using multivariate data-driven approach to link cognitive performance with both structural and functional magnetic resonance imaging. Atrophy and brain network connectivity were compared between groups, in relation to the time from expected symptom onset. Results: There were group differences in brain structure and function, in the absence of differences in cognitive performance. Specifically, we identified behaviorally relevant structural and functional network differences. Structure-function relationships were similar in both groups, but coupling between functional connectivity and cognition was stronger for carriers than for non-carriers, and increased with proximity to the expected onset of disease. Discussion: Our findings suggest that the maintenance of functional network connectivity enables carriers to maintain cognitive performance.

Colour polymorphisms are common among animal species. When combined with genetic and ecological data, these polymorphisms can be excellent systems in which to understand adaptation and the molecular changes underlying phenotypic evolution. The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera, Aphrophoridae), a widespread insect species in the Holarctic region, exhibits a striking dorsal colour/pattern balanced polymorphism. Although experimental crosses have revealed the Mendelian inheritance of this trait, its genetic basis remains unknown. In this study we aimed to identify candidate genomic regions associated with the colour balanced polymorphism in this species.

The malaria parasite Plasmodium obligatorily infects and replicates inside hepatocytes surrounded by a parasitophorous vacuole membrane (PVM), which is decorated by the host-cell derived autophagy protein LC3. We have previously shown that the parasite-derived, PVM-resident protein UIS3 sequesters LC3 to avoid parasite elimination by autophagy from hepatocytes. Here we show that a small molecule capable of disrupting this interaction triggers parasite elimination in a host cell autophagy-dependent manner. Molecular docking analysis of more than 20 million compounds combined with a phenotypic screen identified one molecule, C4 (4-{[4-(4-{5-[3-(trifluoromethyl) phenyl]-1,2,4-oxadiazol-3-yl}benzyl)piperazino]carbonyl}benzonitrile), capable of impairing infection. Using biophysical assays, we established that this impairment is due to the ability of C4 to disrupt UIS3–LC3 interaction, thus inhibiting the parasite’s ability to evade the host autophagy response. C4 impacts infection in autophagy-sufficient cells without harming the normal autophagy pathway of the host cell. This study, by revealing the disruption of a critical host–parasite interaction without affecting the host’s normal function, uncovers an efficient anti-malarial strategy to prevent this deadly disease.

Following publication of the original article [1], it has been brought to the authors' attention that in their paper (Rodrigues et al. 2016) they reported the genome size based on 2C values (diploid genome) when it is more common to present it as 1C value.