The role of novel transcriptional regulation mechanisms in neurogenesis

Subventricular Zone (SVZ) is the main neurogenic niche in adult rodent brain. Neurogenesis is regulated by extracellular or intracellular mechanisms, which include transcriptional factors and epigenetic modifications. C-terminal Binding Proteins (CtBPs) are transcriptional corepressors that interact with transcriptional factors to repress the transcription. Moreover, these proteins are important in the regulation of cellular proliferation, differentiation, and survival. These findings suggest that CtBPs may play a role in the modulation of adult neurogenesis. Therefore, the main aim of this work was to evaluate the effects of CtBPs in SVZ neurogenesis. Herein, we analyze CtBPs expression in the SVZ in vitro and in vivo and its effects on SVZ neurogenesis in vitro. First, CtBPs expression was analyzed by immunostainings in SVZ cell cultures obtained from 1- to 3-day-old C57BL/6J mice and in 8-10-week-old mice in vivo. Both CtBP1 and 2 were expressed in proliferating cells (Ki67+), immature cells (Nestin+ and Sox2+), proliferative neuroblasts (Ki67+ and DCX+), astrocytes (GFAP+) and oligodendrocytes (Olig2+), in the SVZ in vitro and in vivo. Then, a substrate-based inhibitor of CtBPs, the 4-methylthio 2-oxobutyric acid (MTOB) that may have a dual effect acting as an inhibitor at high concentrations but as substrate at low concentrations, was used to assess the effect of CtBPs on neurogenesis in vitro. Our results showed that 1 mM and 2.5 mM MTOB induced cell death as detected by propidium iodide incorporation and nuclear morphology analysis. Moreover, 5 µM, 25 µM and 50 µM of MTOB did not affect the total number of Ki67 proliferating cells while 25 µM MTOB increased the percentage of proliferating neuroblasts. Regarding cell differentiation, 5 µM, 25 µM and 50 µM of MTOB increased the percentage of NeuN-mature neurons and Olig2-oligodendrocytes. Altogether, our results suggest that CtBPs are a good target to regulate the transcriptional mechanisms in SVZ neurogenesis.A Zona Subventricular (SVZ) é o maior nicho neurogénico no cérebro adulto de roedores. A neurogénese é regulada por mecanismos extracelulares e intracelulares, que incluem fatores de transcrição e modificações epigenéticas. As proteínas de ligação ao C-terminal (CtBPs) são corepressores transcricionais que, ao interagirem com fatores de transcrição, reprimem a transcrição. Além disso, estas proteínas são importantes na regulação da proliferação, diferenciação e sobrevivência celular. Estas evidências sugerem que as CtBPs podem ser alvos terapêuticos promissores para regular a neurogénese. Assim, o principal objetivo deste trabalho foi avaliar os efeitos das CtBPs na neurogénese da SVZ. Em particular, analisámos a expressão das CtBPs na SVZ in vitro e in vivo e os efeitos destas proteínas na sobrevivência, proliferação e diferenciação celular. A expressão das CtBPs foi analisada por imunomarcações em culturas de células da SVZ obtidas de murganhos C57BL/6J com 1-3 dias e em murganhos C57BL/6J com 8-10 semanas de idade in vivo. As CtBP1 e 2 são expressas em células proliferativas (Ki67+), células imaturas (Nestin+, Sox2+), neuroblastos em proliferação (Ki67+ e DCX+), astrócitos (GFAP+) e oligodendrócitos (Olig2+), na SVZ in vitro e in vivo. Em seguida, o ácido 4-metiltio-2-oxobutírico (MTOB), um antagonista das CtBPs que pode ter um duplo efeito ao atuar como inibidor a altas concentrações, mas a baixas concentrações como substrato, foi utilizado para avaliar o efeito das CtBPs na neurogénese na SVZ in vitro. Os nossos resultados mostraram que o MTOB a 1 mM e 2.5 mM induzia morte celular, detetada pela incorporação de iodeto de propídio e pela análise da morfologia nuclear. Além disso, 5 µM, 25 µM e 50 µM de MTOB não afetaram o número total de células em proliferação, mas 25 µM de MTOB aumentou a percentagem de neuroblastos em proliferação. Relativamente à diferenciação celular, 5 µM, 25 µM e 50 µM de MTOB aumentaram a percentagem de neurónios maduros (NeuN+) e oligodendrócitos (Olig2+). Em suma, os nossos resultados sugerem que as CtBPs modulam a neurogénese na SVZ, ao promoverem a diferenciação em neurónios e oligodendrócitos, o que poderá ter relevância na reparação de doenças neurodegenerativas

Tempo musical na interpretação de Préludes II de Claude Debussy

Mestrado em MúsicaEste trabalho propõe-se estudar o tempo musical, tendo em conta a forma como é indicado e explorado, no seio da obra para piano Préludes II de Claude Debussy. Tal investigação comporta três perspectivas que se interligam para a definição de resultados: a manifestada pelo compositor na partitura; a tecida por autores, numa perspectiva analítica; e a comprovada por gravações escolhidas realizadas por intérpretes criteriosamente seleccionados.This work proposes to study musical time, given the way it is displayed and explored within the work Préludes II for piano by Claude Debussy. The process is defined from three points of view: as written in the score by the composer; as perceived by scholars, in the analitic way; and, as interpreted by selected performers in specific recordings

Intensifying the manufacture of hiPSC therapy products through metabolic and process understanding

In vitro differentiation of human induced pluripotent stem cells into specific lineages such as cardiomyocytes (hPSC-CM) and hepatocytes (hPCS-Hep) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the implementation of protocols for hPSC expansion and differentiation, there are some technological challenges remaining include the low scalability and differentiation yields. Additionally, generated cells are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype and function. In this study, we aim to overcome this hurdle by devising bioinspired and integrated strategies to improve the generation and functionality of these hiPSC-derivatives. We also applied robust multi-parametric techniques including proteomics, transcriptomics, metabolomics and fluxomics as complementary analytical tools to support bioprocess optimization and product characterization. We cultured hiPSC as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion and used a capacitance probe for in situ monitoring of cell growth/differentiation. After cell expansion, the hepatic differentiation step was integrated by addition of key soluble factors and controlling the dissolved oxygen concentration at various stages of the process to generate populations enriched for definitive endoderm, hepatocyte progenitors and mature hepatocytes. The analyses of hepatic markers expression throughout the stages of the differentiation confirmed that hepatocyte differentiation was improved in 3D spheroids when compared to 2D culture. Noteworthy, these hiPSC-HLC exhibited functional characteristics typical of hepatocytes (albumin production, glycogen storage and CYP450 activity). We also demonstrate the potential of dielectric spectroscopy to monitor cell expansion and hepatic differentiation in STB. For CM differentiation, we relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. We assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. “-Omics” analyses showed that addition of galactose to culture medium and culturing the cells under perfusion improves total oxidative capacity of the cells and ameliorates fatty acid oxidation. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Funding: This work was supported by FCT-funded projects NETDIAMOND (SAICTPAC/0047/2015), MetaCardio (Ref.032566) and FCT/ERA-Net (ERAdicatPH; Ref. E-Rare3/0002/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged

Cuidados inovadores de enfermagem na pandemia de COVID-19: relato de uma experiência extensionista

Among the professional skills necessary in the training of nursing students, there is that associated with working in health services. In this context of training, participation in university extension programs increases learning possibilities and allows approximation of theoretical-practical experience, providing activities for the benefit of the community. The objective of this article is to report the extension experiences of the project “Innovative Nursing Care”, during the COVID-19 pandemic. This is a descriptive, retrospective and cross-sectional report, carried out based on the experience of professors and undergraduate nursing students. It was possible to observe that the experience of nursing students from different undergraduate periods enabled them to learn about clinical care for patients with chronic conditions, providing better quality training for both scholarship students and volunteers during the stoppage of classes generated by the pandemic.Dentre as competências profissionais necessárias na formação do acadêmico de enfermagem, está aquela associada à atuação nos serviços de saúde. Nesse contexto de formação, a participação em programas de extensão universitária aumenta as possibilidades de aprendizado e permite a aproximação da vivência teórico-prática, proporcionando atividades em benefício da comunidade. Objetiva-se com esse artigo relatar as experiências extensionistas do projeto “Cuidados Inovadores de Enfermagem”, durante a pandemia de COVID-19.  Trata-se de um relato descritivo, retrospectivo e transversal, realizado a partir da vivência de docentes e acadêmicos da graduação em Enfermagem. Foi possível observar que a experiência dos acadêmicos do curso de enfermagem de diferentes períodos da graduação possibilitou o aprendizado em cuidados clínicos ao paciente em condições crônicas, proporcionando uma formação de melhor qualidade tanto para o acadêmico bolsista quanto os voluntários durante a paralisação das aulas geradas pela pandemia

Further evidence of possible therapeutic uses of Sambucus nigra L. extracts by the assessment of the In Vitro and In Vivo anti-inflammatory properties of Its PLGA and PCL-Based Nanoformulations

Sambucus nigra L. is widely used in traditional medicine with different applications. However, confirmative studies are strongly required. This study aimed to assess the biological activities of the S. nigra flower’s extract encapsulated into two different types of nanoparticles for optimizing its properties and producing further evidence of its potential therapeutic uses. Different nanoparticles (poly(lactide-co-glycolide, PLGA) and poly-Ɛ-caprolactone (PCL), both with oleic acid, were prepared by emulsification/solvent diffusion and solvent-displacement methods, respectively. Oleic acid was used as a capping agent. After the nanoparticles’ preparation, they were characterized and the biological activities were studied in terms of collagenase, in vitro and in vivo anti-inflammatory, and in vitro cell viability. Rutin and naringenin were found to be the major phenolic compounds in the studied extract. The encapsulation efficiency was higher than 76% and revealed to have an impact on the release of the extract, mainly for the PLGA. Moreover, biochemical and histopathological analyses confirmed that the extract-loaded PLGA-based nanoparticles displayed the highest anti-inflammatory activity. In addition to supporting the previously reported evidence of potential therapeutic uses of S. nigra, these results could draw the pharmaceutical industry’s interest to the novelty of the nanoproducts.Authors also gratefully acknowledge the Régiefrutas and Fundação para a Ciência e a Tecnologia (FCT) (UIDB/04138/2020, UIDB/00100/2020, UIDP/50017/2020+UIDB/50017/2020) and Portugal 2020 to the Portuguese Mass Spectrometry Network (LISBOA-01-0145-FEDER-402-022125) for financial support. Furthermore, the authors are also grateful to the work supported by the FCT and the Portuguese National and Regional Budget, through CCMAR/Multi/04326/2019 project. Finally, Luísa Custódio was supported by the FCT Scientific Employment Stimulus (CEECIND/00425/2017).info:eu-repo/semantics/publishedVersio

Cuidados inovadores de enfermagem na pandemia de COVID-19: relato de uma experiência extensionista

Among the professional skills necessary in the training of nursing students, there is that associated with working in health services. In this context of training, participation in university extension programs increases learning possibilities and allows approximation of theoretical-practical experience, providing activities for the benefit of the community. The objective of this article is to report the extension experiences of the project “Innovative Nursing Care”, during the COVID-19 pandemic. This is a descriptive, retrospective and cross-sectional report, carried out based on the experience of professors and undergraduate nursing students. It was possible to observe that the experience of nursing students from different undergraduate periods enabled them to learn about clinical care for patients with chronic conditions, providing better quality training for both scholarship students and volunteers during the stoppage of classes generated by the pandemic.Dentre as competências profissionais necessárias na formação do acadêmico de enfermagem, está aquela associada à atuação nos serviços de saúde. Nesse contexto de formação, a participação em programas de extensão universitária aumenta as possibilidades de aprendizado e permite a aproximação da vivência teórico-prática, proporcionando atividades em benefício da comunidade. Objetiva-se com esse artigo relatar as experiências extensionistas do projeto “Cuidados Inovadores de Enfermagem”, durante a pandemia de COVID-19.  Trata-se de um relato descritivo, retrospectivo e transversal, realizado a partir da vivência de docentes e acadêmicos da graduação em Enfermagem. Foi possível observar que a experiência dos acadêmicos do curso de enfermagem de diferentes períodos da graduação possibilitou o aprendizado em cuidados clínicos ao paciente em condições crônicas, proporcionando uma formação de melhor qualidade tanto para o acadêmico bolsista quanto os voluntários durante a paralisação das aulas geradas pela pandemia

A newfangled collagenase inhibitor topical formulation based on Ethosomes with Sambucus nigra L. Extract

Sambucus nigra L. (S. nigra) is a shrub widespread in Europe and western Asia, traditionally used in medicine, that has become popular in recent years as a potential source of a wide range of interesting bioactive compounds. The aim of the present work was to develop a topical S. nigra extract formulation based on ethosomes and thus to support its health claims with scientific evidence. S. nigra extract was prepared by an ultrasound-assisted method and then included in ethosomes. The ethosomes were analyzed in terms of their size, stability over time, morphology, entrapment capacity (EC), extract release profile, stability over time and several biological activities. The prepared ethosomes were indicated to be well defined, presenting sizes around 600 nm. The extract entrapment capacity in ethosomes was 73.9 ± 24.8%, with an interesting slow extract release profile over 24 h. The extract-loaded ethosomes presented collagenase inhibition activity and a very good skin compatibility after human application. This study demonstrates the potential use of S. nigra extract incorporated in ethosomes as a potential cosmeceutical ingredient and on further studies should be performed to better understand the impact of S. nigra compounds on skin care over the time.info:eu-repo/semantics/publishedVersio

Sugar-based bactericides targeting phosphatidylethanolamine-enriched membranes

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242839/Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials.The European Union is gratefully acknowledged for the support of the project “Diagnostic and Drug Discovery Initiative for Alzheimer’s Disease” (D3i4AD), FP7-PEOPLE-2013-IAPP, GA 612347. We thank the Management Authorities of the European Regional Development Fund and the National Strategic Reference Framework for the support of the Incentive System - Research and Technological Development Co-Promotion FACIB Project number 21457. Fundação para a Ciência e a Tecnologia is also acknowledged for the support of projects UID/Multi/00612/2013, FCT/UID/ Multi/04046/2013, IF/00808/2013/CP1159/CT0003, PTDC/BBBBQB/6071/2014, as well as for the post-doc grant SFRH/BPD/42567/2007 (A.M.), the Ph.D. grants SFRH/BDE/51998/2012 (C.D.), and SFRH/BDE/51957/2012 (J.P.P.), both co-sponsored by CIPAN, and also for the Ph.D. grant SFRH/BD/116614/2016 (R.N.).info:eu-repo/semantics/publishedVersio