Programa de intervenção nas interacções pais-filhos “Desenvolver a Sorrir” – Estudo exploratório

Este artigo descreve um Programa de intervenção nas interacções pais-filhos, em contextos lúdicos de aprendizagem, o qual é direccionado para famílias “de risco” com crianças até aos 3 anos. Apresenta ainda, um estudo de carácter exploratório onde se avalia a eficácia do programa. O Programa, que tem por objectivo desenvolver competências parentais através da modelagem e experimentação de interacções positivas em sete sessões, foi aplicado a 19 díades pais (Idade M=28.5; DP=9.48) criança (Idade M=21 meses; DP=0.99) em contexto domiciliário. As interacções das 19 díades foram avaliadas com o NCATS – Teaching Scale e o HOME utilizando-se um desenho de avaliação pré-pós teste. Os resultados indicam diferenças significativas em todas as áreas do meio da criança (estimulação diária, materiais lúdicos, ambiente, envolvimento e responsividade emocional e verbal dos pais) e da qualidade da interacção das díades (sensibilidade aos sinais, resposta ao descontentamento, promoção do desenvolvimento, clareza dos sinais e responsividade ao adulto). Não obstante o carácter exploratório do estudo, considera-se que quer a adesão e satisfação dos pais, quer os efeitos positivos do programa nas interacções das díades apontam para a pertinência da prossecução do seu estudo de validação com vista à sua generalização

Simple, sensitive and quantitative bioluminescence assay for determination of malaria pre-patent period

© 2014 Zuzarte-Luis et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stateBackground: The first phase of malaria infection occurs in the liver and is clinically silent. Inside hepatocytes each Plasmodium sporozoite replicate into thousands of erythrocyte-infectious merozoites that when released into the blood stream result in clinical symptoms of the disease. The time between sporozoite inoculation and the appearance of parasites in the blood is defined as the pre-patent period, which is classically analysed by time-consuming and labor-intensive techniques, such as microscopy and PCR. Methods: Luciferase-expressing Plasmodium berghei parasites were used to measure pre-patent period of malaria infection in rodents using a bioluminescence assay that requires only one microliter of blood collected from the tail-vein. The accuracy and sensitivity of this new method was compared with conventional microscopy and PCR based techniques, and its capacity to measure the impact of anti-malarial interventions against the liver evaluated. Results: The described method is very sensitive allowing the detection of parasites during the first cycles of blood stage replication. It accurately translates differences in liver load due to inoculation of different sporozoite doses as well as a result of treatment with different primaquine regimens. Conclusions: A novel, simple, fast, and sensitive method to measure pre-patent period of malaria infection in rodents is described here. The sensitivity and accuracy of this new method is comparable to standard PCR and microscopy-based techniques, respectively.This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) grants PTDC/SAU-MIC/113697/2009 (VZL) and EXCL/IMI-MIC/0056/2012 (MMM).info:eu-repo/semantics/publishedVersio

Cx43 can form functional channels at the nuclear envelope and modulate gene expression in cardiac cells

Conexina 43; Expresión génica; Translocación nuclearConnexina 43; Expressió gènica; Translocació nuclearConnexin 43; Gene expression; Nuclear translocationClassically associated with gap junction-mediated intercellular communication, connexin43 (Cx43) is increasingly recognized to possess non-canonical biological functions, including gene expression regulation. However, the mechanisms governing the localization and role played by Cx43 in the nucleus, namely in transcription modulation, remain unknown. Using comprehensive and complementary approaches encompassing biochemical assays, super-resolution and immunogold transmission electron microscopy, we demonstrate that Cx43 localizes to the nuclear envelope of different cell types and in cardiac tissue. We show that translocation of Cx43 to the nucleus relies on Importin-β, and that Cx43 significantly impacts the cellular transcriptome, likely by interacting with transcriptional regulators. In vitro patch-clamp recordings from HEK293 and adult primary cardiomyocytes demonstrate that Cx43 forms active channels at the nuclear envelope, providing evidence that Cx43 can participate in nucleocytoplasmic shuttling of small molecules. The accumulation of nuclear Cx43 during myogenic differentiation of cardiomyoblasts is suggested to modulate expression of genes implicated in this process. Altogether, our study provides new evidence for further defining the biological roles of nuclear Cx43, namely in cardiac pathophysiology.This work was supported by the European Regional Development Fund (ERDF) through the Operational Program for Competitiveness Factors (COMPETE) under the projects HealthyAging2020 CENTRO-01-0145-FEDER-000012-N2323, CENTRO-01-0145-FEDER-032179, CENTRO-01-0145-FEDER-032414, EXPL/MED-OUT/0590/2021, UIDB/04539/2020 and PPBI-Portuguese Platform of BioImaging (POCI-01-0145-FEDER-022122). This work was also supported by the European Union's Horizon 2020 research and innovation programme under grant agreement MIA-Portugal no. 857524 and the Comissão de Coordenação da Região Centro (CCDRC) through the Centro2020 Program. T.A. acknowledges funding from Instituto de Salud Carlos III, grant PI16/00772 co-financed by the ERDF, and Fundación Científica Asociación Española Contra el Cáncer (IDEAS20039AASE). This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003), and the Microscopy and Bioimaging Lab (iLAB) at the Faculty of Medicine of the University of Coimbra (Coimbra, Portugal). The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. Super-resolution microscopy experiments benefited from access to CBI/IGBMC (Illkirch, France) and were supported by FRISBI (ANR-10-INBS-0005). Financial support was provided by Instruct-ERIC (PID 14677)

Acute invariant NKT cell activation triggers an immune response that drives prominent changes in iron homeostasis

© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/.Iron homeostasis is an essential biological process that ensures the tissue distribution of iron for various cellular processes. As the major producer of hepcidin, the liver is central to the regulation of iron metabolism. The liver is also home to many immune cells, which upon activation may greatly impact iron metabolism. Here, we focus on the role of invariant natural killer T (iNKT) cells, a subset of T lymphocytes that, in mice, is most abundant in the liver. Activation of iNKT cells with the prototypical glycosphingolipid antigen, α-galactosylceramide, resulted in immune cell proliferation and biphasic changes in iron metabolism. This involved an early phase characterized by hypoferremia, hepcidin induction and ferroportin suppression, and a second phase associated with strong suppression of hepcidin despite elevated levels of circulating and tissue iron. We further show that these changes in iron metabolism are fully dependent on iNKT cell activation. Finally, we demonstrate that the biphasic regulation of hepcidin is independent of NK and Kupffer cells, and is initially driven by the STAT3 inflammatory pathway, whereas the second phase is regulated by repression of the BMP/SMAD signaling pathway. These findings indicate that iNKT activation and the resulting cell proliferation influence iron homeostasis.This work was supported by grants from the Canadian Institutes of Health Research (CIHR, Grant no. PJT-159775) and Natural Sciences and Engineering Research Council of Canada (NSERC, Grant RGPIN-2018-06442) to MMS. HH received a PhD scholarship from the NSERC. SL is a Research Scholars Emeritus awardee from the FRQS.info:eu-repo/semantics/publishedVersio

Safety profile of solid lipid nanoparticles loaded with rosmarinic acid for oral use: in vitro and animal approaches

Rosmarinic acid (RA) possesses several protective bioactivities that have attracted increasing interest by nutraceutical/pharmaceutical industries. Considering the reduced bioavailability after oral use, effective (and safe) delivery systems are crucial to protect RA from gastrointestinal degradation. This study aims to characterize the safety profile of solid lipid nanoparticles produced with Witepsol and Carnauba waxes and loaded with RA, using in vitro and in vivo approaches, focused on genotoxicity and cytotoxicity assays, redox status markers, hematological and biochemical profile, liver and kidney function, gut bacterial microbiota, and fecal fatty acids composition. Free RA and sage extract, empty nanoparticles, or nanoparticles loaded with RA or sage extract (0.15 and 1.5 mg/mL) were evaluated for cell (lymphocytes) viability, necrosis and apoptosis, and antioxidant/prooxidant effects upon DNA. Wistar rats were orally treated for 14 days with vehicle (control) and with Witepsol or Carnauba nanoparticles loaded with RA at 1 and 10 mg/kg body weight/d. Blood, urine, feces, and several tissues were collected for analysis. Free and loaded RA, at 0.15 mg/mL, presented a safe profile, while genotoxic potential was found for the higher dose (1.5 mg/mL), mainly by necrosis. Our data suggest that both types of nanoparticles are safe when loaded with moderate concentrations of RA, without in vitro genotoxicity and cytotoxicity and with an in vivo safety profile in rats orally treated, thus opening new avenues for use in nutraceutical applications.info:eu-repo/semantics/publishedVersio

DNA damage precedes apoptosis during the regression of the interdigital tissue in vertebrate embryos

DNA damage independent of caspase activation accompanies programmed cell death in different vertebrate embryonic organs. We analyzed the significance of DNA damage during the regression of the interdigital tissue, which sculpts the digits in the embryonic limb. Interdigit remodeling involves oxidative stress, massive apoptosis and cell senescence. Phosphorylation of H2AX mediated by ATM precedes caspase dependent apoptosis and cell senescence during interdigit regression. The association of ?H2AX with other downstream DNA repair factors, including MDC1, Rad50 and 53BP1 suggests a defensive response of cells against DNA damage. The relative distribution of cells ?H2AX-only positive, TUNEL-only positive, and cells double positive for both markers is consistent with a sequence of degenerative events starting by damage of the DNA. In support of this interpretation, the relative number of ?H2AX-only cells increases after caspase inhibition while the relative number of TUNELonly cells increases after inhibition of ATM. Furthermore, cultured interdigits survived and maintained intense chondrogenic potential, even at advanced stages of degeneration, discarding a previous commitment to die. Our findings support a new biological paradigm considering embryonic cell death secondary to genotoxic stimuli, challenging the idea that considers physiological cell death a cell suicide regulated by an internal death clock that pre-programmes degeneration

Host Cell Phosphatidylcholine Is a Key Mediator of Malaria Parasite Survival during Liver Stage Infection

During invasion, Plasmodium, the causative agent of malaria, wraps itself in a parasitophorous vacuole membrane (PVM), which constitutes a critical interface between the parasite and its host cell. Within hepatocytes, each Plasmodium sporozoite generates thousands of new parasites, creating high demand for lipids to support this replication and enlarge the PVM. Here, a global analysis of the total lipid repertoire of Plasmodium-infected hepatocytes reveals an enrichment of neutral lipids and the major membrane phospholipid, phosphatidylcholine (PC). While infection is unaffected in mice deficient in key enzymes involved in neutral lipid synthesis and lipolysis, ablation of rate-limiting enzymes in hepatic PC biosynthetic pathways significantly decreases parasite numbers. Host PC is taken up by both P. berghei and P. falciparum and is necessary for correct localization of parasite proteins to the PVM, which is essential for parasite survival. Thus, Plasmodium relies on the abundance of these lipids within hepatocytes to support infection.Seventh Framework Programme (European Commission) (Grant Agreement 311502)Fundacao para a Ciencia e a Tecnologia (Grant EXCL/IMI-MIC/0056/2012)Fundacao para a Ciencia e a Tecnologia (Grant PTDC/IMI-MIC/1568/2012

New Claims for Wild Carrot (Daucus carota subsp. carota) Essential Oil

The essential oil of Daucus carota subsp. carota from Portugal, with high amounts of geranyl acetate (29.0%), -pinene (27.2%), and 11 H-himachal-4-en-1 -ol (9.2%), was assessed for its biological potential. The antimicrobial activity was evaluated against several Gram-positive and Gram-negative bacteria, yeasts, dermatophytes, and Aspergillus strains. The minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) were evaluated showing a significant activity towards Gram-positive bacteria (MIC = 0.32-0.64 L/mL), Cryptococcus neoformans (0.16 L/mL), and dermatophytes (0.32-0.64 L/mL). The inhibition of the germ tube formation and the effect of the oil on Candida albicans biofilms were also unveiled. The oil inhibited more than 50% of filamentation at concentrations as low as 0.04 L/mL (MIC/128) and decreased both biofilm mass and cell viability. The antioxidant capacity of the oil, as assessed by two in chemico methods, was not relevant. Still, it seems to exhibit some anti-inflammatory potential by decreasing nitric oxide production around 20% in LPS-stimulated macrophages, without decreasing macrophages viability. Moreover, the oils safety profile was assessed on keratinocytes, alveolar epithelial cells, macrophages, and hepatocytes. Overall, the oil demonstrated a safety profile at concentrations below 0.64 L/mL. The present work highlights the bioactive potential of D. carota subsp. carota suggesting its industrial exploitation

Active APPL1 sequestration by Plasmodium favors liver-stage development

© 2022 The Authors. This is an open access article under the CC BY-NC-ND license http://creativecommons.org/licenses/by-nc-nd/4.0/).Intracellular pathogens manipulate host cells to survive and thrive. Cellular sensing and signaling pathways are among the key host machineries deregulated to favor infection. In this study, we show that liver-stage Plasmodium parasites compete with the host to sequester a host endosomal-adaptor protein (APPL1) known to regulate signaling in response to endocytosis. The enrichment of APPL1 at the parasitophorous vacuole membrane (PVM) involves an atypical Plasmodium Rab5 isoform (Rab5b). Depletion of host APPL1 alters neither the infection nor parasite development; however, upon overexpression of a GTPase-deficient host Rab5 mutant (hRab5_Q79L), the parasites are smaller and their PVM is stripped of APPL1. Infection with the GTPase-deficient Plasmodium berghei Rab5b mutant (PbRab5b_Q91L) in this case rescues the PVM APPL1 signal and parasite size. In summary, we observe a robust correlation between the level of APPL1 retention at the PVM and parasite size during exoerythrocytic development.This work was supported by grants from the la Caixa Banking Foundation and Fundação para a Ciência e a Tecnologia (HR17-00264 and PTDC/SAU-PAR/30751/2017 respectively, both to M.M.M.). A.L and S.S.B were sponsored by Fundação para a Ciência e a Tecnologia fellowships (PD/BD/114036/2015, SFRH/BD/114464/2016, respectively). V.S acknowledges funding from the Science and Engineering Research Board (SERB) (EMR/2016/006810), Department of Science and Technology (DST), Government of India. V.P. and C.S. were supported by GRK2581 (P6) SPHINGOINF of the Growing Spine Foundation (DFG).info:eu-repo/semantics/publishedVersio

Cytotoxicity assessment of endodontic sealers: metabolic activity, morphology and chromosomal alterations

Introduction: Endodontic treatment aims to eliminate infection of the root canals and fill the dental pulp space, being, the obturation of root canals an important step. The study of the toxicity/biocompatibility of the sealers used to fill the root canals is crucial since they are applied into direct contact with periradicular tissues.There are several types of sealers, categorized according to their main chemical constituents. The aim of this study was to evaluate the cytotoxicity of three root canal sealers, AH Plus, Bio MTA+ and Bio C, on immortalized human gingival fibroblasts. Methods: To study the cytotoxicity of the sealers we performed a Methyltetrazolium (MTT) assay, a study of cell's morphology and a cytogenetic study. Cells were placed in contact with material-conditioned media, for 24 h, at three different concentrations (1, 10 and 100 mg/ml) for the MTT assay. Cell morphology and cytogenetic studies were performed at 100 mg/ml. Cells in normal culture medium were analyzed as control group. Results: MTT assay revealed a cytotoxic effect of Bio MTA+ and Bio C with a growing decrease of metabolic activity with increasing compound concentration, reaching 50% with 100 mg/ml. Regarding the cells morphology, Bio C was the compound that showed a more drastic effect, with a decrease in cell confluence and several morphological changes. AH Plus and Bio MTA+ did not seem to affect the cell confluence, however morphology changes were observed, as compromised cell membranes and loss of cell content. Cytogenetic study was thus far only performed with AH Plus. Since there was a severe decrease of mitotic index after treatment, it was not yet possible to obtain sufficient metaphases, even after several cytogenetic harvesting procedures, but, so far, no relevant structural or numerical changes were observed. Discussion: This preliminary study allowed us to verify that these root canal sealers exhibit some cytotoxicity, depending on the concentration used. Although more studies are still needed, this work could be important to both, help in the selection of the most appropriate compounds for clinical practice and to determine the maximum recommended amounts of each sealer.info:eu-repo/semantics/publishedVersio