Locking the GFP Fluorophore to Enhance Its Emission Intensity

Funding Information: Thanks are due to the University of Aveiro, FCT/MEC, Centro 2020 and Portugal2020, the COMPETE program, and the European Union (FEDER program) via the financial support to the LAQV-REQUIMTE (UIDB/50006/2020 and UIDP/50006/2020), to the CICECO-Aveiro Institute of Materials (UID/CTM/50011/2019, UIDB/50011/2020 and UIDP/50011/2020), financed by national funds through the FCT/MCTES, to the Portuguese NMR Network. SG is supported by national funds (OE), through FCT, I.P., in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. JRMF. Thanks FCT and ESF (European Social Fund) through POCH (Programa Operacional Capital Humano) for her PhD grant (UI/BD/151272/2021). Publisher Copyright: © 2022 by the authors.The Green Fluorescent Protein (GFP) and its analogues have been widely used as fluorescent biomarkers in cell biology. Yet, the chromophore responsible for the fluorescence of the GFP is not emissive when isolated in solution, outside the protein environment. The most accepted explanation is that the quenching of the fluorescence results from the rotation of the aryl–alkene bond and from the Z/E isomerization. Over the years, many efforts have been performed to block these torsional rotations, mimicking the environment inside the protein β-barrel, to restore the emission intensity. Molecule rigidification through chemical modifications or complexation, or through crystallization, is one of the strategies used. This review presents an overview of the strategies developed to achieve highly emissive GFP chromophore by hindering the torsional rotations.publishersversionpublishe

Evaluation of dentinogenesis inducer biomaterials: an in vivo study

When exposure of the pulp to external environment occurs, reparative dentinogenesis can be induced by direct pulp capping to maintain pulp tissue vitality and function. These clinical situations require the use of materials that induce dentin repair and, subsequently, formation of a mineralized tissue. Objective: This work aims to assess the effect of tricalcium silicate cements and mineral trioxide aggregate cements, including repairing dentin formation and inflammatory reactions over time after pulp exposure in Wistar rats. Methodology: These two biomaterials were compared with positive control groups (open cavity with pulp tissue exposure) and negative control groups (no intervention). The evaluations were performed in three stages; three, seven and twenty-one days, and consisted of an imaging (nuclear medicine) and histological evaluation (H&E staining, immunohistochemistry and Alizarin Red S). Results: The therapeutic effect of these biomaterials was confirmed. Nuclear medicine evaluation demonstrated that the uptake of 99mTc-Hydroxymethylene diphosphonate (HMDP) showed no significant differences between the different experimental groups and the control, revealing the non-occurrence of differences in the phosphocalcium metabolism. The histological study demonstrated that in mineral trioxide aggregate therapies, the presence of moderate inflammatory infiltration was found after three days, decreasing during follow-ups. The formation of mineralized tissue was only verified at 21 days of follow-up. The tricalcium silicate therapies demonstrated the presence of a slight inflammatory infiltration on the third day, increasing throughout the follow-up. The formation of mineralized tissue was observed in the seventh follow-up day, increasing over time. Conclusions: The mineral trioxide aggregate (WhiteProRoot®MTA) and tricalcium silicate (Biodentine™) present slight and reversible inflammatory signs in the pulp tissue, with the formation of mineralized tissue. However, the exacerbated induction of mineralized tissue formation with the tricalcium silicate biomaterial may lead to the formation of pulp calcifications

Multi-step subcritical water extracts of fucus vesiculosus l. And codium tomentosum stackhouse: Composition, health-benefits and safety

PO-CI-01-0145FEDER-030240Mental health and active aging are two of the main concerns in the 21st century. To search for new neuroprotective compounds, extracts of Codium tomentosum Stackhouse and Fucus vesiculosus L. were obtained through multi-step (four step) subcritical water extraction using a temperature gradient. The safety assessment of the extracts was performed by screening pharmaceutical compounds and pesticides by UHPLC-MS/MS, and iodine and arsenic levels by ICP-MS. Although the extracts were free of pharmaceutical compounds and pesticides, the presence of arsenic and high iodine contents were found in the first two extraction steps. Thus, the health-benefits were only evaluated for the fractions obtained in steps 3 and 4 from the extraction process. These fractions were tested against five brain enzymes implicated in Alzheimer’s, Parkinson’s, and major depression etiology as well as against reactive oxygen and nitrogen species, having been observed a strong enzyme inhibition and radical scavenging activities for the step 4 fractions from both seaweed species. Regarding the variation of the chemical composition during the extraction, step 1 fractions were the richest in phenolic compounds. With the increase in temperature, Maillard reaction, caramelization and thermo-oxidation occurred, and the resulting products positively affected the antioxidant capacity and the neuroprotective effects.publishersversionpublishe

Using a 3-tier Training Model for Effective Exchange of Good Practices in as ERASMUS+ Project

VISIR+ is an Erasmus+ project that aims to develop educational modules for electric and electronic circuits theory and practice following an enquiry-based teaching and learning methodology. The project has installed five new VISIR remote labs in Higher Education Institutions located in Argentina and Brazil, to allow students doing more experiments and hence acquire better experimental skills, through a combination of traditional (hands-on), remote and virtual laboratories. A key aspect for the success of this project was to motivate and train teachers in the underpinning educational methodology. As such, VISIR+ adopted a 3-tier training process to effectively support the use of VISIR in the Institutions that received it. This process is based on the “train the trainer” approach, which required the participating partner institutions to identify and engage a number of associated partners, interested in using their newly installed remote lab. To measure the quality of the training process, the same satisfaction questionnaire was used in all training actions. This paper presents a detailed description of the training actions along with the analysis of the satisfaction questionnaire results. Major conclusions are that the quality level of the training process remained practically the same across all training actions and that trainees sometimes considered the practical use of the VISIR remote lab as difficult, irrespectively of where and when the training action took place.info:eu-repo/semantics/publishedVersio

Wilms Tumor 1b Expression Defines a Pro-regenerative Macrophage Subtype and Is Required for Organ Regeneration in the Zebrafish

Organ regeneration is preceded by the recruitment of innate immune cells, which play an active role during repair and regrowth. Here, we studied macrophage subtypes during organ regeneration in the zebrafish, an animal model with a high regenerative capacity. We identified a macrophage subpopulation expressing Wilms tumor 1b (wt1b), which accumulates within regenerating tissues. This wt1b+ macrophage population exhibited an overall pro-regenerative gene expression profile and different migratory behavior compared to the remainder of the macrophages. Functional studies showed that wt1b regulates macrophage migration and retention at the injury area. Furthermore, wt1b-null mutant zebrafish presented signs of impaired macrophage differentiation, delayed fin growth upon caudal fin amputation, and reduced cardiomyocyte proliferation following cardiac injury that correlated with altered macrophage recruitment to the regenerating areas. We describe a pro-regenerative macrophage subtype in the zebrafish and a role for wt1b in organ regeneration.A.B.G.-R. is supported by the Sara Borrell Program (CD11/00165) and CIBER de Enfermedades Cardiovasculares (CB16/11/00286). H.R. was supported by a short-term EMBO fellowship (EMBOSTF7204). I.J.M. was supported by a Marie-Sklodowska-Curie postdoctoral fellowship (PIEF-GA-2012-330728). N.M. is supported by Swiss National Science Foundation grant 31003A_15972 and the European Research Council (starting grant 337703–zebra–Heart). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacio´ n, y Universidades (MCNU), and the Pro CNIC Foundation AGRADECIENTOS: ProCNIC; Severo Ochoa (SEV-2015-0505)S

The germline mutational landscape of BRCA1 and BRCA2 in Brazil

The detection of germline mutations in BRCA1 and BRCA2 is essential to the formulation of clinical management strategies, and in Brazil, there is limited access to these services, mainly due to the costs/availability of genetic testing. Aiming at the identification of recurrent mutations that could be included in a low-cost mutation panel, used as a first screening approach, we compiled the testing reports of 649 probands with pathogenic/likely pathogenic variants referred to 28 public and private health care centers distributed across 11 Brazilian States. Overall, 126 and 103 distinct mutations were identified in BRCA1 and BRCA2, respectively. Twenty-six novel variants were reported from both genes, and BRCA2 showed higher mutational heterogeneity. Some recurrent mutations were reported exclusively in certain geographic regions, suggesting a founder effect. Our findings confirm that there is significant molecular heterogeneity in these genes among Brazilian carriers, while also suggesting that this heterogeneity precludes the use of screening protocols that include recurrent mutation testing only. This is the first study to show that profiles of recurrent mutations may be unique to different Brazilian regions. These data should be explored in larger regional cohorts to determine if screening with a panel of recurrent mutations would be effective.This work was supported in part by grants from Barretos Cancer Hospital (FINEP - CT-INFRA, 02/2010), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2013/24633-2 and 2103/23277-8), Fundação de Apoio à Pesquisa do Rio Grande do Norte (FAPERN), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Ministério da Saúde, the Breast Cancer Research Foundation (Avon grant #02-2013-044) and National Institute of Health/National Cancer Institute (grant #RC4 CA153828-01) for the Clinical Cancer Genomics Community Research Network. Support in part was provided by grants from Fundo de Incentivo a Pesquisa e Eventos (FIPE) from Hospital de Clínicas de Porto Alegre, by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, BioComputacional 3381/2013, Rede de Pesquisa em Genômica Populacional Humana), Secretaria da Saúde do Estado da Bahia (SESAB), Laboratório de Imunologia e Biologia Molecular (UFBA), INCT pra Controle do Câncer and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). RMR and PAP are recipients of CNPq Productivity Grants, and Bárbara Alemar received a grant from the same agencyinfo:eu-repo/semantics/publishedVersio

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement Nu 241500 (BuruliVac), from Fundacao Calouste Gulbenkian and from Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). JBG, TGM and AGF had a personal grant from the Portuguese Science and Technology Foundation (FCT) (SFRH/BD/33573/2009, SFRH/BD/41598/2007 and SFRH/BPD/68547/2010, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Spreading remote lab usage: A system — A community — A Federation

Experiments have been at the heart of scientific development and education for centuries. From the outburst of Information and Communication Technologies, virtual and remote labs have added to hands-on labs a new conception of practical experience, especially in Science, Technology, Engineering and Mathematics education. This paper aims at describing the features of a remote lab named Virtual Instruments System in Reality, embedded in a community of practice and forming the spearhead of a federation of remote labs. More particularly, it discusses the advantages and disadvantages of remote labs over virtual labs as regards to scalability constraints and development and maintenance costs. Finally, it describes an actual implementation in an international community of practice of engineering schools forming the embryo of a first world wide federation of Virtual Instruments System in Reality nodes, under the framework of a project funded by the Erasmus+ Program.info:eu-repo/semantics/publishedVersio

Chapter 1

Experimenting is fundamental to the training process of all scientists and engineers. While experiments have been traditionally done inside laboratories, the emergence of Information and Communication Technologies added two alter-natives accessible anytime, anywhere. These two alternatives are known as virtual and remote labs, and are sometimes indistinguishably referred as online labs. Sim-ilarly to other instructional technologies, virtual and remote labs require some ef-fort from teachers in integrating them into curricula, taking into consideration sev-eral factors that affect their adoption (i.e. cost) and their educational effectiveness (i.e. benefit). This chapter analyses these two dimensions and sustains the case where only through international cooperation it is possible to serve the large num-ber of teachers and students involved in engineering education. It presents an ex-ample in the area of Electrical and Electronics Engineering, based on a remote lab named Virtual Instruments System in Reality, and it then describes how a number of European and Latin-American institutions have been cooperating under the scope of an Erasmus+ project2, for spreading its use in Brazil and Argentina.info:eu-repo/semantics/publishedVersio