Synthesis of 5H-pyrido[4,3-b]indole by a modification of Pomeranz-Fritsch isoquinoline synthesis.

5H-Pyrido[4,3-b]indole was obtained from 3-formylindole in 16% overall yield by Jackson and Shannon modification of the Pomeranz-Fristch isoquinoline synthesis. The final cyclisation occurred but the removal of the tosyl group and oxidation of the dihydrocompound was not efficient. Changes in the concentration of the acid catalyst gave 29% as the best yield for the last step. An NMR study of the cyclisation is described.Fundação para a Ciência e Tecnologia (FCT) and FEDE

Sustained gene expression in the retina by improved episomal vectors

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.Fundacao para a Ciencia e Tecnologia, Portugal [PEst/OE/EQB-LA 0023/2013, SFRH/BD/76873/2011, SFRH/BD/70318/2010, PTDC/SAU/BEB/098475/2008]; European Union [PIRG-GA-2009-249314

Production de vins mousseux a partir de "Vinhos Verdes" blancs monovarietaux

On a fait l'étude comparative de vins mousseux élaborés à partir de vins de base issus de cinq cépages recommandés pour la Région: Loureiro, Trajadura, Avesso, Pedernã et Azal blanc, en ce qui concerne les propriétés organoleptiques. Les vins de base ont été produits selon le processus utilisé dans la Région -égrappage, pressurage, débourbage et fermentation alcoolique-, ayant été soumis aussi à une fermentation malolactique. La prise de mousse a été effectuée en bouteille, avec des levures immobilisées en billes d'alginate, pendant cinq mois à 14 °C. Les propriétés sensorielles des vins ont été évaluées en utilisant des fiches classificatrices et descriptives, par une chambre de neuf dégustateurs expérimentés. Le traitement statistique des résultats, effectué par le logiciel SPSS, a été fait en recourrant à l'analyse de variance. On a trouvé quelques différences parmi les cinq vins mousseux surtout en ce qui concerne l'aspect du cordon et l'arôme. Cependant, tous ces vins ont obtenu des classifications globales au-dessus de l'acceptable, atteignant parfois l'excellent. Ces résultats préliminaires ainsi obtenus, basés sur les caractéristiques organoleptiques, suggèrent la possibilité d'obtenir des vins mousseux de qualité à partir des cépages de "Vinho Verde"

Current concepts and challenges in osteochondral tissue engineering and regenerative medicine

"Publication Date (Web): February 20, 2015"In the last few years, great progress has been made to validate tissue engineering strategies in preclinical studies and clinical trials on the regeneration of osteochondral defects. In the preclinical studies, one of the dominant strategies comprises the development of biomimetic/bioactive scaffolds, which are used alone or incorporated with growth factors and/or stem cells. Many new trends are emerging for modulation of stem cell fate towards osteogenic and chondrogenic differentiations, but bone/cartilage interface regeneration and physical stimulus have been showing great promise. Besides the matrix-associated autologous chondrocyte implantation (MACI) procedure, the matrix-associated stem cells implantation (MASI) and layered scaffolds in acellular or cellular strategy are also applied in clinic. This review outlines the progresses at preclinical and clinical levels, and identifies the new challenges in osteochondral tissue engineering. Future perspectives are provided, e.g., the applications of extracellular matrix-like biomaterials, computer-aided design/manufacture of osteochondral implant and reprogrammed cells for osteochondral regeneration.The authors thank the Portuguese Foundation for Science and Technology (FCT) through the projects TISSUE2TISSUE (PTDC/CTM/105703/2008) and OsteoCart (PTDC/CTM-BPC/115977/2009). We also acknowledge European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement REGPOT-CT2012-316331-POLARIS. L-P.Y. acknowledges the PhD scholarship from FCT (SFRH/BD/64717/2009). The FCT distinction attributed to J.M.O. and A.L.O. under the Investigator FCT program (IF/00423/2012) and (IF/00411/2013) are also greatly acknowledged

the dilemma of the sample size calculation for sensitivity and specificity estimation

Sample size calculation in biomedical practice is typically based on the problematicWald method for a binomial proportion, with potentially dangerous consequences. This work highlights the need of incorporating the concept of conditional probability in sample size determination to avoid reduced sample sizes that lead to inadequate confidence intervals. Therefore, new definitions are proposed for coverage probability and expected length of confidence intervals for conditional probabilities, like sensitivity and specificity. The new definitions were used to assess seven confidence interval estimation methods. In order to determine the sample size, two procedures-an optimal one, based on the new definitions, and an approximation-were developed for each estimation method. Our findings confirm the similarity of the approximated sample sizes to the optimal ones. R code is provided to disseminate these methodological advances and translate them into biomedical practice.publishersversionpublishe

Multiresponsive spiropyran-based copolymer: synthesis and characterization

Photochromic copolymers carrying spiropyran side groups have attracted particular attention for potential applications in optical devices, photonic memory and photosensing, as well as, fluorescence imaging.[1] These materials allow overcoming many of the limitations inherent to traditional spiropyran doped polymers, such as, phase separation of the colorant and retardation of the decoloration of the open form.[2] The introduction of light sensitive moieties within responsive polymers has led to the development of sophisticated multiresponsive systems.[3] Block copolymers are important self-assembling systems that can assume a diversity of nanometer-scale morphologies due to the incompatibility and the connectivity constrains between the chemically distinct segments. Therefore, self-assembly of photochromic copolymers in the solid state or in solution allows the development of nanostructured materials. [4] The aim of this research study is the synthesis of a multiresponsive spiropyran-based copolymer, poly(styrene)- co-poly([1´,3´,3´-trimethyl-6-methacryloyloxyspiro(2H-1-benzopyran-2,2´-indoline)]4-vinylbenzoic acid) 2. The functionalized block copolymer poly(styrene)-block-poly(4-vinylbenzoic acid) (PSt-b-P4VBA) 1 synthesized by reversible addition fragmentation chain-transfer (RAFT) polymerization was coupled with 1,3,3-trimethyl-6- hydroxyspiro(2H-1-benzopyran-2,2-indoline). The link between the functionalized block copolymer and spiropyran was successfully obtained by Steglich esterification using DMAP/DCC as catalysts (Scheme 1).n-STeP ProjectNORTE-07-0124-FEDER-000039Programa Operacional Regional do Norte (ON.2)PEst-C/CTM/LA0025/2013 (Strategic Project - LA 25 - 2013-2014

Effect of clay mineral addition on properties of bio-based polymer blends

The effect of clay mineral addition to bio-based blends on morphology and physical properties of thermoplastic starch (TPS) and polypropylene grafted with maleic anhydride (PP-g-MA) was investigated. Blends and nanocomposites containing organoclay, Cloisite 30B, were prepared by melt mixing and characterized by several techniques. X-ray diffraction (XRD), scanning and transmission electron microscopy (SEM, STEM) and dynamic mechanical analysis (DMA) demonstrate a very good dispersion of the clay mineral in the polymer matrix, an increase of polymer compatibility and an improvement of mechanical properties. Biodegradation studies performed in compost revealed that Cloisite 30B addition enhanced the matrix biodegradability. Therefore clay minerals, which can be obtained from natural resources, can be efficiently used to improve the properties of bio-based materials and contribute to sustainability.The authors acknowledge the n-STeP - Nanostructured systems for Tail, with reference NORTE-07-0124-FEDER-000039, supported by the Programa Operacional Regional do Norte (ON.2), PEst-C/CTM/LA0025/2013 (Strategic Project - LA 25 - 2013-2014)

Staphylococcus epidermidis biofilms undergo metabolic and matrix remodeling under nitrosative stress

Funding Information: This work was financially supported by Fundação para a Ciência e Tecnologia (FCT) Project – PTDC/BIA-MIC/31566/2017, R&D unit MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020). AO has a fellowship UI/BD/153389/2022 from FCT. The NMR data were acquired from CERMAX, ITQB-NOVA, Oeiras, Portugal, with equipment funded by FCT, project AAC 01/SAICT/2016. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments Publisher Copyright: Copyright © 2023 Oliveira, Saraiva and Carvalho.Staphylococcus epidermidis is a commensal skin bacterium that forms host- and antibiotic-resistant biofilms that are a major cause of implant-associated infections. Most research has focused on studying the responses to host-imposed stresses on planktonic bacteria. In this work, we addressed the open question of how S. epidermidis thrives on toxic concentrations of nitric oxide (NO) produced by host innate immune cells during biofilm assembly. We analyzed alterations of gene expression, metabolism, and matrix structure of biofilms of two clinical isolates of S. epidermidis, namely, 1457 and RP62A, formed under NO stress conditions. In both strains, NO lowers the amount of biofilm mass and causes increased production of lactate and decreased acetate excretion from biofilm glucose metabolism. Transcriptional analysis revealed that NO induces icaA, which is directly involved in polysaccharide intercellular adhesion (PIA) production, and genes encoding proteins of the amino sugar pathway (glmM and glmU) that link glycolysis to PIA synthesis. However, the strains seem to have distinct regulatory mechanisms to boost lactate production, as NO causes a substantial upregulation of ldh gene in strain RP62A but not in strain 1457. The analysis of the matrix components of the staphylococcal biofilms, assessed by confocal laser scanning microscopy (CLSM), showed that NO stimulates PIA and protein production and interferes with biofilm structure in a strain-dependent manner, but independently of the Ldh level. Thus, NO resistance is attained by remodeling the staphylococcal matrix architecture and adaptation of main metabolic processes, likely providing in vivo fitness of S. epidermidis biofilms contacting NO-proficient macrophages.publishersversionpublishe

3-Aminopyrroles and their application in the synthesis ofpyrrolo[3,2-d]pyrimidine (9-deazapurine) derivatives

3-Aminopyrrole derivatives have been synthesized from 3-anilino-2-cyanoacrylonitrile using Thorpe-Ziegler cyclization. These substituted pyrroles are readily converted into 5H-pyrrolo[3,2- d]pyrimidine (9-deazapurines).FEDERFundação para a Ciência e Tecnologia (FCT