Live-cell imaging of transcriptional activity at DNA double-strand breaks

Copyright © 2021 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseDNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.This work was funded by PTDC/MED-OUT/32271/2017, PTDC/BIA-MOL/30438/2017 and PTDC/MED-OUT/4301/2020 from Fundação para a Ciência e a Tecnologia (FCT), Portugal and by LISBOA-01-0145-FEDER-007391, project cofunded by FEDER through POR Lisboa, Portugal 2020-Programa Operacional Regional de Lisboa, and FCT. Funding was also received from EU Horizon 2020 Research and Innovation Programme (RiboMed 857119). M.A. is the recipient of the FCT Ph.D. fellowship 2020.05899.BD.info:eu-repo/semantics/publishedVersio

Tourist events and satisfaction: a product of regional tourism competitiveness

Purpose – This study aims to analyze the contribution of tourist event and satisfaction with tourism events as a product of regional tourism competitiveness, as well as evaluate factors of competitiveness. Design/methodology/approach – The research followed a quantitative methodology by conducting a questionnaire on the four largest tourist events in Madeira (Carnival, Flower Festival, Atlantic Festival and Wine Festival), an insular island located in Portugal. Findings – The sample consisted of 2,262 tourists surveyed during the year 2017 and structural equation models were used as the statistical method. Results showed that satisfaction is reflected in the client’s loyalty to the choice of a tourist destination and also contributes directly and indirectly to the regional tourist competitiveness. Originality/value – This study contributes to the valorization of local and regional events, perceived by the level of satisfaction and loyalty of tourists, as products of regional competitiveness of a tourist destination.info:eu-repo/semantics/publishedVersio

(R)-(1-Ammonio­prop­yl)phospho­nate

The title compound, C3H10NO3P, crystallizes in its zwitterionic form, H3N+CH(C2H5)PO(O−)(OH), with the asymmetric unit being composed by two of such entities (Z′ = 2). The crystal packing leads to a sequence of hydro­phobic and hydro­philic layers. While the hydro­phobic layer comprises the aliphatic substituent groups, the hydro­philic one is held together by a series of strong and rather directional N+—H⋯O and O—H⋯O hydrogen bonds

Mudanças ambientais e interação humana na fachada atlântica ocidental

Fundação para a Ciência e a Tecnologia (FCT

A Three-Dimensional Ply Failure Model for Composite Structures

A fully 3D failure model to predict damage in composite structures subjected to multiaxial loading is presented in this paper. The formulation incorporates shear nonlinearities effects, irreversible strains, damage and strain rate effects by using a viscoplastic damageable constitutive law. The proposed formulation enables the prediction of failure initiation and failure propagation by combining stress-based, damage mechanics and fracture mechanics approaches within an unified energy based context. An objectivity algorithm has been embedded into the formulation to avoid problems associated with strain localization and mesh dependence. The proposed model has been implemented into ABAQUS/Explicit FE code within brick elements as a userdefined material model. Numerical predictions for standard uniaxial tests at element and coupon levels are presented and discussed

Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1)

The response of tobacco plants genetically engineered with the AtTPS1 gene to stress induced by excess Cu and Cd was evaluated in hydroponic solution (100 and 400 lM Cu and 50 and 200 lM Cd) after a 48 h exposure. Two transgenic lines, transformed with the AtTPS1 (trehalose-6-phosphate synthase) gene from Arabidopsis, with different levels of trehalose-6-phosphate synthase expression (B5H, higher and B1F, lower), and a wild type (WT) were investigated. Protein content, antioxidative enzymes (CAT, POD, SOD, and APX), glucose, fructose, lipid peroxidation, hydrogen peroxide and Cd and Cu contents were determined in leaves. The two transgenic lines were differently influenced by Cd and Cu exposure as they induced a different antioxidant enzymatic defense response. B1F and B5H plants showed a better acclimation to Cd and excess Cu compared to WT. Furthermore B1F was more tolerant than B5H to Cd and excess Cu. B1F accumulated less Cd and Cu in leaves, probably due to a more efficient exclusion mechanism. Catalase was shown to be the most important enzyme in the antioxidative system of these plants

Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops

PTDC/BIA-MOL/30438/2017 PTDC/MED-OUT/4301/2020 RiboMed 857119 PD/BD/128292/2017 LCF/PR/HP21/52310016 PTDC/BIA-MOL/6624/2020 PTDC/MED-ONC/7864/2020DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. Depletion of TET enzymes reduced global R-loops in the absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The genome-wide distribution of 5hmC and R-loops shows a positive correlation in mouse and human stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation.publishersversionpublishe

Productiveness Evaluation of a Machine Tool Manual Setup Compared with Automated CNC Machine

The automatic tool change of machine tools affects the productive efficiency in several ways such as starving time reduction, increase/decrease of production rate as well reliability and reduce the process related costs of manufacturing. Based on that background an analysis of experimental scenarios of manual tool change versus automatic tool change was made in order to compare and evaluate its related production rate