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

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

Pawedness trait test (PaTRaT) : a new paradigm to evaluate paw preference and dexterity in rats

Pawedness Trait Test (PaTRaT)-A New Paradigm to Evaluate Paw Preference and Dexterity in RatsIn rodents, dexterity is commonly analyzed in preference paradigms in which animals are given the chance to use either the left or the right front paws to manipulate food. However, paw preference and dexterity at population and individual levels are controversial as results are incongruent across paradigms. We have therefore developed a semi-quantitative method-the pawdeness trait test (PaTRaT)-to evaluate paw preference degree in rats. The PaTRaT consists in a classification system, ranging from +4 to 4 where increasingly positive and negative values reflect the bias for left or right paw use, respectively. Sprague-Dawley male rats were confined into a metal rectangular mesh cylinder, from which they can see, smell and reach sugared rewards with their paws. Due to its size, the reward could only cross the mesh if aligned with its diagonal, imposing additional coordination. Animals were allowed to retrieve 10 rewards per session in a total of four sessions while their behavior was recorded. PaTRaT was repeated 4 and 8 weeks after the first evaluation. To exclude potential bias, rats were also tested for paw fine movement and general locomotion in other behavioral paradigms as well as impulsivity (variable delay-to-signal, VDS), memory and cognitive flexibility (water maze). At the population level 54% of the animals presented a rightward bias. Individually, all animals presented marked side-preferences, >2 and <-2 for left-and right-sided bias, respectively, and this preference was stable across the three evaluations. Inter-rater consistency was very high between two experienced raters and substantial when two additional inexperienced raters were included. Left-and right-biased animals presented no differences in the ability to perform fine movements with any of the forelimbs (staircase) and general locomotor performance. Additionally, these groups performed similarly in executive function and memory tasks. In conclusion, PaTRaT is able to reliably classify rats' pawedness direction and degree.This work has been funded by the European Regional Development Fund (FEDER), through the Competitiveness Factors Operational Programme (COMPETE) and the Northern Portugal Regional Operational Programme (NORTE 2020) under the Portugal 2020 Partnership Agreement (project NORTE-01-0145-FEDER-000023). It was also funded by National funds, through the Foundation for Science and Technology (Fundacao para a Ciencia e a Tecnologia, FCT), under the scope of the projects POCI-01-0145-FEDER-007038 and PTDC/NEU-SCC/5301/2014. Researchers were supported by FCT grant numbers SFRH/BD/109111/2015 (AMC), SFRH/BD/52291/2013 (ME via Inter-University Doctoral Programme in Ageing and Chronic Disease, PhDOC), PD/BD/114120/2015 (SPN via PhDOC), SFRH/BD/89936/2012 (SB), PD/BD/114117/2015 (MRG via PhDOC) and SFRH/BPD/80118/2011 (HL-A).info:eu-repo/semantics/publishedVersio

Deleterious ABCA7 mutations and transcript rescue mechanisms in early onset Alzheimer’s disease

Abstract: Premature termination codon (PTC) mutations in the ATP-Binding Cassette, Sub-Family A, Member 7 gene (ABCA7) have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer's disease (LOAD). High variability, however, is observed in downstream ABCA7 mRNA and protein expression, disease penetrance, and onset age, indicative of unknown modifying factors. Here, we investigated the prevalence and disease penetrance of ABCA7 PTC mutations in a large early onset AD (EOAD)-control cohort, and examined the effect on transcript level with comprehensive third-generation long-read sequencing. We characterized the ABCA7 coding sequence with next-generation sequencing in 928 EOAD patients and 980 matched control individuals. With MetaSKAT rare variant association analysis, we observed a fivefold enrichment (p = 0.0004) of PTC mutations in EOAD patients (3%) versus controls (0.6%). Ten novel PTC mutations were only observed in patients, and PTC mutation carriers in general had an increased familial AD load. In addition, we observed nominal risk reducing trends for three common coding variants. Seven PTC mutations were further analyzed using targeted long-read cDNA sequencing on an Oxford Nanopore MinION platform. PTC-containing transcripts for each investigated PTC mutation were observed at varying proportion (5-41% of the total read count), implying incomplete nonsense-mediated mRNA decay (NMD). Furthermore, we distinguished and phased several previously unknown alternative splicing events (up to 30% of transcripts). In conjunction with PTC mutations, several of these novel ABCA7 isoforms have the potential to rescue deleterious PTC effects. In conclusion, ABCA7 PTC mutations play a substantial role in EOAD, warranting genetic screening of ABCA7 in genetically unexplained patients. Long-read cDNA sequencing revealed both varying degrees of NMD and transcript-modifying events, which may influence ABCA7 dosage, disease severity, and may create opportunities for therapeutic interventions in AD

O SIGNIFICADO DA ADMINISTRAÇÃO APLICADA À ENFERMAGEM SEGUNDO A OPINIÃO DE GRADUANDAS

Este estudo teve por objetivo conhecer as opiniões que as alunas têm, quando chegam para cursar a disciplina Administração aplicada à Enfermagem. Para tanto, coletou-se as opiniões de 30 alunas do 8° semestre do Curso de Graduação da EEUSP. Os resultados mostraram que as alunas consideram a administração como uma forma de organizar o trabalho e que a função administrativa da enfermeira consiste em conciliar a assistência com a burocracia do serviço.The objective of this study was to know the opinions that the students have, when they begin the course of Administration applied of Nursing. The results indicated that the students considered administration a manner how the nurses are organizing their works and that nursing activity administration is to conciliate the assistence with the bureaucratic service