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

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

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

Plano amostral para cálculo de densidade larvária de Aedes aegypti e Aedes albopictus no Estado de São Paulo, Brasil

The Yellow Fever and Dengue Vector Control Program developed by the Superintendency for the Control of Endemic Diseases in the State of S. Paulo recommends Aedes aegypti and Aedes albopictus larval density monitoring in cities with domiciliar infestation. The sampling plan which has been applied in the countries of the Presidente Prudente region (SP- Brazil) since 1987 is described. The infestation is measured by using the Breteau Index. A sample of buildings is drawn, monthly and independently, in the infested cities, in which measurements are to be made. The sample is stratified and the elementary unit selection is made by using two-stage cluster sampling: of blocks and buildings. The sample sizes were defined using the coefficient of variation and the intraclass correlation as estimated for towns covered by the Regional Health Service of S. José do Rio Preto. These sizes must be corrected periodically in accordance with the values of the Breteau Index and its variance obtained in previous months.O Programa de Controle de Vetores de Febre Amarela e Dengue, desenvolvido pela Superintendência de Controle de Endemias do Estado de São Paulo, Brasil, prevê a realização de pesquisa para avaliação da densidade larvária de Aedes aegypti e Aedes albopictus em edificações de municípios com infestação domiciliar. Descreve-se o plano amostral que vem sendo aplicado, desde outubro de 1987, nos municípios da Região de Presidente Prudente. Para acompanhamento da densidade está sendo utilizado o índice de Breteau. São sorteados, nos municípios infestados, mensalmente e de forma independente, amostras de edificações para a obtenção das estimativas do índice. O plano amostral prevê a seleção de conglomerados em 2 estágios: quadras e edificações. O tamanho da amostra foi definido estimando-se o coeficiente da correlação intraconglomerado e variância relativa por elemento através de pesquisas realizadas anteriormente em municípios do Serviço Regional de São José do Rio Preto. O plano propõe que os valores relativos ao tamanho da amostra sejam atualizados periodicamente em função dos valores obtidos para o estimador do Índice de Breteau e sua variância, em meses anteriores

Methyl 2-(4,6-dichloro-1,3,5-triazin-2-yl­amino)acetate

The title compound, C6H6Cl2N4O2, was prepared by the nucleophilic substitution of 2,4,6-trichloro-1,3,5-triazine by glycine methyl ester hydro­chloride, and was isolated from the reaction by using flash chromatography. The crystal structure at 150 K reveals the presence two crystallographically independent mol­ecules in the asymmetric unit which differ in the orientation of the pendant methoxy­carbonyl group. Each mol­ecular unit is engaged in strong and highly directional N—H⋯N hydrogen-bonding inter­actions with a symmetry-related mol­ecule, forming supra­molecular dimers which act as the synthons in the crystal packing

Biologia e competição intra-específica de taquinídeos sobre o hospedeiro, Diatraea saccharalis em condições de laboratório

This assay was carried out at the Laboratório de Controle Biológico do IAA/PANALSUCAR, Coordonadoria Regional - Sul, Araras, SP, Brazil, and its objective was to know some biological parameters and the intraspecific competition of the tachinid flies, Metagonistylum minense Towns., 1927 and Paratheresia claripalpis Wulp, 1896, both parasitoids of the sugarcane borer Diatraea saccharalis (Fabr., 1794). One thousand inoculations with five replications were made for 200 larvae, aiming to observe the biology of these tachinids. The mean larval period for M. minense was 8.49 days and the weight of single pupae (one maggot per larva) was 39.53 mg. The mean larval period for P. claripalpis was 10.48 days and the mean weight of single pupae was 53.73 mg. For the intraspecific competition between these species it was observed that it is significant in P. claripalpis, where the weight of triple pupae was about 50.94% lower than the weight of single pupae whereas for M. minense this reduction was about 42.73%. The mean number of maggots obtained per female for P. claripalpis was 374.46 ± 22.61 and 438.86 ± 22.35 for M. minense.Esta pesquisa foi conduzida no laboratório de Controle Biológico da Coordenadoria Regional Sul do IAA/PLANALSUCAR, Araras, SP. Teve por objetivo determinar alguns dos parâmetros biológicos e a competição intra-específica das moscas Metagonistylum minense Towns., 1927, e Paratheresia claripalpis Wulp, 1896, parasitóides da broca-da-cana, Diatraea saccharalis Fabr., 1794. Foram feitas 1.000 inoculações com cinco repetições de 200 lagartas para cada espécie, visando acompanhar a biologia destes taquinídeos. O período larval médio para M. minense foi de 8,49 dias e o peso médio dos pupários "simples" foi de 39,52 mg. Para a espécie P. claripalpis, o período larval médio foi de 10,48 dias, sendo o peso médio dos pupários "simples" de 53,73 mg. Quanto à competição intra-específica, observou-se que ela é mais acentuada em P. claripalpis, onde o peso médio dos pupários "triplos" foi cerca de 50,94% inferior ao peso de pupários "simples", enquanto para M. minense esta redução foi cerca de 42,73%. O número médio de larvas geradas por fêmea da espécie P. claripalpis foi de 374,46 ± 22,61, e de M. minense 438,86 ± 22,35

Is there cardiac autonomic dysfunction in children and adolescents with exercise-induced bronchospasm?

The pulmonary impairment in patients with bronchoconstriction induced by eucapnic voluntary hyperpnea(EVH) goes beyond the respiratory system, also impairing autonomic nervous modulation. This study aimed to evaluate the behavior of cardiac autonomic modulation in young asthmatics with and without EIB after the EVH test. Research design and methods A cross-sectional study design using 54 asthmatics(51.9% female), aged between 10 and 19 years, investigated with the EVH test. Forced expiratory volume in one second(FEV1) was measured at 5, 10, 15, and 30 min after EVH. Heart rate variability(HRV) measures of time were assessed pre and 30 min-post EVH. The diagnosis of Exercise-Induced bronchoconstriction with underlying clinical asthma(EIBA) was confirmed by a fall in FEV1 ≥10% compared to baseline. Results Thirty(55.5%) asthmatics had EIBA. Subjects with EIBA have reduced mean of the R-R intervals in relation to baseline until 15 minutes after EVH. Individuals without EIBA had increased parasympathetic activity compared to baseline(rMSSD) from 5 min after EVH(p < 0.05). This parasympathetic activity increase in relation to baseline was seen in individuals with EIBA after 25 minutes (rMSSD = 49.9 ± 5.3 vs 63.5 ± 7.2, p < 0.05). Conclusion Young asthmatics with EIBA present a delay in the increase of the parasympathetic component after EVH when compared to asthmatics without EIBA

Glycine methyl ester hydro­chloride

The title compound [systematic name: (methoxy­carbonyl­meth­yl)ammonium chloride], crystallizes as a salt, C3H8NO2 +·Cl−, with the charged species inter­acting mutually via strong and highly directional N+—H⋯Cl− hydrogen bonds which lead to the formation of a supra­molecular tape running parallel to the c axis. Tapes close pack in the solid state mediated by multipoint recognition synthons based on weak C—H⋯O inter­actions and van der Waals contacts between adjacent methyl groups

Bacterial cellulose-based biomaterials on third-degree burns in rats

Burns are cutaneous lesions that present  high rate of morbidity and mortality worldwide. In order to innovate the treatment strategies currently applied new biomaterials are being investigated. The aim of the present study was to evaluate the action of bacterial cellulose in both membrane and gel form, in the treatment of third degree burns in rats. For this, 24 Wistar rats were used, divided into three distinct groups. The lesion was performed with the aid of a soldering iron heated at 150 °C pressed on the back of the animal for 10 seconds. Treatment was performed immediately after wound induction, and skin samples were collected on the tenth day post-injury. Statistical analysis was performed using a significance level of 5% (p?0.05). The histological results show differences in the healing process presented by each group. The group that received bacterial cellulose in the membrane format presented the best results, such as discrete inflammatory infiltrate and better morphological quality of the tissue, characterizing an advanced stage of the healing process, also proven in the collagen quantitative analysis. On the other hand, the group that received the cellulose gel showed characteristics of an inflammatory phase with the presence of evident ulcerations, which corresponds to a delay in the healing process even when compared to CG alone. Thus, it was concluded that before the biomaterials tested cellulose membrane in the format presented more favorable results both in terms of environmental protection as a contribution to an adequate tissue recovery.