Rad9/53BP1 promotes DNA repair via crossover recombination by limiting the Sgs1 and Mph1 helicases

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3\u2032 single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer

Formation and nucleolytic processing of Cas9-induced DNA breaks in human cells quantified by droplet digital PCR

Cas9 endonuclease from S. pyogenes is widely used to induce controlled double strand breaks (DSB) at desired genomic loci for gene editing. Here, we describe a droplet digital PCR (ddPCR) method to precisely quantify the kinetic of formation and 5\u2032-end nucleolytic processing of Cas9-induced DSB in different human cells lines. Notably, DSB processing is a finely regulated process, which dictates the choice between non-homologous end joining (NHEJ) and homology directed repair (HDR). This step of DSB repair is also a relevant point to be taken into consideration to improve Cas9-mediated technology. Indeed, by this protocol, we show that processing of Cas9-induced DSB is impaired by CTIP or BRCA1 depletion, while it is accelerated after down-regulation of DNAPKcs and 53BP1, two DSB repair key factors. In conclusion, the method we describe here can be used to study DSB repair mechanisms, with direct utility for molecularly optimising the knock-out/in outcomes in genome manipulation

Experimental benchmarking of Monte Carlo simulations for radiotherapy dosimetry using monochromatic X-ray beams in the presence of metal-based compounds

The local dose deposition obtained in X-ray radiotherapy can be increased by the presence of metal-based compounds in the irradiated tissues. This finding is strongly enhanced if the radiation energy is chosen in the kiloelectronvolt energy range, due to the proximity to the absorption edge. In this study, we present a MC application developed with the toolkit Geant4 to investigate the dosimetric distribution of a uniform monochromatic X-ray beam, and benchmark it against experimental measurements. Two validation studies were performed, using a commercial PTW RW3 water-equivalent slab phantom for radiotherapy, and a custom-made PMMA phantom conceived to assess the influence of high atomic number compounds on the dose profile, such as iodine and gadolinium at different concentrations. An agreement within 9% among simulations and experimental data was found for the monochromatic energies considered, which were in the range of 30–140 keV; the agreement was better than 5% for depths <60 mm. A dose enhancement was observed in the calculations, corresponding to the regions containing the contrast agents. Dose enhancement factors (DEFs) were calculated, and the highest values were found for energies higher than the corresponding K-edges of iodine and gadolinium. The in-silico results are in line with the empirical findings, which suggest that Geant4 can be satisfactorily used as a tool for the calculation of the percentage depth dose (PDD) at the energies considered in this study in the presence of contrast agents

The scaffold protein SLX4/FANCP plays a conserved role in early steps of homologous recombination DNA repair

Both in yeast and mammals, the scaffold protein SLX4/FANCP has been implicated in late steps of homologous recombination DNA repair, delivering the structure specific nucleases MUS81, SLX1 and XPF/RAD1 onto DNA repair intermediates (such as joint molecules and 3\u2019 non homologous DNA flap). Working with the model organism S. cerevisiae, we showed that Slx4 competes with the 53BP1-ortholog Rad9 for DSB end binding, favoring DNA end resection and homologous recombination repair. To investigate a possible conservation of the pathway, we exploited the AsiSI restriction enzyme and Cas9-based systems to study SLX4 role in controlling DSB resection in U2OS human osteosarcoma cells and FANCP patient derived fibroblasts. We also analyzed homologous recombination DNA repair through standard GFP reporter cassette assays and immunofluorescence foci of specific factors. The obtained results indicate that down regulation of SLX4/FANCP limits DSB resection and repair, supporting an important conserved SLX4/FANCP role in early steps of homologous recombination DNA repair, independently of the nucleases MUS81 and XPF

Pessoas com deficiência visual: esporte e lazer como fator de inclusão

El desafío de este proyecto es vivenciar prácticas de actividades físicas y de ocio en el ambiente de la Universidad, involucrando a personas con discapacidad visual de la APACE / Passo Fundo, en el sentido de repensar nuestras prácticas con miras a contribuir en el proceso de inclusión social. Elementos de lectura de realidad/diagnóstico: El deporte y el ocio en las diversas modalidades posibilitan espacios para diferentes edades, intereses y condiciones. La actuación con portadores de deficiencia visual es un desafío en virtud de las pocas vivencias, informaciones, materiales, desconocimiento en adaptar actividades y la poca motivación de las personas hacia las prácticas, teniendo en cuenta que muchos todavía permanecen en la oscuridad de la sociedad, sea por falta de divulgación o la resistencia a las experimentaciones. Aparentemente existe interés y anhelo e incluso insistencia para involucrarse, pero acaban chocando con el sentimiento de inferioridad, olvidándose de sus capacidades y potencialidades. La sede de APACE es el punto de encuentro de los asociados, todos portadores de alguna deficiencia visual, que revelan anhelos, voluntades y deseos, unidos en un solo propósito, la búsqueda de hacer su vida lo más integrada posible. El espacio no es adecuado para las actividades prácticas, por lo que todas son realizadas en la Universidad. Se percibe que la discapacidad no los hace menos capaces de usufructuar y conducir sus vidas, en el área del deporte y del ocio, no retirando la capacidad de construir su autonomía y su historia. Pero todavía está muy presente signos de inseguridad para enfrentar las situaciones de lo cotidiano. El proyecto viene sirve de oportunidad para la convivencia con videntes mediante las actividades, reafirmando la posibilidad de transformación en el modo de pensar, actuar y sentir, en un proceso que se construye en las relaciones e interrelaciones.The challenge of this project is to experience practices of physical and leisure activities in the University environment, involving people with visual disabilities of the APACE / Passo Fundo, in the sense of rethinking our practices with a view to contributing to the social inclusion process. Elements of reality/diagnosis reading: Sport and leisure in the different modalities allow spaces for different ages, interests and conditions. The performance with carriers of visual impairment is a challenge because of the few experiences, information, materials, ignorance in adapting activities and the low motivation of people towards the practices, taking into account that many still remain in the darkness of society, either for lack of disclosure or resistance to experimentation. Apparently there is interest and longing and even insistence to get involved, but they end up colliding with the feeling of inferiority, forgetting their capabilities and potential. The headquarters of APACE is the meeting point of the associates, all bearers of some visual deficiency, which reveal longings, wills and desires, united in a single purpose, the search to make their life as integrated as possible. The space is not suitable for practical activities, so all are done at the University. It is perceived that disability does not make them less able to enjoy and lead their lives, in the area of sports and leisure, not removing the ability to build their autonomy and their history. But signs of insecurity are still very present to face situations of everyday life. The project is an opportunity for coexistence with seers through activities, reaffirming the possibility of transformation in the way of thinking, acting and feeling, in a process that is built on relationships and interrelationships.O desafio deste Projeto é vivenciar práticas de atividades físicas e de lazer no ambiente da Universidade envolvendo pessoas com deficiência visual da APACE / Passo Fundo no sentido de repensar nossas práticas numa perspectiva de contribuir no processo de inclusão social. Elementos de leitura de realidade/diagnóstico: O esporte e o lazer nas variadas modalidades possibilitam espaços para diferentes idades, interesses e condições. Atuar com portadores de deficiência visual é desafiador em virtude de poucas vivências, informações, materiais, desconhecimento em adaptar atividades e a pouca motivação das pessoas para as práticas, tendo em vista, que muitos ainda permanecem na escuridão da sociedade, seja por falta de divulgação ou ainda a resistência às experimentações. Aparentemente existe interesse e anseio e até insistência para se envolver, mas acabam esbarrando no sentimento de inferioridade, esquecendo-se de suas capacidades e potencialidades. A sede da APACE é o ponto de encontro dos associados, todos portadores de alguma deficiência visual, que revelam anseios, vontades e desejos, unidos em um só propósito, a busca de tornar sua vida a mais integrada possível. O espaço não é adequado para as atividades práticas, por este motivo todas são realizadas na Universidade. Percebe-se que a deficiência não os torna menos capazes de usufruir e conduzir suas vidas, na área do esporte e do Lazer, não retirando a capacidade de construir sua autonomia e sua história. Porém ainda está muito presente sinais de insegurança para enfrentar as situações do cotidiano. O projeto vem para oportunizar o convívio com videntes pelas atividades reafirmando a possibilidade de transformação no modo de pensar, agir e sentir, num processo que se constrói nas relações e inter-relações.peerReviewe

934 Artificial Liver Support in Children with Fulminant Hepatic Failure: Our Experience

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Polos regionais de desenvolvimento do esporte e do lazer: a descentralização da gestão de uma Política de Estado

La implementación del Polo en la UPF es de extrema importancia para el desarrollo regional, permitiendo las políticas públicas de una forma descentralizada y articulada. Se compone de 151 municipios distribuidos en seis micro-regiones correspondientes a las Coordinadoras Regionales de Educación: 7ª, 9ª, 15ª, 20ª, 25ª y 39ª CRE. El polo actúa en sintonía con FUNDERGS como facilitador de acciones, estimulando el desarrollo deportivo en la región e incentivando nuevas alternativas y propuestas volcadas en el deporte y el ocio. En el caso de los municipios de su región, se cuenta con la infraestructura existente en sus dependencias en los diversos eventos propuestos por FUNDERGS y municipios de su región, recibiendo, interpretando, diagnosticando y ayudando a los gestores municipales en sus demandas, siendo el principal interlocutor del Estado. En los dos años de instalación, varias fueron las acciones propuestas por FUNDERGS y desarrolladas por el polo, teniendo públicos y variados objetivos.The implementation of the Pole at UPF is extremely important for regional development, allowing public policies in a decentralized and articulated way. It consists of 151 municipalities distributed in six micro-regions corresponding to the Regional Coordinators of Education: 7th, 9th, 15th, 20th, 25th and 39th CRE. The polo acts in sync with FUNDERGS as a facilitator of actions, stimulating sports development in the region and encouraging new alternatives and proposals focused on sports and leisure. In the case of the municipalities of its region, existing infrastructure in its dependencies is available in the various events proposed by FUNDERGS and municipalities in its region, receiving, interpreting, diagnosing and assisting municipal managers in their demands, the main one being interlocutor of the State. In the two years of installation, several were the actions proposed by FUNDERGS and developed by the pole, having public and varied objectives.A implementação do Polo na UPF é de extrema importância para o desenvolvimento regional, visando as políticas públicas de forma descentralizada e articulada. É composto por 151 municípios distribuídos em 6 micro-regiões correspondente as Coordenadorias Regionais de Educação: 7ª, 9ª, 15ª, 20ª, 25ª e 39ª CRE. O polo atua junto em sintonia com a FUNDERGS como facilitador de ações, estimulando o desenvolvimento esportivo na região e incentivando novas alternativas e propostas voltadas ao esporte e lazer. Disponibiliza a infraestrutura existente nas suas dependências nos diversos eventos propostos pela FUNDERGS, e municípios da sua região, recebendo, interpretando, diagnosticando e auxiliando os gestores municipais em suas demandas, sendo, o principal interlocutor do Estado. Nos dois anos de instalação, várias foram as ações propostas pela FUNDERGS e desenvolvidas pelo polo, tendo público e objetivos variados.peerReviewe

Microbeam Radiation Therapy controls local growth of radioresistant melanoma and treats out-of-field locoregional metastasis.

PURPOSE Synchrotron-generated microbeam radiotherapy (MRT) represents an innovative preclinical type of cancer radiotherapy with an excellent therapeutic ratio. Beyond local control, metastatic spread is another important endpoint to assess the effectiveness of radiotherapy treatment. Currently, no data exists on an association between MRT and metastasis. Here, we evaluated the ability of MRT to delay B16F10 murine melanoma progression and locoregional metastatic spread. METHODS AND MATERIALS We assessed the primary tumor response and the extent of metastasis in sentinel lymph nodes in two cohorts of C57BL/6J mice, one receiving a single MRT and another receiving two MRT delivered with a 10-day interval. We compared these two cohorts with synchrotron broad beam-irradiated and non-irradiated mice. In addition, using multi-plex quantitative platforms, we measured plasma concentrations of 34 pro- and anti-inflammatory cytokines and frequencies of immune cell subsets infiltrating primary tumors that received either one or two MRT treatments. RESULTS Two MRT treatments were significantly more effective for local control than single MRT. Remarkably, the second MRT also triggered a pronounced regression of out-of-radiation field locoregional metastasis. Augmentation of CXCL5, CXCL12 and CCL22 levels after the second MRT indicated that inhibition of melanoma progression could be associated with increased activity of anti-tumor neutrophils and T-cells. Indeed, we demonstrated elevated infiltration of neutrophils and activated T-cells in the tumors following the second MRT. CONCLUSIONS Our study highlights the importance of monitoring metastasis following MRT and provides the first MRT fractionation schedule that promotes local and locoregional control with the potential to manage distant metastasis

Slx4 and Rtt107 control checkpoint signalling and DNA resection at double-strand breaks

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5' strand. Importantly, in slx4\u394 sae2\u394 double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity

TOP BP1(Dpb11) plays a conserved role in homologous recombination DNA repair through the coordinated recruitment of 53BP1(Rad9)

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1-TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair