Cockayne Syndrome: The many challenges and approaches to understand a multifaceted disease

The striking and complex phenotype of Cockayne syndrome (CS) patients combines progeria-like features with developmental deficits. Since the establishment of the in vitro culture of skin fibroblasts derived from patients with CS in the 1970s, significant progress has been made in the understanding of the genetic alterations associated with the disease and their impact on molecular, cellular, and organismal functions. In this review, we provide a historic perspective on the research into CS by revisiting seminal papers in this field. We highlighted the great contributions of several researchers in the last decades, ranging from the cloning and characterization of CS genes to the molecular dissection of their roles in DNA repair, transcription, redox processes and metabolism control. We also provide a detailed description of all pathological mutations in genes ERCC6 and ERCC8 reported to date and their impact on CS-related proteins. Finally, we review the contributions (and limitations) of many genetic animal models to the study of CS and how cutting-edge technologies, such as cell reprogramming and state-of-the-art genome editing, are helping us to address unanswered questions

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Avalia??o da participa??o de processos redox na s?ndrome de Cockayne.

Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. N?cleo de Pesquisas em Ci?ncias Biol?gicas, Pr?-Reitoria de Pesquisa de P?s Gradua??o, Universidade Federal de Ouro Preto.O reparo por Excis?o de Nucleot?deos (NER) ? um importante mecanismo que auxilia na manuten??o da integridade do genoma, sendo respons?vel pelo reparo de les?es que causam distor??es na fita dupla do DNA. Muta??es em algumas prote?nas que participam da via NER est?o associadas ao desenvolvimento de doen?as severas, como a S?ndrome de Cockayne (CS), cujo os principais sintomas s?o neurodegenera??o e envelhecimento precoce. Sabe-se que os processos redox est?o relacionados ao desenvolvimento de doen?as neurodegenerativas como Alzheimer e Parkinson, pela sua capacidade de reagir com biomol?culas, principalmente o DNA. Evid?ncias na literatura mostram que processos redox tamb?m sejam um dos fatores que possam contribuir para a progress?o da S?ndrome de Cockayne (CS). O objetivo desse trabalho foi avaliar a participa??o dos processos redox em CS. Inicialmente, realizamos uma extensa an?lise dos trabalhos j? publicados na literatura com descri??es de muta??es para a s?ndrome, a fim de identificarmos uma poss?vel rela??o entre tais muta??es, funcionalidade das prote?nas e o fen?tipo dos pacientes. Em seguida, a sensibilidade ? indu??o de processos redox foi avaliado em modelos celulares mutados nas prote?nas CSA e CSB, ap?s tratamento com BSO e per?xido de hidrog?nio. Tamb?m, analisamos o perfil redox do c?rebro e f?gado de camundongos envelhecidos por 1,5, 12 e 24 meses deficientes e proficientes na prote?na CSA, por meio da quantifica??o de prote?nas carboniladas, grupos ti?is e atividade da enzima Glutationa Peroxidase. Nossos dados apontam a inexist?ncia de rela??o clara entre muta??es e fen?tipos manifestados pelos pacientes. Al?m disso, mostram que as c?lulas mutadas em CSA e CSB apresentam sensibilidade diferencial entre si e entre o grupo controle, indicando que muta??es diferentes no mesmo gene conferem respostas espec?ficas frente a uma situa??o de indu??o de processos redox. Em rela??o aos experimentos com tecidos dos animais, detectamos redu??o da concentra??o de prote?nas carboniladas no c?rebro de camundongos CSA com o envelhecimento. De maneira geral, os dados indicam uma n?o rela??o entre muta??o e sintomas manifestados pelos pacientes, assim como nos resultados dos in vitro. Ressaltamos tamb?m a necessidade do desenvolvimento de modelos animais e celulares que melhor representem o fen?tipo da doen?a e possibilitem o seu estudo de maneira mais precisa, para que os mecanismos de indu??o e progress?o de CS sejam completamente elucidados.The Nucleotide excision repair (NER) pathway is an important mechanism that helps in the maintenance of the genome integrity, being responsible for the repair of distortive lesions in the DNA double helix. Mutations in some proteins that act on NER pathway are associated to the development of severe diseases as Cockayne Syndrome (CS), which the main symptoms are neurodegeneration and premature ageing. It is known that redox processes are related to the development of neurodegenerative diseases like Alzheimer and Parkinson by it?s potential to react with biomolecules, especially the DNA. Literature reports show that redox processes are also a factor that may contribute to the progression of CS. The aim of this work was to evaluate the participation of redox processes in the CS. Initially, we performed and extensive research of publicated works in the scientific literature with description of mutations that cause this syndrome to identify possible relation between mutations, protein functionality and patients phenotype. Following, the sensibility to redox processes induction was evaluated in cellular models carrying mutations in the CSA and CSB proteins after the treatment with BSO and hydrogen peroxide. We also analysed the redox profile of the brain and liver of aged mice with 1,5, 12 and 24 months old that are deficient and proficient in the CSA protein, by quantification of carbonilated protein levels, thiols groups and Gluthathione Peroxidase enzyme activity. Our data point the inexistence of clear relation between mutations and patients phenotypes. In addition, we show that cells mutated in CSA and CSB exhibit differential sensibility among themselves and control group, pointing that different mutations in the same gene confer specific responses when redox processes are induced. In relation to the experiment with animals tissues, we detect reduction of carbonilated protein content in the brains of CSA?s mice with the age. In general, the data indicates no relation between mutation and patient?s symptoms as the results of the in vitro experiments. We also highlight the importance of the development of animals and cellular models that best represent the disease?s phenotype and enable it?s research in a more precise manner, for the best elucidation of the mechanisms of induction and progression of CS

Antiproliferative and toxicogenomic effects of resveratrol in bladder cancer cells with different TP53 status.

The antitumor activity of resveratrol, a polyphenolic compound found mainly in grapes, has been studied in several types of cancer. In bladder cancer, its antiproliferative effects have already been demonstrated; however, its mechanism of action is not completely understood. The aim of this study was to evaluate resveratrol antitumor activity (12.5, 25, 50, 100, 150, 200, and 250 ?M) and its possible mechanisms of action in bladder tumor cells with different TP53 gene status (RT4, grade 1, TP53 wild type; 5637-grade 2 and T24-grade 3, TP53 mutated). Cell proliferation, clonogenic survival, morphological changes, cell cycle progression, apoptosis rates, genotoxicity, global methylation, immunocytochemistry for p53 and PCNA and relative expression profiles of the AKT, mTOR, RASSF1A, HOXB3, SRC, PLK1, and DNMT1 were evaluated. Resveratrol decreased cell proliferation and induced DNA damage in all cell lines. Regarding the long-term effects, resveratrol reduced the number of colonies in all cell lines; however, TP53 wild type cells were more resistant. Increased rates of apoptosis were found in the TP53 wild type cells and this was accompanied by AKT, mTOR, and SRC downregulation. In addition, the resveratrol antiproliferative effects in wild type TP53 cells were accompanied by modulation of the DNMT1 gene. In the TP53 mutated cells, cell cycle arrest at S phase with PLK1 downregulation was observed. Additionally, there was modulation of the HOXB3/RASSF1A pathway and nuclear PCNA reduction in the highest-grade cells. In conclusion, resveratrol has antiproliferative activity in bladder tumor cells; however, the mechanisms of action are dependent on TP53 status

Antiproliferative and toxicogenomic effects of resveratrol in bladder cancer cells with different TP53

The antitumor activity of resveratrol, a polyphenolic compound found mainly in grapes, has been studied in several types of cancer. In bladder cancer, its antiproliferative effects have already been demonstrated; however, its mechanism of action is not completely understood. The aim of this study was to evaluate resveratrol antitumor activity (12.5, 25, 50, 100, 150, 200, and 250 ?M) and its possible mechanisms of action in bladder tumor cells with different TP53 gene status (RT4, grade 1, TP53 wild type; 5637-grade 2 and T24-grade 3, TP53 mutated). Cell proliferation, clonogenic survival, morphological changes, cell cycle progression, apoptosis rates, genotoxicity, global methylation, immunocytochemistry for p53 and PCNA and relative expression profiles of the AKT, mTOR, RASSF1A, HOXB3, SRC, PLK1, and DNMT1 were evaluated. Resveratrol decreased cell proliferation and induced DNA damage in all cell lines. Regarding the long-term effects, resveratrol reduced the number of colonies in all cell lines; however, TP53 wild type cells were more resistant. Increased rates of apoptosis were found in the TP53 wild type cells and this was accompanied by AKT, mTOR, and SRC downregulation. In addition, the resveratrol antiproliferative effects in wild type TP53 cells were accompanied by modulation of the DNMT1 gene. In the TP53 mutated cells, cell cycle arrest at S phase with PLK1 downregulation was observed. Additionally, there was modulation of the HOXB3/RASSF1A pathway and nuclear PCNA reduction in the highest-grade cells. In conclusion, resveratrol has antiproliferative activity in bladder tumor cells; however, the mechanisms of action are dependent on TP53 status

Assessment of dapagliflozin effect on diabetic endothelial dysfunction of brachial artery (ADDENDA-BHS2 trial): rationale, design, and baseline characteristics of a randomized controlled trial

Endothelial dysfunction (ED) is a hallmark in type 2 diabetes mellitus (T2DM) that favor both atherogenesis and ischemia and reperfusion injury (IRI). Sodium-glucose-2 co-transporter inhibitors (SGLT2i) may hypothetically improve microvascular and macrovascular functions via a broad spectrum of mechanisms, being superior to traditional antidiabetic therapy such as sulfonylurea, even in subjects under equivalent glycemic control. Hence, the present clinical trial was designed to compare the effect of these two treatments on markers of arterial wall function and inflammation in T2DM patients as well as on the potential mediating parameters. ADDENDA-BHS2 is a prospective, single-center, active-controlled, open, randomized trial. Ninety-eight participants (40-70years old) with HbA1c 7-9% were randomized (1:1, stratified by gender, BMI and HbA1c levels) to either dapagliflozin 10mg/day or glibenclamide 5mg/day on top of metformin. The primary endpoint was the change of flow-mediated dilation (FMD) after a 12-week period of treatment evaluated at rest and after IRI between dapagliflozin and glibenclamide arms. Secondary outcomes were defined as the difference between treatments regarding: plasma nitric oxide (NO) change after FMD, plasma isoprostane, plasma levels of vascular inflammatory markers and systemic inflammatory markers, plasma levels of adipokines, anthropometric measures, glucose control parameters, office and ambulatory BP control. Safety endpoints were defined as systolic and diastolic function assessed by echocardiography and retinopathy change. Serious adverse events were recorded. The study protocol was approved by the Independent Scientific Advisory Committee. The ADDENDA-BHS2 trial is an investigator-initiated clinical trial comparing the effect of dapagliflozin versus glibenclamide on several aspects of vascular function in high cardiovascular risk T2DM patients. Besides, a large clinical and biochemical phenotype assessment will be obtained for exploring potential mediations and associations.11CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ301465/2017-

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.13Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt