Genetic and Environmental Modifiers of Somatic Trinucleotide Repeat Dynamics

The expansion of CAG•CTG trinucleotide repeat sequences has been identified as the genetic cause of several human diseases, including myotonic dystrophy type 1, Huntington disease, and an ever-increasing number of spinocerebellar ataxias. Once above a size threshold, the repeats become dramatically unstable in the germline and also throughout the soma, with a marked bias towards further expansion. Such expansions constitute a unique form of dynamic mutation, whose mechanism is poorly understood. While germline instability serves as the molecular basis for genetic anticipation; age-dependent, tissue-specific somatic instability most likely contributes to the tissue specificity, phenotypic variability and progressive nature of these conditions. The study of the mutation mechanism is therefore of major interest, as it may provide valuable clues towards a better understanding of disease pathophysiology. It is generally assumed that the repeat length changes arise through DNA polymerase slippage during DNA replication, however no direct evidence exists to support this hypothesis in mammalian systems. Transgenic mouse models of unstable CAG•CTG repeats have been previously generated, and shown to recreate the dynamic nature of somatic mosaicism observed in humans. Tissues from these mice have now been used in order to establish an in vitro cell culture system, where the repeat dynamics could be investigated under controlled conditions. Monitoring of repeat stability in these cells over long periods of time, and numerous population doublings, has revealed the progressive accumulation of larger alleles, as a result of repeat length changes in vitro, confirmed by single cell cloning. Selection of cells carrying longer repeats was observed during the first few passages of the cultures, and frequent additional selective sweeps were also detected at later stages. The highest levels of instability were observed in cultured kidney cells, whilst the transgene remained relatively stable in eye cells and very stable in lung cells, paralleling previous in vivo observations. More importantly, the levels of repeat instability in cultured cells did not correlate with cell proliferation rates, rejecting a simple association between length change mutations and cell division, and suggesting an important role for additional cell type-specific and possibly environmental trans-acting modifiers of repeat metabolism. The effects of multiple genotoxic agents on the mutational dynamics of expanded trinucleotide repeats were assessed in this tissue culture model of unstable DNA. The drugs tested were selected based on their ability to affect cell cycle progression, DNA polymerase activity, DNA methylation, intracellular levels of oxidative stress or DNA conformational metabolism. The analysis led to the identification of chemicals, such as aspirin, 5-azacytidine and 1-beta-D-arabinofuranosyl-cytosine that resulted in the deceleration of the rate of trinucleotide repeat expansion, particularly in a kidney clonal cell line carrying rapidly expanding repetitive tracts. These observations were reported in the absence of major changes in the rates of cell turnover. In contrast, forced cell cycle progression by exposure to caffeine resulted in a significantly higher rate of triplet repeat expansion. Increased levels of oxidative stress, generated in culture by exposure to a variety of drugs, were associated with reduced levels of repeat size variability, most likely through means of cell selection in culture. Since pathology in CAG'CTG-associated diseases is mediated by a variety of complex and unrelated molecular pathways, drug induced modification of DNA dynamics could present a possible therapeutical route for these disorders. Specifically, chemical treatments that resulted in suppression of somatic repeat expansion would be expected to be beneficial, whilst reversion of the expanded mutant repeat to the normal repeat size range, observed in the general population, would be predicted to be curative. Although preliminary, the findings described in this study may open new avenues in the search for novel therapeutical strategies. Mechanistic models of repeat length mutation based on DNA replication, recombination and repair have been proposed. The latter have implied the involvement of mammalian MutS homologues (Msh2, Msh3 and Msh6). In order to gain further insight into the molecular mechanisms driving trinucleotide repeat mutation, the involvement of a mammalian MutL homologue (Pms2) in the mutation dynamics was investigated. No significant differences were observed between Pms2+/+ and Pms2+/+ mice, suggesting that a single functional Pms2 allele is sufficient to maintain high levels of somatic mosaicism. The levels of Pms2 mRNA and protein in heterozygotes deficient for Pms2 have not yet been investigated. In contrast to what would be predicted by the replication slippage model, lower levels of trinucleotide somatic mosaicism were detected in homozygous Pms2-null mice, compared with age-matched controls, carrying either one or two functional copies of the Pms2 allele. In addition, a higher frequency of rare but large deletion events was detected in Pms2-/- animals. Both results proved statistically significant by single molecule analysis. These findings imply that, not only MMR enzymes that directly bind to DNA, but also proteins that are subsequently recruited by MutS proteins, play a central role in the accumulation of repeat length changes, arguing against a mutation mechanism mediated by stabilisation of alternative DNA secondary structures by MMR proteins. MMR gene polymorphisms and variants might therefore be considered potential determinants of trinucleotide repeat instability in humans, predicted to affect both age of onset and disease progression

Um palacete assobradado: a coexistência entre organizações estatais e instituições de mercado no financiamento habitacional brasileiro = New institutions, old organizations: legal reforms in the Brazilian housing finance

- DOI: http://dx.doi.org/10.1590/2317-617220193

Chronic exposure to cadmium and antioxidants does not affect the dynamics of expanded CAG•CTG trinucleotide repeats in a mouse cell culture system of unstable DNA

More than 30 human disorders are caused by the expansion of simple sequence DNA repeats, among which triplet repeats remain the most frequent. Most trinucleotide repeat expansion disorders affect primarily the nervous system, through mechanisms of neurodysfunction and/or neurodegeneration. While trinucleotide repeat tracts are short and stably transmitted in unaffected individuals, disease-associated expansions are highly dynamic in the germline and in somatic cells, with a tendency towards further expansion. Since longer repeats are associated with increasing disease severity and earlier onset of symptoms, intergenerational repeat size gains account for the phenomenon of anticipation. In turn, higher levels of age-dependent somatic expansion have been linked with increased disease severity and earlier age of onset, implicating somatic instability in the onset and progression of disease symptoms. Hence, tackling the root cause of symptoms through the control of repeat dynamics may provide therapeutic modulation of clinical manifestations. DNA repair pathways have been firmly implicated in the molecular mechanism of repeat length mutation. The demonstration that repeat expansion depends on functional DNA mismatch repair (MMR) proteins, points to MMR as a potential therapeutic target. Similarly, a role of DNA base excision repair (BER) in repeat expansion has also been suggested, particularly during the removal of oxidative lesions. Using a well-characterized mouse cell model system of an unstable CAG•CTG trinucleotide repeat, we tested if expanded repeat tracts can be stabilized by small molecules with reported roles in both pathways: cadmium (an inhibitor of MMR activity) and a variety of antioxidants (capable of neutralizing oxidative species). We found that chronic exposure to sublethal doses of cadmium and antioxidants did not result in significant reduction of the rate of trinucleotide repeat expansion. Surprisingly, manganese yielded a significant stabilization of the triplet repeat tract. We conclude that treatment with cadmium and antioxidants, at doses that do not interfere with cell survival and cell culture dynamics, is not sufficient to modify trinucleotide repeat dynamics in cell culture

ÁGUA SUBTERRÂNEA NO GRÁBEN DE PONTA GROSSA, PR

A cidade de Ponta Grossa situa-se sobre a feição geológica denominada Gráben de Ponta Grossa, um conjunto de blocos tectônicos nas direções NE-SW e NW-SE abatidos no Mesozoico. O gráben aparece nos mapas geológicos na forma de blocos de rochas do Grupo Itararé (Carbonífero-Permiano) delimitados por falhas encaixados em rochas da Formação Ponta Grossa (Devoniano). Expressiva parte do perímetro urbano de Ponta Grossa situa-se dentro dos limites do gráben. É crescente o número de poços tubulares profundos que explotam águas subterrâneas do Grupo Itararé e formações Ponta Grossa e Furnas (Siluriano-Devoniano) na região do gráben. Os dados destes poços indicam desnivelamentos de blocos que ultrapassam200 m. Comparando-se os teores médios de fosfato, sulfato, ferro total, sólidos dissolvidos totais e dureza total das águas constata-se variações significativas em função das unidades explotadas: os teores de fosfato são maiores no Grupo Itararé, o ferro total é maior na Formação Ponta Grossa e sulfato, sólidos dissolvidos e dureza totais são maiores na Formação Furnas. As vazões médias são maiores na Formação Furnas (cerca de 14,1 m³/h), intermediárias no Grupo Itararé (8,5 m³/h) e menores na Formação Ponta Grossa (3,8 m³/h). Os resultados indicam a necessidade de análises locacionais e projetos construtivos de poços criteriosos visando adequar vazões e evitar mistura de águas com qualidades muito distintas provenientes de diferentes unidades geológicas superpostas perfuradas pelos poços

Selective albumin-binding surfaces modified with a thrombin-inhibiting peptide

Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to coagulation, which is explained by its capacity to adsorb albumin in a selective and reversible way. This work opens new perspectives to the improvement of the haemocompatibility of blood-contacting medical devices

Beta-agonist stimulation ameliorates the phenotype of spinal and bulbar muscular atrophy mice and patient-derived myotubes

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease characterized by the loss of lower motor neurons. SBMA is caused by expansions of a polyglutamine tract in the gene coding for androgen receptor (AR). Expression of polyglutamine-expanded AR causes damage to motor neurons and skeletal muscle cells. Here we investigated the effect of β-agonist stimulation in SBMA myotube cells derived from mice and patients, and in knock-in mice. We show that treatment of myotubes expressing polyglutamine-expanded AR with the β-agonist clenbuterol increases their size. Clenbuterol activated the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway and decreased the accumulation of polyglutamine-expanded AR. Treatment of SBMA knock-in mice with clenbuterol, which was started at disease onset, ameliorated motor function and extended survival. Clenbuterol improved muscle pathology, attenuated the glycolytic-to-oxidative metabolic alterations occurring in SBMA muscles and induced hypertrophy of both glycolytic and oxidative fibers. These results indicate that β-agonist stimulation is a novel therapeutic strategy for SBMA

Usando o Geogebra para o Ensino de Sólidos de Revolução

This research was developed based on the principles of the Didactic Engineering, to study and discuss the contributions that the use of the GeoGebra software may offer to understanding of mathematics concepts often used to evaluate areas and volumes of revolution solids. Was applied a didactic intervention using GeoGebra to a group of students to propitiate them knowledge enough to work with a the interactive dynamic geometry interconnecting it to empirical and formal concepts of Spatial Geometry, improving their dimensional view and preparing them for teaching Geometry. After analysis and discussions about the obtained results, were noticed a remarkable progress from the students related to the understanding of the fundamental topics of the Geometry concepts proposed in this work.Este estudo foi desenvolvido baseado nos princípios da Engenharia Didática visando estudar e discutir as contribuições que o uso do GeoGebra pode oferecer para a compreensão de conceitos matemáticos frequentemente usados para o cálculo de áreas e volumes de sólidos de revolução.Foi aplicada uma intervenção didática usando o GeoGebra a um grupo de estudantes visando propiciá-los conhecimento suficiente para trabalhar com a geometria dinâmica interativa de modo interligado aos conceitos empíricos e formais de Geometria Espacial, aperfeiçoando sua visão tridimensional e preparando-os para a prática docente de tal conteúdo.Após análises e discussões sobre os resultados obtidos, constatou-se notável progresso dos estudantes no que se refere ao entendimento dos conceitos fundamentais dos tópicos trabalhados

Usando o Geogebra para o Ensino de Sólidos de Revolução

This research was developed based on the principles of the Didactic Engineering, to study and discuss the contributions that the use of the GeoGebra software may offer to understanding of mathematics concepts often used to evaluate areas and volumes of revolution solids. Was applied a didactic intervention using GeoGebra to a group of students to propitiate them knowledge enough to work with a the interactive dynamic geometry interconnecting it to empirical and formal concepts of Spatial Geometry, improving their dimensional view and preparing them for teaching Geometry. After analysis and discussions about the obtained results, were noticed a remarkable progress from the students related to the understanding of the fundamental topics of the Geometry concepts proposed in this work.Este estudo foi desenvolvido baseado nos princípios da Engenharia Didática visando estudar e discutir as contribuições que o uso do GeoGebra pode oferecer para a compreensão de conceitos matemáticos frequentemente usados para o cálculo de áreas e volumes de sólidos de revolução.Foi aplicada uma intervenção didática usando o GeoGebra a um grupo de estudantes visando propiciá-los conhecimento suficiente para trabalhar com a geometria dinâmica interativa de modo interligado aos conceitos empíricos e formais de Geometria Espacial, aperfeiçoando sua visão tridimensional e preparando-os para a prática docente de tal conteúdo.Após análises e discussões sobre os resultados obtidos, constatou-se notável progresso dos estudantes no que se refere ao entendimento dos conceitos fundamentais dos tópicos trabalhados

Risk factors on the treatment of cecum injuries with primary suture in rats

Purpose: Analyze the results of primary suture in the treatment of cecum traumatic injuries in rats, after the exposure to increasing time intervals between the trauma and the surgery and with different peritonitis degrees. Methods: In a randomized double-blinded study, 96 Wistar male rats, weight ranging between 200 and 250 grams, underwent laparotomy, in which a 5-milimiter-diameter-injury in the contramesenteric edge of the cecum was performed. In 12 animals of the control-group a prompt primary suture was executed, with total and separated stitches, with 7.0 polypropylene thread. In the other groups, with 12 animals each, a laparotomy for repair of the injury was executed after intervals of 30 minutes, 1, 2, 4, 6, 9 and 12 hours. At the time of injury repair, one of its edges was ressected and sent for anatomopathological examination. A daily control after the surgery was done, observing the presence of complications, specially dehiscence of the suture, and the euthanasia of the animals were done in the 1st, 4th, 7th and 14th day after the surgery. Necropsy was executed in all animals, observing the macroscopic and microscopic findings in the area of suture. Results: There was no association between the delay for surgical treatment of the injury and peritonitis degrees. The mortality in the 14 animals with diffuse peritonitis was 100%. Global mortality was 25% (24 animals), and 6 animals (25%) died before treatment. None of the animals treated that evolved to death had complications related to the suture of the injury. These early deaths were due to peritonitis and sepsis. Among the 72 surviving rats, there was dehiscence of the suture in 9 animals (12.5%). This complication was statistically significant greater in animals operated on after the sixth hour following the trauma. The incidence of dehiscence was also greater in the rats presenting more intense fecal contamination. Intensity of the peritonitis at the moment of suture observed in histological examination had no association with the occurrence of complications of the primary suture. Conclusion: The primary suture as a risky procedure to treat rats, after an interval superior to six hours after the trauma or in the period of intense contamination of the cavity by feces.Objetivo: O presente trabalho teve como objetivo avaliar os resultados da sutura primária no tratamento das lesões traumáticas de ceco em ratos, após exposição a intervalos de tempo crescentes entre o trauma e a cirurgia, e com diferentes graus de peritonite. Métodos: Em estudo randomizado, duplo-cego, 96 ratos Wistar, machos, com peso variando de 200 a 250 gramas, foram submetidos a laparotomia, em que se realizava lesão de 5 milímetros de diâmetro na borda contramesentérica do ceco. Em 12 animais do grupo-controle realizava-se de imediato sutura primária com pontos totais, separados, com fio de polipropileno 7.0. Nos demais grupos, com 12 animais cada, a laparotomia para reparo da lesão foi realizada após intervalos de: 30 minutos, 1, 2, 4, 6, 9 e 12 horas. No momento do reparo da lesão, uma das suas bordas era ressecada e enviada para exame anatomopatológico. Foi feito controle diário no pós-operatório, atentando-se para a presença de complicações, em especial deiscência da sutura, sendo a eutanásia dos animais realizada no 1º, 4º, 7º e 14º dia de pós-operatório. Em todos animais foi realizada necropsia, atentando-se aos achados macroscópicos e microscópicos do local da sutura. Resultados: Não houve associação entre a demora para o tratamento cirúrgico da lesão e a evolução para graus mais avançados de peritonite. A mortalidade nos 14 animais com peritonite difusa foi de 100%. A mortalidade global foi de 25% (24 animais), sendo que 6 animais (25% dos óbitos) morreram antes do tratamento. Nenhum dos animais tratados que evoluíram a óbito teve complicação relacionada com a sutura da lesão. Os óbitos foram precoces, decorrentes de peritonite e sepse. Entre os 72 ratos sobreviventes, observou-se deiscência da sutura em 9 animais (12,5%). A ocorrência desta complicação foi maior em animais operados a partir da sexta hora após o trauma, sendo os resultados estatisticamente significativos. A incidência de deiscência também foi maior nos ratos que apresentavam contaminação fecal mais intensa da cavidade peritoneal. A intensidade da peritonite no momento da sutura observada no exame histológico não teve associação com a ocorrência de complicações da sutura primária. Conclusão: A sutura primária é um procedimento de risco para tratar ratos, transcorrido intervalo superior a seis horas após o trauma, ou na vigência de contaminação intensa da cavidade por fezes.61762

What is the Diagnosis?

Patient T.D., 23 years old, female, with corrected transposition of the great arteries, complete atrioventricular block with narrow QRS, submitted to bicameral pacemaker implantation at 13 years of age, with generator replacement three years ago. She has a good functional capacity, but complains of fatigue in the face of intense effort