Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing

Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17-78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium.

FTO influences adipogenesis by regulating mitotic clonal expansion

The fat mass and obesity-associated (FTO) gene plays a pivotal role in regulating body weight and fat mass; however, the underlying mechanisms are poorly understood. Here we show that primary adipocytes and mouse embryonic fibroblasts (MEFs) derived from FTO overexpression (FTO-4) mice exhibit increased potential for adipogenic differentiation, while MEFs derived from FTO knockout (FTO-KO) mice show reduced adipogenesis. As predicted from these findings, fat pads from FTO-4 mice fed a high-fat diet show more numerous adipocytes. FTO influences adipogenesis by regulating events early in adipogenesis, during the process of mitotic clonal expansion. The effect of FTO on adipogenesis appears to be mediated via enhanced expression of the pro-adipogenic short isoform of RUNX1T1, which enhanced adipocyte proliferation, and is increased in FTO-4 MEFs and reduced in FTO-KO MEFs. Our findings provide novel mechanistic insight into how upregulation of FTO leads to obesity.

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

Effects of competition on sexual and clonal reproduction of a tunicate: the importance of competitor identity

Individual fitness and the structure of marine communities are strongly affected by spatial competition. Among the most common space holders are the colonial ascidians, which have the ability to monopolize large areas of hard substrate, overgrowing most other competitors. The effects of competition on colony growth and on gonad production of the ascidian Didemnum perlucidum were studied in southeastern Brazil by experimentally removing surrounding competitors. Colonies of D, perlucidum competing for space exhibited a growth rate 9 times less than that of colonies that were competitor free. Among the colonies subject to competition, growth rates were unrelated to the percentage of colony border that was free of competitors. However, the identity of the competitor was important in the outcome of border contacts. At the beginning of the experiment, most border encounters of D. perlucidum were with solitary organisms, which in most cases were overgrown. These were progressively replaced by colonial ascidians and bryozoans, resulting mostly in stand-off interactions. Besides reducing asexual growth, spatial competition also affected female gonad production. Colonies free of competitors had a significantly higher proportion of zooids with ovaries. Thus, our findings show that spatial competition reduces both ascidian colony size and gonad production.

Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic beta-cell line

Chronic exposure of pancreatic beta-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to beta-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic beta-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to beta-cell incubations at 100 mu M, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Delta(3.5),Delta(2.4)-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P < 0.05]. Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects. Moreover, concomitant incubation of AA with PA dose-dependently attenuated the detrimental effects of the saturated fatty acid, so reducing apoptosis and decreasing parameters of oxidative stress [ROS (reactive oxygen species) and NO levels] while improving the GSH/GSSG ratio. AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-kappa B (nuclear factor kappa B) and the p47 subunit of NADPH oxidase in PA-treated cells. These findings indicate that AA has an important regulatory and protective beta-cell action, which may be beneficial to function and survival in the `lipotoxic` environment commonly associated with Type 2 diabetes mellitus.

Microbiological diversity of generalized aggressive periodontitis by 16S rRNA clonal analysis

Background/aim: The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with generalized aggressive periodontitis by using culture-independent molecular methods based on 16S ribosomal DNA cloning. Methods: Samples from 10 subjects with generalized aggressive periodontitis were selected. DNA was extracted and the 16S rRNA gene was amplified with the universal primer pairs 9F and 1525R. Amplified genes were cloned, sequenced, and identified by comparison with known 16S rRNA sequences. Results: One hundred and ten species were identified from 10 subjects and 1007 clones were sequenced. Of these, 70 species were most prevalent. Fifty-seven percent of the clone (40 taxa) sequences represented phylotypes for which no cultivated isolates have been reported. Several species of Selenomonas and Streptococcus were found at high prevalence and proportion in all subjects. Overall, 50% of the clone libraries were formed by these two genera. Selenomonas sputigena, the species most commonly detected, was found in nine of 10 subjects. Other species of Selenomonas were often present at high levels, including S. noxia, Selenomonas sp. EW084, Selenomonas sp. EW076, Selenomonas FT050, Selenomonas sp. P2PA_80, and Selenomonas sp. strain GAA14. The classical putative periodontal pathogens, such as, Aggregatibacter actinomycetemcomitans, was below the limit of detection and was not detected. Conclusion: These data suggest that other species, notably species of Selenomonas, may be associated with disease in generalized aggressive periodontitis subjects.

Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis

Background and Objective: This study evaluated the prevalence and the molecular diversity of Archaea in the subgingival biofilm samples of subjects with peri-implantitis. Material and Methods: Fifty subjects were assigned into two groups: Control (n = 25), consisting of subjects with healthy implants; and Test (n = 25), consisting of subjects with peri-implantitis sites, as well as a healthy implant. In the Test group, subgingival biofilm samples were taken from the deepest sites of the diseased implant. In both groups, subgingival biofilm was collected from one site with a healthy implant and from one site with a periodontally healthy tooth. DNA was extracted and the 16S ribosomal RNA gene was amplified with universal primer pairs for Archaea. Amplified genes were cloned and sequenced, and the phylotypes were identified by comparison with known 16S ribosomal RNA sequences. Results: In the Control group, Archaea were detected in two and three sites of the implant and the tooth, respectively. In the Test group, Archaea were detected in 12, 4 and 2 sites of diseased implants, healthy implants and teeth, respectively. Diseased implants presented a significantly higher prevalence of Archaea in comparison with healthy implants and natural teeth, irrespective of group. Over 90% of the clone libraries were formed by Methanobrevibacter oralis, which was detected in both groups. Methanobacterium congelense/curvum was detected in four subjects from the Test group and in two subjects from the Control group. Conclusion: Although M. oralis was the main species of Archaea associated with both healthy and diseased implant sites, the data indicated an increased prevalence of Archaea in peri-implantitis sites, and their role in pathogenesis should be further investigated.

OZJ : uma ferramenta para geração de óraculos para teste de software a partir de especificação formal

A literatura sobre Teste de Software apresenta diversas estratégias e metodologias que definem critérios eficazes e automatizáveis para selecionar casos de teste capazes de detectar erros em softwares. Embora eficientes na descoberta de erros, as técnicas de seleção de casos de teste exigem que uma quantidade relativamente grande de testes seja realizada para satisfazer os seus critérios. Essa característica acarreta, em parte, um alto custo na atividade de teste, uma vez que, ao fim de cada teste deve-se verificar se o comportamento do software está ou não de acordo com os seus requisitos. Oráculo para teste de software é um mecanismo capaz de determinar se o resultado de um teste está ou não de acordo com os valores esperados. Freqüentemente, assume-se que o próprio projetista de teste é o responsável por esta tarefa. A automatização da atividade dos oráculos deu origem a oráculos automáticos, os quais são capazes de determinar o bom ou mau funcionamento do software a partir de uma fonte de informação confiável. Ao longo dos anos, a especificação formal vêm sendo largamente utilizada como fonte de informação para oráculos automáticos. Diversas estratégias vêm propondo geradores de oráculos baseados em especificações formais. Dentre as características marcantes dessas estratégias, cita-se aquelas que são aplicáveis a implementações derivadas a partir da estrutura da especificação e aquelas que geram oráculos a partir de técnicas específicas de seleção de casos. Essas características, entretanto, limitam a aplicação abrangente dos oráculos por restringi-los tanto a implementações derivadas diretamente de especificações como ao uso de técnicas específicas de seleção de casos de teste. Este trabalho apresenta um estudo sobre os geradores de oráculos para teste de software, identifica aspectos fundamentais que regem seu processo de construção e propõe uma estratégia que permite a geração de oráculos semi-automaticamente, mesmo para implementações não derivadas diretamente da estrutura da especificação. A estratégia proposta é, também, aplicável aos casos de teste derivados de qualquer técnica de seleção de casos de teste.

Impacto do gerenciamento de reclamações na confiança e lealdade do consumidor, no contexto de trocas relacionais de serviços : construção e teste de um modelo teórico

Esta tese tem como objetivo central investigar o impacto do gerenciamento de reclamações na confiança e lealdade do consumidor, em trocas de serviços relacionais. Para tanto, foi desenvolvido e testado um modelo teórico, que retrata os inter-relacionamentos entre avaliações específicas do processo de reclamação e construtos relacionais. Desta forma, o presente estudo relaciona três importantes correntes de pesquisa - teoria da justiça, gerenciamento de reclamações e estudos sobre confiança. De modo geral, os resultados obtidos através da Modelagem de Equações Estruturais confirmam 10 das 12 hipóteses estabelecidas a partir do modelo teórico proposto. Mais especificamente, os resultados indicam que as percepções de justiça distributiva, interpessoal e processual afetam significativamente a percepção global de justiça e o nível de satisfação do consumidor com o gerenciamento da reclamação. A confiança do consumidor após a reclamação é influenciada diretamente pelo nível de satisfa ção final alcançado e pelas experiências anteriores do consumidor com a empresa. Por fim, o grau de lealdade do consumidor é influenciado pela confiança do consumidor e pelo valor relacional.

Efeito do exercício aeróbio moderado em gestantes com sobrepeso avaliado através de teste de exercício submáximo : um ensaio clínico randomizado

Objetivo: Avaliar os efeitos de um programa de exercício aeróbio sobre o condicionamento cardiorrespiratório em gestantes hígidas, de baixo risco, com sobrepeso. Métodos: 92 mulheres gestantes com sobrepeso (índice de massa corporal 26-31kg/m2), idade ≥ 20 anos, idade gestacional ≤ 20 semanas, com ausência de diabetes e hipertensão, foram alocadas aleatoriamente para realizar exercício aeróbio três vezes por semana com uma hora de duração ou para realizar sessões de relaxamento no grupo controle. Foram realizados dois testes de exercício submáximo em esteira, utilizando protocolo de rampa na entrada do estudo e outro teste após 12 semanas. Resultados: Em teste de exercício submáximo 12 semanas após randomização, o consumo de oxigênio (VO2) no limiar anaeróbio aumentou 17% (± 3) no grupo intervenção enquanto reduziu 16% (± 3) no grupo controle, de modo que após 12 semanas de exercício ajustado através da análise de covariância pelo o VO2 no limiar na linha de base, idade gestacional e idade materna foi de 2,68ml/kg/min (IC 95% 1,32-4,03) maior, P = 0,002. Conclusão: Exercício aeróbio realizado em gestantes com sobrepeso produz um aumento no limiar anaeróbio, sobrepondo os efeitos negativos da gestação sobre o condicionamento cardiorrespiratório em mulheres com estilo de vida sedentário.