Comparison of the early stages of forced unfolding for fibronectin type III modules

The structural changes accompanying stretch-induced early unfolding events were investigated for the four type III fibronectin (FN-III) modules, FN-III7, FN-III8, FN-III9, and FN-III10 by using steered molecular dynamics. Simulations revealed that two main energy barriers, I and II, have to be overcome to initiate unraveling of FN-III's tertiary structure. In crossing the first barrier, the two opposing β-sheets of FN-III are rotated against each other such that the β-strands of both β-sheets align parallel to the force vector (aligned state). All further events in the unfolding pathway proceed from this intermediate state. A second energy barrier has to be overcome to break the first major cluster of hydrogen bonds between adjacent β-strands. Simulations revealed that the height of barrier I varied significantly among the four modules studied, being largest for FN-III7 and lowest for FN-III10, whereas the height of barrier II showed little variation. Key residues affecting the mechanical stability of FN-III modules were identified. These results suggest that FN-III modules can be prestretched into an intermediate state with only minor changes to their tertiary structures. FN-III10, for example, extends 12 Å from the native “twisted” to the intermediate aligned state, and an additional 10 Å from the aligned state to further unfolding where the first β-strand is peeled away. The implications of the existence of intermediate states regarding the elasticity of fibrillar fibers and the stretch-induced exposure of cryptic sites are discussed.

Forced expression of the tumor suppressor adenomatosis polyposis coli protein induces disordered cell migration in the intestinal epithelium.

Mutations of the human adenomatosis polyposis coli (APC) gene are associated with the development of familial as well as sporadic intestinal neoplasia. To examine the in vivo function of APC, 129/Sv embryonic stem (ES) cells were transfected with DNA encoding the wild-type human protein under the control of a promoter that is active in all four of the small intestine's principal epithelial lineages during their migration-associated differentiation. ES-APC cells were then introduced into C57BL/6-ROSA26 blastocysts. Analyses of adult B6-ROSA26<-->129/Sv-APC chimeric mice revealed that forced expression of APC results in markedly disordered cell migration. When compared with the effects of forced expression of E-cadherin, the data suggest that APC-catenin and E-cadherin-catenin complexes have opposing effects on intestinal epithelial cell movement/adhesiveness; augmentation of E-cadherin-beta-catenin complexes produces a highly ordered, "adhesive" migration, whereas augmentation of APC-beta-catenin complexes produces a disordered, nonadhesive migratory phenotype. We propose that APC mutations may promote tumorigenesis by increasing the relative activity of cadherin-catenin complexes, resulting in enhanced adhesiveness and functional anchorage of initiated cells within the intestinal crypt. Our studies also indicate that chimeric mice generated from B6-ROSA26 blastocysts and genetically manipulated ES cells should be useful for auditing gene function in the gastrointestinal tract and in other tissues.

Forced expression of tropomyosin 2 or 3 in v-Ki-ras-transformed fibroblasts results in distinct phenotypic effects.

Transformation of cells in tissue culture results in a variety of cellular changes including alterations in cell growth, adhesiveness, motility, morphology, and organization of the cytoskeleton. Morphological and cytoskeletal changes are perhaps the most readily apparent features of transformed cells. Although a number of studies have documented a decrease in the expression of specific tropomyosin (TM) isoforms in transformed cells, it remains to be determined if the suppression of TM synthesis is essential in the establishment and maintenance of the transformed pheno-type. To address the roles of different TM isoforms in transformed cells we have examined the effects of expressing specific TM isoforms in transformed cells using a Kirsten virus-transformed cell line (ATCC NRK1569) as a model system. In contrast to normal fibroblasts, the NRK 1569 cells contain reduced levels of TM-1 and undetectable levels of TM-2 and TM-3. These cells have a rounded morphology and are devoid of stress fibers. Employing expression plasmids for TM-2 and TM-3, stable cell lines were established from the NRK 1569 cells that express these isoforms individually. We demonstrate that expression of TM-2 or TM-3 leads to increased cell spreading accompanied by the formation of identifiable microfilament bundles, as well as significant restoration of well-defined vinculin-containing focal adhesion plaques, although expression of each isoform exhibited distinct properties. In addition, cells expressing TM-2, but not TM-3, exhibited contact-inhibited cell growth and a requirement for serum.

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.

Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards. We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols. Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection of Escherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 microM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in kcat and up to 6-fold increase in kcat/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme. The results suggest a general strategy for modification of drug- and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells.

Adaptação cultural e validação da Compliance with Standard Precautions Scale (CSPS) para enfermeiros brasileiros

Introdução: As precauções-padrão (PP) constituem um conjunto de medidas que têm como finalidade minimizar o risco de transmissão ocupacional de patógenos, sendo indispensável sua utilização por profissionais de saúde, sobretudo pelos enfermeiros. No entanto, a não adesão às PP constitui problemática amplamente discutida em todo o mundo. Embora haja diversos estudos brasileiros que visem avaliar a adesão às PP , ainda tem-se observado grande fragilidade no processo de construção e de validação dos instrumentos utilizados para avaliação deste construto. Objetivo: Realizar a adaptação cultural e validação da Compliance with Standard Precautions Scale (CSPS) para enfermeiros brasileiros. Metodologia: Trata-se de um estudo metodológico para a adaptação e validação da CSPS. Essa escala é composta por 20 itens com quatro opções de respostas, e destina-se a avaliar a adesão às PP. O processo de adaptação consistiu em Tradução, Consenso entre Juízes, Retrotradução e Validação Semântica. A primeira etapa foi a tradução do idioma original para o português do Brasil. Após foi realizado um comitê composto por sete juízes, a versão de consenso obtida na etapa anterior foi traduzida novamente para o idioma de origem. Foram avaliadas as propriedades psicométricas do instrumento, considerando-se as validades de face e de conteúdo, a validade de construto e a confiabilidade. A versão para o Português do Brasil da CSPS (CSPS-PB) foi aplicada em uma amostra de 300 enfermeiros que atuam na assistência a pacientes em um hospital localizado na cidade de São Paulo/SP. A confiabilidade foi avaliada por meio da consistência interna (alfa de Cronbach) e teste reteste (coeficiente de correlação intraclasse - ICC). Para a validação de construto, foi utilizada a comparação entre grupos diferentes, análise fatorial exploratória e análise fatorial confirmatória, segundo o Modelo de Equações Estruturais (SEM). Utilizou-se o software IBM® SPSS, 19.0. Para a análise fatorial confirmatória foi utilizado o módulo específico Analysis of Moment Structures (IBM® SPSS AMOS). Para a análise paralela utilizou-se o programa RanEigen Syntax. O nível de significância adotado foi ? = 0,05. Todos os aspectos éticos foram contemplados. Resultados: A tradução realizada por tradutores juramentados garantiu qualidade a esse processo. A validação de face e de conteúdo possibilitou a realização de modificações pertinentes e imperativas a fim de atender aos critérios de equivalências conceituais, idiomáticas, culturais e semânticas. Obteve-se ?=0,61 na avaliação da consistência interna, indicando confiabilidade satisfatória. O ICC indicou uma correlação de 0,87 quase perfeita para o teste reteste duas semanas após a primeira abordagem, conferindo estabilidade satisfatória. A validade de construto mostrou que a CSPS-PB foi capaz de discriminar as médias de adesão às PP entre grupos distintos referente à idade (F=5,15 p<=0,01), ao tempo de experiência clínica (F = 8,9 p<= 0,000) e a ter recebido treinamento (t = 2,48 p<=0,01). Na análise fatorial confirmatória, o modelo foi subidentificado. A análise fatorial exploratória indicou que todos os itens apresentaram cargas fatoriais adequadas (>=0,30), sendo identificados quatro fatores pela análise paralela. O total de variância explicada foi de 35,48%. Conclusão: A CSPS-PB, trata-se de um instrumento adequado, confiável e válido para medir a adesão às PP entre enfermeiros brasileiros

Desfechos tardios de sobreviventes de ensaio clínico randomizado controlado (protocolo ARDSnet vs. Open Lung Approach para o manejo ventilatório da síndrome do desconforto respiratório agudo moderado-grave)

Apesar da utilização da ventilação mecânica protetora como estratégia para o tratamento da síndrome do desconforto respiratório agudo, ao menos um quarto dos pacientes com essa síndrome ainda apresentam redução na função pulmonar após 6 meses de seguimento. Não se sabe se esta redução está relacionada com a gravidade da síndrome ou associada com a forma de ventilar o paciente. Nosso objetivo neste trabalho foi avaliar a associação entre alterações funcionais e estruturais do pulmão com parâmetros de gravidade clínica e de ventilação mecânica. Foi realizada uma análise secundária dos dados obtidos em estudo randomizado e controlado que incluiu pacientes com síndrome do desconforto respiratório agudo moderada/grave, internados em seis unidades de terapia intensiva em um hospital terciário da cidade de São Paulo. Foram analisados dados de pacientes que tinham ao menos um teste de função pulmonar no seguimento. O teste funcional incluiu a medida da capacidade vital forçada, volumes pulmonares e a capacidade de difusão do monóxido de carbono após 1, 2 e 6 meses de seguimento. Foram considerados variáveis independentes o volume corrente, a pressão de distensão e a pressão positiva ao final da expiração (todos medidos após 24 horas da randomização) e um sistema de classificação de prognóstico (APACHE II), a relação PaO2/FIO2 e a complacência respiratória estática (todos medidos antes da randomização). Também foi realizada tomografia de alta resolução do tórax juntamente com os testes de função pulmonar, e posterior análise quantitativa das imagens. Na avaliação de 6 meses também foi realizado teste de caminhada de 6 minutos e um questionário de qualidade de vida (SF-36). Um total de 21 pacientes realizaram o teste de função pulmonar após 1 mês e 15 pacientes realizaram após 2 e 6 meses de seguimento. A capacidade vital forçada foi relacionada inversamente com a pressão de distensão na avaliação de 1, 2 e 6 meses (p < 0,01). A capacidade de difusão do monóxido de carbono relacionou-se inversamente com a pressão de distensão e com o APACHE II (ambos p < 0,01) na avaliação de 1 e 2 meses. Após 6 meses de seguimento, houve correlação inversa entre a pressão de distensão e a capacidade vital forçada independente do volume corrente, da pressão de platô e da complacência estática respiratória após ajustes (R2 = 0,51, p = 0,02). A pressão de distensão também se relacionou com o volume pulmonar total, a densidade pulmonar media e a porcentagem de volume pulmonar não aerado ou pobremente aerado medidos através da análise quantitativa da tomografia computadorizada de tórax realizada na avaliação de 6 meses. Também foi observada relação entre a qualidade de vida após 6 meses de seguimento e a pressão de distensão considerando o domínio estado geral de saúde. Nós concluímos que mesmo em pacientes ventilados com reduzido volume corrente e pressão de platô limitada, maiores valores de pressão de distensão relacionaram-se com menores valores de função pulmonar no seguimento de longo prazo

Improving the Success of Wetland Mitigation Banking: Lessons Learned in Monitoring and Compliance

Wetlands that are lost to development are not effectively compensated by the current wetland mitigation banking regulatory program due to inadequate monitoring and compliance. Based on a critical investigation of two wetland mitigation banks in Colorado described herein, recommendations are given to improve the effectiveness of the wetland mitigation banking program. The recommendations to improve mitigation banking are to specify and follow comprehensive monitoring and reporting plans, develop solid contingency and adaptive management plans, utilize specially developed checklists and templates, and impose enforcement when compliance is not met. Implementing these recommendations will assist regulators and bankers in achieving more effective wetland mitigation and will help the United States reach its no net loss of wetlands goal.

Compliance Strategies for Gasoline Distribution Facilities

Gasoline Distribution Generally Available Control Technology (GD GACT) is a Federal environmental regulation that is specifically written and enforced to reduce HAP emissions from gasoline distribution (GD) facilities. The regulation targets four specific types of GD facilities: bulk gasoline terminals, bulk gasoline plants, pipeline breakout stations, and pipeline pumping stations. A GD GACT compliance plan was developed for a particular, representative example of each type of GD facility affected by the regulation. Each facility in the study is owned and operated by a single company. The compliance plans were developed to meet the regulatory requirements contained within GD GACT. The compliance plans will be implemented at each facility prior to the January 10, 2011 compliance date.

Surface and Groundwater Environmental Policy Compliance at the Adams County Regional Park and Fairgrounds

The Adams County Regional Park and Fairgrounds must comply with environmental policies related to surface water and groundwater protection. This paper assesses various methods which have proven to be effective in the reduction of nutrients and other contaminants found in surface and groundwater at comparable livestock-based venues. Data was gathered from other facilities in order to identify specific compliance alternatives and evaluate management options. Empirical research, coupled with GIS mapping technology yielded explicit water quality management recommendations for the Adams County Regional Park and Fairgrounds. The outcome of this research and mapping exercise include twelve management recommendations and two site-specific locations for structural BMPs designed to better control water pollution at the Adams County Regional Park and Fairgrounds.

A Regulatory Compliance Plan for Decommissioning a Sodium Phosphate Manufacturing Plant

Sodium phosphates are a class of chemicals that have been widely employed in commercial and consumer applications. However, declining use of these chemicals due to environmental concerns has lead to restructuring within the industry that has caused, and is likely to continue to cause, reduction of sodium phosphate production capacity. Closure of a sodium phosphate manufacturing plant necessitates decommissioning and decontamination activities that are subject to a variety of federal, state, and local regulations. A compliance plan was developed to provide a blueprint for ensuring that all federal regulatory requirements are met, however, site dependent state and local requirements were excluded. The compliance plan provides a framework that addresses project team formation and project planning, regulatory requirements, identification of affected processing equipment, plant pre-shutdown activities, waste stream identification and waste management facilities, safety, training, and emergency preparedness planning, and project decommissioning remedial actions. This regulatory compliance plan will enable sodium phosphate plant operators to complete decontamination and decommissioning work in a timely, efficient, compliant, and cost effective manner.

Clinical pathway intervention compliance and effectiveness when used in the treatment of patients with severe sepsis and septic shock at an Intensive Care Unit in Spain

The purpose of this quasi-experimental study was to assess levels of compliance with the intervention bundles contained in a clinical pathway used in the treatment of patients with severe sepsis and septic shock, and to analyze the pathway’s impact on survival and duration of hospital stays. We used data on 125 patients in an Intensive Care Unit, divided into a control group (N=84) and an intervention group (N=41). Levels of compliance increased from 13.1% to 29.3% in 5 resuscitation bundle interventions and from 14.3% to 22% in 3 monitoring bundle interventions. In-hospital mortality at 28 days decreased by 11.2% and the duration of hospital stay was reduced by 5 days. Although compliance was low, the intervention enhanced adherence to the instructions given in the clinical pathway and we observed a decline in mortality at 28 days and shorter hospital stays.

Accurate integration of forced and damped oscillators

The new methods accurately integrate forced and damped oscillators. A family of analytical functions is introduced known as T-functions which are dependent on three parameters. The solution is expressed as a series of T-functions calculating their coefficients by means of recurrences which involve the perturbation function. In the T-functions series method the perturbation parameter is the factor in the local truncation error. Furthermore, this method is zero-stable and convergent. An application of this method is exposed to resolve a physic IVP, modeled by means of forced and damped oscillators. The good behavior and precision of the methods, is evidenced by contrasting the results with other-reputed algorithms implemented in MAPLE.