PADRONIZAÇÃO DA PCR-RFLP PARA A GENOTIPAGEM DOS ALELOS CYP2E1 *1A/*5B A PARTIR DE DNA OBTIDO DA MUCOSA ORAL

O metabolismo de xenobióticos é desempenhado por enzimas da família do citocromo P450; entre elas, destaca-se a enzima CYP2E1, que é responsável pelo metabolismo de compostos hidrofílicos de baixo peso molecular, nitrosaminas, benzeno, hidrocarbonetos policíclicos aromáticos, entre outros. Esse gene possui dois alelos, nos quais a presença do alelo tipo mutante (5B) demonstra maior taxa de transcrição e atividade enzimática, causando assim, um desequilíbrio de subprodutos gerados para a fase II do metabolismo. Este trabalho teve como objetivo padronizar a análise dos alelos *1A e *5B do gene CYP2E1 no Laboratório de Doenças Infecciosas e Parasitárias da Unoesc, com o estabelecimento de um protocolo padrão para a extração de DNA em amostras de mucosa oral e análise de RFLP (Analise do polimorfismo pelo tamanho de fragmentos gerados por restrição enzimática) para a determinação do genótipo das amostras. Para tal, amostras da mucosa oral de 20 voluntários foram coletadas e a extração realizada pelo método de salting-out. Para a amplificação do fragmento, foram utilizados 10 pmol de cada iniciador (CYPE2F 5'-CCAGTC GAG TCT ACA TTG TCA-3' e CYPE2R 5'-TTC ATT CTG TCT TCT AAC TGG-3'), 5 U de Taq DNA Polimerase, tampão de reação da enzima (10 mM Tris HCl, pH 8,5; 50 mM KCl) e 1,5 mM de MgCl2 e 2 mM de dNTP e aproximadamente 100 ng de DNA. As condições da PCR foram de 35 ciclos a 92 °C por um minuto, 60 °C por um minuto, e 72 °C por um minuto.  Após a amplificação, 10 μl do produto da PCR foi submetido à digestão durante 12 horas a 37 °C com a enzima de restrição PstI, 90 mM Tris HCl (pH7,5), 50 mM NaCl, 10 mM MgCl2, 10mM DTT, 10ug/μl de BSA e 5 unidades da enzima, seguidas da inativação a 65 °C por 15 minutos. Para a determinação do genótipo do gene CYP2E1 nos indivíduos, é realizada a partir da análise do perfil de restrição obtido, onde a ausência da sítio de restrição para a enzima PstI, banda de 410 pares de base (pb), indica alelo tipo selvagem clivagem (*1A), já no caso da presença do sítio de restrição desta enzima, resultara na clivagem do amplicon em dois fragmentos, 290 pb e 120 pb, demonstrando a presença do alelo tipo mutante (*5B). Desta forma, com base nesta metodologia, podemos realizar a análise da frequência dos alelos *1A e *5B do gene CYP2E1 em trabalhadores expostos a xenobióticos, podendo ser utilizado como marcadores de susceptibilidade ao desenvolvimento de neoplasias relacionado à cabeça e pescoço.Palavras-chave: Citocromo; PCR; Xenobiótico.CONTATO: [email protected]

Protective effects of diet containing rutin against trichlorfon-induced muscle bioenergetics disruption and impairment on fatty acid profile of silver catfish Rhamdia quelen

Trichlorfon is an organophosphate insecticide that is widely used on fish farms to control parasitic infections. It has been detected in freshwater ecosystems as well as in fishery products. There is a growing body of evidence to suggest that certain feed additives may reduce or prevent pesticide-induced toxicity in fish. The aim of the present study was to determine whether acute exposure to trichlorfon would alter bioenergetic homeostasis and alter fatty acid profiles in muscles of silver catfish (Rhamdia quelen). We also sought to determine whether rutin prevents or reduces these effects. Cytosolic and mitochondrial creatine kinase (CK) and activities of complexes II-III and IV in muscle were significantly inhibited by exposure to 11 mg/L trichlorfon for 48 h compared to effects in the unexposed group. Total content of polyunsaturated fatty acids (omega-3 and omega-6) were significantly lower in muscle of silver catfish exposed to 11 mg/L trichlorfon for 48 h than in the unexposed group. Addition of 3 mg rutin/kg feed increased CK activity and prevented inhibition of complex IV activity, as well as preventing all alterations of muscle fatty acid profiles elicited by exposure to trichlorfon. No significant differences were observed between groups with respect to muscle adenylate kinase or pyruvate kinase activities, as well as total content of saturated and monounsaturated fatty acids. Our findings suggest that exposure (48 h) to 11 mg trichlorfon/L water inhibits cytosolic and mitochondrial CK activity in muscle. Trichlorfon also affects activities of complexes II-III and IV in respiratory chain, with important consequences for adenosine triphosphate production. The pesticide alters fatty acid profiles in the fish and endangers human consumers of the product. The most important finding of the present study is that inclusion of rutin improves bioenergetic homeostasis and muscle fatty acid profiles, suggesting that it reduces trichlorfon-induced muscle damage

Anesthesia of Epinephelus marginatus with essential oil of Aloysia polystachya: an approach on blood parameters

This study investigated the anesthetic potential of the essential oil (EO) of Aloysia polystachya in juveniles of dusky grouper (Epinephelus marginatus). Fish were exposed to different concentrations of EO of A. polystachya to evaluate time of induction and recovery from anesthesia. In the second experiment, fish were divided into four groups: control, ethanol and 50 or 300 mu L L-1 EO of A. polystachya, and each group was submitted to induction for 3.5 min and recovery for 5 or 10 min. The blood gases and glucose levels showed alterations as a function of the recovery times, but Na+ and K+ levels did not show any alteration. In conclusion, the EO from leaves of A. polystachya is an effective anesthetic for dusky grouper, because anesthesia was reached within the recommended time at EO concentrations of 300 and 400 mu L L-1. However, most evaluated blood parameters showed compensatory responses due to EO exposure.Fundacao de Amparo a Pesquisa do Estado do Rio Grande do Sul/Programa de Apoio a Nucleos de Excelencia (FAPERGS/PRONEX) [10/0016-8]; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [470964/2009-0]; Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior, Brazil (CAPES)info:eu-repo/semantics/publishedVersio

A cluster randomized trial to improve adherence to evidence-based guidelines on diabetes and reduce clinical inertia in primary care physicians in Belgium: study protocol [NTR 1369]

Contains fulltext : 70617.pdf (publisher's version ) (Open Access)ABSTRACT: BACKGROUND: Most quality improvement programs in diabetes care incorporate aspects of clinician education, performance feedback, patient education, care management, and diabetes care teams to support primary care physicians. Few studies have applied all of these dimensions to address clinical inertia. AIM: To evaluate interventions to improve adherence to evidence-based guidelines for diabetes and reduce clinical inertia in primary care physicians. DESIGN: Two-arm cluster randomized controlled trial. PARTICIPANTS: Primary care physicians in Belgium. INTERVENTIONS: Primary care physicians will be randomly allocated to 'Usual' (UQIP) or 'Advanced' (AQIP) Quality Improvement Programs. Physicians in the UQIP will receive interventions addressing the main physician, patient, and office system factors that contribute to clinical inertia. Physicians in the AQIP will receive additional interventions that focus on sustainable behavior changes in patients and providers. OUTCOMES: Primary endpoints are the proportions of patients within targets for three clinical outcomes: 1) glycosylated hemoglobin < 7%; 2) systolic blood pressure differences </=130 mmHg; and 3) low density lipoprotein/cholesterol < 100 mg/dl. Secondary endpoints are individual improvements in 12 validated parameters: glycosylated hemoglobin, low and high density lipoprotein/cholesterol, total cholesterol, systolic blood pressure, diastolic blood pressure, weight, physical exercise, healthy diet, smoking status, and statin and anti-platelet therapy. PRIMARY AND SECONDARY ANALYSIS: Statistical analyses will be performed using an intent-to-treat approach with a multilevel model. Linear and generalized linear mixed models will be used to account for the clustered nature of the data, i.e., patients clustered withinimary care physicians, and repeated assessments clustered within patients. To compare patient characteristics at baseline and between the intervention arms, the generalized estimating equations (GEE) approach will be used, taking the clustered nature of the data within physicians into account. We will also use the GEE approach to test for differences in evolution of the primary and secondary endpoints for all patients, and for patients in the two interventions arms, accounting for within-patient clustering. TRIAL REGISTRATION: number: NTR 1369

Interdisciplinary diabetes care teams operating on the interface between primary and specialty care are associated with improved outcomes of care: findings from the Leuven Diabetes Project

<p>Abstract</p> <p>Background</p> <p>Type 2 diabetes mellitus is a complex, progressive disease which requires a variety of quality improvement strategies. Limited information is available on the feasibility and effectiveness of interdisciplinary diabetes care teams (IDCT) operating on the interface between primary and specialty care. A first study hypothesis was that the implementation of an IDCT is feasible in a health care setting with limited tradition in shared care. A second hypothesis was that patients who make use of an IDCT would have significantly better outcomes compared to non-users of the IDCT after an 18-month intervention period. A third hypothesis was that patients who used the IDCT in an Advanced quality Improvement Program (AQIP) would have significantly better outcomes compared to users of a Usual Quality Improvement Program (UQIP).</p> <p>Methods</p> <p>This investigation comprised a two-arm cluster randomized trial conducted in a primary care setting in Belgium. Primary care physicians (PCPs, n = 120) and their patients with type 2 diabetes mellitus (n = 2495) were included and subjects were randomly assigned to the intervention arms. The IDCT acted as a cornerstone to both the intervention arms, but the number, type and intensity of IDCT related interventions varied depending upon the intervention arm.</p> <p>Results</p> <p>Final registration included 67 PCPs and 1577 patients in the AQIP and 53 PCPs and 918 patients in the UQIP. 84% of the PCPs made use of the IDCT. The expected participation rate in patients (30%) was not attained, with 12,5% of the patients using the IDCT. When comparing users and non-users of the IDCT (irrespective of the intervention arm) and after 18 months of intervention the use of the IDCT was significantly associated with improvements in HbA1c, LDL-cholesterol, an increase in statins and anti-platelet therapy as well as the number of targets that were reached. When comparing users of the IDCT in the two intervention arms no significant differences were noted, except for anti-platelet therapy.</p> <p>Conclusion</p> <p>IDCT's operating on the interface between primary and specialty care are associated with improved outcomes of care. More research is required on what team and program characteristics contribute to improvements in diabetes care.</p> <p>Trial registration</p> <p>NTR 1369.</p

Integrin α8β1 regulates adhesion, migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK-dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine-glycine-aspartate tripeptide motif)-dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal-derived cell types. Its role in epithelial cells remains unknown

Barriers and facilitators to evidence based care of type 2 diabetes patients: experiences of general practitioners participating to a quality improvement program

Objective To evaluate the barriers and facilitators to high-quality diabetes care as experienced by general practitioners (GPs) who participated in an 18-month quality improvement program (QIP). This QIP was implemented to promote compliance with international guidelines. Methods Twenty out of the 120 participating GPs in the QIP underwent semi-structured interviews that focused on three questions: 'Which changes did you implement or did you observe in the quality of diabetes care during your participation in the QIP?' 'According to your experience, what induced these changes?' and 'What difficulties did you experience in making the changes?' Results Most GPs reported that enhanced knowledge, improved motivation, and a greater sense of responsibility were the key factors that led to greater compliance with diabetes care guidelines and consequent improvements in diabetes care. Other factors were improved communication with patients and consulting specialists and reliance on diabetes nurse educators. Some GPs were reluctant to collaborate with specialists, and especially with diabetes educators and dieticians. Others blamed poor compliance with the guidelines on lack of time. Most interviewees reported that a considerable minority of patients were unwilling to change their lifestyles. Conclusion Qualitative research nested in an experimental trial may clarify the improvements that a QIP may bring about in a general practice, provide insight into GPs' approach to diabetes care and reveal the program's limits. Implementation of a QIP encounters an array of cognitive, motivational, and relational obstacles that are embedded in a patient-healthcare provider relationshipGeert Goderis, Liesbeth Borgermans, Chantal Mathieu, Carine Van Den Broeke, Karen Hannes, Jan Heyrman and Richard Gro

The effect of infliximab plus methotrexate on the modulation of inflammatory disease markers in juvenile idiopathic arthritis: analyses from a randomized, placebo-controlled trial

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