Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.

Avaliação da concentração de monóxido de carbono no ar exalado em tabagistas com DPOC / Evaluation of the exhaled carbon monoxide levels in smokers with COPD

OBJETIVO: Medir os níveis de monóxido de carbono no ar exalado (COex) em tabagistas com e sem DPOC. MÉTODOS: Tabagistas frequentadores dos ambulatórios do Hospital São Lucas em Porto Alegre (RS) entre setembro de 2007 e março de 2009 foram convidados a participar do estudo. Os participantes responderam a um questionário com características demográficas e epidemiológicas e realizaram espirometria, medição de cotinina urinária e de COex. Os participantes foram agrupados conforme a presença de DPOC. RESULTADOS: Foram incluídos 294 tabagistas, 174 (59,18 por cento) diagnosticados com DPOC. Todos os participantes apresentavam níveis de cotinina urinária > 50 ng/mL. Os fumantes com DPOC apresentaram medianas significativamente superiores as do grupo sem DPOC para as variáveis idade e maços-ano (p < 0,001 e p = 0,026, respectivamente). Não houve diferença significativa nas demais variáveis. Quando ajustados para sexo, início do tabagismo, cigarros/dia e cotinina urinária, os valores médios de COex foram mais altos no grupo DPOC que no grupo sem DPOC, mas sem significância estatística (17,8 ± 0,6 ppm e 16,6 ± 0,7 ppm, respectivamente; p = 0,200). As diferenças permaneceram não significativas quando o método de base logarítmica foi usado. Uma ampla dispersão dos valores de COex foi encontrada quando os participantes foram classificados conforme os valores de VEF1 (r = -0,06; p = 0,53) ou o sistema de classificação de Global Initiative for Chronic Obstructive Lung Disease (r = 0,08; p = 0,34). As proporções de resultados falso-negativos para tabagismo foram de 18,4 por cento e 6,7 por cento, respectivamente, nos grupos com e sem DPOC (p = 0,007). CONCLUÕES: Esse estudo mostrou que os valores de COex não apresentaram diferenças significativas em fumantes com ou sem DPOC. Desse modo, parece não haver nenhuma restrição relevante para a sua aplicabilidade em fumantes com DPOC.

OBJECTIVE: To measure exhaled carbon monoxide (COex) levels in smokers with and without COPD. METHODS: Smokers treated at outpatient clinics of São Lucas Hospital in the city of Porto Alegre, Brazil, between September of 2007 and March of 2009 were invited to participate in this study. The participants completed a questionnaire regarding demographic and epidemiologic characteristics and were submitted to spirometry, as well as to determination of COex and urinary cotinine levels. The participants were divided into two groups: those with COPD and those without COPD. RESULTS: The study involved 294 smokers, of whom 174 (59.18 percent) had been diagnosed with COPD. All of the participants presented with urinary cotinine levels > 50 ng/mL. Smokers with COPD presented significantly higher median values for age and pack-years than did those without COPD (p < 0.001 and p = 0.026, respectively). No other statistically significant differences were found. When adjusted for gender, age at smoking onset, number of cigarettes/day and urinary cotinine level, the mean values of COex were higher, but not statistically so, in the COPD group than in the non-COPD group (17.8 ± 0.6 ppm and 16.6 ± 0.7 ppm, respectively; p = 0.200). The differences remained nonsignificant when plotted logarithmically. A wide dispersion of COex values was found when the participants were classified by FEV1 level (r = -0.06; p = 0.53) or by Global Initiative for Chronic Obstructive Lung Disease classification (r = 0.08; p = 0.34). The proportions of false-negative results for smoking were 18.4 percent and 6.7 percent, respectively, in the COPD and non-COPD groups (p = 0.007). CONCLUSIONS: Since COex values did not differ significantly between smokers with COPD and those without, there seem to be no major contraindications to their use in smokers with COPD.

Validação do método para determinação de cotinina em urina por cromatografia líquida de alta eficiência / Validation of a high-performance liquid chromatography method for the determination of cotinine in urine

Tobacco dependence reaches nearly one third of the world population, and is the second leading cause of deth around the world. Cotinine, a major metabolite of nicotine, is conssidered the most appropriate parameter to evaluate tobacco exposure HLPC (High Performance Liquid Chromatography) was choses to analyze cotinine concentrations in urine. Aiming at validating this method of analysis, urine samples with added cotinine were submitted to liquid-liquid extractions using2-phenylimidazole as the internal standard. After evaporation, residue was injected in HLPC with ultraviolet detection and C8 column. The following parameters were stabilishe: linearity, precision, detection and qualification limit, accuracy, ans stability. The calibration curve, determined ian a interval of 10ng/mL and 1000ng/mL, presented linearity (R2 = 0,09979). The method limit of detection was of 5ng/mL, and the limit of quantification was of 10ng/mL. In 50, 500, and 1000ng/mL concentrations, the precision was of 11.2; 12.3, and 8.1 per cent, accuracy found was of 107.0; 101.7, and 97.0 per cent, and loss during the stability test was of 8.1...