RESUMEN
We present a novel centrifugal microfluidic approach for fast and accurate tuberculosis (TB) diagnosis based on the use of standard laboratory equipment. The herein presented workflow can directly be integrated into laboratories with standard equipment and automates complex sample preparation. The system consists of a microfluidic cartridge, a laboratory centrifuge and a standard PCR cycler. The cartridge includes all required reagents and automates collection of bacteria on filter membranes, bacterial lysis, nucleic acid extraction and aliquoting of the DNA extract for PCR analysis. We show that storage of the reagents in aluminium-coated pouches is stable during accelerated storage and transport tests. When the limit of detection was assessed, we found that the cartridge-automated workflow consistently detected 10 CFU ml-1 of mycobacteria in spiked sputum samples. First tests with clinical samples showed a 100% specificity for non-TB specimens. In addition, Mycobacterium tuberculosis (MTB) was re-found in pre-characterized smear microscopy and culture positive sputum samples suggesting a high diagnostic sensitvity. In summary, the novel cartridge-automated workflow enables a flexible and sensitive TB diagnosis without the need to invest in specialized instrumentation.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Laboratorios , Microfluídica , Mycobacterium tuberculosis/genética , Sensibilidad y Especificidad , Esputo , Tuberculosis/diagnósticoRESUMEN
PURPOSE: Elucidation of the molecular mechanism of radiation-induced activation of src kinase, which initiates EGFR internalization and nuclear transport. MATERIAL AND METHODS: Radiation-induced src activation was investigated in the bronchial carcinoma cell line A549. Proteins were Western blotted and quantified by the help of specific antibodies. Residual DNA-damage was quantified with gammaH(2)AX-foci analysis. Radiation-induced lipid peroxidation was prevented by acetyl-cysteine. RESULTS: The radiation-induced src activation and EGFR stabilization could be mimicked by addition of hydroxy-nonenal (HNE), one of the major lipid peroxidation products. Radiation-generated HNE is bound to EGFR and src and correlated with complex formation between both following radiation. Treatment with HNE activated src and stimulated radiation-associated EGFR and caveolin 1 phosphorylations resulting in increased nuclear transport of EGFR. Consequently, radiation-induced phosphorylation and activation of DNA-PK were increased. This phosphorylation was associated with improved removal of residual damage 24h after irradiation. Inhibition of radiation-induced HNE generation by acetyl-cysteine blocked radiation-induced src activation and EGFR phosphorylation. CONCLUSIONS: HNE generated in response to radiation exposure activates src kinase and is involved in regulation of radiation-stimulated DNA-repair processes.