02739nas a2200229   4500008004100000022001400041245013200055210006900187260007300256300001200329490000700341520187900348653001402227653003102241653002102272653000902293100001902302700001902321700002102340700001902361856012902380       2011                        eng d  a0167-701200aNovel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents0 aNovel screening method based on menadione mediated rapid reducti  aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcFEB  a202-2070 v843 a<p>A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 mu M of XTT and 60 mu M of menadione in 250 mu l of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG. M. tuberculosis and M. smegmatis respectively. The calculated Z' factors were &gt; 0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether. XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli. (C) 2010 Elsevier B.V. All rights reserved.</p>10aMenadione10aMycobacterium tuberculosis10aTetrazolium salt10aXRMA1 aSingh, Upasana1 aAkhtar, Shamim1 aMishra, Abhishek1 aSarkar, Dhiman  uhttp://library.ncl.res.in/content/novel-screening-method-based-menadione-mediated-rapid-reduction-tetrazolium-salt-testing-002421nas a2200193   4500008004100000022001400041245012200055210006900177260007600246300001400322490000800336520165600344100001902000700001702019700002602036700002702062700001902089856011902108       2013                        eng d  a0021-919300aNitrite reductase NirBD is induced and plays an important role during in vitro dormancy of mycobacterium tuberculosis0 aNitrite reductase NirBD is induced and plays an important role d  a1752 N ST NW, WASHINGTON, DC 20036-2904 USAbAMER SOC MICROBIOLOGYcOCT  a4592-45990 v1953 a<p>Mycobacterium tuberculosis is one of the strongest reducers of nitrate among all mycobacteria. Reduction of nitrate to nitrite, mediated by nitrate reductase (NarGHJI) of M. tuberculosis, is induced during the dormant stage, and the enzyme has a respiratory function in the absence of oxygen. Nitrite reductase (NirBD) is also functional during aerobic growth when nitrite is the sole nitrogen source. However, the role of NirBD-mediated nitrite reduction during the dormancy is not yet characterized. Here, we analyzed nitrite reduction during aerobic growth as well as in a hypoxic dormancy model of M. tuberculosis in vitro. When nitrite was used as the sole nitrogen source in the medium, the organism grew and the reduction of nitrite was evident in both hypoxic and aerobic cultures of M. tuberculosis. Remarkably, the hypoxic culture of M. tuberculosis, compared to the aerobic culture, showed 32- and 4-fold-increased expression of nitrite reductase (NirBD) at the transcription and protein levels, respectively. More importantly, a nirBD mutant of M. tuberculosis was unable to reduce nitrite and compared to the wild-type (WT) strain had a &gt;2-log reduction in viability after 240 h in the Wayne model of hypoxic dormancy. Dependence of M. tuberculosis on nitrite reductase (NirBD) was also seen in a human macrophage-based dormancy model where the nirBD mutant was impaired for survival compared to the WT strain. Overall, the increased expression and essentiality of nitrite reductase in the in vitro dormancy models suggested that NirBD-mediated nitrite reduction could be critical during the persistent stage of M. tuberculosis.</p>1 aAkhtar, Shamim1 aKhan, Arshad1 aSohaskey, Charles, D.1 aJagannath, Chinnaswamy1 aSarkar, Dhiman  uhttp://library.ncl.res.in/content/nitrite-reductase-nirbd-induced-and-plays-important-role-during-vitro-dormancy-0