Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence.

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E-MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E-MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E-MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, Mtb Delta whiB3 displayed intramacrophage survival defect, which can be rescued by pharmacological inhibition of phagosomal acidification. Last, Mtb Delta whiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.

Voltammetric determination of paracetamol, tramadol and caffeine using poly(Nile blue) modified glassy carbon electrode

A poly(Nile blue) modified glassy carbon electrode (PNBMGCE) was fabricated by electropolymerisation of Nile blue (NB) monomer using cyclic voltammetry (CV) and was used for the determination of paracetamol (ACOP), tramadol (TRA) and caffeine (CAF). The electrochemical investigations showed that PNB - film formed on the surface of glassy carbon electrode (GCE) improved the electroactive surface area and displayed a remarkable increase in the peak current and a substantial decrease in over potential of ACOP, TRA and CAF when compared to bare GCE. The dependence of peak current and potential on pH, sweep rate and concentration were also investigated at the surface of PNBMGCE. It showed good sensitivity and selectivity in a wide linear range from 2.0 x 10(-7) to 1.62 x 10(-5) M, 1.0 x 10(-6) to 3.1 x 10(-4) M and 8.0 x 10(-7) to 2.0 x 10(-5) M, with detection limits of 0.08, 0.5 and 0.1 mu M, for ACOP, TRA and CAF, respectively. The PNBMGCE was also successfully applied for the determination of ACOP, TRA and CAF in pharmaceutical dosage forms. (C) 2016 Elsevier B.V. All rights reserved.

Global Response Sensitivity Analysis of Randomly Excited Dynamic Structures

The response of structural dynamical systems excited by multiple random excitations is considered. Two new procedures for evaluating global response sensitivity measures with respect to the excitation components are proposed. The first procedure is valid for stationary response of linear systems under stationary random excitations and is based on the notion of Hellinger's metric of distance between two power spectral density functions. The second procedure is more generally valid and is based on the l2 norm based distance measure between two probability density functions. Specific cases which admit exact solutions are presented, and solution procedures based on Monte Carlo simulations for more general class of problems are outlined. Illustrations include studies on a parametrically excited linear system and a nonlinear random vibration problem involving moving oscillator-beam system that considers excitations attributable to random support motions and guide-way unevenness. (C) 2015 American Society of Civil Engineers.