RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.
Allergy testing is important for evidence-based care and treatment. In this paper, we report a new clinical laboratory instrument, AdvanSure AlloStation Smart, which is used for a multiple allergen-specific immunoglobulin (IgE) assay. For efficient automation of immunoblot procedures, a novel tilting carousel technology was incorporated. The analyzer showed 100.5-101.7% of pipetting accuracy, no sample carryover, and 0.2-3.2% coefficient of variation of optical precision. Assay performance on the system was comparable with that of a single allergen test, ImmunoCAP for 23 allergens. The slope and the intercept of this comparison were 0.994 and 0.070, respectively. The Pearson correlation coefficient between two methods was r ¼ 0.836. The coefficient of variation of intra-assay precision ranged from 3.2% to 11.1% and that of inter-assay precision varied from 4.0% to 17.6%. The AdvanSure AlloStation Smart provides a simple and reliable multiple assay platform to monitor allergen-specific IgE profiles.
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