To obtain a comprehensive assessment of metabolite levels from extracts of leukocytes, we have recorded ultra-high-resolution 1 H-13 C HSQC NMR spectra of cell extracts, which exhibit spectral signatures of numerous small molecules. However, conventional acquisition of such spectra is time consuming and hampers measurements on multiple samples, which would be needed for statistical analysis of metabolite concentrations. Here we show that the measurement time can be dramatically reduced without loss of spectral quality when using non-linear sampling (NLS) and a new highfidelity Forward Maximum-entropy (FM) reconstruction algorithm. This FM reconstruction conserves all measured time domain data points and guesses the missing data points by an iterative process. This consists of discrete Fourier transformation of the sparse time-domain data set, computation of the spectral entropy, determination of a multidimensional entropy gradient, and calculation of new values for the missing time domain data points with a conjugate gradient approach. Since this procedure does not alter measured data points it reproduces signal intensities with high fidelity and does not suffer from a dynamic-range problem. As an example we measured a natural abundance 1 H-13 C HSQC spectrum of metabolites from granulocyte cell extracts. We show that a high-resolution 1 H-13 C HSQC spectrum with 4k complex increments recorded linearly within 3.7 days can be reconstructed from 1/7 th of the increments with nearly identical spectral appearance, indistinguishable signal intensities and comparable or even lower root mean square (rms) and peak noise patterns measured in signal-free areas. Thus, this approach allows recording of ultra-high resolution 1 H-13 C HSQC spectra in a fraction of the time needed for recording linearly sampled spectra.
The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The DascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain.
ABSTRACT:1 H-NMR (nuclear magnetic resonance)-based chemometric methods have been applied for the first time to investigate changes in the plasma metabolite profiles of Atlantic salmon Salmo salar as a result of exposure to Aeromonas salmonicida subsp. salmonicida, a Gram-negative bacterium that is the etiological agent of furunculosis. Plasma samples were obtained from salmon that survived 21 d post exposure to A. salmonicida, and from a control group maintained under similar conditions. 1D 1 H-NMR spectra were acquired and principal components analysis (PCA) was used to assess differences between the spectral profiles of plasma from salmon that survived an A. salmonicida challenge, and non-infected controls. PCA enables simultaneous comparison of spectra, presenting a simplified overview of the relationship between spectral data, where spectra cluster based on metabolite profile similarities and differences; information regarding the metabolite variations can therefore be readily deciphered. The major metabolite changes responsible for the spectral differences were related to modification in the lipoprotein profile and choline-based residues, with minor changes in carbohydrates, glycerol, trimethylamine-N-oxide and betaine. These changes indicated that exposure to A. salmonicida induced a characteristic biochemical response which could be used to determine the health status of salmon. This study suggests that with further development this metabolite profiling technique may be a useful tool for diagnosis of disease states in salmon and could provide a better understanding of the host-pathogen relationship which at present is poorly understood for A. salmonicida and Atlantic salmon.KEY WORDS: Aeromonas salmonicida · Atlantic salmon · 1 H-nuclear magnetic resonance spectroscopy · Principal components analysis · Salmo salar
Resale or republication not permitted without written consent of the publisherDis Aquat Org 65: [107][108][109][110][111][112][113][114] 2005 large numbers of complex spectra to extract meaningful information relating to the biochemical response to the pathophysiological stimuli or disease processes under investigation. This approach to metabolite profiling, termed 'metabonomics', is defined by Nicholson et al. (1999) as, 'the quantitative measurement of timerelated multiparametric metabolic responses of a living systems to pathophysiological stimuli or genetic modification'. It is being applied increasingly to investigate the metabolic effects of diseases (Brindle et al. 2003), drugs and toxins , Robosky et al. 2002, Watkins et al. 2002, particularly in mammalian systems. NMR based-metabolite profiling of tissues has also been applied in examining the effects of environmental stresses on shellfish (Viant et al. 2003).Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the etiological agent of an infectious disease in salmonids called furunculosis. Furunculosis may manifest itself either as an acute septicaemia, chronic focal myodermonecrosis or as a clinically inap...
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