Gas chromatography mass spectrometry-based metabolite profiling in plants

The concept of metabolite profiling has been around for decades, but technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. Here we provide a detailed protocol for gas chromatography mass spectrometry (GC-MS)-based metabolite profiling that offers a good balance of sensitivity and reliability, being considerably more sensitive than NMR and more robust than liquid chromatography-linked mass spectrometry. We summarize all steps from collecting plant material and sample handling to derivatization procedures, instrumentation settings and evaluating the resultant chromatograms. We also define the contribution of GC-MS-based metabolite profiling to the fields of diagnostics, gene annotation and systems biology. Using the protocol described here facilitates routine determination of the relative levels of 300-500 analytes of polar and nonpolar extracts in approximately 400 experimental samples per week per machine.     

Different impact of staining procedures using visible stains and fluorescent dyes for large-scale investigation of proteomes by MALDI-TOF mass spectrometry

Replicate 2-D gels were stained with four visible or fluorescent dyes using published procedures, and 48 co-detected spots were selected for contrasting values in abundance, M(r) and pI. Success rate of identification and sequence coverage were affected in a dye-dependent manner by the three parameters. Frequency of missed cleavages and recovery of sulfur-containing peptides also depended on the dye. Finally, the dataset was used to predict the number of proteins identifiable when integrating the differential contribution of each parameter. Sypro Ruby appeared to combine several favorable features: no dependence of the identification rate upon the physicochemical properties of proteins, no impact on frequency of missed cleavages, and a higher predicted identification rate.     

Gel-free and gel-based proteomics in Bacillus subtilis: a comparative study

The proteome of exponentially growing Bacillus subtilis cells was dissected by the implementation of shotgun proteomics and a semigel-based approach for a particular exploration of membrane proteins. The current number of 745 protein identifications that was gained by the use of two-dimensional gel electrophoresis could be increased by 473 additional proteins. Therefore, almost 50% of the 2500 genes expressed in growing B. subtilis cells have been demonstrated at the protein level. In terms of exploring cellular physiology and adaptation to environmental changes or stress, proteins showing an alteration in expression level are of primary interest. The large number of vegetative proteins identified by gel-based and gel-free approaches is a good starting point for comparative physiological investigations. For this reason a gel-free quantitation with the recently introduced iTRAQ (isobaric tagging for relative and absolute quantitation) reagent technique was performed to investigate the heat shock response in B. subtilis. A comparison with gel-based data showed that both techniques revealed a similar level of up-regulation for proteins belonging to well studied heat hock regulons (SigB, HrcA, and CtsR). However, additional datasets have been obtained by the gel-free approach indicating a strong heat sensitivity of specific enzymes involved in amino acid synthesis.     

Improved parameterized complexity of the maximum agreement subtree and maximum compatible tree problems

Given a set of evolutionary trees on a same set of taxa, the maximum agreement subtree problem (MAST), respectively, maximum compatible tree problem (MCT), consists of finding a largest subset of taxa such that all input trees restricted to these taxa are isomorphic, respectively compatible. These problems have several applications in phylogenetics such as the computation of a consensus of phylogenies obtained from different data sets, the identification of species subjected to horizontal gene transfers and, more recently, the inference of supertrees, e.g., Trees Of Life. We provide two linear time algorithms to check the isomorphism, respectively, compatibility, of a set of trees or otherwise identify a conflict between the trees with respect to the relative location of a small subset of taxa. Then, we use these algorithms as subroutines to solve MAST and MCT on rooted or unrooted trees of unbounded degree. More precisely, we give exact fixed-parameter tractable algorithms, whose running time is uniformly polynomial when the number of taxa on which the trees disagree is bounded. The improves on a known result for MAST and proves fixed-parameter tractability for MCT.     

A Pressurized Nitrogen Counterbalance to Cortical Glutamatergic Pathway Stimulation

Previous microdialysis studies performed in rats have revealed a decrease of striatal dopamine and glutamate induced by nitrogen narcosis. We sought to establish the hypothetical role of the glutamatergic corticostriatal pathway because of the glutamate deficiency which occurs in the basal ganglia in this hyperbaric syndrome. Retrodialysis with 1 mM of Saclofen and 100 mM of KCl in the prefrontal cortex under normobaric conditions led to an increase in striatal levels of glutamate by 95.2% and no changes in dopamine levels. Under 3 MPa of nitrogen and with the infusion, the rate of striatal glutamate decreased by 51.3%, to a greater extent than under pressurised nitrogen alone (−23.8%). The rate of dopamine decreased, which also occurred under pressurised nitrogen (−36.9 and −31.4%, respectively). In conclusion, the function of the corticostriatal pathway is affected by nitrogen under pressure. This suggests that the nitrogen-induced break point seems to be located at the glutamatergic striatopetal neurons.     

Virtual screening,selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase

Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a K_i of 70 μM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (K_i 38 μM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve π–π stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.     

Influence of spring and autumn phenological transitions on forest ecosystem productivity

We use eddy covariance measurements of net ecosystem productivity (NEP) from 21 FLUXNET sites (153 site-years of data) to investigate relationships between phenology and productivity (in terms of both NEP and gross ecosystem photosynthesis, GEP) in temperate and boreal forests. Results are used to evaluate the plausibility of four different conceptual models. Phenological indicators were derived from the eddy covariance time series, and from remote sensing and models. We examine spatial patterns (across sites) and temporal patterns (across years); an important conclusion is that it is likely that neither of these accurately represents how productivity will respond to future phenological shifts resulting from ongoing climate change. In spring and autumn, increased GEP resulting from an ‘extra’ day tends to be offset by concurrent, but smaller, increases in ecosystem respiration, and thus the effect on NEP is still positive. Spring productivity anomalies appear to have carry-over effects that translate to productivity anomalies in the following autumn, but it is not clear that these result directly from phenological anomalies. Finally, the productivity of evergreen needleleaf forests is less sensitive to phenology than is productivity of deciduous broadleaf forests. This has implications for how climate change may drive shifts in competition within mixed-species stands.     

Factors Likely to Affect the Long-term Results of Ventricular Stimulation After Myocardial Infarction

Background

The results of programmed ventricular stimulation (PVS) may change after myocardial infarction (MI). The objective was to study the factors that could predict the results of a second PVS.

Methods

Left ventricular ejection fraction (LVEF) and QRS duration were determined and PVS performed within 3 to 14 years of one another (mean 7.5±5) in 50 patients studied systematically between 1 and 3 months after acute MI.

Results

QRS duration increased from 120±23 ms to 132±29 (p 0.04). LVEF did not decrease significantly (36±12 % vs 37±13 %). Ventricular tachycardia with cycle length (CL) > 220ms (VT) was induced in 11 patients at PVS 1, who had inducible VT with a CL > 220 ms (8) or < 220 ms (ventricular flutter, VFl) (3) at PVS 2. VFl or fibrillation (VF) was induced in 14 patients at PVS 1 and remained inducible in 5; 5 patients had inducible VT and 4 had a negative 2nd PVS. 2. 25 patients had initially negative PVS; 7 had secondarily inducible VT, 4 a VFl/VF, 14 a negative PVS. Changes of PVS were related to initially increasing QRS duration and secondarily changes in LVEF and revascularization but not to the number of extrastimuli required to induce VFl.

Conclusions

In patients without induced VT at first study, changes of PVS are possible during the life. Patients with initially long QRS duration and those who developed decreased LVEF are more at risk to have inducible monomorphic VT at 2nd study, than other patients.