Combined effects of endurance training and dietary unsaturated fatty acids on physical performance, fat oxidation and insulin sensitivity.

Endurance training improves exercise performance and insulin sensitivity, and these effects may be in part mediated by an enhanced fat oxidation. Since n-3 and n-9 unsaturated fatty acids may also increase fat oxidation, we hypothesised that a diet enriched in these fatty acids may enhance the effects of endurance training on exercise performance, insulin sensitivity and fat oxidation. To assess this hypothesis, sixteen normal-weight sedentary male subjects were randomly assigned to an isoenergetic diet enriched with fish and olive oils (unsaturated fatty acid group (UFA): 52 % carbohydrates, 34 % fat (12 % SFA, 12 % MUFA, 5 % PUFA), 14 % protein), or a control diet (control group (CON): 62 % carbohydrates, 24 % fat (12 % SFA, 6 % MUFA, 2 % PUFA), 14 % protein) and underwent a 10 d gradual endurance training protocol. Exercise performance was evaluated by measuring VO2max and the time to exhaustion during a cycling exercise at 80 % VO2max; glucose homeostasis was assessed after ingestion of a test meal. Fat oxidation was assessed by indirect calorimetry at rest and during an exercise at 50 % VO2max. Training significantly increased time to exhaustion, but not VO2max, and lowered incremental insulin area under the curve after the test meal, indicating improved insulin sensitivity. Those effects were, however, of similar magnitude in UFA and CON. Fat oxidation tended to increase in UFA, but not in CON. This difference was, however, not significant. It is concluded that a diet enriched with fish- and olive oil does not substantially enhance the effects of a short-term endurance training protocol in healthy young subjects.

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase.

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.

Virulent bacterial infection improves aversive learning performance in Drosophila melanogaster.

Virulent infections are expected to impair learning ability, either as a direct consequence of stressed physiological state or as an adaptive response that minimizes diversion of energy from immune defense. This prediction has been well supported for mammals and bees. Here, we report an opposite result in Drosophila melanogaster. Using an odor-mechanical shock conditioning paradigm, we found that intestinal infection with bacterial pathogens Pseudomonas entomophila or Erwinia c. carotovora improved flies' learning performance after a 1h retention interval. Infection with P. entomophila (but not E. c. carotovora) also improved learning performance after 5 min retention. No effect on learning performance was detected for intestinal infections with an avirulent GacA mutant of P. entomophila or for virulent systemic (hemocoel) infection with E. c. carotovora. Assays of unconditioned responses to odorants and shock do not support a major role for changes in general responsiveness to stimuli in explaining the changes in learning performance, although differences in their specific salience for learning cannot be excluded. Our results demonstrate that the effects of pathogens on learning performance in insects are less predictable than suggested by previous studies, and support the notion that immune stress can sometimes boost cognitive abilities.

Effects of 2 different prior endurance exercises on whole-body fat oxidation kinetics: light vs. heavy exercise.

This study aimed to compare the effects of 2 different prior endurance exercises on subsequent whole-body fat oxidation kinetics. Fifteen men performed 2 identical submaximal incremental tests (Incr2) on a cycle ergometer after (i) a ∼40-min submaximal incremental test (Incr1) followed by a 90-min continuous exercise performed at 50% of maximal aerobic power-output and a 1-h rest period (Heavy); and (ii) Incr1 followed by a 2.5-h rest period (Light). Fat oxidation was measured using indirect calorimetry and plotted as a function of exercise intensity during Incr1 and Incr2. A sinusoidal equation, including 3 independent variables (dilatation, symmetry and translation), was used to characterize the fat oxidation kinetics and to determine the intensity (Fat(max)) that elicited the maximal fat oxidation (MFO) during Incr. After the Heavy and Light trials, Fat(max), MFO, and fat oxidation rates were significantly greater during Incr2 than Incr1 (p < 0.001). However, Δ (i.e., Incr2-Incr1) Fat(max), MFO, and fat oxidation rates were greater in the Heavy compared with the Light trial (p < 0.05). The fat oxidation kinetics during Incr2(Heavy) showed a greater dilatation and rightward asymmetry than Incr1(Heavy), whereas only a greater dilatation was observed in Incr2(Light) (p < 0.05). This study showed that although to a lesser extent in the Light trial, both prior exercise sessions led to an increase in Fat(max), MFO, and absolute fat oxidation rates during Incr2, inducing significant changes in the shape of the fat oxidation kinetics.

Exposure to bioaerosols in poultry houses at different stages of fattening: use of real-time PCR for airborne bacterial quantification

Previous studies have demonstrated that poultry-house workers are exposed to very high levels of organic dust and consequently have an increased prevalence of adverse respiratory symptoms. However, the influence of the age of broilers, on bioaerosol concentrations has not been investigated. To evaluate the evolution of bioaerosol concentration during the fattening period, bioaerosol parameters (inhalable dust, endotoxin and bacteria) were measured in 12 poultry confinement buildings in Switzerland, at 3 different stages of the birds' growth; Samples of air taken from within the breathing zones of individual poultry-house employees as they caught the chickens ready to be transported for slaughter, were also analysed. Quantitative PCR (Q-PCR) was used to assess the quantity of total airborne bacteria and total airborne Staphylococcus species. Bioaerosol levels increased significantly during the fattening period of the chickens. During the task of catching mature birds, the mean inhalable dust concentration for a worker was 31 ± 4.7 mg/m3, and endotoxin concentration was 11'080 ± 3436 UE/m3 air, more than ten-fold higher than the Swiss occupational recommended value (1000 UE/m3). The mean exposure level of bird catchers to total bacteria and Staphylococcus species measured by Q-PCR is also very high, respectively reaching values of 72 (± 11) x107 cells/m3 air and 70 (± 16) x106/m3 air. It was concluded that in the absence of wearing protective breathing apparatus, chicken catchers in Switzerland risk exposure beyond recommended limits for all measured bioaerosol parameters. Moreover, the use of Q-PCR to estimate total and specific numbers of airborne bacteria is a promising tool for evaluating any modifications intended to improve the safety of current working practices.

Architectural features of bacterial genomes and their applications

Résumé -Caractéristiques architecturales des génomes bactériens et leurs applications Les bactéries possèdent généralement un seul chromosome circulaire. A chaque génération, ce chromosome est répliqué bidirectionnellement, par deux complexes enzymatiques de réplication se déplaçant en sens opposé depuis l'origine de réplication jusqu'au terminus, situé à l'opposé. Ce mode de réplication régit l'architecture du chromosome -l'orientation des gènes par rapport à la réplication, notamment - et est en grande partie à l'origine des pressions qui provoquent la variation de la composition en nucléotides du génome, hors des contraintes liées à la structure et à la fonction des protéines codées sur le chromosome. Le but de cette thèse est de contribuer à quantifier les effets de la réplication sur l'architecture chromosomique, en s'intéressant notamment aux gènes des ARN ribosomiques, cruciaux pour la bactérie. D'un autre côté, cette architecture est spécifique à l'espèce et donne ainsi une «identité génomique » aux gènes. Il est démontré ici qu'il est possible d'utiliser des marqueurs «naïfs » de cette identité pour détecter, notamment dans le génome du staphylocoque doré, des îlots de pathogénicité, qui concentrent un grand nombre de facteurs de virulence de la bactérie. Ces îlots de pathogénicité sont mobiles, et peuvent passer d'une bactérie à une autre, mais conservent durant un certain temps l'identité génomique de leur hôte précédent, ce qui permet de les reconnaître dans leur nouvel hôte. Ces méthodes simples, rapides et fiables seront de la plus haute importance lorsque le séquençage des génomes entiers sera rapide et disponible à très faible coût. Il sera alors possible d'analyser instantanément les déterminants pathogéniques et de résistance aux antibiotiques des agents pathogènes. Summary The bacterial genome is a highly organized structure, which may be referred to as the genome architecture, and is mainly directed by DNA replication. This thesis provides significant insights in the comprehension of the forces that shape bacterial chromosomes, different in each genome and contributing to confer them an identity. First, it shows the importance of the replication in directing the orientation of prokaryotic ribosomal RNAs, and how it shapes their nucleotide composition in a tax on-specific manner. Second, it highlights the pressure acting on the orientation of the genes in general, a majority of which are transcribed in the same direction as replication. Consequently, apparent infra-arm genome rearrangements, involving an exchange of the leading/lagging strands and shown to reduce growth rate, are very likely artifacts due to an incorrect contig assembly. Third, it shows that this genomic identity can be used to detect foreign parts in genomes, by establishing this identity for a given host and identifying the regions that deviate from it. This property is notably illustrated with Staphylococcus aureus: known pathogenicity islands and phages, and putative ancient pathogenicity islands concentrating many known pathogenicity-related genes are highlighted; the analysis also detects, incidentally, proteins responsible for the adhesion of S. aureus to the hosts' cells. In conclusion, the study of nucleotide composition of bacterial genomes provides the opportunity to better understand the genome-level pressures that shape DNA sequences, and to identify genes and regions potentially related to pathogenicity with fast, simple and reliable methods. This will be of crucial importance when whole-genome sequencing will be a rapid, inexpensive and routine tool.

Iron acquisition with the natural siderophore enantiomers pyochelin and enantio-pyochelin in Pseudomonas species.

The bacterial siderophore pyochelin is composed of salicylate and two cysteine-derived heterocycles, the second of which is modified by reduction and N-methylation during biosynthesis. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation, whereas the second cysteine remains in its L-configuration. Stereochemistry is opposite in the Pseudomonas fluorescens siderophore enantio-pyochelin, in which the first ring originates from L-cysteine and the second ring from D-cysteine. Both siderophores promote growth of the producer organism during iron limitation and induce the expression of their biosynthesis genes by activating the transcriptional AraC-type regulator PchR. However, neither siderophore is functional as an iron carrier or as a transcriptional inducer in the other species, demonstrating that both processes are highly stereospecific. Stereospecificity of pyochelin/enantio-pyochelin-mediated iron uptake is ensured at two levels: (i) by the outer membrane siderophore receptors and (ii) by the cytosolic PchR regulators.

Oxidation of methane at the CH4/H2O-(CO2) transition zone in the external part of the Central Alps, Switzerland: Evidence from stable isotope investigations

With the aim of understanding the mechanisms that control the metamorphic transition from the CH4- to the H2O-(CO2)-dominated fluid zone in the Helvetic domain of the Central Alps of Switzerland, fluid inclusions in quartz, illite ``crystallinity'' index, vitrinite reflectance, and the stable isotope compositions of vein and whole rock minerals and fluids trapped in quartz were investigated along four cross-sections. Increasing temperature during prograde metamorphism led to the formation of dry gas by hydrocarbon cracking in the CH4-zone. Fluid immiscibility in the H2O-CH4-(CO2)-NaCl system resulted in cogenetic, CH4- and H2O-dominated fluid inclusions. In the CH4-zone, fluids were trapped at temperatures <= 270 +/- 5 degrees C. The end of the CH4-zone is markedby a sudden increase of CO2 content in the gas phase of fluid inclusions. At temperatures > 270 +/- 5 degrees C, in the H2O-zone, the total amount of volatiles within the fluid decreased below 1 mol% with no immiscibility. This resulted m total homogenization temperatures of H2O-(CO2-CH4)-NaCl inclusions below 180 degrees C. Hydrogen isotope compositions of methane in fluid inclusion have delta D values of less than -100 parts per thousand in the CH4-zone, typical for an origin through cracking of higher hydrocarbons, but where the methane has not equilibrated with the pore water. delta D values of fluid inclusion water are around -40 parts per thousand., in isotopic equilibrium with phyllosilicates of the whole rocks. Within the CH4 to H2O(CO2) transition zone, delta D(H2O) values in fluid inclusions decrease to -130 parts per thousand interpreted to reflect the contribution of deuterium depleted water from methane oxidation. In the H2O-zone, delta D(H2O) values increase again towards an average of -30 parts per thousand which is again consistent with isotopic equilibrium with host-rock phyllosilicates. delta C-13 values of methane in fluid inclusions from the CH4-zone are around -27 parts per thousand in isotopic equilibrium with calcite in veins and whole rocks. The delta C-13(CH4) values decrease to less than -35 parts per thousand at the transition to the H2O-zone and are no longer in equilibrium with the carbonates in the whole rocks. delta C-13 values of CO, are variable but too low to be in equilibrium with the wall rock fluids, compatible with a contribution of CO2 from closed system oxidation of methane. Differences in isotopic composition between host-rock and Alpine fissure carbonate are generally small, suggesting that the amount of CO2 produced by oxidation of methane was small compared to the C-budget in the rocks and local pore fluids were buffered by the wall rocks during precipitation of calcite within the fissures. (c) 2006 Elsevier B.V. All rights reserved.

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.

Accelerated digestion for high-throughput proteomics analysis of whole bacterial proteomes.

In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.

Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation.

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.

Total and exogenous carbohydrate oxidation in obese prepubertal children.

The aim was to explore whether the origin of carbohydrate oxidation (exogenous compared with endogenous carbohydrate) after consumption of a mixed meal was influenced by obesity in children. Ten obese prepubertal children 8 y of age (44.2 +/- 3.6 kg) were studied over 9.5 h and compared with eight normal-weight, matched control children (28.5 +/- 1.6 kg). They were fed a mixed meal containing naturally enriched [13C]carbohydrate (cane sugar and popcorn) providing 55% of the daily energy requirement as measured by 24-h resting metabolic rate. Total carbohydrate oxidation was calculated by indirect calorimetry (hood system) whereas exogenous carbohydrate oxidation was estimated from carbon dioxide production (VCO2), the isotopic enrichment of breath 13CO2, and the abundance of [13C]carbohydrate in the meal ingested. The time course of 13CO2 in breath-measured over 570 min-followed a similar pattern in both groups. Although total carbohydrate oxidation was not significantly different among the two groups, exogenous carbohydrate utilization was significantly greater (P < 0.03) and endogenous carbohydrate oxidation was significantly lower (P < 0.05) in obese compared with control children. In addition, the rate of exogenous carbohydrate oxidation expressed as a proportion of total carbohydrate oxidation was positively related to the body fat of the children (r = 0.68, P < 0.01). The study suggests that in the postprandial phase, a smaller proportion of carbohydrate oxidation is accounted for by glycogen breakdown in obese children. The sparing of endogenous glycogen may result from decreased glycogen turnover already present at an early age.

Information from single-cell bacterial biosensors: what is it good for?

Bacterial reporter cells (i.e. strains engineered to produce easily measurable signals in response to one or more chemical targets) can principally be used to quantify chemical signals and analytes, physicochemical conditions and gradients on a microscale (i.e. micrometer to submillimeter distances), when the reporter signal is determined in individual cells. This makes sense, as bacterial life essentially thrives in microheterogenic environments and single-cell reporter information can help us to understand the microphysiology of bacterial cells and its importance for macroscale processes like pollutant biodegradation, beneficial bacteria-eukaryote interactions, and infection. Recent findings, however, showed that clonal bacterial populations are essentially always physiologically, phenotypically and genotypically heterogeneous, thus emphasizing the need for sound statistical approaches for the interpretation of reporter response in individual bacterial cells. Serious attempts have been made to measure and interpret single-cell reporter gene expression and to understand variability in reporter expression among individuals in a population.

Oxidation proteomics: the role of thiol modifications

Identification of thiol modifications has gained significant importance. It is increasingly recognized that cysteines play an important role in protein function under both physiological and patho-physiological conditions. Here we reviewed different approaches that are used to identify oxidized proteins and discuss different fluorescent labeling techniques, differential two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization - time of flight identification, in short MALDI-TOF. We illuminate processes that depend on protein oxidation of cysteines and we look into consequences of thiol oxidation during aging and in a variety of diseases, with a special reference to Alzheimer's disease. There is an urgent need for methods that detect specifically oxidized proteins and are able to distinguish different oxidation types.