Oral insecticidal activity of root colonizing Pseudomonas fluorescens CHA0: a biocontrol agent with potential to control plant pests and diseases

The application of microbial biocontrol agents for the control of fungal plant diseases and plant insect pests is a promising approach in the development of environmentally benign pest management strategies. The ideal biocontrol organism would be a bacterium or a fungus with activity against both, insect pests and fungal pathogens. Here we demonstrate the oral insecticidal activity of the root colonizing Pseudomonas fluorescens CHA0, which is so far known for its capacity to efficiently suppress fungal plant pathogens. Feeding assays with CHA0-sprayed leaves showed that this strain displays oral insecticidal activity and is able to efficiently kill larvae of three important insect pests. We further show data indicating that the Fit insect toxin produced by CHA0 and also metabolites controlled by the global regulator GacA contribute to oral insect toxicity.

Quantification of the neurochemical profile using simulated macromolecule resonances at 3 T.

The broad resonances underlying the entire (1) H NMR spectrum of the brain, ascribed to macromolecules, can influence metabolite quantification. At the intermediate field strength of 3 T, distinct approaches for the determination of the macromolecule signal, previously used at either 1.5 or 7 T and higher, may become equivalent. The aim of this study was to evaluate, at 3 T for healthy subjects using LCModel, the impact on the metabolite quantification of two different macromolecule approaches: (i) experimentally measured macromolecules; and (ii) mathematically estimated macromolecules. Although small, but significant, differences in metabolite quantification (up to 23% for glutamate) were noted for some metabolites, 10 metabolites were quantified reproducibly with both approaches with a Cramer-Rao lower bound below 20%, and the neurochemical profiles were therefore similar. We conclude that the mathematical approximation can provide sufficiently accurate and reproducible estimation of the macromolecule contribution to the (1) H spectrum at 3 T. Copyright © 2013 John Wiley & Sons, Ltd.

Endogenous steroid profiling in the athlete biological passport.

The Athlete Biological Passport (ABP) is an individual electronic document that collects data regarding a specific athlete that is useful in differentiating between natural physiologic variations of selected biomarkers and deviations caused by artificial manipulations. A subsidiary of the endocrine module of the ABP, that which here is called Athlete Steroidal Passport (ASP), collects data on markers of an altered metabolism of endogenous steroidal hormones measured in urine samples. The ASP aims to identify not only doping with anabolic-androgenic steroids, but also most indirect steroid doping strategies such as doping with estrogen receptor antagonists and aromatase inhibitors. Development of specific markers of steroid doping, use of the athlete's previous measurements to define individual limits, with the athlete becoming his or her own reference, the inclusion of heterogeneous factors such as the UDPglucuronosyltransferase B17 genotype of the athlete, the knowledge of potentially confounding effects such as heavy alcohol consumption, the development of an external quality control system to control analytical uncertainty, and finally the use of Bayesian inferential methods to evaluate the value of indirect evidence have made the ASP a valuable alternative to deter steroid doping in elite sports. The ASP can be used to target athletes for gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS) testing, to withdraw temporarily the athlete from competing when an abnormality has been detected, and ultimately to lead to an antidoping infraction if that abnormality cannot be explained by a medical condition. Although the ASP has been developed primarily to ensure fairness in elite sports, its application in endocrinology for clinical purposes is straightforward in an evidence-based medicine paradigm.

Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.

Techniques for analytical testing of unconventional samples.

Forensic scientists have long detected the presence of drugs and their metabolites in biological materials using body fluids such as urine, blood and/or other biological liquids or tissues. For doping analysis, only urine has so far been collected. In recent years, remarkable advances in sensitive analytical techniques have encouraged the analysis of drugs in unconventional biological samples such as hair, saliva and sweat. These samples are easily collected, although drug levels are often lower than the corresponding levels in urine or blood. This chapter reviews recent studies in the detection of doping agents in hair, saliva and sweat. Sampling, analytical procedures and interpretation of the results are discussed in comparison with those obtained from urine and blood samples.

Differential allocation and deployment of direct and indirect defences by Vicia sepium along elevation gradients

Dissecting drivers of plant defence investment remains central for understanding the assemblage of communities across different habitats. There is increasing evidence that direct defence strategies against herbivores, including secondary metabolites production, differ along ecological gradients in response to variation in biotic and abiotic conditions. In contrast, intraspecific variation in indirect defences remains unexplored. Here, we investigated variation in herbivory rate, resistance to herbivores, and indirect defences in ant-attracting Vicia species along the elevation gradient of the Alps. Specifically, we compared volatile organic compounds (VOCs) and ant attraction in high and low elevation ecotypes. Consistent with adaptation to the lower herbivory conditions that we detected at higher elevations in the field, high elevation plants were visited by fewer ants and were more susceptible to herbivore attack. In parallel, constitutive volatile organic compound production and subsequent ant attraction were lower in the high elevation ecotypes. We observed an elevation-driven trade-off between constitutive and inducible production of VOCs and ant attraction along the environmental cline. At higher elevations, inducible defences increased, while constitutive defence decreased, suggesting that the high elevation ecotypes compensate for lower indirect constitutive defences only after herbivore attack. Synthesis. Overall, direct and indirect defences of plants vary along elevation gradients. Our findings show that plant allocation to defences are subject to trade-offs depending on local conditions, and point to a feedback mechanism linking local herbivore pressure, predator abundance and the defence investment of plants.

The pollutant diethylhexyl phthalate regulates hepatic energy metabolism via species-specific PPARalpha-dependent mechanisms.

Background: The modulation of energetic homeostasis by pollutants has recently emerged as a potential contributor to the onset of metabolic disorders. Diethylhexyl phthalate (DEHP) is a widely used industrial plasticizer to which humans are widely exposed. Phthalates can activate the three peroxisome proliferatoractivated receptor (PPAR) isotypes on cellular models and induce peroxisome proliferation in rodents.Objectives: In this study, we aimed to evaluate the systemic and metabolic consequences of DEHP exposure that have remained so far unexplored and to characterize the underlying molecular mechanisms of action.Methods: As a proof of concept and mechanism, genetically engineered mouse models of PPARs were exposed to high doses of DEHP, followed by metabolic and molecular analyses.Results: DEHP-treated mice were protected from diet-induced obesity via PPARalpha-dependent activation of hepatic fatty acid catabolism, whereas the activity of neither PPARbeta nor PPARgamma was affected. However, the lean phenotype observed in response to DEHP in wild-type mice was surprisingly abolished in PPARalpha-humanized mice. These species differences are associated with a different pattern of coregulator recruitment.Conclusion: These results demonstrate that DEHP exerts species-specific metabolic actions that rely to a large extent on PPARalpha signaling and highlight the metabolic importance of the species-specific activation of PPARalpha by xenobiotic compounds. Editor's SummaryDiethylhexyl phthalate (DEHP) is an industrial plasticizer used in cosmetics, medical devices, food packaging, and other applications. Evidence that DEHP metabolites can activate peroxisome proliferatoractivated receptors (PPARs) involved in fatty acid oxidation (PPARalpha and PPARbeta) and adiposite function and insulin resistance (PPARgamma) has raised concerns about potential effects of DEHP on metabolic homeostasis. In rodents, PPARalpha activation also induces hepatic peroxisome proliferation, but this response to PPARalpha activation is not observed in humans. Feige et al. (p. 234) evaluated systemic and metabolic consequences of high-dose oral DEHP in combination with a high-fat diet in wild-type mice and genetically engineered mouse PPAR models. The authors report that mice exposed to DEHP gained less weight than controls, without modifying their feeding behavior; they also exhibited lower triglyceride levels, smaller adipocytes, and improved glucose tolerance compared with controls. These effects, which were observed in mice fed both high-fat and standard diets, appeared to be mediated by PPARalpha-dependent activation of hepatic fatty acid catabolism without apparent involvement of PPARbeta or PPARgamma. However, mouse models that expressed human (versus mouse) PPARalpha tended to gain more weight on a high-fat diet than their DHEP-unexposed counterparts. The authors conclude that findings support species-specific metabolic effects of DEHP mediated by PPARalpha activation.

In silico prediction of human carboxylesterase-1 (hCES1) metabolism combining docking analyses and MD simulations.

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted in developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas little has been done to predict the hydrolytic activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES1. The study involves both docking analyses of known substrates to develop predictive models, and molecular dynamics (MD) simulations to reveal the in situ behavior of substrates and products, with particular attention being paid to the influence of their ionization state. The results emphasize some crucial properties of the hCES1 catalytic cavity, confirming that as a trend with several exceptions, hCES1 prefers substrates with relatively smaller and somewhat polar alkyl/aryl groups and larger hydrophobic acyl moieties. The docking results underline the usefulness of the hydrophobic interaction score proposed here, which allows a robust prediction of hCES1 catalysis, while the MD simulations show the different behavior of substrates and products in the enzyme cavity, suggesting in particular that basic substrates interact with the enzyme in their unprotonated form.

Oligodendroglia metabolically support axons and contribute to neurodegeneration.

Oligodendroglia support axon survival and function through mechanisms independent of myelination, and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been proposed. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and use. Here we show that the most abundant lactate transporter in the central nervous system, monocarboxylate transporter 1 (MCT1, also known as SLC16A1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and in mouse models of, amyotrophic lateral sclerosis, suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.

Nous derivats del 1-1'-bisnaftalè en el camp de l'enantioreconeixement: enantiodiferenciació i síntesi enantioselectiva

La línia principal d’investigació del nostre grup de recerca es basa en la síntesi i estudi de nous agents de solvatació quirals (CSAs), és a dir, compostos enantiopurs que s’utilitzen per a determinar la puresa enantiomèrica mitjançant la Ressonància Magnètica Nuclear (RMN). Al present treball volem sintetitzar i estudiar estructuralment la dicetona racèmica 14 amb la qual, mitjançant reaccions de reducció, podem obtenir el diol 12, el nou CSA que volem sintetitzar. També forma part del present treball la resolució de la dicetona racèmica 14, és a dir, aconseguir la separació dels seus dos enantiòmers mitjançant la formació de derivats diastereoisomèrics de 14 amb un reactiu enantiopur.

A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues.

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

DETERMINATION OF CELL-SPECIFIC NEUROTOXICITY OF MALONATE, METHYLMALONATE AND PROPIONATE IN A 3D RAT BRAIN CELL AGGREGATE SYSTEM

Malonate, methylmalonate and propionate are potentially neurotoxic metabolites in branched-chain organic acidurias. Their effects were tested on cultured 3D rat brain cell aggregates, using dosages of 0.1, 1.0 and 10.0 mM with a short but intense (twice a day over 3 days) and a longer but less intense treatment (every 3 rdday over 9 days). CNS cell-specific immunohistochemical stainings allowed the follow-up of neurons (axons, phosphorylated medium-weight neurofilament), astrocytes (glial fibrillary acidic protein) and oligodendrocytes (myelin basic protein). Methylmalonate and malonate were quantified by tandem mass spectrometry. Tandem mass spectrometry analysis of harvested brain cell aggregates revealed clear intracellular accumulation of methylmalonate and malonate. In immunohistochemical stainings oligodendrocytes appeared the most affected brain cells. The MBP signal disappeared already at 0.1 mM treatment with each metabolite. Mature astrocytes were not affected by propionate, while immature astrocytes on intense treatment with propionate developed cell swelling. 1 mM methylmalonate induced cell swelling of both immature and mature astrocytes , while 1 mM malonate only affected mature astrocytes. Neurons were not affected by methylmalonate, but 10.0 mM malonate on less intense treatment and 0.1, 1.0 and 10.0 mM propionate on intense treatment affected axonal growth. Our study shows significant uptake and deleterious effects of these metabolites on brain cells, principally on astrocytes and oligodendrocytes. This may be explained by the absence of the pathway in glial cells, which thus are not able to degrade these metabolites. Further studies are ongoing to elucidate the underlying mechanisms of the observed neurotoxic effects.

On problems of calculating energy expenditure and substrate utilization from respiratory exchange data.

Indirect calorimetry based on respiratory exchange measurement has been successfully used from the beginning of the century to obtain an estimate of heat production (energy expenditure) in human subjects and animals. The errors inherent to this classical technique can stem from various sources: 1) model of calculation and assumptions, 2) calorimetric factors used, 3) technical factors and 4) human factors. The physiological and biochemical factors influencing the interpretation of calorimetric data include a change in the size of the bicarbonate and urea pools and the accumulation or loss (via breath, urine or sweat) of intermediary metabolites (gluconeogenesis, ketogenesis). More recently, respiratory gas exchange data have been used to estimate substrate utilization rates in various physiological and metabolic situations (fasting, post-prandial state, etc.). It should be recalled that indirect calorimetry provides an index of overall substrate disappearance rates. This is incorrectly assumed to be equivalent to substrate "oxidation" rates. Unfortunately, there is no adequate golden standard to validate whole body substrate "oxidation" rates, and this contrasts to the "validation" of heat production by indirect calorimetry, through use of direct calorimetry under strict thermal equilibrium conditions. Tracer techniques using stable (or radioactive) isotopes, represent an independent way of assessing substrate utilization rates. When carbohydrate metabolism is measured with both techniques, indirect calorimetry generally provides consistent glucose "oxidation" rates as compared to isotopic tracers, but only when certain metabolic processes (such as gluconeogenesis and lipogenesis) are minimal or / and when the respiratory quotients are not at the extreme of the physiological range. However, it is believed that the tracer techniques underestimate true glucose "oxidation" rates due to the failure to account for glycogenolysis in the tissue storing glucose, since this escapes the systemic circulation. A major advantage of isotopic techniques is that they are able to estimate (given certain assumptions) various metabolic processes (such as gluconeogenesis) in a noninvasive way. Furthermore when, in addition to the 3 macronutrients, a fourth substrate is administered (such as ethanol), isotopic quantification of substrate "oxidation" allows one to eliminate the inherent assumptions made by indirect calorimetry. In conclusion, isotopic tracers techniques and indirect calorimetry should be considered as complementary techniques, in particular since the tracer techniques require the measurement of carbon dioxide production obtained by indirect calorimetry. However, it should be kept in mind that the assessment of substrate oxidation by indirect calorimetry may involve large errors in particular over a short period of time. By indirect calorimetry, energy expenditure (heat production) is calculated with substantially less error than substrate oxidation rates.

Visualització dinàmica d'un procés metabòlic amb finalitats docents

La incorporació del Moodle com a eina de docència, i l’augment de hores no presencials a les diverses assignatures fa que calgui incorporar les noves tecnologies perquè els alumnes disposin de més material docent al seu abast. Però l’acumulació de material fa que només sigui útil aquell material que es guanyi a l’alumne. En aquest sentit, creiem que les animacions i les eines interactives poden ser materials atractius pels alumnes. En aquest projecte hem creat una eina d’animació interactiva perquè l’estudiant de Bioquímica practiqui i aprengui una de les rutes metabòliques principals: la glucòlisi. Aquesta eina consta d’una pantalla separada en 3 zones: 1) una zona lateral que inclou la ruta metabòlica completa, i en la que l’alumne pot prémer sobre cada un dels passos que estructuren la ruta (finestra del metabolisme); 2) una zona inferior que presenta la reacció individual de la ruta metabòlica, en la que s’observa l’estructura química de les molècules i informació de l’enzim implicat en la reacció (finestra de l’enzim); i 3) una zona central en la que a través d’una animació amb Macromedia Flash MX, l’alumne observa el mecanisme químic de la reacció (finestra del mecanisme químic). Les tres zones són interactives i en prémer sobre elles donen informació, respectivament sobre la ruta completa, l’enzim de cada pas en particular i el mecanisme d’acció. A més, la finestra del mecanisme químic permet aturar en qualsevol moment la animació, tornar enrere i veure els intermediaris. Durant el segon semestre del curs 2006-2007 hem avaluat l’eina amb alumnes de Bioquímica de la Llicenciatura de Química, incorporant-la als dossier electrònics. Creiem que hem assolit els objectius que es proposaven: aprendre una ruta metabòlica, de manera divertida; aprendre el mecanisme de cada un dels passos de la ruta i aprendre l’estructura química dels metabòlits intermediaris de la ruta.

Animació de les rutes glucolítica i gluconeogènica : una eina no presencial per a aprendre de manera amena

La incorporació del Moodle com a eina de docència, i l’augment de hores no presencials a les diverses assignatures fa que calgui incorporar les noves tecnologies perquè els alumnes disposin de més material docent al seu abast. Però l’acumulació de material fa que només sigui útil aquell material que es guanyi a l’alumne. En aquest sentit, creiem que les animacions i les eines interactives poden ser materials atractius pels alumnes. En aquest projecte hem creat una eina d’animació interactiva perquè l’estudiant de Bioquímica practiqui i aprengui dues rutes principals del metabolisme de hidrats de carboni: la glucòlisi i la gluconeogènesi. Aquesta eina consta d’una pantalla separada en 3 zones: 1) una zona lateral que inclou la ruta metabòlica completa, i en la que l’alumne pot prémer sobre cada un dels passos que estructuren la ruta (finestra del metabolisme); 2) una zona inferior que presenta la reacció individual de la ruta metabòlica, en la que s’observa l’estructura química de les molècules i informació de l’enzim implicat en la reacció (finestra de l’enzim); i 3) una zona central en la que a través d’una animació amb Macromedia Flash MX, l’alumne observa el mecanisme químic de la reacció (finestra del mecanisme químic). Les tres zones són interactives i en prémer sobre elles donen informació, respectivament sobre la ruta completa, l’enzim de cada pas en particular i el mecanisme d’acció. A més, la finestra del mecanisme químic permet aturar en qualsevol moment la animació, tornar enrere i veure els intermediaris. Durant el segon semestre del curs 2007-2008 hem avaluat l’eina amb alumnes de Bioquímica de la Llicenciatura de Química, incorporant-la als dossier electrònics i al Moodle. Creiem que hem assolit els objectius que es proposaven: que l’alumne aprengui les dues rutes metabòliques, de manera divertida; el mecanisme de cada un dels passos de les rutes i l’estructura química dels metabòlits intermediaris de les rutes.