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.

Cross-frequency coupling in EEG revealed by adaptive oscillation tracking.

Introduction: Neuronal oscillations have been the focus of increasing interest in the neuroscientific community, in part because they have been considered as a possible integrating mechanism through which internal states can influence stimulus processing in a top-down way (Engel et al., 2001). Moreover, increasing evidence indicates that oscillations in different frequency bands interact with one other through coupling mechanisms (Jensen and Colgin, 2007). The existence and the importance of these cross-frequency couplings during various tasks have been verified by recent studies (Canolty et al., 2006; Lakatos et al., 2007). In this study, we measure the strength and directionality of two types of couplings - phase-amplitude couplings and phase-phase couplings - between various bands in EEG data recorded during an illusory contour experiment that were identified using a recently-proposed adaptive frequency tracking algorithm (Van Zaen et al., 2010). Methods: The data used in this study have been taken from a previously published study examining the spatiotemporal mechanisms of illusory contour processing (Murray et al., 2002). The EEG in the present study were from a subset of nine subjects. Each stimulus was composed of 'pac-man' inducers presented in two orientations: IC, when an illusory contour was present, and NC, when no contour could be detected. The signals recorded by the electrodes P2, P4, P6, PO4 and PO6 were averaged, and filtered into the following bands: 4-8Hz, 8-12Hz, 15-25Hz, 35-45Hz, 45-55Hz, 55-65Hz and 65-75Hz. An adaptive frequency tracking algorithm (Van Zaen et al., 2010) was then applied in each band in order to extract the main oscillation and estimate its frequency. This additional step ensures that clean phase information is obtained when taking the Hilbert transform. The frequency estimated by the tracker was averaged over sliding windows and then used to compare the two conditions. Two types of cross-frequency couplings were considered: phase-amplitude couplings and phase-phase couplings. Both types were measured with the phase locking value (PLV, Lachaux et al., 1999) over sliding windows. The phase-amplitude couplings were computed with the phase of the low frequency oscillation and the phase of the amplitude of the high frequency one. Different coupling coefficients were used when measuring phase-phase couplings in order to estimate different m:n synchronizations (4:3, 3:2, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 and 9:1) and to take into account the frequency differences across bands. Moreover, the direction of coupling was estimated with a directionality index (Bahraminasab et al., 2008). Finally, the two conditions IC and NC were compared with ANOVAs with 'subject' as a random effect and 'condition' as a fixed effect. Before computing the statistical tests, the PLV values were transformed into approximately normal variables (Penny et al., 2008). Results: When comparing the mean estimated frequency across conditions, a significant difference was found only in the 4-8Hz band, such that the frequency within this band was significantly higher for IC than NC stimuli starting at ~250ms post-stimulus onset (Fig. 1; solid line shows IC and dashed line NC). Significant differences in phase-amplitude couplings were obtained only when the 4-8 Hz band was taken as the low frequency band. Moreover, in all significant situations, the coupling strength is higher for the NC than IC condition. An example of significant difference between conditions is shown in Fig. 2 for the phase-amplitude coupling between the 4-8Hz and 55-65Hz bands (p-value in top panel and mean PLV values in the bottom panel). A decrease in coupling strength was observed shortly after stimulus onset for both conditions and was greater for the condition IC. This phenomenon was observed with all other frequency bands. The results obtained for the phase-phase couplings were more complex. As for the phase-amplitude couplings, all significant differences were obtained when the 4-8Hz band was considered as the low frequency band. The stimulus condition exhibiting the higher coupling strength depended on the ratio of the coupling coefficients. When this ratio was small, the IC condition exhibited the higher phase-phase coupling strength. When this ratio was large, the NC condition exhibited the higher coupling strength. Fig. 3 shows the phase-phase couplings between the 4-8Hz and 35-45Hz bands for the coupling coefficient 6:1, and the coupling strength was significantly higher for the IC than NC condition. By contrast, for the coupling coefficient 9:1 the NC condition gave the higher coupling strength (Fig. 4). Control analyses verified that it is not a consequence of the frequency difference between the two conditions in the 4-8Hz band. The directionality measures indicated a transfer of information from the low frequency components towards the high frequency ones. Conclusions: Adaptive tracking is a feasible method for EEG analyses, revealing information both about stimulus-related differences and coupling patterns across frequencies. Theta oscillations play a central role in illusory shape processing and more generally in visual processing. The presence vs. absence of illusory shapes was paralleled by faster theta oscillations. Phase-amplitude couplings were decreased more for IC than NC and might be due to a resetting mechanism. The complex patterns in phase-phase coupling between theta and beta/gamma suggest that the contribution of these oscillations to visual binding and stimulus processing are not as straightforward as conventionally held. Causality analyses further suggest that theta oscillations drive beta/gamma oscillations (see also Schroeder and Lakatos, 2009). The present findings highlight the need for applying more sophisticated signal analyses in order to establish a fuller understanding of the functional role of neural oscillations.

Analysis of cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry

A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These cannabinoids include a dagger(9)-tetrahydrocannabinol (THC), 11-hydroxy-a dagger(9)-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), a dagger(9)-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and a dagger(9)-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal (TM) device. The advantages of performing a liquid-liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5 min on a Kinetex (TM) column packed with 2.6-mu m core-shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000 (TM) triple quadrupole LC-MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50 pg/ml and from 500 to 80 pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method's LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collection

Analysis of sensitivity with respect to a compositional parameter : A comment on 'Local sensitivity analysis for compositional data with application to soil texture in hydrologic modelling' by L. Loosvelt and co-authors

A comment about the article “Local sensitivity analysis for compositional data with application to soil texture in hydrologic modelling” writen by L. Loosvelt and co-authors. The present comment is centered in three specific points. The first one is related to the fact that the authors avoid the use of ilr-coordinates. The second one refers to some generalization of sensitivity analysis when input parameters are compositional. The third tries to show that the role of the Dirichlet distribution in the sensitivity analysis is irrelevant

Simultaneous determination of gross alpha, gross beta and Ra-226 in natural water by liquid scintillation counting.

The determination of gross alpha, gross beta and 226Ra activity in natural waters is useful in a wide range of environmental studies. Furthermore, gross alpha and gross beta parameters are included in international legislation on the quality of drinking water [Council Directive 98/83/EC].1 In this work, a low-background liquid scintillation counter (Wallac, Quantulus 1220) was used to simultaneously determine gross alpha, gross beta and 226Ra activity in natural water samples. Sample preparation involved evaporation to remove 222Rn and its short-lived decay daughters. The evaporation process concentrated the sample ten-fold. Afterwards, a sample aliquot of 8 mL was mixed with 12 mL of Ultima Gold AB scintillation cocktail in low-diffusion vials. In this study, a theoretical mathematical model based on secular equilibrium conditions between 226Ra and its short-lived decay daughters is presented. The proposed model makes it possible to determine 226Ra activity from two measurements. These measurements also allow determining gross alpha and gross beta simultaneously. To validate the proposed model, spiked samples with different activity levels for each parameter were analysed. Additionally, to evaluate the model's applicability in natural water, eight natural water samples from different parts of Spain were analysed. The eight natural water samples were also characterised by alpha spectrometry for the naturally occurring isotopes of uranium (234U, 235U and 238U), radium (224Ra and 226Ra), 210Po and 232Th. The results for gross alpha and 226Ra activity were compared with alpha spectrometry characterization, and an acceptable concordance was obtained.

Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes‑mediated transformation

The objective of this work was to perform the screening of soybean genotypes as to their ability to respond to the induction of hairy roots by Agrobacterium rhizogenes‑mediated transformation. Four Brazilian soybean cultivars (BRSMG 68 Vencedora, BRS 137, Embrapa 48, and MG/BR 46 Conquista) and two North American ones adapted to Brazilian cropping conditions (Bragg and IAS‑5) were screened for their capacity to respond to A. rhizogenes in protocols for in vitro hairy root culture and ex vitro composite plant production. Four‑day‑old seedlings with uniform size were injected with A. rhizogenes harboring the plasmid p35S‑GFP. Seedlings expressing green fluorescent protein (GFP) in at least one hairy root were used to determine the transformation frequency. Using an axenic in vitro protocol, excised cotyledons from four‑day‑old seedlings were infected with A. rhizogenes harboring the pCAMBIA1301 plasmid, containing the gusA reporter gene. The transformation frequency and the number of days for hairy root emergence after bacterial infection (DAI) were evaluated. The transformation frequency and DAI varied according to the genotype. Cultivars MG/BR 46 Conquista and BRSMG 68 Vencedora are more susceptible to A. rhizogenes and can be recommended for transformation experiments.

Fluid flow and dispersion predictions in porous media by utilizing advanced numerical methods

Diplomityön tavoitteena oli tarkastella numeerisen virtauslaskennan avulla virtaukseen liittyviä ilmiöitä ja kaasun dispersiota. Diplomityön sisältö on jaettu viiteen osaan; johdantoon, teoriaan, katsaukseen virtauksen mallinnukseen huokoisessa materiaalissa liittyviin tutkimusselvityksiin, numeeriseen mallinnukseen sekä tulosten esittämiseen ja johtopäätöksiin. Diplomityön alussa kiinnitettiin huomiota erilaisiin kokeellisiin, numeerisiin ja teoreettisiin mallinnusmenetelmiin, joilla voidaan mallintaa virtausta huokoisessa materiaalissa. Kirjallisuusosassa tehtiin katsaus aikaisemmin julkaistuihin puoliempiirisiin ja empiirisiin tutkimusselvityksiin, jotka liittyvät huokoisen materiaalin aiheuttamaan painehäviöön. Numeerisessa virtauslaskenta osassa rakennettiin ja esitettiin huokoista materiaalia kuvaavat numeeriset mallit käyttäen kaupallista FLUENT -ohjelmistoa. Työn lopussa arvioitiin teorian, numeerisen virtauslaskennan ja kokeellisten tutkimusselvitysten tuloksia. Kolmiulotteisen huokoisen materiaalinnumeerisessa mallinnuksesta saadut tulokset vaikuttivat lupaavilta. Näiden tulosten perusteella tehtiin suosituksia ajatellen tulevaa virtauksen mallinnusta huokoisessa materiaalissa. Osa tässä diplomityössä esitetyistä tuloksista tullaan esittämään 55. Kanadan Kemiantekniikan konferenssissa Torontossa 1619 Lokakuussa 2005. ASME :n kansainvälisessä tekniikan alan julkaisussa. Työ on hyväksytty esitettäväksi esitettäväksi laskennallisen virtausmekaniikan (CFD) aihealueessa 'Peruskäsitteet'. Lisäksi työn yksityiskohtaiset tulokset tullaan lähettämään myös CES:n julkaisuun.

Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry.

A highly sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of buprenorphine and its major metabolite norbuprenorphine in human plasma. In order to speed up the process and decrease costs, sample preparation was performed by simple protein precipitation with acetonitrile. To the best of our knowledge, this is the first application of this extraction technique for the quantification of buprenorphine in plasma. Matrix effects were strongly reduced and selectivity increased by using an efficient chromatographic separation on a sub-2μm column (Acquity UPLC BEH C18 1.7μm, 2.1×50mm) in 5min with a gradient of ammonium formate 20mM pH 3.05 and acetonitrile as mobile phase at a flow rate of 0.4ml/min. Detection was made using a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The procedure was fully validated according to the latest Food and Drug Administration guidelines and the Société Française des Sciences et Techniques Pharmaceutiques. Very good results were obtained by using a stable isotope-labeled internal standard for each analyte, to compensate for the variability due to the extraction and ionization steps. The method was very sensitive with lower limits of quantification of 0.1ng/ml for buprenorphine and 0.25ng/ml for norbuprenorphine. The upper limit of quantification was 250ng/ml for both drugs. Trueness (98.4-113.7%), repeatability (1.9-7.7%), intermediate precision (2.6-7.9%) and internal standard-normalized matrix effects (94-101%) were in accordance with international recommendations. The procedure was successfully used to quantify plasma samples from patients included in a clinical pharmacogenetic study and can be transferred for routine therapeutic drug monitoring in clinical laboratories without further development.

Screening for wound-induced oxylipins in Arabidopsis thaliana by differential HPLC-APCI/MS profiling of crude leaf extracts and subsequent characterisation by capillary-scale NMR

A simple non-targeted differential HPLC-APCI/MS approach has been developed in order to survey metabolome modifications that occur in the leaves of Arabidopsis thaliana following wound-induced stress. The wound-induced accumulation of metabolites, particularly oxylipins, was evaluated by HPLC-MS analysis of crude leaf extracts. A generic, rapid and reproducible pressure liquid extraction procedure was developed for the analysis of restricted leaf samples without the need for specific sample preparation. The presence of various oxylipins was determined by head-to-head comparison of the HPLC-MS data, filtered with a component detection algorithm, and automatically compared with the aid of software searching for small differences in similar HPLC-MS profiles. Repeatability was verified in several specimens belonging to different series. Wound-inducible jasmonates were efficiently highlighted by this non-targeted approach without the need for complex sample preparation as is the case for the 'oxylipin signature' procedure based on GC-MS. Furthermore this HPLC-MS screening technique allowed the isolation of induced compounds for further characterisation by capillary-scale NMR (CapNMR) after HPLC scale-up. In this paper, the screening method is described and applied to illustrate its potential for monitoring polar and non-polar stress-induced constituents as well as its use in combination with CapNMR for the structural assignment of wound-induced compounds of interest

Prevalence of iodine inadequacy in Switzerland assessed by the estimated average requirement cut-point method in relation to the impact of iodized salt.

OBJECTIVE: To assess the iodine status of Swiss population groups and to evaluate the influence of iodized salt as a vector for iodine fortification. DESIGN: The relationship between 24 h urinary iodine and Na excretions was assessed in the general population after correcting for confounders. Single-day intakes were estimated assuming that 92 % of dietary iodine was excreted in 24 h urine. Usual intake distributions were derived for male and female population groups after adjustment for within-subject variability. The estimated average requirement (EAR) cut-point method was applied as guidance to assess the inadequacy of the iodine supply. SETTING: Public health strategies to reduce the dietary salt intake in the general population may affect its iodine supply. SUBJECTS: The study population (1481 volunteers, aged ≥15 years) was randomly selected from three different linguistic regions of Switzerland. RESULTS: The 24 h urine samples from 1420 participants were determined to be properly collected. Mean iodine intakes obtained for men (n 705) and women (n 715) were 179 (sd 68.1) µg/d and 138 (sd 57.8) µg/d, respectively. Urinary Na and Ca, and BMI were significantly and positively associated with higher iodine intake, as were men and non-smokers. Fifty-four per cent of the total iodine intake originated from iodized salt. The prevalence of inadequate iodine intake as estimated by the EAR cut-point method was 2 % for men and 14 % for women. CONCLUSIONS: The estimated prevalence of inadequate iodine intake was within the optimal target range of 2-3 % for men, but not for women.

Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.