AMPA receptor translocation and phosphorylation are induced by transcranial direct current stimulation in rats.

Over the last decade, the interest in transcranial direct current stimulation (tDCS) has continued to increase, along with consideration of how it affects neuroplasticity mechanisms in the brain. Both human and animal studies have demonstrated numerous benefits and, although its application has increased, the neurophysiological mechanisms underlying tDCS' beneficial effects remain largely unknown. Recent studies have shown that long-term potentiation (LTP) increases following tDCS. In this work, we utilized a rodent model of tDCS to directly assess changes in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, a critical protein for enhancing synaptic transmission. Animals were subjected to 250 µA of direct current (DC) stimulation for 30 min with immediate tissue collection. Translocation and phosphorylation of AMPA receptors were examined using protein immunoblot analysis following a subcellular fractionation method. Our findings show that a single application of in vivo tDCS can affect both the translocation and phosphorylation of AMPA receptors in the hippocampus while increasing AMPA receptor phosphorylation in the hypothalamus. In the hippocampus, tDCS increased AMPA translocation to the synapse and increased the phosphorylation of the S831 site on GluA1. In the hypothalamus, no statistically significant changes were observed in AMPA translocation while an increase in the phosphorylation of the S831 site was observed. No changes in the phosphorylation of GluA1 at the S845 site were detected in either brain region. In sum, our findings identify specific AMPA receptor changes induced by tDCS, thereby providing further details on the mechanisms by which tDCS could affect the establishment of LTP and modulate neuroplasticity.

Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the ß-oxidative or nonoxidative pathways. The first step in the ß-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the ß-oxidative pathway.

Completion of the core ß-oxidative pathway of benzoic acid biosynthesis in plants.

Despite the importance of benzoic acid (BA) as a precursor for a wide array of primary and secondary metabolites, its biosynthesis in plants has not been fully elucidated. BA formation from phenylalanine requires shortening of the C(3) side chain by two carbon units, which can occur by a non-ß-oxidative route and/or a ß-oxidative pathway analogous to the catabolism of fatty acids. Enzymes responsible for the first and last reactions of the core BA ß-oxidative pathway (cinnamic acid → cinnamoyl-CoA → 3-hydroxy-3-phenylpropanoyl-CoA → 3-oxo-3-phenylpropanoyl-CoA → BA-CoA) have previously been characterized in petunia, a plant with flowers rich in phenylpropanoid/benzenoid volatile compounds. Using a functional genomics approach, we have identified a petunia gene encoding cinnamoyl-CoA hydratase-dehydrogenase (PhCHD), a bifunctional peroxisomal enzyme responsible for two consecutively occurring unexplored intermediate steps in the core BA ß-oxidative pathway. PhCHD spatially, developmentally, and temporally coexpresses with known genes in the BA ß-oxidative pathway, and correlates with emission of benzenoid volatiles. Kinetic analysis of recombinant PhCHD revealed it most efficiently converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA, thus forming the substrate for the final step in the pathway. Down-regulation of PhCHD expression in petunia flowers resulted in reduced CHD enzyme activity, as well as decreased formation of BA-CoA, BA and their derived volatiles. Moreover, transgenic lines accumulated the PhCHD substrate cinnamoyl-CoA and the upstream pathway intermediate cinnamic acid. Discovery of PhCHD completes the elucidation of the core BA ß-oxidative route in plants, and together with the previously characterized CoA-ligase and thiolase enzymes, provides evidence that the whole pathway occurs in peroxisomes.

Profiling hydroxycinnamoyl-coenzyme A thioesters: unlocking the back door of phenylpropanoid metabolism.

In plants, 20 to 30% of photosynthetically fixed carbon is directed toward lignin and other phenylpropanoid compounds for which hydroxycinnamoyl-coenzyme A (CoA) esters are key intermediates. CoA thioesters, ubiquitous metabolites found in all living cells (often at trace levels), have traditionally been challenging to measure. Here we report a hydrophilic interaction liquid chromatography (HILIC) method, coupled with tandem mass spectrometry (MS/MS), that allows simultaneous sensitive quantification of previously undetectable hydroxycinnamoyl-CoA esters and an extended range of acyl-CoAs from plant tissues. This method provides rapid liquid chromatography (LC) analysis (10 min/sample) and the ability for qualitative assessment of acyl-CoAs by MS/MS precursor ion scanning.

Improving Quantification of tabun, sarin, soman, cyclosarin, and sulfur mustard by focusing agents: A field portable gas chromatography-mass spectrometry study.

Commercial gas chromatograph-mass spectrometers, one of which being Inficon's HAPSITE® ER, have demonstrated chemical detection and identification of nerve agents (G-series) and blistering agents (mustard gas) in the field; however most analyses relies on self-contained or external calibration that inherently drifts over time. We describe an analytical approach that uses target-based thermal desorption standards, called focusing agents, to accurately calculate concentrations of chemical warfare agents that are analyzed by gas chromatograph-mass spectrometry. Here, we provide relative response factors of focusing agents (2-chloroethyl ethyl sulfide, diisopropyl fluorophosphate, diethyl methylphosphonate, diethyl malonate, methyl salicylate, and dichlorvos) that are used to quantify concentrations of tabun, sarin, soman, cyclosarin and sulfur mustard loaded on thermal desorption tubes (Tenax® TA). Aging effects of focusing agents are evaluated by monitoring deviations in quantification as thermal desorption tubes age in storage at room temperature and relative humidity. The addition of focusing agents improves the quantification of tabun, sarin, soman, cyclosarin and sulfur mustard that is analyzed within the same day as well as a 14-day period. Among the six focusing agents studied here, diisopropyl fluorophosphate has the best performance for nerve agents (G-series) and blistering agents (mustard gas) compared to other focusing agents in this work and is recommended for field use for quantification. The use of focusing agent in the field leads to more accurate and reliable quantification of Tabun (GA), Sarin (GB), Soman (GD), Cyclosarin (GF) and Sulfur Mustard (HD) than the traditional internal standard. Future improvements on the detection of chemical, biological, radiological, nuclear, and explosive materials (CBRNE) can be safely demonstrated with standards calibrated for harmful agents.

An important role of a BAHD acyl transferase-like protein in plant innate immunity.

Salicylic acid (SA) is an important regulator of plant resistance to biotrophic and hemi-biotrophic pathogens. The enhanced pseudomonas susceptibility 1 (eps1) mutant in Arabidopsis thaliana is hypersusceptible to both virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae. Through positional cloning, the EPS1 gene was isolated and found to encode a novel member of the BAHD acyltransferase superfamily. Pathogen-induced accumulation of SA and expression of pathogenesis-related (PR) genes were compromised in the eps1 mutant. SA could induce PR1 gene expression and restore disease resistance in the eps1 mutant. These results suggest that EPS1 functions upstream of SA and may be involved directly in synthesis of a precursor or a regulatory molecule for SA biosynthesis. Mutations of EPS1 or other genes important for SA accumulation or signaling conferred enhanced resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola in the Nossen-0 background but had little effect in the Columbia-0 background. These results suggest that there is natural variation among Arabidopsis ecotypes with respect to the antagonistic cross-talk between defense signaling pathways against various types of microbial pathogens.

Metabolomic stability of exercise-induced sweat.

Due to increased interest in the use of excreted sweat for biomarker discovery, data must be generated supporting sample collection and handling methods to allow for controlled, large-scale biomarker discovery studies to be performed. In this manuscript, twelve amino acids were quantitated from exercise-induced excreted sweat held at room temperature or a simulated body temperature of 37 °C for up to 90 min. The data illustrate a large dynamic range exists among amino acids in sweat. Additionally, the amino acid quantities vary across individuals and among the same individual under different storage conditions, with alanine, arginine, and threonine showing a significant statistical difference between sampling events (p < 0.05). Furthermore, the results establish amino acids are relatively invariant, at both storage temperatures tested, for up to 90 min illustrated by <10% (15/156) of the amino acids measurements demonstrating change greater than 10% from the time zero value. An untargeted metabolomics approach was also applied to the data set to evaluate global changes to the metabolome. The results show more than 88% of all data points fall within the established limits, regardless of temperature condition and ionization mode. Collectively, this study demonstrates that sweat is largely invariant at two distinct temperatures for up to 90 min. These results establish sweat collection and sample handling is possible for up to 90 min with minimal changes in metabolite abundances.

The proteomic and metabolomic characterization of exercise-induced sweat for human performance monitoring: A pilot investigation.

Sweat is a biofluid with several attractive attributes. However, investigation into sweat for biomarker discovery applications is still in its infancy. To add support for the use of sweat as a non-invasive media for human performance monitoring, volunteer participants were subjected to a physical exertion model using a treadmill. Following exercise, sweat was collected, aliquotted, and analyzed for metabolite and protein content via high-resolution mass spectrometry. Overall, the proteomic analysis illustrates significant enrichment steps will be required for proteomic biomarker discovery from single sweat samples as protein abundance is low in this medium. Furthermore, the results indicate a potential for protein degradation, or a large number of low molecular weight protein/peptides, in these samples. Metabolomic analysis shows a strong correlation in the overall abundance among sweat metabolites. Finally, hierarchical clustering of participant metabolite abundances show trends emerging, although no significant trends were observed (alpha = 0.8, lambda = 1 standard error via cross validation). However, these data suggest with a greater number of biological replicates, stronger, statistically significant results, can be obtained. Collectively, this study represents the first to simultaneously use both proteomic and metabolomic analysis to investigate sweat. These data highlight several pitfalls of sweat analysis for biomarker discovery applications.

Exhaled isoprene for monitoring recovery from acute hypoxic stress.

Hypoxia-like incidents in-flight have increased over the past decade causing severe safety concerns across the aviation community. As a result, the need to monitor flight crews in real-time for the onset of hypoxic conditions is paramount for continued aeronautical safety. Here, hypoxic events were simulated in the laboratory via a reduced oxygen-breathing device to determine the effect of recovery gas oxygen concentration (21% and 100%) on exhaled breath volatile organic compound composition. Data from samples collected both serially (throughout the exposure), prior to, and following exposures yielded 326 statistically significant features, 203 of which were unique. Of those, 72 features were tentatively identified while 51 were verified with authentic standards. A comparison of samples collected serially between recovery and hypoxia time points shows a statistically significant reduction in exhaled breath isoprene (2-methyl-1,3-butadiene, log2 FC -0.399, p = 0.005, FDR = 0.034, q = 0.033), however no significant difference in isoprene abundance was observed when comparing recovery gases (21% or 100% O2, p = 0.152). Furthermore, examination of pre-/post-exposure 1 l bag breath samples illustrate an overall increase in exhaled isoprene abundance post-exposure (log2 FC 0.393, p = 0.005, FDR = 0.094, q = 0.033) but again no significant difference between recovery gas (21% and 100%, p = 0.798) was observed. A statistically significant difference in trend was observed between isoprene abundance and recovery gases O2 concentration when plotted against minimum oxygen saturation (p = 0.0419 100% O2, p = 0.7034 21% O2). Collectively, these results suggest exhaled isoprene is dynamic in the laboratory ROBD setup and additional experimentation will be required to fully understand the dynamics of isoprene in response to acute hypoxic stress.

Quantification of plant volatiles.

Plant volatiles occupy diverse roles as signaling molecules, defensive compounds, hormones, and even waste products. Exponential growth in the related literature coupled with the availability of new analytical and computational technologies has inspired novel avenues of inquiry while giving researchers the tools to analyze the plant metabolome to an unprecedented level of detail. As availability of instrumentation and the need for qualitative and especially quantitative metabolic analysis grow within the scientific community so does the need for robust, adaptable, and widely disseminated protocols to enable rapid progression from experimental design to data analysis with minimal input toward method development. This protocol describes the collection and quantitative analysis of plant volatile headspace compounds. It is intended to guide those with little to no experience in analytical chemistry in the quantification of plant volatiles using gas chromatography coupled to mass spectrometry by describing procedures for calibrating and optimizing collection and analysis of these diverse compounds.

Metabolomics of plant volatiles.

Plants communicate with their surrounding ecosystems using a diverse array of volatile metabolites that are indicative of the physiological status of the emitter. A variety of systems have been adapted to capture, analyze, identify, and quantify airborne metabolites released by plants. Metabolomic experiments typically involve four steps: sample collection, preparation, product separation, and data analysis. To date, two different types of headspace sampling, static and dynamic, are widely used for volatile metabolome investigation. For static headspace analysis, solid-phase microextraction (SPME) is used to sample volatiles while push and pull as well as closed-loop stripping methods are used for dynamic headspace sampling. After collection, volatile blends are most efficiently and routinely separated prior to analysis using gas chromatography (GC). Sample preparation is simplified because derivatization is not needed with volatile metabolites. GC coupled to detection by electron impact mass spectrometry (EI-MS) provides high chromatographic resolution, sensitivity, compound-specific detection, quantitation, and the potential to identify unknowns by characteristic and reproducible fragmentation spectra in addition to retention time. A variety of resources can be used to identify unknown compounds in a given volatile sample including >600,000 compounds with known mass spectra catalogued in searchable mass spectral libraries.

Evolution of Cinnamate/p-coumarate carboxyl methyltransferases and their role in the biosynthesis of methylcinnamate.

Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase-like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings.

Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol.

Petunia flower petals emit large amounts of isoeugenol, which has been shown to be synthesized by isoeugenol synthase (PhIGS1) from an ester of coniferyl alcohol, hypothesized to be coniferyl acetate. This paper describes the identification and characterization of a novel petunia gene encoding an enzyme belonging to the BAHD acyltransferase family whose expression correlates with isoeugenol biosynthesis. RNAi suppression of this gene results in inhibition of isoeugenol biosynthesis. Biochemical characterization of the protein encoded by this gene showed that it has acetyltransferase activity and is most efficient with coniferyl alcohol among the alcohol substrates tested. Overall, these data support the conclusion that coniferyl acetate is the substrate of isoeugenol synthase.