BackgroundPlant natural products have been co-opted for millennia by humans for various uses such as flavor, fragrances, and medicines. These compounds often are only produced in relatively low amounts and are difficult to chemically synthesize, limiting access. While elucidation of the underlying biosynthetic processes might help alleviate these issues (e.g., via metabolic engineering), investigation of this is hindered by the low levels of relevant gene expression and expansion of the corresponding enzymatic gene families. However, the often-inducible nature of such metabolic processes enables selection of those genes whose expression pattern indicates a role in production of the targeted natural product.ResultsHere, we combine metabolomics and transcriptomics to investigate the inducible biosynthesis of the bioactive diterpenoid tanshinones from the Chinese medicinal herb, Salvia miltiorrhiza (Danshen). Untargeted metabolomics investigation of elicited hairy root cultures indicated that tanshinone production was a dominant component of the metabolic response, increasing at later time points. A transcriptomic approach was applied to not only define a comprehensive transcriptome (comprised of 20,972 non-redundant genes), but also its response to induction, revealing 6,358 genes that exhibited differential expression, with significant enrichment for up-regulation of genes involved in stress, stimulus and immune response processes. Consistent with our metabolomics analysis, there appears to be a slower but more sustained increased in transcript levels of known genes from diterpenoid and, more specifically, tanshinone biosynthesis. Among the co-regulated genes were 70 transcription factors and 8 cytochromes P450, providing targets for future investigation.ConclusionsOur results indicate a biphasic response of Danshen terpenoid metabolism to elicitation, with early induction of sesqui- and tri- terpenoid biosynthesis, followed by later and more sustained production of the diterpenoid tanshinones. Our data provides a firm foundation for further elucidation of tanshinone and other inducible natural product metabolism in Danshen.
A diverse array of neurotransmitters and neuromodulators control and affect brain function. A profound understanding of the signaling pathways and the neural circuits underlying behavior is therefore likely to require the tracking of concentration changes of active neurochemicals. In the present study, we demonstrate the feasibility of a method allowing the simultaneous determination of the concentrations of six neurotransmitters: acetylcholine, serotonin, dopamine, g-aminobutyric acid (GABA), glutamate and aspartate, in the extracellular brain fluid (EBF). We used hydrophilic interaction chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS) to analyze the EBF from the monkey brain. A push-pull sampling method was used to collect EBF from the prefrontal cortex (PFC) of conscious monkeys at flow rates in the range of low nL/min. The detection limits of acetylcholine, serotonin, dopamine, GABA, glutamate and aspartate were 0.015, 0.15, 0.3, 1.2, 6 and 15 femtomoles, respectively, allowing us to quantitatively determine the concentrations of these six neurotransmitters simultaneously from 500 nL in vivo samples. We conclude that HILIC/ MS/MS combined with the push-pull sampling method represents a sensitive technique for simultaneous monitoring of neurotransmitters from EBF samples.
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