Analysis of root volatiles and functional characterization of a root-specific germacrene A synthase in Artemisia pallens.

MAIN CONCLUSION: The study demonstrated that Artemisia pallens roots can be a source of terpene-rich essential oil and root-specific ApTPS1 forms germacrene A contributing to major root volatiles. Davana (Artemisia pallens Bess) is a valuable aromatic herb within the Asteraceae family, highly prized for its essential oil (EO) produced in the aerial parts. However, the root volatile composition, and the genes responsible for root volatiles have remained unexplored until now. Here, we show that A. pallens roots possess distinct oil bodies and yields ~ 0.05% of EO, which is primarily composed of sesquiterpenes ß-elemene, neryl isovalerate, ß-selinene, and α-selinene, and trace amounts of monoterpenes ß-myrcene, D-limonene. This shows that, besides aerial parts, roots of davana can also be a source of unique EO. Moreover, we functionally characterized a terpene synthase (ApTPS1) that exhibited high in silico expression in the root transcriptome. The recombinant ApTPS1 showed the formation of ß-elemene and germacrene A with E,E-farnesyl diphosphate (FPP) as a substrate. Detailed analysis of assay products revealed that ß-elemene was the thermal rearrangement product of germacrene A. The functional expression of ApTPS1 in Saccharomyces cerevisiae confirmed the in vivo germacrene A synthase activity of ApTPS1. At the transcript level, ApTPS1 displayed predominant expression in roots, with significantly lower level of expression in other tissues. This expression pattern of ApTPS1 positively correlated with the tissue-specific accumulation level of germacrene A. Overall, these findings provide fundamental insights into the EO profile of davana roots, and the contribution of ApTPS1 in the formation of a major root volatile.

Diacylglycerol kinase alleviates autophagic degradation of the endoplasmic reticulum in SPT10-deficient yeast to enhance triterpene biosynthesis.

A recent study showed that deletion of the gene encoding the transcription regulator SuPpressor of Ty10 (SPT10) increases total phospholipids, and our previous study established a critical link between phospholipids and the mevalonate/ergosterol (MEV/ERG) pathway, which synthesises triterpenes. This study aims to use spt10Δ yeast to improve triterpene production. Though MEV/ERG pathway was highly expressed in spt10Δ yeast, results showed insufficient accumulation of key metabolites and also revealed massive endoplasmic reticulum (ER) degradation. We found a stable, massive ER structure when we overexpressed diacylglycerol kinase1 (DGK1OE ) in spt10Δ yeast. Analyses of ER-stress and autophagy suggest that DGK1OE in the spt10Δ strain decreased autophagy, resulting in increased MEV/ERG pathway activity. Heterologous expression of ß-amyrin synthase showed significant production of the triterpene ß-amyrin in DGK1OE spt10Δ yeast. Overall, our study provides a strategic approach to improve triterpene production by increasing ER biogenesis while limiting ER degradation.

Betulinic acid mitigates oxidative stress-mediated apoptosis and enhances longevity in the yeast Saccharomyces cerevisiae model.

Betulinic acid (BA), a pentacyclic triterpenoid found in certain plant species, has been reported to have several health benefits including antioxidant and anti-apoptotic properties. However, the mechanism by which BA confers these properties is currently unknown. Saccharomyces cerevisiae, a budding yeast with a short life cycle and conserved cellular mechanism with high homology to humans, was used as a model for determining the role of BA in aging and programmed cell death (PCD). Treatment with hydrogen peroxide (H2O2) exhibited significantly increased (30-35%) survivability of antioxidant (sod1Δ, sod2Δ, cta1Δ, ctt1Δ, and tsa1Δ) and anti-apoptotic (pep4Δ and fis1Δ) mutant strains when cells were pretreated with BA (30 µM) as demonstrated in spot and CFU (Colony forming units) assays. Measurement of intracellular oxidation level using the ROS-specific dye H2DCF-DA showed that all tested BA-pretreated mutants exhibited decreased ROS than the control when exposed to H2O2. Similarly, when mutant strains were pretreated with BA and then exposed to H2O2, there was reduced lipid peroxidation as revealed by the reduced malondialdehyde content. Furthermore, BA-pretreated mutant cells showed significantly lower apoptotic activity by decreasing DNA/nuclear fragmentation and chromatin condensation under H2O2-induced stress as determined by DAPI and acridine orange/ethidium bromide staining. In addition, BA treatment also extended the life span of antioxidant and anti-apoptotic mutants by ∼10-25% by scavenging ROS and preventing apoptotic cell death. Our overall results suggest that BA extends the chronological life span of mutant strains lacking antioxidant and anti-apoptotic genes by lowering the impact of oxidative stress, ROS levels, and apoptotic activity. These properties of BA could be further explored for its use as a valuable nutraceutical.

Triglyceride deficiency and diacylglycerol kinase1 activity lead to the upregulation of mevalonate pathway in yeast: A study for the development of potential yeast platform for improved production of triterpenoid.

Besides energy storage and membrane biogenesis, lipids are known for their numerous biological functions. The two essential lipids, diacylglycerol (DG) and phosphatidic acid (PA), are shown to be associated with cell signalling processes. In this study, we examined whether triglyceride-deficient yeast mutants (tgΔ), dga1Δ and dga1Δlro1Δ, may play an important role in mevalonate (MEV) pathway regulation. Our metabolite analyses revealed that tgΔ cells showed high levels of squalene (SQ) and ergosterol (ERG), which are key indicators of MEV pathway activity. In addition, gene expression studies indicated that the MEV pathway genes in tgΔ cells were significantly upregulated. Interestingly, tgΔ cells exhibited high diacylglycerol kinase1 (DGK1) expression. Furthermore, DGK1 overexpression in WT and tgΔ phenotypes causes a substantial elevation in SQ and ERG levels, and we also found a significant increase in transcript levels of MEV pathway genes, confirming the new role of DGK1 in MEV pathway regulation. This suggests that high DG phosphorylation activity increases the PA pool that may induce the upregulation of MEV pathway in tgΔ cells. The induced MEV pathway is one of the key strategies in the field of synthetic biology for improved production of terpenoids in yeast. Thus, to examine whether increased endogenous MEV pathway flux can be redirected to triterpenoid, ß-Amyrin synthase gene was heterologously expressed in DGK1 overexpressing tgΔ cells that led to significant production of ß-Amyrin, a natural triterpenoid. In conclusion, our findings provide a novel strategy to increase MEV pathway precursors by modulating endogenous signal lipids for improved production of terpenoids.

Orbitofrontal cortex activity and connectivity predict future depression symptoms in adolescence.

BACKGROUND: Major depressive disorder is a leading cause of disability worldwide; however, little is known about pathological mechanisms involved in its development. Research in adolescent depression has focused on reward sensitivity and striatal mechanisms implementing it. The contribution of loss sensitivity to future depression, as well as the orbitofrontal cortex (OFC) mechanisms critical for processing losses and rewards, remain unexplored. Furthermore, it is unclear whether OFC functioning interacts with familial history in predicting future depression. METHODS: In this longitudinal study we recorded functional magnetic resonance imaging (fMRI) data while 229 adolescent females with or without parental history of depression completed a monetary gambling task. We examined if OFC blood-oxygen-level-dependent (BOLD) response and functional connectivity during loss and win feedback was associated with depression symptoms concurrently and prospectively (9 months later), and whether this relationship was moderated by parental history of depression. RESULTS: Reduced OFC response during loss was associated with higher depression symptoms concurrently and prospectively, even after controlling for concurrent depression, specifically in adolescents with parental history of depression. Similarly, increased OFC-posterior insula connectivity during loss was associated with future depression symptoms but this relationship was not moderated by parental history of depression. CONCLUSIONS: This study provides the first evidence for loss-related alterations in OFC functioning and its interaction with familial history of depression as possible mechanisms in the development of depression. While the current fMRI literature has mainly focused on reward, the present findings underscore the need to include prefrontal loss processing in existing developmental models of depression.

Functional connectivity during language processing in acute cocaine withdrawal: a pilot study.

Recent research revealed decreased access to semantic and associative networks in acute cocaine withdrawal. In autism, such behavioral outcomes are associated with decreased functional connectivity using functional magnetic resonance imaging. Therefore, we wished to determine whether connectivity is also decreased in acute cocaine withdrawal. Eight subjects in acute cocaine withdrawal were compared to controls for connectivity in language areas while performing a task involving categorization of words according to semantic and phonological relatedness. Acute withdrawal subjects had significantly less overall connectivity during semantic relatedness, and a trend towards less connectivity during phonological relatedness. Of potential future interest is whether this might serve as an imaging marker for treatment in patients.

Effect of propranolol on functional connectivity in autism spectrum disorder--a pilot study.

A decrease in interaction between brain regions is observed in individuals with autism spectrum disorder (ASD), which is believed to be related to restricted neural network access in ASD. Propranolol, a beta-adrenergic antagonist, has revealed benefit during performance of tasks involving flexibility of access to networks, a benefit also seen in ASD. Our goal was to determine the effect of propranolol on functional connectivity in ASD during a verbal decision making task as compared to nadolol, thereby accounting for the potential spurious fMRI effects due to peripheral hemodynamic effects of propranolol. Ten ASD subjects underwent fMRI scans after administration of placebo, propranolol or nadolol, while performing a phonological decision making task. Comparison of functional connectivity between pre-defined ROI-pairs revealed a significant increase with propranolol compared to nadolol, suggesting a potential imaging marker for the cognitive effects of propranolol in ASD.

Network model of decreased context utilization in autism spectrum disorder.

Individuals with autism spectrum disorders (ASD) demonstrate impaired utilization of context, which allows for superior performance on the "false memory" task. We report the application of a simplified parallel distributed processing model of context utilization to the false memory task. For individuals without ASD, experiments support a model wherein presentation of one word, e.g., ''apple,'' strongly activates the neighboring nodes of closely related words such as ''fruit,'' ''tree,'' whereas in ASD these neighboring nodes are relatively less activated. We demonstrate this model to be consistent with the superior performance on recognition testing on the false memory test, but not on free recall. This may have an anatomic basis in diminished hippocampal neuronal arborization and the abnormal minicolumnar pathology in ASD.