Neurobehavioral and molecular changes in a rodent model of ACTH-induced HPA axis dysfunction.

Hypothalamic-pituitary-adrenal (HPA) axis dysregulation is linked to the pathophysiology of depression. Although exogenous adrenocorticotropic hormone (ACTH) is associated with a depressive-like phenotype in rodents, comprehensive neurobehavioral and mechanistic evidence to support these findings are limited. Sprague-Dawley rats (male, n = 30; female, n = 10) were randomly assigned to the control (male, n = 10) or ACTH (male, n = 20; female n = 10) groups that received saline (0.1 ml, sc.) or ACTH (100 µg/day, sc.), respectively, for two weeks. Thereafter, rats in the ACTH group were subdivided to receive ACTH plus saline (ACTH_S; male, n = 10; female, n = 5; 0.2 ml, ip.) or ACTH plus imipramine (ACTH_I; male, n = 10; female, n = 5;10 mg/kg, ip.) for a further four weeks. Neurobehavioral changes were assessed using the forced swim test (FST), the sucrose preference test (SPT), and the open field test (OFT). Following termination, the brain regional mRNA expression of BDNF and CREB was determined using RT-PCR. After two-weeks, ACTH administration significantly increased immobility in the FST (p = 0.03), decreased interaction with the center of the OFT (p < 0.01), and increased sucrose consumption (p = 0.03) in male, but not female rats. ACTH administration significantly increased the expression of BDNF in the hippocampus and CREB in all brain regions in males (p < 0.05), but not in female rats. Imipramine treatment did not ameliorate these ACTH-induced neurobehavioral or molecular changes. In conclusion, ACTH administration resulted in a sex-specific onset of depressive-like symptoms and changes in brain regional expression of neurotrophic factors. These results suggest sex-specific mechanisms underlying the development of depressive-like behavior in a model of ACTH-induced HPA axis dysregulation.

The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones.

Microbial competition within plant tissues affects invading pathogens' fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce's disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography-mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria.

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.

An integrated workflow for phenazine-modifying enzyme characterization.

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.

Exometabolomic Analysis of Cross-Feeding Metabolites.

Microbial consortia have the potential to perform complex, industrially important tasks. The design of microbial consortia requires knowledge of the substrate preferences and metabolic outputs of each member, to allow understanding of potential interactions such as competition and beneficial metabolic exchange. Here, we used exometabolite profiling to follow the resource processing by a microbial co-culture of two biotechnologically relevant microbes, the bacterial cellulose degrader Cellulomonas fimi, and the oleaginous yeast Yarrowia lipolytica. We characterized the substrate preferences of the two strains on compounds typically found in lignocellulose hydrolysates. This allowed prediction that specific sugars resulting from hemicellulose polysaccharide degradation by C. fimi may serve as a cross-feeding metabolites to Y. lipolytica in co-culture. We also showed that products of ionic liquid-treated switchgrass lignocellulose degradation by C. fimi were channeled to Y. lipolytica in a co-culture. Additionally, we observed metabolites, such as shikimic acid accumulating in the co-culture supernatants, suggesting the potential for producing interesting co-products. Insights gained from characterizing the exometabolite profiles of individual and co-cultures of the two strains can help to refine this interaction, and guide strategies for making this an industrially viable co-culture to produce valuable products from lignocellulose material.

A fungal transcription factor essential for starch degradation affects integration of carbon and nitrogen metabolism.

In Neurospora crassa, the transcription factor COL-26 functions as a regulator of glucose signaling and metabolism. Its loss leads to resistance to carbon catabolite repression. Here, we report that COL-26 is necessary for the expression of amylolytic genes in N. crassa and is required for the utilization of maltose and starch. Additionally, the Δcol-26 mutant shows growth defects on preferred carbon sources, such as glucose, an effect that was alleviated if glutamine replaced ammonium as the primary nitrogen source. This rescue did not occur when maltose was used as a sole carbon source. Transcriptome and metabolic analyses of the Δcol-26 mutant relative to its wild type parental strain revealed that amino acid and nitrogen metabolism, the TCA cycle and GABA shunt were adversely affected. Phylogenetic analysis showed a single col-26 homolog in Sordariales, Ophilostomatales, and the Magnaporthales, but an expanded number of col-26 homologs in other filamentous fungal species. Deletion of the closest homolog of col-26 in Trichoderma reesei, bglR, resulted in a mutant with similar preferred carbon source growth deficiency, and which was alleviated if glutamine was the sole nitrogen source, suggesting conservation of COL-26 and BglR function. Our finding provides novel insight into the role of COL-26 for utilization of starch and in integrating carbon and nitrogen metabolism for balanced metabolic activities for optimal carbon and nitrogen distribution.

Seasonal accumulation of major alkaloids in organs of pharmaceutical crop Narcissus Carlton.

Narcissus pseudonarcissus (L.) cv. Carlton is being cultivated as a main source of galanthamine from the bulbs. After galanthamine, haemanthamine and narciclasine are the next most abundant alkaloids in this cultivar. Both these compounds are promising chemical scaffolds for potential anticancer drugs. For further research and drug development, a reliable supply of these compounds will be needed. In this study a field experiment was conducted to investigate the levels of galanthamine, haemanthamine and narciclasine in plants of N. pseudonarcissus cv. Carlton. In a field experiment alkaloids in the bulbs, leaves and roots were analyzed by quantitative (1)H NMR to monitor the variations during the growing season. Major primary and secondary metabolites were identified in the various plant parts. Multivariate data analysis was performed on the (1)H NMR spectra to investigate how metabolites changed in the plant organs over time. The results show that the leaves have relatively high concentrations of the alkaloids before flowering. The bulbs had lower concentrations of the compounds of interest but would have a higher total yield of alkaloids due to bigger biomass. Narcissus pseudonarcissus cv. Carlton represents a good source of galanthamine, and can potentially be a source of the other major alkaloids depending on choice of organ and harvest time.

Effects of fungicides on galanthamine and metabolite profiles in Narcissus bulbs.

Large-scale plant cultivation usually involves the use of pesticides. Apart from eliminating the target organism, the external chemicals may affect the metabolism of the crop plant. This may have implications for plants cultivated for specific medicinal compounds. In this study the effects of diverse fungicides on the metabolism of Narcissus pseudonarcissus cv. Carlton bulbs were investigated. N. pseudonarcissus cv. Carlton is being cultivated for the extraction of the alkaloid galanthamine. Fungicides typically used in Narcissus cultivation were applied in a field experiment. The aim was to determine whether fungicide applications changed the concentration of galanthamine in the bulbs. (1)H NMR spectroscopy allowed quantitative analysis of galanthamine and other metabolites in bulb extracts. Multivariate data analysis revealed changes in bulb metabolite patterns caused by fungicides. Bulbs treated before planting generally had higher levels of alkaloids, while foliar field applications caused lower alkaloid levels but altered carbohydrate metabolism. Within these groups, certain fungicide treatments caused changes in specific metabolites. This study shows that the fungicides used in Narcissus cultivation can cause a change in the metabolome still detectable in the bulbs after harvest. The standard cultivation practices in terms of fungicide treatment were found suitable for the production of N. pseudonarcissus cv. Carlton as raw material for galanthamine extraction. In the cultivation of medicinal plants for secondary metabolites the potential effect of pesticides and other agrochemicals should be taken into account.

Ethnopharmacology in overdrive: the remarkable anti-HIV activity of Artemisia annua.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua contains the well-known antimalarial compound artemisinin, which forms the backbone of the global malaria treatment regime. In African countries a tea infusion prepared from Artemisia annua has been used for the treatment of malaria only for the past 10-20 years. Several informal claims in Africa exist that the Artemisia annua tea infusions are also able to inhibit HIV. Since HIV is a relatively newly emerged disease, the claims, if substantiated, could provide a very good example of "ethnopharmacology in overdrive". The objective of this study was to provide quantitative scientific evidence that the Artemisia annua tea infusion exhibits anti-HIV activity through in vitro studies. A second objective was to determine if artemisinin plays a direct or indirect (synergistic) role in any observed activity. This was done by the inclusion of a chemically closely related species, Artemisia afra, known not to contain any artemisinin in our studies. MATERIALS AND METHODS: Validated cellular systems were used to test Artemisia annua tea samples for anti-HIV activity. Two independent tests with different formats (an infection format and a co-cultivation format) were used. Samples were also tested for cellular toxicity against the human cells used in the assays. RESULTS: The Artemisia annua tea infusion was found to be highly active with IC(50) values as low as 2.0 µg/mL. Moreover we found that artemisinin was inactive at 25 µg/mL and that a chemically related species Artemisia afra (not containing artemisinin) showed a similar level of activity. This indicates that the role of artemisinin, directly or indirectly (synergism), in the observed activity is rather limited. Additionally, no cellular toxicity was seen for the tea infusion at the highest concentrations tested. CONCLUSION: This study provides the first in vitro evidence of anti-HIV activity of the Artemisia annua tea infusion. We also report for the first time on the anti-HIV activity of Artemisia afra although this was not an objective of this study. These results open the way to identify new active pharmaceutical ingredients in Artemisia annua and thereby potentially reduce the cost for the production of the important antimalarial compound artemisinin.

Effect of fertilizers on galanthamine and metabolite profiles in Narcissus bulbs by 1H NMR.

Narcissus bulbs contain the biologically active alkaloid galanthamine, and Narcissus is being developed as a natural source of the molecule for the pharmaceutical industry. The effect of fertilizer on galanthamine production was investigated in a field study using a (1)H nuclear magnetic resonance (NMR) metabolite profiling approach. Galanthamine was quantitated and major metabolites in the bulbs were identified. The application of standard fertilization levels of nitrogen and potassium caused a significant increase in galanthamine as compared to a control. Multivariate data analysis of the (1)H NMR data revealed that applying double the standard level of nitrogen fertilizer resulted in production of more amino acids and citric acid cycle intermediates, but not more galanthamine. The results indicated that standard levels of fertilizer currently applied in The Netherlands are sufficient for optimal galanthamine accumulation in the bulbs. This study shows how (1)H NMR-based metabolic profiling can provide insight into the response of plant metabolism to agricultural practices.

Analysis of metabolic variation and galanthamine content in Narcissus bulbs by 1H NMR.

INTRODUCTION: Galanthamine is a benzazepine alkaloid used as a drug to relieve symptoms of Alzheimer's disease. For pharmaceutical use this natural product has been extracted from the plant Leucojum aestivum (Amaryllidaceae) or produced synthetically. Limited supply of the natural source and high cost of synthetic production has led to a search for alternative sources of galanthamine. The bulbs of Narcissus pseudonarcissus (Amaryllidaceae) have been identified as a potential source of raw material for galanthamine extraction. Since inconsistent chemical composition can be an issue with medicinal plant material, it is of interest to know whether large variations occur between Narcissus bulbs grown in different geographical locations. OBJECTIVE: To evaluate whether large differences exist in the overall metabolic profiles of Narcissus bulbs grown in the two most important cultivation regions. METHODOLOGY: (1)H NMR and principal component analysis were used for an unbiased comparison of the bulb samples. RESULTS: Overall metabolite profiles were quite similar, but galanthamine levels could slightly discriminate samples by geographical region. (1)H NMR was used for quantitation of galanthamine, and was found to be comparable to quantitation by HPLC. Compared with conventional chromatographic methods, sample preparation for (1)H NMR analysis is simple and rapid, and only a small amount of plant material is required. CONCLUSIONS: Since useful qualitative and quantitative information about the metabolic state of Narcissus bulbs can be obtained by (1)H NMR, this method is useful for agricultural applications, and for quality control of raw material used in the pharmaceutical industry.

Comparison of activity of immobilised and soluble metalloporphyrins by NO release of oximes by chemiluminescence.

The ongoing interest in very efficient systems for the imitation of cytochrome P-450-dependent monooxygenase reactions, consisting of metalloporphyrin and oxygen donor, prompted us to develop a method to compare the catalytic activity of soluble metalloporphyrins with those which have been immobilised on different silica surfaces. In this article, we describe a method which allows direct comparison between the catalytic activities of soluble and immobilised metalloporphyrins by measuring the NO release of cyclohexanone oxime. We observed that all metalloporphyrins are able to enhance the NO release of cyclohexanone oxime in the presence of iodosobenzene as oxygen donor. In case of cationic metalloporphyrins, no difference in the catalytic activity was determined. In contrast, for perfluorinated metalloporphyrins we ascertained a major loss of activity after immobilisation. These results led us to the assumption that it is not easy to compare quantitative results of NO releases if soluble and immobilised metalloporphyrins were used.