Doris Engelmeier

mzGroupAnalyzer-Predicting Pathways and Novel Chemical Structures from Untargeted High-Throughput Metabolomics Data

The metabolome is a highly dynamic entity and the final readout of the genotype x environment x phenotype (GxExP) relationship of an organism. Monitoring metabolite dynamics over time thus theoretically encrypts the whole range of possible chemical and biochemical transformations of small molecules involved in metabolism. The bottleneck is, however, the sheer number of unidentified structures in these samples. This represents the next challenge for metabolomics technology and is comparable with genome sequencing 30 years ago. At the same time it is impossible to handle the amount of data involved in a metabolomics analysis manually. Algorithms are therefore imperative to allow for automated m/z feature extraction and subsequent structure or pathway assignment. Here we provide an automated pathway inference strategy comprising measurements of metabolome time series using LC- MS with high resolution and high mass accuracy. An algorithm was developed, called mzGroupAnalyzer, to automatically explore the metabolome for the detection of metabolite transformations caused by biochemical or chemical modifications. Pathways are extracted directly from the data and putative novel structures can be identified. The detected m/z features can be mapped on a van Krevelen diagram according to their H/C and O/C ratios for pattern recognition and to visualize oxidative processes and biochemical transformations. This method was applied to Arabidopsis thaliana treated simultaneously with cold and high light. Due to a protective antioxidant response the plants turn from green to purple color via the accumulation of flavonoid structures. The detection of potential biochemical pathways resulted in 15 putatively new compounds involved in the flavonoid-pathway. These compounds were further validated by product ion spectra from the same data. The mzGroupAnalyzer is implemented in the graphical user interface (GUI) of the metabolomics toolbox COVAIN (Sun & Weckwerth, 2012, Metabolomics 8: 81–93). The strategy can be extended to any biological system.

Synthesis of Side Chain Substituted 3-Butylisocoumarins and Absolute Configurations of Natural Isocoumarins from Artemisia dracunculus

Summary.A series of 3-hydroxy- and 3-bromobutyl-substituted isocoumarins was synthesized. The absolute configurations of three isocoumarin derivatives from Artemisia dracunculus, namely (−)-epoxyartemidin, (−)-2′-methoxydihydroartemidin, and (+)-3′-hydroxyartemidin were determined by chemical means via derivatization, kinetic racemate resolution (Horeau method), and comparison of the circular dichroism (CD) spectra.

System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti-depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity-dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N-phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N-phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.

Combining microdilution with MicroResp™: Microbial substrate utilization, antimicrobial susceptibility and respiration

Pharmacological studies focus on susceptibility of pathogenic microbes against specific drugs or combinations of them, ecological studies on substrate utilization efficiency of variable microbial communities. The MicroResp™ system was especially developed to study soil microbial communities. It was slightly modified to facilitate exploring of microbial growth efficiency in a concentration-dependent fashion (microdilutions of carbohydrate mixtures or specific toxic chemicals). After turbidimetric growth assessment, colorimetric indicator plates (cresol red agar) were mounted to the assay plates. The substrate utilisation design is illustrated by glucose and a plant carbohydrate mixture, the antimicrobial susceptibility design by the naphthoquinone juglone. Dose-response effects are explored by curve fitting of nonlinear models that especially have been developed to detect hormetic effects that are characterized by stimulation at lower followed by inhibition at higher dosages (U- and inverse U-shaped effects). Multivariate analyses are presented utilizing metavariables that were obtained in the curve fitting process of the measured parameters growth and respiration and the factor growth efficiency.

Development of a pharmacodynamic screening model with Crithidia fasciculata

ZusammenfassungDas Genus Crithidia gehört zur Familie der Trypanosomatidae, welche u.a. auch die Genera Leishmania und Trypanosoma umfasst. Mit diesen Genera bestehen Gemeinsamkeiten hinsichtlich biochemischer Vorgänge, wie der Polyaminsynthese und dem Methioninstoffwechsel. Daher wurde Crithidia fasciculata als Modellorganismus für die Untersuchung der antiparasitären Aktivität verschiedener Substanzen herangezogen. C. fasciculata kommt in Insekten und Amphibien natürlich vor, ist aber nicht humanpathogen. Zunächst wurden verschiedene Kulturmedien ausgewertet, von welchen sich TPS am besten für die Stammpassage eignet und E-Medium – eine neue Formulierung – für die eigentliche Sensibilitätsprüfung. Optimales Wachstum von C. fasciculata wurde unter mikro-aerophiler Atmosphäre beobachtet. Schließlich wurde ein System für die Sensibilitätstestung ausgearbeitet und in der Auswertung von Extrakten aus höheren tropischen Pflanzen der Genera Stemona und Aglaia eingesetzt. Extrakte mit signifikanter Crithidienhemmung wurden für die chromatographische Fraktionierung ausgewählt, mit nachfolgender Isolierung, Strukturanalyse und weiterer Aktivitätsbestimmung der reinen Inhaltsstoffe. Ermutigende Ergebnisse wurden mit Extrakten von Aglaia odorata (Blättern), A. elaeagnoidea (Borke) und A. edulis (Blättern) erzielt, mit EC90-Werten von 1213 ng/ml, 1606 ng/ml bezw. 1462 ng/ml.SummaryThe genus Crithidia is a member of the family Trypanosomatidae and is related to the genera Leishmania and Trypanosoma with which it shares a variety of biochemical mechanisms, such as polyamine synthesis and methionin salvage. In consequence, a screening system for antiparasitic candidate material has been developed with Crithidia fasciculata, a parasite naturally occurring in insects and amphibians, but devoid of pathogenicity for humans. Initially a variety of culture media were evaluated of which TPS was best suited for the maintenance of stock cultures, and E-medium – a newly developed formula – for sensitivity testing. Optimal growth of C. fasciculata was observed under microaerophilic conditions. A system for sensitivity testing was developed and applied to the investigation of extracts from higher tropical plants of the genera Stemona and Aglaia for anticrithidial activity. Extracts with significant anti-crithidial activity were scheduled for chromatographic fractionation and the subsequent isolation, purification and structural identification of individual compounds for further sensitivity testing. Encouraging results were obtained with extracts from Aglaia odorata leaves, A. elaeagnoidea stem bark and A. edulis leaves, with EC90 values of 1213 ng/ml, 1606 ng/ml, and 1462 ng/ml, respectively.

Chapter 17 Antifungal natural products: assays and applications

Publisher Summary Various ecological models explain species diversity, and it has been found that microbial biodiversity is closely linked to plant biodiversity. A mathematical exploration of a feedback model among plants, their competitor plants, and the microbial communities of both concludes that negative feedbacks promote the coexistence of species. In this context, the evaluation of antifungal properties of natural products may constitute an important tool to test various hypotheses in exploring extant interactions of plant and fungi in a community scenario. As a consequence, bioassays determining susceptibility of fungal isolates to specific secondary metabolites may become a key technology in this field. As disease outbreaks also occur in natural plant communities, a parallel exploration of agricultural and natural ecosystems has to be carried out to develop new and optimize existing sustainable control mechanisms. It has been reported recently that complex symbiotic communities may be determined by antifungal compounds produced by ant-associated bacteria belonging to genera Streptomyces and Burkholderia .