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Genome-scale metabolic models have become a fundamental tool for examining metabolic principles. However, metabolism is not solely characterized by the underlying biochemical reactions and catalyzing enzymes, but also affected by regulatory events. Since the pioneering work of Covert and co-workers as well as Shlomi and co-workers it is debated, how regulation and metabolism synergistically characterize a coherent cellular state. The first approaches started from metabolic models, which were extended by the regulation of the encoding genes of the catalyzing enzymes. By now, bioinformatics databases in principle allow addressing the challenge of integrating regulation and metabolism on a system-wide level. Collecting information from several databases we provide a network representation of the integrated gene regulatory and metabolic system for Escherichia coli, including major cellular processes, from metabolic processes via protein modification to a variety of regulatory events. Besides transcriptional regulation, we also take into account regulation of translation, enzyme activities and reactions. Our network model provides novel topological characterizations of system components based on their positions in the network. We show that network characteristics suggest a representation of the integrated system as three network domains (regulatory, metabolic and interface networks) instead of two. This new three-domain representation reveals the structural centrality of components with known high functional relevance. This integrated network can serve as a platform for understanding coherent cellular states as active subnetworks and to elucidate crossover effects between metabolism and gene regulation.

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It is well known that the development of chocolate flavor is initiated during cocoa bean fermentation. Storage proteins undergo the most intensive breakdown yielding peptides and free amino acids, which both serve as flavor precursors. A comprehensive analysis of cocoa proteins and oligopeptides of non-fermented and fermented beans from various geographic origins allows the assessment of systematic differences with respect to their origin as well as fermentation status. Protein quantities as well as their profiles derived from two-dimensional gel electrophoresis, showed striking differences for non-fermented beans depending on their geographical origin. From fermented beans, oligopeptides were relatively quantified by utilizing UHPLC-ESI-Q-q-TOF and annotated based on their characteristic fragmentation pattern in the positive-ion mode. With >800 unique oligopeptides, excluding di- and tri-peptides, across 25 different samples, we are herein reporting on the largest collection of cocoa oligopeptides ever observed and identified. The detected diversity of peptides could not be correlated to the geographical origin but rather to the degree of fermentation. Our findings suggest that the variability in peptide patterns depends on the fermentation method applied in the country of origin ultimately indicating diversified proteolytic activities. Furthermore, our results showed that well-fermented and fair-fermented beans can be differentiated from partially fermented and under-fermented ones by higher numbers and total amounts of oligopeptides.

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Fermentation and drying are the two crucial processing steps required to produce cocoa beans with desired properties, especially taste and flavor. To understand their impact on the lipid profile of cocoa, the lipid composition of unfermented raw and fermented dried beans from six different origins was investigated using high-performance liquid chromatography-mass spectrometry methods. While the comparison of triacylglycerol profiles across the different origins showed only small variations in individual compound concentrations, the comparison along the fermentation status showed major differences regarding the occurrence of polar lipids. These compounds may serve as biomarkers for the fermentation status of the beans and a simple analytical method suitable for field trials is proposed. Finally, a hypothesis identifying key unsaturated triacylglycerols contributing to the hardness and softness of cocoa butter is presented.

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Degradation products of proteins produced during fermentation are believed to be the key precursors of a range of Maillard reactions that deliver the characteristic flavor and aroma of cocoa and chocolate. We have utilized UPLC-ESI-Q-q-TOF to identify and relatively quantify the largest collection of cocoa oligopeptides during a spontaneous fermentation time series using Ivory Coast cocoa beans. Peptides were identified, sequenced by tandem mass spectrometry and annotated based on their characteristic fragmentation pattern in the positive-ion mode. This enabled us to quantitatively trace the sequential degradation of the two main cocoa storage proteins, namely, albumin and vicilin. We observed sequential proteolytic degradation forming longer peptides in the early stages of fermentation and an increasing number of shorter peptides at the latter stages of fermentation. Protein degradation is mediated by both endo- and exopeptidases degrading at either peptide termini. In excess of 800 fermentation peptides could be unambiguously identified, providing unprecedented mechanistic details of cocoa fermentation.

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Hydroxycinnamates including free hydroxycinnamic acids and their chlorogenic acid derivatives and glycosides have been profiled in leaf extracts of 98 Rhododendron species using LC-MS techniques. In total, 69 hydroxycinnamic acid derivatives were identified in the leaves of 98 Rhododendron species. Some derivatives serve as unique phytochemical marker for a single species, whereas other compounds are limited to certain subgenera. The distribution of compounds among six different subgenera of Rhododendron was studied using PCA and PLS-DA. This contribution presents data that provide unique metabolomic insight in the distribution of a class of secondary metabolites within a large selection of species from the botanically diverse plant genus Rhododendron.

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Despite being highly interdependent, the major biochemical networks of the living cell-the networks of interacting genes and of metabolic reactions, respectively-have been approached mostly as separate systems so far. Recently, a framework for interdependent networks has emerged in the context of statistical physics. In a first quantitative application of this framework to systems biology, here we study the interdependent network of gene regulation and metabolism for the model organism Escherichia coli in terms of a biologically motivated percolation model. Particularly, we approach the system's conflicting tasks of reacting rapidly to (internal and external) perturbations, while being robust to minor environmental fluctuations. Considering its response to perturbations that are localized with respect to functional criteria, we find the interdependent system to be sensitive to gene regulatory and protein-level perturbations, yet robust against metabolic changes. We expect this approach to be applicable to a range of other interdependent networks.Although networks of interacting genes and metabolic reactions are interdependent, they have largely been treated as separate systems. Here the authors apply a statistical framework for interdependent networks to E. coli, and show that it is sensitive to gene and protein perturbations but robust against metabolic changes.

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INTRODUCTION: The science of metabolomics offers the possibility to measure full secondary plant metabolomes with limited experimental effort to allow identification of metabolome differences using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of liquid chromatography mass spectrometry (LC-MS) data. OBJECTIVE: To demonstrate a bioinformatics driven hypothesis generator for identification of biologically active compounds in plant crude extracts, which is validated by activity guided fractionation. METHODOLOGY: Crude extracts of Rhododendron leaves were tested for their antibacterial activity using agar diffusion and minimum inhibitory concentration assays. Extracts were profiled by LC-MS. PCA and PLS-DA were used for differentiation of bioactive and inactive extracts and their metabolites. Preparative-high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy were used for separation and structure elucidation of pure compound(s) respectively. RESULTS: An antibacterial Rhododendron collettianum was compared to a series of inactive extracts. Three metabolites were found to distinguish R. collettianum from other species indicating the ability of PCA and PLS-DA to suggest potential bioactive substances. An activity-guided fractionation of R. collettianum extracts was carried out and cannabiorcichromenic acid (CCA) was identified as antibacterial compound thereby validating the PCA and PLS-DA generated hypothesis. Four mammalian cell lines were used to estimate possible cytotoxicity of CCA. CONCLUSION: It was shown that bioinformatics tools facilitate early stage identification of a biologically active compound(s) using LC-MS data, which reduce complexity and number of separation steps in bioactive screening. Copyright © 2017 John Wiley & Sons, Ltd.

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The exceptional diversity of the genus Rhododendron has a strong potential for identification, characterization, and production of bioactive lead compounds for health purposes. A particularly relevant field of application is the search for new antibiotics. Here, we present a comparative analysis of nearly 90 Rhododendron species targeted toward the search for such candidate substances. Through a combination of phytochemical profiles with antimicrobial susceptibility and cytotoxicity, complemented by phylogenetic analyses, we identify seven potentially antimicrobial active but non-cytotoxic compounds in terms of mass-to-charge ratios and retention times. Exemplary bioactivity-guided fractionation for a promising Rhododendron species experimentally supports in fact one of these candidate lead compounds. By combining categorical correlation analysis with Boolean operations, we have been able to investigate the origin of bioactive effects in further detail. Intriguingly, we discovered clear indications of systems effects (synergistic interactions and functional redundancies of compounds) in the manifestation of antimicrobial activities in this plant genus.