Multiscale Metabolic Modeling: Dynamic Flux Balance Analysis on a Whole-Plant Scale

Plant metabolism is characterized by a unique complexity on the cellular, tissue, and organ levels. On a whole-plant scale, changing source and sink relations accompanying plant development add another level of complexity to metabolism. With the aim of achieving a spatiotemporal resolution of source-sink interactions in crop plant metabolism, a multiscale metabolic modeling (MMM) approach was applied that integrates static organ-specific models with a whole-plant dynamic model. Allowing for a dynamic flux balance analysis on a whole-plant scale, the MMM approach was used to decipher the metabolic behavior of source and sink organs during the generative phase of the barley (Hordeum vulgare) plant. It reveals a sink-to-source shift of the barley stem caused by the senescence-related decrease in leaf source capacity, which is not sufficient to meet the nutrient requirements of sink organs such as the growing seed. The MMM platform represents a novel approach for the in silico analysis of metabolism on a whole-plant level, allowing for a systemic, spatiotemporally resolved understanding of metabolic processes involved in carbon partitioning, thus providing a novel tool for studying yield stability and crop improvement.Plants are of vital significance as a source of food (Grusak and DellaPenna, 1999; Rogalski and Carrer, 2011), feed (Lu et al., 2011), energy (Tilman et al., 2006; Parmar et al., 2011), and feedstocks for the chemical industry (Metzger and Bornscheuer, 2006; Kinghorn et al., 2011). Given the close connection between plant metabolism and the usability of plant products, there is a growing interest in understanding and predicting the behavior and regulation of plant metabolic processes. In order to increase crop quality and yield, there is a need for methods guiding the rational redesign of the plant metabolic network (Schwender, 2009).Mathematical modeling of plant metabolism offers new approaches to understand, predict, and modify complex plant metabolic processes. In plant research, the issue of metabolic modeling is constantly gaining attention, and different modeling approaches applied to plant metabolism exist, ranging from highly detailed quantitative to less complex qualitative approaches (for review, see Giersch, 2000; Morgan and Rhodes, 2002; Poolman et al., 2004; Rios-Estepa and Lange, 2007).A widely used modeling approach is flux balance analysis (FBA), which allows the prediction of metabolic capabilities and steady-state fluxes under different environmental and genetic backgrounds using (non)linear optimization (Orth et al., 2010). Assuming steady-state conditions, FBA has the advantage of not requiring the knowledge of kinetic parameters and, therefore, can be applied to model detailed, large-scale systems. In recent years, the FBA approach has been applied to several different plant species, such as maize (Zea mays; Dal’Molin et al., 2010; Saha et al., 2011), barley (Hordeum vulgare; Grafahrend-Belau et al., 2009b; Melkus et al., 2011; Rolletschek et al., 2011), rice (Oryza sativa; Lakshmanan et al., 2013), Arabidopsis (Arabidopsis thaliana; Poolman et al., 2009; de Oliveira Dal’Molin et al., 2010; Radrich et al., 2010; Williams et al., 2010; Mintz-Oron et al., 2012; Cheung et al., 2013), and rapeseed (Brassica napus; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011), as well as algae (Boyle and Morgan, 2009; Cogne et al., 2011; Dal’Molin et al., 2011) and photoautotrophic bacteria (Knoop et al., 2010; Montagud et al., 2010; Boyle and Morgan, 2011). These models have been used to study different aspects of metabolism, including the prediction of optimal metabolic yields and energy efficiencies (Dal’Molin et al., 2010; Boyle and Morgan, 2011), changes in flux under different environmental and genetic backgrounds (Grafahrend-Belau et al., 2009b; Dal’Molin et al., 2010; Melkus et al., 2011), and nonintuitive metabolic pathways that merit subsequent experimental investigations (Poolman et al., 2009; Knoop et al., 2010; Rolletschek et al., 2011). Although FBA of plant metabolic models was shown to be capable of reproducing experimentally determined flux distributions (Williams et al., 2010; Hay and Schwender, 2011b) and generating new insights into metabolic behavior, capacities, and efficiencies (Sweetlove and Ratcliffe, 2011), challenges remain to advance the utility and predictive power of the models.Given that many plant metabolic functions are based on interactions between different subcellular compartments, cell types, tissues, and organs, the reconstruction of organ-specific models and the integration of these models into interacting multiorgan and/or whole-plant models is a prerequisite to get insight into complex plant metabolic processes organized on a whole-plant scale (e.g. source-sink interactions). Almost all FBA models of plant metabolism are restricted to one cell type (Boyle and Morgan, 2009; Knoop et al., 2010; Montagud et al., 2010; Cogne et al., 2011; Dal’Molin et al., 2011), one tissue or one organ (Grafahrend-Belau et al., 2009b; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011; Mintz-Oron et al., 2012), and only one model exists taking into account the interaction between two cell types by specifying the interaction between mesophyll and bundle sheath cells in C4 photosynthesis (Dal’Molin et al., 2010). So far, no model representing metabolism at the whole-plant scale exists.Considering whole-plant metabolism raises the problem of taking into account temporal and environmental changes in metabolism during plant development and growth. Although classical static FBA is unable to predict the dynamics of metabolic processes, as the network analysis is based on steady-state solutions, time-dependent processes can be taken into account by extending the classical static FBA to a dynamic flux balance analysis (dFBA), as proposed by Mahadevan et al. (2002). The static (SOA) and dynamic optimization approaches introduced in this work provide a framework for analyzing the transience of metabolism by integrating kinetic expressions to dynamically constrain exchange fluxes. Due to the requirement of knowing or estimating a large number of kinetic parameters, so far dFBA has only been applied to a plant metabolic model once, to study the photosynthetic metabolism in the chloroplasts of C3 plants by a simplified model of five biochemical reactions (Luo et al., 2009). Integrating a dynamic model into a static FBA model is an alternative approach to perform dFBA.In this study, a multiscale metabolic modeling (MMM) approach was applied with the aim of achieving a spatiotemporal resolution of cereal crop plant metabolism. To provide a framework for the in silico analysis of the metabolic dynamics of barley on a whole-plant scale, the MMM approach integrates a static multiorgan FBA model and a dynamic whole-plant multiscale functional plant model (FPM) to perform dFBA. The performance of the novel whole-plant MMM approach was tested by studying source-sink interactions during the seed developmental phase of barley plants.     

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.     

Continuous ECS-indicated recording of the proton-motive charge flux in leaves

Technical features and examples of application of a special emitter–detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550–520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, not only allows steady-state assessment of the proton motive force (pmf) and its partitioning into ΔpH and ΔΨ components, but also continuous recording of the overall charge flux driven by photosynthetic light reactions. The new approach employs a double-modulation technique to derive a continuous signal from the light/dark modulation amplitude of the P515 signal. This new, continuously measured signal primarily reflects the rate of proton efflux via the ATP synthase, which under quasi-stationary conditions corresponds to the overall rate of proton influx driven by coupled electron transport. Simultaneous measurements of charge flux and CO₂ uptake as a function of light intensity indicated a close to linear relationship in the light-limited range. A linear relationship between these two signals was also found for different internal CO₂ concentrations, except for very low CO₂, where the rate of charge flux distinctly exceeded the rate of CO₂ uptake. Parallel oscillations in CO₂ uptake and charge flux were induced by high CO₂ and O₂. The new device may contribute to the elucidation of complex regulatory mechanisms in intact leaves.     

Discovery of a small-molecule inhibitor and cellular probe of Keap1–Nrf2 protein–protein interaction

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1–Nrf2 protein–protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure–activity relationships support its use as a lead for our ongoing optimization     

Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells

The histone methyltransferase G9a is overexpressed in a variety of cancer types, including pancreatic adenocarcinoma, and promotes tumor invasiveness and metastasis. We recently reported the discovery of BRD4770, a small-molecule inhibitor of G9a that induces senescence in PANC-1 cells. We observed that the cytotoxic effects of BRD4770 were dependent on genetic background, with cell lines lacking functional p53 being relatively resistant to compound treatment. To understand the mechanism of genetic selectivity, we used two complementary screening approaches to identify enhancers of BRD4770. The natural product and putative BH3 mimetic gossypol enhanced the cytotoxicity of BRD4770 in a synergistic manner in p53-mutant PANC-1 cells but not in immortalized non-tumorigenic pancreatic cells. The combination of gossypol and BRD4770 increased LC3-II levels and the autophagosome number in PANC-1 cells, and the compound combination appears to act in a BNIP3 (B-cell lymphoma 2 19-kDa interacting protein)-dependent manner, suggesting that these compounds act together to induce autophagy-related cell death in pancreatic cancer cells.     

Natalizumab Treatment Reduces Fatigue in Multiple Sclerosis. Results from the TYNERGY Trial; A Study in the Real Life Setting

Fatigue is a significant symptom in multiple sclerosis (MS) patients. First-generation disease modifying therapies (DMTs) are at best moderately effective to improve fatigue. Observations from small cohorts have indicated that natalizumab, an antibody targeting VLA-4, may reduce MS-related fatigue. The TYNERGY study aimed to further evaluate the effects of natalizumab treatment on MS-related fatigue. In this one-armed clinical trial including 195 MS patients, natalizumab was prescribed in a real-life setting, and a validated questionnaire, the Fatigue Scale for Motor and Cognitive functions (FSMC), was used both before and after 12 months of treatment to evaluate a possible change in the fatigue experienced by the patients. In the treated cohort all measured variables, that is, fatigue score, quality of life, sleepiness, depression, cognition, and disability progression were improved from baseline (all p values<0.0001). Walking speed as measured by the six-minute walk-test also increased at month 12 (p = 0.0016). All patients were aware of the nature of the treatment agent, and of the study outcomes.

Conclusion

Natalizumab, as used in a real-life setting, might improve MS-related fatigue based on the results from this one-armed un-controlled stud. Also other parameters related to patients'' quality of life seemed to improve with natalizumab treatment.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00884481","term_id":"NCT00884481"}}NCT00884481     

Decoupling the retention time of easily degradable and persistent substances using ultrafiltration membranes increases biogas production yield

The decoupling of the retention time of easily degradable and persistent substances relieves the degradation process from inhibitors and increases the biogas yield. Anaerobic digestion of maize silage was investigated in a pilot‐scale plant with a coupled ultrafiltration membrane. The aim of the study was the evaluation of the influence of the membrane‐based relief of the degradation process and the increase of the retention time of persistent substances. For that purpose, the fermenter content was separated into solid and liquid fractions. The solid fraction was recirculated to the fermenter for longer retention time and higher substrate degradation rates. The fermentation process was improved by the removal of the liquid fraction and adding volatile fatty acids. The results showed an increase of the biogas yield by 7.2% in comparison to the anaerobic digestion without membrane filtration.     

Domestication and crop evolution of wheat andbarley: Genes,genomics, and future directions

Wheat and barley are two of the founder crops of the agricultural revolution that took place 10,000 years ago in the Fertile Crescent and both crops remain among the world's most important crops. Domestication of these crops from their wild ancestors required the evolution of traits useful to humans, rather than survival in their natural environment. Of these traits, grain retention and threshability, yield improvement, changes to photoperiod sensitivity and nutritional value are most pronounced between wild and domesticated forms. Knowledge about the geographical origins of these crops and the genes responsible for domestication traits largely pre-dates the era of nextgeneration sequencing, although sequencing will lead to new insights. Molecular markers were initially used to calculate distance(relatedness), genetic diversity and to generate genetic maps which were useful in cloning major domestication genes. Both crops are characterized by large,complex genomes which were long thought to be beyond the scope of whole-genome sequencing. However, advances in sequencing technologies have improved the state of genomic resources for both wheat and barley. The availability of reference genomes for wheat and some of its progenitors,as well as for barley, sets the stage for answering unresolved questions in domestication genomics of wheat and barley.