Browsing by Author "Feuer, Ronny"

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Open Access
Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol
(2012) Feuer, Ronny; Gottlieb, Katrin; Viertel, Gero; Klotz, Johannes; Schober, Steffen; Bossert, Martin; Sawodny, Oliver; Sprenger, Georg; Ederer, Michael
Bacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. A block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in E. coli. The resulting mutant strain was able to grow on a medium containing glycerol and lactate, which served as an exogenous pyruvate source. Heterologous expression of a pyruvate carboxylase gene from Corynebacterium glutamicum was used for anaplerosis of the TCA cycle. Selective conditions were controlled in a continuous culture with limited lactate feed and an excess of glycerol feed. After 200–300 generations pyruvate-prototrophic mutants were isolated. The genomic analysis of an evolved strain revealed that the genotypic basis for the regained pyruvate-prototrophy was not obvious. A constraint-based model of the metabolism was employed to compute all possible detours around the given metabolic block by solving a hierarchy of linear programming problems. The regulatory network was expected to be responsible for the adaptation process. Hence, a Boolean model of the transcription factor network was connected to the metabolic model. Our model analysis only showed a marginal impact of transcriptional control on the biomass yield on substrate which is a key variable in the selection process. In our experiment, microarray analysis confirmed that transcriptional control probably played a minor role in the deregulation of the alternative pathways for the circumvention of the block.
Open Access
Model-based characterization of inflammatory gene expression patterns of activated macrophages
(2016) Rex, Julia; Albrecht, Ute; Ehlting, Christian; Thomas, Maria; Zanger, Ulrich M.; Sawodny, Oliver; Häussinger, Dieter; Ederer, Michael; Feuer, Ronny; Bode, Johannes G.
Macrophages are cells with remarkable plasticity. They integrate signals from their microenvironment leading to context-dependent polarization into classically (M1) or alternatively (M2) activated macrophages, representing two extremes of a broad spectrum of divergent phenotypes. Thereby, macrophages deliver protective and pro-regenerative signals towards injured tissue but, depending on the eliciting damage, may also be responsible for the generation and aggravation of tissue injury. Although incompletely understood, there is emerging evidence that macrophage polarization is critical for these antagonistic roles. To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied. A comprehensive literature-based Boolean model was developed to describe the M1 (LPS-activated) and M2 (IL-4/13-activated) polarization types. The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages. By dynamic modeling of gene expression, the chronology of pathway activation and autocrine signaling was estimated. Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation, indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization.
Open Access
Rapid sampling of Escherichia coli after changing oxygen conditions reveals transcriptional dynamics
(2017) Wulffen, Joachim von; Ulmer, Andreas; Jäger, Günter; Sawodny, Oliver; Feuer, Ronny
Escherichia coli is able to shift between anaerobic and aerobic metabolism by adapting its gene expression, e.g., of metabolic genes, to the new environment. The dynamics of gene expression that result from environmental shifts are limited, amongst others, by the time needed for regulation and transcription elongation. In this study, we examined gene expression dynamics after an anaerobic-to-aerobic shift on a short time scale (0.5, 1, 2, 5, and 10 min) by RNA sequencing with emphasis on delay times and transcriptional elongation rates (TER). Transient expression patterns and timing of differential expression, characterized by delay and elongation, were identified as key features of the dataset. Gene ontology enrichment analysis revealed early upregulation of respiratory and iron-related gene sets. We inferred specific TERs of 89 operons with a mean TER of 42.0 nt/s and mean delay time of 22.4 s. TERs correlate with sequence features, such as codon bias, whereas delay times correlate with the involvement of regulators. The presented data illustrate that at very short times after a shift in oxygenation, extensional changes of the transcriptome, such as temporary responses, can be observed. Besides regulation, TERs contribute to the dynamics of gene expression.
Open Access
Selective gene expression profiling contributes to a better understanding of the molecular pathways underlying the histological changes observed after RHMVL
(2022) Arlt, Janine; Vlaic, Sebastian; Feuer, Ronny; Thomas, Maria; Settmacher, Utz; Dahmen, Uta; Dirsch, Olaf
Background. In previous studies, five vasoactive drugs were investigated for their effect on the recovery process after extended liver resection without observing relevant improvements. We hypothesized that an analysis of gene expression could help to identify potentially druggable pathways and could support the selection of promising drug candidates. Methods. Liver samples obtained from rats after combined 70% partial hepatectomy and right median hepatic vein ligation ( n = 6/group) sacrificed at 0 h, 24 h, 48 h, and 7days were selected for this study. Liver samples were collected from differentially perfused regions of the median lobe (obstruction-zone, border-zone, normal-zone). Gene expression profiling of marker genes regulating hepatic hemodynamics, vascular remodeling, and liver regeneration was performed with microfluidic chips. We used 3 technical replicates from each sample. Raw data were normalized using LEMming and differentially expressed genes were identified using LIMMA. Results. The strongest differences were found in obstruction-zone at 24 h and 48 h postoperatively compared to all other groups. mRNA expression of marker genes from hepatic hemodynamics pathways (iNOS,Ptgs2,Edn1) was most upregulated. Conclusion. These upregulated genes suggest a strong vasoconstrictive effect promoting arterial hypoperfusion in the obstruction-zone. Reducing iNOS expression using selective iNOS inhibitors seems to be a promising approach to promote vasodilation and liver regeneration.
Open Access
Transition of an anaerobic Escherichia coli culture to aerobiosis: balancing mRNA and protein levels in a demand-directed dynamic flux balance analysis
(2016) Wulffen, Joachim von; Sawodny, Oliver; Feuer, Ronny
The facultative anaerobic bacterium Escherichia coli is frequently forced to adapt to changing environmental conditions. One important determinant for metabolism is the availability of oxygen allowing a more efficient metabolism. Especially in large scale bioreactors, the distribution of oxygen is inhomogeneous and individual cells encounter frequent changes. This might contribute to observed yield losses during process upscaling. Short-term gene expression data exist of an anaerobic E. coli batch culture shifting to aerobic conditions. The data reveal temporary upregulation of genes that are less efficient in terms of energy conservation than the genes predicted by conventional flux balance analyses. In this study, we provide evidence for a positive correlation between metabolic fluxes and gene expression. We then hypothesize that the more efficient enzymes are limited by their low expression, restricting flux through their reactions. We define a demand that triggers expression of the demanded enzymes that we explicitly include in our model. With these features we propose a method, demand-directed dynamic flux balance analysis, dddFBA, bringing together elements of several previously published methods. The introduction of additional flux constraints proportional to gene expression provoke a temporary demand for less efficient enzymes, which is in agreement with the transient upregulation of these genes observed in the data. In the proposed approach, the applied objective function of growth rate maximization together with the introduced constraints triggers expression of metabolically less efficient genes. This finding is one possible explanation for the yield losses observed in large scale bacterial cultivations where steady oxygen supply cannot be warranted.