Publications

583 Publications visible to you, out of a total of 583

Abstract (Expand)

Adaptive Laboratory Evolution (ALE) is increasingly being used as a technique for untargeted strain optimization. This work aimed at developing an automated and miniaturized ALE approach based on repetitive batch cultivations in microtiter plates. The new method is applied to the recently published strain Corynebacterium glutamicum pEKEx3-xylXABCDCc, which is capable of utilizing d-xylose via the Weimberg (WMB) pathway. As a result, the significantly improved strain WMB2evo was obtained, showing a specific growth rate of 0.26h-1 on d-xylose as sole carbon and energy source. WMB2evo grows stable during lab-scale bioreactor operation, demonstrating the high potential of this strain for future biorefinery applications. Genome sequencing of cell samples from two different ALE processes revealed potential key mutations, e.g. in the gene cg0196 (encoding for the transcriptional regulator IolR of the myo-inositol metabolism). These findings open up new perspectives for the rational engineering of C. glutamicum towards improved d-xylose utilization.

Authors: A. Radek, N. Tenhaef, M. F. Muller, C. Brusseler, W. Wiechert, J. Marienhagen, T. Polen, S. Noack

Date Published: 30th May 2017

Publication Type: Not specified

Abstract (Expand)

The thermoacidophilic Crenarchaeon Sulfolobus solfataricus is a model organism for archaeal adaptation to extreme environments and renowned for its ability to degrade a broad variety of substrates. It has been well characterised concerning the utilisation of numerous carbohydrates as carbon source. However, its amino acid metabolism, especially the degradation of single amino acids, is not as well understood. In this work, we performed metabolic modelling as well as metabolome, transcriptome and proteome analysis on cells grown on caseinhydrolysate as carbon source in order to draw a comprehensive picture of amino acid metabolism in S. solfataricus P2. We found that 10 out of 16 detectable amino acids are imported from the growth medium. Overall, uptake of glutamate, methionine, leucine, phenylalanine and isoleucine was the highest of all observed amino acids. Our simulations predict an incomplete degradation of leucine and tyrosine to organic acids, and in accordance with this, we detected the export of branched-chain and aromatic organic acids as well as amino acids, ammonium and trehalose into the culture supernatants. The branched-chain amino acids as well as phenylalanine and tyrosine are degraded to organic acids via oxidative Stickland reactions. Such reactions are known for prokaryotes capable of anaerobic growth, but so far have never been observed in an obligate aerobe. Also, 3-methyl-2-butenoate and 2-methyl-2-butenoate are for the first time found as products of modified Stickland reactions for the degradation of branched-chain amino acids. This work presents the first detailed description of branched-chain and aromatic amino acid catabolism in S. solfataricus.

Authors: Helge Stark, Jacqueline Wolf, Andreas Albersmeier, Trong K. Pham, Julia D. Hofmann, Bettina Siebers, Jörn Kalinowski, Phillip C. Wright, Meina Neumann-Schaal, Dietmar Schomburg

Date Published: 29th May 2017

Publication Type: Not specified

Abstract (Expand)

In this article, the four coordinators of neglected tropical disease (NTD) drug development projects funded under the European Commission (EC) Framework Programme 7 argue that the EC should reassess their funding strategy to cover the steps necessary to translate a lead compound into a drug candidate for testing in clinical trials, and suggest ways in which this might be achieved.

Authors: R. J. Pierce, J. MacDougall, R. Leurs, M. P. Costi

Date Published: 23rd May 2017

Publication Type: Journal

Abstract (Expand)

PCB 153 is one of the most abundant PCB congeners detected in biological samples. It is a persistent compound that is still present in the environment despite the ban on production and use of PCBs in the late 1970s. It has strong tendencies to bioaccumulate and biomagnify in biota, and studies have suggested that it is an endocrine and metabolic disruptor. In order to study mechanisms of toxicity, we exposed Atlantic cod (Gadus morhua) to various doses of PCB 153 (0, 0.5, 2 and 8 mg/kg body weight) for two weeks and examined the effects on expression of liver proteins using label-free quantitative proteomics. Label-free liquid chromatography-mass spectrometry analysis of the liver proteome resulted in the quantification of 1272 proteins, of which 78 proteins were differentially regulated in the PCB 153-treated dose groups compared to the control group. Functional enrichment analysis showed that pathways significantly affected are related to the lipid metabolism, cytoskeletal remodeling, cell cycle and cell adhesion. Importantly, the main effects appear to be on lipid metabolism, with up-regulation of enzymes in the de novo fatty acid synthesis pathway, consistent with previous transcriptomics results. Increased plasma triglyceride levels were also observed in the PCB 153 treated fish, in agreement with the induction of the lipogenic genes and proteins. The results suggest that PCB 153 perturbs lipid metabolism in the Atlantic cod liver. Elevated levels of lipogenic enzymes and plasma triglycerides further suggest increased synthesis of fatty acids and triglycerides.

Author: Fekadu Yadetie, Eystein Oveland, Anne Døskeland, Frode Berven Anders Goksøyr, Odd André Karlsen

Date Published: 1st Apr 2017

Publication Type: Not specified

Abstract (Expand)

Neural specificity refers to the degree to which neural representations of different stimuli can be distinguished. Evidence suggests that neural specificity, operationally defined as stimulus-related differences in functional magnetic resonance imaging (fMRI) activation patterns, declines with advancing adult age, and that individual differences in neural specificity are associated with individual differences in fluid intelligence. A growing body of literature also suggests that regular physical activity may help preserve cognitive abilities in old age. Based on this literature, we hypothesized that exercise-induced improvements in fitness would be associated with greater neural specificity among older adults. A total of 52 adults aged 59–74 years were randomly assigned to one of two aerobic-fitness training regimens, which differed in intensity. Participants in both groups trained three times a week on stationary bicycles. In the low-intensity (LI) group, the resistance was kept constant at a low level (10 Watts). In the high-intensity (HI) group, the resistance depended on participants’ heart rate and therefore typically increased with increasing fitness. Before and after the 6-month training phase, participants took part in a functional MRI experiment in which they viewed pictures of faces and buildings. We used multivariate pattern analysis (MVPA) to estimate the distinctiveness of neural activation patterns in ventral visual cortex (VVC) evoked by face or building stimuli. Fitness was also assessed before and after training. In line with our hypothesis, training-induced changes in fitness were positively associated with changes in neural specificity. We conclude that physical activity may protect against age-related declines in neural specificity.

Authors: Maike M. Kleemeyer, Thad A. Polk, Sabine Schaefer, Nils C. Bodammer, Lars Brechtel, Ulman Lindenberger

Date Published: 16th Mar 2017

Publication Type: Journal

Abstract (Expand)

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.

Authors: F. Di Pisa, G. Landi, L. Dello Iacono, C. Pozzi, C. Borsari, S. Ferrari, M. Santucci, N. Santarem, A. Cordeiro-da-Silva, C. B. Moraes, L. M. Alcantara, V. Fontana, L. H. Freitas-Junior, S. Gul, M. Kuzikov, B. Behrens, I. Pohner, R. C. Wade, M. P. Costi, S. Mangani

Date Published: 8th Mar 2017

Publication Type: Journal

Abstract (Expand)

The transketolase from Geobacillus stearothermophilus (TKGst) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non‐α‐hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TKGst based on double‐site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TKGst for various applications in asymmetric carboligation.

Authors: Chaoqiang Zhou, Thangavelu Saravanan, Marion Lorillière, Dongzhi Wei, Franck Charmantray, Laurence Hecquet, Wolf-Dieter Fessner, Dong Yi

Date Published: 2nd Mar 2017

Publication Type: Not specified

Abstract

Not specified

Authors: Wolfgang Müller, Meik Bittkowski, Martin Golebiewski, Renate Kania, Maja Rey, Andreas Weidemann, Ulrike Wittig

Date Published: 1st Mar 2017

Publication Type: Journal

Abstract (Expand)

Standards are essential to the advancement of science and technology. In systems and synthetic biology, numerous standards and associated tools have been developed over the last 16 years. This special issue of the Journal of Integrative Bioinformatics aims to support the exchange, distribution and archiving of these standards, as well as to provide centralised and easily citable access to them.

Authors: F. Schreiber, G. D. Bader, P. Gleeson, M. Golebiewski, M. Hucka, N. Le Novere, C. Myers, D. Nickerson, B. Sommer, D. Walthemath

Date Published: 12th Feb 2017

Publication Type: Not specified

Abstract (Expand)

Summary paragraph Predicting a multicellular organism’s phenotype quantitatively from its genotype is challenging, as genetic effects must propagate up time and length scales. Circadian clocks arelength scales. Circadian clocks are intracellular regulators that control temporal gene expression patterns and hence metabolism, physiology and behaviour, from sleep/wake cycles in mammals to flowering in plants 1–3 . Clock genes are rarely essential but appropriate alleles can confer a competitive advantage 4,5 and have been repeatedly selected during crop domestication 3,6 . Here we quantitatively explain and predict canonical phenotypes of circadian timing in a multicellular, model organism. We used metabolic and physiological data to combine and extend mathematical models of rhythmic gene expression, photoperiod-dependent flowering, elongation growth and starch metabolism within a Framework Model for growth of Arabidopsis thaliana 7–9 . The model predicted the effect of altered circadian timing upon each particular phenotype in clock-mutant plants. Altered night-time metabolism of stored starch accounted for most but not all of the decrease in whole-plant growth rate. Altered mobilisation of a secondary store of organic acids explained the remaining defect. Our results link genotype through specific processes to higher-level phenotypes, formalising our understanding of a subtle, pleiotropic syndrome at the whole-organism level, and validating the systems approach to understand complex traits starting from intracellular circuits.

Authors: Yin Hoon Chew, Daniel D. Seaton, Virginie Mengin, Anna Flis, Sam T. Mugford, Alison M. Smith, Mark Stitt, Andrew J Millar

Date Published: 6th Feb 2017

Publication Type: Tech report

Abstract (Expand)

Predicting a multicellular organism’s phenotype quantitatively from its genotype is challenging, as genetic effects must propagate across scales. Circadian clocks are intracellular regulators that control temporal gene expression patterns and hence metabolism, physiology and behaviour. Here we explain and predict canonical phenotypes of circadian timing in a multicellular, model organism. We used diverse metabolic and physiological data to combine and extend mathematical models of rhythmic gene expression, photoperiod-dependent flowering, elongation growth and starch metabolism within a Framework Model for the vegetative growth of Arabidopsis thaliana, sharing the model and data files in a structured, public resource. The calibrated model predicted the effect of altered circadian timing upon each particular phenotype in clock-mutant plants under standard laboratory conditions. Altered night-time metabolism of stored starch accounted for most of the decrease in whole-plant biomass, as previously proposed. Mobilisation of a secondary store of malate and fumarate was also mis-regulated, accounting for any remaining biomass defect. We test three candidate mechanisms for the accumulation of these organic acids. Our results link genotype through specific processes to higher-level phenotypes, formalising our understanding of a subtle, pleiotropic syndrome at the whole-organism level, and validating the systems approach to understand complex traits starting from intracellular circuits. This work updates the first biorXiv version, February 2017,with an expanded description and additional analysis of the same core data sets and the same FMv2 model, summary tables and supporting, follow-on data from three further studies with further collaborators. This biorXiv revision constitutes the second version of this report.

Authors: Yin Hoon Chew, Daniel D. Seaton, Virginie Mengin, Anna Flis, Sam T. Mugford, Gavin M. George, Michael Moulin, Alastair Hume, Samuel C. Zeeman, Teresa B. Fitzpatrick, Alison M. Smith, Mark Stitt, Andrew J. Millar

Date Published: 6th Feb 2017

Publication Type: Tech report

Abstract (Expand)

Cell activation is a vital step for T-cell memory/effector differentiation as well as for productive HIV infection. To identify novel regulators of this process, we used next-generation sequencing to profile changes in microRNA expression occurring in purified human naive CD4 T cells in response to TCR stimulation and/or HIV infection. Our results demonstrate, for the first time, the transcriptional up-regulation of miR-34c-5p in response to TCR stimulation in naive CD4 T cells. The induction of this miR was further consistently found to be reduced by both HIV-1 and HIV-2 infections. Overexpression of miR-34c-5p led to changes in the expression of several genes involved in TCR signaling and cell activation, confirming its role as a novel regulator of naive CD4 T-cell activation. We additionally show that miR-34c-5p promotes HIV-1 replication, suggesting that its down-regulation during HIV infection may be part of an anti-viral host response.

Authors: A. J. Amaral, J. Andrade, R. B. Foxall, P. Matoso, A. M. Matos, R. S. Soares, C. Rocha, C. G. Ramos, R. Tendeiro, A. Serra-Caetano, J. A. Guerra-Assuncao, M. Santa-Marta, J. Goncalves, M. Gama-Carvalho, A. E. Sousa

Date Published: 1st Feb 2017

Publication Type: Not specified

Abstract (Expand)

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2'-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3-4.2 muM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-beta-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability.

Authors: C. Borsari, N. Santarem, J. Torrado, A. I. Olias, M. J. Corral, C. Baptista, S. Gul, M. Wolf, M. Kuzikov, B. Ellinger, G. Witt, P. Gribbon, J. Reinshagen, P. Linciano, A. Tait, L. Costantino, L. H. Freitas-Junior, C. B. Moraes, P. Bruno Dos Santos, L. M. Alcantara, C. H. Franco, C. D. Bertolacini, V. Fontana, P. Tejera Nevado, J. Clos, J. M. Alunda, A. Cordeiro-da-Silva, S. Ferrari, M. P. Costi

Date Published: 27th Jan 2017

Publication Type: Journal

Abstract (Expand)

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro-proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type-specific proliferation. First, cell type-specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate-limiting for faster cycling cells while slower cell cycles are controlled at the G1-S progression. The integrated mathematical model of Epo-driven proliferation explains cell type-specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti-proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

Authors: L. Adlung, S. Kar, M. C. Wagner, B. She, S. Chakraborty, J. Bao, S. Lattermann, M. Boerries, H. Busch, P. Wuchter, A. D. Ho, J. Timmer, M. Schilling, T. Hofer, U. Klingmuller

Date Published: 24th Jan 2017

Publication Type: Journal

Abstract (Expand)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2-phenoxy-1,4-naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2-phenoxy-1,4-naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2-phenoxy-1,4-naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non-covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2-phenoxy-1,4-naphthoquinone displays selectivity for GAPDHs over two other cysteine-dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.

Authors: S. Bruno, E. Uliassi, M. Zaffagnini, F. Prati, C. Bergamini, R. Amorati, G. Paredi, M. Margiotta, P. Conti, M. P. Costi, M. Kaiser, A. Cavalli, R. Fato, M. L. Bolognesi

Date Published: 13th Jan 2017

Publication Type: Journal

Abstract (Expand)

Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.

Authors: Z. Xu, Y. Wang, K. F. Chater, H. Y. Ou, H. H. Xu, Z. Deng, M. Tao

Date Published: 8th Jan 2017

Publication Type: Not specified

Abstract (Expand)

In this review, we present our most recent understanding of key biomolecular processes that underlie two motor neuron degenerative disorders, amyotrophic lateral sclerosis, and spinal muscular atrophy. We focus on the role of four multifunctional proteins involved in RNA metabolism (TDP-43, FUS, SMN, and Senataxin) that play a causal role in these diseases. Recent results have led to a novel scenario of intricate connections between these four proteins, bringing transcriptome homeostasis into the spotlight as a common theme in motor neuron degeneration. We review reported functional and physical interactions between these four proteins, highlighting their common association with nuclear bodies and small nuclear ribonucleoprotein particle biogenesis and function. We discuss how these interactions are turning out to be particularly relevant for the control of transcription and chromatin homeostasis, including the recent identification of an association between SMN and Senataxin required to ensure the resolution of DNA-RNA hybrid formation and proper termination by RNA polymerase II. These connections strongly support the existence of common pathways underlying the spinal muscular atrophy and amyotrophic lateral sclerosis phenotype. We also discuss the potential of genome-wide expression profiling, in particular RNA sequencing derived data, to contribute to unravelling the underlying mechanisms. We provide a review of publicly available datasets that have addressed both diseases using these approaches, and highlight the value of investing in cross-disease studies to promote our understanding of the pathways leading to neurodegeneration.

Authors: M. Gama-Carvalho, M. L Garcia-Vaquero, F. R Pinto, F. Besse, J. Weis, A. Voigt, J. B. Schulz, J. De Las Rivas

Date Published: 6th Jan 2017

Publication Type: Not specified

Abstract (Expand)

The FAIRDOMHub is a repository for publishing FAIR (Findable, Accessible, Interoperable and Reusable) Data, Operating procedures and Models (https://fairdomhub.org/) for the Systems Biology community. It is a web-accessible repository for storing and sharing systems biology research assets. It enables researchers to organize, share and publish data, models and protocols, interlink them in the context of the systems biology investigations that produced them, and to interrogate them via API interfaces. By using the FAIRDOMHub, researchers can achieve more effective exchange with geographically distributed collaborators during projects, ensure results are sustained and preserved and generate reproducible publications that adhere to the FAIR guiding principles of data stewardship.

Authors: K. Wolstencroft, O. Krebs, J. L. Snoep, N. J. Stanford, F. Bacall, M. Golebiewski, R. Kuzyakiv, Q. Nguyen, S. Owen, S. Soiland-Reyes, J. Straszewski, D. D. van Niekerk, A. R. Williams, L. Malmstrom, B. Rinn, W. Muller, C. Goble

Date Published: 4th Jan 2017

Publication Type: Journal

Abstract (Expand)

The FAIRDOMHub is a repository for publishing FAIR (Findable, Accessible, Interoperable and Reusable) Data, Operating procedures and Models (https://fairdomhub.org/) for the Systems Biology community. It is a web-accessible repository for storing and sharing systems biology research assets. It enables researchers to organize, share and publish data, models and protocols, interlink them in the context of the systems biology investigations that produced them, and to interrogate them via API interfaces. By using the FAIRDOMHub, researchers can achieve more effective exchange with geographically distributed collaborators during projects, ensure results are sustained and preserved and generate reproducible publications that adhere to the FAIR guiding principles of data stewardship.

Authors: K. Wolstencroft, O. Krebs, J. L. Snoep, N. J. Stanford, F. Bacall, M. Golebiewski, R. Kuzyakiv, Q. Nguyen, S. Owen, S. Soiland-Reyes, J. Straszewski, D. D. van Niekerk, A. R. Williams, L. Malmstrom, B. Rinn, W. Muller, C. Goble

Date Published: 4th Jan 2017

Publication Type: Journal

Abstract (Expand)

Organisms use circadian clocks to generate 24-h rhythms in gene expression. However, the clock can interact with other pathways to generate shorter period oscillations. It remains unclear how these different frequencies are generated. Here, we examine this problem by studying the coupling of the clock to the alternative sigma factor sigC in the cyanobacterium Synechococcus elongatus. Using single-cell microscopy, we find that psbAI, a key photosyn- thesis gene regulated by both sigC and the clock, is activated with two peaks of gene expression every circadian cycle under constant low light. This two-peak oscillation is dependent on sigC, without which psbAI rhythms revert to one oscillatory peak per day. We also observe two circadian peaks of elongation rate, which are dependent on sigC, suggesting a role for the frequency doubling in modulating growth. We propose that the two-peak rhythm in psbAI expression is generated by an incoherent feedforward loop between the clock, sigC and psbAI. Modelling and experiments suggest that this could be a general network motif to allow frequency doubling of outputs.

Authors: Bruno MC Martins, Arijit K Das, Liliana Antunes, James CW Locke

Date Published: 22nd Dec 2016

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders. RESULTS: First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients' metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach. CONCLUSION: We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.

Authors: K. van Eunen, C. M. Volker-Touw, A. Gerding, A. Bleeker, J. C. Wolters, W. J. van Rijt, A. M. Martines, K. E. Niezen-Koning, R. M. Heiner, H. Permentier, A. K. Groen, D. J. Reijngoud, T. G. Derks, B. M. Bakker

Date Published: 7th Dec 2016

Publication Type: Journal

Abstract (Expand)

Pseudomonas is a highly versatile genus containing species that can be harmful to humans and plants while others are widely used for bioengineering and bioremediation. We analysed 432 sequenced Pseudomonas strains by integrating results from a large scale functional comparison using protein domains with data from six metabolic models, nearly a thousand transcriptome measurements and four large scale transposon mutagenesis experiments. Through heterogeneous data integration we linked gene essentiality, persistence and expression variability. The pan-genome of Pseudomonas is closed indicating a limited role of horizontal gene transfer in the evolutionary history of this genus. A large fraction of essential genes are highly persistent, still non essential genes represent a considerable fraction of the core-genome. Our results emphasize the power of integrating large scale comparative functional genomics with heterogeneous data for exploring bacterial diversity and versatility.

Authors: J. J. Koehorst, J. C. van Dam, R. G. van Heck, E. Saccenti, V. A. Dos Santos, M. Suarez-Diez, P. J. Schaap

Date Published: 6th Dec 2016

Publication Type: Journal

Abstract (Expand)

Sucrose translocation between plant tissues is crucial for growth, development and reproduction of plants. Systemic analysis of these metabolic and underlying regulatory processes allow a detailed understanding of carbon distribution within the plant and the formation of associated phenotypic traits. Sucrose translocation from ‘source’ tissues (e.g. mesophyll) to ‘sink’ tissues (e.g. root) is tightly bound to the proton gradient across the membranes. The plant sucrose transporters are grouped into efflux exporters (SWEET family) and proton-symport importers (SUC, STP families). To better understand regulation of sucrose export from source tissues and sucrose import into sink tissues, there is a need for a metabolic model that takes in account the tissue organisation of Arabidopsis thaliana with corresponding metabolic specificities of respective tissues in terms of sucrose and proton production/utilization. An ability of the model to operate under different light modes (‘light’ and ‘dark’) and correspondingly in different energy producing modes is particularly important in understanding regulatory modules.

Authors: Maksim Zakhartsev, Irina Medvedeva, Yury Orlov, Ilya Akberdin, Olga Krebs, Waltraud X. Schulze

Date Published: 1st Dec 2016

Publication Type: Journal

Abstract (Expand)

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.

Authors: P. Dalle Pezze, S. Ruf, A. G. Sonntag, M. Langelaar-Makkinje, P. Hall, A. M. Heberle, P. Razquin Navas, K. van Eunen, R. C. Tolle, J. J. Schwarz, H. Wiese, B. Warscheid, J. Deitersen, B. Stork, E. Fassler, S. Schauble, U. Hahn, P. Horvatovich, D. P. Shanley, K. Thedieck

Date Published: 21st Nov 2016

Publication Type: Journal

Abstract (Expand)

Ataxia telangiectasia (A-T) is a rare autosomal recessive disease characterized by progressive neurodegeneration and cerebellar ataxia. A-T is causally linked to defects in ATM, a master regulator of the response to and repair of DNA double-strand breaks. The molecular basis of cerebellar atrophy and neurodegeneration in A-T patients is unclear. Here we report and examine the significance of increased PARylation, low NAD+, and mitochondrial dysfunction in ATM-deficient neurons, mice, and worms. Treatments that replenish intracellular NAD+ reduce the severity of A-T neuropathology, normalize neuromuscular function, delay memory loss, and extend lifespan in both animal models. Mechanistically, treatments that increase intracellular NAD+ also stimulate neuronal DNA repair and improve mitochondrial quality via mitophagy. This work links two major theories on aging, DNA damage accumulation, and mitochondrial dysfunction through nuclear DNA damage-induced nuclear-mitochondrial signaling, and demonstrates that they are important pathophysiological determinants in premature aging of A-T, pointing to therapeutic interventions.

Authors: Evandro Fei Fang, Henok Kassahun, Deborah L. Croteau, Morten Scheibye-Knudsen, Krisztina Marosi, Huiming Lu, Raghavendra A. Shamanna, Sumana Kalyanasundaram, Ravi Chand Bollineni, Mark A. Wilson, Wendy B. Iser, Bradley N. Wollman, Marya Morevati, Jun Li, Jesse S. Kerr, Qiping Lu, Tyler B. Waltz, Jane Tian, David A. Sinclair, Mark P. Mattson, Hilde Nilsen, Vilhelm A. Bohr

Date Published: 1st Oct 2016

Publication Type: Not specified

Abstract (Expand)

Archaea are characterised by a complex metabolism with many unique enzymes that differ from their bacterial and eukaryotic counterparts. The thermoacidophilic archaeon Sulfolobus solfataricus is known for its metabolic versatility and is able to utilize a great variety of different carbon sources. However, the underlying degradation pathways and their regulation are often unknown. In this work, we analyse growth on different carbon sources using an integrated systems biology approach. The comparison of growth on L-fucose and D-glucose allows first insights into the genome-wide changes in response to the two carbon sources and revealed a new pathway for L-fucose degradation in S. solfataricus. During growth on L-fucose we observed major changes in the central carbon metabolic network, as well as an increased activity of the glyoxylate bypass and the 3-hydroxypropionate/4-hydroxybutyrate cycle. Within the newly discovered pathway for L-fucose degradation the following key reactions were identified: (i) L-fucose oxidation to L-fuconate via a dehydrogenase, (ii) dehydration to 2-keto-3-deoxy-L-fuconate via dehydratase, (iii) 2-keto-3-deoxy-L-fuconate cleavage to pyruvate and L-lactaldehyde via aldolase and (iv) L-lactaldehyde conversion to L-lactate via aldehyde dehydrogenase. This pathway as well as L-fucose transport shows interesting overlaps to the D-arabinose pathway, representing another example for pathway promiscuity in Sulfolobus species. This article is protected by copyright. All rights reserved.

Authors: J. Wolf, H. Stark, K. Fafenrot, A. Albersmeier, T. K. Pham, K. B. Muller, B. Meyer, L. Hoffmann, L. Shen, S. P. Albaum, T. Kouril, K. Schmidt-Hohagen, M. Neumann-Schaal, C. Brasen, J. Kalinowski, P. C. Wright, S. V. Albers, D. Schomburg, B. Siebers

Date Published: 10th Sep 2016

Publication Type: Not specified

Abstract (Expand)

MOTIVATION: A major goal of drug development is to selectively target certain cell types. Cellular decisions influenced by drugs are often dependent on the dynamic processing of information. Selective responses can be achieved by differences between the involved cell types at levels of receptor, signaling, gene regulation or further downstream. Therefore, a systematic approach to detect and quantify cell type-specific parameters in dynamical systems becomes necessary. RESULTS: Here, we demonstrate that a combination of nonlinear modeling with L1 regularization is capable of detecting cell type-specific parameters. To adapt the least-squares numerical optimization routine to L1 regularization, sub-gradient strategies as well as truncation of proposed optimization steps were implemented. Likelihood-ratio tests were used to determine the optimal regularization strength resulting in a sparse solution in terms of a minimal number of cell type-specific parameters that is in agreement with the data. By applying our implementation to a realistic dynamical benchmark model of the DREAM6 challenge we were able to recover parameter differences with an accuracy of 78%. Within the subset of detected differences, 91% were in agreement with their true value. Furthermore, we found that the results could be improved using the profile likelihood. In conclusion, the approach constitutes a general method to infer an overarching model with a minimum number of individual parameters for the particular models. AVAILABILITY AND IMPLEMENTATION: A MATLAB implementation is provided within the freely available, open-source modeling environment Data2Dynamics. Source code for all examples is provided online at http://www.data2dynamics.org/ CONTACT: bernhard.steiert@fdm.uni-freiburg.de.

Authors: B. Steiert, J. Timmer, C. Kreutz

Date Published: 3rd Sep 2016

Publication Type: Not specified

Abstract (Expand)

In systems biology, one of the major tasks is to tailor model complexity to information content of the data. A useful model should describe the data and produce well-determined parameter estimates and predictions. Too small of a model will not be able to describe the data whereas a model which is too large tends to overfit measurement errors and does not provide precise predictions. Typically, the model is modified and tuned to fit the data, which often results in an oversized model. To restore the balance between model complexity and available measurements, either new data has to be gathered or the model has to be reduced. In this manuscript, we present a data-based method for reducing non-linear models. The profile likelihood is utilised to assess parameter identifiability and designate likely candidates for reduction. Parameter dependencies are analysed along profiles, providing context-dependent suggestions for the type of reduction. We discriminate four distinct scenarios, each associated with a specific model reduction strategy. Iterating the presented procedure eventually results in an identifiable model, which is capable of generating precise and testable predictions. Source code for all toy examples is provided within the freely available, open-source modelling environment Data2Dynamics based on MATLAB available at http://www.data2dynamics.org/, as well as the R packages dMod/cOde available at https://github.com/dkaschek/. Moreover, the concept is generally applicable and can readily be used with any software capable of calculating the profile likelihood.

Authors: T. Maiwald, H. Hass, B. Steiert, J. Vanlier, R. Engesser, A. Raue, F. Kipkeew, H. H. Bock, D. Kaschek, C. Kreutz, J. Timmer

Date Published: 3rd Sep 2016

Publication Type: Not specified

Abstract (Expand)

Absolute measurements of protein abundance are important in the understanding of biological processes and the precise computational modeling of biological pathways. We developed targeted LC-MS/MS assays in the selected reaction monitoring (SRM) mode to quantify over 50 mitochondrial proteins in a single run. The targeted proteins cover the tricarboxylic acid cycle, fatty acid beta-oxidation, oxidative phosphorylation, and the detoxification of reactive oxygen species. Assays used isotopically labeled concatemers as internal standards designed to target murine mitochondrial proteins and their human orthologues. Most assays were also suitable to quantify the corresponding protein orthologues in rats. After exclusion of peptides that did not pass the selection criteria, we arrived at SRM assays for 55 mouse, 52 human, and 51 rat proteins. These assays were optimized in isolated mitochondrial fractions from mouse and rat liver and cultured human fibroblasts and in total liver extracts from mouse, rat, and human. The developed proteomics approach is suitable for the quantification of proteins in the mitochondrial energy metabolic pathways in mice, rats, and humans as a basis for translational research. Initial data show that the assays have great potential for elucidating the adaptive response of human patients to mutations in mitochondrial proteins in a clinical setting.

Authors: J. C. Wolters, J. Ciapaite, K. van Eunen, K. E. Niezen-Koning, A. Matton, R. J. Porte, P. Horvatovich, B. M. Bakker, R. Bischoff, H. P. Permentier

Date Published: 2nd Sep 2016

Publication Type: Journal

Abstract (Expand)

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 muM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 muM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

Authors: C. Borsari, R. Luciani, C. Pozzi, I. Poehner, S. Henrich, M. Trande, A. Cordeiro-da-Silva, N. Santarem, C. Baptista, A. Tait, F. Di Pisa, L. Dello Iacono, G. Landi, S. Gul, M. Wolf, M. Kuzikov, B. Ellinger, J. Reinshagen, G. Witt, P. Gribbon, M. Kohler, O. Keminer, B. Behrens, L. Costantino, P. Tejera Nevado, E. Bifeld, J. Eick, J. Clos, J. Torrado, M. D. Jimenez-Anton, M. J. Corral, J. M. Alunda, F. Pellati, R. C. Wade, S. Ferrari, S. Mangani, M. P. Costi

Date Published: 25th Aug 2016

Publication Type: Journal

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