Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria

<p>Abstract</p> <p>Background</p> <p>Fluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells. However, mathematical models are necessary for extracting biologically meaningful quantities from the primary data.</p> <p>Results</p> <p>We present a rigorous method for deriving relative protein synthesis rates (mRNA concentrations) and protein concentrations by means of kinetic models of gene expression. We experimentally and computationally validate this approach in the case of the protein Fis, a global regulator of transcription in <it>Escherichia coli</it>. We show that the mRNA and protein concentration profiles predicted from the models agree quite well with direct measurements obtained by Northern and Western blots, respectively. Moreover, we present computational procedures for taking into account systematic biases like the folding time of the fluorescent reporter protein and differences in the half-lives of reporter and host gene products. The results show that large differences in protein half-lives, more than mRNA half-lives, may be critical for the interpretation of reporter gene data in the analysis of the dynamics of regulatory systems.</p> <p>Conclusions</p> <p>The paper contributes to the development of sound methods for the interpretation of reporter gene data, notably in the context of the reconstruction and validation of models of regulatory networks. The results have wide applicability for the analysis of gene expression in bacteria and may be extended to higher organisms.</p

ALA, EPA and DHA differentially Modulate Palmitate-induced Lipotoxicity through Alterations of its Metabolism and Storage in C12C12 Muscle Cells

On that occasion, the two French societies dedicated to lipid science and technology, GERLI and SFEL, will combine their efforts to assist the scientific committee to establish an attractive program for the Euro Fed Lipid congress.Since few decades, incidence of obesity and type 2 diabetes (T2D) is increasing. Excessive intake of energy leads to fat overload and formation of lipotoxic compounds mainly derived from the saturated fatty acid palmitate in insulin-sensitive tissues (muscle, liver and white adipose tissue), promoting insulin resistance (IR, a well-known metabolic disorder in T2D). Supplementation with n-3 fatty acids (n-3FA) is suggested to reduce lipotoxicity and IR. We hypothesized that, according to the n-3FA used, differential and specific effects on palmitate metabolism in muscle cells will be demonstrated. C2C12 myotubes were treated with 500 µM of palmitate without or with 50 µM of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16 hours and collected for measurement of membrane fluidity using diphenyl-hexatriene, ceramide content, insulin-dependent Akt protein phosphorylation (as an index of IR). The assessment of the intracellular metabolism and incorporation of palmitate into lipid fractions (triglycerides, phospholipids, diglycerides) was performed after treatment for 3 hours with [1-14C]-palmitate. As expected, palmitate-induced IR was restored by EPA and DHA supplementation whereas ALA had no effect compared to palmitate alone. EPA and DHA significantly improved C2C12 membrane fluidity compared to palmitate alone (+8.5% and +13% respectively, p<0.05). Furthermore, palmitate incorporation into the diglyceride fraction was decreased by 31 and 47% by EPA and DHA vs. palmitate, respectively (p=0.05). However, DHA significantly increased the ratio of diglycerides to total lipids vs. palmitate alone (p<0.05), whereas EPA did not. Finally, EPA was more potent to decrease palmitate-induced ceramide accumulation (+174%, p<0.05 vs. control) compared to DHA (-50% and -29% repectively, p<0.05). In conclusion and contrary to ALA, EPA and DHA treatment improved the insulin signalling pathway by differently modulating membrane fluidity and lipid and palmitate metabolism, thus demonstrating that n-3FA have different metabolic impacts on C2C12 lipid metabolism

Effets comparatifs des acides gras omega-3 (ALA, EPA, DHA) sur la sensibilité à l’insuline des cellules musculaires C2C12 dans un contexte lipotoxique

Objectifs :Etudier le rôle des ω3 sur la lipotoxicité induite par l’acide gras saturé palmitate (PAL, C16:0) dans un modèle de cellule musculaire C2C12.Identifier les effets propres de chaque w3 (ALA, EPA et DHA) à dose équivalente sur la fluidité des membranes et la réponse à l’insuline.Suivre le devenir intracellulaire du [1-14C]-palmitate en présence d’un w3 et définir les classes de lipides altérées.Rechercher les voies de signalisation impliquées dans la modulation de la réponse à l’insuline

Impact differentiel des acides gras N-3, ALA, EPA et DHA sur la lipotoxicité induite par le palmitate dans le muscle

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Oméga-3, Obésité et Syndrome Métabolique

National audienceDans un contexte d’augmentation de la prévalence de l’obésité et des maladies métaboliques associées, la qualité des lipides alimentaires fait l’objet de nombreux travaux et études épidémiologiques. Parmi ces lipides, les acides gras polyinsaturés oméga-3 (ω3) sont apportés en quantité insuffisante dans l’alimentation occidentale. L’augmentation de l’apport en ω3 favoriserait la prévention de l’obésité par différents effets démontrés : réduction de l’accumulation de lipides au niveau hépatique et musculaire ; amélioration de la sensibilité à l’insuline des tissus insulino-sensibles (notamment du muscle squelettique). Cependant, les données in vitro, in vivo ou cliniques, n’aboutissent pas à un consensus. La durée, la dose et la composition fine du traitement expliqueraient l’hétérogénéité des résultats. L’objectif de mon travail de thèse est de comparer les effets respectifs des acides gras alpha-linolénique (ALA), docosahexaénoïque (DHA) et éicosapenténoïque (EPA) dans la prévention de l’obésité et du syndrome métabolique. Pour cela, des souris seront mises sous régime riche en graisse et en sucrose, supplémenté ou non en ALA, EPA ou DHA durant 16 semaines. Cette étude permettra d’évaluer l’impact de chaque acide gras ω3 sur la composition corporelle, la sensibilité à l’insuline et l’inflammation systémique. Le modèle de cellules musculaires C2C12 nous permettra d’évaluer l’effet propre de chaque ω3 sur le devenir intracellulaire des acides gras (oxydation, estérification, production de dérivés lipotoxiques) et d’explorer les liens unissant les adaptations métaboliques et la sensibilité à l’insuline. Ce projet d’étude translationnelle (de la cellule au corps entier) contribuera à la meilleure compréhension des effets propres à chaque ω3 sur des voies importantes participant à la physiopathologie de l’obésité et du syndrome métabolique

N−3PUFA differentially modulate palmitate-induced lipotoxicity through alterations of its metabolism in C2C12 muscle cells

We gratefully acknowledge financial support from Avril (23000596) for the PhD thesis of A. Pinel and our research. We also thank Lesieur for their scientific supportExcessive energy intake leads to fat overload and the formation of lipotoxic compounds mainly derived from the saturated fatty add palmitate (PAL), thus promoting insulin resistance (IR) in skeletal muscle. N-3 polyunsaturated fatty acids (n - 3PUFA) may prevent lipotoxicity and IR. The purpose of this study was to examine the differential effects of n 3PUFA on fatty acid metabolism and insulin sensitivity in muscle cells. C2C12 myotubes were treated with 500 mu M of PAL without or with 50 mu M of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16h. PAL decreased insulin-dependent AKT activation and glucose uptake and increased the synthesis of ceramides and diglycerides (DG) derivatives, leading to protein kinase CO activation. EPA and DMA, but not ALA, prevented PAL-decreased AKT activation but glucose uptake was restored to control values by all n 3PUFA vs. PAL. Total DG and ceramide contents were decreased by all n 3PUFA, but only EPA and DMA increased PAL beta-oxidation, decreased PAL incorporation into DG and reduced protein kinase CO activation. EPA and DMA emerge as better candidates than ALA to improve fatty acid metabolism in skeletal muscle cells, notably via their ability to increase mitochondrial beta-oxidatio

Green autofluorescence, a double edged monitoring tool for bacterial growth and activity in micro-plates

International audienceThe intrinsic green autofluorescence of an Escherichia coli culture has long been overlooked and empirically corrected in green fluorescent protein (GFP) reporter experiments. We show here, by using complementary methods of fluorescence analysis and HPLC, that this autofluorescence, principally arise from the secreted flavins in the external media. The cells secrete roughly 10 times more than what they keep inside. We show next that the secreted flavin fluorescence can be used as a complementary method in measuring the cell concentration particularly when the classical method, based on optical density measure, starts to fail. We also demonstrate that the same external flavins limit the dynamical range of GFP quantification and can lead to a false interpretation of lower global dynamic range of expression than what really happens. In the end we evaluate different autofluorescence correction methods to extract the real GFP signal

Inference of quantitative models of bacterial promoters from time-series reporter gene data

International audienceThe inference of regulatory interactions and quantitative models of gene regulation from time-series transcriptomics data has been extensively studied and applied to a range of problems in drug discovery, cancer research, and biotechnology. The application of existing methods is commonly based on implicit assumptions on the biological processes under study. First, the measurements of mRNA abundance obtained in transcriptomics experiments are taken to be representative of protein concentrations. Second, the observed changes in gene expression are assumed to be solely due to transcription factors and other specific regulators, while changes in the activity of the gene expression machinery and other global physiological effects are neglected. While convenient in practice, these assumptions are often not valid and bias the reverse engineering process. Here we systematically investigate, using a combination of models and experiments, the importance of this bias and possible corrections. We measure in real time and in vivo the activity of genes involved in the FliA-FlgM module of the E. coli motility network. From these data, we estimate protein concentrations and global physiological effects by means of kinetic models of gene expression. Our results indicate that correcting for the bias of commonly-made assumptions improves the quality of the models inferred from the data. Moreover, we show by simulation that these improvements are expected to be even stronger for systems in which protein concentrations have longer half-lives and the activity of the gene expression machinery varies more strongly across conditions than in the FliA-FlgM module. The approach proposed in this study is broadly applicable when using time-series transcriptome data to learn about the structure and dynamics of regulatory networks. In the case of the FliA-FlgM module, our results demonstrate the importance of global physiological effects and the active regulation of FliA and FlgM half-lives for the dynamics of FliA-dependent promoters

Les acides gras omega-3 à longue chaîne réduisent la lipotoxicité induite par le palmitate en modifiant son métabolisme et son stockage dans la cellule musculaire C2C12

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