The Experts below are selected from a list of 1095 Experts worldwide ranked by ideXlab platform
Jeancharles Portais - One of the best experts on this subject based on the ideXlab platform.
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recent advances in high throughput 13c Fluxomics
Current Opinion in Biotechnology, 2017Co-Authors: Stephanie Heux, Cecilia Berges, Pierre Millard, Jeancharles Portais, Fabien LetisseAbstract:The rise of high throughput (HT) strain engineering tools accompanying the area of synthetic biology is supporting the generation of a large number of microbial cell factories. A current bottleneck in process development is our limited capacity to rapidly analyze the metabolic state of the engineered strains, and in particular their intracellular fluxes. HT 13C-Fluxomics workflows have not yet become commonplace, despite the existence of several HT tools at each of the required stages. This includes cultivation and sampling systems, analytics for isotopic analysis, and software for data processing and flux calculation. Here, we review recent advances in the field and highlight bottlenecks that must be overcome to allow the emergence of true HT 13C-Fluxomics workflows.
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Ultrafast quantitative 2D NMR: an efficient tool for the measurement of specific isotopic enrichments in complex biological mixtures.
Analytical Chemistry, 2011Co-Authors: Patrick Giraudeau, Jeancharles Portais, Stephane Massou, Edern Cahoreau, Yoann Robin, Serge AkokaAbstract:Two-dimensional nuclear magnetic resonance (2D NMR) is a promising tool for studying metabolic fluxes by measuring 13C-enrichments in complex mixtures of 13C-labeled metabolites. However, the methods reported so far are hampered by very long acquisition durations limiting the use of 2D NMR as a quantitative tool for Fluxomics. In this paper, we propose a new approach for measuring specific 13C-enrichments in a very fast way, by using new experiments based on ultrafast 2D NMR. Two homonuclear 2D experiments (ultrafast COSY and zTOCSY) are proposed to measure 13C-enrichments in a single scan. Their advantages and limitations are discussed, and their high analytical potentialities are highlighted. Both methods are characterized by an accuracy of 1−2%, an average precision of 3%, and an excellent linearity. The analytical performance is equivalent or better than any of the conventional methods previously reported. The two ultrafast experiments are applied to the measurement of 13C-enrichments on a biomass hyd...
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nmr based Fluxomics quantitative 2d nmr methods for isotopomers analysis
Phytochemistry, 2007Co-Authors: Stephane Massou, Jeancharles Portais, Fabien Letisse, Cecile NicolasAbstract:Abstract We have investigated the reliability of 2D-COSY and 2D-TOCSY experiments to provide accurate measurements of 13 C-enrichments in complex mixtures of 13 C-labelled metabolites. This was done from both theoretical considerations and experimental investigations. The results showed that 2D-TOCSY but not 2D-COSY could provide accurate measurements of 13 C-enrichments, provided efficient zero-quantum filters were applied during the mixing period. This approach extends the range of NMR methods applicable in 13 C-labelling experiments and is suitable to investigating the dynamic behaviour of metabolic systems.
Fabien Letisse - One of the best experts on this subject based on the ideXlab platform.
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increasing field strength versus advanced isotope labeling for nmr based Fluxomics
Magnetic Resonance in Chemistry, 2020Co-Authors: Mickael Dinclaux, Pierre Millard, Fabien Letisse, Edern Cahoreau, Guy LippensAbstract:: Nuclear magnetic resonance (NMR)-based Fluxomics seeks to measure the incorporation of isotope labels in selected metabolites to follow kinetically the synthesis of the latter. It can however equally be used to understand the biosynthetic origin of the same metabolites. We investigate here different NMR approaches to optimize such experiments in terms of resolution and time requirement. Using the isoleucine biosynthesis as an example, we explore the use of different field strengths ranging from 500 MHz to 1.1 GHz. Because of the different field dependence of chemical shift and heteronuclear J couplings, the spectra change at different field strengths. We equally explore the approach to silence the leucine/valine methyl signals through the use of a suitable deuterated precursor, thereby allowing selective observation of the Ile 13 C labeling pattern. Combining both approaches, we arrive at an efficient procedure for the NMR-based exploration of Ile biosynthesis.
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recent advances in high throughput 13c Fluxomics
Current Opinion in Biotechnology, 2017Co-Authors: Stephanie Heux, Cecilia Berges, Pierre Millard, Jeancharles Portais, Fabien LetisseAbstract:The rise of high throughput (HT) strain engineering tools accompanying the area of synthetic biology is supporting the generation of a large number of microbial cell factories. A current bottleneck in process development is our limited capacity to rapidly analyze the metabolic state of the engineered strains, and in particular their intracellular fluxes. HT 13C-Fluxomics workflows have not yet become commonplace, despite the existence of several HT tools at each of the required stages. This includes cultivation and sampling systems, analytics for isotopic analysis, and software for data processing and flux calculation. Here, we review recent advances in the field and highlight bottlenecks that must be overcome to allow the emergence of true HT 13C-Fluxomics workflows.
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nmr based Fluxomics quantitative 2d nmr methods for isotopomers analysis
Phytochemistry, 2007Co-Authors: Stephane Massou, Jeancharles Portais, Fabien Letisse, Cecile NicolasAbstract:Abstract We have investigated the reliability of 2D-COSY and 2D-TOCSY experiments to provide accurate measurements of 13 C-enrichments in complex mixtures of 13 C-labelled metabolites. This was done from both theoretical considerations and experimental investigations. The results showed that 2D-TOCSY but not 2D-COSY could provide accurate measurements of 13 C-enrichments, provided efficient zero-quantum filters were applied during the mixing period. This approach extends the range of NMR methods applicable in 13 C-labelling experiments and is suitable to investigating the dynamic behaviour of metabolic systems.
Julian L. Griffin - One of the best experts on this subject based on the ideXlab platform.
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An introduction to biological nuclear magnetic resonance spectroscopy
Biological Reviews, 2011Co-Authors: John H.f. Bothwell, Julian L. GriffinAbstract:Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques available to biology. This review is an introduction to the potential of this method and is aimed at readers who have little or no experience in acquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, rather than imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which to address a number of biological problems. Using detailed examples, we discuss the use of 1H NMR spectroscopy in mixture analysis and metabolomics, the use of 13C NMR spectroscopy in tracking isotopomers and determining the flux through metabolic pathways (‘Fluxomics’) and the use of 31P NMR spectroscopy in monitoring ATP generation and intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probe the physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and how it can determine macromolecular structures by measuring the bonds and distances which separate individual atoms. We finish by outlining some of the key challenges which remain in NMR spectroscopy and we highlight how recent advances—such as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisation—are opening new avenues for today's biological NMR spectroscopists.
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Applications of metabolomics and proteomics to the mdx mouse model of Duchenne muscular dystrophy: lessons from downstream of the transcriptome
Genome Medicine, 2009Co-Authors: Julian L. Griffin, Christine Des RosiersAbstract:Functional genomic studies are dominated by transcriptomic approaches, in part reflecting the vast amount of information that can be obtained, the ability to amplify mRNA and the availability of commercially standardized functional genomic DNA microarrays and related techniques. This can be contrasted with proteomics, metabolomics and metabolic flux analysis (Fluxomics), which have all been much slower in development, despite these techniques each providing a unique viewpoint of what is happening in the overall biological system. Here, we give an overview of developments in these fields 'downstream' of the transcriptome by considering the characterization of one particular, but widely used, mouse model of human disease. The mdx mouse is a model of Duchenne muscular dystrophy (DMD) and has been widely used to understand the progressive skeletal muscle wasting that accompanies DMD, and more recently the associated cardiomyopathy, as well as to unravel the roles of the other isoforms of dystrophin, such as those found in the brain. Studies using proteomics, metabolomics and Fluxomics have characterized perturbations in calcium homeostasis in dystrophic skeletal muscle, provided an understanding of the role of dystrophin in skeletal muscle regeneration, and defined the changes in substrate energy metabolism in the working heart. More importantly, they all point to perturbations in proteins, metabolites and metabolic fluxes reflecting mitochondrial energetic alterations, even in the early stage of the dystrophic pathology. Philosophically, these studies also illustrate an important lesson relevant to both functional genomics and the mouse phenotyping in that the knowledge generated has advanced our understanding of cell biology and physiological organization as much as it has advanced our understanding of the disease.
Patrick Giraudeau - One of the best experts on this subject based on the ideXlab platform.
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nmr based metabolomics and Fluxomics developments and future prospects
Analyst, 2020Co-Authors: Patrick GiraudeauAbstract:NMR spectroscopy is an essential analytical technique in metabolomics and Fluxomics workflows, owing to its high structural elucidation capabilities combined with its intrinsic quantitative nature. However, routine NMR “omic” analytical methods suffer from several drawbacks that may have limited their use as a method of choice, in particular when compared to another widely used technique, mass spectrometry. This review describes, in a critical and perspective discussion, how some of the most recent developments emerging from the NMR community could act as real game changers for metabolomics and Fluxomics in the near future. Advanced developments to make NMR metabolomics more resolutive, more sensitive and more accessible are described, as well as new approaches to improve the identification of biomarkers. We hope that this review will convince a broad end-user community of the increasing role of NMR in the “omic” world at the beginning of the 2020s.
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Ultrafast quantitative 2D NMR: an efficient tool for the measurement of specific isotopic enrichments in complex biological mixtures.
Analytical Chemistry, 2011Co-Authors: Patrick Giraudeau, Jeancharles Portais, Stephane Massou, Edern Cahoreau, Yoann Robin, Serge AkokaAbstract:Two-dimensional nuclear magnetic resonance (2D NMR) is a promising tool for studying metabolic fluxes by measuring 13C-enrichments in complex mixtures of 13C-labeled metabolites. However, the methods reported so far are hampered by very long acquisition durations limiting the use of 2D NMR as a quantitative tool for Fluxomics. In this paper, we propose a new approach for measuring specific 13C-enrichments in a very fast way, by using new experiments based on ultrafast 2D NMR. Two homonuclear 2D experiments (ultrafast COSY and zTOCSY) are proposed to measure 13C-enrichments in a single scan. Their advantages and limitations are discussed, and their high analytical potentialities are highlighted. Both methods are characterized by an accuracy of 1−2%, an average precision of 3%, and an excellent linearity. The analytical performance is equivalent or better than any of the conventional methods previously reported. The two ultrafast experiments are applied to the measurement of 13C-enrichments on a biomass hyd...
Wolf B Frommer - One of the best experts on this subject based on the ideXlab platform.
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comparison of quantitative metabolite imaging tools and carbon 13 techniques for Fluxomics
Methods of Molecular Biology, 2009Co-Authors: Totte Niittylae, Bhavna Chaudhuri, Uwe Sauer, Wolf B FrommerAbstract:The recent development of analytic technologies allows fast analysis of metabolism in real time. Fluxomics aims to define the genes involved in regulation of flux through a metabolic or signaling pathway. Flux through a metabolic or signaling pathway is determined by the activity of its individual components; regulation can occur at many levels, including transcriptional, posttranslational, and allosteric levels. Currently two technologies are used to monitor fluxes. The first is pulse labeling of the organism with a tracer such as C13, followed by mass spectrometric analysis of the partitioning of label into different compounds. The second approach is based on the use of flux sensors, proteins that respond with a conformational change to ligand binding. Fluorescence resonance energy transfer (FRET) detects the conformational change and serves as a proxy for ligand concentration. Both methods provide high time resolution. In contrast to mass spectrometry assays, FRET nanosensors monitor only a single compound, but the advantage of FRET nanosensors is that they yield data with cellular and subcellular resolution.
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Fluxomics mass spectrometry versus quantitative imaging
Current Opinion in Plant Biology, 2007Co-Authors: Wolfgang Wiechert, Oliver Schweissgut, Hitomi Takanaga, Wolf B FrommerAbstract:The recent development of analytic high-throughput technologies enables us to take a bird's view of how metabolism is regulated in real time. We have known for a long time that metabolism is highly regulated at all levels, including transcriptional, posttranslational and allosteric controls. Flux through a metabolic or signaling pathway is determined by the activity of its individual components. Fluxomics aims to define the genes involved in regulation by following the flux. Two technologies are used to monitor fluxes. Pulse labeling of the organism or cell with a tracer, such as 13 C, followed by mass spectrometric analysis of the partitioning of label into different compounds provides an efficient tool to study flux and to compare the effect of mutations on flux. The second approach is based on the use of flux sensors, proteins that respond with a conformational change to ligand binding. Fluorescence resonance energy transfer (FRET) detects the conformational change and serves as a proxy for ligand concentration. In contrast to the mass spectrometry assays, FRET nanosensors monitor only a single compound. Both methods provide high time resolution. The major advantages of FRET nanosensors are that they yield data with cellular and subcellular resolution and the method is minimally invasive.
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Fluxomics with ratiometric metabolite dyes
Plant Signaling & Behavior, 2007Co-Authors: Bhavna Chaudhuri, Totte Niittyla, Friederike Hormann, Wolf B FrommerAbstract:Today’s major excitement in biology centers on signaling: How can a cell or organism measure the myriad of environmental cues, integrate it, and acclimate to the new conditions? Hormonal signals and second messengers are in the focus of most of these studies, e.g. regulation of glucose transporter GLUT4 cycling by insulin, or regulation of plant growth by auxin or brassinosteroids.1-3 In comparison, we generally assume that we know almost everything about basic metabolism since it has been studied for many decades; for example we know since the early 80s that allosteric regulation by fructose-2,6-bisphophate plays an important role in regulating glycolysis in plants and animals. 4 This may be the reason why studies of metabolism appear to be a bit out of fashion. But if we look to other organisms such as E. coli or yeast, we rapidly realize that metabolism is controlled by complex interconnected signaling networks, and that we understand little of these signaling networks in humans and plants. 5,6 As it t...
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A novel analytical method for in vivo phosphate tracking
FEBS Letters, 2006Co-Authors: Hong Gu, Sylvie Lalonde, Sakiko Okumoto, Loren L. Looger, Anne Marie Scharff-poulsen, Arthur R. Grossman, Jens Kossmann, Iver Jakobsen, Wolf B FrommerAbstract:Abstract Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (Pi) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows Pi-dependent increases in FRET efficiency. FLIPPi affinity mutants cover Pi changes over eight orders of magnitude. COS-7 cells co-expressing a low-affinity FLIPPi and a Na+/Pi co-transporter exhibited FRET changes when perfused with 100 μM Pi, demonstrating concentrative Pi uptake by PiT2. FLIPPi sensors are suitable for real-time monitoring of Pi metabolism in living cells, providing a new tool for Fluxomics, analysis of pathophysiology or changes of Pi during cell migration.