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Gallagher F.A.,University of Cambridge | Kettunen M.I.,University of Cambridge | Day S.E.,University of Cambridge | Hu D.-E.,University of Cambridge | And 5 more authors.
Magnetic Resonance in Medicine | Year: 2011

Dynamic nuclear polarization can be used to increase the sensitivity of solution state 13C magnetic resonance spectroscopy by four orders of magnitude. We show here that [1- 13C]glutamate can be polarized to 28%, representing a 35,000-fold increase in its sensitivity to detection at 9.4 T and 37°C. The metabolism of hyperpolarized glutamate to α-ketoglutarate, catalyzed by the enzyme alanine transaminase, was detected in vitro in human hepatoma cells (HepG2). Incubation of the cells with sodium pyruvate increased the level of the hyperpolarized label in the α-ketoglutarate pool, with an associated increase in the apparent rate constant describing flux of hyperpolarized 13C label between glutamate and α-ketoglutarate. The metabolism of hyperpolarized glutamate was observed in vivo following coadministration of pyruvate in a murine lymphoma model. This represents a new method to probe glutamate metabolism and citric acid cycle activity in vivo; as glutamate is an endogenous molecule, it has the potential to be used in the clinic. Magn Reson Med, 2011. © 2011 Wiley-Liss, Inc. Copyright © 2011 Wiley-Liss, Inc. Source


Schroeder M.A.,University of Oxford | Swietach P.,University of Oxford | Atherton H.J.,University of Oxford | Gallagher F.A.,University of Cambridge | And 5 more authors.
Cardiovascular Research | Year: 2010

Aims: Technological limitations have restricted in vivo assessment of intracellular pH (pHi) in the myocardium. The aim of this study was to evaluate the potential of hyperpolarized [1-13C]pyruvate, coupled with 13C magnetic resonance spectroscopy (MRS), to measure pH i in the healthy and diseased heart. Methods and results: Hyperpolarized [1-13C]pyruvate was infused into isolated rat hearts before and immediately after ischaemia, and the formation of 13CO2 and H13CO3 - was monitored using 13C MRS. The HCO3 -/CO 2 ratio was used in the Henderson-Hasselbalch equation to estimate pHi. We tested the validity of this approach by comparing 13C-based pHi measurements with 31P MRS measurements of pHi. There was good agreement between the pHi measured using 13C and 31P MRS in control hearts, being 7.12 ± 0.10 and 7.07 ± 0.02, respectively. In reperfused hearts, 13C and 31P measurements of pHi also agreed, although 13C equilibration limited observation of myocardial recovery from acidosis. In hearts pre-treated with the carbonic anhydrase (CA) inhibitor, 6-ethoxyzolamide, the 13C measurement underestimated the 31P-measured pHi by 0.80 pH units. Mathematical modelling predicted that the validity of measuring pHi from the H 13CO3 -/13CO2 ratio depended on CA activity, and may give an incorrect measure of pHi under conditions in which CA was inhibited, such as in acidosis. Hyperpolarized [1-13C]pyruvate was also infused into healthy living rats, where in vivo pHi from the H13CO3 -/ 13CO2 ratio was measured to be 7.20 ± 0.03. Conclusion: Metabolically generated 13CO2 and H 13CO3 - can be used as a marker of cardiac pHi in vivo, provided that CA activity is at normal levels. © The Author 2009. Source


Trappmann B.,University of Cambridge | Gautrot J.E.,University of Cambridge | Connelly J.T.,University of Cambridge | Connelly J.T.,Queen Mary, University of London | And 14 more authors.
Nature Materials | Year: 2012

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal- related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions. © 2012 Macmillan Publishers Limited. All rights reserved. Source


Aldridge S.,CRUK Cambridge Research Institute | Hadfield J.,CRUK Cambridge Research Institute
Methods in Molecular Biology | Year: 2012

MicroRNA analysis has been widely adopted for basic and applied science. The tools and technologies available for quantifying and analysing miRNAs are still maturing. Here, we give an introductory overview of the main tools and the challenges in their use. We also discuss the importance of basic experimental design, sample handling and analysis methods as the impact of these can be as profound as the choice of miRNA analysis platform. Whether the reader is interested in a gene-by-gene or genome-wide approach choosing the platform to use is not trivial. Careful thought given before starting an experiment will make the execution much easier. © 2012 Springer Science+Business Media, LLC. Source


Massie C.E.,CRUK Cambridge Research Institute | Massie C.E.,Cambridge Institute for Medical Research | Mills I.G.,CRUK Cambridge Research Institute | Mills I.G.,University of Oslo
Methods in Molecular Biology | Year: 2012

Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method. Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method. Source

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