scholarly journals Radial Flow Perfusion Enables Real-Time Profiling of Cellular Metabolism at Low Oxygen Levels with Hyperpolarized 13C NMR Spectroscopy

Metabolites ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 576
Author(s):  
Anthony Mancuso ◽  
Mehrdad Pourfathi ◽  
Ryan M. Kiefer ◽  
Michael C. Noji ◽  
Sarmad Siddiqui ◽  
...  
Keyword(s):  
Radial Flow ◽  
Cell Metabolism ◽  
Cell Mass ◽  
Axial Flow ◽  
13C Nmr Spectroscopy ◽  
Resonance Spectroscopy ◽  
Low Oxygen ◽  
Hyperpolarized 13C ◽  
Flow Perfusion ◽  
Flow Configurations

In this study, we describe new methods for studying cancer cell metabolism with hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS) that will enable quantitative studies at low oxygen concentrations. Cultured hepatocellular carcinoma cells were grown on the surfaces of non-porous microcarriers inside an NMR spectrometer. They were perfused radially from a central distributer in a modified NMR tube (bioreactor). The oxygen level of the perfusate was continuously monitored and controlled externally. Hyperpolarized substrates were injected continuously into the perfusate stream with a newly designed system that prevented oxygen and temperature perturbations in the bioreactor. Computational and experimental results demonstrated that cell mass oxygen profiles with radial flow were much more uniform than with conventional axial flow. Further, the metabolism of HP [1-13C]pyruvate was markedly different between the two flow configurations, demonstrating the importance of avoiding large oxygen gradients in cell perfusion experiments.

Imaging tumour cell metabolism using hyperpolarized 13C magnetic resonance spectroscopy

2010 ◽  
Vol 38 (5) ◽  
pp. 1220-1224 ◽  
Author(s):  
Timothy H. Witney ◽  
Kevin M. Brindle
Keyword(s):  
Magnetic Resonance ◽  
Treatment Response ◽  
Tumour Cell ◽  
Cell Metabolism ◽  
Resonance Spectroscopy ◽  
Early Assessment ◽  
Drug Induced ◽  
Hyperpolarized 13C ◽  
New Treatments

Patients with similar tumour types frequently show different responses to the same therapy. The development of new treatments would benefit, therefore, from imaging methods that allow an early assessment of treatment response in individual patients, allowing rapid selection of the most effective treatment. We have been using 13C MRSI (magnetic resonance spectroscopic imaging) of tumour cell metabolism, using hyperpolarized 13C-labelled cellular metabolites, to detect treatment response. Nuclear spin hyperpolarization can increase sensitivity in the magnetic resonance experiment >10000 times, allowing us to image labelled cell substrates in vivo and their subsequent metabolism. We showed that exchange of hyperpolarized 13C label between lactate and pyruvate, catalysed by lactate dehydrogenase, was decreased in treated tumours undergoing drug-induced cell death, and that tissue pH could be imaged from the ratio of the signal intensities of hyperpolarized H13CO3− and 13CO2 following intravenous injection of hyperpolarized H13CO3. Tumour cell glutaminase activity, a potential measure of cell proliferation, can be determined using hyperpolarized [5-13C]glutamine, and treatment-induced tumour cell necrosis can be imaged in vivo from measurements of the conversion of hyperpolarized [1,4-13C2]fumarate into malate. Since these substrates are endogenous and, in some cases, have already been safely infused into patients, these techniques have the potential to translate to the clinic.

scholarly journals Metabolic Signatures of Cryptosporidium parvum-Infected HCT-8 Cells and Impact of Selected Metabolic Inhibitors on C. parvum Infection under Physioxia and Hyperoxia

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 60
Author(s):  
Juan Vélez ◽  
Zahady Velasquez ◽  
Liliana M. R. Silva ◽  
Ulrich Gärtner ◽  
Klaus Failing ◽  
...  
Keyword(s):  
Host Cell ◽  
Cryptosporidium Parvum ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Glucose Consumption ◽  
Host Cells ◽  
Metabolic Inhibitors ◽  
Low Oxygen ◽  
Metabolic Signatures

Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C.parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.

scholarly journals Assessment of Metabolic Fluxes in the Mouse Brain in Vivo Using 1H-[13C] NMR Spectroscopy at 14.1 Tesla

2015 ◽  
Vol 35 (5) ◽  
pp. 759-765 ◽  
Author(s):  
Lijing Xin ◽  
Bernard Lanz ◽  
gxia Lei ◽  
Rolf Gruetter
Keyword(s):  
Mouse Models ◽  
Mouse Brain ◽  
Conversion Rate ◽  
Tca Cycle ◽  
Compartment Model ◽  
13C Nmr Spectroscopy ◽  
Resonance Spectroscopy ◽  
Metabolic Fluxes ◽  
Short Echo Time

13C magnetic resonance spectroscopy (MRS) combined with the administration of 13C labeled substrates uniquely allows to measure metabolic fluxes in vivo in the brain of humans and rats. The extension to mouse models may provide exclusive prospect for the investigation of models of human diseases. In the present study, the short-echo-time (TE) full-sensitivity 1H-[13C] MRS sequence combined with high magnetic field (14.1 T) and infusion of [U-13C6] glucose was used to enhance the experimental sensitivity in vivo in the mouse brain and the 13C turnover curves of glutamate C4, glutamine C4, glutamate+glutamine C3, aspartate C2, lactate C3, alanine C3, γ-aminobutyric acid C2, C3 and C4 were obtained. A one-compartment model was used to fit 13C turnover curves and resulted in values of metabolic fluxes including the tricarboxylic acid (TCA) cycle flux VTCA (1.05 ± 0.04 μmol/g per minute), the exchange flux between 2-oxoglutarate and glutamate Vx (0.48 ± 0.02 μmol/g per minute), the glutamate-glutamine exchange rate Vgln (0.20 ± 0.02 μmol/g per minute), the pyruvate dilution factor Kdil (0.82 ± 0.01), and the ratio for the lactate conversion rate and the alanine conversion rate VLac/ VAla (10 ± 2). This study opens the prospect of studying transgenic mouse models of brain pathologies.

scholarly journals Combined XRD-paramagnetic 13C NMR spectroscopy of 1,2,3-triazoles for revealing copper traces in a Huisgen click-chemistry cycloaddition. A model case

2019 ◽  
Vol 25 (1) ◽  
pp. 98-106
Author(s):  
Daniel Canseco-González ◽  
José Luis Rodríguez de la O ◽  
José Enrique Herbert-Pucheta
Keyword(s):  
Click Chemistry ◽  
13C Nmr Spectroscopy ◽  
Model Systems ◽  
Paramagnetic Nmr ◽  
Resonance Spectroscopy ◽  
X Ray Diffraction ◽  
Model Case ◽  
Purification Methods ◽  
Paramagnetic Metal ◽  
Torsional Angles

AbstractCopper-catalyzed Alkyne-Azide Cycloaddition (CuAAC) click chemistry robustness has been demonstrated over recent years to produce 1,2,3-triazoles with excellent yields at mild conditions with simple purification methods. However, the consequences of having copper paramagnetic traces in final products, which complicate spectroscopic assignments and can produce inaccurate conclusions, has been scarcely discussed. Herein we present a strategy that combines X-Ray Diffraction (XRD) with 13C- paramagnetic Nuclear Magnetic Resonance spectroscopy, in order to demonstrate the presence of paramagnetic metal traces at standard Huisgen synthesis and purification conditions. We also demonstrate that the derivatization of 1,4-disubstituted-1,2,3-triazoles to produce 1,3,4,-trisubstituted-1,2,3.triazolium salts, promotes an efficient removal of Cu(II/I) moieties. Evidence of paramagnetic metal moieties is given using XRD structural analysis of abnormalities in torsional angles between substituents and the 1,2,3-triazole center, in parallel to 13C- paramagnetic NMR chemical shift and line width analysis. As model systems to demonstrate the importance of characterizing paramagnetic traces, we present the synthesis of novel 1-((3s,5s,7s)-adamantan-1-yl)-4-cyclopropyl-1H-1,2,3-triazole and its derivatized 1-((3s,5s,7s)-adamantan-1-yl)-4-cyclopropyl-3-methyl-1H-[1,2,3]-triazol-3-ium triflate salt.

scholarly journals Hypoxic-response elements in the oncolytic parvovirus Minute virus of mice do not allow for increased vector production at low oxygen concentration

2006 ◽  
Vol 87 (5) ◽  
pp. 1197-1201 ◽  
Author(s):  
Charlotte Servais ◽  
Perrine Caillet-Fauquet ◽  
Marie-Louise Draps ◽  
Thierry Velu ◽  
Yvan de Launoit ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Consensus Sequence ◽  
High Titre ◽  
Host Cells ◽  
Low Oxygen ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Responsive Element ◽  
Low Oxygen Concentration

Vectors derived from the autonomous parvovirus Minute virus of mice, MVM(p), are promising tools for the gene therapy of cancer. The validation of their in vivo anti-tumour effect is, however, hampered by the difficulty to produce high-titre stocks. In an attempt to increase vector titres, host cells were subjected to low oxygen tension (hypoxia). It has been shown that a number of viruses are produced at higher titres under these conditions. This is the case, among others, for another member of the family Parvoviridae, the erythrovirus B19 virus. Hypoxia stabilizes a hypoxia-inducible transcription factor (HIF-1α) that interacts with a ‘hypoxia-responsive element’ (HRE), the consensus sequence of which (A/GCGTG) is present in the B19 and MVM promoters. Whilst the native P4 promoter was induced weakly in hypoxia, vector production was reduced dramatically, and adding HRE elements to the P4 promoter of the vector did not alleviate this reduction. Hypoxia has many effects on cell metabolism. Therefore, even if the P4 promoter is activated, the cellular factors that are required for the completion of the parvoviral life cycle may not be expressed.

Blade Loadings for Hovercraft and other Radial Flow Fans

1967 ◽  
Vol 9 (4) ◽  
pp. 265-277 ◽  
Author(s):  
A. D. S. Carter
Keyword(s):  
Radial Flow ◽  
Axial Flow ◽  
Maximum Temperature ◽  
Centrifugal Fan ◽  
Blade Loading ◽  
Guide Vanes ◽  
Inlet Guide Vanes ◽  
Flow Compressor ◽  
High Work ◽  
Outside Diameter

The layout of a hovercraft leads naturally to the choice of a radial outward flow fan, but the aerodynamic requirements are more stringent than those normally associated with industrial fans. In this paper a blade loading criterion used extensively in axial flow compressor practice has been adapted to the more general case of radial flow fans. Using this criterion maximum fluid deflections and maximum temperature rise coefficients have been calculated. It is shown that fluid deflections in radial fans should be substantially lower than those in axial flow machines. For high work output the ratio of rotor outside diameter to rotor inside diameter should be as close to unity as is mechanically possible. Inlet guide vanes would be of no benefit to the conventional industrial type centrifugal fan, but for such applications as hovercraft inlet guide vanes could be most beneficial. The paper outlines those areas in which further research is necessary fully to confirm the approach, and hence the quantitative values, given in this paper.

EXTH-46. MRS BASED BIOMARKERS OF IDH1 MUTANT GLIOMA RESPONSE TO THE IDH INHIBITOR BAY-1436032

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi173-vi173
Author(s):  
Donghyun Hong ◽  
Noriaki Minami ◽  
Céline Taglang ◽  
Georgios Batsios ◽  
Anne Marie Gillespie ◽  
...  
Keyword(s):  
Animal Models ◽  
Animal Studies ◽  
Response To Treatment ◽  
Resonance Spectroscopy ◽  
Low Grade ◽  
Isocitrate Dehydrogenase 1 ◽  
1H Mrs ◽  
Hyperpolarized 13C ◽  
13C Mrs ◽  
Mutant Idh1

Abstract Gliomas are the most prevalent type of brain tumor in the central nervous system. Mutations in the cytosolic enzyme isocitrate dehydrogenase 1 (IDH1) are a common feature of primary low-grade gliomas, catalyzing the conversion of α-ketoglutarate (αKG) to the oncometabolite 2-hydroxyglutarate (2HG), and mutant IDH1 is a therapeutic target for these tumors. Several mutant IDH inhibitors are currently in clinical trials, nonetheless, complementary non-invasive early biomarkers to assess drug delivery and potential therapeutic response are still needed. The goal of this study was therefore to determine the potential of 1H and hyperpolarized 13C magnetic resonance spectroscopy (MRS)-based biomarkers as indicators of mutant IDH1 low-grade glioma response to treatment with the clinically-relevant IDH1 inhibitor BAY-1436032 in cells and animal models. Immortalized human astrocytes engineered to express mutant IDH1 were treated with 500nM (IC50 value) of BAY-1436032 and BT257 tumors implanted in rats were treated with 150mg/kg of BAY-1436032. To assess steady-state metabolite levels, 1H MRS spectra were acquired on a 500 MHz MRS cancer for cells and a 3 T scanner for animal studies. To assess metabolic fluxes, we used hyperpolarized 13C MRS and probed the fate of hyperpolarized [1-13C]αKG. 1H MRS showed a significant decrease in 2HG as well as a significant increase in glutamate (Glu) and phosphocholine (PCh) following BAY-1436032 treatment in both cell and animal models compared to controls. Furthermore, hyperpolarized 13C MRS showed that hyperpolarized 2HG production from hyperpolarized [1-13C]αKG was decreased and hyperpolarized glutamate production from hyperpolarized [1-13C]αKG was increased in the BAY-1436032 treated groups compared to controls. These findings are consistent with our previous study, which investigated the MRS-detectable consequences of two other mutant IDH inhibitors: AG120 and AG881. Collectively, our work identifies translatable MRS-based metabolic biomarkers of mutant IDH1 inhibition.

scholarly journals Evaluation of LDH-A and Glutaminase Inhibition In Vivo by Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy of Tumors

2013 ◽  
Vol 73 (14) ◽  
pp. 4190-4195 ◽  
Author(s):  
Prasanta Dutta ◽  
Anne Le ◽  
David L. Vander Jagt ◽  
Takashi Tsukamoto ◽  
Gary V. Martinez ◽  
...  
Keyword(s):  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Hyperpolarized 13C
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