High-Speed Tracer Analysis of Metabolism (HS-TrAM)

Tracing the fate of stable isotopically-enriched nutrients is a sophisticated method of describing and quantifying the activity of metabolic pathways. Nuclear Magnetic Resonance (NMR) spectroscopy offers high resolution data in terms of resolving metabolic pathway utilisation. Despite this, NMR spectroscopy is under-utilised due to length of time required to collect the data, quantification requiring multiple samples and complicated analysis. Here we present two techniques, quantitative spectral filters and enhancement of the splitting of 13C signals due to homonuclear 13C,13C or heteronuclear 13C,15N J-coupling in 1H,13C-HSQC NMR spectra. Together, these allow the rapid collection of NMR spectroscopy data in a quantitative manner on a single sample. The reduced duration of HSQC spectra data acquisition opens up the possibility of real-time tracing of metabolism including the study of metabolic pathways in vivo. We show how these techniques can be used to trace the fate of labelled nutrients in a whole organ model of kidney preservation prior to transplantation using a porcine kidney as a model organ. In addition, we show how the use of multiple nutrients, differentially labelled with 13C and 15N, can be used to provide additional information with which to profile metabolic pathways.

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High-Speed Tracer Analysis of Metabolism (HS-TrAM)

Wellcome Open Research ◽

10.12688/wellcomeopenres.13387.1 ◽

2018 ◽

Vol 3 ◽

pp. 5 ◽

Cited By ~ 1

Author(s):

Thomas Brendan Smith ◽

Kamlesh Patel ◽

Haydn Munford ◽

Andrew Peet ◽

Daniel A. Tennant ◽

...

Keyword(s):

Metabolic Pathways ◽

High Speed ◽

Kidney Preservation ◽

High Resolution Data ◽

Additional Information ◽

Quantitative Manner ◽

Organ Model ◽

Time Required ◽

Multiple Samples

Tracing the fate of stable isotopically-enriched nutrients is a sophisticated method of describing and quantifying the activity of metabolic pathways. Nuclear Magnetic Resonance (NMR) offers high resolution data, yet is under-utilised due to length of time required to collect the data, quantification requiring multiple samples and complicated analysis. Here we present two techniques, quantitative spectral filters and enhancement of the splitting due to J-coupling in 1H,13C-HSQC NMR spectra, which allow the rapid collection of NMR data in a quantitative manner on a single sample. The reduced duration of HSQC spectra data acquisition opens up the possibility of real-time tracing of metabolism including the study of metabolic pathways in vivo. We show how these novel techniques can be used to trace the fate of labelled nutrients in a whole organ model of kidney preservation prior to transplantation using a porcine kidney as a model organ, and also show how the use of multiple nutrients, differentially labelled with 13C and 15N, can be used to provide additional information with which to profile metabolic pathways.

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Metabolic pathways for ketone body production. Carbon-13 NMR spectroscopy of rat liver in vivo using carbon-13-multilabeled fatty acids

Biochemistry ◽

10.1021/bi00370a011 ◽

1986 ◽

Vol 25 (22) ◽

pp. 6799-6807 ◽

Cited By ~ 29

Author(s):

Claudia Pahl-Wostl ◽

Joachim Seelig

Keyword(s):

Fatty Acids ◽

Nmr Spectroscopy ◽

Rat Liver ◽

Metabolic Pathways ◽

Ketone Body ◽

Body Production

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Metabolism of nitrate and ammonium in seedlings of Norway spruce (Picea abies) measured by in vivo 14N and 15N NMR spectroscopy

Physiologia Plantarum ◽

10.1034/j.1399-3054.1995.940302.x ◽

1995 ◽

Vol 94 (3) ◽

pp. 384-390 ◽

Cited By ~ 1

Author(s):

Halvor Aarnes ◽

Aud B. Eriksen ◽

Timothy E. Southon

Keyword(s):

Nmr Spectroscopy ◽

Picea Abies ◽

Norway Spruce ◽

15N Nmr ◽

15N Nmr Spectroscopy

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Biological NMR Spectroscopy

10.1093/oso/9780195094688.001.0001 ◽

1997 ◽

Keyword(s):

Nuclear Magnetic Resonance ◽

Nmr Spectroscopy ◽

Magnetic Resonance ◽

Magnetic Resonance Spectroscopy ◽

State Of The Art ◽

Critical Assessment ◽

Resonance Spectroscopy ◽

History Of ◽

Structure Of Proteins

This book presents a critical assessment of progress on the use of nuclear magnetic resonance spectroscopy to determine the structure of proteins, including brief reviews of the history of the field along with coverage of current clinical and in vivo applications. The book, in honor of Oleg Jardetsky, one of the pioneers of the field, is edited by two of the most highly respected investigators using NMR, and features contributions by most of the leading workers in the field. It will be valued as a landmark publication that presents the state-of-the-art perspectives regarding one of today's most important technologies.

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High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

Nature Communications ◽

10.1038/s41467-020-19851-1 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Jiang Lan Fan ◽

Jose A. Rivera ◽

Wei Sun ◽

John Peterson ◽

Henry Haeberle ◽

...

Keyword(s):

Blood Flow ◽

High Speed ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Microscopy ◽

Fluorescence Signal ◽

Imaging Method ◽

Spatiotemporal Resolution ◽

Two Photon

AbstractUnderstanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

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Integrated multi-omics analysis of RB-loss identifies widespread cellular programming and synthetic weaknesses

Communications Biology ◽

10.1038/s42003-021-02495-2 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Swetha Rajasekaran ◽

Jalal Siddiqui ◽

Jessica Rakijas ◽

Brandon Nicolay ◽

Chenyu Lin ◽

...

Keyword(s):

Metabolic Pathways ◽

Human Cells ◽

Cellular Reprogramming ◽

Hallmarks Of Cancer ◽

Compensatory Mechanisms ◽

Response Factors ◽

Cancer Data ◽

Cell Stress Response ◽

Metabolic Output

AbstractInactivation of RB is one of the hallmarks of cancer, however gaps remain in our understanding of how RB-loss changes human cells. Here we show that pRB-depletion results in cellular reprogramming, we quantitatively measured how RB-depletion altered the transcriptional, proteomic and metabolic output of non-tumorigenic RPE1 human cells. These profiles identified widespread changes in metabolic and cell stress response factors previously linked to E2F function. In addition, we find a number of additional pathways that are sensitive to RB-depletion that are not E2F-regulated that may represent compensatory mechanisms to support the growth of RB-depleted cells. To determine whether these molecular changes are also present in RB1−/− tumors, we compared these results to Retinoblastoma and Small Cell Lung Cancer data, and identified widespread conservation of alterations found in RPE1 cells. To define which of these changes contribute to the growth of cells with de-regulated E2F activity, we assayed how inhibiting or depleting these proteins affected the growth of RB1−/− cells and of Drosophila E2f1-RNAi models in vivo. From this analysis, we identify key metabolic pathways that are essential for the growth of pRB-deleted human cells.

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Sampling Method Affects HR-MAS NMR Spectra of Healthy Caprine Brain Biopsies

Metabolites ◽

10.3390/metabo11010038 ◽

2021 ◽

Vol 11 (1) ◽

pp. 38

Author(s):

Annakatrin Häni ◽

Gaëlle Diserens ◽

Anna Oevermann ◽

Peter Vermathen ◽

Christina Precht

Keyword(s):

Nmr Spectroscopy ◽

Sampling Method ◽

Magic Angle Spinning ◽

Mas Nmr ◽

Magic Angle ◽

1H Nuclear Magnetic Resonance ◽

Rapid Changes ◽

Tissue Degradation ◽

Angle Spinning

The metabolic profiling of tissue biopsies using high-resolution–magic angle spinning (HR-MAS) 1H nuclear magnetic resonance (NMR) spectroscopy may be influenced by experimental factors such as the sampling method. Therefore, we compared the effects of two different sampling methods on the metabolome of brain tissue obtained from the brainstem and thalamus of healthy goats by 1H HR-MAS NMR spectroscopy—in vivo-harvested biopsy by a minimally invasive stereotactic approach compared with postmortem-harvested sample by dissection with a scalpel. Lactate and creatine were elevated, and choline-containing compounds were altered in the postmortem compared to the in vivo-harvested samples, demonstrating rapid changes most likely due to sample ischemia. In addition, in the brainstem samples acetate and inositols, and in the thalamus samples ƴ-aminobutyric acid, were relatively increased postmortem, demonstrating regional differences in tissue degradation. In conclusion, in vivo-harvested brain biopsies show different metabolic alterations compared to postmortem-harvested samples, reflecting less tissue degradation. Sampling method and brain region should be taken into account in the analysis of metabolic profiles. To be as close as possible to the actual situation in the living individual, it is desirable to use brain samples obtained by stereotactic biopsy whenever possible.

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