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Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.
PURPOSE: A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells.
Hyperpolarized singlet lifetimes of pyruvate in human blood and in the mouse.
Hyperpolarized NMR is a promising technique for non-invasive imaging of tissue metabolism in vivo. However, the pathways that can be studied are limited by the fast T1 decay of the nuclear spin order. In metabolites containing pairs of coupled nuclear spins-1/2, the spin order may be maintained by exploiting the non-magnetic singlet (spin-0) state of the pair. This may allow preservation of the hyperpolarization in vivo during transport to tissues of interest, such as tumors, or to detect slower metabolic reactions. We show here that in human blood and in a mouse in vivo at millitesla fields the (13)C singlet lifetime of [1,2-(13)C2]pyruvate was significantly longer than the (13)C T1, although it was shorter than the T1 at field strengths of several tesla. We also examine the singlet-derived NMR spectrum observed for hyperpolarized [1,2-(13)C2]lactate, originating from the metabolism of [1,2-(13)C2]pyruvate.
(13) C magnetic resonance spectroscopy measurements with hyperpolarized [1-(13) C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo.
PURPOSE: Dissolution dynamic nuclear polarization can increase the sensitivity of the (13) C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize (13) C-labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.1.1), which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. METHODS: Pyruvate decarboxylase from Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using (13) C MRS measurements of the conversion of hyperpolarized [1-(13) C] pyruvate to H(13) CO3-. RESULTS: Induction of zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two-fold increase in the H(13) CO3-/[1-(13) C] pyruvate signal ratio following intravenous injection of hyperpolarized [1-(13) C] pyruvate. CONCLUSION: We have demonstrated the feasibility of using zmPDC as an in vivo reporter gene for use with hyperpolarized (13) C MRS. Magn Reson Med 76:391-401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
(13) C magnetic resonance spectroscopic imaging of hyperpolarized [1-(13) C, U-(2) H5 ] ethanol oxidation can be used to assess aldehyde dehydrogenase activity in vivo.
PURPOSE: Aldehyde dehydrogenase (ALDH2) is an emerging drug target for the treatment of heart disease, cocaine and alcohol dependence, and conditions caused by genetic polymorphisms in ALDH2. Noninvasive measurement of ALDH2 activity in vivo could inform the development of these drugs and accelerate their translation to the clinic. METHODS: [1-(13) C, U-(2) H5 ] ethanol was hyperpolarized using dynamic nuclear polarization, injected into mice and its oxidation in the liver monitored using (13) C MR spectroscopy and spectroscopic imaging. RESULTS: Oxidation of [1-(13) C, U-(2) H5 ] ethanol to [1-(13) C] acetate was observed. Saturation of the acetaldehyde resonance, which was below the level of detection in vivo, demonstrated that acetate was produced via acetaldehyde. Irreversible inhibition of ALDH2 activity with disulfiram resulted in a proportional decrease in the amplitude of the acetate resonance. CONCLUSION: (13) C magnetic resonance spectroscopy measurements of hyperpolarized [1-(13) C, U-(2) H5 ] ethanol oxidation allow real-time assessment of ALDH2 activity in liver in vivo.
Detection of transgene expression using hyperpolarized 13C urea and diffusion-weighted magnetic resonance spectroscopy.
PURPOSE: To assess the potential of a gene reporter system, based on a urea transporter (UTB) and hyperpolarized [(13) C]urea. METHODS: Mice were implanted subcutaneously with either unmodified control cells or otherwise identical cells expressing UTB. After injection of hyperpolarized [(13) C]urea, a spin echo sequence was used to measure urea concentration, T1 , and diffusion in control and UTB-expressing tissue. RESULTS: The apparent diffusion coefficient of hyperpolarized urea was 21% lower in tissue expressing UTB, in comparison with control tissue (P < 0.05, 1-tailed t-test, n = 6 in each group). No difference in water apparent diffusion coefficient or cellularity between these tissues was found, indicating that they were otherwise similar in composition. CONCLUSION: Expression of UTB, by mediating cell uptake of urea, lowers the apparent diffusion coefficient of hyperpolarized (13) C urea in tissue and thus the transporter has the potential to be used as a magnetic resonance-based gene reporter in vivo. Magn Reson Med 73:1401-1406, 2015. © 2014 Wiley Periodicals, Inc.
Imaging Tumor Metabolism to Assess Disease Progression and Treatment Response.
Changes in tumor metabolism may accompany disease progression and can occur following treatment, often before there are changes in tumor size. We focus here on imaging methods that can be used to image various aspects of tumor metabolism, with an emphasis on methods that can be used for tumor grading, assessing disease progression, and monitoring treatment response. Clin Cancer Res; 22(21); 5196-203. ©2016 AACR.
USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation.
The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.
Compact and contactless reflectance confocal microscope for neurosurgery
Visual guidance at the cellular level during neurosurgical procedures is essential for complete tumour resection. We present a compact reflectance confocal microscope with a 20 mm working distance that provided <1.2 μm spatial resolution over a 600 μm × 600 μm field of view in the near-infrared region. A physical footprint of 200 mm × 550 mm was achieved using only standard off-the-shelf components. Theoretical performance of the optical design was first evaluated via commercial Zemax software. Then three specimens from rodents: fixed brain, frozen calvaria and live hippocampal slices, were used to experimentally assess system capability and robustness. Results show great potential for the proposed system to be translated into use as a next generation label-free and contactless neurosurgical microscope.
z-STED Imaging and Spectroscopy to Investigate Nanoscale Membrane Structure and Dynamics.
Super-resolution stimulated emission depletion (STED) microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy) or axially (z). Two-dimensional STED has been extensively used to elucidate the nanoscale membrane structure and dynamics via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of ∼100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles, or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively.
Electrically-tunable positioning of topological defects in liquid crystals.
Topological defects are a consequence of broken symmetry in ordered systems and are important for understanding a wide variety of phenomena in physics. In liquid crystals (LCs), defects exist as points of discontinuous order in the vector field that describes the average orientation of the molecules in space and are crucial for explaining the fundamental behaviour and properties of these mesophases. Recently, LC defects have also been explored from the perspective of technological applications including self-assembly of nanomaterials, optical-vortex generation and in tunable plasmonic metamaterials. Here, we demonstrate the fabrication and stabilisation of electrically-tunable defects in an LC device using two-photon polymerisation and explore the dynamic behaviour of defects when confined by polymer structures laser-written in topologically discontinuous states. We anticipate that our defect fabrication technique will enable the realisation of tunable, 3D, reconfigurable LC templates towards nanoparticle self-assembly, tunable metamaterials and next-generation spatial light modulators for light-shaping.
Somatostatin secretion by Na+-dependent Ca2+-induced Ca2+ release in pancreatic delta-cells.
Pancreatic islets are complex micro-organs consisting of at least three different cell types: glucagon-secreting α-, insulin-producing β- and somatostatin-releasing δ-cells1. Somatostatin is a powerful paracrine inhibitor of insulin and glucagon secretion2. In diabetes, increased somatostatinergic signalling leads to defective counter-regulatory glucagon secretion3. This increases the risk of severe hypoglycaemia, a dangerous complication of insulin therapy4. The regulation of somatostatin secretion involves both intrinsic and paracrine mechanisms5 but their relative contributions and whether they interact remains unclear. Here we show that dapagliflozin-sensitive glucose- and insulin-dependent sodium uptake stimulates somatostatin secretion by elevating the cytoplasmic Na+ concentration ([Na+]i) and promoting intracellular Ca2+-induced Ca2+ release (CICR). This mechanism also becomes activated when [Na+]i is elevated following the inhibition of the plasmalemmal Na+-K+ pump by reductions of the extracellular K+ concentration emulating those produced by exogenous insulin in vivo6. Islets from some donors with type-2 diabetes hypersecrete somatostatin, leading to suppression of glucagon secretion that can be alleviated by a somatostatin receptor antagonist. Our data highlight the role of Na+ as an intracellular second messenger, illustrate the significance of the intraislet paracrine network and provide a mechanistic framework for pharmacological correction of the hormone secretion defects associated with diabetes that selectively target the δ-cells.
Reduced somatostatin signalling leads to hypersecretion of glucagon in mice fed a high-fat diet.
OBJECTIVES: Elevated plasma glucagon is an early symptom of diabetes, occurring in subjects with impaired glucose regulation. Here, we explored alpha-cell function in female mice fed a high-fat diet (HFD). METHODS: Female mice expressing the Ca2+ indicator GCaMP3 specifically in alpha-cells were fed a high-fat or control (CTL) diet. We then conducted in vivo phenotyping of these mice, as well as experiments on isolated (ex vivo) islets and in the in situ perfused pancreas. RESULTS: In HFD-fed mice, fed plasma glucagon levels were increased and glucagon secretion from isolated islets and in the perfused mouse pancreas was also elevated. In mice fed a CTL diet, increasing glucose reduced intracellular Ca2+ ([Ca2+]i) oscillation frequency and amplitude. This effect was also observed in HFD mice; however, both the frequency and amplitude of the [Ca2+]i oscillations were higher than those in CTL alpha-cells. Given that alpha-cells are under strong paracrine control from neighbouring somatostatin-secreting delta-cells, we hypothesised that this elevation of alpha-cell output was due to a lack of somatostatin (SST) secretion. Indeed, SST secretion in isolated islets from HFD-fed mice was reduced but exogenous SST also failed to suppress glucagon secretion and [Ca2+]i activity from HFD alpha-cells, in contrast to observations in CTL mice. CONCLUSIONS: These findings suggest that reduced delta-cell function, combined with intrinsic changes in alpha-cells including sensitivity to somatostatin, accounts for the hyperglucagonaemia in mice fed a HFD.
Validation of the foot profile score.
BACKGROUND: There are numerous static measures of foot posture but there is no published score of dynamic foot motion. Three-dimensional gait analysis can include a multi-segment foot model like the Oxford Foot Model (OFM) to comprehensively quantify foot kinematic deviations across the gait cycle but it lacks an overall score, like the Gait Profile score (GPS), used to summarize the quality of lower extremity motion. RESEARCH QUESTION: This paper introduces the Foot Profile Score (FPS), a single number, analogous to the GPS but based on kinematic data of the OFM. The aim of this study is to validate the FPS by studying its properties and design, and analyse it against a clinical assessment of foot deformity. METHODS: Concurrent validity was established for the FPS analysing the relationship with Clinical Foot Deformity Score (CFDS) in 60 subjects with a condition affecting the lower limbs globally Content validity was established for the six Foot Variable Scores (FVS) that make up the FPS using a multiple regression of the CFDS on the 6 FVS in the 60 subjects. Predictive validity was established analysing the relationship of the FPS and GPS comparing 60 global involvement subjects with 60 subjects with isolated foot deformity. RESULTS: Pearson correlation between the FPS and CFDS was significant at 0.62 (p < 0.001). Each element of FVS contributes positively to predicting the CFDS with R2 = 0.456 (p < 0.001). FPS contributed independently to the prediction of CFDS (t = 3.9, p < 0.001). The correlation between the GPS and FPS in the global involvement group was significant at r = 0.64 (p < 0.001), while there was no correlation found with r = 0.08 (p = 0.54) in the foot deformity group. SIGNIFICANCE: The FPS is the first validated score of dynamic foot motion.