Publications by authors named "Thomas Jue"

38 Publications

Hyperpolarized C MR Spectroscopy Depicts in Vivo Effect of Exercise on Pyruvate Metabolism in Human Skeletal Muscle.

Radiology 2021 Jun 22:204500. Epub 2021 Jun 22.

From the Advanced Imaging Research Center (J.M.P., C.E.H., J.M., J.C., J.R., J.L., G.D.R., A.C., C.R.M.), Department of Radiology (J.M.P., A.C., C.R.M.), Department of Neurology and Neurotherapeutics (R.G.H.), and Department of Internal Medicine (C.R.M.), University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-8568; Department of Electrical and Computer Engineering, University of Texas at Dallas, Dallas, Tex (J.M.P.); Department of Diagnostic Imaging and Radiology, Developing Brain Institute, Children's National Hospital, Washington, DC (Z.Z.); Department of Pediatrics and Radiology, George Washington University, Washington, DC (Z.Z.); GE Healthcare, Dallas, Tex (G.D.R.); Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, Calif (T.J.); and Veterans Affairs North Texas Healthcare System, Dallas, Tex (C.R.M.).

Background Pyruvate dehydrogenase (PDH) and lactate dehydrogenase are essential for adenosine triphosphate production in skeletal muscle. At the onset of exercise, oxidation of glucose and glycogen is quickly enabled by dephosphorylation of PDH. However, direct measurement of PDH flux in exercising human muscle is daunting, and the net effect of covalent modification and other control mechanisms on PDH flux has not been assessed. Purpose To demonstrate the feasibility of assessing PDH activation and changes in pyruvate metabolism in human skeletal muscle after the onset of exercise using carbon 13 (C) MRI with hyperpolarized (HP) [1-C]-pyruvate. Materials and Methods For this prospective study, sedentary adults in good general health (mean age, 42 years ± 18 [standard deviation]; six men) were recruited from August 2019 to September 2020. Subgroups of the participants were injected with HP [1-C]-pyruvate at resting, during plantar flexion exercise, or 5 minutes after exercise during recovery. In parallel, hydrogen 1 arterial spin labeling MRI was performed to estimate muscle tissue perfusion. An unpaired test was used for comparing C data among the states. Results At rest, HP [1-C]-lactate and [1-C]-alanine were detected in calf muscle, but [C]-bicarbonate was negligible. During moderate flexion-extension exercise, total HP C signals (tC) increased 2.8-fold because of increased muscle perfusion ( = .005), and HP [1-C]-lactate-to-tC ratio increased 1.7-fold ( = .04). HP [C]-bicarbonate-to-tC ratio increased 8.4-fold ( = .002) and returned to the resting level 5 minutes after exercise, whereas the lactate-to-tC ratio continued to increase to 2.3-fold as compared with resting ( = .008). Conclusion Lactate and bicarbonate production from hyperpolarized (HP) [1-carbon 13 {C}]-pyruvate in skeletal muscle rapidly reflected the onset and the termination of exercise. These results demonstrate the feasibility of imaging skeletal muscle metabolism using HP [1-C]-pyruvate MRI and the sensitivity of in vivo pyruvate metabolism to exercise states. © RSNA, 2021
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http://dx.doi.org/10.1148/radiol.2021204500DOI Listing
June 2021

Localization of myoglobin in mitochondria: implication in regulation of mitochondrial respiration in rat skeletal muscle.

Physiol Rep 2021 03;9(5):e14769

Graduate School of Human and Socio-Environmental Studies, Kanazawa University, Ishikawa, Japan.

Mitochondria play a principal role in metabolism, and mitochondrial respiration is an important process for producing adenosine triphosphate. Recently, we showed the possibility that the muscle-specific protein myoglobin (Mb) interacts with mitochondrial complex IV to augment the respiration capacity in skeletal muscles. However, the precise mechanism for the Mb-mediated upregulation remains under debate. The aim of this study was to ascertain whether Mb is truly integrated into the mitochondria of skeletal muscle and to investigate the submitochondrial localization. Isolated mitochondria from rat gastrocnemius muscle were subjected to different proteinase K (PK) concentrations to digest proteins interacting with the outer membrane. Western blotting analysis revealed that the PK digested translocase of outer mitochondrial membrane 20 (Tom20), and the immunoreactivity of Tom20 decreased with the amount of PK used. However, the immunoreactivity of Mb with PK treatment was better preserved, indicating that Mb is integrated into the mitochondria of skeletal muscle. The mitochondrial protease protection assay experiments suggested that Mb localizes within the mitochondria in the inner membrane from the intermembrane space side. These results strongly suggest that Mb inside muscle mitochondria could be implicated in the regulation of mitochondrial respiration via complex IV.
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http://dx.doi.org/10.14814/phy2.14769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923563PMC
March 2021

Impaired skeletal muscle mitochondrial bioenergetics and physical performance in chronic kidney disease.

JCI Insight 2020 03 12;5(5). Epub 2020 Mar 12.

Division of Nephrology, Department of Medicine, School of Medicine, UCD, Sacramento, California, USA.

The maintenance of functional independence is the top priority of patients with chronic kidney disease (CKD). Defects in mitochondrial energetics may compromise physical performance and independence. We investigated associations of the presence and severity of kidney disease with in vivo muscle energetics and the association of muscle energetics with physical performance. We performed measures of in vivo leg and hand muscle mitochondrial capacity (ATPmax) and resting ATP turnover (ATPflux) using 31phosphorus magnetic resonance spectroscopy and oxygen uptake (O2 uptake) by optical spectroscopy in 77 people (53 participants with CKD and 24 controls). We measured physical performance using the 6-minute walk test. Participants with CKD had a median estimated glomerular filtration rate (eGFR) of 33 ml/min per 1.73 m2. Participants with CKD had a -0.19 mM/s lower leg ATPmax compared with controls but no difference in hand ATPmax. Resting O2 uptake was higher in CKD compared with controls, despite no difference in ATPflux. ATPmax correlated with eGFR and serum bicarbonate among participants with GFR <60. ATPmax of the hand and leg correlated with 6-minute walking distance. The presence and severity of CKD associate with muscle mitochondrial capacity. Dysfunction of muscle mitochondrial energetics may contribute to reduced physical performance in CKD.
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http://dx.doi.org/10.1172/jci.insight.133289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141399PMC
March 2020

A mouse model and F NMR approach to investigate the effects of sialic acid supplementation on cognitive development.

FEBS Lett 2020 01 3;594(1):135-143. Epub 2019 Aug 3.

Department of Biochemistry & Molecular Medicine, Cell Biology, University of California Davis, CA, USA.

Researchers have observed that a sialic acid (Sia)-supplemented neonatal diet leads to improved cognition in weanling piglets. However, whether cognitive improvement appears with different physiological backgrounds and persists into adulthood is not known. Here, we have established a convenient mouse model and used an F NMR approach to address these questions, test the conditionally essential nutrient hypothesis about Sia supplementation, and assess the prospect of measuring Sia metabolism directly in vivo. Indeed, the neonatal mouse brain uptakes more Sia than the adult brain, and Sia supplementation of neonatal mice improves the cognitive performance of adult mice. The non-invasive F NMR approach and viable mouse model opens unique opportunities for clarifying the interplay of nutritional supplementation, metabolism, and cognitive development.
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http://dx.doi.org/10.1002/1873-3468.13548DOI Listing
January 2020

Comparative NMR and NIRS analysis of oxygen-dependent metabolism in exercising finger flexor muscles.

Am J Physiol Regul Integr Comp Physiol 2017 Dec 6;313(6):R740-R753. Epub 2017 Sep 6.

Biochemistry and Molecular Medicine, University of California Davis, Davis, California; and

Muscle contraction requires the physiology to adapt rapidly to meet the surge in energy demand. To investigate the shift in metabolic control, especially between oxygen and metabolism, researchers often depend on near-infrared spectroscopy (NIRS) to measure noninvasively the tissue O Because NIRS detects the overlapping myoglobin (Mb) and hemoglobin (Hb) signals in muscle, interpreting the data as an index of cellular or vascular O requires deconvoluting the relative contribution. Currently, many in the NIRS field ascribe the signal to Hb. In contrast, H NMR has only detected the Mb signal in contracting muscle, and comparative NIRS and NMR experiments indicate a predominant Mb contribution. The present study has examined the question of the NIRS signal origin by measuring simultaneously the H NMR, P NMR, and NIRS signals in finger flexor muscles during the transition from rest to contraction, recovery, ischemia, and reperfusion. The experiment results confirm a predominant Mb contribution to the NIRS signal from muscle. Given the NMR and NIRS corroborated changes in the intracellular O, the analysis shows that at the onset of muscle contraction, O declines immediately and reaches new steady states as contraction intensity rises. Moreover, lactate formation increases even under quite aerobic condition.
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http://dx.doi.org/10.1152/ajpregu.00203.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814692PMC
December 2017

Differential Interaction of Myoglobin with Select Fatty Acids of Carbon Chain Lengths C8 to C16.

Lipids 2017 08 21;52(8):711-727. Epub 2017 Jun 21.

Department of Biochemistry and Molecular Medicine, University of California Davis, Davis, CA, 95616-8635, USA.

Previous studies have shown that palmitic acid (PAM) and oleic acid (OLE) can bind myoglobin (Mb). How fatty acids (FA) with different carbon chain lengths and sulfate substitution interact with Mb remains uncertain. Indeed, C8:0 and C10:0 fatty acids do not perturb the intensities of the H-NMR MbCN signal intensity at FA:Mb ratios below 2:1. Starting with C12:0, C12:0-C16:0, FA induce a noticeable spectral change. C12:0 and C14:0 FA affect both the 5- and 8-heme methyl signals, whereas the C16:0 FA perturbs only the 8-heme methyl signal. All C12:0-C16:0 saturated FA induce upfield shifts in the -CH peak of different FA in the presence of Mb. Increasing the apparent solubility with a sulfate group substitution enhances the FA interaction of lauric sulfate (LAU 1-SO) but not palmitate sulfate acid (PAM 1-SO). The detergent (DET) property of FA has no significant contribution. Common positive, neutral, and negative DET at DET:Mb ratio of 1:1 induce no perturbation of the MbCN spectra. The experiment observations establish a basis to investigate the molecular mechanism underlying the FA interaction with Mb.
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http://dx.doi.org/10.1007/s11745-017-4272-zDOI Listing
August 2017

Effect of fatty acid interaction on myoglobin oxygen affinity and triglyceride metabolism.

J Physiol Biochem 2016 Aug 29;73(3):359-370. Epub 2017 Mar 29.

Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX, 77843, USA.

Recent studies have suggested myoglobin (Mb) may have other cellular functions in addition to storing and transporting O. Indeed, NMR experiments have shown that the saturated fatty acid (FA) palmitate (PA) can interact with myoglobin (Mb) in its ligated state (MbCO and MbCN) but does not interact with Mb in its deoxygenated state. The observation has led to the hypothesis that Mb can also serve as a fatty acid transporter. The present study further investigates fatty acid interaction with the physiological states of Mb using the more soluble but unsaturated fatty acid, oleic acid (OA). OA binds to MbCO but does not bind to deoxy Mb. OA binding to Mb, however, does not alter its O affinity. Without any Mb, muscle has a significantly lower level of triglyceride (TG). In Mb knock-out (MbKO) mice, both heart and skeletal muscles have lower level of TG relative to the control mice. Training further decreases the relative TG in the MbKO skeletal muscle. Nevertheless, the absence of Mb and lower TG level in muscle does not impair the MbKO mouse performance as evidenced by voluntary wheel running measurements. The results support the hypothesis of a complex physiological role for Mb, especially with respect to fatty acid metabolism.
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http://dx.doi.org/10.1007/s13105-017-0559-zDOI Listing
August 2016

Intracellular oxygen tension limits muscle contraction-induced change in muscle oxygen consumption under hypoxic conditions during Hb-free perfusion.

Physiol Rep 2017 Jan;5(2)

Faculty of Human Sciences, Kanazawa University, Kanazawa, Japan

Under acute hypoxic conditions, the muscle oxygen uptake (mV˙O) during exercise is reduced by the restriction in oxygen-supplied volume to the mitochondria within the peripheral tissue. This suggests the existence of a factor restricting the mV˙O under hypoxic conditions at the peripheral tissue level. Therefore, this study set out to test the hypothesis that the restriction in mV˙O is regulated by the net decrease in intracellular oxygen tension equilibrated with myoglobin oxygen saturation (∆PO) during muscle contraction under hypoxic conditions. The hindlimb of male Wistar rats (8 weeks old, n = 5) was perfused with hemoglobin-free Krebs-Henseleit buffer equilibrated with three different fractions of O gas: 95.0%O, 71.3%O, and 47.5%O The deoxygenated myoglobin (Mb) kinetics during muscle contraction were measured under each oxygen condition with a near-infrared spectroscopy. The ∆[deoxy-Mb] kinetics were converted to oxygen saturation of myoglobin (SO), and the PO was then calculated based on the SO and the O dissociation curve of the Mb. The SO and PO at rest decreased with the decrease in O supply, and the muscle contraction caused a further decrease in SO and PO under all O conditions. The net increase in mV˙O from the muscle contraction (∆mV˙O) gradually decreased as the ∆PO decreased during muscle contraction. The results of this study suggest that ΔPO is a key determinant of the ΔmV˙O.
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http://dx.doi.org/10.14814/phy2.13112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5269414PMC
January 2017

Myoglobin and the regulation of mitochondrial respiratory chain complex IV.

J Physiol 2016 Jan 20;594(2):483-95. Epub 2015 Dec 20.

Faculty of Human Sciences, Kanazawa University, Kanazawa, 920-1192, Japan.

Key Points: Mitochondrial respiration is regulated by multiple elaborate mechanisms. It has been shown that muscle specific O2 binding protein, Myoglobin (Mb), is localized in mitochondria and interacts with respiratory chain complex IV, suggesting that Mb could be a factor that regulates mitochondrial respiration. Here, we demonstrate that muscle mitochondrial respiration is improved by Mb overexpression via up-regulation of complex IV activity in cultured myoblasts; in contrast, suppression of Mb expression induces a decrease in complex IV activity and mitochondrial respiration compared with the overexpression model. The present data are the first to show the biological significance of mitochondrial Mb as a potential modulator of mitochondrial respiratory capacity.

Abstract: Mitochondria are important organelles for metabolism, and their respiratory capacity is a primary factor in the regulation of energy expenditure. Deficiencies of cytochrome c oxidase complex IV, which reduces O2 in mitochondria, are linked to several diseases, such as mitochondrial myopathy. Moreover, mitochondrial respiration in skeletal muscle tissue tends to be susceptible to complex IV activity. Recently, we showed that the muscle-specific protein myoglobin (Mb) interacts with complex IV. The precise roles of mitochondrial Mb remain unclear. Here, we demonstrate that Mb facilitates mitochondrial respiratory capacity in skeletal muscles. Although mitochondrial DNA copy numbers were not altered in Mb-overexpressing myotubes, O2 consumption was greater in these myotubes than that in mock cells (Mock vs. Mb-Flag::GFP: state 4, 1.00 ± 0.09 vs. 1.77 ± 0.34; state 3, 1.00 ± 0.29; Mock: 1.60 ± 0.53; complex 2-3-4: 1.00 ± 0.30 vs. 1.50 ± 0.44; complex IV: 1.00 ± 0.14 vs. 1.87 ± 0.27). This improvement in respiratory capacity could be because of the activation of enzymatic activity of respiratory complexes. Moreover, mitochondrial respiration was up-regulated in myoblasts transiently overexpressing Mb; complex IV activity was solely activated in Mb-overexpressing myoblasts, and complex IV activity was decreased in the myoblasts in which Mb expression was suppressed by Mb-siRNA transfection (Mb vector transfected vs. Mb vector, control siRNA transfected vs. Mb vector, Mb siRNA transfected: 0.15 vs. 0.15 vs. 0.06). Therefore, Mb enhances the enzymatic activity of complex IV to ameliorate mitochondrial respiratory capacity, and could play a pivotal role in skeletal muscle metabolism.
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http://dx.doi.org/10.1113/JP270824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713734PMC
January 2016

Hyperpolarized 13C NMR observation of lactate kinetics in skeletal muscle.

J Exp Biol 2015 Oct 7;218(Pt 20):3308-18. Epub 2015 Sep 7.

Biochemistry and Molecular Medicine, University of California Davis, Davis, CA 95616, USA

The production of glycolytic end products, such as lactate, usually evokes a cellular shift from aerobic to anaerobic ATP generation and O2 insufficiency. In the classical view, muscle lactate must be exported to the liver for clearance. However, lactate also forms under well-oxygenated conditions, and this has led investigators to postulate lactate shuttling from non-oxidative to oxidative muscle fiber, where it can serve as a precursor. Indeed, the intracellular lactate shuttle and the glycogen shunt hypotheses expand the vision to include a dynamic mobilization and utilization of lactate during a muscle contraction cycle. Testing the tenability of these provocative ideas during a rapid contraction cycle has posed a technical challenge. The present study reports the use of hyperpolarized [1-(13)C]lactate and [2-(13)C]pyruvate in dynamic nuclear polarization (DNP) NMR experiments to measure the rapid pyruvate and lactate kinetics in rat muscle. With a 3 s temporal resolution, (13)C DNP NMR detects both [1-(13)C]lactate and [2-(13)C]pyruvate kinetics in muscle. Infusion of dichloroacetate stimulates pyruvate dehydrogenase activity and shifts the kinetics toward oxidative metabolism. Bicarbonate formation from [1-(13)C]lactate increases sharply and acetyl-l-carnitine, acetoacetate and glutamate levels also rise. Such a quick mobilization of pyruvate and lactate toward oxidative metabolism supports the postulated role of lactate in the glycogen shunt and the intracellular lactate shuttle models. The study thus introduces an innovative DNP approach to measure metabolite transients, which will help delineate the cellular and physiological role of lactate and glycolytic end products.
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http://dx.doi.org/10.1242/jeb.123141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4644943PMC
October 2015

Interaction of myoglobin with oleic acid.

Chem Phys Lipids 2015 Oct 26;191:115-22. Epub 2015 Jul 26.

Biochemistry and Molecular Medicine, University of California, Davis, Davis CA 95616-8635, United States. Electronic address:

Previous studies have shown that palmitate (PA) can interact with myoglobin (Mb). The present study has investigated the interaction of the more soluble unsaturated fatty acid, oleic acid (OA). Indeed, (1)H NMR measurements of the Mb signal during OA titration also show signal changes consistent with specific and non-specific binding. At OA:Mb ratio<4:1, OA perturbs selectively the 8-heme methyl signal, consistent with a local and specific fatty acid-protein interaction. As OA:Mb ratio increases from 4:1 to 40:1, all hyperfine shifted MbCN signals decrease, consistent with a non-selective, global perturbation of the protein. The pH titration analysis indicates that the observed OA methylene signal in the presence of Mb reflects a non-specific interaction and does not originate from a shift in the lamella-micelle equilibrium. Given the OA interaction with Mb, a fatty acid flux model suggests that Mb can play a fatty acid transport role under certain physiological conditions.
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http://dx.doi.org/10.1016/j.chemphyslip.2015.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633329PMC
October 2015

Endurance training facilitates myoglobin desaturation during muscle contraction in rat skeletal muscle.

Sci Rep 2015 Mar 24;5:9403. Epub 2015 Mar 24.

Faculty of Human Sciences, Kanazawa University, Kanazawa 920-1192, Japan.

At onset of muscle contraction, myoglobin (Mb) immediately releases its bound O2 to the mitochondria. Accordingly, intracellular O2 tension (PmbO2) markedly declines in order to increase muscle O2 uptake (mVO2). However, whether the change in PmbO2 during muscle contraction modulates mVO2 and whether the O2 release rate from Mb increases in endurance-trained muscles remain unclear. The purpose of this study was, therefore, to determine the effect of endurance training on O2 saturation of Mb (SmbO2) and PmbO2 kinetics during muscle contraction. Male Wistar rats were subjected to a 4-week swimming training (Tr group; 6 days per week, 30 min × 4 sets per day) with a weight load of 2% body mass. After the training period, deoxygenated Mb kinetics during muscle contraction were measured using near-infrared spectroscopy under hemoglobin-free medium perfusion. In the Tr group, the VmO2peak significantly increased by 32%. Although the PmbO2 during muscle contraction did not affect the increased mVO2 in endurance-trained muscle, the O2 release rate from Mb increased because of the increased Mb concentration and faster decremental rate in SmbO2 at the maximal twitch tension. These results suggest that the Mb dynamics during muscle contraction are contributing factors to faster VO2 kinetics in endurance-trained muscle.
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http://dx.doi.org/10.1038/srep09403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371155PMC
March 2015

Palmitate interaction with physiological states of myoglobin.

Biochim Biophys Acta 2014 Jan;1840(1):656-66

Background: Previous studies have shown that palmitate (PA) can bind specifically and non-specifically to Fe(III)MbCN. The present study has observed PA interaction with physiological states of Fe(II)Mb, and the observations support the hypothesis that Mb may have a potential role in facilitating intracellular fatty acid transport.

Methods: 1H NMR spectra measurements of the Mb signal during PA titration show signal changes consistent with specific and non-specific binding.

Results: Palmitate (PA) interacts differently with physiological states of Mb. Deoxy Mb does not interact specifically or non-specifically with PA, while the carbonmonoxy myoglobin (MbCO) interaction with PA decreases the intensity of selective signals and produces a 0.15ppmupfield shift of the PAmethylene peak. The selective signal change upon PA titration provides a basis to determine an apparent PA binding constant,which serves to create a model comparing the competitive PA binding and facilitated fatty acid transport of Mb and fatty acid binding protein(FABP).

Conclusions: Given contrasting PA interaction of ligated vs. unligated Mb, the cellular fatty acid binding protein(FABP) and Mb concentration in the cell, the reported cellular diffusion coefficients, the PA dissociation constants from ligated Mb and FABP, a fatty acid flux model suggests that Mb can compete with FABP transporting cellular fatty acid.

General Significance: Under oxygenated conditions and continuous energy demand, Mb dependent fatty acid transport could influence the cell's preference for carbohydrate or fatty acid as a fuel source and regulate fatty acid metabolism.
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http://dx.doi.org/10.1016/j.bbagen.2013.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934850PMC
January 2014

Reply toPancheva, Panchev, and Pancheva.

J Appl Physiol (1985) 2013 Jul;115(1):151

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http://dx.doi.org/10.1152/japplphysiol.00449.2013DOI Listing
July 2013

EPR assessment of protein sites for incorporation of Gd(III) MRI contrast labels.

Contrast Media Mol Imaging 2013 May-Jun;8(3):252-64

Department of Experimental Medical Science, Lund University, Lund, Sweden.

We have engineered apolipoprotein A-I (apoA-I), a major protein constituent of high-density lipoprotein (HDL), to contain DOTA-chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism and regulation in vivo. This protein contrast agent was obtained by attaching the thiol-reactive Gd[MTS-ADO3A] label at Cys residues replaced at four distinct positions (52, 55, 76 and 80) in apoA-I. MRI of infused mice previously showed that the Gd-labeled apoA-I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoA-I are superior when the ADO3A moiety is located at position 55, compared with the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave X-band (9 GHz) electron paramagnetic resonance (EPR) spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipid-free with the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipid-free and rHDL states correlate to the product of the X-band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDL-associated apoA-I containing the Gd[MTS-ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by X-band EPR. While room temperature EPR requires >1 m m Gd(III)-labeled and only >10 µ m nitroxide-labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5 µl). Thus, X-band EPR provides a practical assessment for the suitability of imaging candidates containing the site-directed ADO3A contrast probe.
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http://dx.doi.org/10.1002/cmmi.1518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633150PMC
November 2013

Interaction between myoglobin and mitochondria in rat skeletal muscle.

J Appl Physiol (1985) 2013 Feb 29;114(4):490-7. Epub 2012 Nov 29.

Faculty of Human Sciences, Kanazawa University, Kanazawa, Japan.

The mechanisms underlying subcellular oxygen transport mediated by myoglobin (Mb) remain unclear. Recent evidence suggests that, in the myocardium, transverse diffusion of Mb is too slow to effectively supply oxygen to meet the immediate mitochondrial oxygen demands at the onset of muscle contractions. The cell may accommodate the demand by maintaining the distribution of Mb to ensure a sufficient O(2) supply in the immediate vicinity of the mitochondria. The present study has verified the co-localization of Mb with mitochondria by using biochemical histological and electron microscopy analyses. Immunohistochemical and electron microscopy analysis indicates a co-localization of Mb with mitochondria. Western blotting confirms the presence of Mb colocalizes with the mitochondrial fraction and appears more prominently in slow-twitch oxidative than in fast-twitch glycolytic muscle. In particular, Mb interacts with cytochrome c oxidase-subunit IV. These results suggest that a direct Mb-mediated O2 delivery to the mitochondria, which may play a potentially significant role for respiration.
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http://dx.doi.org/10.1152/japplphysiol.00789.2012DOI Listing
February 2013

Imaging apolipoprotein AI in vivo.

NMR Biomed 2011 Aug 24;24(7):916-24. Epub 2011 Jan 24.

Biochemistry and Molecular Medicine, University of California Davis, Davis, CA 95616-8635, USA.

Coronary disease risk increases inversely with high-density lipoprotein (HDL) level. The measurement of the biodistribution and clearance of HDL in vivo, however, has posed a technical challenge. This study presents an approach to the development of a lipoprotein MRI agent by linking gadolinium methanethiosulfonate (Gd[MTS-ADO3A]) to a selective cysteine mutation in position 55 of apo AI, the major protein of HDL. The contrast agent targets both liver and kidney, the sites of HDL catabolism, whereas the standard MRI contrast agent, gadolinium-diethylenetriaminepentaacetic acid-bismethylamide (GdDTPA-BMA, gadodiamide), enhances only the kidney image. Using a modified apolipoprotein AI to create an HDL contrast agent provides a new approach to investigate HDL biodistribution, metabolism and regulation in vivo.
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http://dx.doi.org/10.1002/nbm.1650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726305PMC
August 2011

'It's hollow': the function of pores within myoglobin.

J Exp Biol 2010 Aug;213(Pt 16):2748-54

Department of Chemistry and Materials Science, Tokyo Institute of Technology, Meguro-ku, Tokyo, 152-8551, Japan.

Despite a century of research, the cellular function of myoglobin (Mb), the mechanism regulating oxygen (O(2)) transport in the cell and the structure-function relationship of Mb remain incompletely understood. In particular, the presence and function of pores within Mb have attracted much recent attention. These pores can bind to Xe as well as to other ligands. Indeed, recent cryogenic X-ray crystallographic studies using novel techniques have captured snapshots of carbon monoxide (CO) migrating through these pores. The observed movement of the CO molecule from the heme iron site to the internal cavities and the associated structural changes of the amino acid residues around the cavities confirm the integral role of the pores in forming a ligand migration pathway from the protein surface to the heme. These observations resolve a long-standing controversy - but how these pores affect the physiological function of Mb poses a striking question at the frontier of biology.
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http://dx.doi.org/10.1242/jeb.042994DOI Listing
August 2010

Myoglobin's old and new clothes: from molecular structure to function in living cells.

J Exp Biol 2010 Aug;213(Pt 16):2713-25

Zentrum Physiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.

Myoglobin, a mobile carrier of oxygen, is without a doubt an important player central to the physiological function of heart and skeletal muscle. Recently, researchers have surmounted technical challenges to measure Mb diffusion in the living cell. Their observations have stimulated a discussion about the relative contribution made by Mb-facilitated diffusion to the total oxygen flux. The calculation of the relative contribution, however, depends upon assumptions, the cell model and cell architecture, cell bioenergetics, oxygen supply and demand. The analysis suggests that important differences can be observed whether steady-state or transient conditions are considered. This article reviews the current evidence underlying the evaluation of the biophysical parameters of myoglobin-facilitated oxygen diffusion in cells, specifically the intracellular concentration of myoglobin, the intracellular diffusion coefficient of myoglobin and the intracellular myoglobin oxygen saturation. The review considers the role of myoglobin in oxygen transport in vertebrate heart and skeletal muscle, in the diving seal during apnea as well as the role of the analogous leghemoglobin of plants. The possible role of myoglobin in intracellular fatty acid transport is addressed. Finally, the recent measurements of myoglobin diffusion inside muscle cells are discussed in terms of their implications for cytoarchitecture and microviscosity in these cells and the identification of intracellular impediments to the diffusion of proteins inside cells. The recent experimental data then help to refine our understanding of Mb function and establish a basis for future investigation.
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http://dx.doi.org/10.1242/jeb.043075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912754PMC
August 2010

NIRS measurement of O(2) dynamics in contracting blood and buffer perfused hindlimb muscle.

Adv Exp Med Biol 2010 ;662:323-8

Kanazawa University, Kanazawa 920-1192, Japan.

In order to obtain evidence that Mb releases O(2) during muscle contraction, we have set up a buffer-perfused hindlimb rat model and applied NIRS to detect the dynamics of tissue deoxygenation during contraction. The NIRS signal was monitored on hindlimb muscle during twitch contractions at 1 Hz, evoked via electrostimulator at different submaximal levels. The hindlimb perfusion was carried out by perfusion of Krebs Bicarbonate buffer. The NIRS still detected a strong signal even under Hb-free contractions. The deoxygenation signal (Delta[deoxy]) was progressively increased at onset of the contraction and reached the plateau under both blood- and buffer-perfused conditions. However, the amplitude of Delta[deoxy] during steady state continued to significantly increase as tension increased. The tension-matched comparison of the Delta[deoxy] level under buffer-perfused and blood perfused conditions indicate that Mb can contribute approximately 50% to the NIRS signal. These results clarify the Mb contribution to the NIRS signal and show a falling intracellular PO(2) as workload increases.
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http://dx.doi.org/10.1007/978-1-4419-1241-1_46DOI Listing
March 2010

Quantification of myoglobin deoxygenation and intracellular partial pressure of O2 during muscle contraction during haemoglobin-free medium perfusion.

Exp Physiol 2010 May 15;95(5):630-40. Epub 2010 Jan 15.

Faculty of Human Sciences, Kanazawa University, Kakuma-machi, Kanazawa-city, Ishikawa, 920-1192, Japan.

Although the O(2) gradient regulates O(2) flux from the capillary into the myocyte to meet the energy demands of contracting muscle, intracellular O(2) dynamics during muscle contraction remain unclear. Our hindlimb perfusion model allows the determination of intracellular myoglobin (Mb) saturation ( ) and intracellular oxygen tension of myoglobin ( ) in contracting muscle using near infrared spectroscopy (NIRS). The hindlimb of male Wistar rats was perfused from the abdominal aorta with a well-oxygenated haemoglobin-free Krebs-Henseleit buffer. The deoxygenated Mb ([deoxy-Mb]) signal was monitored by NIRS. Based on the value of [deoxy-Mb], and were calculated, and the time course was evaluated by an exponential function model. Both and started to decrease immediately after the onset of contraction. The steady-state values of and progressively decreased with relative work intensity or muscle oxygen consumption. At the maximal twitch rate, and were 49% and 2.4 mmHg, respectively. Moreover, the rate of release of O(2) from Mb at the onset of contraction increased with muscle oxygen consumption. These results suggest that at the onset of muscle contraction, Mb supplies O(2) during the steep decline in , which expands the O(2) gradient to increase the O(2) flux to meet the increased energy demands.
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http://dx.doi.org/10.1113/expphysiol.2009.050344DOI Listing
May 2010

Oximetry with the NMR signals of hemoglobin Val E11 and Tyr C7.

Eur J Appl Physiol 2009 Oct 21;107(3):325-33. Epub 2009 Jul 21.

Department of Biochemistry and Molecular Medicine, University of California Davis, 95616-8635, USA.

The NMR visibility of the signals from erythrocyte hemoglobin (Hb) presents an opportunity to assess the vascular PO(2) (partial pressure of oxygen) in vivo to gather insight into the regulation of O(2) transport, especially in contracting muscle tissue. Some concerns, however, have arisen about the validity of using the Val E11 signal as an indicator of PO(2), since its intensity depends on tertiary structural changes, in contrast to the quaternary structure changes associated with relaxed (R) and tense (T) transition during O(2) binding. We have examined the Val E11 and Tyr C7 signal intensity as a function of Hb saturation by developing an oximetry system, which permits the comparative analysis of the NMR and spectrophotometric measurements. The spectrophotometric assay defines the Hb saturation level at a given PO(2) and yields standard oxygen-binding curves. Under defined PO(2) and Hb saturation values, the NMR measurements have determined that the Val E11 signal, as well as the Tyr C7 signal, tracks closely Hb saturation and can therefore serve as a vascular oxygen biomarker.
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http://dx.doi.org/10.1007/s00421-009-1125-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753772PMC
October 2009

Blood flow and metabolic regulation in seal muscle during apnea.

J Exp Biol 2008 Oct;211(Pt 20):3323-32

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093, USA.

In order to examine myoglobin (Mb) function and metabolic responses of seal muscle during progressive ischemia and hypoxemia, Mb saturation and high-energy phosphate levels were monitored with NMR spectroscopy during sleep apnea in elephant seals (Mirounga angustirostris). Muscle blood flow (MBF) was measured with laser-Doppler flowmetry (LDF). During six, spontaneous, 8-12 min apneas of an unrestrained juvenile seal, apneic MBF decreased to 46+/-10% of the mean eupneic MBF. By the end of apnea, MBF reached 31+/-8% of the eupneic value. The t(1/2) for 90% decline in apneic MBF was 1.9+/-1.2 min. The initial post-apneic peak in MBF occurred within 0.20+/-0.04 min after the start of eupnea. NMR measurements revealed that Mb desaturated rapidly from its eupenic resting level to a lower steady state value within 4 min after the onset of apnea at rates between 1.7+/-1.0 and 3.8+/-1.5% min(-1), which corresponded to a muscle O(2) depletion rate of 1-2.3 ml O(2) kg(-1) min(-1). High-energy phosphate levels did not change with apnea. During the transition from apnea to eupnea, Mb resaturated to 95% of its resting level within the first minute. Despite the high Mb concentration in seal muscle, experiments detected Mb diffusing with a translational diffusion coefficient of 4.5 x 10(-7) cm(2) s(-1), consistent with the value observed in rat myocardium. Equipoise P(O(2)) analysis revealed that Mb is the predominant intracellular O(2) transporter in elephant seals during eupnea and apnea.
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http://dx.doi.org/10.1242/jeb.018887DOI Listing
October 2008

Interaction of fatty acid with myoglobin.

FEBS Lett 2008 Oct 7;582(25-26):3643-9. Epub 2008 Oct 7.

Department of Biochemistry and Molecular Medicine, University of California Davis, Davis, CA 95616-8635, USA.

Upon titration with palmitate, the (1)H NMR spectra of metmyoglobin cyanide (MbCN) reveal a selective perturbation of the 8 heme methyl, consistent with a specific interaction of myoglobin (Mb) with fatty acid. Other detectable hyperfine shifted resonances of the heme group remain unchanged. Mb also enhances fatty acid solubility, as reflected in a more intense methylene peak of palmitate in Mb solution than in Tris buffer. Ligand binding analysis indicates an apparent palmitate dissociation constant (K(d)) of 43microM. These results suggest that Mb can bind fatty acid and may have a role in facilitating fatty acid transport in the cell.
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http://dx.doi.org/10.1016/j.febslet.2008.09.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2591068PMC
October 2008

Cardioselective dominant-negative thyroid hormone receptor (Delta337T) modulates myocardial metabolism and contractile efficiency.

Am J Physiol Endocrinol Metab 2008 Aug 3;295(2):E420-7. Epub 2008 Jun 3.

Children's Hospital and Regional Medical Center MSW 4841, 4800 Sand Point Way NE, Seattle, WA 98105, USA.

Dominant-negative thyroid hormone receptors (TRs) show elevated expression relative to ligand-binding TRs during cardiac hypertrophy. We tested the hypothesis that overexpression of a dominant-negative TR alters cardiac metabolism and contractile efficiency (CE). We used mice expressing the cardioselective dominant-negative TRbeta(1) mutation Delta337T. Isolated working Delta337T hearts and nontransgenic control (Con) hearts were perfused with (13)C-labeled free fatty acids (FFA), acetoacetate (ACAC), lactate, and glucose at physiological concentrations for 30 min. (13)C NMR spectroscopy and isotopomer analyses were used to determine substrate flux and fractional contributions (Fc) of acetyl-CoA to the citric acid cycle (CAC). Delta337T hearts exhibited rate depression but higher developed pressure and CE, defined as work per oxygen consumption (MVo(2)). Unlabeled substrate Fc from endogenous sources was higher in Delta337T, but ACAC Fc was lower. Fluxes through CAC, lactate, ACAC, and FFA were reduced in Delta337T. CE and Fc differences were reversed by pacing Delta337T to Con rates, accompanied by an increase in FFA Fc. Delta337T hearts lacked the ability to increase MVo(2). Decreases in protein expression for glucose transporter-4 and hexokinase-2 and increases in pyruvate dehydrogenase kinase-2 and -4 suggest that these hearts are unable to increase carbohydrate oxidation in response to stress. These data show that Delta337T alters the metabolic phenotype in murine heart by reducing substrate flux for multiple pathways. Some of these changes are heart rate dependent, indicating that the substrate shift may represent an accommodation to altered contractile protein kinetics, which can be disrupted by pacing stress.
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http://dx.doi.org/10.1152/ajpendo.90329.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519753PMC
August 2008

Determination of myoglobin concentration in blood-perfused tissue.

Eur J Appl Physiol 2008 Sep 31;104(1):41-8. Epub 2008 May 31.

Faculty of Human Sciences, Institute of Human and Social Science, Kanazawa University, Kanazawa, Japan.

The standard method for determining the myoglobin (Mb) concentration in blood-perfused tissue often relies on a simple but clever differencing algorithm of the optical spectra, as proposed by Reynafarje. However, the underlying assumptions of the differencing algorithm do not always lead to an accurate assessment of Mb concentration in blood-perfused tissue. Consequently, the erroneous data becloud the understanding of Mb function and oxygen transport in the cell. The present study has examined the Mb concentration in buffer and blood-perfused mouse heart. In buffer-perfused heart containing no hemoglobin (Hb), the optical differencing method yields a tissue Mb concentration of 0.26 mM. In blood-perfused tissue, the method leads to an overestimation of Mb. However, using the distinct (1)H NMR signals of MbCO and HbCO yields a Mb concentration of 0.26 mM in both buffer- and blood-perfused myocardium. Given the NMR and optical data, a computer simulation analysis has identified some error sources in the optical differencing algorithm and has suggested a simple modification that can improve the Mb determination. Even though the present study has determined a higher Mb concentration than previously reported, it does not alter significantly the equipoise PO(2), the PO(2) where Mb and O(2) contribute equally to the O(2) flux. It also suggests that any Mb increase with exercise training does not necessarily enhance the intracellular O(2) delivery.
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http://dx.doi.org/10.1007/s00421-008-0775-xDOI Listing
September 2008

A voice over the smoke for academic freedom.

Authors:
Thomas Jue

Science 2007 Feb;315(5814):937

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http://dx.doi.org/10.1126/science.315.5814.937bDOI Listing
February 2007

Anisotropy and temperature dependence of myoglobin translational diffusion in myocardium: implication for oxygen transport and cellular architecture.

Biophys J 2007 Apr 11;92(7):2608-20. Epub 2007 Jan 11.

Department of Biochemistry and Molecular Medicine, University of California Davis, Davis, California 95616-8635, USA.

Pulsed field gradient NMR methods have determined the temperature-dependent diffusion of myoglobin (Mb) in perfused rat myocardium. Mb diffuses with an averaged translational diffusion coefficient (DMb) of 4.24-8.37x10(-7)cm2/s from 22 degrees C to 40 degrees C and shows no orientation preference over a root mean-square displacement of 2.5-3.5 microm. The DMb agrees with the value predicted by rotational diffusion measurements. Based on the DMb, the equipoise diffusion PO2, the PO2 in which Mb-facilitated and free O2 diffusion contribute equally to the O2 flux, varies from 2.72 to 0.15 in myocardium and from 7.27 to 4.24 mmHg in skeletal muscle. Given the basal PO2 of approximately 10 mmHg, the Mb contribution to O2 transport appears insignificant in myocardium. In skeletal muscle, Mb-facilitated diffusion begins to contribute significantly only when the PO2 approaches the P50. In marine mammals, the high Mb concentration confers a predominant role for Mb in intracellular O2 transport under all physiological conditions. The Q10 of the DMb ranges from 1.3 to 1.6. The Mb diffusion data indicate that the postulated gel network in the cell must have a minimum percolation cutoff size exceeding 17.5 A and does not impose tortuosity within the diffusion root mean-square displacement. Moreover, the similar Q10 for the DMb of solution versus cell Mb suggests that any temperature-dependent alteration of the postulated cell matrix does not significantly affect protein mobility.
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http://dx.doi.org/10.1529/biophysj.106.094458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864849PMC
April 2007

Myoglobin translational diffusion in rat myocardium and its implication on intracellular oxygen transport.

J Physiol 2007 Jan 12;578(Pt 2):595-603. Epub 2006 Oct 12.

Department of Biochemistry and Molecular Medicine, University of California Davis, Davis, CA 95616-8635, USA.

Current theory of respiratory control invokes a role of myoglobin (Mb)-facilitated O2 diffusion in regulating the intracellular O2 flux, provided Mb diffusion can compete effectively with free O2 diffusion. Pulsed-field gradient NMR methods have now followed gradient-dependent changes in the distinct 1H NMR gamma CH3 Val E11 signal of MbO2 in perfused rat myocardium to obtain the endogenous Mb translational diffusion coefficient (D(Mb)) of 4.24 x 10(-7) cm2 s(-1) at 22 degrees C. The D(Mb) matches precisely the value predicted by in vivo NMR rotational diffusion measurements of Mb and shows no orientation preference. Given values in the literature for the Krogh's free O2 diffusion coefficient (K0), myocardial Mb concentration and a partial pressure of O2 that half saturates Mb (P50), the analysis yields an equipoise diffusion P(O2) of 1.77 mmHg, where Mb and free O2 contribute equally to the O2 flux. In the myocardium, Mb-facilitated O2 diffusion contributes increasingly more than free O2 diffusion when the P(O2) falls below 1.77 mmHg. In skeletal muscle, the P(O2) must fall below 5.72 mmHg. Altering the Mb P50 induces modest change. Mb-facilitated diffusion has a higher poise in skeletal muscle than in myocardium. Because the basal P(O2) hovers around 10 mmHg, Mb does not have a predominant role in facilitating O2 transport in myocardium but contributes significantly only when cellular oxygen falls below the equipoise diffusion P(O2).
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http://dx.doi.org/10.1113/jphysiol.2006.116061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2075141PMC
January 2007

Investigation of bioactive NO-scavenging role of myoglobin in myocardium.

Pflugers Arch 2006 Apr 9;452(1):36-42. Epub 2006 Feb 9.

Department of Biochemistry and Molecular Medicine, University of California Davis, Davis, CA 95616-8635, USA.

Because nitric oxide (NO) can react with myoglobin (Mb) to oxidize the heme Fe(II) to Fe(III), the appearance of metmyoglobin (metMb) during bradykinin stimulation underpins the hypothesis that Mb acts as an NO scavenger in the cell. Although some experiments have detected the reporter metMb signal in the -3.7 ppm spectral region, others have not corroborated the finding. Because metMb also has characteristic hyperfine-shifted signals in the 40-100 ppm spectral region, detection of these signals would confirm the presence of metMb. Perfused rat myocardium study has examined this spectral region in a range of bradykinin infusion protocols. Although bradykinin elicits a set of physiological responses, consistent with the induction of NO, the (1)H nuclear magnetic resonance spectra in all experiments reveal no detectable metMb signals. Moreover, in the perfused myocardium model, the bradykinin-induced decline in myocardial oxygen consumption does not appear to arise only from NO binding to cytochrome oxidase.
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http://dx.doi.org/10.1007/s00424-005-0011-zDOI Listing
April 2006
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