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Retinal Müller (glial) cells metabolize glucose to lactate, which is preferentially taken up by photoreceptor neurons as fuel for their oxidative metabolism. We explored whether lactate supply to neurons is a glial function controlled by neuronal signals. For this, we used subcellular fluorescence imaging and either amperometric or optical biosensors to monitor metabolic responses simultaneously from mitochondrial and cytosolic compartments of individual Müller cells from salamander retina. Our results demonstrate that lactate production and release is controlled by the combined action of glutamate and NH(4)(+), both at micromolar concentrations. Transport of glutamate by a high-affinity carrier can produce in Müller cells a rapid rise of glutamate concentration. In our isolated Müller cells, glutamine synthetase (GS) converted transported glutamate to glutamine that was released. This reaction, predominant when enough NH(4)(+) is available, was limited at micromolar concentrations of NH(4)(+), and more glutamate entered then as substrate into the mitochondrial tricarboxylic acid cycle (TCA). Increased production of glutamine by GS leads to increased utilization of ATP, some of which is generated glycolytically. Methionine sulfoximine, a specific inhibitor of GS, suppressed the stimulatory effect of glutamate and NH(4)(+) on glycolysis and induced massive entry of glutamate into the TCA cycle. The rate of glutamine production also determined the amount of pyruvate transaminated by glutamate to alanine. Lactate, alanine, and glutamine can be taken up and metabolized by photoreceptor neurons. We conclude that a major function of Müller glial cells is to nourish retinal neurons and to metabolize the neurotoxic ammonia and glutamate.

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PURPOSE: The development of extended areas of nonperfused capillaries after branch vein occlusion (BVO) is correlated to the secondary constriction of the arteriole crossing the occluded area. The decrease in nitric oxide (NO) in tissue that occurs early after BVO accounts for the secondary arteriolar constriction. The present study shows that the administration of an NO donor can reverse the secondary arteriolar vasoconstriction observed after BVO. METHODS: Simultaneous preretinal NO profiles and arteriolar diameter measurements were performed in miniature pigs after experimental BVO. The effect of preretinal microinjections of the NO donor sodium nitroprusside (SNP) on the arteriolar diameter was studied. RESULTS: Significant arteriolar vasoconstriction (mean arteriolar diameter, 92.1% +/- 3.3% of control; n = 7; P = 7.4 x 10(-5)) and a simultaneous decrease in the preretinal NO concentration ([NO]) (preretinal [NO], 20% +/- 15.6% of control; n = 5; P = 0.0003) were observed 4 hours after BVO. Microinjection of the NO donor SNP (1 mM applied by puffer) near the constricted retinal arteriole caused a segmental, reversible arteriolar dilation that reached its maximum 20 minutes after the injection (mean arteriolar diameter; 110.8% +/- 7.5% of control; n = 6; P = 0.02) and was completely reversed 60 minutes later (n = 6). CONCLUSIONS: Local administration of NO donors may contribute to the restoration of the retinal arteriolar blood flow after BVO and thus may improve the supply of oxygen and nutrients to the injured tissue.

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Photoreceptors need the support of pigment epithelial (PE) and Müller glial cells in order to maintain visual sensitivity and neurotransmitter resynthesis. In rod outer segments (ROS), all-trans-retinal is transformed to all-trans-retinol by retinol dehydrogenase using NADPH. NADPH is restored in ROS by the pentose phosphate pathway utilizing high amounts of glucose supplied by choriocapillaries. The retinal formed is transported to PE cells where regeneration of 11-cis-retinal occurs. Müller cells take up and metabolize glucose predominantly to lactate which is massively released into the extracellular space (ES). Lactate is taken up by photoreceptors, where it is transformed to pyruvate which, in turn, enters the Krebs cycle in mitochondria of the inner segment. Stimulation of neurotransmitter release by darkness induces 130% rise in the amount of glutamate released into ES. Glutamate is transported into Müller cells where it is predominantly transformed to glutamine. Stimulation of photoreceptors induces an eightfold increase in glutamine formation. It appears, therefore, that there is a signaling function in the transfer of amino acids from Müller cells to photoreceptors. Work on the model-system of the honeybee retina demonstrated that photoreceptors release NH4+ and glutamate in a stimulus-dependent manner which, in turn, contribute to the biosynthesis of alanine in glia. Alanine released into the extracellular space is taken up and used by photoreceptors. Glial cells take glutamate by high-affinity transporters. This uptake induces a transient change in glial cell metabolism. The transformation of glutamate to glutamine is possibly also controlled by the uptake of NH4+ which directly affects cellular metabolism.

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To determine if lactate is produced during aerobic metabolism in peripheral nerve, we incubated pieces of rabbit vagus nerve in oxygenated solution containing D-[U-14C]glucose while stimulating electrically. After 30 min, nearly all the radioactivity in metabolites in the nerve was in lactate, glucose 6-phosphate, glutamate, and aspartate. Much lactate was released to the bath: 8.2 pmol (microg dry wt)(-1) from the exogenous glucose and 14.2 pmol (microg dry wt)(-1) from endogenous substrates. Lactate release was not increased when bath PO2 was decreased, indicating that it did not come from anoxic tissue. When the bath contained [U-14C]lactate at a total concentration of 2.13 mM and 1 mM glucose, 14C was incorporated in CO2 and glutamate. The initial rate of formation of CO2 from bath lactate was more rapid than its formation from bath glucose. The results are most readily explained by the hypothesis that has been proposed for brain tissue in which glial cells supply lactate to neurons.

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PURPOSE: Following retinal branch vein occlusion (b.v.o.), the arteriole crossing the occluded territories is often constricted. This constriction persists up to several weeks and is correlated with the development of extended territories of non-perfused capillaries. We present here results of an investigation supporting the hypothesis that decrease in the production of nitric oxide (NO) accounts for the observed arteriolar constriction. METHODS: Preretinal [NO] was measured using an NO microprobe in the anesthethized miniature pigs, before and within the first 4 hours following experimental b.v.o. Modifications of arteriolar diameter were correlated to preretinal [NO] changes. The retinal arteriolar sensitivity to constitutive NO was checked by performing preretinal puff injections of nitro-1-arginine (L-NA) after both systemic hypoxia and b.v.o. RESULTS: Two hours after b.v.o. there was 73.7 +/- 4% decrease in preretinal [NO] and a simultaneous 25.4 +/- 3.4% decrease in the diameter of the arteriole in the affected territory. Both persisted for at least 4 hours after b.v.o. Puffing L-NA over an arteriole previously dilated by systemic hypoxia induced a vasoconstriction. However no arteriolar constriction was observed when puffing was performed on an arteriole after b.v.o. CONCLUSIONS: These results show that experimental b.v.o. induced in the affected retina an impairment in the release of constitutive NO and an arteriolar constriction, which in turn, contribute to the development of tissue hypoxia and neuronal swelling and death in the inner retina.

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The idea of a metabolic coupling between neurons and astrocytes in the brain has been entertained for about 100 years. The use recently of simple and well-compartmentalized nervous systems, such as the honeybee retina or purified preparations of neurons and glia, provided strong support for a nutritive function of glial cells: glial cells transform glucose to a fuel substrate taken up and used by neurons. Particularly, in the honeybee retina, photoreceptor-neurons consume alanine supplied by glial cells and exogenous proline. NH4+ and glutamate are transported into glia by functional plasma membrane transport systems. During increased activity a transient rise in the intraglial concentration of NH4+ or of glutamate causes a net increase in the level of reduced nicotinamide adenine dinucleotides [NAD(P)H]. Quantitative biochemistry showed that this is due to activation of glycolysis in glial cells by the direct action of NH4+ and of glutamate, probably on the enzymatic reactions controlled by phosphofructokinase alanine aminotransferase and glutamate dehydrogenase. This activation leads to a massive increase in the production and release of alanine by glia. This constitutes an intracellular signal and it depends upon the rate of conversion of NH4+ and of glutamate to alanine and alpha-ketoglutarate, respectively, in the glial cells. Alanine and alpha-ketoglutarate are released extracellularly and then taken up by neurons where they contribute to the maintenance of the mitochondrial redox potential. This signaling raises the novel hypothesis of a tight regulation of the nutritive function of glia.

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PURPOSE: After retinal branch vein occlusion (BVO), the arteriole crossing the occluded territories is often constricted. This constriction persists up to several weeks and is correlated with the development of extended territories of nonperfused capillaries. These are results of an investigation supporting the hypothesis that decrease in the production of nitric oxide (NO) accounts for the observed arteriolar constriction. METHODS: Preretinal [NO] was measured using an NO microprobe in the anesthetized miniature pigs, before and during the first 4 hours after experimental branch vein occlusion. Modifications of arteriolar diameter were correlated to preretinal [NO] changes. The retinal arteriolar sensitivity to constitutive NO was checked by applying preretinal puff injections of nitro-L-arginine (L-NA) after both systemic hypoxia and branch vein occlusion. RESULTS: Two hours after branch vein occlusion there was a 73.7 +/- 4% decrease in preretinal [NO] and a simultaneous 25.4 +/- 3.4% decrease in the diameter of the arteriole in the affected territory. Both persisted for at least 4 hours after branch vein occlusion. Applying a puff of L-NA to an arteriole previously dilated by systemic hypoxia induced a vasoconstriction. However, no arteriolar constriction was observed when a puff was applied to an arteriole after branch vein occlusion. CONCLUSIONS: These results show that experimental branch vein occlusion induces in the affected retina an impairment in the release of constitutive NO and an arteriolar constriction, which, in turn, contributes to the development of hypoxia in tissue and neuronal swelling and death in the inner retina.

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Glial cells transform glucose to a fuel substrate taken up and used by neurons. In the honeybee retina, photoreceptor neurons consume both alanine supplied by glial cells and exogenous proline. Ammonium (NH4+) and glutamate, produced and released in a stimulus-dependent manner by photoreceptor neurons, contribute to the biosynthesis of alanine in glia. Here we report that NH4+ and glutamate are transported into glia and that a transient rise in the intraglial concentration of NH4+ or of glutamate causes a net increase in the level of reduced nicotinamide adenine dinucleotides [NAD(P)H]. Biochemical measurements indicate that this is attributable to activation of glycolysis in glial cells by the direct action of NH4+ and glutamate on at least two enzymatic reactions: those catalyzed by phosphofructokinase (PFK; ATP:D-fructose-6-phosphotransferase, EC2.7.1.11) and glutamate dehydrogenase (GDH; L-glutamate:NAD oxidoreductase, deaminating; EC1.4.1.3). This activation leads to an increase in the production and release of alanine by glia. This signaling, which depends on the rate of conversion of NH4+ and glutamate to alanine and alpha-ketoglutarate, respectively, in the glial cells, raises the novel possibility of a tight regulation of the nutritive function of glia.

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A novel method of microbiosensor fabrication is described. It is based on the electrochemical polymerization of an enzyme-amphiphilic pyrroleammonium solution on the surface of a microelectrode in the absence of supporting electrolyte. By trapping glutamate oxidase (GMO) or polyphenol oxidase (PPO) in such polypyrrole films, we made microbiosensors for the amperometric determination of glutamate or dopamine, respectively. The response of the GMO microelectrode to glutamate was based on the amperometric detection of the enzymically generated hydrogen peroxide at 0.6 V vs SCE. The detection limit and sensitivity of this microbiosensor were 1 µM and 32 mA M(-1) cm(-2), respectively. The response of the PPO microelectrode to dopamine was based on the amperometric detection of the enzymically generated quinoid product at -0.2 V. The calibration range for dopamine measurement was 5 × 10(-8)-8 × 10(-5) M and the detection limit and sensitivity were 5 × 10(-8) M and 59 mA M(-1) cm(-2), respectively.

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We made simultaneous measurements of light-induced changes in the rate of oxygen consumption (QO2) and transmembrane current of single salamander rod photoreceptors. Since the change of PO2 was suppressed by 2 mM Amytal, an inhibitor of mitochondrial respiration, we conclude that it is mitochondrial in origin. To identify the cause of the change of QO2, we measured, in batches of rods, the concentrations of ATP and phosphocreatine (PCr). After 3 min of illumination, when the QO2 had decreased approximately 25%, ATP levels did not change significantly; in contrast, the amount of PCr had decreased approximately 40%. We conclude that either the light-induced decrease of QO2 is not caused by an increase in [ATP] or [PCr], or that the light-induced change of [PCr] is highly heterogeneous in the rod cell.

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PURPOSE: We present hereby some results indicating that there is a significant decrease in the release of NO by the retina immediately after branch-venous occlusion (BVO). METHODS: Using an NO microprobe we measured (NO) in the preretinal vitreous of miniature pigs before and two hours aRer bvo. Conventional and electronic microscopy were performed on the affected retina. RESULTS: At the retinal surface (NO) was reduced to 25% of the previous value two hours after BVO, at the same time significative oedema of the inner retina was observed. CONCLUSIONS: Immediately after BVO there is a significative decrease in the amount of NO released by the affected retina, parallel to the first evidences of an oedema of the inner retinal layers.

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PURPOSE: Experimental evidence indicates that the retinal microcirculation is mainly controlled by factors released from the tissue surrounding the arterioles. This study explores whether nitric oxide (NO), a possible factor, is released in the retina and controls the arteriolar tone. METHODS: Using a NO microprobe, the authors measured [NO] in the preretinal vitreous of miniature pigs as a function of distance from the retinal surface. Additionally, the NO-synthase inhibitor nitro-L-arginine was pressure injected. Finally, the retinal pool size of arginine and its biosynthesis from 14C(U)-glucose were biochemically assessed on retinal tissue and acutely isolated Müller cells. RESULTS: At the retinal surface, [NO] measured 6 to 9 microM, and, in the vitreous, it fell to zero approximately 180 microns away from the retina. Therefore, NO is degraded faster in the vitreous (65 to 80 microM.minute-1) than in aqueous solution. Light flicker stimulation of the dark-adapted retina induced a reversible increase of [NO] (approximately 1.6 microM). Preretinal juxta-arteriolar microinjections of nitro-L-arginine (0.6 mM) induced a segmental and reversible arteriolar vasoconstriction of 45%; in contrast, intravenous infusion of nitro-L-arginine had no measurable effect on arteriolar diameter. The retinal pool size of arginine was small (< or = 200 microM), but there was an important rate of arginine biosynthesis in Müller cells. CONCLUSIONS: These results strongly suggest that cells in the retina, other than endothelial cells, produce and release NO, which in turn controls the basal dilating arteriolar tone in the inner retina.

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The nature of fuel molecules trafficking between mammalian glial cells and neurons was explored using acute retinal cell preparations of solitary Müller glial cells, Müller cells still attached to photoreceptors (the "cell complex"), and solitary photoreceptors. 14C-Molecules in the cell complex, Müller cells, and respective baths were quantitated following 30 min incubation in bicarbonate-buffered Ringer's solution carrying 5 mM 14C(U)-glucose, and substrate preference by solitary photoreceptors was assessed by measuring 14CO2 production. Müller cells alone metabolized 14C-glucose predominantly to carbohydrate intermediates, while the presence of photoreceptors raised proportionately the amount of radiolabeling in amino acids. 14C-Lactate was the major carbohydrate found in the bath. However, in the presence of photoreceptors, its amount was 70% less than that for Müller cells alone. This decrease matched the expected production of 14CO2 by photoreceptor oxidative metabolism and was antagonized by the addition of unlabeled lactate. Moreover, while solitary photoreceptors consumed both exogenous 14C-lactate and 14C-glucose, lactate was a better substrate for their oxidative metabolism. In the cell complex, the metabolism of amino acids increased and illumination affected primarily glutamate and glutamine production: the specific activity of glutamate changed in parallel with that of lactate, and that of glutamine increased by eightfold in darkness. These results demonstrate transfer of lactate from Müller cells to photoreceptors and underscore a photoreceptor-dependent modulation of lactate and amino acid metabolism. We propose that net production and release of lactate by Müller cells serves to maintain their glycolysis elevated and to fuel mitochondrial oxidative metabolism and glutamate resynthesis in photoreceptors.

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PURPOSE: NO is a nonpolar gas, which diffuses across cell membranes in an isotropic fashion. In relatively large arteries NO has been identified as the endothelium-derived releasing factor and the current hypothesis is that NO controls the vascular tone. We are investigating an alternative hypothesis proposing that the retinal tissues surrounding the arterioles contribute in the process of NO production. METHODS: We performed flicker light stimulation in anesthetized minipigs (hypnodyl 100 mg/hr, tubocurarine 0.1 mg/hr und N2O) in steady-state of normoxia-normocapnia. An NO-microprobe mounted on a micromanipulator was introduced into the eye through the pars plana and positioned on the preretinal space in a zone free of visible vessels. Preretinal NO gradient and recording of NO release variations in response to flicker light stimulation were performed so as periarteriolar microinjections of nitro-L-arginine. RESULTS: When advancing the NO-probe from the vitreous towards the retina we recorded an NO-gradient corresponding to an efflux of 7.1 pMol x min-1 x cm-2 (N = 9). With the microprobe positioned close to the retina and after 60 min dark adaptation we recorded a mean increase in NO release of 1.59 muMol +/- 0.13 SE. After periarteriolar microinjection of nitro-Larginine we recorded a transitory reversible vasoconstriction. CONCLUSIONS: These results indicate that a process occurring in the extravascular tissue of the retina modulates the production of NO and that constitutional NO release by the retina is a major determinant of the basal retinal arteriolar tone.

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Nitric oxide (NO) is one of the most important messenger molecules dilating blood vessels in response to neurotransmitters and serving itself as a neurotransmitter in the brain. We explored its role in the retinal vascular regulation using a sensitive and specific NO microsensor and local perivascular microinjections of NO-synthase inhibitor (nitro-L-arginine in the intact eye of anesthetized miniature pigs. Our findings suggest that NO release is involved in the regulation of basal vascular tone in the inner retina. In contrast hypercapnia and hypoxia induce vasodilatation in an NO independent mechanism.

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Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.

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The retina of honeybee drone is a nervous tissue with a crystal-like structure in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments. The phosphorylation of glucose and its subsequent incorporation into glycogen occur in glia, whereas O2 consumption (QO2) occurs in the photoreceptors. Experimental evidence showed that glia phosphorylate glucose and supply the photoreceptors with metabolic substrates. We aimed to identify these transferred substrates. Using ion-exchange and reversed-phase HPLC and gas chromatography-mass spectrometry, we demonstrated that more than 50% of 14C(U)-glucose entering the glia is transformed to alanine by transamination of pyruvate with glutamate. In the absence of extracellular glucose, glycogen is used to make alanine; thus, its pool size in isolated retinas is maintained stable or even increased. Our model proposes that the formation of alanine occurs in the glia, thereby maintaining the redox potential of this cell and contributing to NH3 homeostasis. Alanine is released into the extracellular space and is then transported into photoreceptors using an Na(+)-dependent transport system. Purified suspensions of photoreceptors have similar alanine aminotransferase activity as glial cells and transform 14C-alanine to glutamate, aspartate, and CO2. Therefore, the alanine entering photoreceptors is transaminated to pyruvate, which in turn enters the Krebs cycle. Proline also supplies the Krebs cycle by making glutamate and, in turn, the intermediate alpha-ketoglutarate. Light stimulation caused a 200% increase of QO2 and a 50% decrease of proline and of glutamate. Also, the production of 14CO2 from 14C-proline was increased. The use of these amino acids would sustain about half of the light-induced delta QO2, the other half being sustained by glycogen via alanine formation. The use of proline meets a necessary anaplerotic function in the Krebs cycle, but implies high NH3 production. The results showed that alanine formation fixes NH3 at a rate exceeding glutamine formation. This is consistent with the rise of a glial pool of alanine upon photostimulation. In conclusion, the results strongly support a nutritive function for glia.

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PURPOSE: The authors investigated the hypothesis that the retinal vasomotor effect of acute hypoxia is mediated by lactate. METHODS: Retinal vasomotor arteriolar response was measured in the intact eyes of miniature pigs after systemic administration and after local preretinal juxta-arteriolar microinjection of lactate. RESULTS: Injection of L-lactate (physiologically produced lactate) into the systemic circulation decreased the arterial blood pH but did not dilate the retinal arterioles. By contrast, microinjections of L-lactate (0.5 mol/l, pH 2) into the juxta-arteriolar vitreous induced a reversible segmental vasodilation of 32 +/- 4% (standard deviation). This vasodilation did not depend on periarteriolar pH lowering because microinjections of a 0.5 mol/l L-lactate at neutral pH also dilated segmentally the retinal arterioles (37 +/- 5.5%). The effect of lactate was stereospecific because microinjections of the isomer D-lactate (0.5 mol/l, pH 2) did not affect the arteriolar caliber (P = 0.63). Perfusion of the eye with the cyclo-oxygenase inhibitor indomethacin, through cannulization of the sublingual artery, caused a generalized reversible arteriolar vasoconstriction of 51 +/- 9.8% but did not inhibit the segmental vasodilator effect of locally microinjected L-lactate. CONCLUSIONS: It is known that acute hypoxia in the isolated retina causes an increase in lactate production. In the intact eye, there is a retinal vasodilation, which is not inhibited by indomethacin. Hence, it was concluded that retinal, but not blood, lactate is a possible mediator of the acute hypoxia-induced vasodilation.

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Among the variety of roles and diverse possible functions that have been attributed to glial cells, the nutritive function is strongly supported by direct experimental evidence obtained in a model of the honeybee drone retina. We have shown that in this nervous tissue, with crystal-like structure, in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments, glial cells transform glucose to alanine and, with proline, fuel the mitochondria of the photoreceptors. Proline supplies the Krebs cycle by making glutamate. The use of proline implies high ammonia production. Pyruvate transamination in the glia fixes ammonia at a rate exceeding glutamine formation. We favor the hypothesis that ammonia rather than K+ is the metabolic signal trafficking between neuron and glial cells.

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