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Calcium Oxalate Stone Formation in the Inner Ear as a Result of an Slc26a4 Mutation*

Open AccessPublished:May 04, 2010DOI:https://doi.org/10.1074/jbc.M110.120188
      Calcium oxalate stone formation occurs under pathological conditions and accounts for more than 80% of all types of kidney stones. In the current study, we show for the first time that calcium oxalate stones are formed in the mouse inner ear of a genetic model for hearing loss and vestibular dysfunction in humans. The vestibular system within the inner ear is dependent on extracellular tiny calcium carbonate minerals for proper function. Thousands of these biominerals, known as otoconia, are associated with the utricle and saccule sensory maculae and are vital for mechanical stimulation of the sensory hair cells. We show that a missense mutation within the Slc26a4 gene abolishes the transport activity of its encoded protein, pendrin. As a consequence, dramatic changes in mineral composition, size, and shape occur within the utricle and saccule in a differential manner. Although abnormal giant carbonate minerals reside in the utricle at all ages, in the saccule, a gradual change in mineral composition leads to a formation of calcium oxalate in adult mice. By combining imaging and spectroscopy tools, we determined the profile of mineral composition and morphology at different time points. We propose a novel mechanism for the accumulation and aggregation of oxalate crystals in the inner ear.

      Introduction

      Biomineralization processes in the human body normally occur in a variety of different tissues, including bones, teeth, and otoconia within the vestibular system of the inner ear. The vestibular system is comprised of five sensory organs. Three cristae connected to semicircular canals are sensitive for angular movement, and the saccule and utricle are sensitive for linear acceleration and gravity. Otoconia are small highly dense calcitic minerals that associate exclusively with the saccule and utricle. Thousands of otoconia, partially embedded in a gelatinous matrix, are supported on the sensory epithelium and serve as an inertial mass that is critical for mechanical stimulation (
      • Thalmann R.
      • Ignatova E.
      • Kachar B.
      • Ornitz D.M.
      • Thalmann I.
      ,
      • Carlström D.
      ) Movement of the otoconial layer through action of gravitational or inertial forces activate the underlying mechanosensory hair cells to generate action potentials that are transmitted to the brain.
      The biomineralization process, such as in otoconia formation, involves organic and inorganic components and results in biominerals that differ significantly in morphology and mechanical properties from similar synthetic or geological minerals (
      • Berman A.
      • Addadi L.
      • Weiner S.
      ,
      • Lowenstam H.
      • Weiner S.
      ). Otoconia formation occurs outside the cells and therefore depends on secretion of the required assembly components into the endolymphatic spaces (
      • Hughes I.
      • Thalmann I.
      • Thalmann R.
      • Ornitz D.M.
      ). Otoconia seeding in mice begins as early as embryonic day (E)14.5
      The abbreviations used are: E
      embryonic day
      P
      postnatal day
      BPPV
      benign paroxysmal positional vertigo
      PDS
      pendrin
      SEM
      scanning electron microscopy
      D-PBS
      Dulbecco's phosphate-buffered saline
      EYFP
      enhanced yellow fluorescent protein
      WT
      wild-type
      FTIR
      Fourier transform infrared.
      and initiates extensive mineral growth, with the highest rate of calcification at E15–16 (
      • Anniko M.
      ). By postnatal day (P)7, otoconia achieve their final size and are maintained at progressive ages with a low rate of calcium turnover (
      • Erway L.C.
      • Purichia N.A.
      • Netzler E.R.
      • D'Amore M.A.
      • Esses D.
      • Levine M.
      ). The main inorganic fraction of otoconia in birds and mammals is calcite (CaCO3), a polymorph of calcium carbonate (
      • Carlström D.
      ,
      • Ross M.D.
      • Peacor D.R.
      ). The organic fraction of otoconia contains several matrix proteins that are critical for the nucleation and mineralization of otoconia. The major core protein is otoconin-90 (Oc90) (also known as otoconin-95; Oc95) (
      • Verpy E.
      • Leibovici M.
      • Petit C.
      ) and accounts for more than 90% of the organic phase of otoconia. Oc90 is characterized by a high abundance of negatively charged amino acid residues and has two regions of homology with secretory phospholipase A2 (PLA2) (
      • Wang Y.
      • Kowalski P.E.
      • Thalmann I.
      • Ornitz D.M.
      • Mager D.L.
      • Thalmann R.
      ). The PLA2 domain lacks enzymatic activity but retains calcium binding function (
      • Pote K.G.
      • Hauer 3rd, C.R.
      • Michel H.
      • Shabanowitz J.
      • Hunt D.F.
      • Kretsinger R.H.
      ). It has been proposed that Oc90 is responsible for determining the calcium carbonate polymorph type of otoconia, namely calcite (
      • Pote K.G.
      • Ross M.D.
      ). The delicate balance between the organic and inorganic components of otoconia, including their spatial and temporal distribution, determines the growth rate, shape, and composition of the mineral.
      Otoconia are subjected to morphological and compositional changes by diverse environmental and genetic factors. Prolonged exposure to medications such as streptomycin results in formation of abnormal giant otoconia (
      • Takumida M.
      • Zhang D.M.
      • Yajin K.
      • Harada Y.
      ,
      • Harada Y.
      • Sugimoto Y.
      ). Age-related otoconia degeneration is highly abundant and increases the risk for free floating particles (
      • Ross M.D.
      • Peacor D.
      • Johnsson L.G.
      • Allard L.F.
      ). Dislocation of otoconia or their broken particles outside their native position can lead to severe vestibular dysfunction in humans. Benign paroxysmal positional vertigo (BPPV) patients suffer from severe dizziness. This clinical condition affects up to 9% of the population older than 65 years of age (
      • Oghalai J.S.
      • Manolidis S.
      • Barth J.L.
      • Stewart M.G.
      • Jenkins H.A.
      ). The phenotype manifests itself when otoconia migrate to one of the cristae (cupulolithiasis) or the semicircular canals (canalithiasis) and hinders its mechanical sensory properties (
      • House M.G.
      • Honrubia V.
      ). BPPV is one of the major clinical conditions attributed to dislocated otoconia.
      In the current study, we identified and characterized a new recessive mouse mutant named loop (gene symbol Slc26a4loop), which carries a recessive missense mutation in the Slc26a4 gene encoding pendrin. Human mutations in SLC26A4 lead to a non-syndromic (DFNB4) and syndromic form of deafness with enlargement of the thyroid gland (Pendred syndrome) (
      • Coyle B.
      • Coffey R.
      • Armour J.A.
      • Gausden E.
      • Hochberg Z.
      • Grossman A.
      • Britton K.
      • Pembrey M.
      • Reardon W.
      • Trembath R.
      ,
      • Sheffield V.C.
      • Kraiem Z.
      • Beck J.C.
      • Nishimura D.
      • Stone E.M.
      • Salameh M.
      • Sadeh O.
      • Glaser B.
      ). Mimicking the human pathology and similar to the knock-out mouse model (Pds−/−) (
      • Everett L.A.
      • Belyantseva I.A.
      • Noben-Trauth K.
      • Cantos R.
      • Chen A.
      • Thakkar S.I.
      • Hoogstraten-Miller S.L.
      • Kachar B.
      • Wu D.K.
      • Green E.D.
      ), loop mice are profoundly deaf and show abnormal vestibular behavior. Here we present the discovery of a new type of giant mineralized bodies with the composition of calcium oxalate within the Slc26a4loop/loop saccule. These mineralized bodies are highly distinct from calcitic wild-type otoconia and have not been previously described in the inner ear. Interestingly, these unique pathological oxalate ear stones are only formed within one out of the two separate compartments of otoconia formation, the utricle and saccule. In our study, we analyzed the wide spectrum of mineral composition in the Slc26a4loop/loop inner ear that undergoes dramatic differential changes and provide insight into an abnormal mineralization mechanism in the presence of a genetic mutation.

      DISCUSSION

      The small size of normal otoconia, together with their distribution pattern over the saccule and utricle, is crucial for generating an optimal mass over each individual hair cell, which is essential for their stimulation. Thus, the formation of giant minerals in Slc26a4loop/loop mutants leads to a differential mass over the sensory cells. Although some hair cells lack any otoconia load, other hair cells are overloaded with giant stones. Hence, both populations of hair cells are missing their native stimulation source and cannot serve their vital role in vestibular function and perception. Furthermore, the giant minerals in Slc26a4loop mice are no longer restricted to the saccule or utricle, as compared with normal otoconia. Ectopic giant stones were found in other components of the vestibular system such as the semicircular canals and their cristae. The variability of the defective vestibular behavior in Slc26a4loop mutant mice, ranging from very mild to severe, can be partially explained by the variable position of dislocated mineralized bodies. It would be interesting to test this hypothesis and learn about possible correlations between the position of the stone and a specific vestibular defect. Displacement of otoconia in humans is associated with BPPV and leads to severe dizziness, imbalance, and nausea (
      • Epley J.M.
      ). The severe vertigo episodes are usually of a short duration and elicited by the position of the patient. A common way to treat BPPV is by maneuvering the free floating otoconia outside the semicircular canal and crista, providing the patient with relief (
      • Beynon G.J.
      ,
      • Epley J.M.
      ,
      • Parnes L.S.
      • Agrawal S.K.
      • Atlas J.
      ). Hence, Slc26a4loop mice can be tested as a platform for validating pioneering therapeutic approaches with respect to vestibular dysfunction as a result of otoconia dislocation.
      Otoconia formation depends on organic and inorganic components that are secreted into an extracellular fluid and assembled to create a myriad of otoconia. The orchestrated expression of the genes involved in otoconia formation, together with the spatial and temporal abundance of the chemical elements, is crucial for proper otoconia assembly. Hence, depletion of components from the organic and inorganic parts of this equation can lead to otoconia defects. For example, deletion of the PMCA2 Ca2+ channel, a key player in establishing endolymph calcium homeostasis, results in a lack of otoconia formation (
      • Kozel P.J.
      • Friedman R.A.
      • Erway L.C.
      • Yamoah E.N.
      • Liu L.H.
      • Riddle T.
      • Duffy J.J.
      • Doetschman T.
      • Miller M.L.
      • Cardell E.L.
      • Shull G.E.
      ). Alternatively, deletion of otoconin-90, the mammalian otoconial matrix protein, leads to formation of giant calcite minerals that lack the major organic fraction of otoconia (
      • Verpy E.
      • Leibovici M.
      • Petit C.
      ,
      • Zhao X.
      • Yang H.
      • Yamoah E.N.
      • Lundberg Y.W.
      ).
      Pendrin is known to be expressed in the transitional cell layer surrounding the sensory epithelium of the saccule and utricle (
      • Royaux I.E.
      • Belyantseva I.A.
      • Wu T.
      • Kachar B.
      • Everett L.A.
      • Marcus D.C.
      • Green E.D.
      ). Pendrin is a member in the SLC family of transporters and transports several different ions, including Cl, I, HCO3, and formate (
      • Scott D.A.
      • Karniski L.P.
      ,
      • Scott D.A.
      • Wang R.
      • Kreman T.M.
      • Sheffield V.C.
      • Karniski L.P.
      ,
      • Soleimani M.
      • Greeley T.
      • Petrovic S.
      • Wang Z.
      • Amlal H.
      • Kopp P.
      • Burnham C.E.
      ). In the inner ear, pH measurements of the endolymph within the utricle of Pds−/− mice show acidification of the fluid (
      • Wangemann P.
      • Nakaya K.
      • Wu T.
      • Maganti R.J.
      • Itza E.M.
      • Sanneman J.D.
      • Harbidge D.G.
      • Billings S.
      • Marcus D.C.
      ,
      • Nakaya K.
      • Harbidge D.G.
      • Wangemann P.
      • Schultz B.D.
      • Green E.D.
      • Wall S.M.
      • Marcus D.C.
      ). Following these studies, pendrin was proposed to function as a Cl/HCO3 transporter in the inner ear that actively secretes bicarbonate into the endolymph, which neutralizes H+ and participates in buffering the normal physiological pH. Hence, control of appropriate pH levels is crucial for mineral assembly and stabilization. Pendrin may also be involved in otoconia formation by supplying HCO3 that is essential to form the calcite (CaCO3) crystals of otoconia. However, the extensive mineral growths observed in the utricle of Slc26a4loop mutants early in development suggests that the reservoir of bicarbonate for otoconia assembly was not reduced drastically. A putative compensation for loss of bicarbonate is achieved by the activity of carbonic anhydrase due to its production of HCO3 and H+ from CO2 and H2O. This enzyme is known to be widely expressed in sensory and non-sensory epithelia of the utricle and saccule (
      • Shiao J.C.
      • Lin L.Y.
      • Horng J.L.
      • Hwang P.P.
      • Kaneko T.
      ,
      • Lim D.J.
      • Karabinas C.
      • Trune D.R.
      ).
      This work has demonstrated how a missense mutation (S408F) within a highly conserved region of the pendrin protein leads to impaired transport activity. We also report that this defective mutation leads to differential defects in otoconia formation between the utricle and saccule of Slc26a4loop mutants. Although abnormal giant calcite minerals always reside in Slc26a4loop/loop utricles, a gradual change in mineral composition is observed in the saccule, with formation of calcium oxalate stones in adult mice. These giant minerals, irrespective of shape and composition, can no longer serve their vital and elementary role in proper hair cell stimulation and vestibular function. Interestingly, these differential mineralization defects occur despite the similar pattern of pendrin expression in the saccule and utricle. Our findings also suggest that the minerals in the utricle and saccule are exposed to different endolymphatic environments. We predict that abolished activity of pendrin in Slc26a4loop mutants leads to different levels of acidification in each of the these two compartments. Hence, anatomical and histological differences between utricle and saccule, together with pH-sensitive proteins that are differentially expressed in these maculae, may be the key for understanding the mechanism underlying this complex pathology.

      Initial Formation of Giant Calcium Carbonate Stones in Slc26a4loop/loop Vestibule

      The similarity between the utricle and saccule is striking. Both are sensitive for linear acceleration, have otoconia as an inertial mass for stimulation initiation, and contain the same type of sensory hair cells. However, several distinct anatomical and histological characteristics are unique to each of these maculae. The differences between utricle and saccule are illustrated (Fig. 8, top panel) and include two main aspects pertaining to the current discussion. First, the endolymph of the utricle is connected to the semicircular canals and the crista ampullaris and share fluid circulation with these vestibular components, as opposed to the saccule, which lacks this direct connection. Second, a pronounced difference is the large abundance of the pigmented vestibular dark cells in the utricle but absent in the saccule.
      Figure thumbnail gr8
      FIGURE 8Proposed working model for calcium oxalate stone formation in the inner ear. The anatomical differences (top panels) between the utricle (green) and saccule (red) are illustrated and summarized in a table. A, under normal conditions, otoconia nucleation begins around E16.5 and reaches its maturation at P7. To support the biomineralization events, otoconia proteins such as Oc90 are secreted to the endolymph prior to and during otoconia nucleation and maturation. B, in Slc26a4loop mice, the organic fraction of otoconia, including Oc90, is secreted normally to the extracellular space, but the homeostasis of the endolymph is impaired. Pendrin activity is depleted due to the S408F mutation, and the lack of HCO3 supply leads to acidification of the endolymphatic fluids. This acidification abolishes the reabsorption of calcium by the pH-sensitive calcium channels, TRPV5 and TRPV6 (
      • Nakaya K.
      • Harbidge D.G.
      • Wangemann P.
      • Schultz B.D.
      • Green E.D.
      • Wall S.M.
      • Marcus D.C.
      ). The localization of TRPV5 and TRPV6 in the semicircular canal duct epithelium and in the vestibular dark cells, which share fluid circulation with the utricle, leads to a higher calcium concentration in the utricle as compared with the saccule. The excess calcium ions in the endolymph of the utricle are sequestered by large amounts of Oc90 and deposited into oversized calcite minerals, whereas in the saccule, significantly smaller minerals are formed. C, at progressive ages, wild-type otoconia are maintained with low calcium turnover, whereas in Slc26a4loop mice, a differential process between saccule and utricle occurs. In the utricle, giant calcitic minerals reside all along the lifespan of the mouse. In the saccule, a gradual change in mineral morphology and composition from calcite into highly disordered calcite at the age of 7 months is observed. The ultrastructural morphology of the highly disordered mineral, a pitted and fissured surface, resembles the morphology of calcite mineral after treatment with an acidic solution. Moreover, this stone contained domains that resembled the morphology of calcium oxalate in the form of weddellite. Between the age of 7 and 10 months, the highly disordered calcite dissolved, and giant calcium oxalate minerals in the form of weddellite were generated. The symmetrical morphology of the weddellite resembles the classical calcium oxalate geological mineral, which is more stable at lower pH as compared with the calcium carbonate. In summary, constant acidification of the saccule leads to dissolution of the calcite mineral that is tied with favorable conditions for calcium oxalate stone formation in the inner ear.
      Previous studies on the Pds−/− mice show that the acidification of the vestibular endolymph inhibits the pH-sensitive calcium channels, TRPV5 and TRPV6 (
      • Nakaya K.
      • Harbidge D.G.
      • Wangemann P.
      • Schultz B.D.
      • Green E.D.
      • Wall S.M.
      • Marcus D.C.
      ,
      • Yeh B.I.
      • Sun T.J.
      • Lee J.Z.
      • Chen H.H.
      • Huang C.L.
      ). Both TRPV5 and TRPV6 were shown to be expressed in the vestibular dark cells and in the epithelial cells of the semicircular canal duct epithelium (
      • Takumida M.
      • Ishibashi T.
      • Hamamoto T.
      • Hirakawa K.
      • Anniko M.
      ,
      • Yamauchi D.
      • Raveendran N.N.
      • Pondugula S.R.
      • Kampalli S.B.
      • Sanneman J.D.
      • Harbidge D.G.
      • Marcus D.C.
      ). The abolished calcium reabsorbance of these channels leads to elevation in calcium levels in the endolymph to which the utricle is exposed. Under normal conditions, the calcium concentration in the endolymph is very low as compared with other physiological fluids. The low calcium level is essential for the mechanotransduction machinery of the sensory hair cells (
      • Marquis R.E.
      • Hudspeth A.J.
      ). One way to maintain this environment is achieved by calcium absorbance of the surrounding epithelial cells. Another way to control calcium levels is by recruiting calcium for otoconia formation, which was proposed to be a method for providing a calcium reservoir (
      • Ross M.D.
      ). A key player in this respect is Oc90, which sequesters calcium ions. In wild-type mice, Oc90 is secreted into the endolymph prior to otoconia nucleation and participates in the growth of these tiny minerals (
      • Thalmann R.
      • Ignatova E.
      • Kachar B.
      • Ornitz D.M.
      • Thalmann I.
      ,
      • Verpy E.
      • Leibovici M.
      • Petit C.
      ). Under normal conditions, the secreted amount of Oc90 competes with a normally low level of calcium ions and is thus essential for the initial nucleation of otoconia. The level of Oc90 expression and secretion into the endolymph of Slc26a4loop mutants is most likely not affected by the pendrin S408F mutation. However, there is a dramatic increase in the amount of Oc90 that is observed in the giant mineral of Slc26a4loop/loop mice as compared with the wild-type otoconia. This discrepancy can be explained by the presumed higher calcium levels in the endolymph of Slc26a4loop mutants, which leads to rapid trapping of any residual Oc90 into a giant crystal.
      Taken together, elevation of calcium levels in the endolymph fluid, within the common compartment of the utricle, cristae, and semicircular canals, leads to a differential calcium concentration between utricle and saccule, with higher calcium levels in the utricle. The formation of giant calcitic stones in the utricle and significantly smaller stones in the saccule can be attributed to differences in calcium concentration. Furthermore, this postulated difference in the level of calcium may be connected to the observation that one giant mineralized body forms in the mutants, instead of many small uniformly sized and shaped otoconia in the wild-type mice. It is conceivable that because of the high calcium concentrations in the Slc26a4loop/loop endolymph, the first nucleated otoconia continues to grow rapidly and sequesters most of the calcium. Any other small crystals that may subsequently form would redissolve because of the lack of calcium in their microenvironment.
      The data presented in this study are confined to postnatal stages. Several models have been proposed in the literature for early otoconia seeding and maturation (
      • Hughes I.
      • Thalmann I.
      • Thalmann R.
      • Ornitz D.M.
      ,
      • Lundberg Y.W.
      • Zhao X.
      • Yamoah E.N.
      ). Although one model suggests secretion of vesicles with otoconia substances as a seeding platform (
      • Suzuki H.
      • Ikeda K.
      • Takasaka T.
      ,
      • Suzuki H.
      • Ikeda K.
      • Furukawa M.
      • Takasaka T.
      ), a second model excludes involvement of vesicles as otoconia precursors and proposes that perimacular seeding occurs following temporal and spatial secretion of individual otoconia components (
      • Lundberg Y.W.
      • Zhao X.
      • Yamoah E.N.
      ). The core structure of the giant calcitic mineralized bodies in Slc26a4loop mutants suggests that these giant stones are developing from a single nucleation core with continued growth rather than multiple assembly of thousands of mature otoconia particles. Observation of E15.5 utricles reveals that the aberrant giant mineral already exists at this stage (data not shown) and supports our assumption. Further characterization of the early developmental steps that initiate the growth of these giant minerals can provide important clues with respect to the proposed models of otoconia development.

      Drastic Changes in Mineral Morphology and Composition in Slc26a4loop/loop Saccule

      Mineral properties are affected by different environmental changes, including temperature and pH. Kinetics of mineral dissolution is dependent on pH levels, which affects its surface hardness and microstructure (
      • Sjöberg E.L.
      • Rickard D.T.
      ). As a consequence of endolymph circulation in the utricle, the volume of endolymph to which the utricle is exposed to is larger than that of the saccule. Therefore, the reduction in endolymphatic pH, following abnormal activity of pendrin, is presumed to be mild in the utricle where the endolymph is circulated frequently. However, in the saccule of Slc26a4loop mutants, which lacks this pronounced circulation, a lower pH level is expected to develop. We revealed a progressive gradual change in mineral morphology and composition in the saccule of Slc26a4loop mutants. Furthermore, the structure of these minerals suggests that a gradual reduction in pH of the saccule endolymph develops with age. In newborns, small calcium carbonate minerals in the form of calcite rarely developed. However, at the age of 7 months, a giant, highly disordered calcium carbonate mineral in the form of calcite was apparent. An ultrastructural analysis of this mineral revealed a pitted and fissured surface, resembling the pattern of calcite mineral after exposure to an acidic solution. Significantly, this stone contained weddellite. At the age of 10 months, the highly disordered calcite was no longer found, and instead, giant calcium oxalate stones in the form of weddellite were detected. Biosynthesis of oxalate from carbonate is not known in vitro. Hence, we suggest that the highly disordered calcite is unstable due to the constant acidification of the environment, and this results in mineral dissolution. Consequently, the endolymphatic environment is enriched with calcium ions, which now bind to oxalate ions and subsequently precipitate as calcium oxalate crystals in the form of weddellite. The symmetrical morphology of the weddellite resembles the classical calcium oxalate geological mineral, which is more stable at lower pH as compared with the calcium carbonate. Thus, acidification of the saccule fluids is in favor of calcium oxalate mineral formation (summarized in Fig. 8). Pendrin is capable of transporting formate but not oxalate (
      • Scott D.A.
      • Karniski L.P.
      ). We hypothesize that the S408F mutation might alter the affinity of pendrin transport to oxalate, which is chemically closely related to formate. Additional experiments we performed with the fast fluorometric approach to test this hypothesis suggest that the S408F mutation does not impose a new function of oxalate transport by the pendrin protein in vitro (data not shown). Formation of calcium oxalate minerals is also known in kidney pathology and accounts for more than 80% of kidney stones (). Thus, because pendrin is widely expressed in the cortical collecting duct of the kidney (
      • Royaux I.E.
      • Wall S.M.
      • Karniski L.P.
      • Everett L.A.
      • Suzuki K.
      • Knepper M.A.
      • Green E.D.
      ), we investigated the possibility of kidney stone formation using Pizzolato's staining for oxalate deposits (
      • Pizzolato P.
      ). Histological analysis of Slc26a4loop/loop kidney up to the age of 10 months did not reveal oxalate stone accumulation (data not shown). In healthy individuals, most of the crystals that are formed in the renal tubules are discharged in the urine. However, the inner ear is a closed fluid-filled compartment that lacks fluid circulation with other systems in the body. Thus, when oxalate crystals are precipitated in the inner ear, they are trapped within the endolymph, and their accumulation and aggregation are inevitable. This aberrant genetic-dependent mineralization process that leads to calcium oxalate stones formation is described in the mammalian ear for the first time.

      Acknowledgments

      We thank Jørgen Frøkiær and Christine Petit for antibodies and Leonid Mittelman for training in confocal microscopy.

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