The Experts below are selected from a list of 1257 Experts worldwide ranked by ideXlab platform
Gary R Lewin - One of the best experts on this subject based on the ideXlab platform.
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Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch
Nature Communications, 2014Co-Authors: Kate Poole, Liudmila Lapatsina, Regina Herget, Ha-duong Ngo, Gary R LewinAbstract:The Stomatin domain protein STOML3 is required for the sensation of touch. Here, Poole et al. show that STOML3 enhances the activity of mechanosensitive Piezo1 and Piezo2 ion channels by reducing their activation thresholds, and that it achieves this through its Stomatin domain. In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of Stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other Stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure–function experiments localize the Piezo modulatory activity of STOML3 to the Stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.
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tuning piezo ion channels to detect molecular scale movements relevant for fine touch
Nature Communications, 2014Co-Authors: Kate Poole, Liudmila Lapatsina, Regina Herget, Gary R LewinAbstract:The Stomatin domain protein STOML3 is required for the sensation of touch. Here, Poole et al. show that STOML3 enhances the activity of mechanosensitive Piezo1 and Piezo2 ion channels by reducing their activation thresholds, and that it achieves this through its Stomatin domain.
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tuning piezo ion channels to detect molecular scale movements relevant for fine touch
Nature Communications, 2014Co-Authors: Kate Poole, Liudmila Lapatsina, Regina Herget, Ha-duong Ngo, Gary R LewinAbstract:In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of Stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other Stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure-function experiments localize the Piezo modulatory activity of STOML3 to the Stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.
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Stomatin domain protein interactions with acid sensing ion channels modulate nociceptor mechanosensitivity
The Journal of Physiology, 2013Co-Authors: Rabih Moshourab, Christiane Wetzel, Carlos Martinezsalgado, Gary R LewinAbstract:Key points • Gene deletion studies revealed that membrane proteins Stomatin and STOML3, as well as the acid-sensing ion channels ASIC2 and ASIC3, regulate mechanosensory transduction. • Both Stomatin and STOML3 interact with ASIC proteins and we asked if deletion of two interacting proteins has a more than additive effect on the mechanosensitivity of cutaneous sensory afferents. • A detailed electrophysiological comparison of sensory afferent phenotypes observed in asic3−/−:Stomatin−/−, asic3−/−:stoml3−/− and asic2−/−:Stomatin−/− mutant mice compared to their respective single gene mutants revealed especially strong effects on the mechanosensitivity of thinly myelinated mechanonociceptors in double mutants. • Deletion of the asic3 gene or pharmacological blockade of this channel decreased adaptation rates specifically in rapidly adapting mechanoreceptors, an effect not exacerbated by deletion of Stomatin-domain genes. • This study reveals that loss of Stomatin–ASIC interactions can have profound effects on mechanosensitivity in specific subsets of skin afferents; interfering with such interactions could have potential for treating mechanical pain. Abstract Acid-sensing ion channels (ASICs) and their interaction partners of the Stomatin family have all been implicated in sensory transduction. Single gene deletion of asic3, asic2, Stomatin, or stoml3 all result in deficits in the mechanosensitivity of distinct cutaneous afferents in the mouse. Here, we generated asic3−/−:Stomatin−/−, asic3−/−:stoml3−/− and asic2−/−:Stomatin−/− double mutant mice to characterize the functional consequences of Stomatin–ASIC protein interactions on sensory afferent mechanosensitivity. The absence of ASIC3 led to a clear increase in mechanosensitivity in rapidly adapting mechanoreceptors (RAMs) and a decrease in the mechanosensitivity in both Aδ- and C-fibre nociceptors. The increased mechanosensitivity of RAMs could be accounted for by a loss of adaptation which could be mimicked by local application of APETx2 a toxin that specifically blocks ASIC3. There is a substantial loss of mechanosensitivity in stoml3−/− mice in which ∼35% of the myelinated fibres lack a mechanosensitive receptive field and this phenotype was found to be identical in asic3−/−:stoml3−/− mutant mice. However, Aδ-nociceptors showed much reduced mechanosensitivity in asic3−/−:stoml3−/− mutant mice compared to asic3−/− controls. Interestingly, in asic2−/−:Stomatin−/− mutant mice many Aδ-nociceptors completely lost their mechanosensitivity which was not observed in asic2−/− or Stomatin−/− mice. Examination of Stomatin−/−:stoml3−/− mutant mice indicated that a Stomatin/STOML3 interaction is unlikely to account for the greater Aδ-nociceptor deficits in double mutant mice. A key finding from these studies is that the loss of Stomatin or STOML3 in asic3−/− or asic2−/− mutant mice markedly exacerbates deficits in the mechanosensitivity of nociceptors without affecting mechanoreceptor function.
Rainer Prohaska - One of the best experts on this subject based on the ideXlab platform.
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Structure-function analysis of human Stomatin: A mutation study.
PloS one, 2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:Stomatin is an ancient, widely expressed, oligomeric, monotopic membrane protein that is associated with cholesterol-rich membranes/lipid rafts. It is part of the SPFH superfamily including Stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins and erlins. Biochemical features such as palmitoylation, oligomerization, and hydrophobic "hairpin" structure show similarity to caveolins and other integral scaffolding proteins. Recent structure analyses of the conserved PHB/SPFH domain revealed amino acid residues and subdomains that appear essential for the structure and function of Stomatin. To test the significance of these residues and domains, we exchanged or deleted them, expressed respective GFP-tagged mutants, and studied their subcellular localization, molecular dynamics and biochemical properties. We show that Stomatin is a cholesterol binding protein and that at least two domains are important for the association with cholesterol-rich membranes. The conserved, prominent coiled-coil domain is necessary for oligomerization, while association with cholesterol-rich membranes is also involved in oligomer formation. FRAP analyses indicate that the C-terminus is the dominant entity for lateral mobility and binding site for the cortical actin cytoskeleton.
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Binding of [3H]photocholesterol to wildtype and mutant Stomatin.
2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:COS-7 cells were transiently transfected with WT or mutant Stomatin constructs. Subsequently they were incubated with a photoactivatable, radioactive cholesterol derivative ([3H]photocholesterol) and irradiated with UV light to crosslink [3H]photocholesterol to respective binding proteins. The cells were solubilized and Stomatin was immunoprecipitated by monoclonal anti-Stomatin antibody GARP-50. (A) SDS-PAGE and autoradiography revealed cholesterol-binding to WT and mutant Stomatin. (B) The expression level of the constructs was determined by immunoblotting with monoclonal anti-Stomatin antibody GARP-50.
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Oligomerization and DRM-association of GFP-tagged wildtype and mutant Stomatin.
2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:A431 cells stably expressing GFP-tagged WT or mutant Stomatin were solubilized and subjected to linear density gradient centrifugation to estimate molecular size (left panel) or step density gradient centrifugation to determine DRM-association (right panel). Gradient fractions were analyzed by SDS-PAGE and proteins were identified by immunoblotting with anti-GFP. The linear 15–50% sucrose gradient was verified by refractometry and calibrated by marker proteins. SDS-PAGE was performed by running molecular weight markers in parallel. GFP-tagged Stomatin constructs showed values of about 70 kDa except for the Pro47Ser mutant, which was estimated at 80 kDa, as predicted due to glycosylation [40].
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Schematic structure of GFP-tagged wildtype and mutant Stomatin.
2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:(A) Schematic model of wildtype (WT) Stomatin, composed of the N-terminal region (N-ter), intramembrane domain (IM), cholesterol recognition/interaction amino acid consensus (CRAC)-like motif (CL), prohibitin homology domain (PHB), also known as Stomatin, prohibitin, flotillin, HflK/C (SPFH) domain, coiled-coil domain (CC), oligomerization and lipid raft-association domain (ORA), and C-terminal domain (C-ter). Palmitate residues bound to Cys-30 and Cys-87 are symbolized by zigzag lines. Stomatin mutants are shown that are deleted at the N-terminus (ΔN), C-terminus (ΔC), and coiled-coil domain (ΔCC), respectively. The positions of exchanged amino acid residues in point mutants are marked. Exchange of Cys-30 or Cys-87 for Ser abolished palmitate bonding. (B) Hypothetical model of a monomeric wildtype Stomatin in association with a biological membrane. Sidedness is marked by “in” (cytoplasmic) and “out” (extracellular or luminal). The color code denotes the domains as illustrated in (A). The green ball at the N-terminal region symbolizes the phosphorylation site at Ser-10; the “P” at the kink within the hydrophobic IM domain marks residue Pro-47, which is responsible for the monotopic membrane protein structure. The model is roughly drawn according to known and estimated sizes; the N-terminal region is α-helical (E. Umlauf, unpublished results), the PHB/SPFH core domain is 5 nm in length and 2 nm in height, while the coiled-coil domain is 6 nm long [47]. CARC denotes a reversed CRAC motif; there are three CARC motifs, two overlapping with the CRAC-like (CL) and one overlapping with the ORA motif. Schematic models of the most remarkable mutants are shown in S4 Fig.
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Ability of GFP-tagged Stomatin mutants to target the plasma membrane, form oligomers, and/or associate with DRMs.
2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:Ability of GFP-tagged Stomatin mutants to target the plasma membrane, form oligomers, and/or associate with DRMs.
Kate Poole - One of the best experts on this subject based on the ideXlab platform.
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tuning piezo ion channels to detect molecular scale movements relevant for fine touch
Nature Communications, 2014Co-Authors: Kate Poole, Liudmila Lapatsina, Regina Herget, Gary R LewinAbstract:The Stomatin domain protein STOML3 is required for the sensation of touch. Here, Poole et al. show that STOML3 enhances the activity of mechanosensitive Piezo1 and Piezo2 ion channels by reducing their activation thresholds, and that it achieves this through its Stomatin domain.
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Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch
Nature Communications, 2014Co-Authors: Kate Poole, Liudmila Lapatsina, Regina Herget, Ha-duong Ngo, Gary R LewinAbstract:The Stomatin domain protein STOML3 is required for the sensation of touch. Here, Poole et al. show that STOML3 enhances the activity of mechanosensitive Piezo1 and Piezo2 ion channels by reducing their activation thresholds, and that it achieves this through its Stomatin domain. In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of Stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other Stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure–function experiments localize the Piezo modulatory activity of STOML3 to the Stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.
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tuning piezo ion channels to detect molecular scale movements relevant for fine touch
Nature Communications, 2014Co-Authors: Kate Poole, Liudmila Lapatsina, Regina Herget, Ha-duong Ngo, Gary R LewinAbstract:In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of Stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other Stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure-function experiments localize the Piezo modulatory activity of STOML3 to the Stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.
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a Stomatin dimer modulates the activity of acid sensing ion channels
The EMBO Journal, 2012Co-Authors: Janko Brand, Kate Poole, Ewan St. John Smith, David Schwefel, Liudmila Lapatsina, Alexey Kozlenkov, Joachim Behlke, Damir Omerbasic, Gary R LewinAbstract:Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, we determined three crystal structures of the conserved Stomatin domain of mouse Stomatin that assembles into a banana-shaped dimer. We show that dimerization is crucial for the repression of acid-sensing ion channel 3 (ASIC3) activity. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 activity. In one crystal form, Stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerization surfaces are also essential for the inhibition of ASIC3-mediated currents. The assembly mode of Stomatin uncovered in this study might serve as a model to understand oligomerization processes of related membrane-remodelling proteins, such as flotillin and prohibitin.
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A Stomatin dimer modulates the activity of acid‐sensing ion channels
The EMBO journal, 2012Co-Authors: Janko Brand, Gary R Lewin, Kate Poole, Ewan St. John Smith, David Schwefel, Liudmila Lapatsina, Damir Omerbašić, Alexey Kozlenkov, Joachim Behlke, Oliver DaumkeAbstract:Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, we determined three crystal structures of the conserved Stomatin domain of mouse Stomatin that assembles into a banana-shaped dimer. We show that dimerization is crucial for the repression of acid-sensing ion channel 3 (ASIC3) activity. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 activity. In one crystal form, Stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerization surfaces are also essential for the inhibition of ASIC3-mediated currents. The assembly mode of Stomatin uncovered in this study might serve as a model to understand oligomerization processes of related membrane-remodelling proteins, such as flotillin and prohibitin.
Liudmila Lapatsina - One of the best experts on this subject based on the ideXlab platform.
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tuning piezo ion channels to detect molecular scale movements relevant for fine touch
Nature Communications, 2014Co-Authors: Kate Poole, Liudmila Lapatsina, Regina Herget, Gary R LewinAbstract:The Stomatin domain protein STOML3 is required for the sensation of touch. Here, Poole et al. show that STOML3 enhances the activity of mechanosensitive Piezo1 and Piezo2 ion channels by reducing their activation thresholds, and that it achieves this through its Stomatin domain.
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Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch
Nature Communications, 2014Co-Authors: Kate Poole, Liudmila Lapatsina, Regina Herget, Ha-duong Ngo, Gary R LewinAbstract:The Stomatin domain protein STOML3 is required for the sensation of touch. Here, Poole et al. show that STOML3 enhances the activity of mechanosensitive Piezo1 and Piezo2 ion channels by reducing their activation thresholds, and that it achieves this through its Stomatin domain. In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of Stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other Stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure–function experiments localize the Piezo modulatory activity of STOML3 to the Stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.
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tuning piezo ion channels to detect molecular scale movements relevant for fine touch
Nature Communications, 2014Co-Authors: Kate Poole, Liudmila Lapatsina, Regina Herget, Ha-duong Ngo, Gary R LewinAbstract:In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of Stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other Stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure-function experiments localize the Piezo modulatory activity of STOML3 to the Stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.
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a Stomatin dimer modulates the activity of acid sensing ion channels
The EMBO Journal, 2012Co-Authors: Janko Brand, Kate Poole, Ewan St. John Smith, David Schwefel, Liudmila Lapatsina, Alexey Kozlenkov, Joachim Behlke, Damir Omerbasic, Gary R LewinAbstract:Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, we determined three crystal structures of the conserved Stomatin domain of mouse Stomatin that assembles into a banana-shaped dimer. We show that dimerization is crucial for the repression of acid-sensing ion channel 3 (ASIC3) activity. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 activity. In one crystal form, Stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerization surfaces are also essential for the inhibition of ASIC3-mediated currents. The assembly mode of Stomatin uncovered in this study might serve as a model to understand oligomerization processes of related membrane-remodelling proteins, such as flotillin and prohibitin.
Ellen Umlauf - One of the best experts on this subject based on the ideXlab platform.
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Structure-function analysis of human Stomatin: A mutation study.
PloS one, 2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:Stomatin is an ancient, widely expressed, oligomeric, monotopic membrane protein that is associated with cholesterol-rich membranes/lipid rafts. It is part of the SPFH superfamily including Stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins and erlins. Biochemical features such as palmitoylation, oligomerization, and hydrophobic "hairpin" structure show similarity to caveolins and other integral scaffolding proteins. Recent structure analyses of the conserved PHB/SPFH domain revealed amino acid residues and subdomains that appear essential for the structure and function of Stomatin. To test the significance of these residues and domains, we exchanged or deleted them, expressed respective GFP-tagged mutants, and studied their subcellular localization, molecular dynamics and biochemical properties. We show that Stomatin is a cholesterol binding protein and that at least two domains are important for the association with cholesterol-rich membranes. The conserved, prominent coiled-coil domain is necessary for oligomerization, while association with cholesterol-rich membranes is also involved in oligomer formation. FRAP analyses indicate that the C-terminus is the dominant entity for lateral mobility and binding site for the cortical actin cytoskeleton.
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Binding of [3H]photocholesterol to wildtype and mutant Stomatin.
2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:COS-7 cells were transiently transfected with WT or mutant Stomatin constructs. Subsequently they were incubated with a photoactivatable, radioactive cholesterol derivative ([3H]photocholesterol) and irradiated with UV light to crosslink [3H]photocholesterol to respective binding proteins. The cells were solubilized and Stomatin was immunoprecipitated by monoclonal anti-Stomatin antibody GARP-50. (A) SDS-PAGE and autoradiography revealed cholesterol-binding to WT and mutant Stomatin. (B) The expression level of the constructs was determined by immunoblotting with monoclonal anti-Stomatin antibody GARP-50.
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Molecular size distribution of GFP-tagged wildtype and mutant Stomatin.
2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:Molecular size distribution of GFP-tagged wildtype and mutant Stomatin.
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Comparison of Stomatin structural changes and functional consequences referring to wildtype.
2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:Comparison of Stomatin structural changes and functional consequences referring to wildtype.
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Densitometric analysis of [3H]photocholesterol-binding to wildtype and mutant Stomatin.
2017Co-Authors: Stefanie Rungaldier, Ulrich Salzer, Ellen Umlauf, Mario Mairhofer, Christoph Thiele, Rainer ProhaskaAbstract:Densitometric analysis of [3H]photocholesterol-binding to wildtype and mutant Stomatin.
