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Role of the hydrophobic core in cytoskeleton protein: cardiac myosin binding protein C

Role of the hydrophobic core in cytoskeleton protein: cardiac myosin binding protein C IntroductionCardiac myosin binding protein C (MYBPC3) is a component of the sarcomere, a functional unit of cardiomyocyte. The sarcomeres conceal the basic mechanism of active muscle contraction. Cardiac myosin binding protein C belongs to the intracellular immunoglobulin superfamily and is the part of the thick filaments. It has structural functions for the sarcomere, as it is a link between myosin thick filaments and titin super-thin filaments [1], [2], [3], [4]. Apart from the structural function, it also acts as a regulator in relation to actin F and S1 myosin subfragment. In its sequence, there are four sites that are phosphorylated by cAMP-dependent protein kinase and calmodulin-activated protein kinase. The activation of these sites leads to changes in the dynamics of myocardial contractility [5].The cardiac myosin binding protein C gene locates on the 11q13-11p13 chromosome, consists of 35 exons, and encodes a 137-kDa molecular polypeptide. The expression of the mutant protein leads to the disorganization in the muscle fibers and the development of hypertrophic cardiomyopathy (HCM) [6]. HCM is characterized by primary left ventricular thickening, which is not associated with abnormal hemodynamic cardiac stress (e.g. hypertension and aortic stenosis). This pathology is an important cause of disability and death among people of all ages but most often causes sudden cardiac death in young population under 30 years old, often athletes. It occurs with a frequency of 1 in 500 in population, which makes it one of the most common genetic diseases. Approximately 60% of the cases are reported as dominant autosomal features and result from mutations in genes encoding cardiac sarcomere proteins. Probably the most common variant is the mutation of the cardiac myosin binding protein C gene.HCM with the MYBPC3 gene is characterized by late clinical manifestation and very poor prognosis associated with sudden cardiac death in the course of cardiac arrhythmias. In the histological examination, the disorder is characterized by pathological hypertrophy (cardiomyocyte hyperplasia), interstitial fibrosis, and the thickening of the coronary arteries. The mechanism of cardiomyopathy is not fully understood. It is suggested that mutations in sarcomere proteins lead to a decrease in cardiomyocyte contractility. This in turn leads to increased cellular tension and, consequently, to the production of tropic and mitotic factors that cause hypertrophy (hyperplasia), cardiomyocyte disorder, and fibrosis. According to another hypothesis, the development of HCM occurs as a result of disturbances in the energy management of the myocardial cell. Cellular energy deprivation impairs pump activity, which is responsible for the uptake of calcium ions into the endoplasmic reticulum, and prolonged calcium retention in the cytoplasm may be a stimulus for cardiomyocyte hyperplasia. Hyperplasia, the disorganization of the muscle fibers, and interstitial fibrosis are adaptations to the inability to generate sufficient contraction power that is needed to maintain adequate cardiac output. In the case of HCM, the above-mentioned adaptive mechanisms are quite good for maintaining normal resting heart volume. On the contrary, under the influence of physical effort, symptoms of failure occur due to an abnormal ratio of cardiomyocytes (which have undergone hyperplasia) to coronary vascularization (which has not increased or even pathological changes in the coronary arteries are observed) and defect of myosin ATPase activity. Physical exercise therefore induces myocardial hypoxia events. Hypoxia and the electrical instability of the heart caused by cardiomyocyte fibrosis induce ventricular arrhythmias. They are believed to be the leading cause of death in patients with HCM [7].The protein under consideration belongs to the immunoglobulin-like superfamily representing the supersecondary structural form called sandwich. Many different proteins of different biological activities belong to this group, including structural proteins, enzymes, and obviously almost all immunoglobulin domains. The high structural similarity does not necessarily represents the similar structure of the hydrophobic core. The significant diversity is observed for immunoglobulin-like domains. The comparison of different hydrophobic core structures is shown in Ref. [8]: the whole spectrum of highly ordered hydrophobic core construction from very high ordering in titin (RD=0.382 1TIT) to very low structuralization (RD=0.722 for lyase 1CTN). The localization of less fitted β-structural fragments is also different in compared domains. Titin as the example of lowest RD for immunoglobulin-like domains seems to represent the status strongly related to biological function. Titin is the fragment of muscle cytoskeleton. These proteins are under external forces that act periodically. The main point for such protein is to return to its relaxed form after the release. The highly ordered hydrophobic core seems to be the factor ensuring the reorganization of the highly ordered hydrophobic core. The same effect is observed in distrophin, where the additional domain in the interface represents highly ordered hydrophobic core organization [9].The work shown in this paper is to check whether all immunoglobulin-like domains present in muscle cells represent a similar ordered hydrophobic core. The consequences of point mutation introduced to human cardiac myosin binding protein C are also discussed.Materials and methodsDataThe protein selected for analysis is myosin binding protein C in two forms: WT (2MQ0) and mutant R502W (2MQ3) [10]. The length of this protein is 95 amino acids. The protein represents the domain of the form of a sandwich. It belongs to immunoglobulin-like domains.Fuzzy oil drop model to characterize the structure of the hydrophobic coreThe fuzzy oil drop model assumes the structure of the hydrophobic core in protein to be in the form of 3D Gauss function. It assumes the concentration of hydrophobicity in the center of the molecule (called T). The hydrophobicity decreases according to the distance versus the center reaching the zero level on the surface. Surface is understood in the distance of 3σ for each direction. σ is the Gauss function parameter. The three-σ rule defines the size of ellipsoid. The protein molecule encapsulated in 3D Gauss function with σ parameters appropriate for protein under consideration. The values of 3D Gauss function in any point of ellipsoid expresses the theoretical idealized hydrophobicity density. The observed hydrophobicity in any point of protein is the effect of interresidual hydrophobic interaction (called O). This is why it does not necessary follow the idealized distribution. The proteins of highly similar hydrophobic distribution are found and described in many papers [8], [9], [11], [12]. The local discordance of hydrophobicity appears to be related to biological activity. The local hydrophobicity deficiency identifies often the ligand-binding cavity [11]. The local hydrophobicity excess, particularly when localized on the surface of the protein, may suggest area for protein-protein complexation [12].The Kullback-Leibler entropy is introduced to measure the differences [13]. Two reference distributions are introduced. One of them is the Gauss-like distribution representing the idealized distribution (T) and the second one is the unified distribution (called R) when each residue represents equal hydrophobicity. This distribution represents the status deprived of any form of hydrophobicity concentration. The value of DKL indicates the distance between the observed distribution and the theoretical distribution (O|T). Its value cannot be interpreted as the DKL is of the entropy category. This is why the relative distance is introduced measuring the distance versus the theoretical distribution versus the sum of distances versus the theoretical and unified distribution (O|R):RD=O|TO|T+O|R.${\rm{RD}} = {{{\rm{O}}|{\rm{T}}} \over {{\rm{O}}|{\rm{T}} + {\rm{O}}|{\rm{R}}}}.$In consequence, RD values range from 0 to 1. This is why the RD value below 0.5 indicates the status of the hydrophobic core close to the theoretical one. The RD value above 0.5 indicates the distance between the observed distribution and the unified distribution. The status of the domain (protein) described by RD>0.5 is interpreted as the molecule without the ordered hydrophobic core.The RD value may be calculated for complex, chain, or domain. For each of these units, the individual ellipsoid is calculated. The status of the selected polypeptide chain fragment can also be characterized by the RD value after the prior normalization of Ti, Oi and Ri of residues belonging to the selected fragment.The fuzzy oil drop model is described in detail in many papers [13], [14]. The description given above is necessary to understand the results discussed in this paper.Programs usedThe RD values are calculated using our own program. The 3D presentation of the protein structure is possible due to the VMD program [15] (http://www.ks.uiuc.edu/Research/vmd/), which allows the visualization of structures.ResultsThe parameters describing the status of the hydrophobic core in proteins under consideration are given in Table 1.Table 1:Parameters describing the status of myosin binding protein C in two forms: WT (2MQ0) and mutant R502W (2MQ3).SecondaryFragmentRD2MQ02MQ3Complete1449–5430.4490.482β2465–4670.3220.6063539–5410.2160.1514500–5060.3550.4555508–5150.3010.3346471–4730.3180.3027475–4770.3558532–5380.3310.4719523–5290.6500.49110485–4890.5820.67111491–4930.7150.584Sandwich120.4730.491The RD values are given for the complete domain, β-structural fragments, and the complete sandwich. The values of RD>0.5 are given in bold.The status of the complete domain suggests the presence of the ordered hydrophobic core in both forms of the domain; however, the status in the mutant is described with a higher value. It means that the order is lower in the domain of the mutant.The hydrophobic core in sandwich is also more ordered in the WT form than in the mutant.The interpretation of the structural stability suggests that the WT form is higher. The high order of the hydrophobicity distribution in the domain suggests a higher stability of the domain. The mutation introduces a lowering of stability, as it is understood based on the fuzzy oil drop model. The hydrophobic interaction (together with the SS-bond system) in proteins is treated as the factor of tertiary structure stabilization. This is why the higher order (lower RD value) is interpreted as the higher stabilization of the domain under consideration.The analysis of the selected β-structural fragments in WT and mutant identifies different β-structural fragments in these two structural forms.The localization of the β-strands of the status expressed by RD>0.5 in WT and mutant is shown in Figure 1.Figure 1:RD values for the complete domain (1), sandwich (12), and β-structural fragments (as given in Table 1, positions 2–11).The red line distinguished the discrimination threshold above of which the irregularity of the hydrophobic core is absent.ConclusionsThe human cardiac myosin binding protein C with an HCM-related mutation R502W is the object of the analysis. The domain under consideration is the element of muscle construction. The physiology of the muscle is to react to external forces. The external force causes the structural reorganization, including also the destruction of the low-energy structure. The second condition for the muscle to function correctly is to return to the standard structure after the external force removal. The disability to return to the initial structural form makes the muscle malfunction. The localization of the low ordered β-strand in WT (Figure 2) suggests the possible deformation as the result of the external force field acting on the N- and C-terminal residues. The extending force in WT seems to introduce disorder in the localized area (three β-strands). The mutant structure reveals other β-strands as the localization of a potential structural deformation. The mutation introduced in 2MQ3 causes the increase of the RD value, which is interpreted as the lowering of hydrophobic core stabilization. Thus, the return to the relaxed form may be of lower probability than in the WT molecule. The localization of less stable β-fragments in sandwich may introduce different local disorders in the sandwich under consideration. The presence of lower stability β-fragments may be the coded form of specific deformations (Figure 2). If the deformation is different from the one coded by evolution, the physiological functioning of the domain may cause pathological phenomena.Figure 2:3D structural form with β-structural fragments of the status RD>0.5 distinguished in red.The yellow residue: position 502 – in WT R in mutant W. Visualization program [15] (http://www.ks.uiuc.edu/Research/vmd/).Titin, the example of other muscle-related domains, was found to represent the very low RD value (RD=0.382) [8]. The interpretation of this high stability (as understood using the fuzzy oil drop model as the criteria) is also related to the physiological function as the protein undergoing the stretching external force field and possible return to initial (low-energy) structural form after the release. The role and characteristics of the protein under consideration in this paper is interpreted in a similar way as titin was presented in [16], [17].Immunoglobulin domains reveal very differentiated status with respect to the fuzzy oil drop model. The localization of β-strands of higher values of RD seems to be related to the biological function of immunoglobulin domains, which also undergo structural deformations in conditions related to antigen binding and immunological signal transduction. This very subtle differentiation of low stability among the β-strands seems to be highly related to the biological function of immunoglobulins [8].The simulation of molecular dynamics with external extended forces introduced may reveal the structural changes. The simulation of molecular dynamics with stepwise release of external force field could simulate the deformation and return to initial relaxed structural forms. The molecular dynamics simulation in conditions as described above could support or abolish the interpretation as given above. Such simulation to verify the hypothesis given above is in plans of the group presenting this paper.Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.Research funding: This work was financially supported by Jagiellonian University Medical College grant system K/ZDS/006363.Employment or leadership: None declared.Honorarium: None declared.Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.References1.Morita H, Rehm HL, Menesses A, McDonough B, Roberts AE, Kucherlapati R, et al. Shared genetic causes of cardiac hypertrophy in children and adults. 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Role of the hydrophobic core in cytoskeleton protein: cardiac myosin binding protein C

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de Gruyter
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©2017 Walter de Gruyter GmbH, Berlin/Boston
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1896-530X
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1896-530X
DOI
10.1515/bams-2017-0018
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Abstract

IntroductionCardiac myosin binding protein C (MYBPC3) is a component of the sarcomere, a functional unit of cardiomyocyte. The sarcomeres conceal the basic mechanism of active muscle contraction. Cardiac myosin binding protein C belongs to the intracellular immunoglobulin superfamily and is the part of the thick filaments. It has structural functions for the sarcomere, as it is a link between myosin thick filaments and titin super-thin filaments [1], [2], [3], [4]. Apart from the structural function, it also acts as a regulator in relation to actin F and S1 myosin subfragment. In its sequence, there are four sites that are phosphorylated by cAMP-dependent protein kinase and calmodulin-activated protein kinase. The activation of these sites leads to changes in the dynamics of myocardial contractility [5].The cardiac myosin binding protein C gene locates on the 11q13-11p13 chromosome, consists of 35 exons, and encodes a 137-kDa molecular polypeptide. The expression of the mutant protein leads to the disorganization in the muscle fibers and the development of hypertrophic cardiomyopathy (HCM) [6]. HCM is characterized by primary left ventricular thickening, which is not associated with abnormal hemodynamic cardiac stress (e.g. hypertension and aortic stenosis). This pathology is an important cause of disability and death among people of all ages but most often causes sudden cardiac death in young population under 30 years old, often athletes. It occurs with a frequency of 1 in 500 in population, which makes it one of the most common genetic diseases. Approximately 60% of the cases are reported as dominant autosomal features and result from mutations in genes encoding cardiac sarcomere proteins. Probably the most common variant is the mutation of the cardiac myosin binding protein C gene.HCM with the MYBPC3 gene is characterized by late clinical manifestation and very poor prognosis associated with sudden cardiac death in the course of cardiac arrhythmias. In the histological examination, the disorder is characterized by pathological hypertrophy (cardiomyocyte hyperplasia), interstitial fibrosis, and the thickening of the coronary arteries. The mechanism of cardiomyopathy is not fully understood. It is suggested that mutations in sarcomere proteins lead to a decrease in cardiomyocyte contractility. This in turn leads to increased cellular tension and, consequently, to the production of tropic and mitotic factors that cause hypertrophy (hyperplasia), cardiomyocyte disorder, and fibrosis. According to another hypothesis, the development of HCM occurs as a result of disturbances in the energy management of the myocardial cell. Cellular energy deprivation impairs pump activity, which is responsible for the uptake of calcium ions into the endoplasmic reticulum, and prolonged calcium retention in the cytoplasm may be a stimulus for cardiomyocyte hyperplasia. Hyperplasia, the disorganization of the muscle fibers, and interstitial fibrosis are adaptations to the inability to generate sufficient contraction power that is needed to maintain adequate cardiac output. In the case of HCM, the above-mentioned adaptive mechanisms are quite good for maintaining normal resting heart volume. On the contrary, under the influence of physical effort, symptoms of failure occur due to an abnormal ratio of cardiomyocytes (which have undergone hyperplasia) to coronary vascularization (which has not increased or even pathological changes in the coronary arteries are observed) and defect of myosin ATPase activity. Physical exercise therefore induces myocardial hypoxia events. Hypoxia and the electrical instability of the heart caused by cardiomyocyte fibrosis induce ventricular arrhythmias. They are believed to be the leading cause of death in patients with HCM [7].The protein under consideration belongs to the immunoglobulin-like superfamily representing the supersecondary structural form called sandwich. Many different proteins of different biological activities belong to this group, including structural proteins, enzymes, and obviously almost all immunoglobulin domains. The high structural similarity does not necessarily represents the similar structure of the hydrophobic core. The significant diversity is observed for immunoglobulin-like domains. The comparison of different hydrophobic core structures is shown in Ref. [8]: the whole spectrum of highly ordered hydrophobic core construction from very high ordering in titin (RD=0.382 1TIT) to very low structuralization (RD=0.722 for lyase 1CTN). The localization of less fitted β-structural fragments is also different in compared domains. Titin as the example of lowest RD for immunoglobulin-like domains seems to represent the status strongly related to biological function. Titin is the fragment of muscle cytoskeleton. These proteins are under external forces that act periodically. The main point for such protein is to return to its relaxed form after the release. The highly ordered hydrophobic core seems to be the factor ensuring the reorganization of the highly ordered hydrophobic core. The same effect is observed in distrophin, where the additional domain in the interface represents highly ordered hydrophobic core organization [9].The work shown in this paper is to check whether all immunoglobulin-like domains present in muscle cells represent a similar ordered hydrophobic core. The consequences of point mutation introduced to human cardiac myosin binding protein C are also discussed.Materials and methodsDataThe protein selected for analysis is myosin binding protein C in two forms: WT (2MQ0) and mutant R502W (2MQ3) [10]. The length of this protein is 95 amino acids. The protein represents the domain of the form of a sandwich. It belongs to immunoglobulin-like domains.Fuzzy oil drop model to characterize the structure of the hydrophobic coreThe fuzzy oil drop model assumes the structure of the hydrophobic core in protein to be in the form of 3D Gauss function. It assumes the concentration of hydrophobicity in the center of the molecule (called T). The hydrophobicity decreases according to the distance versus the center reaching the zero level on the surface. Surface is understood in the distance of 3σ for each direction. σ is the Gauss function parameter. The three-σ rule defines the size of ellipsoid. The protein molecule encapsulated in 3D Gauss function with σ parameters appropriate for protein under consideration. The values of 3D Gauss function in any point of ellipsoid expresses the theoretical idealized hydrophobicity density. The observed hydrophobicity in any point of protein is the effect of interresidual hydrophobic interaction (called O). This is why it does not necessary follow the idealized distribution. The proteins of highly similar hydrophobic distribution are found and described in many papers [8], [9], [11], [12]. The local discordance of hydrophobicity appears to be related to biological activity. The local hydrophobicity deficiency identifies often the ligand-binding cavity [11]. The local hydrophobicity excess, particularly when localized on the surface of the protein, may suggest area for protein-protein complexation [12].The Kullback-Leibler entropy is introduced to measure the differences [13]. Two reference distributions are introduced. One of them is the Gauss-like distribution representing the idealized distribution (T) and the second one is the unified distribution (called R) when each residue represents equal hydrophobicity. This distribution represents the status deprived of any form of hydrophobicity concentration. The value of DKL indicates the distance between the observed distribution and the theoretical distribution (O|T). Its value cannot be interpreted as the DKL is of the entropy category. This is why the relative distance is introduced measuring the distance versus the theoretical distribution versus the sum of distances versus the theoretical and unified distribution (O|R):RD=O|TO|T+O|R.${\rm{RD}} = {{{\rm{O}}|{\rm{T}}} \over {{\rm{O}}|{\rm{T}} + {\rm{O}}|{\rm{R}}}}.$In consequence, RD values range from 0 to 1. This is why the RD value below 0.5 indicates the status of the hydrophobic core close to the theoretical one. The RD value above 0.5 indicates the distance between the observed distribution and the unified distribution. The status of the domain (protein) described by RD>0.5 is interpreted as the molecule without the ordered hydrophobic core.The RD value may be calculated for complex, chain, or domain. For each of these units, the individual ellipsoid is calculated. The status of the selected polypeptide chain fragment can also be characterized by the RD value after the prior normalization of Ti, Oi and Ri of residues belonging to the selected fragment.The fuzzy oil drop model is described in detail in many papers [13], [14]. The description given above is necessary to understand the results discussed in this paper.Programs usedThe RD values are calculated using our own program. The 3D presentation of the protein structure is possible due to the VMD program [15] (http://www.ks.uiuc.edu/Research/vmd/), which allows the visualization of structures.ResultsThe parameters describing the status of the hydrophobic core in proteins under consideration are given in Table 1.Table 1:Parameters describing the status of myosin binding protein C in two forms: WT (2MQ0) and mutant R502W (2MQ3).SecondaryFragmentRD2MQ02MQ3Complete1449–5430.4490.482β2465–4670.3220.6063539–5410.2160.1514500–5060.3550.4555508–5150.3010.3346471–4730.3180.3027475–4770.3558532–5380.3310.4719523–5290.6500.49110485–4890.5820.67111491–4930.7150.584Sandwich120.4730.491The RD values are given for the complete domain, β-structural fragments, and the complete sandwich. The values of RD>0.5 are given in bold.The status of the complete domain suggests the presence of the ordered hydrophobic core in both forms of the domain; however, the status in the mutant is described with a higher value. It means that the order is lower in the domain of the mutant.The hydrophobic core in sandwich is also more ordered in the WT form than in the mutant.The interpretation of the structural stability suggests that the WT form is higher. The high order of the hydrophobicity distribution in the domain suggests a higher stability of the domain. The mutation introduces a lowering of stability, as it is understood based on the fuzzy oil drop model. The hydrophobic interaction (together with the SS-bond system) in proteins is treated as the factor of tertiary structure stabilization. This is why the higher order (lower RD value) is interpreted as the higher stabilization of the domain under consideration.The analysis of the selected β-structural fragments in WT and mutant identifies different β-structural fragments in these two structural forms.The localization of the β-strands of the status expressed by RD>0.5 in WT and mutant is shown in Figure 1.Figure 1:RD values for the complete domain (1), sandwich (12), and β-structural fragments (as given in Table 1, positions 2–11).The red line distinguished the discrimination threshold above of which the irregularity of the hydrophobic core is absent.ConclusionsThe human cardiac myosin binding protein C with an HCM-related mutation R502W is the object of the analysis. The domain under consideration is the element of muscle construction. The physiology of the muscle is to react to external forces. The external force causes the structural reorganization, including also the destruction of the low-energy structure. The second condition for the muscle to function correctly is to return to the standard structure after the external force removal. The disability to return to the initial structural form makes the muscle malfunction. The localization of the low ordered β-strand in WT (Figure 2) suggests the possible deformation as the result of the external force field acting on the N- and C-terminal residues. The extending force in WT seems to introduce disorder in the localized area (three β-strands). The mutant structure reveals other β-strands as the localization of a potential structural deformation. The mutation introduced in 2MQ3 causes the increase of the RD value, which is interpreted as the lowering of hydrophobic core stabilization. Thus, the return to the relaxed form may be of lower probability than in the WT molecule. The localization of less stable β-fragments in sandwich may introduce different local disorders in the sandwich under consideration. The presence of lower stability β-fragments may be the coded form of specific deformations (Figure 2). If the deformation is different from the one coded by evolution, the physiological functioning of the domain may cause pathological phenomena.Figure 2:3D structural form with β-structural fragments of the status RD>0.5 distinguished in red.The yellow residue: position 502 – in WT R in mutant W. Visualization program [15] (http://www.ks.uiuc.edu/Research/vmd/).Titin, the example of other muscle-related domains, was found to represent the very low RD value (RD=0.382) [8]. The interpretation of this high stability (as understood using the fuzzy oil drop model as the criteria) is also related to the physiological function as the protein undergoing the stretching external force field and possible return to initial (low-energy) structural form after the release. The role and characteristics of the protein under consideration in this paper is interpreted in a similar way as titin was presented in [16], [17].Immunoglobulin domains reveal very differentiated status with respect to the fuzzy oil drop model. The localization of β-strands of higher values of RD seems to be related to the biological function of immunoglobulin domains, which also undergo structural deformations in conditions related to antigen binding and immunological signal transduction. This very subtle differentiation of low stability among the β-strands seems to be highly related to the biological function of immunoglobulins [8].The simulation of molecular dynamics with external extended forces introduced may reveal the structural changes. The simulation of molecular dynamics with stepwise release of external force field could simulate the deformation and return to initial relaxed structural forms. 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Bio-Algorithms and Med-Systemsde Gruyter

Published: Sep 26, 2017

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