http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#Head http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 http://www.nanopub.org/nschema#hasAssertion http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#assertion http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 http://www.nanopub.org/nschema#hasProvenance http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#provenance http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 http://www.nanopub.org/nschema#hasPublicationInfo http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#pubinfo http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 http://www.w3.org/1999/02/22-rdf-syntax-ns#type http://www.nanopub.org/nschema#Nanopublication http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#assertion https://w3id.org/aopdo/BiologicalContext https://w3id.org/aopdo/hasBiologicalOrganizationLevel Cell https://w3id.org/aopdo/BiologicalContext https://w3id.org/aopdo/hasOrganTerm nervous system https://w3id.org/aopdo/Components https://w3id.org/aopdo/hasAction Decreased https://w3id.org/aopdo/Components https://w3id.org/aopdo/hasObject acetylcholinesterase https://w3id.org/aopdo/Components https://w3id.org/aopdo/hasProcess acetylcholinesterase activity https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasAOPName AChE Inhibition Leading to Neurodegeneration https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasAOPName AChE inhibition - acute mortality via predation https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasAOPName AChE inhibition - acute mortality https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasAOPName Inhibition of acetylcholinesterase (AChE), arrhythmias https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasAOPName Organo-Phosphate Chemicals leading to impaired cognitive function https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasAOPName Organo-Phosphate Chemicals leading to sensory axonal peripheral neuropathy and mortality https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasAOPName elavl3, sox10, mbp induced neuronal effects https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasAuthorStatus Under Development: Contributions and Comments Welcome https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasAuthorStatus Under development: Not open for comment. Do not cite https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasOECDStatus Under Development https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasPointOfContact Dan Villeneuve https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasPointOfContact Donggon Yoo https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasPointOfContact Karen Watanabe https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasPointOfContact Kristie Sullivan https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasPointOfContact SAROJ AMAR https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasPointOfContact Young Jun Kim https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasRoleInAOP KeyEvent https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasRoleInAOP MolecularInitiatingEvent https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/hasRoleInAOP MolecularInitiatingEvent https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasApplicabilityDomain AChE is present in all life stages of both vertebrate and invertebrate species (Lu et al 2012). Acetylcholinesterase associated with cholinergic responses in most insects is coded by the ace1 gene and in vertebrates by the ace gene (Lu et al 2012; Taylor 2011. Plants have AChE but it is most likely involved in regulation of membrane permeability and the ability of a leaf to unroll (Tretyn and Kendrick 1991). The primary amino acid sequence of the AChE enzyme is relatively well conserved across vertebrate and invertebrate species, suggesting that chemicals are likely to interact with the enzyme in a similar manner across a wide range of animals. From the sequence similarity analyses, the taxonomic domain of applicability of this MIE likely includes species belonging to many lineages, including branchiopoda (crustaceans, e.g., daphnids), insecta (insects), arachnida (arachnids, e.g., spiders, ticks, scorpions), cephalopoda (molluscans, e.g., octopods, squids), lepidosauria (reptiles, e.g., snakes, lizards), chondrichthyes (cartilaginous fishes, e.g., sharks), amphibia (amphibians), mammalian (mammals), aves (birds), actinopterygii (bony fish), ascidiacea (sac-like marine invertebrates), trematoda (platyhelminthes, e.g., flatworms), and gastropoda (gastropods, e.g., snails and slugs) Species within these taxonomic lineages and others are predicted to be intrinsically susceptible to chemicals that target functional orthologs of the daphnid AChE (Russom, 2014). Advanced computational approaches such as crystal structures of the enzyme and transcriptomics have provided empirical evidence of the enzyme structure, relevant binding sites, and function across species (Lushington et al., 2006; Lu et al., 2012; Wallace 1992). Studies have found that AChE activity increases as the organism develops. Prakesh and Kaur 1982 looked at AChE inhibition across three insect species; controls and those exposed to DDVP. They saw little difference in the larval stages but did see increased inhibition in pupal and adult stages (greatest inhibition). Karanth and Pope 2003 looked at AChE and acetylcholine synthesis in rat striatum in controls and animals exposed to 0.3 and 1 times the maximum tolerated dose. Although these doses are below the lethal concentrations and they mention that not observed cholinergic responses were observed, they do provide differences related to life stages of the rodents. Grue et al 1981 present baseline (no toxicity exposure) in wild starlings (both sexes) of brain cholinesterase and found activity increased as birds aged from 1-20 days until it reached a steady state at adulthood. A study with Red Flour Beetle found that the gene associated with cholinergic functions (Ace1) was expressed at all life-stages, with increases as the organism developed from egg to larva to pupa to adult. (Lu et al., 2012 cited in Russom et al 2014.) In mammals and birds, studies have determined that skeletal muscles of immature birds and mammals contain both butyrylcholinesterase and AChE, with butyrylcholinesterase decreasing and AChE increasing as the animal develops (Tsim et al. 1988; Berman et al, 1987). Another study found that changes in AChE within the developing pig brain were dependent on the area of the brain, and life stage of the animal, with significant decreases in activity within the pons and hippocampus from birth to 36 months, and no significant change in activity in the cerebellum, where activity increased up to four months of age, leveling off thereafter (Adejumo and Egbunike, 2004). https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasBiologicalContext https://w3id.org/aopdo/BiologicalContext https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasComponents https://w3id.org/aopdo/Components https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasDescription Acetylcholinesterase is found primarily in blood, brain, and muscle, and regulates the level of the neurotransmitter ACh [acetylcholine] at cholinergic synapses of muscarinic and nicotinic receptors. Acetylcholinesterase features an anionic site (glutamate residue), and an esteratic site (serine hydroxyl group) (Wilson, 2010; Soreq, 2001). In response to a stimulus, ACh is released into the synaptic cleft and binds to the receptor protein, resulting in changes to the flow of ions across the cell, thereby signaling nerve and muscle activity. The signal is stopped when the amine of ACh binds at the anionic site of AChE, and aligns the ester of ACh to the serine hydroxyl group of the enzyme. Acetylcholine is subsequently hydrolyzed, resulting in a covalent bond with the serine hydroxyl group and the subsequent release of choline, followed by a rapid hydrolysis of the enzyme to form free AChE and acetic acid (Wilson, 2010; Soreq, 2001)." [From Russom et al. 2014. Environ. Toxicol. Chem. 33: 2157-2169] Molecular target gene symbol: ACHE KEGG enzyme: EC 3.1.1.7 https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasFormData { "ID": "12", "objectID": "7fea0e6e-7938-4890-b279-71e95ad85e1e", "Components": [ { "Process": "acetylcholinesterase activity\t", "Object": "acetylcholinesterase\t", "Action": "Decreased" } ], "Overview": { "IncludedInAOP": [ { "AOPName": "AChE inhibition - acute mortality", "RoleInAOP": "MolecularInitiatingEvent\t", "PointOfContact": "Dan Villeneuve", "AuthorStatus": "Under Development: Contributions and Comments Welcome", "OECDStatus": "Under Development" }, { "AOPName": "AChE Inhibition Leading to Neurodegeneration", "RoleInAOP": "MolecularInitiatingEvent\t", "PointOfContact": "Karen Watanabe", "AuthorStatus": "Under development: Not open for comment. Do not cite" }, { "AOPName": "AChE inhibition - acute mortality via predation", "RoleInAOP": "MolecularInitiatingEvent\t", "PointOfContact": "Kristie Sullivan", "AuthorStatus": "Under development: Not open for comment. Do not cite" }, { "AOPName": "Organo-Phosphate Chemicals leading to impaired cognitive function", "RoleInAOP": "MolecularInitiatingEvent\t", "PointOfContact": "SAROJ AMAR", "AuthorStatus": "Under development: Not open for comment. Do not cite" }, { "AOPName": "Organo-Phosphate Chemicals leading to sensory axonal peripheral neuropathy and mortality", "RoleInAOP": "MolecularInitiatingEvent", "PointOfContact": "SAROJ AMAR", "AuthorStatus": "Under development: Not open for comment. Do not cite" }, { "AOPName": "elavl3, sox10, mbp induced neuronal effects", "RoleInAOP": "KeyEvent", "PointOfContact": "Donggon Yoo " }, { "AOPName": "Inhibition of acetylcholinesterase (AChE), arrhythmias", "RoleInAOP": "MolecularInitiatingEvent\t", "PointOfContact": "Young Jun Kim", "AuthorStatus": "Under development: Not open for comment. Do not cite" } ], "TaxonomicApplicability": [], "LifeStages": [ { "LifeStage": "All life stages\t", "LifeStagesEvidence": "High" } ], "SexApplicability": [ { "SexApplicabilityEvidence": "High", "Sex": "Female" } ] }, "References": [ { "Title": "Assay methods for cholinesterases", "DOI": "https://doi.org/10.1002/9780470110218.ch1" } ], "Title": "Acetylcholinesterase (AchE) Inhibition", "ShortName": "AchE Inhibition", "BiologicalContext": { "BiologicalOrganizationLevel": "Cell", "OrganTerm": "nervous system" }, "Description": "Acetylcholinesterase is found primarily in blood, brain, and muscle, and regulates the level of the neurotransmitter ACh [acetylcholine] at cholinergic synapses of muscarinic and nicotinic receptors. Acetylcholinesterase features an anionic site (glutamate residue), and an esteratic site (serine hydroxyl group) (Wilson, 2010; Soreq, 2001). In response to a stimulus, ACh is released into the synaptic cleft and binds to the receptor protein, resulting in changes to the flow of ions across the cell, thereby signaling nerve and muscle activity. The signal is stopped when the amine of ACh binds at the anionic site of AChE, and aligns the ester of ACh to the serine hydroxyl group of the enzyme. Acetylcholine is subsequently hydrolyzed, resulting in a covalent bond with the serine hydroxyl group and the subsequent release of choline, followed by a rapid hydrolysis of the enzyme to form free AChE and acetic acid (Wilson, 2010; Soreq, 2001).\" [From Russom et al. 2014. Environ. Toxicol. Chem. 33: 2157-2169]\n\nMolecular target gene symbol: ACHE\n\nKEGG enzyme: EC 3.1.1.7", "MeasurementAndDetectionTechniques": "Direct measures of AChE activity levels can be made using the modified Ellman method, although selective inhibitors that remove other cholinesterases not directly related to cholinergic responses (e.g., butyrylcholinesterase) are required [45,46].\nRadiometric methods have been identified as better for measuring inhibition because of carbamylation (carbamate exposure) [20,46,47].\nTOXCAST: NVS_ENZ_hAChE\nA direct measure of cholinesterase activity levels can be made within the relevant tissues after in vivo exposure, specifically the brain as well as red blood cells in mammals. Some analytical methods used to measure cholinesterase activity may not distinguish between butyrylcholinesterase, which is found with AChE in plasma and some skeletal and muscle tissues. Although the structure of butyrylcholinesterase is very similar to AChE, its biological function is not clear, and its activity is not associated with cholinergic response covered under this AOP (Lushington et al., 2006). Therefore experimental procedures used to measure cholinesterase as well as the tissue analyzed should be considered when evaluating studies reporting AChE inhibition (Wilson 2010; Wilson and Henderson 2007). For measuring AChE levels, the Ellman method is recommended with some modifications (Ellman et al., 1961; Wilson et al., 1996) while radiometric methods have been identified as better for measuring inhibition due to carbamylation (carbamate exposure) (see Wilson 2010; Wilson et al., 1996; Johnson and Russell 1975).\nIn order to effectively bind to the AChE enzyme, thion forms of OPs (i.e., RO)3P=S) must first undergo a metabolic activation via mixed function oxidases to yield the active, oxon form (Fukuto 1990). Estimating the potential toxicity in whole organisms based on in vitro data may be problematic since metabolic activation may be required (e.g., phosphorothionates) and may not be reflected in the in vitro test result (Guo et al. 2006; Lushington et al. 2006).\nTypically, carbamates do not require metabolic activation in order to bind to the enzyme, although some procarbamates (e.g., carbosulfan) have been developed that are not direct inhibitors of AChE, but take advantage of metabolic distinctions between taxa, resulting in a toxic form in invertebrates (e.g., carbofuran) but not vertebrate species (Stenersen 2004). Therefore in vitro assays measuring AChE inhibition for procarbamates in invertebrate species will not account for metabolic activation and therefore may not represent the actual enzyme activity.", "ApplicabilityDomain": "AChE is present in all life stages of both vertebrate and invertebrate species (Lu et al 2012).\n\nAcetylcholinesterase associated with cholinergic responses in most insects is coded by the ace1 gene and in vertebrates by the ace gene (Lu et al 2012; Taylor 2011.\n\nPlants have AChE but it is most likely involved in regulation of membrane permeability and the ability of a leaf to unroll (Tretyn and Kendrick 1991).\n\nThe primary amino acid sequence of the AChE enzyme is relatively well conserved across vertebrate and invertebrate species, suggesting that chemicals are likely to interact with the enzyme in a similar manner across a wide range of animals. From the sequence similarity analyses, the taxonomic domain of applicability of this MIE likely includes species belonging to many lineages, including branchiopoda (crustaceans, e.g., daphnids), insecta (insects), arachnida (arachnids, e.g., spiders, ticks, scorpions), cephalopoda (molluscans, e.g., octopods, squids), lepidosauria (reptiles, e.g., snakes, lizards), chondrichthyes (cartilaginous fishes, e.g., sharks), amphibia (amphibians), mammalian (mammals), aves (birds), actinopterygii (bony fish), ascidiacea (sac-like marine invertebrates), trematoda (platyhelminthes, e.g., flatworms), and gastropoda (gastropods, e.g., snails and slugs) Species within these taxonomic lineages and others are predicted to be intrinsically susceptible to chemicals that target functional orthologs of the daphnid AChE (Russom, 2014).\n\nAdvanced computational approaches such as crystal structures of the enzyme and transcriptomics have provided empirical evidence of the enzyme structure, relevant binding sites, and function across species (Lushington et al., 2006; Lu et al., 2012; Wallace 1992).\n\nStudies have found that AChE activity increases as the organism develops.\n\nPrakesh and Kaur 1982 looked at AChE inhibition across three insect species; controls and those exposed to DDVP. They saw little difference in the larval stages but did see increased inhibition in pupal and adult stages (greatest inhibition). \n\nKaranth and Pope 2003 looked at AChE and acetylcholine synthesis in rat striatum in controls and animals exposed to 0.3 and 1 times the maximum tolerated dose. Although these doses are below the lethal concentrations and they mention that not observed cholinergic responses were observed, they do provide differences related to life stages of the rodents. \n\nGrue et al 1981 present baseline (no toxicity exposure) in wild starlings (both sexes) of brain cholinesterase and found activity increased as birds aged from 1-20 days until it reached a steady state at adulthood.\n\nA study with Red Flour Beetle found that the gene associated with cholinergic functions (Ace1) was expressed at all life-stages, with increases as the organism developed from egg to larva to pupa to adult. (Lu et al., 2012 cited in Russom et al 2014.)\n\nIn mammals and birds, studies have determined that skeletal muscles of immature birds and mammals contain both butyrylcholinesterase and AChE, with butyrylcholinesterase decreasing and AChE increasing as the animal develops (Tsim et al. 1988; Berman et al, 1987). \n\nAnother study found that changes in AChE within the developing pig brain were dependent on the area of the brain, and life stage of the animal, with significant decreases in activity within the pons and hippocampus from birth to 36 months, and no significant change in activity in the cerebellum, where activity increased up to four months of age, leveling off thereafter (Adejumo and Egbunike, 2004)." } https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasID 12 https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasMeasurementAndDetectionTechniques Direct measures of AChE activity levels can be made using the modified Ellman method, although selective inhibitors that remove other cholinesterases not directly related to cholinergic responses (e.g., butyrylcholinesterase) are required [45,46]. Radiometric methods have been identified as better for measuring inhibition because of carbamylation (carbamate exposure) [20,46,47]. TOXCAST: NVS_ENZ_hAChE A direct measure of cholinesterase activity levels can be made within the relevant tissues after in vivo exposure, specifically the brain as well as red blood cells in mammals. Some analytical methods used to measure cholinesterase activity may not distinguish between butyrylcholinesterase, which is found with AChE in plasma and some skeletal and muscle tissues. Although the structure of butyrylcholinesterase is very similar to AChE, its biological function is not clear, and its activity is not associated with cholinergic response covered under this AOP (Lushington et al., 2006). Therefore experimental procedures used to measure cholinesterase as well as the tissue analyzed should be considered when evaluating studies reporting AChE inhibition (Wilson 2010; Wilson and Henderson 2007). For measuring AChE levels, the Ellman method is recommended with some modifications (Ellman et al., 1961; Wilson et al., 1996) while radiometric methods have been identified as better for measuring inhibition due to carbamylation (carbamate exposure) (see Wilson 2010; Wilson et al., 1996; Johnson and Russell 1975). In order to effectively bind to the AChE enzyme, thion forms of OPs (i.e., RO)3P=S) must first undergo a metabolic activation via mixed function oxidases to yield the active, oxon form (Fukuto 1990). Estimating the potential toxicity in whole organisms based on in vitro data may be problematic since metabolic activation may be required (e.g., phosphorothionates) and may not be reflected in the in vitro test result (Guo et al. 2006; Lushington et al. 2006). Typically, carbamates do not require metabolic activation in order to bind to the enzyme, although some procarbamates (e.g., carbosulfan) have been developed that are not direct inhibitors of AChE, but take advantage of metabolic distinctions between taxa, resulting in a toxic form in invertebrates (e.g., carbofuran) but not vertebrate species (Stenersen 2004). Therefore in vitro assays measuring AChE inhibition for procarbamates in invertebrate species will not account for metabolic activation and therefore may not represent the actual enzyme activity. https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasOverview https://w3id.org/aopdo/Overview https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasReferences https://w3id.org/aopdo/References https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasShortName AchE Inhibition https://w3id.org/aopdo/KeyEvent https://w3id.org/aopdo/hasTitle Acetylcholinesterase (AchE) Inhibition https://w3id.org/aopdo/LifeStages https://w3id.org/aopdo/hasLifeStage All life stages https://w3id.org/aopdo/LifeStages https://w3id.org/aopdo/hasLifeStagesEvidence High https://w3id.org/aopdo/Overview https://w3id.org/aopdo/hasIncludedInAOP https://w3id.org/aopdo/IncludedInAOP https://w3id.org/aopdo/Overview https://w3id.org/aopdo/hasLifeStages https://w3id.org/aopdo/LifeStages https://w3id.org/aopdo/Overview https://w3id.org/aopdo/hasSexApplicability https://w3id.org/aopdo/SexApplicability https://w3id.org/aopdo/References https://w3id.org/aopdo/hasDOI https://doi.org/10.1002/9780470110218.ch1 https://w3id.org/aopdo/References https://w3id.org/aopdo/hasTitle Assay methods for cholinesterases https://w3id.org/aopdo/SexApplicability https://w3id.org/aopdo/hasSex Female https://w3id.org/aopdo/SexApplicability https://w3id.org/aopdo/hasSexApplicabilityEvidence High http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#provenance http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#activity http://www.w3.org/1999/02/22-rdf-syntax-ns#type https://aopsketchpad.com/supervisedActivity http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#activity http://www.w3.org/ns/prov#wasAssociatedWith https://aopsketchpad.com/ http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#assertion http://www.w3.org/ns/prov#generatedAtTime 2024-12-07T13:05:28.670760 https://aopsketchpad.com/ http://www.w3.org/1999/02/22-rdf-syntax-ns#type http://www.w3.org/ns/prov#SoftwareAgent https://aopsketchpad.com/ http://www.w3.org/ns/prov#actedOnBehalfOf https://orcid.org/0000-0003-0593-2598 https://orcid.org/0000-0003-0593-2598 http://www.w3.org/ns/prov#wasAttributedTo https://orcid.org/0000-0003-0593-2598 https://orcid.org/0000-0003-0593-2598 http://xmlns.com/foaf/0.1/name Saurav Kumar http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#pubinfo http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#sig http://purl.org/nanopub/x/hasAlgorithm RSA http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#sig http://purl.org/nanopub/x/hasPublicKey MIIBIjANBgkqhkiG9w0BAQEFAAOCAQ8AMIIBCgKCAQEAkNDnQ1etSGFS/NJHpdsE HC3ePO0broUEus7O8b2ZMg7u91UyKV/BZwMskm1apekS9cPXP3BNApdt0pTkQJA5 QMCmimgqBQ8Gtk4b5mHwvSgwsXOrepT/J2u3Qhvs4pHHytkA4yeSAZd5eIYUWixK f90HICEb2LG3zbbLFqqfNohNjTvQ+1RbJ2Y3AnzNgiGbjvLmugwyQdpV/jE+D/Rq K+1Hhb3b8p8qkpHDBvDAJMydzl5tnPk+0jfMsLAPLOeaTKbI0N02ljjStcsCjocM oJEeKjl5dJMNy7YRsT+xWTsfDa7ak2LaOky+CJvr7XpN08x4mTE/IL5wwfJUrHxK WQIDAQAB http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#sig http://purl.org/nanopub/x/hasSignature jxIAZAQHK/jFZlXM6CRALaZFBqFFMEExYb593ffewXp3dscgGDpW45zLoovU5OQWlSSIedfcnDilp3dKx9a7Lu+b1BrottZaOH8t2bANEyzrMZDIIVih6UEHdNmZCWxlQf7uSzZJOYRhO9cKOKySYqKhV2dp9DMNe4XvU6gTIXUy6rA6KJNwpLV/vX863eMnGdrIAPW2/JrsPKwlH1bDjERRqkGEolPMnFB3FOevyz5C6ExOUnLWgFPFnWZ6897BsLBCCZGwRvQt4SJbG+pE424P5lM5VSh4E54YPrrhFtdET3hexSNDjXNNv5SYIGZkZYzGidBpA6rOII0GHewDGw== http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8#sig http://purl.org/nanopub/x/hasSignatureTarget http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 http://purl.org/dc/terms/creator https://aopsketchpad.com http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 http://purl.org/dc/terms/dateSubmitted 2024-12-07 http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 http://purl.org/dc/terms/license https://creativecommons.org/licenses/by/4.0/ http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 http://www.w3.org/ns/prov#wasAttributedTo https://orcid.org/0000-0003-0593-2598 http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 https://w3id.org/np/o/ntemplate/wasCreatedFromProvenanceTemplate https://w3id.org/np/RAp_-kdLEx25ZkR8QSG2MZpV5ajv8W2xM0TLoD7Wc76gg http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 https://w3id.org/np/o/ntemplate/wasCreatedFromPubinfoTemplate https://w3id.org/np/RAp_-kdLEx25ZkR8QSG2MZpV5ajv8W2xM0TLoD7Wc76gg http://purl.org/np/RA6gME7jsrG9K31SYj1OJoce9nOB9ZgS3HJlNgfUzkSv8 https://w3id.org/np/o/ntemplate/wasCreatedFromTemplate https://w3id.org/np/RAp_-kdLEx25ZkR8QSG2MZpV5ajv8W2xM0TLoD7Wc76gg