Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_A.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_B.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_B.mzid.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_A.mzid.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_A.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_B.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_B.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_A.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_A.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/10/PXD005699/Sample_B.pride.mztab.gzprimaryOK200002150David.Archer@Nottingham.ac.ukJolanda van MunsterMass SpectrometryShotgun proteomicsNot availableLignocellulosePlant-polysaccharide degrading enzymesAspergillus nigerCazymeshttp://www.ebi.ac.uk/pride/archive/projects/PXD005699fungus Aspergillus niger strain AB4.119 was grown on potato dextrose agar slopes at 28°C until spores were produced. All AB4.1 cultures were supplemented with 10 mM uridine. Spores were harvested with 0.1 % (v/v) Tween 20 and liquid cultures of 100 ml aspergillus minimal medium (AMM)9 with 1 % glucose were inoculated with 106 spores ml-1 and incubated at 28°C, 150 RPM for 48 h. Mycelium was harvested using Miracloth, washed with AMM without carbon source, and 1.5 g (wet weight) was transferred to cultures of 100 ml AMM with 1 % (w/v) ball milled wheat straw and incubated for 24 h at 28°C, 150 rpm.Mycelium and culture filtrate were separated by filtration through Miracloth. proteins were precipitated by adding 5 ml trichloroacetic acid to 20 ml culture filtrate, incubating for 1 h at 4°C and spinning at 16060 g for 10 min. Pellets were washed twice with 200 µl acetone, loaded onto a 10% SDS-PAGE gel and electrophoresis performed until the dye front had moved approximately 1 cm into the separating gel. Each gel lane was then cut into a single slice, on which in-gel tryptic digestion was performed using a DigestPro automated digestion unit (Intavis Ltd). Peptides were analysed generally as described by reference with the following exceptions: an Acclaim PepMap C18 nano-trap column (Thermo Scientific) was used for injection. Peptides were resolved with a 7 segment gradient ( 1-6 % solvent B over 1 min, 6-15 % B over 58 min, 15-32 % B over 58 min, 32-40 % B over 5 min, 40-90 % B over 1 min, held at 90 % B for 6 min and then reduced to 1 % B over 1 min), Tandem mass spectra were acquired in the mass range m/z 300 to 2000, followed by MS/MS for the top twenty multiply charged ions. MS as in Matia-Gonzalez, A. M., Laing, E. E., Gerber, A. P. Conserved mRNA-binding proteomes in eukaryotic organisms. Nat Struct Mol Biol 22, 1027-1033 (2015).PrideNot availableCarbamidomethylOxidationData were processed with Proteome Discoverer software v1.4 (Thermo Scientific), searched against the A. niger SwissProt database (downloaded 23-03-16) using the SEQUEST algorithm, with a 10 ppm peptide precursor tolerance and a maximum of 1 missed cleavage, and peptide data was filtered to meet a false discovery rate of 1 % while retaining only proteins identified by ≥ 2 unique peptides.ProteomicsDavid ArcherLTQ Orbitrap VelosUniversity of Nottingham, School of Life SciencesCOMPLETEAspergillus Niger28220903 van Munster JM, Thomas B, Riese M, Davis AL, Gray CJ, Archer DB, Flitsch SL. Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger. Sci Rep. 2017 7:43117 10.1038/srep43117Jolandavanmunster@gmail.comTechnicalUniversity of Manchester10.6019/PXD005699Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.van Munster Jolanda M JM, Thomas Baptiste B, Riese Michel M, Davis Adrienne L AL, Gray Christopher J CJ, Archer David B DB, Flitsch Sabine L SL11alpha, Polypaecilum, ion, TalANAc, Solanum tuberosum subsp. tuberosum, bone marrow fibrosis, Glukose, Monohydrate, nip, SDH, (DL)-Isomer, myeloid metaplasia, Solvent, trichloro-, P270, 2-Propanone, peptide, actin-dependent regulator of chromatin subfamily F member 1, SDS, Cut, Polypeptides, BAF250a, serial element, peptido, Carbon-12, l(1)7Ba, l(1)7Bb, Dextrose, B1, SWI|SNF-related, Emericella, l(2)SH1330, Min, Injectables, present in fewer numbers in organism, WMS, DmelCG42273, 1-beta-D-ribofuranosylpyrimidine-2, Uridin, C, Divorced, CANDF6, peptides, Allo-Uridine, Rubidium, hypoplasia, min, mAPC, 35Bb, Polysorbate 20, Divorces, proteins, Myceliums, wheat, ecotype, DL-glucose, Trichloroacetic, CG11387, Tricum aestivum, common wheat, CIMF, AI047805, glucose, 13E, decreased, SOLO DANCERS, Carbon, D1, n, dye, CT, idiopathic myelofibrosis, Sodium Trichloroacetate, ML1, u, GPHYSD2, Aspergillus niger Tiegh., ELD, ATP, SGS, hOSA1, moved, Molds, Z-300, Triticum vulgare, Fungi, Ct, Osa homolog 1, Dmel_CG7826, AI836084, segment, Chaetosartorya, somatic, 2-(acetylamino)-2-deoxy-, Thermo Scientific, not genetically inherited, Mold, Fsrg1, ACMICD, myelosclerosis with myeloid metaplasia, Vitreous, RING3, l(2)br3, l35Bb, weight, Dm1, Ionen, FSRG1, 1-beta-D-ribofuranosyluracil, B120, cultivar, BcDNA:GH10590, Uridine, (alpha-D)-Isomer, Acetic acid, Dmel_CG7835, Mnb, MNB, l(2)SH2 1330., D-Glucose, Acid, potatoes, megakaryocytic myelosclerosis, Aspergillus lacticoffeatus Frisvad & Samson, protein_coding_transcript, URIDINE, 6C, OSA1, 2004, myelosclerosis, MASS, N-(3-(3-(piperidinomethyl)phenoxy)-propyl)-2-(2-hydroxyethyl-1-thio)acetamido.2-(4-hydroxybenzoyl)benzoate, 15S)-, AW124434, Polyoxyethylene (20) sorbitan monolaurate, Vitreous Carbon, Filamentous, D Glucose, Acide, (5Z, carbonium, Polypeptide, BG:DS01219.1, Glucose Monohydrate, Polypaecilum <Aspergillus>, Proteomes, Fsrg-1, Polysorbate, chronic idiopathic myelofibrosis, NAT, Nat, osteomyelofibrosis, gluco-hexose, F10B6_15, Acide trichloracetique, l(1)VE614, D17H6S113E, alpha-L-Talopyranuronic acid, Glucose, mol, Petromyces, aleukemic myelosis, FBN, Gene, alpha-L-N-acetyltalosaminuronic acid, carbon, antibiotic A 7, IB, Triticum aestivum subsp. aestivum, FSH, DmelCG4482, CG7826, F10B6.15, myelofibrosis, culture filtrate, Electrophoreses, hELD, reduced, Filtrations, Injection, subnumerary, ECTOL1, SWI-like protein, polysorbatum, Tween 20, Gene Products, CG7835, CG42273, tiny, trichloracetique, Separated, l(2)br23, polisorbato, Pilz, Filamentous Fungi, polysorbate, Urd, Agnogenic myeloid metaplasia, iones, Triticum aestivum8, ligand, Triticum aestivam, BAF250, 4733401P19Rik, messenger RNA, retruded, potato, Filamentous Fungus, Ring3, decreased number, 3H)-dione, ions, kf, OCTD, Injectable, Allo Uridine, Kohlenstoff, template RNA, bread wheat, DmelCG11387, Polyoxyethylene sorbitan monolaurate, BRG1-associated factor 250, peptidos, liquid, fungi, Aspergillus lacticoffeatus, Anhydrous, PTHB1, small, A. niger, Spore, primary myelofibrosis, DYRK1, An4, culture medium, SMARCF1, (beta-D)-Isomer, BM029, CG4482, Proteins, Fungus, l(2)35Bb, Mycota, carbone, beta-Uridine, Peptide, strain, AMM, carbono, polypeptide, ML-1, C18, A-7, AW228947, Canadian hard winter wheat, Protein, Trichloroacetate, INSDC_feature:mRNA, 4(1H, MFS1, Sodium, Rubidium Trichloroacetate, Dyrk1, mKIAA4005, myelofibrosis with myeloid metaplasia, WMS2, AU020952, MRD14, Separation, BAF250A, C1orf4, 10^[-9], D6S113E, underdeveloped, mRNA, culture supernatant, Separations, Rnf3, RNF3, uridine, CC1, Frg-1, fungus, SWDS, CG15268, CGI-97, SWI|SNF complex protein p270, Allouridine, Anhydrous Dextrose, Carbon 12, ME-IV, Protein Gene Products, incised, xnup93, Gene Proteins, matrix-associated, br3, IL-17F, ARID domain-containing protein 1A, BRG1-associated factor 250a, Ion, ATPIB, SSKS, idiopathic bone marrow fibrosis, Peptid, NIP, alpha-L-2-N-acetylamino-2-desoxytaluronic acid, Phialosimplex, GlcA. niger, Molds, Activity, Biocatalysts, carbohydrate, Pflanze, Fungi, Fungus, saccharide, glycans, hydrates de carbone, saccharides, Glykan, enzyme activity, Spectrum Analyses, Mycota, glucide, Spectrum Analysis, Mold, viridiplantae, carbohydrates, Glykane, Spectroscopy, polisacarido, Aspergillus niger Tiegh.., glucido, Mass, Analysis, glucides, Mass Spectrum Analyses, Kohlenhydrat, Mass Spectroscopy, Polysaccharide, saccharidum, Mass Spectrum Analysis, plantae, Mass Spectrum, Pilz, polisacaridos, Filamentous Fungi, Aspergillus lacticoffeatus Frisvad & Samson, Kohlenhydrate, enzymes, Analyses, Glycans, Spectrometry, Plant, Glycane, fungus, 2004, Filamentous Fungus, carbohidratos, Carbohydrate, polysaccharides, carbohidrato, Enzyme, Filamentous, Glycan, Biocatalyst, fungi, a carbohydrate, glucidos, Aspergillus lacticoffeatus, General activityApplications Software, Open Source, A. niger, data, Computer Software, False, immune system tolerance, Proteins, cleavage, Gene, mEET, Computer, precursor, Thermo Scientific, Source Softwares, Peptide, Software Tools, Tool, Programs, polypeptide, peptide, Program, Computer Applications, Polypeptides, Software Tool, Drug Tolerance, ClvPrd, peptido, Immune Tolerance, Algorithm, Computer Applications Software, NEDD4 WW domain-binding protein 3, Protein, Computer Applications Softwares, Gene Products, Immunologic Tolerance, Software Engineering, Softwares, Software, Computer Program, Application, Data Base, parent ion, Open Source Softwares, proportion, Software Applications, F, peptides, Source Software, Software Application, Open, Engineering, proportionality, Open Source Software, Computer Programs and Programming, rate, proteins, LPS-induced TNF-alpha factor homolog, Computer Software Application, LITAF-like protein, Protein Gene Products, Computer Programs, Gene Proteins, Applications, Applications Softwares, Self Tolerance, Tools, 10^[-6], precursor ion, Tolerance, ppm, quotient, Peptid, peptidos, Polypeptide, Aspergillus niger Tiegh., Peptide., drug tolerance, Estrogen-enhanced transcript protein, Computer Software Applications, Immunological Tolerance, ratiomulticellular organismal catabolic process, single-organism catabolic process, Sectors, Activity, Cyp306a1, Biocatalysts, Pflanze, carbohydrate, T3H13.3, Tertiary Sectors, STK10, FEZL, saccharide, CG6578, glycans, hydrates de carbone, Glykan, Spectrum Analyses, Triticum aestivum subsp. aestivum, glucide, exo, viridiplantae, carbohydrates, mXrn1, method, polisacarido, culture filtrate, Fodder, Dhm2, TOF, method used in an experiment, glucido, Mass, Endo, Analysis, Animal, CD105, ZNF312, Mass Spectroscopy, Polysaccharide, ASD2, T3H13_3, plantae, Mass Spectrum Analysis, Industries, Pilz, Filamentous Fungi, Kohlenhydrate, Tertiary., enzymes, Analyses, Glycans, availability, TACHD, catabolism, Triticum aestivum8, Dendo-A, Triticum aestivam, Glycane, Filamentous Fungus, wheat, free, Fast, Tricum aestivum, common wheat, AI528660, reaction, Enzyme, bread wheat, ATP:Fas-activated serine/threonine protein phosphotransferase activity, Glycan, D-endo, S-endoglin, fungi, SIMPLE, FEZ, Aspergillus lacticoffeatus, Aspergillus niger Tiegh., Cyp306a1-Dm, FAST, Feeds, TP53I7, AI662476, A. niger, culture medium, Molds, degradation, CYP306A1, Triticum vulgare, Fungi, Fungus, Fodders, saccharides, enzyme activity, FASTK, Mycota, Spectrum Analysis, Mold, Feed, Glykane, Spectroscopy, Oligosaccharide, Tertiary, DmelCG6578, Endometriosis, PIG7, Canadian hard winter wheat, D-endoA, glucides, Foods, Mass Spectrum Analyses, Kohlenhydrat, saccharidum, VSD1, Mass Spectrum, polisacaridos, Aspergillus lacticoffeatus Frisvad & Samson, breakdown, Animal Feeds, 306a1, culture supernatant, Plant, Spectrometry, fungus, 2004, carbohidratos, Carbohydrate, Glycoside, endo, CG14296, Sugar, plan specification, polysaccharides, Biotechnologies, carbohidrato, ZFP312, MALDI, Sector, Filamentous, Biocatalyst, DmelCG14296, microarray, Tertiary Sector, EXORDIUM, a carbohydrate, glucidos, General activitymulticellular organismal catabolic process, single-organism catabolic process, Sectors, Activity, Cyp306a1, Biocatalysts, Pflanze, carbohydrate, T3H13.3, Tertiary Sectors, STK10, FEZL, saccharide, CG6578, glycans, hydrates de carbone, Glykan, Spectrum Analyses, Triticum aestivum subsp. aestivum, glucide, exo, viridiplantae, carbohydrates, mXrn1, method, polisacarido, culture filtrate, Fodder, Dhm2, TOF, method used in an experiment, glucido, Mass, Endo, Analysis, Animal, CD105, ZNF312, Mass Spectroscopy, Polysaccharide, ASD2, T3H13_3, plantae, Mass Spectrum Analysis, Industries, Pilz, Filamentous Fungi, Kohlenhydrate, Tertiary., enzymes, Analyses, Glycans, availability, TACHD, catabolism, Triticum aestivum8, Dendo-A, Triticum aestivam, Glycane, Filamentous Fungus, wheat, free, Fast, Tricum aestivum, common wheat, AI528660, reaction, Enzyme, bread wheat, ATP:Fas-activated serine/threonine protein phosphotransferase activity, Glycan, D-endo, S-endoglin, fungi, SIMPLE, FEZ, Aspergillus lacticoffeatus, Aspergillus niger Tiegh., Cyp306a1-Dm, FAST, Feeds, TP53I7, AI662476, A. niger, culture medium, Molds, degradation, CYP306A1, Triticum vulgare, Fungi, Fungus, Fodders, saccharides, enzyme activity, FASTK, Mycota, Spectrum Analysis, Mold, Feed, Glykane, Spectroscopy, Oligosaccharide, Tertiary, DmelCG6578, Endometriosis, PIG7, Canadian hard winter wheat, D-endoA, glucides, Foods, Mass Spectrum Analyses, Kohlenhydrat, saccharidum, VSD1, Mass Spectrum, polisacaridos, Aspergillus lacticoffeatus Frisvad & Samson, breakdown, Animal Feeds, 306a1, culture supernatant, Plant, Spectrometry, fungus, 2004, carbohidratos, Carbohydrate, Glycoside, endo, CG14296, Sugar, plan specification, polysaccharides, Biotechnologies, carbohidrato, ZFP312, MALDI, Sector, Filamentous, Biocatalyst, DmelCG14296, microarray, Tertiary Sector, EXORDIUM, a carbohydrate, glucidos, General activityA. niger, Molds, Activity, Biocatalysts, carbohydrate, Pflanze, Fungi, Fungus, saccharide, glycans, hydrates de carbone, saccharides, Glykan, enzyme activity, Spectrum Analyses, Mycota, glucide, Spectrum Analysis, Mold, viridiplantae, carbohydrates, Glykane, Spectroscopy, polisacarido, Aspergillus niger Tiegh.., glucido, Mass, Analysis, glucides, Mass Spectrum Analyses, Kohlenhydrat, Mass Spectroscopy, Polysaccharide, saccharidum, Mass Spectrum Analysis, plantae, Mass Spectrum, Pilz, polisacaridos, Filamentous Fungi, Aspergillus lacticoffeatus Frisvad & Samson, Kohlenhydrate, enzymes, Analyses, Glycans, Spectrometry, Plant, Glycane, fungus, 2004, Filamentous Fungus, carbohidratos, Carbohydrate, polysaccharides, carbohidrato, Enzyme, Filamentous, Glycan, Biocatalyst, fungi, a carbohydrate, glucidos, Aspergillus lacticoffeatus, General activity215trueApplication of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus nigerRenewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.2018-10-262017-01-12PXD0056992806551646099180498295358511145133352442249215684342024527872842188090368093270429330845811363336351524260119834300662758385NCBITaxon:12806888856189611143011872635555711452623649346136403195206512695736664923916197467073853883641135461582158231066568895390531136364762721123294422291180992323122586365458333460792519365914320597129386558195851149315242592637371250232NCBITaxon:12721119577481866148858431043002451358672256807617554373592540816918045426683429531945305861266835201089149822505814963674452158554528113208147956410039237514636942049433208964379504208963445974137979138712728384400667203275453260293529367156759388036478434643867716052165994048321659721659545503230981315282693042165915627161696833916243218321729204277944762376454317203094980197525556978159509439843948840479NCBITaxon:2157525338551531051067439899037122857759515205968762262311210884555588845587077918178081736309431796758144571698936160488409859NCBITaxon:371621047826794015059071483643204210613204913816936303555432046632633311994565453511025411103217459216129264201288676410026420369456941016763410210761277351524867131658239254727933428146135843014172987609174633457744491675476048932128735675684030135062191726518130194111929439367110411310239320255598632582788396591047594120347495109716049016529983552527313366366817585439801102432608663102459808757417555183201964471452680457966313670190832331156114155355104219325552113834397634981812359871069955529NCBITaxon:4751850298335476113615741465477180066556484337677144137929976798251021699823897641400820840741400821137351155711089725482444474153NCBITaxon:5055789969844675251000364653493341471474935480194675129911435974127647313708632654787656589363983532215958936698381920111067757727103481985234878026555422286380966479464098609958615718717668599196337157295137221171101292921076878349741986020280950253924991488406334249234235152335985898598865591324974649987039202111072943311711393635272159874190850371170827117082841823502414511396929319021978224746360461471237472269402375939723287581722532566481219071NCBITaxon:573905079452659600131972211915853101774208588596NCBITaxon:6157505210283079895155864110519899723750599897576131720002634196102641088878176179212190659407121906772275885815689964975151950582873757244NCBITaxon:22023717NCBITaxon:170361220199985705060506172407476792745062724915961533355433355418400524643213686150596487844612286662941786661347515363291018194301086731018510184NCBITaxon:1763573039635586331492464372862876705801321650852718481865368595105135553482260710185687294296110091416333NCBITaxon:619184023210007348824123363630710470058127187878645159812894555663577553470951451225786425011449447260704NCBITaxon:13356262607052607076584574054163677483167233150561007133705654195NCBITaxon:305550445791399878168119011928752829126806315327341478281055524NCBITaxon:960613585846900811116781502283051930283672859300852900561742NCBITaxon:569382688306401794699151011849952622543525875913151590144824283811139442258441604193704759272120441437086042328167728503515819901195195326738637013702492405360883858162248839432186156227748817562615835583715333760572671193501998086119350216787910857913711371272407190650371475917262981288057125627457572709030903113335503728142433747924910078711082NCBITaxon:96157243359463318586260799141262200298147802492670NCBITaxon:1263719281249212528303856838569591169266100658137352375612750454266625515197881356638375064422336009412934979054632245552536352472140125751511519976499085151514389923745374744544641524323593760592022288705158059553655508815793271602938515781149786338922938211620894535936479437509173NCBITaxon:68544127542251177529915901090134167387158955210129661030612967158515845158493765939343271591002263767338891479235904614234512876895911983329664670414647929365761642803411602216413315992803828035369091089176881498946218121410803481793922878892950271131332952828294456861749346819112652699722732189038210760124719040072715973699782811431140206411981087135547743658142450722953379698204232124223129163218033698223470871453720916457332153581575462669404013799287770202060328114319338293550833291292211486437457252649997283035739087183087267271037908550717629970767892683704155737292140536513882941281607839563924314262654898227377561155962073220865958827355516936053307972551811354914321371597495350261159556143213840607258781414473352150949334919496340161433472800593604631646104660762903381518454744850035670974772656697719512401748418556013357555479204274158053607699468810376201488741627632972610059412003758289093164354282683142568500712303839900138946494083828351343074941442423221978542337742171598921409212205853939621157781154756929793244704749906604162103591905158114452749907312987779913112273170187546991103589915NCBITaxon:372961036098334187104911307981336795316407299467103401578658264530333733063830113064139925306423636505911696547885469811211620343176526161033728612994044586207340115357111952965261174553111471613386541313166343055213231201798360253237312761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