Metabolomic analysis with 1H-NMR for non-invasive diagnosis of hepatic fibrosis degree in patients with chronic hepatitis C

ContentslistsavailableatScienceDirect

Digestive

and

Liver

Disease

j o u r n a l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / d l d

Liver,

Pancreas

and

Biliary

Tract

Metabolomic

analysis

with

1

H-NMR

for

non-invasive

diagnosis

of

hepatic

fibrosis

degree

in

patients

with

chronic

hepatitis

C

Tommaso

Gabbani

_,

_Maria

_Marsico

_,

_Patrizia

_Bernini

_,

_Elisabetta

_Lorefice

_,

Cecilia

Grappone

_,

_Maria

_Rosa

_Biagini

_,

_Stefano

_Milani

_,

_Vito

_Annese

a_{GastroenterologyUnit,Morgagni-PierantoniHospital,AUSLRomagna,Forlì,Italy} b_{GastroenterologyUnit,ModenaUniversityHospital,Modena,Italy}

c_{CERM,ChemistryDepartment,FlorenceUniversity,SestoFiorentino,Italy} d_{GastroenterologyUO,AziendaUSL11,Empoli,Italy}

e_{GastroenterologySOD,AOUCareggiUniversityHospital,Florence,Italy} f_{GastroenterologyDepartment,ValiantClinic,Dubai,UAE}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10January2017

Receivedinrevisedform18May2017 Accepted20May2017

Availableonline13June2017 Keywords:

ChronichepatitisC Hepaticfibrosis Metabolomics

Protonnuclearmagneticresonance Spectroscopy

a

b

s

t

r

a

c

t

Background:Theassessmentoffibrosisdegreeinliverdiseasesisbasedonseveralnon-invasive

tech-niques,butnonehasbeenaccurate.

Aim:Thisstudyemployedprotonnuclearmagneticresonancespectroscopytoidentifymetabolicprofiles

inserumandurine,specificfordifferentfibrosisdegreeinchronichepatitisCpatients.

Method:71plasma,73serum,and578urinesampleswerecollected.Allsampleswereanalyzedusing

1_{H-NMRspectroscopytechniqueandthreedifferentNMRspectrawereacquiredforeachserum/plasma}

sample.Thedataanalyseswereperformedbypartialleastsquareregression,principalcomponent

anal-ysis,andMonteCarlocross-validationinasupervisedmethodology.

Results:Thecross-validationtestcorrectlyassignedeachsampletoitsspecificdonorwith98.44%accuracy

forurinesamplesand65%forserum/plasmasamples.Advancedfibrosisandcirrhosiswererecognized

with71%sensitivityforCPMGplasmaspectraand69%specificityforNOESYserumspectra.Accuracy

forNOESYserumspectrawas68%.Noesyspectrarecognizedadvancedfibrosisandcirrhosiswith71%

sensitivity,30%specificity,and50%accuracyinurinesamples.

Conclusion:Metabolomicanalysisofurinespectrausing1_{H-NMRspectroscopycanrecognizeaspecific}

individualprofileinallpatientswithchronichepatitisC.However,thismethodcannotyetdifferentiate

withsufficientaccuracy,patientswithadvancedfibrosisfrompatientswithmilderdisease.

©2017EditriceGastroenterologicaItalianaS.r.l.PublishedbyElsevierLtd.Allrightsreserved.

1. Introduction

Theabilitytoverifythepossibledeteriorationofchronic hepati-tis,andthusdevelopmentofliverfibrosis,isstilllargelyrelatedto theassessmentofclinicalandinstrumentalexamswithamediocre diagnosticpowerintermsofsensitivityandspecificity.Sofar,the searchforserummarkersornon-invasiveimagingtechniques, pre-dictors ofliverfibrosis degree,hasnot ledtothedevelopment ofaccuratetests.ThemetabolomicapproachwithNuclear Mag-neticResonance(NMR)spectroscopy,capableofidentifyingthefull spectrumofmetabolitesintheurineandintheperipheralblood samplesofsubjects[1],canprovideausefulsolutiontoachievea non-invasivediagnosisoffibrosis,ormayperhapsallowthe

identi-∗ Correspondingauthorat:GastroenterologyUnit,Morgagni-PierantoniHospital, ViaCarloForlanini,34,47121Forlì,Italy.

E-mailaddress:tom.gabbani@gmail.com(T.Gabbani).

ficationofmetabolitesmarkersofhepaticfibrosisthatmaypossibly beexploitedformoresimplebiochemicaltests[2–5].Theaimof thisstudywastoidentifythemetabolicprofiles,inserumandurine samples,relatedtodifferentstagesofhepaticfibrosisinpatients withchronichepatitisCinfection(HCV)complicatedbyliver fibro-sis.

2. Materialsandmethods

Thirty-threeconsecutivepatientswithchronicHCVinfection and those undergoing different stages of hepatic fibrosis were enrolled:10patientswereaffectedbylivercirrhosis(Child-Pugh score A), 23 patients were affected by chronic active hepatitis withamild ormoderatehepaticfibrosis.Informedconsentwas obtainedfromeachpatientincludedinthestudy.Thestudy pro-tocolconformstotheethicalguidelinesofthe1975Declarationof Helsinki(6threvision,2008).Thestudywasapprovedbythe eth-icalcommitteeoftheGastroenterologyUnit.Hepaticfibrosiswas

http://dx.doi.org/10.1016/j.dld.2017.05.018

T.Gabbanietal./DigestiveandLiverDisease49(2017)1338–1344 1339

investigatedusingliverbiopsy(gradingandstaging)orafibroscan meanvalue>13.5kPaonatleast10observations.Patientswithany pathologicalconditionthatmightinfluencethemetabolicprofileof thepatients(HIVand/orHBVco-infection,pregnancyandlactation, active liver autoimmune diseases, thyroid disorders, malignant neoplasia,renal disease,diabetesmellitus,alcohol consumption >50g/dieordrugsconsumption)wereexcludedfromthestudy. Thestudywasconductedoveraperiodof3months,duringwhich patientshadtorun3samplesofserum/plasma(time0,time1and time2),distributedaccordingtoatimeintervalof30days.A num-berofurinesamples(between12and24)within3monthswere alsoobtained,withweeklyorbiweeklyfrequency.Thepatientalso hadtoprovideadetaileddietdiaryonthedaypriortoeach collec-tionofserum/plasmaorurine.Theurinesampleswerecollected incollectiontubeswithscrewcapFalcon,15ml.Theplasmawas collectedinEDTAK2tubesfor2–6ml,seruminsiliconizedtubes 2–6ml. Allurine, plasma, and serumtubes were stored at4◦C beforetransfertothelaboratory.Samplesoffreshserum-plasma werecentrifugedat3500rpmfor10minandRT31000␮laliquots ofeachplasmaandserumsamplewerepreparedincryovialsand storedat−80◦_{C(afterindicationofthecode,patientnameanddate}

ofanalysis).Thefreshurinesamplesweredividedintofive,1800_␮l aliquotsforeachsample,andthenpreparedincryovialsandstored at−80◦_{C.Eachstoredcryovialwascategorizedaccordingtoa}

spe-cificalphanumeric codingsystem.Foreach bloodsample, some biochemicalparameters weremeasuredtoidentifythepossible developmentofmetabolicabnormalitieswhichmightsignificantly altertheresultsofthespectrometerduringthestudyperiod.The serum/plasmasamplewasfirstthawedatroomtemperatureand then300␮lweretransferredto300Eppendorftubesandmixedby inversionbeforethepreparationforNMRanalysis.Eachsamplewas dilutedto1:1withsodiumphosphatebuffer(70mMNa2HPO4,20% (v/v)2H2O,0.025%(w/v)NaN3,0.8%(TSP),pH7.4)andthen180ml of this mixture wereplaced in NMR tubes of 2.50mm (Bruker BioSpinLtd.).Inturn,itwasinsertedinto5mmTeflontubing(New Era)andintoanother5-mmdiameterouterglasstube(NewEra). Thehomogeneityofthemagneticfieldwaspreviouslyevaluated. Theurinesampleswerethawedatroomtemperatureandmixed byinversion.Ofeachsample,630␮lwerecentrifugedataspeedof 14,000rpmfor5minat4◦Ctopelletthesedimentpresent. Subse-quently,540␮lofsupernatantwererecoveredandaddedto60␮lof sodiumphosphatebuffer(0.2MNa2HPO4,0.2MNaH2PO4in100% 2H2O,pH7.0),containingsodiumtrimethylsilyl[2,2,3,3-2H4] pro-pionate(TSP)10mM,toreferencetheNMRspectrumtozeroonce acquired,and30mMNaN3.450␮lofthismixturewasplacedin NMRtubesof4.25mm(BrukerBioSpinLtd.)andthenanalyzed. AllProtonNuclear MagneticResonance(1_{H-NMR)spectrawere}

acquiredusingaBrukerBioSpin600.13MHzspectrometerwitha 5mmdiametercryo-probeCPTCI1H-13C/31P-2Hwithcoilforthe gradientalongtheZaxis,automatictuning-matching(ATM)and automaticsampler.AthermocouplePT100allowsthestabilization ofthesampletemperaturewithanaccuracyof0.1K.Each mea-surementsamplesareleftforthreeminutesinsidethemagnetin ordertoallowthestabilizationofthetemperature(300.0K–310.0K fortheurineandserumandplasma).Foreachurinesample, one-dimensionalspectra(1-D)withwatersignalsuppressionusinga standard pulse sequence (NOESYpresat, Bruker), wereacquired with64scans,64.000pointsanda12.019Hzspectral width,an acquisitiontime of2.7s, arelaxation delayof4s, anda mixing timeof100ms.NOESYpresat,Carr-Purcell-Meiboom-Gill(CPMG, Bruker)spectraandmodifieddiffusionsequencesusingadiffusion timeof 120mswereacquiredforeach serumandplasma sam-ple.Inthiscase,NOESYpresatspectraconsistedof64scans,98k datapoints,aspectralwidthof18,028Hz,anacquisitiontimeof 2.7s,arelaxationdelayof4s,andamixingtimeof100ms.CPMG spectraconsistedof64scans,74kdatapoints,aspectralwidth

of12,019Hz,anacquisitiontimeof3.1s,arelaxationdelayof4s, anda mixingtimeof100ms.Inordertoclarifywhich datacan bederivedfromusingaparticularspectrumratherthananother,it shouldbestressedthatinaNOESYspectrumofserum/plasma sam-plesbothsignalsfromsmallandlargemetabolitescanbeseen;ina CPMGspectra,signalsfromlowmolecularweightmetabolitesare predominant(albuminsignalsarecutoffbyplayingonthe differ-entrelaxationtimeaswellasthoserelatedtolipoproteins,which areverysmall).Fordiffusionspectra,signalsfromlowmolecular metabolitesareeliminatedinfavoroflargemolecularmetabolites. Inthiscase,diversityofdiffusiongradientsbetweenlargeandsmall moleculesisused.

To process spectra,the FID (Free Induction Decays) of each experimenthasbeenmultipliedbyanexponentialfunctionbefore applicationoftheFouriertransform.Afterthetransformationof thespectrausingacorrectionautomaticprogramimplementedin TopSpin(version2.1,Bruker),thedistortionofthebaselinephase wascorrected.ThecalibrationwasperformedonthesignalofTMSP calibratedon0.00ppm.Arecalibrationonlactatesignalplacedat 1333ppmwasperformedforserumandplasmasamples.Thisis becausetheTMSPinbloodderivatives,inwhichalbuminispresent, tendstoshiftbecauseofalbuminbinding.Theportionofeach spec-trum,rangedbetween0.02ppmand10.00ppm,wassegmented intobucketsofwidthof0.02ppmandthecorrespondingspectral regionwasintegratedusingAMIXsoftware(BrukerBioSpin).The regionofthespectraofurinebetween6.0and4.5ppmcontaining theresidualwatersignalandthesignalforureahasbeen elimi-nated.Forserumandplasma,duetolowerefficiencyinthewater signalsuppression,aregionbetween6.0and4.2ppmwasremoved. Thetotalareaofthespectrumhasbeencalculatedontheremaining bucketsthatwerenormalizedbeforethestatisticalanalysis[6].

3. Statisticalanalysis

TheregressionPLS(PartialLeastSquares)wasappliedtothedata matrixandthecomponentsthathelptoexplain99%ofthevariance havebeensubjectedtoanadditionalCA(CanonicalAnalysis),to furtherreducethenumberofvariables.

Thistypeofstatisticalmodelresultsfrominformedstatistical technique.Theinformationonmembershipwasusedonthegroup ofpatientswithhepatitisCorHCVrelatedcirrhosis.Tovalidate thismodel,adoublecross-validationschemewasused.The origi-nalmatrixofdatawasdividedintoatrainingset(90%ofsamples) andatestset(theremaining10%);whilethestatisticalmodelwas builtusingthePLS-AConthetrainingset.Samplesbelongingtothe testsethavebeenusedasSVMmethod(SupportVectorMachines). Theentireprocedurewasrepeated100times,asrequiredby cross-validationschemeofMonteCarlo.Toavoidpossiblebiasduetothe presenceinthetestsetandtrainingsetofsamplesfromthesame patient,thevalidationschemewasbuiltsothatthesetofsamples ofserum,plasmaorurine,asappropriate,ofthesamepatientwas presentineverystepofthevalidation;onlyinthetrainingsetorin thetestset,neverinboth.

Thesensitivitywascalculatedbytheaveragerecognition val-ues obtainedfromeachsamplebelongingtopatientsdiagnosedas healthy.Specificitywascalculatedbytheaveragerecognition val-ues obtainedfromeachsamplebelongingtopatientsdiagnosed assick.Theaccuracywascalculatedbyaveragingrecognition val-ues obtainedfromeachsample.Thetestperformancevalueswere representedinaconfusionmatrix.

4. Results

In order toverify theaccuracy in the identification of spe-cificmetabolicprofiles,thesystem’sabilitytoidentifyaspecific metabolicprofileforeachpatientfrommultiplesamplesofurine,

Fig.1. (A)PlotofthefirsttwocanonicalcomponentsofthePLS-CArelativetothespectrumofserumCPMG.(B)PlotofthefirsttwocanonicalcomponentsofthePLS-CA relativetothespectrumofserumNOESY.

serumandplasmawasanalyzed.Thestatisticalmodelconstructed byanalysisPLS-CAhadanexcellentclusteringofurinesamples. Onlyafewspectraweremisclassifiedbythesystem.Overall,the methodshowedanaccuracyof98.44%intheassignmentofthe indi-vidualpatienturinesamplesshowingthatwith8–20urinesamples,

itispossibletoobtainthemetabolicprofileofaspecificpatient. Thishighaccuracyimpliedthevalidationofthemethodofanalysis usedtodiscriminatethemetabolicprofileandtoassignaspecific metabolicprofiletoeachpatient.Theanalysisfortherecognition ofthepatientdescribedabovefortheurineperformedonmultiple

T.Gabbanietal./DigestiveandLiverDisease49(2017)1338–1344 1341

Fig.2.PlotofthetwocomponentsofthecanonicalPLS-CArelatingtoCPMGspectra(A)andDiffusion(B)plasma.

samplesofserumandplasma,gavelessaccurateresults.The accu-racywasinfact50%forpatientwithonlytwosamplesofserum andplasma,and65%forpatientswiththreesamplesofserumand

plasma.Whenvalidationofthemethodwasdone,thestatistical modelonsamplesofurine,serum,andplasmawasappliedto

eval-Fig.3. PlotofthefirsttwocanonicalcomponentsofthePLS-CArelativetothespectrumofplasmaNOESY.

uatethepossibilityofdistinguishingpatientswithlivercirrhosis fromthosewithonlyhepatitis.

TheanalysisofserafortheCPMGspectrumaccordingtothe diagnosisofhepatitisorcirrhosisshowedagoodclusteringofthe samplesbelongingtothetwocategoriesofpatients.Onlya lim-itednumberofoutlayerswereintheplot(Fig.1A).Theanalysis oftheNOESYspectrumshowedaloweraccuracyinclusteringthe samplesfromdifferentgroups,withasignificantlyhighernumber ofsamplesmisclassified(Fig.1B).Theanalysisofthespectrawith CPMG,Diffusion,andNOESYmethod,onplasmasamplesshowed goodclusteringofdatainthetwo groupsofpatients.However, inthis casetherewasevidenceofnumerousoutlierswith over-lappingofthetwogroupsofpatients.Thisphenomenonappeared tobelessapparentwithdiffusionanalysis,inwhichthesignalof micromoleculesisdeleted(Figs.2AandBand3).

The analysis of urine samples, where the concentration of metabolitesismuchlowercomparedtothebloodsamples,was per-formedonthefullspectrumsincethesignalsuppressionofcertain classesofmoleculesreducedtheabilityofthediscriminativetest drastically.TheanalysistechniquewithNOESYshowedan exten-siveoverlapofthespectraofthesamplesfromthetwogroupsof patients,withagoodpolarizationonbothsidesduetothelarge numberofsamples(Fig.4).Themethodofanalysisperformedby calculatingthepercentageoftimesthatthesystemcorrectly recog-nizedthesampleasbelongingtotherespectivegroupofpatients, showedratherunsatisfactoryvalues.Inall,thevaluesofrecognition forcirrhoticpatientsarequitelow,infactonlytwoof10patients hadahighdegreeofrecognition,notlessthan80%.Foratleast 4patients,ahighvariabilitywasdemonstratedonrecognitionof individualsamplecloseto90%forsomesamplesand0%forsome othersamplesobtainedfromthesamepatient.

Thedataconcerningtherecognitionofsamplesofpatientswith chronichepatitiswerebetterobtained.Therecognitionvalueper

patient wasnever below 50% and more than half of thecases showedavaluecloseto90%.ThesensitivityforbothCPMGand NOESYspectrumoftheserumwasapproximately60%.The speci-ficitywaslimitedto50%fortheCPMGspectrumand69%forthe NOESYspectrum.Theaccuracyofthetestonserumhasamounted to56%fortheCPMGspectrumand48%fortheNOESYspectrum. Theanalysisofplasmasamplesshowedsensitivityof71%anda specificityof50%fortheCPMGspectrum.TheDiffusionspectrum providedasensitivityof42%andaspecificityof67%.TheNOESY spectrumshowedsensitivityof66%andaspecificityof52%.The overallaccuracyofanalysisonplasmasamples were50%forthe CPMGspectrum,67%fortheDiffusionspectrum,and52%forNOESY spectrum.Theperformancecharacteristicsoftestonurinesamples showedasensitivityof71%andaspecificityof30%withanoverall accuracyof50%(Table1A-B-C-D).

5. Discussion

Thisstudyrepresentedthefirstapplicationofhigh-field1_HNMR

metabolomicsfordetectionofaspecificmetabolicprofile,which allowdifferentiationbetweenmild/moderatefibrosisand cirrho-sis.Thedataofthisstudysuggestedthatthemetabolomicsanalysis with1_{H-NMRspectroscopycanbeappliedtostudyviralhepatitis,}

obtainingwithaconsiderableaccuracyaurinarymetabolomic pro-filefingerprint.Asexpected,theaccuracywaslowerinserumand plasmasamplesduetofewernumbersofsamplesavailable. How-ever,thedatasuggestedthatthedistinctionbetweennon-cirrhotic hepatitisandcirrhosisisnotveryaccuratewiththissystemandthat theclusteringobservedwasduetothetendencytoclustersamples belongingtothesamepatient.Thelowestdiscriminativepowerin termsofspecificityhasbeenachievedwiththeNOESYspectrumof theurine.Thehighvariabilityofdailyurinarymetabolitesrelated todiethadanegativeimpactontheperformanceofthetest,while

T.Gabbanietal./DigestiveandLiverDisease49(2017)1338–1344 1343

Fig.4.PlotofthefirsttwocanonicalcomponentsofthePLS-CArelatingtoNOESYspectraofurine.

Table1

thespectrumoftheurineprovidedthehighestvalueofsensitivity ofthetest.Theexpectationofgreatestdispersionofdatarelated toalargenumberofurinesamplesanalyzedwasnotconfirmed; insteadthetestshowedagoodabilitytodiscriminatepatientsby analysisofurine.Thissuggestedthatitwasusefultoinvestigate theurinarymetabolitesthatallowedthisclusterization.According tothediagnosis,thediffusionspectrumofplasmasampleswith suppressionofsignalofmacromoleculesandtheNOESYspectrum ofserumprovidedbetterspecificityvaluefordistinguishingthe samples,althoughstillunsatisfactory.TheNOESYspectrum analy-sisofserumsamples,however,showedapoorsensitivitywhen compared tothespectrum oftheCPMG plasmawhich showed asensitivityvalueof71%.Thisobservationwasprobablydueto lossofdatarelatingtomicromoleculesduetothephenomenonof superpositionofthecurvescausedbytheanalysisofthecomplete spectrum.Accordingtoliterature[7,8],thestudyshowedthe fun-damentalroleofsmallmoleculesincapturingthesubtlechanges thataffectthemetabolismofdifferentsubjectsandpathological conditions.Anaccuracyof62%wasobtainedonCPMGspectraof plasma.Performanceindicesofourtestindicatedalowspecificity. Itwaspossiblethatthislimitwasinfluencedbytherelativelylow sizeinourseries.Themetabolomicexaminationperformedwith differentmethodsondifferentbiologicalsubstrateswasunableto distinguishnon-cirrhoticpatientsfromcirrhoticpatientswith suf-ficientaccuracy.Itispossiblethatthis inabilitydepends onthe limitedvariationofthemetabolicprofileassociatedwithinitial cir-rhosis,comparedtochronichepatitiswithmildfibrosis.Itiswell knownthatthesetwogroupsofpatientsdifferonlyonthebasis ofthehistopathologicalfindings,demonstratingthepresenceof regenerativenodulesintheliver,whilstotherindicesoffunction and hepatocellular damageare substantially identical. Cirrhotic patientswithadvanceddisease,inwhichthefunctionalchangesof theliveraremoremarked,wereexcluded.Thiscontributedtothe difficultytodifferentiatingthetwogroups.Thefindingsfromthe testsarequitesimilarwiththosereportedintheliteratureforother non-invasivetestsbasedondirectandindirectbiochemical param-eters,whoseabilitytodifferentiateinitialcirrhosiscomparedtoa moderateoradvancedfibrosisisverylimited[9].

Thedataareindisagreementwiththosereportedinastudyof guineapigs,inwhichhepatitisandcirrhosiswereinducedacutely (within about4 months) through theadministration of thioac-etamide [10]. In this study, the method of NMR spectroscopy appliedonsamplesofmouseplasma,showedahigh discrimina-tivepotentialbetweendifferentstagesofdisease.However,itis questionablewhetherthisexperimentalmodelofanimalcirrhosis reallyreflectswhathappensinhumandiseaseinwhichthe evo-lutiontocirrhosisdevelopsoveraperiodofdecadesandnotina periodoffewweeksafteranacuteinjury.Arecentstudyshowed thatthe1_{H-NMRmetabolomicstechniquedemonstratedexcellent}

discriminativeabilityincomparinggroupsofhealthysubjectsand patientswithcirrhosisandHCC[11].Unfortunately,theresultsof thisstudyarenotdirectlycomparablewithourviewsofthe dif-ferentcharacteristicsofthecasesconsidered.Nevertheless,they suggesttheexistenceofametabolicprofilecharacteristicof cir-rhoticpatients;while in this study,cirrhoticpatientswere not

sufficientlydifferentiatedfromthosewithhepatitis.Thisfactisnot asurpriseconsideringthatthetransitionfromchronichepatitisto cirrhosisoccursalongacontinuumofmetabolicchangesthatthey areclearlydistinguishableattheirends,butnotatallintermediate stages.

6. Conclusion

Inconclusion, thisstudydemonstratedthatthetechniqueof metabolomics analysis by 1_{H-NMR spectroscopy is a powerful}

toolfordeterminingthespecificmetabolicspectrumofasubject mainlyfromurinesamples.Themethodmanagestoidentifyan individualfingerprint,despitethedailyvariabilityof physiologi-calmetabolism.However,themethodhasnotachievedsufficient diagnosticaccuracytodifferentiatepatientswithchronicHCVfrom patientswithcompensatedcirrhosis.Itremainsanopenquestion astowhetherthisincapacityislinkedtotheobjectivedifficulty ofdifferentiatingtwodiseasestateslinkedbyaslowand continu-ousprogression,ortothelimitsofthemethodologyincapturing thesubtlevariationsofmetabolicstructureassociatedwiththese conditions.

Conflictofinterest

Nonedeclared.

Acknowledgment

We particularly thank Dr. Andrew Jamieson, MB ChB(Hons) BSc(Hons) PhD MRCP(UK) FRCPGlasg Consultant Physician and Endocrinologist,HonorarySeniorLecturerinMedicineforrevision ofEnglishstyleandgrammar.

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