MicrobiomeBiomarkers-

PostMortemInterval

By

MaitriSolanki

AThesissubmittedinfulfillmentoftherequirementsforthedegreeof

MasterofForensicScience(ProfessionalPractice)

In

TheSchoolofVeterinaryandLifeSciences

MurdochUniversity

AcademicSupervisor:AssociateProfessorJamesSpeers

Semester1,2019

ii

Declaration

I declare that this thesis does not contain any material submitted previously for the

award of any other degree or diploma at any university or other tertiary institution.

Furthermore, to thebestofmyknowledge, itdoesnot containanymaterialpreviously

publishedorwrittenbyanotherindividual,exceptwhereduereferencehasbeenmadein

thetext.Finally, Ideclarethatallreportedexperimentationsperformedinthisresearch

were carried out bymyself, except that any contribution by others,withwhom I have

workedisexplicitlyacknowledged.

Signed:MaitriSolanki

iii

Acknowledgements

Firstandforemost,IwouldliketothankAssociateProfessorJamesSpeersforhissupport,

guidance, mentorship, and constructive feedback offered throughout this process. I

sincerelyappreciatethegenerositywithwhichyouhavesharedyourtime.

I would also like to thankmy family and friends for their constant support, guidance,

patience, and encouragement. Your contributions throughout this process have been

invaluable.

iv

TableofContents

TitlePage…………………………………………………………………………………………………………i

Declaration………………………………………………………………………………………………………ii

Acknowledgements………………………………………………………………………………………....iii

TableofContents……………………………………………………………………………………………..iv

PartOne

LiteratureReview…………………………………………………………………………………………….1-14

TitlePage………………………………………………………………………………………………………….i

Abstract…………………………………………………………………………………………………………….ii

TableofContents……………………………………………………………………………………………...iii

ListofAbbreviations…………………………………………………………………………………………..iv

1.Introduction…………………………………………………………………………………………………..1

2.HistoryofMicrobiomesinForensicScience……………………………………………………4

3.SoilMicrobiomes…………………………………………………………………………………………….4

4.SkinMicrobiomes……………………………………………………………………………………………4

5.MicrobiomeevidenceinCriminalJusticeSystem…………………………………………….5

6.PhysicalStagesofDecomposition……………………………………………………………………6

7.PMIestimationusingmiRNAandcircRNA……………………………………………………….8

8.PMIestimationusing18S-rRNAandmicroRNA……………………………………………..10

9.AdvancesinMicrobialForensics……………………………………………………………………..11

10.Metabolomics……………………………………………………………………………………………….11

11.Micro-RNAinForensics………………………………………………………………………………….12

v

12.ConclusionandFurtherResearch……………………………………………………………………13

References…………………………………………………………………………………………………………...14

PartTwo

Manuscript……………………………………………………………………………………………………………1-17

Titlepage…………………………………………………………………………………………………………………i

Abstract……………………………………………………………………………………………………………………ii

ListofAbbreviations…………………………………………………………………………………………………iii

1.Introduction…………………………………………………………………………………………………………..1

Discussion………………………………………………………………………………………………………………….2

2.StagesofDecomposition….…………………………………………………………………………………….6

3.EstimationofpostmortemintervalusingBiochemicalmarkers……………………………..7

4.BiochemicalmarkersofPMI……………………………………………………………………………………8

5.RoleofmicrobesinPMIdetermination…………………………………………………………………..9

6.ProfilingofRNAdegradationforestimationofPMI………………………………………………..10

7.IdentificationofMolecularBiomarkersforestimationofPMIusingbloodsamples..11

8.DeterminationofPMIintheadvancestageusingmiRNAsandcircRNAs…………………12

9.Postmortemintervaldeterminationusing18S-rRNAandmicroRNA……………………….13

10.Conclusion….…………………………………………………………………………………………………………15

11.Limitations/researchgap….…………………………………………………………………………………..16

12.References…………………………………………………………………………………………………………....17

vi

PartOne:LiteratureReview

MicrobiomeBiomarkers–PostMortemInterval

i

ABSTRACT

Microbiomeisthecatalogofmicrobesandtheirgenes.Andabiomarkerisasubstance

thatcanbeobjectivelymeasuredandwhichcanactasanindicatorofpathogenic

processes,biologicalprocessesorpharmacologicalresponsestoatherapeutic

intervention.Also,thetimebetweenphysiologicaldeathandtheexaminationofthedead

bodyisknownasthePostmortemInterval(PMI).DeterminationofPMIcanbeacomplex

problemduetoitbeinginfluencedbyanumberofintrinsicandextrinsicfactors.Someof

theintrinsicfactorsincludeage,sex,andpathologicalandphysiologicalstatesofthe

corpse.Whiletheextrinsicfactorsincludetemperature,humidityandinsectactivity.

Recently,molecularchangessuchasprotein,RNAandDNAdegradationhavebeen

studiedquietwidelyandareseemtobeproducingpromisingresultsinthefieldofPMI

estimation.Morespecifically,studyingRNAdegradationafterdeathisconsideredquiet

usefulforprecisePMIestimation.SomeofthedifferenttypesofRNAthataidinPMI

estimationincludemiRNAs,circRNAs,18S-rRNAandsoon.Thisstudyfocusesonthe

potentialforestimatingPMIusingmicrobiomebiomarkersasatoolratherthanthe

traditionPMIestimationbystudyingvariousstagesofdecomposition.

ii

TableofContents

TitlePage…………………………………………………………………………………………………………i

Declaration………………………………………………………………………………………………………ii

Acknowledgements………………………………………………………………………………………....iii

TableofContents……………………………………………………………………………………………..iv

PartOne

LiteratureReview…………………………………………………………………………………………….1-17

TitlePage………………………………………………………………………………………………………….i

Abstract……………………………………………………………………………………………………………ii

TableofContents……………………………………………………………………………………………..iii

ListofAbbreviations………………………………………………………………………………………….iv

1.Introduction………………………………………………………………………………………………….1

2.HistoryofMicrobiomesinForensicScience…………………………………………………..4

3.SoilMicrobiomes……………………………………………………………………………………………4

4.SkinMicrobiomes…………………………………………………………………………………………..4

5.MicrobiomeevidenceinCriminalJusticeSystem……………………………………………5

6.PhysicalStagesofDecomposition…………………………………………………………………..6

7.PMIestimationusingmiRNAandcircRNA………………………………………………………8

8.PMIestimationusing18S-rRNAandmicroRNA……………………………………………..10

9.AdvancesinMicrobialForensics……………………………………………………………………..11

10.Metabolomics……………………………………………………………………………………………….11

11.Micro-RNAinForensics………………………………………………………………………………….12

iii

12.ConclusionandFurtherResearch…………………………………………………………………..13

References……………………………………………………………………………………………………………14

iv

LISTOFABBREVIATIONS

ATPAdenosineTriphosphate

DNADeoxyribonucleicAcid

circRNACircularRNA

miRNAMicroRNA

PMIPostMortemInterval

RFLPRestrictionFragmentLengthPolymorphism

RNARibonucleicacid

16S-rRNA16SRibosomalRNA

18S-rRNA18SRibosomalRNA

RT-qPCRQuantitativeReverseTranscriptionPCR

v

1

1.0INTRODUCTION

Microbiome:

Microbiota,ofteninterchangeablyusedinplaceofmicrobiomereferstothemicrobial

taxaassociatedwithhuman.Microbiomeisthecatalogofthesemicrobesandtheirgenes

(1).“Metagenomics“,originallyreferredtoshotguncharacterizationoftotalDNA,

althoughnowitisincreasinglybeingappliedtostudiesofmarkergenessuchasthe16S

rRNAgene(1).

Biomarker:

AbiomarkerasdefinedbytheNationalInstituteofHealthBiomarkersDefinitions

WorkingGroupin1998isasubstancethatcanbeobjectivelymeasuredandwhichcanact

asanindicatorofpathogenicprocesses,biologicalprocessesorpharmacological

responsestoatherapeuticintervention(2).

Inotherwordsitcanalsobedescribedasasubstanceorastructureinthebodywho

itselforitsproductscanbemeasured(2).

PostMortemInterval(PMI):

Thetimebetweenphysiologicaldeathandtheexaminationofthedeadbodyisknownas

thePostmortemInterval(PMI)(3).ThethreestagesthatPMIcanbedividedintoinclude

EPMI,PMIintheadvancedstageandskeletonizedremains(4).Thetraditionalmethodsof

PMIestimationarenotconsideredtobesoaccurateduringadvancedstagesasthe

corpsewouldhaveeventuallyextensivelydestroyed(4).Alotofcaseinformationwould

2

havedestroyedatthisadvancedstageandeventhecrimescenewouldhavechangeddue

tothedelayinPMI(4).

Biologicalmarkerscannowbedetectedmorepreciselywiththeadvanceinmolecular

biologytechnologies.Astudyby(4),havediscoveredpotentialcorrelationbetweenRNA

degradationinacorpseandPMI.AgoodmethodtodetectRNAprofilesisqPCR(4).

DeterminationofPMIcanbeacomplexproblemduetoitbeinginfluencedbyanumber

ofintrinsicandextrinsicfactors(5).Someoftheintrinsicfactorsincludeage,sex,and

pathologicalandphysiologicalstatesofthecorpse.Whiletheextrinsicfactorsinclude

temperature,humidityandinsectactivity.

Inpreviousyears,PMIestimationwasdoneusingtraditionalmethodsthatconsidered

physiologicalchangessuchasalgormortis,livormortis,rigormortisandsupravital

activitywhichcouldonlyprovidearoughestimationofPMIandalsocreatechances

whereoneindicationwascontradictedbytheother(5).Becauseofthesedrawbacksthat

scientistsfaced,recently,anothermethodofPMIestimationhasbeengeneratedthat

makesuseofnucleicaciddegradation(5).

Recently,molecularchangessuchasprotein,RNAandDNAdegradationhavebeen

studiedquietwidelyandareseemtobeproducingpromisingresultsinthefieldofPMI

estimation(5).Morespecifically,studyingRNAdegradationafterdeathisconsidered

quietusefulforprecisePMIestimation.ThemRNAprofilesarewidelydetectedusing

ReverseTranscriptionreal-timequantitativepolymerasechainreaction(RT-qPCR)

becauseitbeinghighlysensitive,widelyusedanditsassay’sbeingreadilyavailable.Also,

accuratetargetgeneexpressionactivity,endogenousreferencegenesarewidelyusedas

internalcontrolswhenperformingdataanalysis(5).

3

SomeofthedifferenttypesofRNAthataidinPMIestimationincludethefollowing:

miRNAsandcircRNAs:

miRNAsbelongtotheclassofnon-codingsinglestrandedRNAmolecules.Theyare

encodedbyendogenousgenesofapproximately22nucleotidesandcomeunderthe

categoryofsatisfactorystablemoleculesthatareminimallyaffectedbyenvironmental

conditions,whichinterferewithPMIestimation(4).

CircRNAsalsofallunderthecategoryofstableRNAsduetothemhavingastable

structurethatconsistsofacovalentbondlinking3’to5’endsthroughbacksplicing(4).

CircRNAsareauniqueclassofnon-codingRNAs,whichpossesscharacteristicsofhigh

stability,abundanceandtissue-specificexpressionpatternsthatmakethemvaluablefor

theestimationofPMIinadvancedstage(4).

18S-rRNAandmicroRNA:

18S-rRNAisoneofthekeyribosomalRNAthatisapartofribosomalproteincomplex(5).

microRNAontheotherhandoneconsistsof21-25nucleotidesandisapartofaclassof

smallnon-codingsinglestrandedRNA.microRNAsarefoundtobepresentinmany

differentspecies(5).

4

2.0HistoryofMicrobesinForensicScience

Sincethelate19thcentury,Microbeshavebeenusedasevidence.Duringthisperiod,

microbiologywasusedasaforensictoolfordeterminingthepathogenicityandthe

causesofdeathinhumansandanimals(6).Alotofthisearlyworkcanbecorrelatedwith

someofthewell-knownscientistslikeLouisPasteur,RobertKochandJosephLister(6).

Alsoduringthisperiod,studieswerebeingcarriedoutbyEdmondLocardinorderto

justifythatmicrobescanalsobeusedastraceevidence.‘Everycontactleavesatrace'is

oneoftheverywellknownsayingsfromLocard(6).

3.0SoilMicrobiomesasEvidence

Initially,themethodusedbycourtsystemforcapturingmicrobialdiversitywasthrough

theuseofterminalrestrictionfragment-lengthpolymorphism.RFLPscleavesDNAby

usingrestrictionenzymesonspecificsetsofnucleotidebases(7).Microbiomesofthesoil

areagreatsourceofevidencewhenestimatingPMI,locatingclandestinegravesand

linkingobjects,humansandlocationsthroughtraceevidence(7).

4.0Skinmicrobiomesastraceevidence

Skinmicrobiomeshaveaveryuniquepropertyofbeingextremelyindividualistic(6).The

microbesfoundonthehandsoftwoindividualscandiffermorethan80percent.Skin

microbiomeservesgreatpurposeastraceevidenceduetoithavingpropertieslike

5

uniquebetweenindividuals,beingtransferredascloudsofmicrobesandremainstable

overtime(6).Itisalsoofbestuseifcollectedatthesametime(sameseasonoftheyear)

asthesurfacesample.Thepositioningoftheskinmicrobiomewithinthefrictionridges

providesahighchanceofpositiveidentificationoftheperpetrator(6).

Skinmicrobiomescanalsoserveotherimportantpurposessuchasidentificationofa

person’sgenderandlifestyle,whethertheywerelocatedi.e.whetherinanurbanorrural

settingandalsoifindividualswerecohabitating(8).Moreover,skinmicrobiomescanalso

providelotsofotherinformationaboutanindividualsuchasbeautyproductsusedby

them,theirdiseasestatus,foodconsumedbythemandactivitiestheyhavebeenpartof.

Theinformationgatheredcanbeofgreatuseinacriminalinvestigation(8).

5.0Microbiomeevidenceinthecriminaljusticesystem

Formicrobiomestobeusedasevidenceincourt,thereisaneedforittobepresentedby

eitheraninvestigatororalawyerwhocansupporttherelevanceandreliabilityofthe

microbialevidence(6).Formicrobiomeevidencetobepresentedincourt,itshouldbe

thoroughlyresearchedanderrorratesshouldbeknownthroughquantitativemachine-

learningmethods(6).

6

6.0TraditionalmethodsforestimatingPMI-thephysicalstagesofdecomposition

(Physiologicalchanges)

Theprocessofdecompositioninvolvesthedegradationofsoftandhardtissuesofa

humanbodyinbasiccomponentsthroughvariousphysiochemicalchanges.Anumberof

externalfactorssuchastemperaturecanhaveasignificantimpactontherateof

degradation(9).Thetimethatamammalianbodytakestodecomposeisusuallyconstant

overallthebodiesexceptfortheeffectthattemperaturepossessonit.Temperaturecan

vastlyaffectthetimeittakesforabodytogettoskeletonizationstagefromfreshstage

(9).

Stagesofdecompositionarecategorizedinsuchawaythathelpsinvestigatorsexplain

theprocessandestimatePMI.Theprocessofdecompositionusuallycommencesstraight

afterdeathunlesstheenvironmentalconditionsdon’tallowittoproceed(9).Thetwo

mainstagesofdecompositionarepreandpostskeletonizationwherethepre

skeletonizationstagecanbesubsequentlydividedinfivesub-stagesnamely:fresh,bloat,

activedecay,advanceddecayanddry.

FreshStage:

Autolysisandputrefactionarethetwoprocessesthatoccursimultaneouslyduring

biochemicaldecomposition.Theinitialprocessthatcanbeidentifiedduringfreshstageof

decompositionisAutolysis(alsoknownasenzymaticself-digestion).Autolysisleadsto

lossofmembranestructureinthemostmetabolicallyactivecellsofthebodybecauseof

7

thelackofenergyproductionintheformofadenosinetriphosphate(ATP)(9).

Temperaturecanaffecttheprocessofdecompositiondifferentlyatdifferentlocations.

Placeswithhighambienttemperatureacceleratesautolysis,andtheprocessisveryslow

atplaceswheretemperaturesarecooler.Attimes,inregionswithfreezing,theprocess

ofdecompositioncanalsobesuspendedduetotheinactivationoftheenzymes(9).

Anumberofprocessesareinvolvedinthereleaseofnutrient-richfluidfromthebody(9).

Theseinclude,thelooseningofepidermisfromthedermis,marblingoftheskinasa

resultofautolysisofredbloodcellsandanumberofpostmortemblisters(bullae),which

causesthecellularmembranetodissolve.Thisfluidthatisreleasedfromthebody,isa

greatenergysourceforthemicrobesaidingputrefaction(9).

Theextenttowhichfreshstageextendsisstartingfromdeathuntiltheskeletonbloatsat

thebloatingstage(10).Asaresultoftheproductionofgaseousbyproductsduetothe

microbialmetabolicactivity,thecarriongetsinflatedandattractsnecrophagousinsects

towardscarcassduringthebloatstage(10).Thenextstagefollowingbloatingistheactive

decaywhereimmenseinsectactivitycausesrapiddecomposition(10).

Theentomologicalactivityissignificantlydecreasedduringadvancedecay(10).

Thestagesofdecompositionaregenerallydefinedbythepresenceandactivityofcertain

insects,whichareusedtocalculatePMI.Hencetheprocessofdecompositionisa

continummandnotdiscreteseriesofevents(10).

8

7.0EstimationofPMIinadvancedstageusingmiRNAsandcircRNAs

ItisverychallengingtoestimateaccuratePMIafterabout24hoursofdeathi.e.inthe

advancedstage,becauseoftheinfluenceofexternalfactorsonthecorpse.

Theexternalfactorsincludetemperaturebeingthemostcommon,humidityand

microorganism(4).However,nucleicacidslikeDNAandRNAareagreatsourcefor

determinationofaccuratePMIduetothembeingleastaffectedbytheexternalfactors.

Thisisbecausethenucleicacidsareprotectedinsidethecellnucleus(4).Inorderto

preciselydeterminePMIusingRNA,itisgenerallypreferredmeasuringthepostmortem

RNAdegradationquantitativelyfollowedbybuildingmathematicalmodels.Thereason

forthisisthatRNAthatispresentintissuesdegradeswithadelayinPMI(4).

RNAprofilescanbedetectedusingvariousmethods.However,Realtimequantitative

polymerasechainreaction(qPCR)isoneofthebestonesused.anumberofmRNA

markersareusedasendogenouscontrolmarkersnamelyGapdh,Beta-actinandRps18

(4).AdrawbackforusingtheseasreferenceforquantitationofdegradedRNAsisthat

theirefficacyisreducedastheydegradeduringadvancedstageofPMI.Accordingtoso

andso,thesemarkersareonlystableduring8daysafterdeath.Asolutiontothisissueis

tousestableandconservedRNAssuchasmicroRNAs(miRNAs)andcircularRNAs

(circRNAs)asnormalreferencegenes(4).

Basedonthestudydoneby(10),concludedthathousekeepinggeneshaveaveryclose

relationshipwithestimatingPMIandhencemakesthempotentialbiomarkersforPMI

9

estimation(10).Thisissobecauseitwasobservedthathousekeepinggeneswere

observeddegradingwithPMIextensionandhencewereknowntohaveaclose

relationshipwithPMI(10).Housekeepinggenesarepresentandstableexpressedinall

thecellsofthebodyandtheirproductsplayaveryimportantroleinthecorrect

functioningofbasiccellactivities(10).

Certainreferencegenesareknowntobeoptimalinspecifictissues,forexample,miR-

122,miR-133aand18Sarethereferencegenesforhearttissues,LC-Ogdh,circ-AFF1and

miR-122areforlivertissuesandmiR-133a,circ-AFF1areforskeletalmuscletissues(4).

Furthermore,basedonthestudyresultsof(12),itwasfoundthatthetargetbiomarkers

inheartandlivertissuesareU6andRps18,whilethatinskeletalmuscletissuesareU6

andbeta-actin(12).ThisstudyalsoshowsU6,whichbelongstoasmallnuclearRNA

familytohavethehighestcorrelationwithPMIas,theU6transcriptwasnoticedtohave

beendegradedalongsidePMIextension(12).TheadvantagethatU6hasisthatitgets

protectedfromexternalfactorsthroughtheribonucleoproteins,butthiseventually

vanisheswithdelayindeathtime.WhileRps18,aribosomalproteinwithhighexpression

stabilityisobservedtobedegradingduringlatePMIandthusisconsideredappropriate

asabiomarkerintheheartandlivertissuesofacorpse.Whenconsideringtheskeletal

muscles,Beta-actinisproventobetherightbiomarkerwhenestimatingPMI(4).

10

8.0Postmortemintervaldeterminationusing18S-rRNAandmicroRNA

ResearchsuggeststhatmRNAisefficientforpostmortemanalysisevenonextreme

conditionswherethecorpseisleftatroomtemperatureforseveraldays(5).

18S-rRNAisfoundinabundanceincellsofthebody.ItisprotectedfromtheRNAenzyme

insidetheribosomalcomplex(5).Thequantityof18S-rRNAincreasesasitisreleased

fromthecomplexwiththeextensionofpostmortemintervalandthedegradationof

proteins(5).Basedontheexperimentsof(5),18S-rRNAlevelsarenotedtobegradually

increasingduringearlystages,howeverpeaksaround96hoursafterdeath(5).

Oneofthestablemarker’susedthatcanbeusedasaninternalstandardismiR-1.The

MicroRNA(miR)isahighlyconservedsmallnon-codingsinglestrandedRNAthatisonly

21-21nucleotideslong.miRiscapableofsilencingagenebybindingtoitstargetmRNA

(5).ThedynamictemporalandspatialexpressionpatternsofmicroRNAscanbedisrupted

duetodevelopmental/physiologicalabnormalities.WhencomparedwithmRNA,

microRNAisshortinlengthandconsideredtobemorestablethanmRNA(5).

-RT-qPCRinPMIestimation:

InanattempttodeterminePMI,RT-qPCRhasbeenseentohaveamajorrolewhen

consideringsensitivityandspecificity.Itishighlyrecommendedconsideringfactorssuch

astemperature,age,sex,causeofdeathandpathologicalstatewhenanalyzingRNA

levelsfromautopsysamples(5).

11

9.0AdvancesinMicrobialForensics:

Microbiomeforensicsisthefieldwheremicrobialcommunitiescanbestudiesin

unprecedenteddepthduetotheadvancesinsequencingplatformsandcomputational

pipelines(6).Ontheotherhand,microbialforensicstargetsandidentifiesindividualtaxa

ofinterestthroughtheuseofvarioustechniques.Forexample,microbialforensics

identifiesspecificstrainsofmicrobesusingfine-scalevariationwithinindividualgenomes,

whereasmicrobiomeforensicsstudiesthedifferenceincommunityofmicrobesthrough

microbialpostmortemchangesandtraceevidence(6).Themicrobiomedatacanbeused

foranumberofreasonsnamelyforcalculatingPMI,locatingclandestinegravesand

linkingobjectsorspacesbyusingskinmicrobes(6).Patternsinmicrobialcommunities

canbestudiedbyusingnext-generationsequencingofphylogeneticallyand

taxonomicallyinformativegenemarkerssuchas16SrRNA,18SrRNAandITS(6).

10.0Metabolomics:

AlongsideRNA,metabolomicsisanotheremergingfieldthathasgreatpotentialfor

determinationofPMIinvariousforensicinvestigations(13).Thisisthroughidentifying

newbiomarkersthatarerelatedtochronologicalchangesafterdeath.Thecauseofdeath

andthechangesfollowingdeathinhumanscanbedeterminedusingmetaboliteprofiling.

Ontopofjustthechemistryofthecorpse,themetabolomicspatternsofthesoilunder

thedecomposinghumancorpsecanalsobeidentified.Distinctivepatternslikefattyacid

signaturesassociatedwithPMIcanalsobederivedusingmetabolicprofilingapproach

(13).

12

11.0Micro-RNAINForensics:

Alotofpotentialforforensicapplicationshavebeenobservedthroughpromisingresults

obtainedfrommiRNAandsmallnon-codingRNAs(14).

Thereexists800-1000miRNAsinthehumangenomeoutofwhichmorethan700miRNAs

havebeenidentified.Thisforms2-3%ofallprotein-codinggenes(14).

13

12.0ConclusionandFurtherResearch:

Basedontheresearchtilldate,thefactorsaffectingRNAdegradationinadeadbody

includePMIandtemperature.Theeffectofotherminuteparameterscanbeeliminated

throughcontrollingotherexperimentalconditionsintheresearch.

PMIestimationcansometimesbetrickyduetomanyfactorsbeinginvolvedandthese

factorsimpactindifferentways.Itisdifficulttoreplicatethisinalaboratorysetting,as

realscenarioisquietdifferentandmoredifficultasmanymorefactorsareinvolved.A

standardandwell-constructedprotocolshouldbeusedforobtainingqualityresultsfrom

RT-qPCRexperiments.TheexperimentaldesignshouldincludeRNAextraction,gDNA

removal,targetgeneinformation,completereactionconditions,primerdesignand

appropriatecontrolsforqPCRassayfollowedbydataanalysis.Furtherresearchneedsto

becarriedoutusingmoretypesofRNAinanattempttofigureoutifmoreaccurate

resultsareobtained.Theexperimentsshouldbestrictlycontrolledandawell-structured

protocolshouldbeused.

14

13.0REFERENCES

1.UrsellL,MetcalfJ,ParfreyL,KnightR.Definingthehumanmicrobiome.Nutrition

Reviews.2012;70:S38-S44.DOI:10.1111/j.1753-4887.2012.00493.x

2.StrimbuK,TavelJ.Whatarebiomarkers?.CurrentOpinioninHIVandAIDS.

2010;5(6):463-466.DOI:10.1097/COH.0b013e32833ed177

3.WellsJ,LaMotteL.TheRoleofaPMI-PredictionModelinEvaluatingForensic

EntomologyExperimentalDesign,theImportanceofCovariates,andtheUtility

ofResponseVariablesforEstimatingTimeSinceDeath.Insects.2017;8(2):47.

DOI:10.3390/insects8020047

4.TuC,DuT,YeX,ShaoC,XieJ,ShenY.UsingmiRNAsandcircRNAstoestimatePMIin

advancedstage.LegalMedicine.2019;38:51-57.Availablefrom

https://doi.org/10.1016/j.legalmed.2019.04.002

5.LiW,MaK,LvY,ZhangP,PanH,ZhangHetal.Postmortemintervaldetermination

using18S-rRNAandmicroRNA.Science&Justice.2014;54(4):307-310.Available

fromhttps://doi.org/10.1016/j.scijus.2014.03.001

6.MetcalfJ,XuZ,BouslimaniA,DorresteinP,CarterD,KnightR.MicrobiomeToolsfor

ForensicScience.TrendsinBiotechnology.2017;35(9).Availablefrom

http://dx.doi.org/10.1016/j.tibtech.2017.03.006

7.Concheri,G.etal.ChemicalelementaldistributionandsoilDNAfingerprintsprovide

thecriticalevidenceinmurdercaseinvestigation.(2011).PLoSOne6,e20222

8.Meadow,J.F.etal.(2014)Bacterialcommunitiesonclassroomsurfacesvarywith

humancontact.Microbiome2,7

15

9.NolanA-N’D,MeadRJ,MakerG,SpeersSJ.Areviewofthebiochemicalproducts

producedduringmammaliandecompositionwiththepurposeofdetermining

thepost-morteminterval.AustralianJournalofForensicSciences.2019.

Availablefromhttps://doi-

org.libproxy.murdoch.edu.au/10.1080/00450618.2019.1589571

10.FinleySJ,BenbowME,JavanGT.Microbialcommunitiesassociatedwithhuman

decompositionandtheirpotentialuseaspostmortemclocks.IntJLegalMed.

2015;129:623-632.DOI10.1007/s00414-014-1059-0

11.ShiCaihua,YangFengshan,Du.ZhuXun,YangYutingErxia,WangShaoli,etal.

Evaluationofhousekeepinggenesforquantitativereal-timePCRanalysisof

Bradysiaodoriphaga(Diptera:Sciaridae).Int.J.Mol.Sci.,17(7)(2016),p.1034

12.J.E.Burke,D.G.Sashital,X.Zuo,Y.X.Wang,S.E.Butcher.StructureoftheyeastU2/U6

snRNAcomplexRNA.(2012).18(4),pp.673-683

13.Vass,A.A.etal.Timesincedeathdeterminationsofhumancadaversusingsoil

solution.J.ForensicSci.(1992).37,1236–1253

14.CourtsC,MadeaB.Micro-RNA–Apotentialforforensicscience?.ForensicScience

International.2010;203(1-3):106-111.Availablefrom

doi:10.1016/j.forsciint.2010.07.002

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17

Part2:Manuscript

MicrobiomeBiomarkers–PostMortemInterval

i

Contents

Manuscript…………………………………………………………………………………………………………….1-17

Titlepage…………………………………………………………………………………………………………………i

Abstract……………………………………………………………………………………………………………………ii

ListofAbbreviations…………………………………………………………………………………………………iii

1.Introduction…………………………………………………………………………………………………………..1

Discussion………………………………………………………………………………………………………………….2

2.StagesofDecomposition….…………………………………………………………………………………….6

3.EstimationofpostmortemintervalusingBiochemicalmarkers……………………………..7

4.BiochemicalmarkersofPMI……………………………………………………………………………………8

5.RoleofmicrobesinPMIdetermination…………………………………………………………………..9

6.ProfilingofRNAdegradationforestimationofPMI………………………………………………..10

7.IdentificationofMolecularBiomarkersforestimationofPMIusingbloodsamples..11

8.DeterminationofPMIintheadvancestageusingmiRNAsandcircRNAs…………………12

9.Postmortemintervaldeterminationusing18S-rRNAandmicroRNA………………………..13

10.Conclusion….………………………………………………………………………………………………………….15

11.Limitations/researchgap….…………………………………………………………………………………….16

12.References………………………………………………………………………………………………………………17

ii

Abstract:

PostMortemIntervalalsoknownasPMIreferstothetimebetweenphysiologicaldeath

andtheexaminationofthebody.PMIestimationcanbedoneusingcommunicationas

evidence,microbiomes,biomarkers,chemicals,andnucliecacids.Eachofthesemethods

havethecapabilitytocontributetoessentialinformationrelatedtoaninvestigation.

Microbeshavelongbeenusedasaphysicalevidenceinforensicsciencebecausetheyare

ubiquitousinnatureandalsobecausetheyhavepredictableecologies.Anotherformof

estimatingPMIisthroughtheuseofbiomarkers.AsidentifiedbythenationalInstituteof

HealthBiomarkersDefinitionsWorkinggroupin1998,abiomarkerisasubstancewhich

canbeobjectivelymeasuredandwhichcanactasanindicatorofpathogenicprocesses,

biologicalprocessesorpharmacologicalresponsestotherapeuticintervention.Thestages

ofdecompositionaidtodeterminethecauseofdeath,thetimeofdeath,etc.Thestages

ofdecompositionarealsoaffectedbyextrinsicandintrinsicfactorsofthedeadbody.The

rateofdecompositionandhencethemicrobialsuccessioncanbeinfluencedby

environmentalvariablesliketemperateandpresenceofsoil.PMIregressionmodels

shouldtakeintoconsiderationenvironmentalparametersthataffectmicrobial

succession.Asaresultoftheadvancesinsequencingplatformsandcomputational

pipelines,microbialcommunitiescanbestudiedingreatdepthwhenconcernedwith

microbialforensics.

iii

Listofabbreviations

DNA-DeoxyribonucleicAcid

circRNA-CircularRNA

miRNA-MicroRNA

PMI-PostMortemInterval

RFLP-RestrictionFragmentLengthPolymorphism

RNA-Ribonucleicacid

16S-rRNA-16SRibosomalRNA

18S-rRNA-18SRibosomalRNA

RT-qPCR-QuantitativeReverseTranscriptionPCR

1

1.Introduction:

PostMortemIntervalalsoknownasPMIreferstothetimebetweenphysiologicaldeath

andtheexaminationofthedeadbody(1).PMIestimationcanbedoneusingvarious

methodssuchascommunications(cellphoneactivity,electronicorhardcopy

communications,visualsightings),biologicalevidenceconcernedwiththehumanbody

andenvironmentwhichincludesrigormortis,lividityandinsectactivity(1).Eachofthese

methodshavethecapabilitytocontributetoessentialinformationrelatedtoan

investigation.Alloftheabovementionedmethodshavetheirlimitationsrelatingtoits

applicabilityanditsaccuracybasedontheconditionsandtimeframesofPMI(i.e.

months,weeks,days)(1).Oneofthemethodsinrecenterathathasproventogenerate

promisingresultsforPMIestimationincludestheuseofdegradationofnucleicacidslike

DNA,RNAandproteins.Researchalsosuggeststhatinthefuture,thetraditionalmethods

forPMIestimationcanbereplacedbymethodsusingdegradednucleicacidsforthe

estimationofPMI(1).

PMIestimationintheinitialhoursafterdeathisbasedontherateofphysicalobservable

modificationssuchasalgor,rigorandlivormortis.Accordingto(1),themethodsbasedon

physicalobservablemodificationsarenotconsideredveryreliableandaccurate.Asa

result,avaluabletechniquehasbeendevelopedwhichusesRNAasatoolinforensic

pathologyinordertostudythemechanismsofdeath,estimatetheageofbiological

stainsandidentifythebodilyfluids(2).

3

Microbiotaisoftenusedinplaceofmicrobiomewhichreferstothemicrobialtaxa

associatedwithhuman(3).Furthermore,microbiomeisthecatalogofmicrobesandtheir

genes(3).Microbeshavelongbeenusedasaphysicalevidenceinforensicscience

becauseofthembeingubiquitousinnatureandalsobecausetheyhavepredictable

ecologies(3).

AnotherformofestimatingPMIisthroughtheuseofbiomarkers.Asidentifiedbythe

nationalInstituteofHealthBiomarkersDefinitionsWorkinggroupin1998,abiomarkeris

asubstancewhichcanbeobjectivelymeasuredandwhichcanactasanindicatorof

pathogenicprocesses,biologicalprocessesorpharmacologicalresponsestotherapeutic

intervention(4).Therecentadvancesinmolecularbiologytechnologieshavemadeit

possibleforpreciselydetectingbiologicalmarkers(4).

SomeofthedifferenttypesofRNAthataidinPMIestimationincludemiRNA,circular

RNAand18S-rRNA.miRNAsarenon-codingsinglestrandedRNAmolecules(4).CircRNAs

arestableRNAsthathaveastablestructureconsistingofcovalentbondslinking3’to5’

endsthroughbacksplicing(4).While18S-rRNAisakeyribosomalRNAwhichisapartof

theribosomalproteincomplex(5).

Thisdissertationfocusesonhowvarioustechniquescanbeusedtoestimatethepost

morteminterval.Itassessesthedifferenttraditionalandmoderndaytechniquesby

analysingtheirlimitations,researchgapsandhowthesetechniquescanbebeneficialfor

PMIestimation.Themainfocusisontechniquesthatusesmicrobiomesandbiomarkers

forpostmortemintervalestimation.

6

2.StagesofDecomposition

Thestagesofdecompositionaidtodeterminethecauseofdeath,thetimeofdeath,etc.

Thestagesofdecompositionarealsoaffectedbyextrinsicandintrinsicfactorsofthe

deadbody.Itisalsoknownthatthesestagesdonothaveaspecificdurationandhenceis

notasusefulindeterminingtheexacttimeofdeath(6).Thefourstagesofdecomposition

includepallormortis,algormortis,rigormortisandlivormortis(6).

PallorMortis:theinitialstageafterdeathwherepalenessisobservedinthefaceandon

otherpartsofthebody.Itiscausedduetostopinbloodflowingthroughcapillary

circulation.Itoccurswithin15-30minutesofdeath(6).

AlgorMortis:Afterthedeathofanindividual,theyareunabletomaintaintheinside

bodytemperatureandhencethebodyitselfstartstoeithercoolorheatbasedonthe

outsidetemperature(6).ThisstagecanbehelpfulindeterminingPMIbyassessingthe

rateatwhichthebodytemperatureacclimatizewiththeoutsideenvironment(6).There

aremanyfactorsthataffectPMIdeterminationwhichincludevariationsintheoutside

temperature,thethicknessofclothingonthedeaceasedandthelocationofthecorpse

(6).

RigorMortis:afterthedeathofanindividual,themusclesbecomeweakandbody

stiffenswherebythemusclesinthebodycontractandstayinthesameposition(6).The

stiffeningofthesemusclesandthebodyisalsoknownasrigormortis.Itoccursaftera

coupleofhoursafterdeathandcompletesafteraroundeighthoursafterdeath.Thetime

7

thatthebodywillstayinthispositiondependsuponfactorssuchasambienttemperature

andtherateofdecompositionofthebody(6).

LiverMortis:itisthefinalstageofdeath.Theaccumulationofbloodinthebodyresultsin

theskinappearingbluishincolour.ThisinotherwordsisalsoknownasLivormortis(6).

Althoughthestagesofdecompositionstartseparatelybuttheycontinuetooccur

simultaneouslyandareoftenoverlappingintheiroccurrence(6).

3.EstimationofpostmortemintervalusingBiochemicalmarkers

Someoftheearlybiochemicalmethodshavenotbeentakenintoconsiderationwhen

determiningPMIbecausetheyhavefailedtoproducereliable,preciseandrapidresults

neededforforensicanalysis(7).Recentresearchhashoweveridentifiedmoresensitive

andspecificbiochemicalmethods,whichcanbeassessedstatisticallyandcanalsobe

standardisedtoproduceaccurateresults(7).

Someoftheveryusefulinformationaboutphaseslikeagonalperiod,supravitalreactions,

leakagefromcelldegradation,diffusionanddecompositionofbiochemicalmarkerscan

beobtainedfromthebiochemicalandmetabolicprofilesgeneratedfrombodyfluids

afterdeath(7).Thisthusresultsinidentifyingthechangingmetabolicenvironmentofthe

host.Theidentificationofrelevantpost-mortembiomarkerscanalsoaidinidentifying

potentialcauseofdeathandPMIestimation.Accordingto(7),biomarkersinrecent

studiesareidentifiedusingNuclearmagneticresonance(NMR)andmassspectrometry

(MS)thatultimatelyresultinprecisePMIestimations.

8

4.BiochemicalmarkersofPMI:

Therearethreefactorsthatbiochemicalchangescanbeattributedtonamely,

biochemicalchangesduringearlypostmortemperiod,theagonalperiodofanoxiaand

distributionofdiffusiblesubstancesintoerythrocytes,plasma,interstitialfluid,tissue

cellsandblood(7).Thevariousbiochemicalmarkersareasfollows:

Metabolites-

SodiumchloridenotsoreliableforPMIestimationastheantemortemserum

concentrationsofsodiumchloridevarybasedonhydrationstatus,kidneyfunctionsand

illness(7).

PotassiumisnotsoreliablebiochemicalmarkerforPMIcalculationsbecauseitisvery

difficultandnearlyimpossibletocalculatepotassium’sserumante-mortem

concentration.Thisisbecauseitisimmediatelyreleasedfromthecellsafterdeath(7).

Decompositionconstitutesofenzymaticbreakdownoflipids,carbohydratesandproteins

(8).

InordertodeterminePMIandunderstandthedecompositionprocess,itismustto

identifythebiochemicalproductspresentinthedecompositionfluidandthetimewhenit

wasproduced(8).

Decompositionconsistsofcomplexreactionsthatcausesthebreakdownofproteinsto

aminoacids,nucleicacidstonucleotidesandcarbodydratestomonosaccharidesandlipid

basedmacromoleculestofattyacids(8).

9

5.RoleofmicrobesinPMIdetermination:

Microbesbeingubiquitousandhavingpredictableecologiesmakethemidealasphysical

evidencewhenconcernedwithforensicscience(3).Advancementinresearchhasleadto

thedevelopmentofnewmicrobialbasedtoolsforuseinforensicscience(3).Thishas

beenpossibleduetotheboostinmicrobiomescienceandtheadvancementofnext

generationsequencingtechnology(3).Microbeshavebeenproventobeverypromising

whenitcomestothedeterminationofPMI.Regressionmodelsmadeusingmicrobiome

datafrompostmortemsampleswithknownPMIareusedtodevelopthemicrobialclock

ofdeath(3).Microbialclock’sareusedinadeathinvestigationwhereinmicrobesare

profilesusingDNAsequencingfromsamplesthatarecollectedwhichisthenmatchedto

apointontheclock(3).Thesignificanttophonomiclandmarksofdecompositioncanbe

organizedusingsomearbitarystages,asdecompositionbeingacontinuousprocess.

Cadaverdecompositoncanbeclassifiedintostagesstartingfromfreshstage,bloat,active

decay,advancedecay,skeletonization(3).

Insituationswhereinsectsareabletogettoacorpse,theblowflieslayeggsthat

eventuallydevelopintomaggotsandblowflies.Thelifestyleoftheoldestmaggotscanbe

usedtogenerateagrowthcurvebasedontemperaturewhichcanthenbeusedto

estimatePMI(3).Thereareanumberoflimitationstothisapproachwhichisthatthis

methodisonlyusefulinPMIdeterminationwithintheinitialtwoweeksof

decomposition,temperatureisabiginfluence,itismainlypreferredonlyifthebodyis

presentintheoutenvironmentwhereinsectcanhaveeasyaccesstothecorpse.Thisis

10

alsoahighchanceofmisinterpretationofmaggotsaswhatstagearetheyintheir

lifecycle(3).

EnvironmentalinfluenceonPMIwhenusingmicrobialmodels:

Therateofdecompositionandhencethemicrobialsuccessioncanbeinfluencedby

environmentalvariablesliketemperateandpresenceofsoil.PMIregressionmodels

shouldtakeintoconsiderationenvironmentalparametersthataffectmicrobialsuccession

(3).

MicrobialevidenceintheJusticesystem:

Developingandtransitioningnewforensicsciencetechnologiesintothejusticesystem

requiresovercomingscientific,investigative,andlegalhurdles.Ifanewtechnologyisto

beintroducedintothejustice,anumberofproceduresneedstobeimplemented.These

includeidentifyingneed,basicresearch,prototypedevelopment,validation,acceptance

andadoption(3).

Aninvestigatororalawyerwhocansupporttherelevanceandreliabilityofthemicrobial

evidenceisrequiredinordertousemicrobiomesasanevidenceincourt.Microbial

evidenceshouldbefullyresearchedanditserrorratesshouldbeunderstoodthrough

quantitativemachine-learningmethodsforittobepresentedincourt.

11

6.ProfilingofRNAdegradationforestimationofPMI

RNAhasthepotentialforPMIestimation,includingidentificationoffluids,woundage

estimationbystudyingtimedependentexpressionoftroponin1mRNAfromskeletal

musclesbeingoneofthepossiblemarker(1).

ItisquiteusefultostudyRNAdecayasameanstoestimatePMIestimationasRNA

degradationorlossofRNAtranscriptafterdeatharerapidandtimedependent(1).RNA

inthecellsofanindividualafterdeathisdegradedbyribonucleasesthatarepresent

withinthecellorfromthoseoriginatingfrombacteriaorotherenvironmental

contamination(1).Apartfromtherolethatendogenousandexogenousribonucleases

playininvivoRNAdegradation,therearealsootherfactorspresentthatcaninfluence

degradationwhichincludeenvironmental,chemicalandthermalfactorsastheyalso

immediatelyinfluenceRNAdegradationafterdeath(1).

7.IdentificationofMolecularBiomarkersforestimationofPMIusingbloodsamples

ThedegradationofRNAinthebodyafterdeathisconsideredtobeaveryusefulmarker

foraccuratePMIestimation.Basedon(9)study,RNAgenerallygetsrapidlydegraded

comparedtoDNAafterdeathandalsoinvitroasaresultofubiquitousribonuclease

activity.IthasbeenresearchedthatRNAmarkersdecreaseinatimedependentmanner

whileRNAlevelscanstillbedetectedafter30daysofcollectionofsample(9).

Thestudyby(9)alsoconfirmedthatcertainRNAmarkersatcertaintemperatures

degradeinatimedependentfashionthrough30daysafteritwasfirstcollected.Someof

12

theminclude18SrRNAandHPRT1,B2MandGAPDHmRNAsactivelydegradeat25

degreesCelsiuswhileGAPDHmRNAdegradeat4degreeCelsius(9).

AnumberofdifferentfactorsalsoaffectthesemRNAswhichincludeintracellular

substanceslikeenzymesandproteinsthatarereleasedfromthehemolysisofRBCsin

wholeblood(9).

8.DeterminationofPMIintheadvancestageusingmiRNAsandcircRNAs:

AsthenucleicacidslikeDNAandRNAarepresentinthecellnucleus,theyarelesslikely

tobedamagedbyexternalfactorsandthushavetopotentialtoestimatedmoreprecise

PMI.Accordingto(4)RNApresentinthetissuesdegradewithdelayinPMI.Itisalso

figuredoutthatoneoftheprecisemethodsforPMIestimationatthispointintimeis

throughtheuseofRNAdegradationmeasuredquantitativelyandthroughmathematical

modelsthatpotrayrelationshipbetweenRNAdegradationandPMI(4).

OneofthegoodmethodsfordetectingRNAprofilesisRealtimequantitativepolymerase

chainreaction(qPCR).Whenconsideringbiochemicalresearch,someofthemRNA

markersthatareconsideredtobeappropriateendogenouscontrolmarkersareGapdh,

betaactinandRps18(4).

WhenconsideringRNAsforPMIestimation,ithasbeenresearchby(4)thatmicroRNAs

andcircularRNAshaveproventobemorestableasreferencegeneswhencomparedto

otherRNAs.Theirresearchalsosuggeststhatreal-timequantitativePolymeraseChain

Reaction(qPCR)accuratelypredictsPMIinitsadvancedstagewithlowerrorratesandby

usingeffectivereferencegenesandtargetbiomarkers(4).

13

CircularRNAsarehighlystableandabundantwithpossessingtissuespecificexpression

patternsbecauseofwhichtheyareconsideredtobeveryvaluablewhenestimatingPMI

inadvancedstage(4).Also,someofthehousekeepinggenesarefoundtobeveryclosely

relatedtotoPMIandhencehaveproventobepotentialtargetbiomarkersforthe

determinationofPMI(4).SomeofthesetargetbiomarkersincludeU6andRps18inheart

andlivertissues,thatofskeletalmuscletissueincludeU6andBetaactin(4).

SomeofthelimitationsincludetheeffectoftemperatureonRNAdegradationbutare

controlledtoacertainextentunderexperimentalconditioninresearch(4).

9.Postmortemintervaldeterminationusing18S-rRNAandmicroRNA:

Basedonrecentresearch,mRNAcanalsobeusedforpostmortemanalysisevenifthe

corpseisleftatroomtemperatureforseveraldays(5).18S-rRNAisprotectedfromRNA

enzymesinsidetheribosomalcomplexandisfoundinabundanceinthebody.Whenthe

extensionofPMIandthedegradationofproteins,thequantity18S-rRNAincreasesandis

releasedfromtheribosomalcomplex.Accordingto(5),duringtheearlystagesafter

death,18S-rRNAlevelsgraduallyincreaseandpeaksaround96hoursafterdeath.

AdvancesinMicrobialForensics:

Asaresultoftheadvancesinsequencingplatformsandcomputationalpipelines,

microbialcommunitiescanbestudiedingreatdepthwhenconcernedwithmicrobial

forensics(10).Therearetwodifferentfieldsinforensicswhenconcernedwithmicrobes

whichincludemicrobialforensicsandmicrobiomeforensics(10).Microbialforensics

relatetotheidentificationofspecificstrainsofmicrobes.OntheotherhandMicrobiome

14

forensicstudiesthedifferenceinmicrobialcommunitiesthroughthemicrobialPM

changesandtraceevidence(10).Thereareanumberofreasonswheremicrobiomedata

canbeusednamelyforcalculatingPMI,locatingclandestinegravesandusingskin

microbestolinkobjectsorspaces.Nextgenerationsequencingofphylogeneticand

taxanomicinformativegenemarkerslike16SrRNA,18SrRNAandITScanbeusedto

studypatternsinmicrobialcommunities(10).

15

10.Conclusion:

IthasbeenresearchedthatthefactorsaffectingRNAdegradationinadeadbodyarePMI

andtemperature.OtherfactorstoohaveaneffectontheestimationofPMIbutitcanbe

eliminatedbycontrollingotherexperimentalconditionsinresearch.Thetraditional

methodsofPMIestimationthatusesmicroorganismsandstagesofdecompositionare

consideredonlytobeusefulwhenestimatingPMIduringtheearlystagesafterdeath.

Howeverastimeelapses,researchsuggeststhatnucleicacidssuchasDNAandRNAarea

goodsourceforPMIestimationastheyareprotectedinsidethenuclearenvelopeandare

leastaffectedbyfactorssuchastemperature.AdvancesinMolecularBiological

techniqueshavealsoproventobeveryusefulwhenanalysingthenucleicacids.

16

11.Limitations/ResearchGaps/FurtherResearch:

PMIestimationthattakesintoconsiderationrigormortisandthedecompositionprocess

inthebodyhasmanyfactorsthatinfluenceitsuchastheoutsideandinside

environmentalconditions.

Detectionofpostmortemchangesinprotein,RNAandDNAmarkershasnotyetbeen

systematicallystudied.

Studiesshouldincorporateenvironmentvariablessuchasoxygen,humidity,precipitation

andpresenceofinsectswhenestimatingPMIusingregressionmodels.

Infuturestudies,thekindsandnumbersofbiomarkersandtissuestoestimatePMIinits

advancedstageshouldbeincreased.

Furtherresearchisneededintohowsuccessionofmicrobesindifferentenvironments

liketerrestrial,aquaticandindooraffectmammaliandecomposition.

17

12.References

1.Sampaio-SilvaF,MagalhãesT,CarvalhoF,Dinis-OliveiraR,SilvestreR.ProfilingofRNA

DegradationforEstimationofPostMortermInterval.PLoSONE.2013;8(2):e56507.

2.SameerGomaaM,MohamadAbdEl-KhalekA,MohamadSameerM.Therelationship

betweenthepostmortemintervalandtheDNAdegradationinbrainandliverofadult

albinorats.JournalofAmericanScience.2013;9(5):535-540.

3.MetcalfJ.Estimatingthepostmortemintervalusingmicrobes:Knowledgegapsanda

pathtotechnologyadoption.ForensicScienceInternational:Genetics.2019;38:211-218.

4.TuC,DuT,YeX,ShaoC,XieJ,ShenY.UsingmiRNAsandcircRNAstoestimatePMI

inadvancedstage.LegalMedicine.2019;38:51-57

5.LiW,MaK,LvY,ZhangP,PanH,ZhangHetal.Postmorteminterval

determinationusing18S-rRNAandmicroRNA.Science&Justice.

2014;54(4):307-310.

6.JalanM.WhataretheFourPostMortemstagesofdeath?.ScienceABC.2018.

7.DonaldsonA,LamontI.Estimationofpost-mortemintervalusingbiochemicalmarkers.

AustralianJournalofForensicSciences.2013;46(1):8-26.

18

8.NolanA,MeadR,MakerG,SpeersS.Areviewofthebiochemicalproductsproduced

duringmammaliandecompositionwiththepurposeofdeterminingthepost-mortem

interval.AustralianJournalofForensicSciences.2019;:1-12.

9.LeeH.IdentiicationofMolecularBiomarkersforEstimationofPostmortemInterval

UsingBloodSamples-APilotStudy.JJForenSci.2015,1(1):002

10.MetcalfJ,XuZ,BouslimaniA,DorresteinP,CarterD,KnightR.MicrobiomeToolsfor

ForensicScience.TrendsinBiotechnology.2017;35(9).