Microbiome Biomarkers- Post Mortem Interval · physiological changes such as algor mortis, livor...
Transcript of Microbiome Biomarkers- Post Mortem Interval · physiological changes such as algor mortis, livor...
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MicrobiomeBiomarkers-
PostMortemInterval
By
MaitriSolanki
AThesissubmittedinfulfillmentoftherequirementsforthedegreeof
MasterofForensicScience(ProfessionalPractice)
In
TheSchoolofVeterinaryandLifeSciences
MurdochUniversity
AcademicSupervisor:AssociateProfessorJamesSpeers
Semester1,2019
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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
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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.
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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
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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
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PartOne:LiteratureReview
MicrobiomeBiomarkers–PostMortemInterval
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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.
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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
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12.ConclusionandFurtherResearch…………………………………………………………………..13
References……………………………………………………………………………………………………………14
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LISTOFABBREVIATIONS
ATPAdenosineTriphosphate
DNADeoxyribonucleicAcid
circRNACircularRNA
miRNAMicroRNA
PMIPostMortemInterval
RFLPRestrictionFragmentLengthPolymorphism
RNARibonucleicacid
16S-rRNA16SRibosomalRNA
18S-rRNA18SRibosomalRNA
RT-qPCRQuantitativeReverseTranscriptionPCR
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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
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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).
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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).
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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
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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).
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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
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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).
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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
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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).
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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).
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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).
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11.0Micro-RNAINForensics:
Alotofpotentialforforensicapplicationshavebeenobservedthroughpromisingresults
obtainedfrommiRNAandsmallnon-codingRNAs(14).
Thereexists800-1000miRNAsinthehumangenomeoutofwhichmorethan700miRNAs
havebeenidentified.Thisforms2-3%ofallprotein-codinggenes(14).
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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.
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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
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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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Part2:Manuscript
MicrobiomeBiomarkers–PostMortemInterval
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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
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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.
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Listofabbreviations
DNA-DeoxyribonucleicAcid
circRNA-CircularRNA
miRNA-MicroRNA
PMI-PostMortemInterval
RFLP-RestrictionFragmentLengthPolymorphism
RNA-Ribonucleicacid
16S-rRNA-16SRibosomalRNA
18S-rRNA-18SRibosomalRNA
RT-qPCR-QuantitativeReverseTranscriptionPCR
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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).
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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.
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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
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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.
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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).
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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
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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.
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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
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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).
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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
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forensicstudiesthedifferenceinmicrobialcommunitiesthroughthemicrobialPM
changesandtraceevidence(10).Thereareanumberofreasonswheremicrobiomedata
canbeusednamelyforcalculatingPMI,locatingclandestinegravesandusingskin
microbestolinkobjectsorspaces.Nextgenerationsequencingofphylogeneticand
taxanomicinformativegenemarkerslike16SrRNA,18SrRNAandITScanbeusedto
studypatternsinmicrobialcommunities(10).
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10.Conclusion:
IthasbeenresearchedthatthefactorsaffectingRNAdegradationinadeadbodyarePMI
andtemperature.OtherfactorstoohaveaneffectontheestimationofPMIbutitcanbe
eliminatedbycontrollingotherexperimentalconditionsinresearch.Thetraditional
methodsofPMIestimationthatusesmicroorganismsandstagesofdecompositionare
consideredonlytobeusefulwhenestimatingPMIduringtheearlystagesafterdeath.
Howeverastimeelapses,researchsuggeststhatnucleicacidssuchasDNAandRNAarea
goodsourceforPMIestimationastheyareprotectedinsidethenuclearenvelopeandare
leastaffectedbyfactorssuchastemperature.AdvancesinMolecularBiological
techniqueshavealsoproventobeveryusefulwhenanalysingthenucleicacids.
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11.Limitations/ResearchGaps/FurtherResearch:
PMIestimationthattakesintoconsiderationrigormortisandthedecompositionprocess
inthebodyhasmanyfactorsthatinfluenceitsuchastheoutsideandinside
environmentalconditions.
Detectionofpostmortemchangesinprotein,RNAandDNAmarkershasnotyetbeen
systematicallystudied.
Studiesshouldincorporateenvironmentvariablessuchasoxygen,humidity,precipitation
andpresenceofinsectswhenestimatingPMIusingregressionmodels.
Infuturestudies,thekindsandnumbersofbiomarkersandtissuestoestimatePMIinits
advancedstageshouldbeincreased.
Furtherresearchisneededintohowsuccessionofmicrobesindifferentenvironments
liketerrestrial,aquaticandindooraffectmammaliandecomposition.
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12.References
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2.SameerGomaaM,MohamadAbdEl-KhalekA,MohamadSameerM.Therelationship
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8.NolanA,MeadR,MakerG,SpeersS.Areviewofthebiochemicalproductsproduced
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