Molecular Biomarkers: Tools of Medicine

148 Pages·2013·17.18 MB·English

BioMed Research International
Molecular Biomarkers: Tools of
Medicine
Guest Editors: Prabir K. Mandal, Shivani Soni, R. Renee Reams,
Tiziano Verri, and Sudhish Mishra
Molecular Biomarkers: Tools of Medicine
BioMed Research International
Molecular Biomarkers: Tools of Medicine
Guest Editors: Prabir K. Mandal, Shivani Soni, R. Renee Reams,
Tiziano Verri, and Sudhish Mishra
Copyright©2013HindawiPublishingCorporation.Allrightsreserved.
Thisisaspecialissuepublishedin“BioMedResearchInternational.”AllarticlesareopenaccessarticlesdistributedundertheCreative
CommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginal
workisproperlycited.
Contents
MolecularBiomarkers:ToolsofMedicine,PrabirK.Mandal,ShivaniSoni,R.ReneeReams,TizianoVerri,
AnitaMandal,andSudhishMishra
Volume2013,ArticleID595496,2pages
DefectsinBaseExcisionRepairSensitizeCellstoManganeseinS.cerevisiae,AdrienneP.Stephenson,
TryphonK.Mazu,JanaS.Miles,MilesD.Freeman,R.ReneeReams,andHernanFlores-Rozas
Volume2013,ArticleID295635,9pages
PracticalGuidanceforImplementingPredictiveBiomarkersintoEarlyPhaseClinicalStudies,
MatthewJ.MartonandRussellWeiner
Volume2013,ArticleID891391,9pages
CirculatingmicroRNAsandKallikreinsbeforeandafterRadicalProstatectomy:AreTheyReallyProstate
CancerMarkers?,MariaGiuliaEgidi,GiovanniCochetti,MariaRitaServa,GabriellaGuelfi,Danilo
Zampini,LucaMechelli,andEttoreMearini
Volume2013,ArticleID241780,11pages
ANovelDifferentialPredictModelBasedonMatrix-AssistedLaserIonizationTime-of-FlightMass
SpectrometryandSerumFerritinforAcuteGraft-versus-HostDisease,Chun-yanZhang,
Shu-hongWang,Wen-rongHuang,Guang-hongGuo,Zhu-hongZhang,Wen-junMou,LiYu,
andYa-PingTian
Volume2013,ArticleID563751,14pages
TheuseofMultidimensionalDatatoIdentifytheMolecularBiomarkerforPancreaticDuctal
Adenocarcinoma,LiweiZhuang,YueQi,YunWu,NannanLiu,andYiliFu
Volume2013,ArticleID798054,6pages
ClinicalEvaluationandCost-EffectivenessAnalysisofSerumTumorMarkersinLungCancer,
RongWang,GuoqingWang,NanZhang,XueLi,andYundeLiu
Volume2013,ArticleID195692,7pages
ImmuneParametersinThePrognosisandTherapyMonitoringofCutaneousMelanomaPatients:
Experience,Role,andLimitations,MonicaNeagu,CarolinaConstantin,andSabinaZurac
Volume2013,ArticleID107940,13pages
ComparativeGeneExpressionProfilinginHumanCumulusCellsaccordingtoOvarianGonadotropin
Treatments,SaidAssou,DelphineHaouzi,Herve´ Dechaud,AnnaGala,AliceFerrie`res,
andSamirHamamah
Volume2013,ArticleID354582,13pages
Aptamers:NovelMoleculesasDiagnosticMarkersinBacterialandViralInfections?,Fla´viaM.Zimbres,
AttilaTa´rnok,HenningUlrich,andCarstenWrenger
Volume2013,ArticleID731516,7pages
DistributionofABOBloodGroupandMajorCardiovascularRiskFactorswithCoronaryHeartDisease,
SantanuBiswas,PradipK.Ghoshal,BhubaneswarHalder,andNripendranathMandal
Volume2013,ArticleID782941,5pages
ImmunomodulatoryEffectofContinuousVenovenousHemofiltrationduringSepsis:PreliminaryData,
GiuseppeServillo,MariaVargas,AntonioPastore,AlfredoProcino,MicheleIannuzzi,AlfredoCapuano,
AndreaMemoli,EleonoraRiccio,andBrunoMemoli
Volume2013,ArticleID108951,6pages
AcetylcholinesteraseasaBiomarkerinEnvironmentalandOccupationalMedicine:NewInsightsand
FuturePerspectives,MariaGiuliaLionetto,RobertoCaricato,AntonioCalisi,MariaElenaGiordano,
andTrifoneSchettino
Volume2013,ArticleID321213,8pages
PolyisoprenylatedMethylatedProteinMethylEsteraseIsBothSensitivetoCurcuminandOverexpressed
inColorectalCancer:ImplicationsforChemopreventionandTreatment,FelixAmissah,
RandolphDuverna,ByronJ.Aguilar,RosemaryA.Poku,andNazariusS.Lamango
Volume2013,ArticleID416534,13pages
EmergingTherapeuticBiomarkersinEndometrialCancer,PeixinDong,MasanoriKaneuchi,
YosukeKonno,HidemichiWatari,SatokoSudo,andNoriakiSakuragi
Volume2013,ArticleID130362,11pages
MouseProstateEpithelialLuminalCellsLineageOriginateintheBasalLayerWherethePrimitive
Stem/EarlyProgenitorCellsReside:ImplicationsforIdentifyingProstateCancerStemCells,
JianjunZhou,LionelFeigenbaum,CaroleYee,HongbinSong,andClaytonYates
Volume2013,ArticleID913179,8pages
HindawiPublishingCorporation
BioMedResearchInternational
Volume2013,ArticleID595496,2pages
http://dx.doi.org/10.1155/2013/595496
Editorial
Molecular Biomarkers: Tools of Medicine
PrabirK.Mandal,1ShivaniSoni,2R.ReneeReams,3TizianoVerri,4
AnitaMandal,1andSudhishMishra5
1DepartmentofBiology,EdwardWatersCollege,1658KingsRoad,Jacksonville,FL32209,USA
2DepartmentofBiologicalSciences,AlabamaStateUniversity,RoomNo.325,LifeScienceBuilding1627,
HallStreet,Montgomery,AL36104,USA
3CollegeofPharmacy&PharmaceuticalSciences,FloridaA&MUniversity,1520MLKingBoulevard,Tallahassee,FL32307,USA
4LaboratoryofGeneralPhysiology,DepartmentofBiologicalandEnvironmentalSciencesandTechnologies,
UniversityofSalento,I-73100Lecce,Italy
5DepartmentofTranslationalScienceandMolecularMedicine,MSUCollegeofHumanMedicineandVanAndelInstitute,
333BostwickAvenueNE,GrandRapids,MI49503,USA
CorrespondenceshouldbeaddressedtoPrabirK.Mandal;[email protected]
Received11October2013;Accepted11October2013
Copyright©2013PrabirK.Mandaletal. This is an open access article distributed under the Creative Commons Attribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Molecularbiomarkersareemergingasthekeyindicesforthe showed a significant decrease, highlighting the potential
managementofpatientswithsignificantdiseases.Motivated usefulnessofkallikreinsapartfromPSAaspotentialprostate
bythesystematicefforttodefinethehumangenome,thecre- cancermarkers.
ation of rapid analytic technologies for evaluating nucleic In the paper entitled “A novel differential predict model
acids and proteins has provided the technological “boom” basedonmatrixx-assistedlaserionizationtimeofflightmass
forthedevelopmentofmolecularbiomarkers.Collaboration spectrometry and serum ferritin for acute graft-versus-host
andcooperationbetweenstakeholdersinvolvedinbiomarker disease,”. C.-Y. Zhang et al. investigated the possibility of
development, application, and regulation may be the most pre warning the risk of an acute graft-versus-host disease
expeditiousroutetowardthetranslationoflaboratorydiscov- (aGVHD)beforeandafterallogeneichematopoieticstemcell
eryintopatientmanagement.Insummary,intensiveresearch transplantation (allo-HSCT) by serum profiling combining
hasoriginatedmultiplefactorsorbiomarkersthatarelikelyto with serum ferritinin. Their joint prewarning model could
behelpfulindiagnosis,characterization,andtherapyselec- predicttheriskofaGVHD,especiallysevereaGVHDbefore
tion of different patients. A thorough understanding of the transplantwhichprovideareliablemethodtocontinuously
relevance of each biomarker will be the key to efficiently monitortheconditionofpatients.
diagnoseadiseaseanddirectthepatientstowardsthedrugs In the paper entitled “The use of multidimensional data
morelikelytobeofbenefitbasedontheirparticularprofile. to identify the molecular biomarker for pancreatic ductal
The papers selected for this special issue represent an adenocarcinoma,” L. Zhuang et al. presented that they have
excellent panel for addressing the molecular biomarkers as adopted an integrative approach to simultaneously identify
thefuturetoolsofmedicine.Thisspecialissuecontainsthir- biomarker and generate testable hypothesis from multidi-
teenpapers. mensionalomicsdata.TheyhavefoundthatPER2expression
Inthepaperentitled“CirculatingmicroRNAsandkallik- washighlyassociatedwiththesurvivaldata,thusrepresent-
reins before and after radical prostatectomy: are they really ing a novel biomarker for earlier detection of pancreatic
prostate cancer markers?,” M. G. Egidi et al. presented 38 ductaladenocarcinoma(PDAC).
patientswithprostatecancer.TheysuggestedthattwomiR- In the paper entitled “Clinical evaluation and cost-
NAs(miR-21andmiR-141)couldbeinvolvedinpostsurgi- effectivenessanalysisofserumtumormarkersinlungcancer,”
calinflammatoryprocesses.Postoperativeserumkallikreins R. Wang et al. showed that combinations of four tumor
2 BioMedResearchInternational
markers(SCCA,NSE,CEA,andCYFRA21-1)improvedthe and inhibition are a human biological marker of pesticide
sensitivityforlungcanceranddifferentcombinationpanels poisoning.Thus,itisusedtostudytheeffectsoftheexposure
had their own usefulness. NSE, CEA, and CYFRA21-1 were toorganophosphateandcarbamatepesticidesonNSinoccu-
theoptimalcombinationpanelwithhighestYouden’sindex pational and environmental medicine. This paper reviews
(0.64), higher sensitivity(75.76%), and specificity (88.57%), anddiscussestherecentfindingsaboutAChE,includingits
whichcanaidinclinicaldiagnosisoflungcancer. sensitivity to other pollutants and expression of different
Inthepaperentitled“Immuneparametersintheprognosis splicevariants.Theseinsightsopennewperspectivesforthe
and therapy monitoring of cutaneous melanoma patients: use of this biomarker in environmental and occupational
experience, role, and limitations,” M. Neagu et al. reported humanhealthmonitoring.
the follow-up for 36 months of the immune parameters of In the paper entitled “Polyisoprenylated methylated pro-
patients diagnosed in stages I–IV, namely, pre- and post- tein methyl esterase is both sensitive to curcumin and over-
surgery immunecirculatingperipheralcells andcirculating expressed in colorectal cancer: implications for chemopreven-
intercommunicatingcytokines. tion and treatment,” F. Amissah et al. discussed polyiso-
Inthepaperentitled“Comparativegeneexpressionprofil- prenylatedmethylatedproteinmethylesterase(PMPMEase)
inginhumancumuluscellsaccordingtoovariangonadotropin which cocatalyzes the polyisoprenylation pathway required
treatments,”S.Assouetal.providedanexclusivestudychar- toprocessvariousmonomericGproteins.Mutationofthese
acterizing gene expression profiles in cumulus cells (CCs) G proteins is considered to be responsible for 50% of col-
ofperiovulatoryfolliclesfrompatientsundergoingHP-hMG orectalcancers.Thisinterestingfindingsuggeststhatelevated
andrFSHgonadotropintreatmentsduringinvitrofertiliza- PMPMEaseactivityanditsoverexpressioncanbeoneofthe
tioncycles. Thisprojecthascharacterizedtheexpression of candidate markers for early diagnosis of colorectal cancer.
thesegenesasbiomarkersofinvitroembryoquality. Susceptibility of this enzyme to curcumin also suggests
Inthepaperentitled“Aptamers:novelmoleculesasdiag- that PMPMEase can be a potential candidate for targeted
nosticmarkersinbacterialandviralinfections?,”F.M.Zimbres anticancertherapy.
et al. urged an urgent need to discover novel diagnostic In the paper, entitled “Emerging therapeutic biomarkers
as well as therapeutic tools against infectious agents. They in endometrial cancer,” P. Dong et al. reviewed the current
viewedthatthesystematicevolutionofligandsbyexponential status of molecular therapies tested in clinical trials and
enrichment (SELEX) represents a powerful technology to mainlydiscussedthepotentialtherapeuticcandidatesthatare
targetselectivepathogenicfactorsaswellasentirebacteriaor possiblyusedtodevelopmoreeffectiveandspecifictherapies
viruses. SELEXuses a large combinatorialoligonucleic acid againstendometrialcancerprogressionandmetastasis.
library(DNAorRNA)whichisprocessedbyahigh-fluxin In the paper entitled “Mouse prostate epithelial luminal
vitroscreenofiterativecycles. cells lineages originate in the basal layer where the primitive
In the paper entitled “Distribution of ABO blood group stem/early progenitor cells reside: implications for identifying
and major cardiovascular risk factors with coronary heart prostate cancer stem cells” J. Zhou et al. have developed an
disease,” S. Biswas et al. viewed that the AB blood group in vivo cell fate tracing mouse model and an in vivo slow-
decreases the risk of CHD in healthy controls; it might be cyclingcelllabelmousemodeltoprovidefurtherinsightinto
due to the higher concentration of high density lipoprotein thisquestion.Throughgeneticmanipulationintheanimals,
cholesterol(HDL-c),whiletheObloodgroupincreasesthe theirfindingsindicatethatthebasalcelllineagecanproduce
riskofCHDduetolowerHDL-clevelsinBengalipopulation moredifferentiatedluminalcells;theputativemouseprostate
ofeasternpartofIndia. stemcells(whichareslow-cyclingandresponsiblefortissue
In the paper entitled “Immunomodulatory effect of con- maintenance)likelyresideinthebasallayer.
tinuousveno-venoushemofiltrationduringsepsis:preliminary Thoughtheselectedtopicsandpapersarenotanexhaus-
data,”G.Servilloetal.reportedthatseveresepsisandseptic tiverepresentationoftheentireareaofmolecularbiomarkers
shockaretheprimarycausesofmultipleorgandysfunction andtoolsofmedicine,yettheyrepresenttherichandmany-
syndrome (MODS), which is the most frequent cause of facetedknowledgethatwehavetheprivilegeofsharingwith
death in intensive care unit patients. Many pro- and anti- thereaders.
inflammatorymediators,suchasinterleukin-6(IL-6),playa
key role in septic syndrome. Continuous renal replacement Acknowledgments
therapy (CRRT) removes in a nonselective way pro- and
anti-inflammatory mediators. The authors investigate the Wewouldliketothanktheauthorsfortheirexcellentcontri-
effectsofcontinuousvenovenoushemofiltration(CVVH)as butionsandpatience.Lastbutnottheleast,thefundamental
immunomodulatorytreatmentofsepsisinaprospectiveclin- workofallreviewersonthesepapersisalsogreatlyacknowl-
icalstudy. edged.
In the paper entitled “Acetylcholinesterase as biomarker
PrabirK.Mandal
in environmental and occupational medicine: new insights
ShivaniSoni
and future perspectives,” M. G. Lionetto et al. viewed that
R.ReneeReams
Acetylcholinesterase(AChE)isakeyenzymeinthenervous
TizianoVerri
system(NS),sinceitterminatesnerveimpulsesbycatalyzing
Anita Mandal
thehydrolysisofacetylcholine.Asaspecificmoleculartarget
Sudhish Mishra
oforganophosphateandcarbamatepesticides,AChEactivity
HindawiPublishingCorporation
BioMedResearchInternational
Volume2013,ArticleID295635,9pages
http://dx.doi.org/10.1155/2013/295635
Research Article
Defects in Base Excision Repair Sensitize Cells to
S. cerevisiae
Manganese in
AdrienneP.Stephenson,TryphonK.Mazu,JanaS.Miles,MilesD.Freeman,
R.ReneeReams,andHernanFlores-Rozas
FloridaA&MUniversity,CollegeofPharmacy&PharmaceuticalSciences,1520MartinLutherKingBoulevard,
DysonBuildingRoom221,Tallahassee,FL32307,USA
CorrespondenceshouldbeaddressedtoHernanFlores-Rozas;[email protected]
Received1July2013;Accepted10September2013
AcademicEditor:ShivaniSoni
Copyright©2013AdrienneP.Stephensonetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Manganese(Mn)isessentialfornormalphysiologicfunctioning;therefore,deficienciesandexcessintakeofmanganesecanresult
2+
indisease.Inhumans,prolongedexposuretomanganesecausesneurotoxicitycharacterizedbyParkinson-likesymptoms.Mn has
beenshowntomediateDNAdamagepossiblythroughthegenerationofreactiveoxygenspecies.Inarecentpublication,weshowed
thatMninducedoxidativeDNAdamageandcausedlesionsinthymines.Thisstudyfurtherinvestigatesthemechanismsbywhich
2+ 2+
cellsprocessMn -mediatedDNAdamageusingtheyeastS.cerevisiae.ThestrainsmostsensitivetoMn werethosedefective
2+
inbaseexcisionrepair,glutathionesynthesis,andsuperoxidedismutasemutants.Mn causedadose-dependentincreaseinthe
accumulationofmutationsusingtheCAN1andlys2-10Amutatorassays.ThespectrumofCAN1mutantsindicatesthatexposureto
2+
Mnresultsinaccumulationofbasesubstitutionsandframeshiftmutations.ThesensitivityofcellstoMn aswellasitsmutagenic
2+ 2+
effectwasreducedbyN-acetylcysteine,glutathione,andMg .ThesedatasuggestthatMn causesoxidativeDNAdamagethat
requiresbaseexcisionrepairforprocessingandthatMninterfereswithpolymerasefidelity.Thestatusofbaseexcisionrepairmay
provideabiomarkerforthesensitivityofindividualstomanganese.
1.Introduction in the agriculture and forest industries [8] as well as in the
case of miners, smelters, welders, and workers in battery
Manganese(Mn)isatraceelementthathasbeenextensively factories[9].Theincreaseinatmosphericlevelscouldresult
documentedforitsvariedroleinthebody’shomeostasis.As inpotentialhealthrisks.
anessentialnutrient,Mnisrequiredforthenormalfunction At elevated levels of exposure, Mn has been shown to
and development of the brain [1], metabolism of proteins, cause manganism, which is an excess of manganese in the
lipids,andcarbohydrates[2–4],andalsoasafunctionalunit basalganglia[10].Manganismischaracterizedbyneurologi-
for many enzymes [3–5]. Therefore, deficiencies that affect calsymptomsresemblingthedystonicmovementassociated
fetal development [6] and excess Mn (environmentalexpo- withParkinson’sdisease(PD)[11–13]andthereforeisarisk
sureand/orelevateddietaryMn[7]),canresultindisorders factorforidiopathicParkinson’sdisease(IPD).AlthoughMn
anddisease. hasbeenstudiedforyears,themechanismbywhichitcauses
There is increasing concern for the use of organic neuronaldamageisnotwellunderstood.Studiessuggestthat
compoundscontainingmanganese in industrialsettings. In neurotoxicity is not caused by a single factor but that it
recentyears,methylcyclopentadienylmanganesetricarbonyl appears to be regulated by a number of factors including
(MMT) gained approval for use in the United States as an apoptosis,oxidativeinjury,DNAdamage,mitochondrialdys-
octaneenhancingfueladditiveusedinunleadedautomotive function,andneuroinflammation[14–18].
gasoline.ExposuretoMnhasalsoincreasedthroughoccupa- The mutagenicity of Mn has been extensively docu-
tionandenvironmentalsettings.Thisincludesagrochemicals mented [19]. Mn has been shown to cause damage to DNA
suchasthefungicides,manebandmancozeb,andpesticides in multiple cell-based assays [18, 20], to interfere with the
2 BioMedResearchInternational
fidelityofDNAreplication[21],toactivatetheDNAdamage Table1:Strainsusedinthisstudy.
response [22], to induce mutations in T4 phage replication
[23]andyeastmitochondriareplication[24,25],and,inhibit Gene ORF Function
repairfactorPARPinhumancells[26],albeitnotscoringas HSP104 YLL026W Proteindisaggregase
a direct mutagen in the Ames test [27]. Despite its muta- Nucleotideexcisionrepair
RAD2 YGR258C
genicity,Mnisnotclassifiedasacarcinogeninhumans.The endonuclease
reasonsforthisdiscrepancyarestillnotclear. RAD52 YML032C Homologousrecombination
Research on manganese toxicity has increased in recent
SOD2 YHR008C Mitochondrialsuperoxidedismutase
years. However, the mechanisms underlying its multiple
RAD18 YCR066W Postreplicationrepair
toxicities(neurotoxicity,genotoxicity,mutagenicity,etc.)[19]
CTA1 YDR256C Catalaseactivity
remainamystery.Itispossiblethatredundantmechanisms
ofDNArepairexistwhichareeffectivetohandlethelevelsof SOD1 YJR104C Superoxidedismutaseactivity
Mntowhichcellsareexposed. MLH1 YMR167W Mismatchrepair
The goal of the current study is to gain insight into GSH2 YOL049W Glutathionesynthetaseactivity
the pathways that are involved in DNA damage/repair that
GSH1 YJL101C Glutamate-cysteineligaseactivity
contributetoprotectingcellsfromthetoxicityofmanganese
APN1 YKL114C Baseexcisionrepair
(Mn).TheyeastS.cerevisiaewasutilizedasamodelsystemto
UBC13 YDR092W DNApostreplicationrepair
study the genotoxic effects of Mn. Yeast has proven to be
anexcellenteukaryoticmodelforstudyingmetal,andplayers RAD27 YKL113C Baseexcisionrepair,DNAreplication
identified through genetic studies virtually all have homo- RAD30 YDR419W BypasssynthesisDNApolymerase
loguesinhumans.Inourstudy,weusetwowell-established NTG1 YAL015C Baseexcisionrepair
mutator assays. The CAN1 assay was used to measure the Wildtype:strainBY4741(MATahis3Δ1leu2Δ0met15Δ0ura3Δ0).
induction of forward mutations, and the lys2-10A reversion RDKY3590(MATa,ura3-52,leu2D1,trp1D63,hom3-10;lys210A).
assay was used to assess replication fidelity. Furthermore,
thisstudyexaminestheprotectiveeffectsoftheantioxidants
2+
N-acetylcysteine and glutathione, as well as Mg on Mn- glutathione (GSH) at the concentrations indicated in each
inducedtoxicityandmutagenesis. figure.Survivalwascalculatedasdescribedabove.
2.MaterialsandMethods 2+
2.4.MutationAnalysis. TheeffectofMn ontheaccumula-
tionofmutationswasassessedbytheCAN1forwardmutation
2.1. General Genetic Methods and Strains. Yeast extract/
assay and the lys2-10A mutation reversion as previously
peptone/dextrose (YPD, 1% yeast extract, 2% peptone, 2%
described[30,31].Mutationratesweredeterminedbyfluctu-
dextrose,2%agar)andsyntheticcomplete(SC,0.67%yeast
ationanalysisusingatleastfiveindependentcolonies[29,32].
nitrogenbasewithoutaminoacid,0.087%aminoacidmix-
Each fluctuation test was repeated at least three times. The
ture, 2% dextrose, 2% agar) media or the corresponding
CAN1 forward mutation assay relies on the introduction of
drop-outmediawereasdescribedin[28,29].Homozygous
mutations on the CAN1 gene which encodes the arginine
haploid deletion strains library (Parental strain BY4741:
MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was obtained from permeaseallowingmutantcellstogrowonplatescontaining
thetoxicarginineanalog,canavanine.Thelys2-10Areversion
ThermoScientific(Pittsburgh,PA,USA).
assayisbasedontherestorationoftheopen-readingframein
2.2. Chemicals. Manganese chloride tetrahydrate (MnCl2- amononucleotiderunof10adenineswithinthelys2alleleof
4H2O),N-acetylcysteine(NAC),glutathione(GSH),canava- strainRDKY3590(Table1),allowingmutantcellstogrowon
nine, and yeast media were purchased from Sigma-Aldrich plateslackinglysine.
(St.Louis,MO,USA).
2.5.DNASequenceAnalysis. Spectrumanalysiswascarried
2+ 𝑟
2.3. Sensitivity of Strains to Mn and Effect of NAC and outbyselectingmutants(Can )onselectiveminimummedia
2+
GSH. The concentration of Mn for strain exposure was drop-out plates containing canavanine [29]. Chromosomal
determined experimentally using the wild type parental DNAwasisolatedfromthemutantsandtherelevantregionof
strain,BY4741.Briefly,singlecoloniesweregrownfor16hon CAN1wasamplifiedbyPCRandsequenced[30].Sequence
YPD with or without Mn2+ at 30∘C with shaking. Cells was carried out at MCLAB (San Francisco, CA, USA).
were then washed with and resuspended in sterile water. Sequence analysis was carried out using Sequencher (Gene
Serial dilutions were spotted onto YPD and plates were Codes,AnnArbor,MI,USA).
∘
incubated at 30 C. Cell growth was monitored daily and
sensitivity was scored after 3 days. Colonies were counted 2.6. Statistical Analysis. Data analysis and graphing were
and survival (in percentage) was calculated relative to the performed using the GraphPad Prism 4 software package.
untreated control. Each strain was tested using at least five Specific analysis for each experiment is indicated in each
2+
independent colonies for each Mn concentration tested. figure legend. In most cases, the mean of at least three
To determine the effect of thiol-based antioxidants, cells experimentsisplottedtogetherwiththestandarddeviation.
2+
were cotreated with Mn and N-acetylcysteine (NAC) or Differencesbetweenmeanvaluesandmultiplegroupswere