Assessing Barriers for Antimicrobial Penetration in Complex Asymmetric Bacterial Membranes A Case Study with Thymol - Sharma et al. - U

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AssessingBarriersforAntimicrobialPenetrationinComplexAsymmetricBacterialMembranes:ACaseStudywithThymolPradyumnSharma,y,?SrividhyaParthasarathi,z,?NiveditaPatil,{MorrisWaskar,{JanhaviS.Raut,{MrinaliniPuranik,{K.GanapathyAyappa,,x,kandJaydeepKumarBasu,zyDepartmentofChemicalEngineering,IndianInstituteofScience,Bangalore560012,IndiazDepartmentofPhysics,IndianInstituteofScience,Bangalore560012,India{UnileverRDBangalore,64,MainRoad,Whitefield,Bangalore560066,IndiaxDepartmentofChemicalEngineering,IndianInstituteofScience,Bangalore560012,IndiakCentreforBiosystemsScienceandEngineering,IndianInstituteofScience,Bangalore560012,India?ContributedequallytothisworkE-mail:ayappa@iisc.ac.in;basu@iisc.ac.inPhone:+91-80-22932769;+91-80-229332811

1E.coliO111:B4E.coliO6:R1GALNACO-AntigenMANGLCMANGLCNACGLCGLCGALGALPPHEPGLCCoresaccharidesPPHEPHEPOOCKDOKDOCOOPPPPGLCNGLCNLipidAFigureS1:AschematicrepresentationofE.coliO111:B4andO6:R1LPSstructureusedinexperimentandsimulationrespectively.(KDO:2-keto-3-deoxyoctulosonate;HEP:L-glycero-D-mannoheptose;MAN:D-mannose;Glc:D-glucose;GAL:D-galactose;GLCN:D-glucosamine;GLCNAC:N-acetyl-D-glucosamine;GALNAC:N-acetyl-D-galactosamine);COL:3,6-Dideoxy-L-xylo-hexopyranose(L3,6dxylHex).2

2MaterialsandMethods-SupportedLipidBilayer(SLB)PreparationofSLBTheLangmuirBlodgett(LB)techniqueenableshomogeneousdepositionofmonolayersoverlargeareaswithprecisecontroloverthemonolayerthickness.Glasssubstrates(0.17mmthickness,20x20mm2fromGlaswarenfabrikKARLHECHTGmbH&CoKG)usedinthebilayerstudieswerecleanedusingtheRCAcleaningmethod(5:1:1-mixtureofH2O:NH4OH:H2O2)tomakethesubstrateshydrophilic.Thesymmetricinnermembrane(IM)wasdepositedusingLBtechniquefroma0.7mg/mLofE.colitotallipidextractsolutioninchloroform.Monolayersweretransferredatapressureof32mN/matatemperatureof23Cataspeedof5mm/minute(upstroke)and3mm/minute(downstroke).RepresentativeisothermsareillustratedinFigureS2A.Theasymmetricoutermembranemodelconsistsofaninnerphospholipidlayer(innerleaflet)andanouterLPSlayer(outerleaflet)wastrans-ferredusingLBandlangmuirschaefer(LS)techniquesrespectively.Theinnerleafletwastransferredatpressureof32mN/mandtheouterleafletcontainingsmoothLPS(S-LPS),(dissolvedin60%CH3Cl,39%MeOHand1%H2Ov/v)wastransferredatapressureof28mNm1at23C(FigureS2B)inthesubphaseof100mMofCaCl.1,2Theisothermswere2reproducibleat23Candhencealltheexperimentswerecarriedoutat23C.Thebilayerswerecarefullytakenoutfromtheteflon-wellandstucktothe300lwaterwellforimagingandFCSprofileswerecollectedusingPicoHarp300.3

3AB23C3030S-LPSisotherm20C15C202023C(mN/m)(mN/m)1010E.colilipidisothermo100mM-CaClsubphaseat23C002608010012010015020025022MMA(Å)Trougharea(cm)FigureS2:(A)Pressure-areaisothermsforE.colitotallipidatthreedifferenttemperatures(B)Pressure-barrierpositionisothermsforS-LPSat23C.BilayerCharacterizationXray-Reflectivity(XR):GrazingincidenceX-rayreflectivity(XR)measurementswereperformedatIndianbeamline,PhotonFactory,KEK,Japanatthewavelength=0.708Å.Measurementswerecarriedoutwithinaclosedcellmaintainingahumidenvironmentat23C.XRmeasurementsgiveinformationaboutthickness,roughnessandelectrondensity.WehaveperformedXRmea-surementsforboththeIMandOM.X-rayreflectivityprofilesareshowninFigureS3,wherethedataisplottedasQversusRQ4.HereRrepresentsthemeasuredreflectivityagainstzzthemomentumtransferalongtheperpendiculardirectionofthefilm,Qz.AfterfittingthereflectivityprofilestoapropermodelusingIgorfit,weextractinformationaboutthethick-nessofsamplealongwiththescatteringlengthdensity(SLD).Parrattformalismwasusedtoanalyzethereflectivitydata.3–54

41E-7InnermembraneOutermembrane4z1E-8R*Q1E-90.10.30.50.7-1Q(Å)zFigureS3:RQ4versusQgraphofreflectivityprofilesofE.coliIMandE.coliOMSLBszzalongwiththerespectivefits.BlueandredlinesrepresentthefittedlinesforIMandOMrespectively.Thicknessvaluesarecalculatedtobe44Åand69ÅforIMandOMrespectively.XRmodelselectedforfittingforbothIMandOMisgiveninFigureS4.Fromthefitting,thicknessofIMandOMarecalculatedas44Åand69Å(withoutO-antigen)respectively.FittingparametersofIMandOMaregiveninTableS1andS2.TableS1:Thicknessandscatteringlengthdensity(SLD)forinnermembrane(IM)ofE.coli.Sample-E.coliIMThickness(Å)SLD(106Å2)FrontingH20-9.40Head6.5110.75Tail13.158.84Tail15.748.48Head8.5011.02H2Olayer2.059.40SiO28.1519.47Backing-Si-20.15

5AInnermembraneBOutermembraneLPSE.colilipidE.colilipidH2OSiO2H2OSiO2SiSi20InnermembraneOutermembrane20))-2-2Å16Å16-6-6LPS10WaterLipidBilayer10(WaterLipidBilayer(12Sisubstrate12SisubstrateSLDSLD88-200204060-20020406080Distancefromtheinterface(Å)Distancefromtheinterface(Å)FigureS4:(A)ModeloftheIMandthecorrespondingscatteringlengthdensity(SLD)extractedfromthefits.(B)OMmodelandthecorrespondingSLDextractedfromthefits.TableS2:Thicknessandscatteringlengthdensity(SLD)foroutermembrane(OM)ofE.coli.Sample-E.coliOMThickness(Å)SLD(106Å2)FrontingH2O-9.40CoreSaccharideofLPS23.059.83Head6.7311.94Tail12.478.43Tail18.508.06Head8.2711.11H2Olayer2.159.40SiO29.3519.41Backing-Si-20.16

6MaterialsandMethods-GiantUnilamellarVesicles(GUVs)GUVpreparationandfixingE.colitotallipidextractwastaggedwithATTO488andCy5-NHSEsterdissolvedintrisbufferwasusedtofilltheGUVs.E.coliIMGUVswerepreparedbytheelectroformationmethod.Briefly,2mMofE.colilipidwithAtto488DMPEandBiotinylated-DMPE,werespreaduniformlyonacleanedindiumtinoxide(ITO)coatedglassslide.Thelipidsweredriedbydesiccatingtheslidesinavacuumdesiccatorfor2hours.1.5mLofdeionized(DI)waterwith250mMsucrosewasaddedwith200nMCy5andaddedbetweentheITOslidesattachedwiththepolydimethylsiloxane(PDMS)spacer.ITOslideswereconnectedtothefrequencygeneratorsettoasinewaveatafrequencyof10Hzandoutputvoltageof1Voltwithnophasedifference.Theslideswereplacedinsidetheincubatorata55Cfor90min.Usinga1mLpipette,thesolutionwascarefullymixedandstoredinthemicro-centrifugetubesat4Cuntilimaging.ThisprocedureyieldedagoodnumberofGUVs.InordertofixtheGUVsontheglasssubstrate,aDPPCbilayerwaspreparedbythevesiclefusionmethodwithBiotinylPEintheratio500:1.SUVsof1mMDPPCwerepreparedbysonicationandaddedtotheplasmatreatedcoverslip.SUVswereincubatedat55Cfor1hourandthentheslideswerewashedwithwatermanytimestoremoveunfusedvesicles.100lof0.2g/mLstreptavidinwasaddedtotheslideandlefttoincubatefor30minatroomtemperature.TheunboundstreptavidinwaswashedwithDIwater.GUVswereaddedtotheslideandfixedtotheglasssubstratethroughthecovalentinteractionsbetweenstreptavidin-biotinylatedPE.Thymolwasaddedsequentiallyfrom0.01mMto33mM(finalconcentrationinthe300lwell)totheGUVsandmonitoredcontinuously.Aftertheadditionofthymol,theslidewiththymolwasmonitoredfor10minutesbeforeaddingthenextconcentration.WedidnotobserveanyeffectontheGUVstilltheeffectiveconcentrationofthymolinthewellwas33mM.Atandabove33mMofthymol,GUVsrupturedslowlyasshownintheFigureS5.ThroughouttheexperimentwedidnotobserveanyleakagefromtheGUVs.7

7BeforeadditionDuringaddition-0s11s15s19s(a)(b)(c)(d)(e)FigureS5:Time-lapseimagesoftheGUVstakenontheequatorialplanebeforeandaftertheadditionofthymolofconcentration33mM(finalconcentrationinthe300lwell).Additionof33mMandabovewasfoundtorupturetheGUVs.Noleakageoftheencapsulateddyewasobserved.FluorescenceCorrelationSpectroscopyonGUVsSincetherupturingconcentrationofthymolforE.coliIMGUVswas33mM,wehavechosenathymolconcentrationof4.4mMandperformedFCSmeasurements.TheautocorrelationfunctionversuslagtimeisplottedinFigure-S6A.Adecreaseintherelaxationtimeisclearlyobservedatathymolconcentrationof4.4mM.TheseprofilesarefittedtoEquation1anddiffusivity(D)valuesareextractedfromEquation2(seemainmanuscript).Aminimumof10GUVswereselectedandFCSprofileswerecollectedonthepolarregionoftheGUVs.Figure-S6BillustratesthehistogramofDvaluesbeforeandaftertheadditionofthymol.AdistinctdoublingintheDvaluesisobserveduponthymoladdition.QualitativelysimilarresultswereobservedinbothIMmodelofSLBandGUVmodelsystems.8

8ABE.colilipidE.colilipid1.0E.colilipid+4.4mMthymolE.colilipid+4.4mMthymol250.7)(15G0.4Occurences50.1-5-4-3-2-110101010104681012142lagtime(seconds)D(m/s)FigureS6:(A)ThefluorescenceautocorrelationfunctionG()plottedasafunctionoflagtimeforE.coliIMGUVsbeforeandafteradditionof4.4mMthymol.Solidlinesrepre-sentfitstoautocorrelationdata(Equation1inmainmanuscript).(B)Histogramsofdiffusioncoefficients(D)valuesestimatedusingEquation2(mainmanuscript).Thehistogramsrep-resentdatacollectedfrom40-50measurementsfrom10differentGUVsonthepolarregionoftheGUVs.Confocalimages:E.colilipidbilayerandtheS-LPSlayerweretaggedwithATTO488DMPEandATTO647NDMPErespectivelyandthedyeconcentrationsforallthelipidandS-LPSsampleswereapproximately5103mol%.ConfocalimagesoftheIMSLBexcitedat488nmand647nmareshowninFigureS7.Uniformfluorescenceintensityindicatesthatthedepositedfilmsarehomogeneousinnature.Howeverwiththeadditionofthymol,intensitiesshowagradualdecreaseasshowninFigures-S7B&E.Intensitywascalculatedbyselectingaregionofinterestof50m50m.Apartfromchangesobservedinthediffusivityvalues,additionofthymolwasalsofoundtoreducetheintensityoftheSLB.9

9A4040TimeVsintensityforbarebilayer(B)LipidChannel363230IMIntensity(arbunits)050100150Time(sec)20Intensity(arbunits)IntensitydependenceVsConcentration100246810X(mM)OMthy6045Redchannellipidintensity(647)CD(E)3050Greenchannellipidintensity(488)1540Intensity(arbunits)004080120160Time(sec)302010Intensity(arbunits)RedchannellipidintensityLipidChannelLPSChannel0Greenchannellipidintensity02468X(mM)thyFigureS7:(A)ConfocalimageofE.colilipidIMexcitedwith488nm,insetrepresentsthechangeinintensityaftertheadditionof8.8mMthymol.(B)IntensityversusXthyforIM,insetrepresentstimeversusintensitytoshownobleachingeffectisobserved.(CandD)ConfocalimagesofOMexcitedwith488nm(innerleaflet)and647nm(outerleaflet)respectively,insetsrepresentthechangeinintensityaftertheadditionof8.8mMthymol).(E)IntensityversusXthyforOM,insetrepresentstimeversusintensitytoshownobleaching.Inboththecasesitcanbeclearlyseenfromthefiguresthatthereissignificantreductionintheintensitiesaftertheadditionofthymol.10

10FCSprofiles-FitparametersforIMtransferredat32mN/mOneoftheimportantphysicalparameterswhichisusedtoquantifyphasepropertiesandextentoforderoflipidbilayersisthelipiddiffusioncoefficient.FCSmeasurementsarerou-tinelyusedtoextractingthetimescalesofdiffusioninbilayers.InitiallyFCSwasperformedonabilayertransferredat32mN/mfortheIMandinteractionofthymolwasstudied.TheautocorrelationdataaregiveninFigure2Aofthemainmanuscript.Correlationcurvesob-tainedfromthesemeasurementsprovideinformationonthetransittimeD,thetimetakenforamoleculetodiffusethroughthefocalspot.Forlipidsdiffusingina2Dplanarmem-brane,correlationprofileswerefittedusingtheEquation1giveninthemainmanuscript.OnceDisknown,DcanbecalculatedfromEquation2(mainmanuscript).FitparametersfortheprofilesgiveninFigure2(mainmanuscript)aregiveninTableS3.R2valuesfortheprofilesgivenbelowareapproximately0.990.005.TableS3:FittedparametersforG()datapresentedinthemainmanuscript(Figure2)fittedusingEquation1(mainmanuscript)SampledetailsDiffusiontime(D)msG1tE.coliIMSLB6.100.937-0.0024.47IM+1.1mMthymol4.560.9450.0005.59IM+2.2mMthymol4.090.9130.0032.72IM+4.4mMthymol3.120.9550.0008.33IM-FCSat26mN/mTostudytheeffectofpressureontheactionofthymol,SLBsweretransferredatalowerpressureof26mN/m.ItcanbeseenfromFigureS8thattheconcentrationofthymolatwhichthediffusivityincreaseoccursisapproximatelysameasSLBtransferredat3211

11mN/m.Above8.8mMthymolconcentration,thediffusivitycurvesaturatessuggestingthatamaximumnumberofthymolmoleculeshadenteredthelipidmembrane.ThecomparisongraphshowninFigureS8C,suggeststhatchangesarequalitativelysimilar.A35E.colilipidE.colilipid+4.4mMthymol2515Occurences523452D(m/s)B6E.colilipiddiffusivityat26mN/m5)/s2m4(D32024681012X(mM)CthyE.coliIM-26mN/m2.5E.coliIM-32mN/m2.0thy=0D/D1.51.0024681012X(mM)thyFigureS8:Histogramsfor(A)E.coliIMSLBpreparedat26mN=mbeforeandafteradditionof4.4mMthymol-DiffusivityvaluesareestimatedfromEquation2.ofthemainmanuscript.(B)Diffusioncoefficientvaluesplottedasafunctionofconcentrationofthymol.Dvaluessaturateaboveathymolconcentrationof8.8mM.(C)ComparisonofnormalisedDvaluesversusXforthefilmstransferredat26mN/mand32mN/m,hereD=2.00m2s1thythy=0andD=1.52m2s1for26mN/mand32mN/mrespectively.thy=012

12Outerlealet-FCShistogramsFCSexperimentswerecarriedoutwiththeinnerleafletoftheOMtaggedwithATTO488andtheouterleafletofOMtaggedwithATTO647NDMPE.Histogramsofthediffusivitiesfortheinnerleafletaregiveninthemainmanuscript(Figures3BandC).ThediffusivityintheouterleafletoftheOMcontainingonlyS-LPSisshowninFigureS9AandfortheOMcontainingamixtureofS-LPSandPOPEisshownintheFigureS9B.ThedataclearlyillustratesthatthelipiddiffusivityintheouterleafletisunchangedinthecaseoftheOMcontainingonlyS-LPS,whereasinthecaseoftheOMcontainingmixtureofS-LPSandPOPEasmallbutperceptibleincreaseinthelipiddiffusivityisobservedintheouterleaflet.WeobtainD=0.67m2s10.10forthebaremembraneandD=0.75m2s10.11forthemembraneexposedtothymol.InterestinglyalargerincreaseindiffusivitywasobservedinthelowerleafletoftheOM(Figure4Binmainmanuscript)whencomparedwiththesmallerincreaseobservedfortheouterleaflet(FigureS9B).ABOLlipid30OLLipid21OLLipid+4.4mMthymolOLLipid+4.4mMthymolOLLipid+8.8mMthymolOLLipid+8.8mMthymol162011Occurences10Occurences6100.51.00.51.01.52.0D(m2/s)D(m2/s)FigureS9:(A)Outerleaflet(OL)lipiddiffusivityofOMexcitedwith647NDMPEwhentheOMcontainsonlyS-LPS(B)Outerleaflet(OL)diffusivitiesfortheOMcontainingamixtureofS-LPS+POPE.E.colilipidsattheinnerleafletoftheOMweretransferredat32mN/mandS-LPS/S-LPS+POPEattheouterleafletwastransferredat28mN/m.13

13MolecularDynamicsSimulationsABCD1:161:81:41:2FigureS10:Datafromallatommoleculardynamicssimulations.(A)Areaperlipidfortheinnermembranesystems.(B)Thicknessoftheinnermembranesystemswherethick-nessisdefinedasthedistancebetweencenterofmassofphosphorusatomsinupperandlowerleaflets.(C)AreaperLPSandthicknessoftheoutermembranewherethicknessisdefinedasthedistancebetweencenterofmassofphosphorusatomsinupperleafletLipid-Aandlowerleafletphospholipids.(D)Side-viewsnapshotoftheinnermembranesystems.DOPE(green),DOPG(red)andTOCL(blue).PhosphorusatomsandthymolmoleculesarerepresentedbyvanderWaalsrepresentationinpinkandwhiterespectively.14

14InnermembraneOutermembraneFigureS11:Umbrellasamplinghistogramsobtainedwhilecomputingthepotentialofmeanforcetodeterminethetranslocationfreeenergiesforthymolinsertionintotheinner(left)andouter(right)membrane.Umbrellasamplingwindowswerecreatedatthespacingof0.1nmandeachwindowwassimulatedfor100ns.ThepotentialofmeanforcefortheinnerandoutermembranesareillustratedinFigure7inthemainmanuscript.15

15A0ns10ns20ns500nsB20nsB20ns100nsUpperLeafletUpperleafletLowerleafletLowerLeafletFigureS12:Datafromallatommoleculardynamicssimulationsoftheinnermembraneexposedto512thymolmoleculesequallydistributedonthetwoleaflets.(A)Side-viewsnapshotoftheinitialsystemhaving512thymolmoleculesillustratingtherapidaggregationofthymolat10nsfollowedbyinsertionintothemembraneat20ns.At500nsallthethymolmoleculesaredispersedwithinthemembrane.DensitydistributionsforthelipidandthymolareillustratedinFigure7Binthemainmanuscript.(B)Correspondingtopviewofthemembraneat20nsillustratingtheformationofporestoenablethymolaccessintothemembraneandat100nstoillustratetheclosingoftheporeoncethemoleculesareinsidethemembrane.Poresarehighlightedwithblackcircle.Onlyphosphorusatomsareshownforclarity.16

16DOPGSN1DOPGSN20.250.25BaremembraneBaremembrane1:161:160.21:80.21:81:41:41:21:21:10.151:10.15cdcdSS0.10.10.050.0500051015051015CarbonnumberCarbonnumberTOCLATOCLB0.250.25BaremembraneBaremembrane1:161:160.21:80.21:81:41:41:21:20.151:10.151:1cdcdSS0.10.10.050.0500051015051015CarbonnumberCarbonnumberTOCLCTOCLD0.250.25BaremembraneBaremembrane1:161:160.21:80.21:81:41:41:21:20.151:10.151:1cdcdSS0.10.10.050.0500051015051015CarbonnumberCarbonnumberFigureS13:DeuteriumorderparameterforDOPGandTOCL1tails.Themembranebe-comesmoredisorderedastheconcentrationofthymolisincreased,similartothetrendobservedinDOPElipids(Figure5Dinmainmanuscript).17

17DOPESN1DOPESN20.250.25BaremembraneBaremembrane1:161:161:81:80.2local0.2local4:5124:512locallocal1:161:16locallocal1:81:80.150.15cdcdSS0.10.10.050.0500051015051015CarbonnumberCarbonnumberFigureS14:DeuteriumorderparameterforlipidtailsofDOPEcalculatedforthelipidmoleculesinthelocalenvironmentofthymolmoleculesforthymoltolipidratiosof4:512,1:16and1:8.Onlylipidmoleculeswhichliewithinaradiusof1nmofathymolmoleculewereincludedinthecomputation.Correspondingvaluesaveragedacrosstheentiremembrane,forthebaremembrane,1:16and1:8systemhavebeengivenforcomparison.20ThymolOAntigen15CoresaccharidesUpperLeafletLowerLeafletWaterwall10z(nm)Thymol3Thymol4Thymol5Thymol6500100200300400500Time(ns)Thymol7Thymol8Thymol9Thymol10FigureS15:Trajectories(zdirection)ofthethymolmoleculesfrommoleculardynamicssimulationsoftheS-LPS.Initially5thymolmolecules(4,5,6,8and9)areplacedintheaqueousregionabovetheO-antigen,2thymolmolecules(3and7)areplacedatthecoresaccharides-O-antigeninterface,andthymol(10)isplacedbelowthelowerleaflet.Inallcases(3-9)weobservethatthymolspontaneouslyaccessestheO-antigenregionoftheS-LPS,howeveraccessintothecoresaccharideisnotobservedoverthe0.5ssimulation.Thethymolmolecule(10)placedbelowthelowerleafletisfoundtospontaneouslyenterthelowerleaflet.18

18CoresaccharidesOantigenFigureS16:Orientationaldistributions(top)andtrajectoryofthymolinx-yplane(bottom)fromarepresentativeumbrellasamplingwindow.TheanglecalculatedisdefinedasAngle1inFigure6Aofthemainmanuscript.Angledistributionsshowthatthymollosesrotationaldegreesonfreedomincore-saccharides(topleft)whereitshowsapreferentialorientationdepictedbythenarrowdistributioninthisregion,whileintheO-antigenregionthemoleculeisfreetorotateasobservedfromthewiderangulardistribution(topright).Thetrajectoryofthymolinx-yplaneshowsthelossoftranslationaldegreeoffreedomincore-saccharides(bottomleft)regioncomparedO-antigenregion(bottomright).19

19A6.29nmB6.29nm6.29nm6.29nmFigureS17:Top-viewsnapshotoftheoutermembrane.(A)Core-saccharides(B)O-antigen.ThelooserpackingandvoidspacecanbeobservedintheO-antigenunitswhichfacilitatesthemovementofmoleculesacrosstheO-antigenregion.References(1)Clifton,L.A.;Holt,S.A.;Hughes,A.V.;Daulton,E.L.;Arunmanee,W.;Heinrich,F.;Khalid,S.;Jefferies,D.;Charlton,T.R.;Webster,J.R.etal.AnaccurateinvitromodeloftheE.colienvelope.AngewandteChemieInternationalEdition2015,54,11952–11955.(2)Clifton,L.;Skoda,M.;Daulton,E.;Hughes,A.;LeBrun,A.;Lakey,J.;Holt,S.;Hughes,V.Gram-Negativebacterialoutermembranemimicasymmetricphospholipid:Lipopolysaccharidebilayers;aGram-Negativebacterialoutermembranemimic.JRSoc.Interface2013,10(89),0810.(3)Biswas,N.;Bhattacharya,R.;Saha,A.;Jana,N.R.;Basu,J.K.Interplayofelectrostat-icsandlipidpackingdeterminesthebindingofchargedpolymercoatednanoparticlestomodelmembranes.PhysicalChemistryChemicalPhysics2015,17,24238–24247.20

20(4)Basu,J.K.GrazingincidenceX-rayscatteringanddiffraction.Resonance2014,19,1158–1176.(5)Basu,J.K.;Sanyal,M.K.OrderingandgrowthofLangmuir–Blodgettfilms:X-rayscatteringstudies.PhysicsReports2002,363,1–84.21