accepted manuscript - Bioscience Reports



BIOSCIENCE REPORTS

ACCEPTED MANUSCRIPT ͸‘†—Žƒ–‡•’”‘Ž‹ˆ‡”ƒ–‹‘‘ˆŠ—ƒ’ƒ…”‡ƒ–‹…‡—”‘‡†‘…”‹‡Ǧͳ –—‘”…‡ŽŽ• Ǥ”œ›’•‹ǡǤǤ‘Ï‘†œ‹‡Œ•‹ǡǤ‡”‰Ž‡” ǡǤŠƒŒƒ˜‹ ǡǤǤ‘™ƒǡǤǤ–”‘™•

‹‰ŠŽ›ƒʹΪ’‡”‡ƒ„Ž‡”‡…‡’–‘”’‘–‡–‹ƒŽ…Šƒ‡Ž˜ƒ‹ŽŽ‘‹†–›’‡͸ȋ͸Ȍ ‘†—Žƒ–‡•ƒ˜ƒ”‹‡–›‘ˆ„‹‘Ž‘‰‹…ƒŽˆ—…–‹‘•‹…Ž—†‹‰…ƒŽ…‹—Ǧ†‡’‡†‡–…‡ŽŽ‰”‘™–Š ƒ†ƒ’‘’–‘•‹•Ǥ‘ˆƒ”ǡ–Š‡”‘Ž‡‘ˆ͸‹…‘–”‘ŽŽ‹‰‰”‘™–Š‘ˆ’ƒ…”‡ƒ–‹… ‡—”‘‡†‘…”‹‡–—‘”ȋȌ…‡ŽŽ•‹•—‘™Ǥ‡”‡ǡ™‡…Šƒ”ƒ…–‡”‹œ‡–Š‡‡š’”‡••‹‘ ‘ˆ͸‹’ƒ…”‡ƒ–‹…Ǧͳƒ† Ǧͳ…‡ŽŽ•Ǥ —”–Š‡”‘”‡ǡ™‡‡˜ƒŽ—ƒ–‡–Š‡ ‹’ƒ…–‘ˆ͸‘‹–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—ǡ–Š‡ƒ…–‹˜‹–›‘ˆ—…Ž‡ƒ”ˆƒ…–‘”‘ˆƒ…–‹˜ƒ–‡†Ǧ …‡ŽŽ•ȋ Ȍƒ†’”‘Ž‹ˆ‡”ƒ–‹‘‘ˆǦͳ…‡ŽŽ•Ǥ͸‡š’”‡••‹‘™ƒ•ƒ••‡••‡†„› ”‡ƒŽǦ–‹‡ƒ†‡•–‡”„Ž‘–Ǥ͸‡š’”‡••‹‘ƒ†’”‘–‡‹’”‘†—…–‹‘ ™‡”‡†‘™”‡‰—Žƒ–‡†„›•‹ǤŠƒ‰‡•‹‹–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—Ž‡˜‡Ž•™‡”‡†‡–‡…–‡† „›ˆŽ—‘”‡•…‡…‡…ƒŽ…‹—‹ƒ‰‹‰ȋˆ—”ƒǦʹȀȌǤ ƒ…–‹˜‹–›™ƒ••–—†‹‡†„›  ”‡’‘”–‡”ƒ••ƒ›Ǣ…‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘„›”†ǡƒ†’”‘’‹†‹—‹‘†‹‡•–ƒ‹‹‰Ǥ ͸ƒ†’”‘–‡‹ƒ”‡’”‡•‡–‹Ǧͳƒ† ǦͳǦ…‡ŽŽ•Ǥ ‘™”‡‰—Žƒ–‹‘‘ˆ͸ƒ––‡—ƒ–‡•Ǧͳ…‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘Ǥ͸ †‘™”‡‰—Žƒ–‹‘‹•ƒ••‘…‹ƒ–‡†™‹–Š†‡…”‡ƒ•‡†ƒʹΪ”‡•’‘•‡’ƒ––‡”ƒ†”‡†—…‡†  ƒ…–‹˜‹–›Ǥ …‘…Ž—•‹‘ǡ͸‹•‡š’”‡••‡†‹’ƒ…”‡ƒ–‹…•ƒ†‘†—Žƒ–‡• …‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘˜‹ƒƒʹΪǦ†‡’‡†‡–‡…Šƒ‹•ǡ™Š‹…Š‹•ƒ……‘’ƒ‹‡†„›  ƒ…–‹˜ƒ–‹‘Ǥ

Cite as Bioscience Reports (2016) DOI: 10.1042/BSR20160106

Copyright 2016 The Author(s). This is an Accepted Manuscript; not the final Version of Record. You are encouraged to use the final Version of Record that, when published, will replace this manuscript and be freely available under a Creative Commons licence. All other rights reserved.

7539 PRGXODWHV SUROLIHUDWLRQ RI KXPDQ SDQFUHDWLF QHXURHQGRFULQH %21 WXPRU FHOOV  0 6NU]\SVNLD 3 $ .RáRG]LHMVNLDE 6 0HUJOHUF 1 .KDMDYLG .: 1RZDND 0= 6WURZVNLEH 

D

'HSDUWPHQW RI $QLPDO 3K\VLRORJ\ DQG %LRFKHPLVWU\ 3R]QDĔ 8QLYHUVLW\ RI /LIH 6FLHQFHV

 3R]QDĔ 3RODQG

E

'HSDUWPHQW RI +HSDWRORJ\ DQG *DVWURHQWHURORJ\ DQG

,QWHUGLVFLSOLQDU\ &HQWUH RI 0HWDEROLVP (QGRFULQRORJ\ F'HSDUWPHQW RI 2SKWKDOPRORJ\ &KDULWp 8QLYHUVLW\ 0HGLFLQH %HUOLQ *HUPDQ\'LDEHWHV DQG 0HWDEROLVP &KDULWp8QLYHUVLW\ 0HGLFLQH %HUOLQ  %HUOLQ *HUPDQ\

G

,QVWLWXWH IRU ([SHULPHQWDO 3HGLDWULF

(QGRFULQRORJ\&KDULWH8QLYHUVLW\0HGLFLQH%HUOLQ%HUOLQ*HUPDQ\H0HGLFDO&OLQLF 'HSDUWPHQWRI*DVWURHQWHURORJ\(OEODQGNOLQLN0HLVVHQ*HUPDQ\         &RUUHVSRQGLQJDXWKRU 0DWKLDV=6WURZVNL0'3K' 'HSDUWPHQW RI +HSDWRORJ\ DQG *DVWURHQWHURORJ\  WKH ,QWHUGLVFLSOLQDU\ &HQWUH RI 0HWDEROLVP(QGRFULQRORJ\'LDEHWHVDQG0HWDEROLVP &KDULWp8QLYHUVLW\0HGLFLQH%HUOLQ &DPSXV9LUFKRZ.OLQLNXP $XJXVWHQEXUJHU3ODW] %HUOLQ*HUPDQ\ 7HO )D[ (PDLOPDWKLDVVWURZVNL#FKDULWHGH ϭ 

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Ź 7539 PRGXODWHV &D LQIOX[ LQ %21 FHOOV Ź 7539 UHJXODWHV 1)$7 DFWLYLW\ LQ %21 FHOOV Ź7539 GRZQUHJXODWLRQ DWWHQXDWHV%21FHOOJURZWK 6KRUWWLWOH7539PRGXODWHVSDQFUHDWLF1(7VSUROLIHUDWLRQ .H\ZRUGV7539%214*3QHXURHQGRFULQHWXPRUSUROLIHUDWLRQFDOFLXP $EEUHYLDWLRQV %UG8EURPRGHR[\XULGLQH&FQGF\FOLQ'&FQGF\FOLQ'*$3'+JO\FHUDOGHK\GH SKRVSKDWH

GHK\GURJHQDVH

077

GLPHWK\OWKLD]RO\O GLSKHQ\OWHWUD]ROLXP

EURPLGH 1(7 QHXURHQGRFULQH WXPRU 7539 WUDQVLHQW UHFHSWRU SRWHQWLDO FDWLRQ FKDQQHO YDQLOORLGVXEIDPLO\PHPEHUQWQRQWDUJHWLQJVL51$1)$7QXFOHDUIDFWRURIDFWLYDWHG7 FHOOV  

Ϯ 

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ϯ 

0DWHULDODQGPHWKRGV  0DWHULDOV $OO FHOO FXOWXUH PHGLD DQG VXSSOHPHQWV ZHUH SXUFKDVHG IURP %LRFKURP $* %HUOLQ *HUPDQ\ 8QOHVVRWKHUZLVHVWDWHGDOORWKHUUHDJHQWVZHUHIURP6LJPD$OGULFK'HLVHQKRIHQ *HUPDQ\  3ULPDU\ UDEELW DQWL7539 DQWLERG\ ZDV SXUFKDVHG IURP 6DQWD &UX] %LRWHFKQRORJ\ 'DOODV 7H[DV 86$  0RXVH ȕDFWLQ DQG DOO VHFRQGDU\ DQWLERGLHV ZHUH SXUFKDVHGIURP6LJPD$OGULFK'HLVHQKRIHQ*HUPDQ\  &HOOFXOWXUH %21FHOOVZHUHIURP'U&RXUWQH\07RZQVHQG-U8QLYHUVLW\RI7H[DV0HGLFDO%UDQFK 7H[DV 86$  4*3 FHOOV ZHUH IURP -DSDQHVH +HDOWK 6FLHQFHV )RXQGDWLRQ 2VDND -DSDQ %21FHOOVZHUHFXOWXUHGLQ'0(0+DP V)4*3FHOOVDQG/&&LQ530,PHGLXP DWƒ&LQDKXPLGLILHGDWPRVSKHUH&2DLU $OOH[SHULPHQWVZHUHSHUIRUPHGLQ PHGLXP FRQWDLQLQJ  )%6  N8O SHQLFLOOLQ DQG  PJO VWUHSWRP\FLQ  VL51$WUDQVIHFWLRQ %21 FHOOV ZHUH WUDQVIHFWHG ZLWK VL51$ XVLQJ +L3HUIHFW UHDJHQW 4LDJHQ +LOGHQ *HUPDQ\ DFFRUGLQJWRWKHPDQXIDFWXUHU¶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—JRI51$XVLQJ+LJKFDSDFLW\F'1$UHYHUVHWUDQVFULSWLRQNLW /LIHWHFKQRORJLHV*UDQG,VODQG1&D@LZHUHFRQILUPHGGXULQJWKHILUVWPLQ IROORZHGE\DQLVRRVPRWLFUHSODFHPHQWZLWKD&DIUHH5LQJHUOLNHVROXWLRQP0(*7$  $IWHUPLQXWHVP0&DZDVDGGHGWRLQFUHDVH>&D@L7KHUHYHUVLELOLW\RI&DFKDQJHV LVDQLQGLFDWRURIFHOOYLDELOLW\DQGIXQFWLRQDOUHOHYDQFHRIWKH&DVHQVLQJYLD&DFKDQQHOV VXFKDV7539 5HVXOWVDUHSUHVHQWHGDVPHDQWUDFHVRIII“6(0 

ϱ 

'HWHUPLQDWLRQRI1)$7DFWLYLW\ 7KHFRQVHTXHQFHVRI7539GRZQUHJXODWLRQLQ%21FHOOVRQ1)$7DFWLYLW\ZHUHDVVHVVHG XVLQJ1)$7UHSRUWHUDVVD\4LDJHQ+LOGHQ*HUPDQ\ KDIWHU7539VL51$WUDQVIHFWLRQ DVSUHYLRXVO\GHVFULEHGLQRXUHDUOLHUVWXG\  'HWHUPLQDWLRQRIFHOOSUROLIHUDWLRQ &HOO SUROLIHUDWLRQ ZDV DVVHVVHG XVLQJ D &HOO 3UROLIHUDWLRQ (/,6$ %UG8 FRORULPHWULF NLW 5RFKH'LDJQRVWLFV3HQ]EHUJ*HUPDQ\ ,QEULHI%21FHOOVZHUHVHHGHGLQZHOOSODWHV DQGWUDQVIHFWHGZLWKQWRU7539VL51$$IWHURUK%UG8VROXWLRQ—0 ZDV DGGHG DQG FHOOV ZHUH LQFXEDWHG IRU  K 7KH DPRXQW RI %U'8 LQFRUSRUDWLRQ LQWR '1$ ZDV GHWHUPLQHGDFFRUGLQJWRPDQXIDFWXUHU¶VLQVWUXFWLRQ 'HWHUPLQDWLRQRIFHOOYLDELOLW\ 7R GHWHUPLQH YLDEOH FHOOV 077 DVVD\ ZDV SHUIRUPHG &HOOV WUDQVIHFWHG HLWKHU ZLWK QW RU 7539VL51$ZHUHDQDO\]HGXVLQJ077DVVD\077VROXWLRQZDVDGGHGWRWKH ZHOOV PJPO   K DIWHU WUDQVIHFWLRQ RI FHOOV HLWKHU ZLWK QW RU 7539 VL51$ 7KHQ FHOOV ZHUH LQFXEDWHG ZLWK 077 IRU  K 7KHUHDIWHU PHGLXP ZDV UHPRYHG IURP ZHOOV DQG IRUPD]DQ FU\VWDOVZHUHGLVVROYHGLQ—O'0622SWLFDOGHQVLW\RIVDPSOHVZDVPHDVXUHGDWDQG  QP ZDYH OHQJWKV XVLQJ 6\QHUJ\  0XOWL0RGH 0LFURSODWH 5HDGHU %LR7HN :LQRRVNL 9786$   &HOOF\FOHDQDO\VLV 7KHFRQVHTXHQFHVRI7539GRZQUHJXODWLRQLQ%21FHOOVRQFHOOF\FOHZHUHGHWHUPLQHG XVLQJSURSLGLXPLRGLGH3, VWDLQLQJKDIWHUVL51$WUDQVIHFWLRQDVGHVFULEHG   6WDWLVWLFDODQDO\VLV 'DWDZHUHDQDO\]HGXVLQJ$129$IROORZHGE\WKH%RQIHUURQLWHVW3YDOXHV S  

 7KH 6WXGHQW¶V WWHVW SDUDPHWULF WZRWDLOHG WWHVW  ZDV XVHG IRU VWDWLVWLFDO VLJQLILFDQFH GHWHUPLQDWLRQ EHWZHHQ WZR VHWV RI GDWD )RU WKH HYDOXDWLRQ RI FDOFLXP LPDJLQJ H[SHULPHQWVVLJQLILFDQFHZDVGHWHUPLQHGXVLQJ6WXGHQW¶VWWHVWIRUSDLUHGDQGXQSDLUHGGDWD SYDOXHV WZRWDLOHG  SURYLGHG WKH\ SDVVHG D QRUPDOLW\ WHVW DFFRUGLQJ WR .ROPRJRURY 6PLUQRY,IWKHQRUPDOLW\WHVWIDLOHGQRQSDUDPHWULFWHVWVZHUHXVHG3UREDELOLWLHVRIS ϲ 

LQGLFDWHG E\ DVWHULVNV   DQG KDVK WDJV   ZHUH FRQVLGHUHG WR EH VLJQLILFDQW 5HVXOWV DUH VKRZQDVPHDQV“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“V WR“Q SW V LQQWVL51$WUDQVIHFWHG%21FHOOV)LJ$% ,QWKHSUHVHQFH RIP0H[WUDFHOOXODU&DIILQFUHDVHGDERYHWKHEDVHOLQH“Q W   V  ,Q FHOOV ZLWK GRZQUHJXODWHG 7539 QR FKDQJH LQ II ZDV GHWHFWHG LQ WKH &DIUHHVROXWLRQXQWLOVDQGRQO\DYHU\VOLJKWGHFUHDVHWR“ZDVUHFRUGHGDW VQ  $IWHUUHSODFHPHQWZLWKWKH&DVROXWLRQWKHIOXRUHVFHQFHUDWLRLQFUHDVHGEDFN WR WKH EDVHOLQH 7KXV FKDQJHV RI >&D@LLQ D &DIUHH DQG D &D FRQWDLQLQJ VROXWLRQ ZHUH FRPSOHWHO\LQKLELWHGLQ7539VL51$WUDQVIHFWHGFHOOVDVFRPSDUHGWRQWWUDQVIHFWHG%21 FHOOVQ S   ϳ 

7539PRGXODWHVSDQFUHDWLF%211(7FHOOSUROLIHUDWLRQ 1H[W ZH H[DPLQHG WKH HIIHFWV RI 7539 GRZQUHJXODWLRQ RQ %21 FHOO SUROLIHUDWLRQ $V VKRZQ LQ )LJ $ DQG % GRZQUHJXODWLRQ RI 7539 SURWHLQ SURGXFWLRQ DWWHQXDWHG %21 FHOOSUROLIHUDWLRQ7RIXUWKHUFRQILUPWKHUROHRI7539LQFRQWUROOLQJ%21FHOOJURZWKZH DQDO\]HG FHOO F\FOH LQ QW DQG 7539 VL51$WUDQVIHFWHG FHOOV $V VKRZQ LQ )LJ & WKH QXPEHU RI FHOOV LQ * SKDVH LQFUHDVHG DIWHU GRZQUHJXODWLRQ RI 7539 ,Q FRQWUDVW D GHFUHDVHGQXPEHURIFHOOVLQ6DQG*0SKDVHVZDVGHWHFWHGDIWHUH[SHULPHQWDOUHGXFWLRQ RI7539SURWHLQSURGXFWLRQ 1H[WZHHYDOXDWHGWKHHIIHFWVRI7539GRZQUHJXODWLRQRQF\FOLQ'&&1' F\FOLQ' &&1'  DQG F\FOLQGHSHQGHQW NLQDVH  &'.  ,PSRUWDQWO\ WKHVH JHQHV DUH UHOHYDQW IRU WKHUHJXODWLRQRIFDOFLXPGHSHQGHQWFHOOSUROLIHUDWLRQ :HIRXQGWKDW7539VL51$ WUDQVIHFWHGFHOOVKDGORZHUH[SUHVVLRQRI&&1'DQG&'.ZKHUHDVH[SUHVVLRQRI&&1' UHPDLQHG VWDEOH DV FRPSDUHG ZLWK QW VL51$WUDQVIHFWHG FHOOV )LJ ')  2YHUDOO WKHVH UHVXOWVLQGLFDWHWKDW7539VWLPXODWHV%21FHOOSUROLIHUDWLRQ  1)$7PRGXODWHV%21FHOOJURZWKDQGYLDELOLW\ 3UHYLRXV VWXGLHV LQGLFDWHG WKDW 7539 PRGXODWHV SUROLIHUDWLRQ RI /1&D3 KXPDQ SURVWDWH DGHQRFDUFLQRPDRU,16(FHOOVYLD1)$7GHSHQGHQWPHFKDQLVPV $VGHPRQVWUDWHG LQ)LJ$%21DQG/&&FHOOVH[SUHVVDOOFDOFLXPVHQVLWLYH1)$7LVRIRUPV6LQFHWKH UROH RI 1)$7 DW FRQWUROOLQJ 1(7 FHOO SUROLIHUDWLRQ LV XQNQRZQ ZH DVVHVVHG ZKHWKHU WZR GLIIHUHQWZHOOFKDUDFWHUL]HGSKDUPDFRORJLFDOLQKLELWRUVRI1)$7DFWLYLW\F\FORVSRULQH$DQG ).    FDQ LQIOXHQFH %21 FHOO JURZWK $V H[SHFWHG ERWK F\FORVSRULQH DV ZHOO DV ). DWWHQXDWHG 1)$7 DFWLYLW\ )LJ %  )XUWKHUPRUH ERWK 1)$7 LQKLELWRUV UHGXFHG %21 FHOO SUROLIHUDWLRQ LQ D GRVHGHSHQGHQW IDVKLRQ )LJ &)  ,Q VXPPDU\ WKHVH GDWD VKRZWKDW1)$7VWLPXODWHV%21FHOOJURZWK 7539PRGXODWHV1)$7DFWLYLW\EXWQRW1)$7H[SUHVVLRQ 7539FDQPRGXODWH1)$7DFWLYLW\LQ&DFRKXPDQHSLWKHOLDOFRORUHFWDODGHQRFDUFLQRPD  /1&D3 DQG ,16( FHOOV     7KHUHIRUH ZH H[DPLQHG WKH HIIHFW RI 7539 GRZQUHJXODWLRQ RQ 1)$7 DFWLYLW\ LQ %21 FHOOV $V D UHVXOW 7539 VL51$WUDQVIHFWHG %21 FHOOV KDG GHFUHDVHG 1)$7 DFWLYLW\ DV FRPSDUHG WR QW VL51$WUDQVIHFWHG FHOOV )LJ $  ,Q FRQWUDVW H[SUHVVLRQ RI 1)$7V ZDV QRW DIIHFWHG E\ 7539 VL51$ GRZQUHJXODWLRQ )LJ %  7R FRQILUP ZKHWKHU 7539 PRGXODWHV %21 FHOO SUROLIHUDWLRQ YLD 1)$7 ϴ 

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

ϵ 

7KHUH LV FRQYLQFLQJ HYLGHQFH LQGLFDWLQJ WKDW 7539 PRGXODWHV 1)$7 DFWLYLW\    7UDQVFULSWLRQ IDFWRUV RI WKH1)$7 IDPLO\ 1)$7V  LQLWLDOO\ GHVFULEHG LQ LPPXQH FHOOV DUH UHTXLUHGIRUFDOFLXPGHSHQGHQWLPPXQHUHVSRQVHV 0RUHRYHU1)$7VZHUHDOVRGHWHFWHG LQRWKHUWLVVXHVLQFOXGLQJKHDUWEUDLQDQGSDQFUHDV $OWKRXJKLWZDVDOUHDG\UHSRUWHG WKDW 1)$7 H[SUHVVLRQ DQG DFWLYLW\ DUH UHTXLUHG IRU SDQFUHDWLF ȕ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ϭϬ 

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ȕHVWUDGLRO ,PSRUWDQWO\ȕHVWUDGLROZDVGHPRQVWUDWHG WRLQFUHDVHEUHDVWFDQFHUFHOOSUROLIHUDWLRQ ZKLFKVKRZH[WUHPHO\KLJKGHQVLW\RI7539 H[SUHVVLRQ 7KHUHIRUHLWLVSRVVLEOHWKDWȕHVWUDGLROPD\SOD\DUROHLQUHJXODWLQJFHOO JURZWKDOVRLQSDQFUHDWLF1(7FHOOV )XUWKHU VWXGLHV XVLQJ SULPDU\ 1(7V DUH UHTXLUHG WR HYDOXDWH WKH SRWHQWLDO FOLQLFDO UHOHYDQFH RI RXU UHVXOWV )RU H[DPSOH D OLQN EHWZHHQ 7539 DQG YDULRXV JURZWK IDFWRUV UHOHYDQWLQ1(7VSUROLIHUDWLRQVKRXOGEHDVVHVVHGLQWKHIXWXUH

ϭϭ 

,QVXPPDU\RXUVWXG\VKRZVIRUWKHILUVWWLPHWKDW7539LVH[SUHVVHGLQSDQFUHDWLF 1(7V ZKHUH LW PRGXODWHV LQWUDFHOOXODU FDOFLXP FRQFHQWUDWLRQ )XUWKHUPRUH ZH VKRZ WKDW VXSSUHVVLRQ RI 7539 SURWHLQ SURGXFWLRQ LV DVVRFLDWHG ZLWK LPSDLUHG SDQFUHDWLF 1(7 FHOO JURZWK  $FNQRZOHGJPHQW :HDFNQRZOHGJH3URI'U%HUWUDP:LHGHQPDQQDQG'U&DUVWHQ*U|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ϭϮ 

5HIHUHQFHV ϭ͘ ŚƵ͕>ƵŽ͕DĂ^͕>ŝƵ͘dZWĐŚĂŶŶĞůƐĂŶĚƚŚĞŝƌŝŵƉůŝĐĂƚŝŽŶƐŝŶŵĞƚĂďŽůŝĐĚŝƐĞĂƐĞƐ͘WĨůƵŐĞƌƐ ƌĐŚŝǀ͗ƵƌŽƉĞĂŶũŽƵƌŶĂůŽĨƉŚLJƐŝŽůŽŐLJ͘ϮϬϭϭ͖ϰϲϭ;ϮͿ͗ϮϭϭͲϮϯ͘ Ϯ͘ ^ŵĂŶŝ d͕ ^ŚĂƉŽǀĂůŽǀ '͕ ^ŬƌLJŵĂ Z͕ WƌĞǀĂƌƐŬĂLJĂ E͕ ZŽƐĂĚŽ :͘ &ƵŶĐƚŝŽŶĂů ĂŶĚ ƉŚLJƐŝŽƉĂƚŚŽůŽŐŝĐĂů ŝŵƉůŝĐĂƚŝŽŶƐŽĨdZWĐŚĂŶŶĞůƐ͘ŝŽĐŚŝŵŝĐĂ Ğƚ ďŝŽƉŚLJƐŝĐĂ ĂĐƚĂ͘ϮϬϭϱ͖ϭϴϱϯ;ϴͿ͗ϭϳϳϮͲ ϴϮ͘ ϯ͘ zĂŶŐz͕zĂŶŐ,͕tĂŶŐ͕DĞƌŐůĞƌ^͕tŽůŽƐŝŶ:D͕ZĞŝŶĂĐŚW^͘&ƵŶĐƚŝŽŶĂůdZWsϭĞdžƉƌĞƐƐŝŽŶŝŶ ŚƵŵĂŶĐŽƌŶĞĂůĨŝďƌŽďůĂƐƚƐ͘džƉĞƌŝŵĞŶƚĂůĞLJĞƌĞƐĞĂƌĐŚ͘ϮϬϭϯ͖ϭϬϳ͗ϭϮϭͲϵ͘ ϰ͘ DĞƌŐůĞƌ^͕^ŬƌnjLJƉƐŬŝD͕^ĂƐƐĞŬD͕WŝĞƚƌnjĂŬW͕WƵĐĐŝ͕tŝĞĚĞŶŵĂŶŶ͕ĞƚĂů͘dŚĞƌŵŽͲƐĞŶƐŝƚŝǀĞ ƚƌĂŶƐŝĞŶƚ ƌĞĐĞƉƚŽƌ ƉŽƚĞŶƚŝĂů ǀĂŶŝůůŽŝĚ ĐŚĂŶŶĞůͲϭ ƌĞŐƵůĂƚĞƐ ŝŶƚƌĂĐĞůůƵůĂƌ ĐĂůĐŝƵŵ ĂŶĚ ƚƌŝŐŐĞƌƐ ĐŚƌŽŵŽŐƌĂŶŝŶ  ƐĞĐƌĞƚŝŽŶ ŝŶ ƉĂŶĐƌĞĂƚŝĐ ŶĞƵƌŽĞŶĚŽĐƌŝŶĞ KEͲϭ ƚƵŵŽƌ ĐĞůůƐ͘ ĞůůƵůĂƌ ƐŝŐŶĂůůŝŶŐ͘ ϮϬϭϮ͖Ϯϰ;ϭͿ͗ϮϯϯͲϰϲ͘ ϱ͘ tĂŶŝŶŐ:͕sƌŝĞŶƐ:͕KǁƐŝĂŶŝŬ'͕^ƚƵǁĞ>͕DĂůůLJ^͕&ĂďŝĂŶ͕ĞƚĂů͘ŶŽǀĞůĨƵŶĐƚŝŽŶŽĨĐĂƉƐĂŝĐŝŶͲ ƐĞŶƐŝƚŝǀĞdZWsϭĐŚĂŶŶĞůƐ͗ŝŶǀŽůǀĞŵĞŶƚŝŶĐĞůůŵŝŐƌĂƚŝŽŶ͘ĞůůĐĂůĐŝƵŵ͘ϮϬϬϳ͖ϰϮ;ϭͿ͗ϭϳͲϮϱ͘ ϲ͘ >ĞŚĞŶΖŬLJŝs͕&ůŽƵƌĂŬŝƐD͕^ŬƌLJŵĂZ͕WƌĞǀĂƌƐŬĂLJĂE͘dZWsϲĐŚĂŶŶĞůĐŽŶƚƌŽůƐƉƌŽƐƚĂƚĞĐĂŶĐĞƌ ĐĞůůƉƌŽůŝĨĞƌĂƚŝŽŶǀŝĂĂ;ϮнͿͬE&dͲĚĞƉĞŶĚĞŶƚƉĂƚŚǁĂLJƐ͘KŶĐŽŐĞŶĞ͘ϮϬϬϳ͖Ϯϲ;ϱϮͿ͗ϳϯϴϬͲϱ͘ ϳ͘ WĞŶŐ:͕ƌŽǁŶD͕,ĞĚŝŐĞƌD͘ƉŝƚŚĞůŝĂůĂϮнĞŶƚƌLJĐŚĂŶŶĞůƐ͗ƚƌĂŶƐĐĞůůƵůĂƌĂϮнƚƌĂŶƐƉŽƌƚ ĂŶĚďĞLJŽŶĚ͘dŚĞ:ŽƵƌŶĂůŽĨƉŚLJƐŝŽůŽŐLJ͘ϮϬϬϯ͖ϱϱϭ;WƚϯͿ͗ϳϮϵͲϰϬ͘ ϴ͘ WĞŶŐ:͕ŚĞŶy͕ĞƌŐĞƌhs͕sĂƐƐŝůĞǀWD͕dƐƵŬĂŐƵĐŚŝ,͕ƌŽǁŶD͕ĞƚĂů͘DŽůĞĐƵůĂƌĐůŽŶŝŶŐ ĂŶĚ ĐŚĂƌĂĐƚĞƌŝnjĂƚŝŽŶ ŽĨ Ă ĐŚĂŶŶĞůͲůŝŬĞ ƚƌĂŶƐƉŽƌƚĞƌ ŵĞĚŝĂƚŝŶŐ ŝŶƚĞƐƚŝŶĂů ĐĂůĐŝƵŵ ĂďƐŽƌƉƚŝŽŶ͘ dŚĞ :ŽƵƌŶĂůŽĨďŝŽůŽŐŝĐĂůĐŚĞŵŝƐƚƌLJ͘ϭϵϵϵ͖Ϯϳϰ;ϯϮͿ͗ϮϮϳϯϵͲϰϲ͘ ϵ͘ WĞŶŐ :͕ ŚƵĂŶŐ >͕ ĞƌŐĞƌ hs͕ ĚĂŵ ZD͕ tŝůůŝĂŵƐ :͕ ƌŽǁŶ D͕ Ğƚ Ăů͘ Ădϭ ĞdžƉƌĞƐƐŝŽŶ ĐŽƌƌĞůĂƚĞƐ ǁŝƚŚ ƚƵŵŽƌ ŐƌĂĚĞ ŝŶ ƉƌŽƐƚĂƚĞ ĐĂŶĐĞƌ͘ ŝŽĐŚĞŵŝĐĂů ĂŶĚ ďŝŽƉŚLJƐŝĐĂů ƌĞƐĞĂƌĐŚ ĐŽŵŵƵŶŝĐĂƚŝŽŶƐ͘ϮϬϬϭ͖ϮϴϮ;ϯͿ͗ϳϮϵͲϯϰ͘ ϭϬ͘ ZĂƉŚĂĞůD͕>ĞŚĞŶΖŬLJŝs͕sĂŶĚĞŶďĞƌŐŚĞD͕ĞĐŬ͕ĞŚĞŶΖŬLJŝs͕ ZĂƉŚĂĞů D͕ KƵůŝĚŝ ͕&ůŽƵƌĂŬŝƐ D͕ĞŽ ͕ DĂůŽƌŶŝ >͘ LJĐůŝŶͲĚĞƉĞŶĚĞŶƚ ŬŝŶĂƐĞ ϰͬϲ ŝŶŚŝďŝƚŽƌƐ ŝŶ ďƌĞĂƐƚ ĐĂŶĐĞƌ ƚŚĞƌĂƉLJ͘ƵƌƌĞŶƚŽƉŝŶŝŽŶŝŶŽŶĐŽůŽŐLJ͘ϮϬϭϰ͖Ϯϲ;ϲͿ͗ϱϲϴͲϳϱ͘ Ϯϳ͘ ZĂŽ ͕ >ƵŽ ͕ ,ŽŐĂŶ W'͘ dƌĂŶƐĐƌŝƉƚŝŽŶ ĨĂĐƚŽƌƐ ŽĨ ƚŚĞ E&d ĨĂŵŝůLJ͗ ƌĞŐƵůĂƚŝŽŶ ĂŶĚ ĨƵŶĐƚŝŽŶ͘ ŶŶƵĂůƌĞǀŝĞǁŽĨŝŵŵƵŶŽůŽŐLJ͘ϭϵϵϳ͖ϭϱ͗ϳϬϳͲϰϳ͘ Ϯϴ͘ DĂĐŝĂŶ&͘E&dƉƌŽƚĞŝŶƐ͗ŬĞLJƌĞŐƵůĂƚŽƌƐŽĨdͲĐĞůůĚĞǀĞůŽƉŵĞŶƚĂŶĚĨƵŶĐƚŝŽŶ͘EĂƚƵƌĞƌĞǀŝĞǁƐ /ŵŵƵŶŽůŽŐLJ͘ϮϬϬϱ͖ϱ;ϲͿ͗ϰϳϮͲϴϰ͘ Ϯϵ͘ WĞƚƌŽŶŝůůŝ s͕ EŝĐŽůůŝ ͕ ŽƐƚĂŶƚŝŶŝ W͕ ŽůŽŶŶĂ Z͕ ĞƌŶĂƌĚŝ W͘ ZĞŐƵůĂƚŝŽŶ ŽĨ ƚŚĞ ƉĞƌŵĞĂďŝůŝƚLJ ƚƌĂŶƐŝƚŝŽŶƉŽƌĞ͕ĂǀŽůƚĂŐĞͲĚĞƉĞŶĚĞŶƚŵŝƚŽĐŚŽŶĚƌŝĂůĐŚĂŶŶĞůŝŶŚŝďŝƚĞĚďLJĐLJĐůŽƐƉŽƌŝŶ͘ŝŽĐŚŝŵŝĐĂĞƚ ďŝŽƉŚLJƐŝĐĂĂĐƚĂ͘ϭϵϵϰ͖ϭϭϴϳ;ϮͿ͗ϮϱϱͲϵ͘ ϯϬ͘ >ĂďůĂŶĐŚĞ ^͕ ŽƚƚĞƚͲZŽƵƐƐĞůůĞ ͕ >ĂŵĂƌĐŚĞ &͕ ĞŶŚĂŵŽƵ Wz͕ ,Ăůŝŵŝ ^͕ >ĞǀĞƌǀĞ y͕ Ğƚ Ăů͘ WƌŽƚĞĐƚŝŽŶŽĨƉĂŶĐƌĞĂƚŝĐ/E^ͲϭďĞƚĂͲĐĞůůƐĨƌŽŵŐůƵĐŽƐĞͲĂŶĚĨƌƵĐƚŽƐĞͲŝŶĚƵĐĞĚĐĞůůĚĞĂƚŚďLJŝŶŚŝďŝƚŝŶŐ ŵŝƚŽĐŚŽŶĚƌŝĂů ƉĞƌŵĞĂďŝůŝƚLJ ƚƌĂŶƐŝƚŝŽŶ ǁŝƚŚ ĐLJĐůŽƐƉŽƌŝŶ  Žƌ ŵĞƚĨŽƌŵŝŶ͘ Ğůů ĚĞĂƚŚ Θ ĚŝƐĞĂƐĞ͘ ϮϬϭϭ͖Ϯ͗Ğϭϯϰ͘ ϯϭ͘ tƵE͕>ŝtE͕^ŚƵtY͕>ǀz͕:ŝĂ>͘ůŽĐŬŝŶŐƚŚĞŵŝƚŽĐŚŽŶĚƌŝĂůƉĞƌŵĞĂďŝůŝƚLJƚƌĂŶƐŝƚŝŽŶƉŽƌĞ ǁŝƚŚ ĐLJĐůŽƐƉŽƌŝŶĞͲ ĐĂŶ ƌĞƐƚŽƌĞ ĐĂƌĚŝŽƉƌŽƚĞĐƚŝŽŶ ŽĨ ŝƐĐŚĞŵŝĐ ƉŽƐƚĐŽŶĚŝƚŝŽŶŝŶŐ ŝŶ ŚLJƉĞƌĐŚŽůĞƐƚĞƌŽůĞŵŝĐ ƌĂƚ ŚĞĂƌƚ͘ ƵƌŽƉĞĂŶ ƌĞǀŝĞǁ ĨŽƌ ŵĞĚŝĐĂů ĂŶĚ ƉŚĂƌŵĂĐŽůŽŐŝĐĂů ƐĐŝĞŶĐĞƐ͘ ϮϬϭϱ͖ϭϵ;ϯͿ͗ϰϰϲͲϱϰ͘ ϯϮ͘ &D@LZDVSHUIRUPHGDIWHUOLJKWJUD\ EDUV DQGVGDUNJUD\EDUV  )LJ  (IIHFWV RI 7539 GRZQUHJXODWLRQ RQ %21 FHOOV SUROLIHUDWLRQ DQG FHOO F\FOH $ %21FHOOSUROLIHUDWLRQDVVHGDQGKDIWHUWUDQVIHFWLRQZLWKVL51$%7KHQXPEHU RIYLDEOH%21FHOOVVWXGLHGKDIWHUWUDQVIHFWLRQZLWKVL51$XVLQJ077DVVD\&%21 FHOOF\FOHDQDO\VLVKDIWHUVL51$WUDQVIHFWLRQ')([SUHVVLRQRI&&1'&&1'DQG &'.LQFHOOVWUDQVIHFWHGZLWKVL51$IRUK5HVXOWVDUHWKHPHDQ“6(0REWDLQHGIURP DWOHDVWQ  )LJ(IIHFWVRI1)$7VXSSUHVVLRQRQ%21FHOOVSUROLIHUDWLRQ$([SUHVVLRQRI1)$7VLQ %21DQG/&&FHOOV%1)$7DFWLYLW\LQ%21FHOOVWUHDWHGZLWK—0).RU —0F\FORVSRULQH$&V$ IRUK%21FHOOSUROLIHUDWLRQWUHDWHGZLWK).& RU&V$ ' IRUK7KHQXPEHURIYLDEOH%21FHOOVDVVHGDIWHULQFXEDWLRQLQWKHSUHVHQFHRI ).( RU&V$) 5HVXOWVDUHWKHPHDQ“6(0REWDLQHGIURPDWOHDVWQ  )LJ  1)$7 DFWLYLW\ DQG H[SUHVVLRQ LQ %21 FHOOV ZLWK GRZQUHJXODWHG 7539 1)$7 ϭϱ 

DFWLYLW\ $  DQG H[SUHVVLRQ %  HYDOXDWHG LQ FHOOV WUDQVIHFWHG HLWKHU ZLWK QW ZKLWH EDUV  RU 7539VL51$JUD\EDUV IRUK&(IIHFWVRIKWUHDWPHQWZLWK).RQ%21FHOOV WUDQVIHFWHGZLWKQWZKLWHEDUV RU7539VL51$JUD\EDUV IRUK5HVXOWVDUHWKHPHDQ“ 6(0Q  

ϭϲ